GENETIC DETERMINANTS OF REPRODUCTIVE ISOLATION IN XENOPUS
by
JOHN H. MALONE
Presented to the Faculty of the Graduate School of
The University of Texas at Arlington in Partial Fulfillment
of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
THE UNIVERSITY OF TEXAS AT ARLINGTON
August 2007
Copyright © by John H. Malone 2007
All Rights Reserved
DEDICATION
To the wonder of the biological world
ACKNOWLEDGEMENTS
There are many people to thank that have helped me to fulfill my lifelong dream
of becoming a professional scientist. This dream to be performed in the context of
current society requires the acquistion of advanced degrees and as such with this
process has come tremendous sacrifice. I thank the five folks that influenced and
fostered my pursuit of biology and consequently have stripped any possibility of me
becoming financial wealthy and probably any hope of normality:
my 7th grade
scienceteacher David Yaffie for thought provoking discussion of tuataras and living
fossils that to this day I still think about, Paul Freed, who showed me the wonders of
amphibians and reptiles by allowing me to interact with professional herpetologists at
the Houston Zoo, Ted Hollingsworth and Dennis Jones who both provided me my first
jobs in herpetology through numerous contract projects with the Texas Parks and
Wildlife Department, and finally Dr. James R. Dixon, the reason I went to Texas A&M
Univerisy and who has provided infinite inspiration and guidance that only comes from
a wise old sage. I have benefited greatly from knowing the following folks that I
consider the best of friends; Jacob Malcom, David Laurencio, and Toby Hibbitts, have
all contributed to my professional development in uncountable ways during the
incredible times that we’ve spent exploring the truth and beauty of nature.
Most
important however, I thank my family for putting up with my incessant desire to see the
world at the expense of not being there as much as I should.
iv
Certainly the best and worst of times of my life have occurred while being a
student at UTA. I have been hugely fortunate to have had the opportunity to continue
fulfilling my passion for field work in many countries while a student at UTA. Some
may regard this as counterproductive but I disagree; seeing biology in action at scales
that I’m comfortable with has influenced my thinking in what I perceive as positive
ways. I thank Jonathan Campbell for initially taking me on as a student and providing
me with much inspiration and guidance during my tenere at UTA. I hope that I can be
half the scientist as Jonathan Campbell. I thank my committee members, Pawel
Michalak, Jonathan Campbell, Ester Betran, Paul Chippindale, Esther Betrán, Cédric
Feschotte, Jermany Marshall, and Andre Pires da Silva for serving on my committee
and providing their precious time to help foster my professional development. I would
like to especially thank my supervising professor/boss, Pawel Michalak for taking a big
risk by taking on a piddly little herpetologist to pursue the broad questions of
evolutionary biology. I have learned a lot from Pawel and I hope that my prescence in
his lab was more benefical than deteramental. I thank all my lab members and Pawel for
putting up with my intense nature. I thank Doyle Hawkins for his statistical prowess and
the awesome microarray analysis discussions we had and for a great collaboration. I
especially thank Professor R. C. Tinsley for donating specimens of Xenopus for use in
this study. He was the only person that responded to my e-mail requests for frogs and
his kindness has made everything here possible.
v
This work was conducted following the protocols of the University of TexasArlington Animal Care Committee (Protocol No. A05.001). Funding was provided by a
National Science Foundation Dissertation Improvement Grant (DEB-0508882) and two
Texas Academy of Science Student Research Grants to JHM.
July 30, 2007
vi
ABSTRACT
GENETIC DETERMINANTS OF REPRODUCTIVE ISOLATION IN XENOPUS
Publication No. ______
John H. Malone, PhD.
The University of Texas at Arlington, 2007
Supervising Professor: Dr. Pawel Michalak
Haldane’s rule states that in F1 interspecific hybrids the heterogametic sex (XY
or ZW) suffers the most dysfunctional effects of hybridization. Frogs of the genus
Xenopus have ZW sex determination yet hybrid males are completely sterile and hybrid
females are fertile, a pattern exactly opposite of Haldane’s rule. One of the most
compelling hypotheses to explain this pattern is faster male evolution. The faster male
evolution hypothesis suggests that hybrid male sterility is more common than hybrid
female sterility because of faster divergence of male reproductive genes (due to sexual
selection), or inherent sensitivity with the process of spermatogenesis. This hypothesis
emphasizes the sex of the individual regardless of which sex is heterogametic and
provides an explanation for examples that may go against Haldane’s rule. Microarray
expression data reveal numerically more male-biased genes but female-biased genes
vii
have a larger divergence in expression between species. Hybrid males (X. laevis x X.
muelleri) have a dramatically lower abundance of motile sperm, increased numbers of
undifferentiated sperm cells, and larger mature sperm cells compared to parental
species. The gene expression pattern for hybrid males shows a striking asymmetric
pattern in that relatively few genes are misexpressed between hybrids and the maternal
species (X. laevis) whereas there are dramatically more genes misexpressed between
hybrid males and the paternal species, X. muelleri. microRNA expression profiles of
hybrid males provide several microRNAs that are misregulated in hybrids and these
may play a role in the sterility pattern. Expression analysis of hybrid females reveals
about 20 times more misexpression compared to hybrid males. A novel sex-reversal
experiment shows that the sterility phenotype does not depend on genetic background
but rather on physiological sex; hybrid males are sterile and hybrid females are fertile
irrespective of their genetic background. Gene expression profiles match these adult
phenotypes in that no genes were detected to be differentially expressed. Our results
suggest that faster male evolution and in particular, the sensitive spermatogenesis
component, is the most likely mechanism operating to produce patterns of reproductive
isolation in Xenopus.
viii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS...................................................................................
iv
ABSTRACT .........................................................................................................
vii
LIST OF ILLUSTRATIONS.................................................................................
xiii
LIST OF TABLES ................................................................................................
xvii
Chapter
Page
1. FERTILITY AND MORPHOLOGICAL
CHARACTERISTICS OF PARENTAL
XENOPUS LAEVIS, XENOPUS MUELLERI
AND F1 HYBRIDS…… ..........................................................................
1
1.1 Abstract…………….. ..........................................................................
1
1.2 Introduction…………..........................................................................
2
1.3 Materials and Methods.........................................................................
4
1.3.1 Crosses……. .........................................................................
4
1.3.2 Fertility of Hybrids ................................................................
6
1.3.3 Karyotyping ..........................................................................
6
1.3.4 Morphological Variaation… ..................................................
7
1.4 Results………………..........................................................................
10
1.5 Discussion………….. ..........................................................................
15
ix
2. SEX-BIASED GENE EXPRESSION IN A ZW SEX
DETERMINATION SYSTEM ..................................................................
23
2.1 Abstract…………….. ..........................................................................
23
2.2 Introduction………….. ........................................................................
24
2.3 Materials and Methods.........................................................................
27
2.3.1 Animals……... ......................................................................
27
2.3.2 RNA Extraction and Microarray Hybridization .....................
27
2.3.3 Data Analysis….. ..................................................................
28
2.4 Results……………………… ..............................................................
36
2.5 Discussion…………….. ......................................................................
42
3. STERILITY AND GENE EXPRESSION IN HYBRID
MALES OF XENOPUS LAEVIS AND X. MUELLERI...............................
52
3.1 Abstract……………………….............................................................
52
3.2 Introduction…………….. ....................................................................
53
3.3 Materials and Methods.........................................................................
56
3.3.1 Experimental Crosses…….....................................................
56
3.3.2 Sperm Assays……….. ..........................................................
56
3.3.3 Microarray Analysis .............................................................
59
3.3.4 Data Analysis .......................................................................
61
3.4 Results………………………… ..........................................................
62
3.4.1 Sperm Abundance……..........................................................
62
3.4.2 Sperm Motility……...............................................................
67
3.4.3 Sperm Morphology…............................................................
68
x
3.4.4 Gene Expression…................................................................
70
3.5 Discussion……………….. ..................................................................
80
3.5.1 Species-Specific Transcription Factor Hypothesis……..........
90
3.5.2 Enchancer Imbalance Hypothesis……...................................
92
3.5.3 Chromatin Imprinting Hypothesis……..... .............................
94
4. TESTIS-DERIVED MICRORNA PROFILES OF
OF AFRICAN CLAWED FROGS (XENOPUS)
AND THEIR STERILE HYBRIDS ...........................................................
98
4.1 Abstract……………………….............................................................
98
4.2 Introduction……………….. ................................................................
99
4.3 Materials and Methods.........................................................................
102
4.3.1 RNA Extraction……. ............................................................
102
4.3.2 miRCURYTM LNA microRNA Arrays…...............................
102
4.3.3 Microarray Data Processing…….. .........................................
103
4.3.4 Quantitative Real-Time PCR Validation…….. ......................
105
4.3.5 miRNA Targe Predication……..............................................
105
4.4 Results…………………………. .........................................................
106
4.4.1 Differenital Expression…….. ................................................
106
4.4.2 Quantitative Real-Time PCR Analysis…… ...........................
107
4.4.3 Predicted Targets of Candidate miRNAs…............................
108
4.5 Discussion………………………….. ...................................................
112
GENE EXPRESSION ANALYSIS OF THE OVARY
OF FERTILE HYBRID FEMALES OF X. LAEVIS
AND X. MUELLERI………….................................................................
117
5.
xi
6.
5.1 Abstract……………… .....................................................................
117
5.2 Introduction……………. ..................................................................
118
5.3 Materials and Methods......................................................................
119
5.3.1 Microarray Experiments ........................................................
119
5.3.2 Data Analysis ........................................................................
120
5.4 Results……………………. ...............................................................
121
5.5 Discussion……………… ..................................................................
129
A TEST OF THE FASTER MALE EVOLUTION
HYPOTHESIS FOR HALDANE’S RULE USING
SEX REVERSED HYBRIDS OF XENOPUS……................................... . 137
6.1 Abstract…………. ..........................................................................
137
6.2 Introduction …….. ..........................................................................
138
6.3 Materials and Methods ....................................................................
140
6.3.1 Crosses and Sex-reversal Treatment ......................................
140
6.3.2 Test Crosses ..........................................................................
143
6.3.3 RNA Extraction and Microarray Analysis..............................
143
6.4 Results……………………..............................................................
145
6.5 Discussion………………… ............................................................
146
A. SUPPLEMENTAL TABLES FOR CHAPTER 1.......................................
149
B. SUPPLEMENTAL TABLES FOR CHAPTER 4......................................
158
REFERENCES .....................................................................................................
176
BIOGRAPHICAL INFORMATION .....................................................................
225
Appendix
xii
LIST OF ILLUSTRATIONS
Figure
Page
1.1 Description of morphological characters used assay
variation in morphology among Xenopus laevis, X.
laevis x X. muelleri hybrid, X. muelleri x X. laevis
hybrid, and X. muelleri. (A) Dorsal view. (B) Ventral
view. SVL=Snout to vent length; TL=Tibiofibula length;
HW=Head width; HL=Head length; RUL=Distance of
radioulna to 3rd finger; HUL=Humerus length; FL=Femur
length; IOD=Interocular distance; IND=Internaris distance;
W=Weight in grams; LTENTL=Left tentacle length;
RTENTL=Right tentacle length.................................................................... 8
1.2
Example karyotype of backcross X. laevis x X. muelleri................................ 14
1.3
Principal component analysis of morphological
variables for Xenopus laevis, X. muelleri and F1
hybrids of both directions. The first and second
principal components explain 71.2 % and
15.7% respectively of the morphological variance. ....................................... 17
2.1
Principal components analysis of each sample
used in the analysis. The first two principal components
explained approximately 87% of the variation in
gene expression (PC1, 44.9%; PC2, 42.2%).
Closed circles, muelleri males; open circles,
laevis males; closed squares, muelleri females;
open squares, laevis females......................................................................... 31
xiii
2.2
Vector analysis of expression responses of
males vs. females in Xenopus laevis and X. muelleri
for those genes recovered as different between species
in the LPE analysis. Each point represents the sum vector
from the 288 pairwise comparisons (16 replicates in
X. laevis and 18 replicates in X. muelleri) and only
those vectors that meet the significance criteria of
α = 0.05 are included in the plot (2314 out of 2437
transcripts recovered as different between species in
the LPE analysis). Genes toward the periphery of the
plot represent the most consistent behavior among
the replicates. The total number of genes upregulated
in males vs. females are shown in red squares and the
total number of genes downregulated in males vs.
females (alternatively upregulated in females but
downregulated in males) are shown in green circles.
Black circles are for those genes that show opposite
patterns of regulation between species. There are more
male biased genes compared to female biased genes.................................... 39
3.1 Visualization of live (green) and dead (red)
sperm in sperm density assay using dual emission filter
for SYBR14 and propidium iodide. Xenopus laevis (A);
hybrid (B); X. muelleri (C); and brightfield combined
with fluorescent image of undifferentiated cells of
hybrids (D). ................................................................................................ 64
3.2 The number of total sperm cells in
Xenopus laevis (black circles) and hybrids
(striped squares) in uninjected sexually
mature males and hCG injected sexually
mature males. Error bars represent ± 1 standard error. ................................. 65
3.3
Proportion of live (green circles), dead
(red squares), and undifferentiated (black triangles)
sperm cells in Xenopus laevis (A) and hybrids (B)
in normal/uninjected compared to hCG injected
males. Error bars represent ± 1 standard error............................................. 66
3.4
Numbers of motile (green circles) and immotile
(red x’s) sperm in Xenopus laevis and hybrids in
normal/uninjected compared to hCG injected males.
Error bars represent ± 1 standard error........................................................ 67
xiv
3.5
Comparison of sperm area (µm2) in Xenopus laevis,
X. muelleri, and hybrids and representative brightfield
images of sperm. Error bars represent ± 1 standard error
and * denotes significance from a Bonferroni multiple
comparison test. Hybrids have larger sperm compared to
X. laevis and X. muelleri but sperm area does not differ
between X. laevis and X. muelleri. ............................................................. 69
3.6
Number of probesets remaining at various thresholds
from comparing the hybridization intensity of
Xenopus muelleri vs. X. laevis. .................................................................. 71
3.7
Volcano plots from FDR corrected t-tests of statistical
significance (vertical axis) against magnitude of
expression change (horizontal axis), where each point
corresponds to a gene/transcript. Expression change
(fold-change) is defined as a log2-transformed ratio of
mean nonhybrid to mean hybrid expression level.
(A) Xenopus laevis vs. Hybrids; (B) Xenpous muelleri
vs. Hybrids. The red line denotes FDR adjusted alpha 0.05.
The horizontal deviation from 0 towards the right or left
reflects hybrid underexpression or overexpression,
respectively. .............................................................................................. 73
4.1
Hy5 vs Hy3 scatterplots for the spike-in
controls. These diagrams show that two
different dyes used in spike-in controls
produced similar fluorescence signal. The
black trendline represents X=Y..................................................................104
4.2
Hierarchical clustering visualizing miRNA
overerepresentation (red color) in the parental
X. laevis relative to hybrids. Only differentially
expressed (q-value < 0.2) miRNAs were included. .....................................107
xv
5.1 Volcano plots from FDR corrected t-tests of
statistical significance (vertical axis) against
magnitude of expression change (horizontal axis),
where each point corresponds to a gene/transcript.
Expression change (fold-change) is defined as a log2transformed ratio of mean nonhybrid to mean hybrid
expression level. (A) Xenopus laevis (L) vs. Hybrids (H);
(B) Xenpous muelleri (M) vs. Hybrids (H). The red line
denotes FDR adjusted alpha 0.05. The horizontal deviation
from 0 towards the right or left reflects hybrid underexpression
or overexpression, respectively...................................................................123
6.1
Flowchart of one replicate of the sex-reversal experiment. ..........................141
6.2
A sample of unambiguous genotype determination
of sex-reversed individuals. Double bands indicate
the presence of W specific marker as amplified from
PCR reactions and indicated ZW genotype. Single band
represents Z chromosome marked and is amplified only
in ZZ genotypes. λ is 100 bp DNA ladder. Neg=Negative
control........................................................................................................142
6.3
Hierarchical clustering of gene expression data
for normal and sex-reversed hybrids of Xenopus
laevis x X. muelleri. FDR corrected moderated
t tests reveal no difference in gene expression
between normal and sex-reversed females and
normal and sex-reversed males. ♀=normal female
(ZW); ♂= normal male (ZZ); SR ♀ = sex-reversed
female (ZZ); SR ♂ = sex-reversed male (ZW). ............................................146
xvi
LIST OF TABLES
Table
Page
1.1 The proportion of eggs fertilized, days until
metamorphosis, and % of adults with developmental
defects for the four types of crosses. SD=Standard
deviation. N=sample size. Results from separate
one-way ANOVAs testing a taxa effect for each
dependent variable are shown below. ..........................................................11
1.2 The proportion of eggs fertilized, days until
metamorphosis, and clutch size for conspecific
and F1 X. laevis x X. muelleri hybrids. Only
one X. muelleri x X. laevis hybrid females was
available and these data are shown but were not
included in statistical analyses. SD=Standard deviation.
N=sample size. Results from separate one-way
ANOVAs testing a taxa effect for each dependent
variable are shown below ...........................................................................11
1.3
Summary statistics for morphological characters
in X. laevis, X. laevis x X. muelleri hybrid, X. muelleri
x X. laevis hybrid, and X. muelleri. Results are
presented as Mean ± SD. L=X. laevis;
M=muelleri. P values for SVL are derived from
one-way ANOVA testing the effect of sex
separately for each taxa. The remaining variables
were tested using an ANCOVA with SVL as the
covariate....................................................................................................13
1.4 Results of ANOVA and ANCOVA (with SVL
as covariate) analyses to examine Taxa,
Sex, and Taxa*Sex interaction effects for each
of the morphological variables....................................................................16
xvii
2.1
Estimates of π0 (the fraction of true hypotheses) and
the false discovery rate (FDR) at several levels of
alpha (the marginal Type I probability) in the
Xenopus dataset..........................................................................................33
2.2 The number of genes comparatively over
and underexpressed from least pooled error (LPE)
tests for all possible simultaneous comparisons
between sex and between species in Xenopus laevis (L)
and X. muelleri (M). The logical operators “>”, “<”,
and “=” represent over, under, and equal expression
respectively in all comparisons. Cells in white are for
genes that are different between species and cells in grey
are for genes not different between species. Cells in black
are comparisons rendered illogical and thus have no possible
result .......................................................................................................... 37
2.3 Results of vector analysis for the fold change
in males vs. females for patterns of expression
behavior in Xenopus laevis and Xenopus muelleri.
Up denotes upregulation in males (or male biased)
and down denotes down regulation in males
(female biased). ♂ denotes those genes that are
male biased ................................................................................................ 40
2.4
The number of transcripts for all possible
gene expression behaviors in males and
females of Xenopus laevis and X. muelleri
and the magnitude of fold change in
expression between species. M=male-biased;
F=female-biased; U=unbiased; Y=differentially
expressed between species; N=expressed
the same in both species ............................................................................ 44
3.1
Top 30 candidate transcripts upregulated
in X. laevis and differentially expressed
between X. laevis and hybrid. Expression
values are in log2 scale; SD=standard deviation
of expression values. P values are adjusted
according to FDR moderated t-tests........................................................... 74
xviii
3.2 Top 30 candidate transcripts upregulated in hybrids
and differentially expressed between X. laevis and
hybrid. Expression values are in log2 scale;
SD=standard deviation of expression values.
P values are adjusted according to FDR moderated
t-tests......................................................................................................... 75
3.3
Top 30 candidate transcripts upregulated in
X. muelleri and differentially expressed
between X. muelleri and hybrid. Expression values
are in log2 scale; SD=standard deviation of expression
values. P values are adjusted according to FDR
moderated t-tests........................................................................................ 76
3.4 Top 30 candidate transcripts upregulated in hybrids
and differentially expressed between X. muelleri
and hybrid. Expression values are in log2 scale;
SD=standard deviation of expression values.
P values are adjusted according to FDR
moderated t-tests......................................................................................... 77
3.5
Candidate loci (56 total) common to both contrasts
(X. laevis vs hybrids; X. muelleri vs. hybrids)
divided according to corresponding expression
behavior. Expression values are in log2 scale.
P values are FDR adjusted values. .............................................................. 78
3.6
Comparison in the overlap of transcripts recovered
as differentially expressed from the two contrasts
with hybrids (Xenopus laevis vs. hybrids and
X. muelleri vs. hybrids) and the interspecies
contrast (Xenopus laevis vs. X. muelleri). The
congruence between patterns of expression behavior
in hybrids compared to the interspecies comparison
suggests a model of semidominance where hybrids
have an intermediate level of expression compared
to the two parental species. ......................................................................... 81
4.1 Top five candidate miRNAs differentially expressed
between hybrid and non-hybrid testes. Fold change
is a mean ratio between X. laevis and hybrid expression
values........................................................................................................108
4.2 ANOVA analysis of TaqMan RT-PCR results. ...........................................109
xix
4.3 MIRANDA-predicted top candidate targets of
miR-202*, miR-125b, and xtr-miR-100. ...................................................110
5.1
Top 30 candidate transcripts upregulated in
X. laevis and differentially expressed between
females of X. laevis and hybrids. Expression values
are in log2 scale; SD=standard deviation of expression
values. P values are adjusted according to FDR
moderated t-tests. .....................................................................................124
5.2
Top 30 candidate transcripts upregulated in hybrids
and differentially expressed between females of
X. laevis and hybrids. Expression values are in
log2 scale; SD=standard deviation of expression
values. P values are adjusted according to FDR
moderated t-tests. .....................................................................................125
5.3
Top 30 candidate transcripts upregulated
in X. muelleri and differentially expressed between
females of X. muelleri and hybrids. Expression values
are in log2 scale; SD=standard deviation of expression
values. P values are adjusted according to FDR
moderated t-tests. ....................................................................................126
5.4
Top 30 candidate transcripts upregulated in hybrids
and differentially expressed between females of
X. muelleri and hybrids. Expression values are in
log2 scale; SD=standard deviation of expression
values. P values are adjusted according to FDR
moderated t-tests. ....................................................................................127
5.5
Comparison in the overlap of transcripts recovered
as differentially expressed from the two contrasts
with hybrids (Xenopus laevis vs. hybrids and
X. muelleri vs. hybrids) and the interspecies contrast
(Xenopus laevis vs. X. muelleri). The congruence
between patterns of expression behavior in hybrids
compared to the interspecies comparison suggests a
model of semidominance where hybrids have an
intermediate level of expression compared to the two
parental species. ......................................................................................128
xx
6.1
Fertility results from test crosses of ZW
and ZZ individuals from the same phenotypic
sex. ZW and ZZ females have the same fertility
characteristics and both ZW and ZZ phenotypic
males are completely sterile......................................................................145
xxi
CHAPTER 1
FERTILITY AND MORPHOLOGICAL CHARACTERISTICS OF PARENTAL
XENOPUS LAEVIS, XENOPUS MUELLERI AND F1 HYBRIDS
1.1 Abstract
We examined patterns of fertility and morphological characteristics in hybrids of
X. laevis and X. muelleri. Crossing these two species results in about a 50% reduction in
the number of fertilized eggs suggesting a postmating, prezygotic reproductive isolation
barrier. Development time is similar in both types of hybrids and X. laevis but much
shorter compared to X. muelleri. Adults of X. muelleri x X. laevis suffer from severe
development defects that are not apparent in the reciprocal or conspecific crosses. Adult
males of hybrid X. laevis x X. muelleri and hybrid X. muelleri x X. laevis are completely
sterile. Eggs from hybrid females in both types of crosses are just as fertilizable as
conspecific species and hatching success of fertilized embryos is the same in hybrids
compared to parental species. Hybrid females suffer from reduced clutch sizes and this
may be due to dysfunctions associated with hormonal regulation of oogenesis.
Morphological characteristics of both types of adult hybrids is highly similar and both
types of hybrids have greater similarity in morphology to X. muelleri compared to X.
laevis. Our results demonstrate that Xenopus fail to conform to Haldane’s rule and
provide hypotheses about the genetic control of morphological traits in hybrid Xenopus.
1
1.2 Introduction
Frogs of the genus Xenopus have served as model organisms in developmental
biology (Cannatella and de Sa, 1993; Callery, 2006) and consequently these efforts have
lead to breakthroughs in understanding the development of the early vertebrate embryo.
There has been considerably less effort spent on the evolutionary biology of this group
and in particular, the characteristics of speciation; however recent efforts are
transforming Xenopus to not just a model of developmental biology but also a model for
evolutionary and speciation genetics (Evans et al., 2004; 2005; Bos and Waldman,
2006; Chain and Evans, 2006; Malone et al., 2006; 2007; Evans, 2007).
Xenopus are comprised of more than 20 species distributed throughout subSahara Africa (Kobel et al., 1996; Evans et al. 2004; 2005). Xenopus are remarkable
among vertebrates in that the group is characterized by increased levels of ploidy.
Extant species range in ploidy from the diploid X. tropicalis (2n=20) to the dodecaploid
X. ruwenzoriensis (2n=108) and it is known that much of this increased ploidy is due to
hybridization among different polyploid lineages or allopolyploidization (Kobel, 1996;
Evans et al., 2005).
Workers at the University of Geneva and University of Bristol lead by H. R.
Kobel and R. C. Tinsley provided an important glimpse of the overall pattern of
reproductive isolation in Xenopus. Artificial hybridization studies reveal that Xenopus
do not conform to one of the most widespread generalizations in evolutionary biology;
Haldane’s rule. Female Xenopus have heterogametic sex chromosomes (ZW) and males
are homogametic (ZZ) yet in over 100 reciprocal crosses the males are sterile and
2
females are fertile (Kobel and Du Pasquier, 1975; Kobel et al. 1981; Picker, 1985;
Picker and de Villiers, 1989; Kobel, 1996; Kobel et al., 1996, Picker et al., 1996;
Fischer et al., 2000). Hybrids in general possess well developed gonads and
reproductive tracts. Sperm from male hybrids are aneuploid and either die or are
abnormally developed due to failure of chromosome pairing during meiosis (Kobel,
1996). Hybrid females reportedly produce two types of eggs. Univalent meiosis in some
eggs causes aneuploidy and subsequent death in advanced stages of embryogenesis
(Müller, 1977; Kobel et al., 1979). The second type is a “big” egg formed of
endoreduplicated polyploidized oocytes. These eggs contain bivalents corresponding to
the somatic chromosome number of the mother and are formed by an extra replication
of all chromosomes (Kobel and Du Pasquier, 1975; Müller, 1977). Segregation in
meiosis proceeds normally and produces an egg that has a complete genome of both
parental species. The frequency of “big” eggs varies with the combination of species but
can be greater than 90% in a clutch (Kobel, 1996).
Despite these claims, there is little quantitative data available on the fertility
characteristics of hybrids other than hybrids of X. laevis x X. gilli. Most of the
conclusions regarding the overall pattern of reproductive isolation appear to be drawn
from intensive study of crosses between X. laevis and X. gilli which are sister taxa.
Therefore, we would like to know if these conclusions extend to other species pairs. In
particular, the remarkable finding concerning endoreduplicated eggs is critical for
understanding patterns of allopolyploidization and diversification within Xenopus.
Finally, analyses of hybrid morphology, which can reveal the appearance of dominant
3
characters present in parental species that are found in hybrids, can generate hypotheses
about the nature of species level genetic dominance.
We examined patterns of fertility in Xenopus laevis, X. muelleri, and F1 hybrids
of X. laevis x X. muelleri and X. muelleri x X. laevis and make comparisons of these
results to what has been concluded about the fertility pattern in Xenopus. Additionally,
we analyzed morphological characteristics of hybrids compared to the two parental
species to determine how morphological characters are shaped in a hybrid genetic
background and to develop hypotheses about the nature of dominance for
morphological characters. Our analyses of fertility and morphology in hybrid X. laevis x
X. muelleri advance our understanding of reproductive isolation in the group by 1)
confirming that Xenopus do not conform to Haldane’s rule, 2) hybrid females are fertile
yet show reduced fertility in terms of the number of eggs laid relative to parental
species, and 3) dominant morphological characteristics are apparent in F1 hybrids
suggesting the presence of maternal effects as important factors towards generating
morphology in a hybrid genetic background.
1.3 Materials and Methods
1.3.1 Crosses
We produced crosses of Xenopus laevis x X. laevis, X. laevis x X. muelleri, X.
muelleri x X. laevis, and X. muelleri x X. muelleri to determine fertility characteristics
for each cross. Xenopus laevis individuals came from two sources; either purchased
from Xenopus Express or provided by J. Roberts from the University of Rochester.
4
Both populations originate from the Cape of South Africa. Xenopus muelleri were wild
caught in Swaziland and kindly provided by R. C. Tinsley.
Crosses were produced by injecting females with 500 IU and males with 150 IU
hCG hormone and then placing pairs in 10 L of artificial pond water. Artificial pond
water was produced by creating one stock solution (A) with 1.50 M NaCl and 0.16 M
CaCl2, and another stock solution (B) of 0.03 M NaHCO3. To make 10 L of artificial
pond water, 40 ml of stock solution A and 40 ml of stock solution B was combined with
10 L of water.
Samples of eggs were examined for fertilization efficiency by taking samples of
embryos from each clutch and then counting the number of fertilized and unfertilized
embryos. For conspecific crosses, the total number of eggs laid by females was counted.
Tadpoles were raised in 34 L plastic containers and fed daily with tadpole food from
Xenopus Express. The day on which the first tadpole metamorphosed was recorded for
each clutch. Metamorphosed frogs were raised to sexual maturity under 12L:12D
photoperiod at 25°C air temperature in 57 L containers and fed every two days with
Xenopus pellets from Xenopus Express. For each cross, the number of adults that had
external deformities (limb abnormalities or missing eyes) was counted compared to the
number of adults that had no external deformities.
We tested the null hypothesis that there was no difference in the proportion of
fertilized eggs, the number of days until metamorphosis, and the proportion of adult
frogs with deformities for each of the four crosses. The number of days until
metamorphosis was square root transformed to achieve normality. A one way ANOVA
5
in R software was used to test each dependent variable and a Tukey multiple
comparison procedure was used to control the family wise error rate in pairwise
comparisons at alpha equals 0.05.
1.3.2 Fertility of Hybrids
Females and males of X. laevis x X. muelleri were examined for fertility
characteristics. Only one female of X. muelleri x X. laevis produced eggs and therefore
we report observations for this single individual. Test crosses were made following the
methodology described above and hybrids were paired with X. laevis. The total number
of eggs laid by each female was counted and samples of eggs were examined for
fertilization efficiency as described above. Hatching success was determined by
counting the number of fertilized eggs that hatched into swimming tadpoles (Stage 31;
Nieuwkoop and Faber, 1956).
We tested the null hypothesis that there was no difference in the proportion of
fertilized eggs, the proportion of fertilized eggs that hatched into tadpoles, and the total
number of eggs laid by females for X. laevis, X. laevis x X. muelleri, and X. muelleri.
The total number of eggs laid by females was square root transformed to achieve
normality. A one way ANOVA in R software was used to test each dependent variable
and a Tukey multiple comparison procedure was used to control the family wise error
rate in pairwise comparisons at alpha equals 0.05.
1.3.3 Karyotyping
Tadpoles from six crosses with F1 female X. laevis x X. muelleri to X. laevis
were karyotyped following protocols from the Grainger lab to determine ploidy levels
6
in the first generation of backcrossing to X. laevis. From each cross, 10 Nieuwkoop and
Faber Stage 26-34 tadpoles were placed into DI water. A razor blade was used to
remove the ventral portion of tadpoles Dorsal portions of tadpoles were placed for 20
minutes into fresh DI water and then transferred into an Eppendorf tube containing 0.2
ml of 60% acetic acid for 5 minutes. All tissue was then transferred, minimizing the
amount of acetic acid to a positively charged slide (Superfrost Plus, Fisher, Inc.). A
large coverslip was pressed firmly on top of the tadpole tissue and the slide and
coverslip was placed on dry ice for 5 minutes. The slide was then removed from dry ice
and the coverslip was then removed from the slide. Nuclei were stained with 1 ul of
0.1mg/ml Hoechst 33342 for 5 minutes and then the slide was tipped to allow Hoechst
stain to run off the slide. A drop of PBS was placed on the slide and a large coverslip
was added and sealed with nail polish. Stained chromosome spreads were then
examined on high power using epifluorescence microscopy (Olympus BH-2) and
chromosomes counted to determine ploidy number.
1.3.4 Morphological Variation
Morphological variation was assayed using 123 specimens of sexually mature
Xenopus from each of the four cross types for the following 12 variables (Fig. 1.1), (1)
snout to vent length (SVL); (2) tibiofibula length (TL); (3) head width (HW); (4) head
length (HL); distance of radioulna to longest finger (RUL); (5) humerus length (HUL);
(6) femur length (FL); (7) interocular distance (IOD); (8) internaris distance (IND); (10)
weight (W); (11) length of the left tentacle (LTENTL); (12) length of the right tentacle
(RTENTL). The length of each ocular tentacle was selected because the presence of a
7
8
SVL
IOD
IND
RTENTL
FL
HUL
HL
HW
8
Figure 1.1. Description of morphological characters used assay variation in morphology among Xenopus laevis, X.
laevis x X. muelleri hybrid, X. muelleri x X. laevis hybrid, and X. muelleri. (A) Dorsal view. (B) Ventral view.
SVL=Snout to vent length; TL=Tibiofibula length; HW=Head width; HL=Head length; RUL=Distance of radioulna to
3rd finger; HUL=Humerus length; FL=Femur length; IOD=Interocular distance; IND=Internaris distance; W=Weight
in grams; LTENTL=Left tentacle length; RTENTL=Right tentacle length.
TL
RUL
LTENTL
tentacle is a character that varies across species of Xenopus and is a conspicuous
character in X. muelleri (Kobel et al., 1996). Each variable was measured in millimeters
using dial calipers and weight was measured in grams using either a 60 g or 1000 g
Pesola scale.
We explored patterns of sexual dimorphism in adults within and between each
taxa (X. laevis, X. laevis x X. muelleri, X. muelleri x X. laevis, and X. muelleri). Body
size (SVL) was highly sexual dimorphic for each taxa (X. laevis: F
1, 23
= 313.31; P
<0.00001; X. laevis x X. muelleri: F 1, 73 = 296.27; P<0.00001; X. muelleri x X. laevis: F
1,4
= 40.783; P=0.0031; X. muelleri: F
1,15
= 104.77; P<0.00001) and we therefore
performed ANCOVAs using SVL as a covariate to test for sexual dimorphism within
each taxa while controlling for the effect of body size for each of the remaining 11
morphological variables. To examine if there were differences in SVL between each
taxa, we used an ANOVA with taxa and sex as factors. For all other morphological
characters, we used an ANCOVA with sex and taxa as factors and SVL as a covariate.
These analyses allowed us to examine for each morphological variable whether there
were differences between the taxa regardless of sex, differences between the sexes
while ignoring the effect of taxa, and finally sexually dimorphic differences between the
taxa (the interaction effect of taxa*sex).
Principal component analysis (PCA) of the correlation matrix for the 12
morphological variables was performed to summarize the morphological variance
among the taxa. The first four principal components explained about 96% of the
variance and these four principal components were used in subsequent analyses. We
9
used discriminate function analysis (DFA) in SYSTAT 8.0 to classify taxa based on the
four principal components. To ascertain the influence of body size compared to shape
characteristics we performed DFA with or without PC1. This strategy allowed us to
explore the importance of either body size or shape related PCs on the ability to
discriminate males and females of each of the four types of taxa.
1.4 Results
The proportion of eggs fertilized was substantially lower in both interspecific
crosses compared to conspecific crosses. There was no difference in the proportion of
fertilized eggs from crosses of either maternal X. laevis with X. muelleri or maternal X.
muelleri with X. laevis but both hybrid crosses had about 50% fewer fertilized eggs
compared to conspecific crosses (Table 1.1). The time to metamorphosis was about 2
times longer for X. muelleri compared to X. laevis and both types of hybrid crosses but
there was no difference in development time for either type of hybrid cross compared to
conspecific X. laevis. There was a substantially greater number of adults frogs with
developmental abnormalities in the maternal X. muelleri with X. laevis cross and these
mainly consisted of front limb abnormalities whereas there were very few
developmental defects in adults of the other three types of crosses (Table 1.1).
Analysis of fertility of F1 hybrid X. laevis x X. muelleri yielded definitive
patterns with regard to Haldane’s rule; hybrid males are completely sterile and hybrid
females are fertile. All 36 test crosses of F1 X. laevis x X. muelleri hybrid males with X.
laevis yielded no fertilized eggs and four test crosses with F1 X. muelleri x X. laevis
hybrid males crossed with X. laevis females provided no fertilized eggs. Three attempts
10
11
Days until Metamorphosis
Mean
SD
N
69.3
9.81
3
61.2
13.5
9
60.5
2.12
2
125.7
25.7
3
F 3, 13 =12.636; P=0.0003757
% Deformities
Mean
SD
N
0.023
0.029
3
0.050
0.112
5
0.808
0.272
2
0.00
0.00
3
F 3, 8= 21.372; P=0.000355
X. laevis
X. laevis x X. muelleri x X. laevis
X. muelleri x X. laevis x X. laevis
X. muelleri
ANOVA
11
% Fertilized Eggs
Mean
SD
N
0.617
0.210
3
0.533
0.262
26
0.787
1
0.633
0.287
3
F 2, 29 =0.3041; P=0.7401
% Hatched into Tadpoles
Mean
SD
N
0.573
0.424
3
0.514
0.289
16
0.412
1
0.869
0.055
3
F 2, 19 = 1.8776; P=0.1803
Clutch Size
Mean
SD
N
3485.2
1827.3
24
1781.5
1238.4
26
1350
1
4101.0
1993.5
9
F 2, 56 = 9.7029; P=0.0002410
Table 1.2. The proportion of eggs fertilized, days until metamorphosis, and clutch size for
conspecific and F1 X. laevis x X. muelleri hybrids. Only one X. muelleri x X. laevis hybrid
females was available and these data are shown but were not included in statistical analyses.
SD=Standard deviation. N=sample size. Results from separate one-way ANOVAs testing a
taxa effect for each dependent variable are shown below.
X. laevis x X. laevis
X. laevis x X. muelleri
X. muelleri x X. laevis
X. muelleri x X. muelleri
ANOVA
% Eggs Fertilized
Mean
SD
N
0.62
0.21
3
0.10
0.15
5
0.13
0.15
5
0.63
0.29
3
F 3, 12=8.9076 ; P=0.002225
Table 1.1. The proportion of eggs fertilized, days until metamorphosis, and % of adults with
developmental defects for the four types of crosses. SD=Standard deviation. N=sample size.
Results from separate one-way ANOVAs testing a taxa effect for each dependent variable are
shown below.
at in vitro fertilization of X. laevis eggs using dissected testes of X. laevis x X. muelleri
hybrids also did not result in any fertilized embryos. Conversely, eggs of F1 X. laevis x
X. muelleri hybrid females had the same fertilization efficiency as conspecific crosses
(Table 1.2). Only one F1 X. muelleri x X. laevis was available and this individual had
79% of eggs fertilized when crossed with X. laevis. There was also no difference in the
hatching success of fertilized eggs because the same proportion of fertilized eggs
hatched into swimming tadpoles compared to conspecific crosses. Clutch size however
was substantially different in F1 X. laevis x X. muelleri hybrid females. Hybrid females
laid about half the number of eggs compared to either of the two parental species (Table
1.2).
Analysis of tadpole karyotypes from backcrosses of X. laevis x X. muelleri x X.
laevis provided no evidence of abnormal ploidy levels. All karyotypes examined had a
chromosomal number of 36 chromosomes providing no evidence of abnormal ploidy
during oogensis of F1 hybrid females (Fig. 1.2).
There are distinct morphological differences between the two parental species
and both types of F1 hybrids (Table 1.3). Xenopus laevis is larger than X. muelleri and
both types of hybrids are smaller compared to X. laevis but similar in body size
compared to X. muelleri. Both types of hybrids posses a tentacle that is smaller in size
compared to X. muelleri but is completely absent in X. laevis.
Morphological variables also show distinct patterns of sexually dimorphism.
Body size (SVL) was strongly sexually dimorphic in all taxa with males having a
smaller body size compared to females. Incorporating body size as a covariate in
12
13
113.0 ± 7.79
W
0.0 ± 0.0
3.33 ± 0.356
IND
RTENTL
8.40 ± 1.20
IOD
0.0 ± 0.0
30.9 ± 2.37
LTENTL
12.5 ± 0.926
FL
28.0 ± 3.17
RUL
HUL
19.8 ± 1.05
HL
0.0 ± 0.0
0.0 ± 0.0
43.7 ± 14.0
4.40 ± 0.279
12.8 ± 0.525
38.9 ± 2.38
14.3 ± 0.773
35.2 ± 1.44
27.1 ± 1.24
29.7 ± 1.30
39.1 ± 1.19
30.8 ± 1.79
23.6 ± 1.25
TL
100.7 ± 3.88
X. laevis
♀ (n=10)
68.0 ± 4.25
HW
SVL
♂ (n=15)
N/A
N/A
0.154
0.833
0.050
0.010
0.497
0.470
0.226
0.990
0.084
<0.0001
P value
1.76 ± 0.271
1.80 ± 0.302
24.8 ± 6.14
2.99 ± 0.283
7.03 ± 0.558
27.1 ± 2.29
11.0 ± 2.71
25.2 ± 1.97
18.3 ± 1.87
19.5 ± 2.03
27.0 ± 1.91
58.8 ± 5.05
♂ (n=45)
2.30 ± 0.341
2.35 ± 0.331
61.6 ± 15.0
3.65 ± 0.266
9.55 ± 0.737
34.4 ± 1.92
13.0 ± 1.33
31.5 ± 2.23
23.9 ± 1.92
25.0 ± 2.59
33.5 ± 2.19
82.7 ± 6.62
L x M hybrid
♀ (n=30)
13
0.167
0.004
0.215
0.146
0.012
0.213
0.017
0.450
0.671
0.001
0.005
<0.0001
P value
1.75 ± 0.129
1.70 ± 0.082
19.1 ± 4.77
2.83 ± 0.330
7.31 ± 0.519
25.4 ± 2.07
11.8 ± 5.25
23.3 ± 1.81
15.9 ± 0.874
17.0 ± 1.65
24.6 ± 2.03
53 ± 4.16
♂ (n=4)
2.20 ± 0.283
2.25 ± 0.071
46.0 ± 11.3
3.90 ± 0.0
8.67 ± 0.990
30.7 ± 6.15
11.7 ± 0.495
30.5 ± 1.41
22.4 ± 0.212
22.0 ± 0.212
32.9 ± 1.91
78.8 ± 3.96
M x L hybrid
♀ (n=2)
0.864
0.252
0.058
0.955
0.885
0.218
0.422
0.052
0.182
0.619
0.003
0.0031
P value
2.67 ± 0.198
2.70 ± 0.271
19.1 ± 1.62
3.34 ± 0.223
6.83 ± 0.285
23.2 ± 1.49
9.40 ± 0.277
22.9 ± 0.315
17.1 ± 0.752
18.3 ± 1.02
24.1 ± 0.404
57.9 ± 2.43
♂ (n=7)
Table 1.3. Summary statistics for morphological characters in X. laevis, X. laevis x X. muelleri
hybrid, X. muelleri x X. laevis hybrid, and X. muelleri. Results are presented as Mean ± SD.
L=X. laevis; M=muelleri. P values for SVL are derived from one-way ANOVA testing the effect
of sex separately for each taxa. The remaining variables were tested using an ANCOVA with
SVL as the covariate.
2.92 ± 0.155
2.93 ± 0.125
40.2 ± 6.85
3.65 ± 0.151
10.1 ± 0.483
25.7 ± 2.14
10.3 ± 0.604
26.1 ± 1.81
18.8 ± 1.15
20.0 ± 1.39
27.4 ± 1.90
72.64 ± 3.33
X. muelleri
♀ (n=10)
P value
0.424
0.384
0.151
0.358
0.087
0.449
0.484
0.643
0.989
0.242
0.340
<0.0001
Figure 1.2. Example karyotype of backcross X. laevis x X. muelleri.
subsequent analyses we found that (1) femur length (FL) and interocular distance (IOD)
were sexually dimorphic in X. laevis, (2) there were no differences in any
morphological variable between males and females of X. muelleri, (3) tibiofibula length
(TL), head width (HW), head length (HL), humerus length (HUL), interocular distance
(IOD), and left tentacle length (LTENTL) were all sexually dimorphic in F1 X. laevis x
X. muelleri, and (4) tibiofibula length (TL) was sexually dimorphic in F1 X. muelleri x
X. laevis (Table 3). All morphological variables examined had a significant taxa effect
suggesting distinct differences in body size and shape among the parental species and
their F1 hybrid. There was a distinct sex effect for tibiofibula length (TL), head width
(HW), humerus length (HUL), femur length (FL), interocular distance (IOD), weight
(W), and the length of both tentacles (LTENTL, RTENTL) revealing differences
between males and females for these specific morphological variables. Finally, there
were differences in sexual dimorphism between the four taxa for head width (HW),
14
femur length (FL), interocular distance (IOD), weight (W), and both tentacle lengths
(LTENTL, RTENTL; Table 1.4).
The first four principal components explain 96% of the variation in morphology
(PC I: 71.3%; PC II: 15.7%; PC III: 5.7%; PC IV: 3.3%). Plotting results for PC I and
PC II shows that males and females of both types of hybrids and the two parental
species separate into distinct clusters (Fig. 1.3). These results also show that males and
females of both types of hybrids are highly similar to each other as there is substantial
overlap in the two hybrids. Finally, hybrids are more similar in external morphology to
X. muelleri than they are to X. laevis as evidenced by the distance among the clusters.
Discriminate function analyses (DFA) confirmed these patterns. DFA using all
four PCs was significant (Wilks’ Lambda = 0.0022; F
28, 405
= 64.14; P<0.0001) and
classified each sex and taxon 90% of the time. Misclassifications in this DFA all result
only from difficulty in discriminating the two types of hybrids (X. laevis x X. muelleri
and X. muelleri x X. laevis). A second DFA using only PCs II-IV was also highly
significant (Wilks’ Lambda = 0.0449; F
21, 325
= 30.14; P<0.0001) and classified each
sex and taxon 75% of the time. Misclassifications were distributed across all males and
females for each of the four taxa and these results suggest that the body size associated
PC I is important towards correct discrimination of each group.
1.5 Discussion
Generating F1 hybrids is more difficult than breeding conspecific species of
Xenopus. Our methodology using hCG to stimulate reproduction in males and females
15
16
F
F
F
F
F
HUL
FL
IOD
IND
W
F
F
RUL
RTENTL
F
HL
3,114
= 18.096
= 8.219
= 10.616
= 53.235
= 4.411
= 9.566
= 9.879
= 973.392
= 1168.224
3,114
3,114
3,114
3,114
3,114
3,114
3,114
= 8.704
= 50.765
= 46.692
Stat
3,114
3,114
3,115
3,114
F
HW
F
F
TL
LTENTL
F
SVL
Character
Taxa
F
F
F
F
F
F
F
F
F
F
F
0.000
0.000
0.000
0.006
0.000
0.000
0.000
0.000
0.000
0.000
16
1, 114
1, 114
1, 114
1, 114
1, 114
1, 114
1, 114
1, 114
1, 114
1, 114
1, 114
= 7.510
= 15.885
= 4.199
= 3.877
= 3.984
= 15.609
= 9.752
= 1.353
= 0.098
= 19.390
= 8.830
0.007
0.000
0.043
0.051
0.048
0.000
0.002
0.247
0.755
0.000
0.004
0.000
F 1, 115 =
273.768
0.000
0.000
P value
Stat
P value
Sex
F
F
F
F
F
F
F
F
F
F
F
F
3,114
3,114
3,114
3,114
3,114
3,114
3,114
3,114
3,114
3,114
3,114
3,115
= 12.572
= 15.503
= 9.671
= 2.364
= 3.848
= 3.885
= 1.428
= 0.252
= 2.115
= 2.803
= 0.913
= 4.437
Stat
Taxa*Sex
0.000
0.000
0.000
0.075
0.011
0.011
0.238
0.860
0.102
0.043
0.437
0.005
P value
Table 1.4. Results of ANOVA and ANCOVA (with SVL as covariate) analyses
to examine Taxa, Sex, and Taxa*Sex interaction effects for each of the morphological
variables.
4
Principal Component 2
3
HybridF
HybridM
laevisF
laevisM
muelleriF
muelleriM
ReciHybF
RecHybM
2
1
0
-1
-2
-3
-4
-10
-5
0
5
10
Principal Component 1
Figure 1.3. Principal component analysis of morphological variables for
Xenopus laevis, X. muelleri and F1 hybrids of both directions. The first and
second principal components explain 71.2 % and 15.7% respectively of the
morphological variance.
nearly always resulted in males amplexing females during in vivo crosses revealing that
there is no barrier at the level of amplexus. Both in vivo crosses with either X. laevis x
X. muelleri or X. muelleri x X. laevis result in about a 50% reduction in the number of
eggs fertilized suggesting a postmating, prezygotic reproductive isolation barrier
(Coyne and Orr, 2004). Brun and Kobel (1977) noted that spermatozoa of X. laevis
become immobilized at the inner jelly layer of X. borealis and it is possible that
isolation at the level of jelly layers may be operating between X. laevis and X. muelleri,
17
particularly since X. muelleri is closely related to X. borealis (Evans et al., 2004; 2005).
However this isolating barrier is not complete because on average between 10-13% of
eggs are fertilizable by heterospecific sperm and this would imply that variation exists
in the extent of this putative barrier to gene flow. The mechanisms that produce this
postmating, prezygotic isolation barrier remain unknown and would be an appropriate
direction for future research.
Fertilization alone is not the best criteria for determining reproductive isolation
as even despite fertilization all embryos could die due to incompatibilities expressed
during ontogeny. The number of days until metamorphosis was not different between X.
laevis and the two types of hybrids but X. muelleri developed much more slowly
compared to all other types of crosses. Developmental abnormalities in adults were
nearly ubiquitous in the X. muelleri x X. laevis cross and this result suggests that
maternal effects are important towards generating external morphology and that there is
massive incompatibility between X. muelleri x X. laevis but little problems with
generating adult X. laevis x X. muelleri hybrids. Our results collectively demonstrate
that viability in hybrid X. laevis and X. muelleri is high but X. muelleri x X. laevis
hybrids have severe developmental defects consisting of abnormally formed front
limbs.
We examined fertility of both types of hybrids and provided definitive
conclusions with regards to Haldane’s rule. Haldane’s rule does not apply to this pair of
Xenopus and this confirms the general observations of Kobel (1996). All males of both
types of hybrids failed to fertilize a single egg both in vivo and in vitro. Previous
18
analyses of spermatogenesis suggest that hybrid X. laevis x X. muelleri have a
dramatically lower abundance of sperm compared to the two parental species, most
sperm cells are in a state of developmental arrest, and the few mature sperm that do
occur are capable of motility (Malone et al., 2007). Mature sperm are also dramatically
larger compared to conspecific sperm and this may be consistent with the notion that
hybrid sperm are aneuploid as reported by Kobel (1996). These data show that hybrid
males are completely sterile and that spermatogenesis is severely perturbed in hybrid
males.
Hybrid females, however, are completely fertile; there was no difference in the
number of eggs fertilized during in vivo crosses with males of X. laevis and no
difference in the number of eggs that hatched into tadpoles. However, there was about a
50% reduction in the number of eggs that were laid by hybrid females. Again it is
important to keep in mind our methodology for producing in vivo crosses. We injected
females with hCG hormone which is a standard technique for inducing ovulation in
Xenopus and the next day pairs were separated and the eggs were counted. It is difficult
to interpret our result in the context of the hCG injection as analyses of hybrid
spermatozoa showed that hybrid males failed to respond to hCG treatment in the same
fashion compared to conspecifics (Malone et al., 2007). It is possible then that hybrid
females also suffer dysfunction at the level of hormones and receptors that precludes
ovulation from occurring in the same way as conspecifics. This would be possible if the
hybrid genetic background provides receptor dysfunction for hCG hormone. This
remains an intriguing hypothesis for reproductive isolation research in general as
19
hormones play a critical role in controlling both oogeneisis and spermatogenesis. The
main fitness variables of interest are clutch size, number of eggs that are fertilized, and
the number of individuals that reach sexual maturity. Clutch size is difficult to interpret
given our results and the methodology used to generate in vivo crosses but does suggest
that we need to understand hormone interactions with oogenesis in hybrids due to the
fact that hybrid females lay about half the number of eggs as these two conspecific
species. In general though, we can conclude that the fertility pattern is exactly opposite
to the prediction of Haldane’s rule; in this case hybrid females are fertile (when they
should be sterile) and hybrid males are sterile (when they should be fertile.
Karyotype analyses provided no evidence for the endoreduplication mechanism
described by Kobel (1996). We examined karyotypes from F2 tadpoles produced from
crosses of hybrid females to X. laevis. Hybrid females are alleged to produce two types
of eggs, a big egg that contains an endoreduplicated genome and a small egg that is
aneuploid. Big eggs are fertilizable whereas small eggs are not, therefore if our F1
hybrid females had an abnormal ploidy level this would be manifested when examining
karyotypes of F2 crosses. First, we found little evidence for big and small eggs when
examining clutches of hybrid eggs. Second, our karyotype analysis does not confirm
abnormal ploidy levels in F1 hybrids as all the F2 tadpole karyotpes we examined had
36 chromosomes which is the diploid number for X. laevis and X. muelleri. Most of the
work on endoreduplicated eggs stems from observations in hybrids of X. laevis x X.
gilli. These two species are sister taxa and our data suggests that this endoreduplication
mechanism may be more common when hybridizing closely related species and the
20
mechanism producing endoreduplication fails to operate in crosses between more
distantly related species.
Our analyses of morphological characteristics in hybrids compared to parental
species show strong patterns of sexual dimorphism and an overall greater similarity of
hybrids to Xenopus muelleri compared to X. laevis. Body size is strongly sexually
dimorphic in all taxa but both types of hybrids have more similarity to X. muelleri
compared to X. laevis. Sexually dimorphic traits occur even while controlling for the
effect of body size and many of these traits differ between the four types of taxa
suggesting that sexual dimorphisms occur in unique ways between X. laevis, X.
muelleri, and F1 hybrids. Both types of hybrids are extremely similar in terms of the 12
morphological variables that we examined but the two parental species are dramatically
different. Additionally, both types of hybrids bear much greater resemblance to X.
muelleri as evidenced by the presence of a tentacle and the much greater similarly in
size and shape. These results suggest that the genome of X. muelleri has a dominating
effect on the control of morphology compared to the X. laevis genome and suggests
possible mechanisms for how genes are expressed in hybrids compared to the two
parental species.
Our analyses provide important conclusions regarding hybridization and
postzygotic isolation in Xenopus. Hybridizing X. laevis and X. muelleri results in
completely sterile hybrid males and fertile hybrid females. These results go against the
prediction of Haldane’s rule and this is likely due to faster male evolution (Malone et
al., 2006). There is an intriguing postmating, prezygotic isolation barrier in that
21
fertilization between these two species does not provide the same fertilization rate as
when crossing conspecific species. The mechanism for this barrier remains unknown
but could be due to interactions between sperm and egg jelly. Reciprocal cross hybrids
(X. muelleri x X. laevis) suffer much more developmental abnormalities compared to X.
laevis x X. muelleri hybrids suggesting that incompatibilities at the level of maternal
effects are important between these two species of Xenopus. Hybrid females lay fewer
eggs compared to conspecifics and this could be due to dysfunction at the level of
hormones and receptors to properly regulate ovulation of eggs. Morphology of adult
hybrids is much more similar to X. muelleri compared to X. laevis. Our results provide
important directions for reproductive isolation research in Xenopus and show the
promise of this exciting new system for evolutionary biology.
22
CHAPTER 2
SEX-BIASED GENE EXPRESSION IN A ZW SEX DETERMINATION SYSTEM
2.1 Abstract
Studies of the transcriptome have shown that a substantial fraction of
interspecific differences in gene expression is the result of sex-biased gene expression.
These results suggest that sex-dependent selection may be an important force in
generating differences between species but to date all studies have focused on
Drosophila. We examined a sample of the transcriptome in the gonads of two species of
Xenopus to provide an additional test of how sex-biased gene expression may contribute
to differences in gene expression between species. In contrast to Drosophila, Xenopus
provides an example of a ZW system with morphologically indistinguishable sex
chromosomes. About 81% of the transcriptome was differentially expressed between X.
laevis and X. muelleri and there were more genes that were male-biased compared to
the number of genes that were female-biased or non-sex-biased. While there were more
genes up-regulated in males of Xenopus, the largest magnitude difference in expression
between species occurred in female-biased genes, and male-biased genes were
proportionally more abundant for the smallest changes in expression between species.
Our results suggest that more genes have a role in the function of the testis compared to
the ovary and female-biased genes play a principle role in expression divergence
between species. These results differ from those in the Drosophila XY system in that
23
more female-biased genes had >4-fold difference of expression between species than
male-biased genes, suggesting that ZW sex chromosomes may facilitate enhanced gene
expression divergence between species.
2.2 Introduction
Studies of the transcriptome have established that gene expression differs
between males and females. Over 12% of the genome is sex-regulated or exclusively
enriched in the germline of Caenorhabditis elegans (Reinke et al. 2000; Jiang et al.
2001). More than half of the genome is differentially expressed between the sexes in
Drosophila (Jin et al. 2001; Ranz et al. 2003), and in mouse 4% of the genome is
differentially expressed in the gonads (Rinn et al. 2004). While several genes are
differentially expressed in most tissues examined thus far (e.g., brain, liver, kidney,
hypothalamus [Rinn et al. 2004; Nishida et al.2005]), the largest contribution to
dimorphic gene expression is found in the gonad of Drosophila (Parisi et al. 2003).
Unique patterns of gene expression in the gonad parallel previous studies of
substitution divergence in genes involved in reproduction. Genes encoding reproductive
proteins rather than non-sex-related genes often diverge rapidly between species and
have higher rates of nonsynonymous to synonymous substitutions indicating positive
Darwinian selection (Civetta and Singh 1995; Wycoff et al. 2000; Swanson et al. 2001;
Swanson and Vacquier 2002a, b; Zhang et al. 2005; Zhang and Parsch 2005). Recent
work on sexual dimorphism of the transcriptome between two species of Drosophila
has implicated sex-dependent selection as a major force driving divergence within and
between species (Ranz et al. 2003; Meiklejohn et al. 2003). Half of the transcriptome
24
was found to be differentially expressed between D. melanogaster and D. simulans and
83% of these differences were due to sex-biased gene expression. Of particular interest
is the fact that the majority of genes contributing to interspecific differences in
expression were male-biased. Male-biased genes were also preferentially misexpressed
in F1 sterile male hybrids of Drosophila (Michalak and Noor 2003) and the contribution
of male-biased genes to interspecific differences in expression suggest that faster male
evolution may be an important force in the process of reproductive isolation. At present,
it is unclear how faster male evolution may shape gene expression divergence in other
organisms and additional examples are needed to determine what may be an important
force in the evolution of new species.
We examined the transcriptome in males and females of two species of frogs of
the genus Xenopus to reveal patterns of sexual dimorphism in the transcriptome and to
examine the contribution of sex-biased genes to the differences in gene expression
between species. Xenopus are excellent organisms to address dimorphic gene
expression and the putative importance of faster male evolution. Sex-reversal
experiments and sex ratios from backcross progeny have established that a dominant
allele in females of X. laevis and X. muelleri determines sex and therefore these two
species have ZW sex determination (Mikamo and Witschi 1963, 1964; Witschi 1971;
Kobel 1985; Hayes 1998; Miyata et al. 1999). No morphologically distinct Z or W sex
chromosomes have been identified thus far and therefore Xenopus have homomorphic
sex chromosomes (Tymowska and Kobel 1972; Schmid and Steinlein 1991).
Additionally, in crosses between these and other species of Xenopus, males are sterile
25
and females are fertile (Kobel and Du Pasquier 1975; Kobel et al. 1981, 1996; Picker
1985; Picker and de Villiers 1989; Kobel 1996; Picker et al. 1996; Fischer et al. 2000).
Xenopus therefore do not follow one of the most pervasive generalizations in
evolutionary biology (Haldane’s rule) because the homogametic sex suffers the most
dysfunctional effects from hybridization. Because females are the heterogametic sex in
Xenopus, dominance effects, or the expression of deleterious recessive alleles in the
heterogametic sex, cannot be a viable explanation for the sterility pattern in Xenopus.
Faster male evolution can, since the process of spermatogenesis may be inherently
sensitive to gene misregulation in hybrid males and/or sexual selection may have caused
accelerated divergence of reproductive genes in males (Wu and Davis 1993; Wu et al.
1996). Therefore, we can predict a priori that the transcriptome of males should be more
divergent to that of females in interspecific comparisons. This not only would entail an
additional example of faster male evolution but also, given the established sterility
pattern in Xenopus hybrids, would be consistent with faster male evolution as an
explanation for reproductive isolation.
We found massive differential expression of genes between Xenopus laevis and
X. muelleri and there were more genes that were male-biased compared to female- and
non-sex-biased genes. While there were numerically more male-biased genes, femalebiased genes had the largest magnitude differences in expression between species,
suggesting a role for female-biased genes in interspecific expression divergence. Our
results are consistent with faster male evolution occurring in Xenopus in that the process
of spermatogenesis and/or the function of the testis may require more genes that could
26
be disrupted in a hybrid background compared to the process of oogenesis and/or the
function of the ovary. Additionally, an interesting role for female-biased genes, possibly
related to the ZW sex determination system, may be involved in facilitating differences
in expression between species of Xenopus.
2.3 Materials and Methods
2.3.1 Animals
We obtained sexually mature F1 lab-bred Xenopus laevis laevis males and
females from the University of Rochester Medical Center (courtesy of Dr. J. Robert)
and Xenopus Express. Males and females of Xenopus laevis were about 2 years old and
the parents of these frogs originate from the southern Cape region of South Africa.
Sexually mature males and females of Xenopus muelleri were collected from
Swaziland, Africa, in 2000 and kindly provided by Prof. R.C. Tinsley. The effects of
age did not alter the conclusions found in this study and therefore we did not include
age effects in our analysis of gene expression.
2.3.2 RNA Extraction and Microarray Hybridization
RNA was extracted from adult ovary and testis in Xenopus laevis (n = 4
females, n = 4 males) and X. muelleri (n = 6 females, n = 3 males) using GeneHunter
and Ambion RiboPure total RNA kits. RNA extractions took place over the course of 2
months but there was no significant time effect in subsequent analyses. Adults were
euthanized with MS-222 and gonadal tissue was immediately extracted and placed in
RNA extraction solution. Tissue was homogenized using a hand-held mortar until the
solution was homogeneous and manufacturer protocols were followed to produce
27
purified total RNA. Samples of RNA were checked for quality by examination of the
28S and 18S ribosomal RNA bands from denaturing gel electrophoresis, by 260/280
ratios from scans with a Nanodrop ND 1000 spectrophotometer, and by readouts of the
Agilent Bioanalyzer. Total RNA samples were prepared and then hybridized to
Affymetrix Xenopus laevis GeneChip Genome Arrays at the University of Texas
Southwestern Medical Center Microarray Array Core Facility following standard
Affymetrix protocols. Affymetrix Microarray Analysis Suite (MAS) v.5.0 was used to
scan and process each microarray chip. The signals of quality control and poly(A)
transcripts were carefully checked and revealed that hybridizations were of high quality
in all chips. Quality control probe sets (i.e., spike in and housekeeping genes) were
removed in subsequent statistical analyses. Pairwise comparisons of the probe level data
for chips within each species-by-sex combination revealed similar correlation
coefficients for X. laevis males (0.915 ± 0.014), X. laevis females (0.963 ± 0.010), X.
muelleri males (0.90 ± 0.060), and X. muelleri females (0.982 ± 0.005), suggesting that
reproducibility was high among the hybridizations for both X. laevis and X. muelleri.
2.3.3 Data Analysis
Two types of data relating to gene expression can be obtained from Affymetrix
microarrays. First, a qualitative assessment of whether a gene is expressed or not
(present vs. absent) and second, a quantitative measure of gene expression which is the
value calculated based on the hybridization of labeled cRNA to perfect match and
mismatch probes. In the Xenopus genome array, probes for a particular transcript are
present in 32 cells on the array chip. Half of those cells possess perfect match (PM)
28
sequences that are complementary to the reference transcript. The other half possess
mismatch (MM) sequences that are complementary to the reference transcript except for
a homomeric base mismatch at the 13th position. The number of instances in which the
PM hybridization signal is larger than the MM signal is computed along with the
average of the logarithm of the PM:MM ratio (after background subtraction) for each
probe set using Microarray Analysis Suite (MAS) 5.0 software (Affymetrix, Inc. 2001).
The software detection algorithm calculates the discrimination score from each probe
pair (PM vs. MM), assesses probe saturation, calculates a detection P value from a
onesided Wilcoxon signed rank test, and assigns a present (P), marginal (M), or absent
(A) call in relation to a threshold value τ = 0.015. Hybridizing RNA from one species
onto a microarray designed for another can introduce bias due to the effect of sequence
divergence between species (Nuzhdin et al. 2004; Gilad et al. 2005). To minimize the
impact of sequence divergence, we selected genes based on the following criteria; (1) P
in all X. muelleri chips (n = 2761); (2) P in all males and A in all females (n = 53); (3) P
in all females and A in all males (n = 68); (4) P in all X. muelleri females and A in all
other chips (n = 6); and (5) P in all X. muelleri males and A in all other chips (n = 3).
This resulted in a dataset consisting of gene expression values for 2891 genes which we
regard as the most conserved between species and thus likely minimize the impact of
sequence divergence.
The method of normalization can have a dramatic affect on higher-level
analyses of microarray data (Li and Wong 2001; Hoffman et al. 2002; Bolstad et al.
2003; Irizarray et al. 2005). We performed two different normalization procedures
29
(global scaling and RMA) and carried out statistical analyses on each of these datasets
as a way to provide a robust inference of the patterns observed in our dataset. Global
scaling was performed using MAS 5.0 software by scaling each chip to a common total
intensity of 10,000. Robust multichip averaging (RMA) normalization was performed
using RMAExpress (Bolstad 2005) using default parameters (background correction
and quantile normalization). For both normalization procedures we used only those
genes based on the five criteria as explained above. Expression values were transformed
to the log2 scale to make the expression distributions closer to a normal distribution in
subsequent analyses. There was little difference in the overall pattern between the two
normalization methods (Supplemental Tables 1–3) but the expression levels for each
gene were more conservatively estimated for the RMA normalization dataset. The
major patterns did not differ between the two normalization methods and we chose to
report results based on the RMA dataset due to the more conservative estimate of
expression values and because RMA is now a more widely used method for
normalizing Affymetrix microarray data. The quantitative expression data for each
sample were analyzed using principal components analysis (PCA) in Avadis 4.1 (Strand
Life Sciences, Inc.) software to heuristically identify how each sample clustered with
the remaining samples. The first two principal components explained 87% of the
variation in gene expression and revealed that males and females of X. laevis and X.
muelleri fell within distinct clusters. The results of the PCA suggest that the expression
data provide biologically meaningful results (Fig. 2.1).
30
PC2
400
300
200
100
0
-600
-400
-200
0
200
400
600
PC1
-100
-200
-300
-400
Figure 2.1. Principal components analysis of each sample used in the analysis.
The first two principal components explained approximately 87% of the
variation in gene expression (PC1, 44.9%; PC2, 42.2%). Closed circles, muelleri
males; open circles, laevis males; closed squares, muelleri females; open
squares, laevis females.
We used local-pooled error (LPE) tests to identify genes differentially expressed
among each species-by-sex combination (Jain et al. 2003). Ordinary two-sample t tests
require a large number of replicate arrays to reasonably estimate the within-gene
standard error. Our Xenopus dataset, like most microarray datasets, suffers from low
replication, and for ordinary t tests this creates a low-power framework for testing
hypotheses. Specifically, error estimates constructed for genes with low sample sizes
results in low power in detecting differential expression and an increased number of
false positives (i.e., inflated Type I error). The LPE approach pools variance estimates
for genes with similar expression intensities from replicated arrays within treatment
conditions (Jain et al. 2003). By borrowing strength from variance estimates for genes
31
with similar intensities, LPE reduces the problem of low power due to low sample size.
We implemented the LPE approach using SAS v. 8.0 (SAS Institute) by first estimating
the variance for each gene over the replicates for each species (X. laevis and X.
muelleri) by sex (female and male) comparison. Next, for each species-by-sex
combination, we used the LOESS procedure with a smoothing parameter of 0.01 to fit a
nonparametric model to the log variance versus the mean expression value in order to
obtain smoothed variance estimates for each gene. These smooth variances were then
used to calculate the local-pooled-error test statistic (z statistic) for the following five
comparisons: (1) a species effect (X. muelleri vs. X. laevis); (2) a test for males (♂ X.
muelleri vs. ♂ X. laevis); (3) a test for females (♀ X. muelleri vs. ♀ X. laevis); (4) a
within X. muelleri test (♂ X. muelleri vs. ♀ X. muelleri); and (5) a within X. laevis test
(♂ X. laevis vs. ♀ X. laevis). We estimated the number of false positives using the false
discovery rate (FDR) procedure of Storey et al. (2004) for each test in our dataset.
Using a marginal significance level of alpha = 0.05, the FDR ranged from 0.00765 to
0.018126 (Table 2.1), so the issue of false positives is of no practical concern.
To examine how patterns of sex-biased gene expression contribute to
differences between species, we first selected genes that were considered different for
the species effect. These 2353 transcripts were then examined for patterns of sex-biased
gene expression using the remaining four tests described above. Additionally, we
subjected genes that were not different between species to the same tests in order to
compare the number of sex-biased genes different between species to those not different
between species. Sex-biased genes were defined as follows: a male-biased gene was
32
defined as a gene that had a higher level of expression in males compared to females
and a female-biased gene had a higher level of expression in females compared to
males. A non-sex biased/unbiased gene had the same level of expression in males and
Table 2.1. Estimates of π0 (the fraction of true hypotheses) and the false
discovery rate (FDR) at several levels of alpha (the marginal Type I probability)
in the Xenopus dataset.
Test
Species effect
(laevis vs. muelleri)
Male effect
(laevis ♂ vs. muelleri ♂)
Female effect
(laevis ♀ vs. muelleri ♀)
Sex within laevis
(laevis ♂ vs. laevis ♀)
Sex within muelleri
(muelleri ♂ vs. muelleri ♀)
π0
0.124524
0.231754
0.127292
0.153580
0.246282
33
Alpha
FDR
0.01
0.001673
0.05
0.007650
0.15
0.021652
0.01
0.003857
0.05
0.017014
0.15
0.045599
0.01
0.001664
0.05
0.007837
0.15
0.022213
0.01
0.002174
0.05
0.009906
0.15
0.027589
0.01
0.004166
0.05
0.018126
0.15
0.048282
females. Our statistical tests consider how a gene was expressed in the two species
simultaneously in order to identify evolutionary changes in expression behavior
between the two species. In this analysis, each gene can have the following nine types
of expression behavior: (1) male-biased in X. laevis and male-biased in X. muelleri
(male-biased in both species); (2) female-biased in X. laevis and female-biased in X.
muelleri (female-biased in both species); (3) unbiased in X. laevis and unbiased in X.
muelleri (unbiased in both species); (4) male-biased in X. laevis, unbiased in X. muelleri
(a gain of male-biased expression in X. laevis or a loss of sex-biased expression in X.
muelleri); (5) female-biased in X. laevis, unbiased in X. muelleri (a gain of femalebiased expression in X. laevis or a loss of sex-biased expression in X. muelleri); (6)
male-biased in X. muelleri, unbiased in X. laevis (a gain of male-biased expression in X.
muelleri or a loss of sex-biased expression in X. laevis); (7) female-biased in X.
muelleri, unbiased in X. laevis (a gain of female-biased expression in X. muelleri or a
loss of sex-biased expression in X. laevis); (8) male-biased in X. laevis, female-biased in
X. muelleri (a reversal in sex-biased expression between species); or (9) male-biased in
X. muelleri, female-biased in X. laevis (a reversal in sex-biased expression between
species). Genes were considered sex-biased if at least one of the species showed
evidence of sex-biased gene expression but reversals were not included in the analysis
of the total number of sex-biased genes, as these genes showed opposite patterns of sexbiased expression between species.
Vector analysis was used to analyze gene expression differences between males
and females within both species to extract patterns of sex-biased gene expression based
34
on conventional fold change criteria for microarray data (Breitling et al. 2005). Fold
changes were defined as the average level of expression for males divided by the
average level of expression for females and fold changes were calculated for each
species separately (X. muelleri, 3 males and 6 females = 18 replicates; X. laevis, 4 males
and 4 females = 16 replicates). Fold changes were then transformed to the log2 scale for
use in vector analysis. A Windows executable version of the perlscript provided by
Breitling et al. (2005) was used to calculate the average length vector, the sum vectors,
the representative vectors, the vector sum lengths, and the vector angles for the fold
change of males vs. females for X. muelleri and X. laevis. Each gene was then assigned
a behavior state based on the angle between the representative vector and the other
prototype vectors. p-values for the sum vectors were calculated by randomly sampling
the expression fold changes 10,000 times and then calculating the sum vector
lengths for these random datasets. The sum vectors for p<0.05 for X. laevis and X.
muelleri were then plotted using Excel to represent patterns of expression between the
sexes in the two species. Goodness of fit tests (G), based on the difference between the
observed and the expected (under the null hypothesis of equal class probabilities)
number of genes, were performed for both the LPE and the vector analysis datasets to
test whether male- or female- biased genes were overrepresented compared to non-sexbiased genes. A Williams correction was applied when the total number of genes
exceeded 200 to better approximate the chi-squared distribution (Sokal and Rohlf
2001). We examined the relationship between the type of sex-biased gene expression
(male or female) and the divergence in gene expression between species to test whether
35
male- or female-biased genes had greater divergences in expression between species.
First, for each list of sex-biased genes that were differentially expressed between
species, we partitioned each set into four categories of fold change: > 4-, > 3- to ≤ 4-, >
3- to ≤ 2-, and < 2-fold change. For each gene in this analysis, fold change was
determined by calculating the mean expression value of X. laevis divided by the mean
expression value for X. muelleri. We then calculated the absolute value log2 values for
the fold change and ordered the genes from the highest to lowest. We tallied the number
of genes that fell within each category of fold change and performed an analysis of
variance using the CATMOD procedure in SAS to test the association between the type
of sex-biased genes (male or female) and the magnitude of expression divergence
between species.
2.4 Results
Results from LPE analysis revealed that a majority of transcripts (81% =
2353/2891) were expressed at different levels between X. laevis and X. muelleri, and,
additionally, most of these transcripts showed evidence of sex-biased gene expression
(Table 2.2; see also Supplementary Table 4 for a list of the most divergent transcripts).
There were about 2.7 (683/251) times more male-biased genes (Gadj = 207.5; df = 1; P <
0.001) and about 2 (513/251) times more female-biased genes (Gadj = 91.64; df = 1; P <
0.001) compared to the number of unbiased genes in both species (Table 2.2). For the
19% of transcripts that had no difference in expression between species, a majority of
36
37
L♂=M♂
L♂<M♂
L♂>M♂
7
L♀=M♀
68
37
683
163
♂-biased
(both sp.)
164
3
11
L♀>M♀
L♀<M♀
0
9
0
3
L♀=M♀
0
L♀>M♀
8
273
62
L♀=M♀
44
0
L♀<M♀
35
122
L♀<M♀
L♀>M♀
M♂>M♀
1
0
0
4
0
54
5
Reversal
19
22
3
7
3
M♂<M♀
L♂>L♀
254
0
52
8
23
0
0
0
3
18
♂-biased
(laevis)
0
67
0
0
75
42
17
53
M♂=M♀
0
0
0
14
0
37
72
14
Reversal
13
20
20
11
8
M♂>M♀
51
15
17
6
0
32
3
0
0
513
124
♀-biased
(both sp.)
2
27
60
56
203
57
8
0
100
M♂<M♀
L♂<L♀
7
0
21
4
0
30
0
0
245
62
♀-biased
(laevis)
1
0
74
35
63
13
0
59
M♂=M♀
14
0
7
3
0
2
0
0
152
26
♂-biased
(muelleri)
1
0
20
31
67
1
0
32
M♂>M♀
129
0
0
0
4
5
0
10
10
29
♀-biased
(muelleri)
0
45
1
0
61
8
2
12
M♂<M♀
L♂=L♀
Table 2.2. The number of genes comparatively over and underexpressed from least pooled error (LPE)
tests for all possible simultaneous comparisons between sex and between species in Xenopus laevis (L) and
X. muelleri (M). The logical operators “>”, “<”, and “=” represent over, under, and equal expression
respectively in all comparisons. Cells in white are for genes that are different between species and cells in
grey are for genes not different between species. Cells in black are comparisons rendered illogical and thus
have no possible result.
54
2
5
2
0
0
0
251
63
Non-sexbiased
3
36
18
8
118
2
66
M ♂=M ♀
transcripts also showed evidence of sex-biased gene expression. There were about 2.6
times (163/63) more male-biased genes (Gadj = 45.8; df = 1; P < 0.001) and about 2
times (124/63) more female-biased genes (Gadj = 20.2; df = 1; P < 0.001) than the
number of unbiased genes in both species.
Examining the number of male- compared to female-biased genes in both
species and the total number of sex-biased transcripts revealed that there were more
male- compared to female-biased genes. For genes that were differentially expressed
between species, there were about 1.3 (683/513) times more male-biased compared to
female-biased transcripts in both species (Gadj = 24.24; df = 1; P < 0.001) and more than
55% (1089/1976) of total sex-biased genes had a male-biased expression pattern (Gadj =
20.7; df = 1; P < 0.001). For genes that had the same expression level between species,
there were more male- compared to female-biased transcripts but only for genes that
had the same expression behavior in both species. There were 1.3 (163/124) times more
male-biased compared to female-biased transcripts in both species but there was no
difference in the total number of male- or female-biased genes (Gadj = 1.48; df = 1; P >
0.05). Results from vector analysis provide additional evidence for a preponderance of
male- compared to female-biased genes in Xenopus (Fig. 2.2). For genes that were
differentially expressed between species, there were about 1.3 (590/457) times more
genes upregulated in males compared to genes upregulated in females of both species
(Gadj = 16.9; df = 1; P < 0.001) and about 1.2 (1138/937) times more total genes
upregulated in males compared to genes that were upregulated in females (Gadj = 19.4;
df = 1; P < 0.001; Table 2.3). For genes that were expressed at the same level between
38
Xenopus laevis
400
300
200
100
Xenopus muelleri
0
-400
-300
-200
-100
0
100
200
300
400
-100
-200
-300
-400
Figure 2.2. Vector analysis of expression responses of males vs. females in
Xenopus laevis and X. muelleri for those genes recovered as different between
species in the LPE analysis. Each point represents the sum vector from the 288
pairwise comparisons (16 replicates in X. laevis and 18 replicates in X. muelleri)
and only those vectors that meet the significance criteria of α = 0.05 are included
in the plot (2314 out of 2437 transcripts recovered as different between species
in the LPE analysis). Genes toward the periphery of the plot represent the most
consistent behavior among the replicates. The total number of genes upregulated
in males vs. females are shown in red squares and the total number of genes
downregulated in males vs. females (alternatively upregulated in females but
downregulated in males) are shown in green circles. Black circles are for those
genes that show opposite patterns of regulation between species. There are more
male biased genes compared to female biased genes.
39
species, there were about 1.3 (149/111) times more genes upregulated in males
compared to genes upregulated in females of both species (Gadj = 5.60; df = 1; P <
0.025), but there was no difference in the total number of genes upregulated in males
Table 2.3. Results of vector analysis for the fold change in males vs.
females for patterns of expression behavior in Xenopus laevis and
Xenopus muelleri. Up denotes upregulation in males (or male biased)
and down denotes down regulation in males (female biased). ♂ denotes
those genes that are male biased.
Regulation
Number of Transcripts
Different btw Species Not different btw Species
421
81
368
99
127
24
112
19
590
149
X. laevis and X. muelleri down
457
111
X. laevis up, X. muelleri down
68
18
X. muelleri up, X. laevis down
109
20
X. laevis specific up
♂
X. laevis specific down
X. muelleri specific up
♂
X. muelleri specific down
X. laevis and X. muelleri up
♂
40
compared to genes that were upregulated in females (Gadj = 1.30; df = 1; P > 0.05;
Table 2.3). Patterns of evolutionary change in expression bias, specifically a gain in one
species or a loss in the other, did not have signatures of male- or female-biased
expression. For genes expressed differently between species, there were equal numbers
of transcripts that showed a gain in expression bias in males compared to females in X.
laevis (Gadj = 0.1621; df = 1; P > 0.05). For X. muelleri, there were 152 genes that
showed a gain in expression bias for males compared to 129 for gains in expression bias
for females, but this tendency toward more male-biased genes was not different (Gadj =
1.873; df = 1; P > 0.05) (Table 2.3).
For genes not different between species there was no difference between gains
in expression bias between males and females of X. laevis (G = 0.878; df = 1; P >0.05)
or X. muelleri (G = 0.163; df = 1; P >0.05). Patterns of reversals in expression bias
between species, specifically upregulation for a gene in one sex in one species with the
opposite pattern of expression in the other species did not reveal evidence of sexbiased
gene expression. For genes that showed a reversal in sex-biased expression, there were
54 genes that had male-biased expression in X. laevis with a corresponding femalebiased expression in X. muelleri and 72 genes that had female-biased expression in X.
laevis and male-biased expression in X. muelleri but these were not statistically
different (G = 2.58; df = 1; P > 0.05).
While numerically there were more male-biased than female-biased genes in
both species, there was a greater proportion of female-biased genes with larger
magnitudes of expression divergence between species than both male and non-sex41
biased genes (Table 2.4). There were about 1.8 (0.072/0.040) times more female-biased
genes with a >4-fold difference in expression between species, compared to male-biased
genes (X2 = 5.71; df = 1; P < 0.017) and nearly 3 (0.072/0.024) times more femalebiased genes com-pared to unbiased genes (X2 = 10.41; df = 1; P < 0.00013). There
were also proportionally more female-biased genes for >3- to ≤ 4 fold change in
expression between species, with 1.5 (0.092/0.060) times more female-biased genes
compared to malebiased genes (X2 = 4.08; df = 1; P < 0.044) and about 3 (0.092/0.03)
times more female-biased genes compared to unbiased genes (X2 = 15.03; df = 1; P <
0.0001). For genes expressed between species at the >2- to ≤ 3-fold level, there was no
difference in the proportions of female (0.24), male (0.22), or unbiased (0.22) genes (X2
= 0.54; df = 2; P = 0.76).
For the smallest category of fold change (<2) there were 1.1 (0.68/0.60) times
more male-biased genes compared to female-biased genes (X2 = 8.40; df = 1; P <
0.0038) and 1.2 (0.73/0.60) times more unbiased genes compared to the proportion of
male-biased genes (X2 = 13.09; df = 1; P < 0.0003).
2.5 Discussion
We recovered a male-biased gene expression pattern and a role for femalebiased genes in interspecific expression divergence for a portion of the transcriptome
that arguably is the most conserved between these two species of Xenopus. This
malebiased expression pattern consists of numerically more genes overexpressed in
males compared to the number of genes overexpressed in females and to the number of
genes that had no sex bias in both species. Female-biased genes were proportionally
42
more abundant for the largest differences in expression between species, and malebiased genes were more represented in the smallest changes in expression between
species. One question that arises is how sequence divergence might have affected the
results we found.
Sequence divergence has recently been shown to be a major problem in cDNA
microarray studies where RNA from a heterospecific species is hybridized onto a
microarray designed for another species (Gilad et al. 2005). However, we do not think
that sequence divergence is generating the patterns we found in Xenopus.
Xenopus are an ancient lineage of frogs and the estimated time of divergence
between X. laevis and X. muelleri is more than 30 million years (Evans et al. 2004,
2005). Exploratory analyses of our dataset revealed strong signatures of sequence
divergence. To lessen this impact we guarded against transcripts that would be most
dramatically affected by sequence divergence by using the Affymetrix detection calls.
We selected transcripts based on five specific criteria (see Methods) to ensure that we
captured transcripts that were detectable in all replicates of X. muelleri—the species for
which bias due to sequence divergence will be introduced. The Affymetrix detection
algorithm uses the raw cell intensities to determine if a transcript is below the threshold
of detection and therefore is not biased by scaling or other normalization methods that
may introduce additional bias into the expression data (Affymetrix, Inc. 2001). We
compared the proportions of significant and nonsignificant transcripts for X. laevis and
X. muelleri within species tests (males vs. females) for our filtered dataset (n = 2891
43
44
24
463
683
≤2
Total
54
15
6
9
152
27
>4
Y
>3 ≤ 2
Y
Species
Effect
F
41
M
X. muelleri
M
>4 ≤ 3
M
X. laevis
254
159
53
26
16
Y
U
M
513
306
123
47
37
Y
F
F
72
48
14
6
4
Y
M
F
245
151
56
15
23
Y
U
F
44
251
182
56
7
6
Y
U
U
152
109
31
7
5
Y
M
U
129
81
36
7
5
Y
F
U
163
156
6
1
0
N
M
M
5
5
0
0
0
N
F
M
52
52
0
0
0
N
U
M
124
123
0
1
0
N
F
F
14
14
0
0
0
N
M
F
1
0
0
N
U
F
62
61
Table 2.4. The number of transcripts for all possible gene expression behaviors in males and
females of Xenopus laevis and X. muelleri and the magnitude of fold change in expression
between species. M=male-biased; F=female-biased; U=unbiased; Y=differentially expressed
between species; N=expressed the same in both species.
63
63
0
0
0
N
U
U
26
26
0
0
0
N
M
U
29
29
0
0
0
N
F
U
transcripts) to the total number of Xenopus transcripts on the X. laevis microarray (n =
15,491) and to the number of transcripts on the array minus the genes in our filtered
dataset (n = 12,600). For the filtered dataset, there were similar numbers of significant
and nonsignificant transcripts in X. laevis (significant = 78%; not significant = 22%)
compared to X. muelleri (significant = 68%; not significant = 32%). The proportion of
nonsignificant transcripts increased dramatically in X. muelleri (53%) when we
examined the total transcripts present on the microarray. The proportion of
nonsignificant calls in X. muelleri increased even further when we examined all
transcripts on the microarray minus the transcripts used in our study (significant = 42%;
not significant = 58%). However, the proportions of significant and nonsignificant
transcripts for X. laevis remained similar for these two datasets (total, significant = 73%
and not significant = 27%; total transcripts minus genes in the filtered dataset,
significant = 72% and not significant = 28%). We believe that these disparate patterns
in the proportion of significant and nonsignificant transcripts provide evidence that the
transcripts used in the analysis were the best set available in a comparison of sex-biased
gene expression in these species of Xenopus.
These patterns of significant and nonsignificant transcripts for the two species
also suggest that sequence divergence in X. muelleri decreases the ability to reject the
null hypothesis of no difference in expression between males and females and therefore
decreases the ability to detect sex-biased expression between species even if such a
pattern was real. Contrary to this expectation, we in fact recover strong evidence for
more male-biased genes between the two species and conclude that the impact of
45
sequence divergence is minimal for the genes that we used in the analysis. Additionally,
if we inadvertently excluded genes that had a higher level of divergence due to our
filtering criteria, then there should be more unbiased genes in both X. laevis and X.
muelleri for genes differentially expressed between species compared to the number of
unbiased genes within each species. Instead, for genes that were differentially expressed
between species, there were fewer unbiased genes in both X. laevis and X. muelleri
(17% = 251/1447) compared to the number of unbiased genes within each species (X.
laevis = 22% [650/2891]; X. muelleri = 32% [927/2891]). This provides additional
support to the notion that the genes we used in the analysis are useful for analyzing
patterns of sexbiased expression between these two species of Xenopus.
Another question that has to be considered is whether the set of transcripts that
we selected is a good representation of the real expression pattern in Xenopus. To
address this question, we normalized with RMA all replicates of X. laevis and tested the
entire microarray (n = 15,491 transcripts) for patterns of sex-biased gene expression.
Since the microarray is designed for X. laevis, we detect without bias the expression
pattern present between males and females. This analysis reveals that 72% of the
transcriptome is differentially expressed between males and females and of this, 57% is
upregulated in males compared to 43% in females. This supports the notion that the
general pattern of expression is more represented by genes with male-limited expression
and that we did not happen to sample a portion of the transcriptome that was malebiased. In general, we believe that our methods for controlling common sources of error
associated with microarray data are appropriate but additional experiments using real46
time PCR will provide unequivocal confirmation for the patterns that we recovered in
these two species of Xenopus.
Xenopus are a unique system to consider patterns of sex-biased gene expression
because Xenopus have ZW sex determination, have homomorphic sex chromosomes,
and provide an important exception to Haldane’s rule (Haldane, 1922; Kobel and Du
Pasquier, 1975; Kobel et al., 1981; 1996; Picker, 1985; Picker and de Villiers, 1989;
Kobel, 1996; Picker et al., 1996; Fischer et al., 2000). Faster male evolution through
sexual selection of male-expressed genes or inherent sensitivity to the process of
spermatogenesis provides one way in which Haldane’s rule may be overridden. In terms
of the number of malebiased genes, our results are consistent with the faster male
evolution hypothesis in that there were more genes involved in the process of
spermatogenesis compared to oogenesis. However, female-biased genes contributed
more to larger divergences in expression between species suggesting the possibility that
ZW sex determination could play an important role in creating expression differences
between species.
There is much debate about the role of neutral evolution in producing changes
in the transcriptome (Rifkin et al. 2003; Khaitovich et al. 2004; Jordan et al. 2005).
While stochastic processes may be operating in the transcriptome of Xenopus, our
results are inconsistent with purely neutral evolution. We recovered a pattern of malebiased expression for genes expressed differently between species but not for genes
with the same level of expression between species. Additionally, genes that had a
pattern of reversal in expression bias were the least represented in this portion of the
47
Xenopus transcriptome. These facts suggest that selective forces acting within and
between species may be more important at producing changes in the transcriptome of
Xenopus.
Our analysis of sexually dimorphic gene expression is the first such analysis
between two species of vertebrates and the first for an organism with ZW sex
determination and homomorphic sex chromosomes. Previous studies of sex-biased gene
expression have used taxa with heteromorphic XY sex chromosomes (Anopheles and
Drosophila; Meiklejohn et al. 2003; Ranz et al. 2003; Connallon and Knowles 2005;
Hahn and Lanzaro 2005; Marinotti et al. 2006). In Drosophila, Ranz et al. (2003)
provided evidence of male-biased gene expression between D. melanogaster and D.
simulans in that the magnitude of differences in expression for males exceeded the
change in expression between females and there were more male-biased genes with >4fold expression divergence between species compared to either female- or non-sexbiased genes. Ranz et al. (2003) show that male-biased genes were most divergent
between species and suggest that partially dominant mutations may explain the
overrepresentation of female-biased genes on the X chromosome.
A within-species comparison of transcription in males and females of Anopheles
gambiae has recently been made. Anopheles gambiae have, like Drosophila,
heteromorphic XY sex chromosomes, however, there is a pronounced pattern of femalebiased expression, suggesting that sex-biased expression is not completely related to the
heterogametic sex (Hahn and Lanzaro 2005). It appears that the physiological roles
played by females of Anopheles (blood feeding and antigen-related defense) may drive
48
the expression pattern between the sexes. This raises the important question if current
expression studies are merely measuring the effects rather than the causes of sexually
dimorphic expression. This female-biased example in Anopheles also suggests a need
for a theoretical treatment of sex-biased gene expression in order to provide predictions
for when patterns of sex-biased expression may be skewed to one sex or the other.
We compared RNA from testes and ovary extracted from two different species
of Xenopus because the gonad has been shown to have massive differential expression
between the sexes. It will be interesting to compare other tissues between males and
females of Xenopus in the future, as it is likely that differential expression between the
sexes and between species will occur for other tissues (Parisi et al. 2004). In Drosophila
melanogaster, there is massive differential expression between the testis and the ovary
but also a large degree of differential expression for somatic tissues (e.g., Parisi et al.
2003, 2004).
Sexual conflict theory predicts a role for the sex chromosomes in facilitating
sex-limited expression (Rice 1984). In the case of ZW systems such as Xenopus, theory
predicts that Z-linked recessive mutations that favor females at the expense of males are
more likely to become fixed while Z-linked mutations that favor males at the expense of
females will fix if these mutations are partially dominant (Rice, 1984). In agreement
with this hypothesis, spermatogonial genes are overrepresented on the X chromosome
of mice (Wang et al., 2001) and sex- and reproduction-related genes are overrepresented
on the human X chromosome (Saifi and Chandra, 1999; Lercher et al., 2003). However,
a fine temporal and spatial analysis of gene expression in mice has revealed that genes
49
expressed early in spermatogenesis are enriched on the X chromosome, whereas genes
expressed late in spermatogenesis are depleted (Khil et al., 2004). Similarly,
underrepresentation of male-biased genes on the X chromosome has been found in C.
elegans and Drosophila (Reinke et al. 2000; Parisi et al. 2003; Ranz et al. 2003). This
X-chromosome demasculinization can be attributed to inactivation of the X
chromosome in male meiosis (Wu and Xu 2003; Khil et al. 2004).
Our results suggest a role for dominant mutations in Xenopus because males are
the homogametic sex and we found more male- compared to female-biased genes. Such
dominant mutations could be germane to the function of the testis or the process of
spermatogenesis (Kleene, 2005). We also found that female-biased genes had the
largest magnitude changes between species and we speculate that these changes might
be associated with the dominance pattern within the ZW system, as theory predicts the
ease by which recessive mutations can fix when located on the Z chromosome. We
would like to examine the genomic location of sex-biased genes in Xenopus; however,
there is little information pertaining to chromosomal location of genes in the Xenopus
genome, although high resolution linkage mapping is currently under way for Xenopus
(Silurana) tropicalis. These data may be helpful for identifying the location of genes in
other species of Xenopus, and in the future these data can be used to test the
chromosome distribution prediction formulated by Rice (1984).
In this study, we found extensive divergence in a sample of the transcriptome of
the adult gonad between two species of Xenopus. This divergence can mainly be
attributed to sex-biased gene expression likely the result of sex-dependent selection.
50
About 81% (2353/2891) of genes were differentially expressed between the two species
and more than 84% (1976/2353) of these interspecific changes had patterns of sexbiased gene expression. We recovered a male-biased expression pattern in both species,
with 57% (683/1196) of sex-biased genes having a malebiased expression pattern, but at
the same time female-biased genes contributed more to the magnitude of expression
difference between species. Additionally, genes expressed the same between species did
not reveal strong evidence for sex-bias expression and this suggests a role for sexdependent selection driving divergence between species. Our results are consistent
with faster male evolution occurring in a ZW system with morphologically
indistinguishable sex chromosomes in terms of the number of male-biased compared
to female- and non-sex-biased genes. However, the simultaneous contribution of
female-biased genes to divergence in expression between species necessitates new
studies that will address dominance effects of sex-biased genes and their spatial and
temporal patterns of expression.
51
CHAPTER 3
STERILITY AND GENE EXPRESSION IN HYBRID MALES OF XENOPUS
LAEVIS AND X. MUELLERI
3.1 Abstract
Reproductive isolation is a defining characteristic of populations that represent
unique biological species, yet we know very little about the gene expression basis for
reproductive isolation. The advent of powerful molecular biology tools provides the
ability to identify genes involved in reproductive isolation and focuses attention on the
molecular mechanisms that separate biological species. Herein we quantify the sterility
pattern of hybrid males in African Clawed Frogs (Xenopus) and apply microarray
analysis of the expression pattern found in testes to identify genes that are misexpressed
in hybrid males relative to their two parental species (Xenopus laevis and X. muelleri).
Phenotypic characteristics of spermatogenesis in sterile male hybrids (X. laevis x X.
muelleri) were examined using a novel sperm assay that allowed quantification of live,
dead, and undifferentiated sperm cells, the number of motile vs. immotile sperm, and
sperm morphology. Hybrids exhibited a dramatically lower abundance of mature sperm
relative to the parental species. Hybrid spermatozoa were larger in size and
accompanied by numerous undifferentiated sperm cells. Microarray analysis of gene
expression in testes was combined with a correction for sequence divergence derived
52
from genomic hybridizations to identify candidate genes involved in the sterility
phenotype. Analysis of the transcriptome revealed a striking asymmetric pattern of
misexpression. There were only about 140 genes misexpressed in hybrids compared to
X. laevis but nearly 4,000 genes misexpressed in hybrids compared to X. muelleri. Our
results provide an important correlation between phenotypic characteristics of sperm
and gene expression in sterile hybrid males. The broad pattern of gene misexpression
suggests intriguing mechanisms creating the dominance pattern of the X. laevis genome
in hybrids. These findings significantly contribute to growing evidence for allelic
dominance in hybrids and have implications for the mechanism of species
differentiation at the transcriptome level.
3.2 Introduction
Biological species remain cohesive by a lack of gene flow between interspecific
populations and the mechanisms that maintain this pattern are manifest by various
forms of reproductive isolation. Postzygotic reproductive isolation is characterized by
dysfunctional phenotypes observed in F1 interspecific hybrids including but not limited
to inviability and/or sterility resulting in decreased gene flow between species. Haldane
(1922) observed that the heterogametic sex (XY or ZW) typically suffers the most
dysfunctional effects of hybridization. Haldane’s rule has been shown to be broadly
applicable across diverse groups of animals suggesting that understanding the basis for
Haldane’s rule provides a key towards understanding the mechanisms for how species
become and/or remain reproductively isolated. Much work, using mainly forward
genetic approaches, has focused on the genetic basis of postzygotic reproductive
53
isolation and Haldane’s rule. These studies have provided support for both dominance
effects and faster male evolution as the main mechanisms generating Haldane’s rule;
however only recently have studies focused on gene expression as related to sterile
hybrids and this opens a new avenue towards understanding the proximate causes of
reproductive isolation (Reiland and Noor, 2002; Michalak and Noor, 2003; Ranz et al.,
2004; Barbash and Lorigan, 2006; Haerty and Singh, 2006; Hegarty et al., 2006;
Josefsson et al., 2006; Lai et al., 2006; Michalak, 2006; Moehring et al., 2007; OrtízBarrientos et al., 2007). By starting at the level of phenotype, analyzing the
transcriptome found in both species and compared to the dysfunctional hybrid, this
reverse genetics approach provides the candidate loci that contribute to reproductive
isolation, identifies targets that have evolved disparately between the two species, and
allows a test of what evolutionary forces create gene misexpression in dysfunctional
hybrids. Although correlational in nature, this approach advances our understanding of
reproductive isolation by generating testable hypotheses for which future functional
experiments can be designed.
Frogs of the genus Xenopus offer an exciting new system to explore the
expression basis of reproductive isolation. Xenopus are characterized genomically by
allopolyploidization and range from diploid (n=20) to dodecaploid (n=108) numbers of
chromosomes (Kobel and Du Pasquier, 1986). Most species can be crossed to produce
viable progeny and males from lab produced and wild caught hybrids are sterile
whereas hybrid females are fertile (Kobel HR and Du Pasquier, 1975; Kobel et al.,
1981; Picker, 1985; Picker and de Villiers, 1989; Kobel, 1996; Kobel et al., 1996;
54
Picker et al., 1996; Fischer et al., 2000). Sex-reversal experiments and sex ratios from
backcross progeny have established that a dominant allele in females of X. laevis, X.
muelleri, and other species determines sex and therefore Xenopus have ZW sex
determination (Mikamo and Witschi, 1963; 1964; Witschi, 1971; Kobel, 1985; Hayes,
1998; Miyata et al., 1999). Additionally, no morphologically distinct Z or W sex
chromosomes have been identified in Xenopus and therefore Xenopus have
homomorphic sex chromosomes (Tymowska and Kobel, 1972; Schmid and Steinlein,
1991). These observations suggest that Xenopus are an exception to Haldane’s rule and,
given Xenopus as a model organism, provide an excellent system to address gene
expression and reproductive isolation.
We explored gene expression and reproductive isolation in hybrids of Xenopus
laevis and X. muelleri, both tetraploid species (2n=36) which hybridize in the wild
(Fischer et al., 2000). To do so, we focused on the phenotypic characteristics of
spermatogenesis in sterile male hybrids (X. laevis x X. muelleri). Affymetrix Xenopus
laevis Genome Arrays were used to assay the transcriptome in testes combined with a
correction for sequence divergence from genomic hybridizations that allowed discovery
of the broad pattern of misexpression as well as the identification of candidate genes
involved in the sterility phenotype. This approach importantly allows a correlation of
the broad gene expression pattern to the phenotypic characteristics observed for sperm
in sterile hybrids and identifies the loci misexpressed in hybrids relative to the two
parental species.
55
3.3 Materials and Methods
3.3.1 Experimental Crosses
Four biological replicate hybrid clutches were produced at The University of
Texas at Arlington. The female parent for each of these crosses was Xenopus l. laevis
and these originate from the Cape region of South Africa or from the laboratory of
Jacques Robert at The University of Rochester. The male parents were X. muelleri and
originate from Nkambeni area of Swaziland and were kindly provided by R. C. Tinsley.
Crosses were conducted in the following manner: each female was injected with
500 IU human chorionic gonadotropin (hCG; Choluron or Carolina Biological Supply)
and placed into a container with 10 L of dechlorinated water. Males were injected with
200 IU hCG and then paired with individual females. Each hybrid clutch was raised to
sexual maturity and clutches were maintained in 12L:12D photoperiod at 25°C air
temperature. Animals were fed Xenopus pellets from Xenopus Express Inc. every two
days.
3.3.2 Sperm Assays
We quantified characteristics of the sperm in hybrids and the two parental
species. From each of three biological replicate clutches we selected two sexually
mature hybrids. Sexual maturity was determined by the presence of dark colored nuptial
pads indicative of circulating androgens and characteristic of sexual maturity (Kelley,
1996). One hybrid was injected with 150 IU hCG and the other was not injected with
hCG in order to test the effect of hCG treatment on sperm abundance.
56
For X. laevis, we used F1 males from Xenopus Express and whose parents
originate from the Cape of South Africa and/or the lab of J. Roberts at the University of
Rochester and selected animals based on sexual maturity. Again, one of the two animals
was injected with 150 IU hCG and then left overnight in 10 L of dechlorinated water.
For X. muelleri we selected one F1 male X. muelleri that was produced at the University
of Texas at Arlington and whose parents originate from the Nkambeni area of
Swaziland. This male was injected with 150 IU hCG. Males were killed by immersion
in MS-222 and testes were harvested and placed in 500 µl of DeBoer’s Solution (110
mM NaCl; 1.3 mM CaCl2; 1.3 mM KCl). Testes were homogenized using a handheld
pestle to form a stock sperm solution. Ten µl of the stock sperm solution was aliquoted
and further diluted 3:1 with distilled water. The 3:1 dilution ratio was found to provide
the maximum number of motile sperm in X. laevis (Christensen et al., 2004) and was
used to assay for sperm motility.
Motile sperm and nonmotile sperm were enumerated during a 2 minute interval.
This procedure was repeated four subsequent times/individual to provide a metric of
sperm motility. Two individuals per treatment (injected and uninjected) and per taxa (X.
laevis and hybrids) were used for sperm motility assays.
We assayed the abundance of live and dead sperm cells using the LIVE/DEAD
Sperm Viability Kit from Molecular Probes (Invitrogen, Inc.). The LIVE/DEAD Sperm
Kit is designed specifically for assaying live and dead sperm cells. Five µl of a 50-fold
dilution of the stock SYBR 14 dye and 5 µl of 2.4 mM propidium iodide were added to
the stock sperm suspension followed by a 10 minute incubation at 36°C for each dye.
57
Stained cells were collected on black polycarbonate filters (1.0 µm pore-size, GE
Osmonics) and enumerated at a magnification of 165X using epifluorescence
microscopy (Olympus BH-2). Briefly, 10 µl of stained sperm were added to 2 ml of
particle-free distilled-water (0.2 µm pore-size filtered) contained in the filter tower of a
10- place filtration unit (Hoeffer). Following filtration, filters were placed on slides, a
drop of immersion oil added to the surface, and a cover slip placed on top. Live and
dead sperm cells were enumerated from fifteen separate, randomly-selected visual
fields. Images were captured using a digital camera (Olympus DP70).
We measured the area of sperm acrosomes in square micrometers as a proxy for
the size for spermatozoa using images captured from placing 10 ul of sperm stock
solution onto a microscope slide. Thirteen sperm cells from hybrids, 21 sperm cells
from X. laevis, and 20 sperm cells from X. muelleri were selected at random from the
images and the area in square micrometers of each sperm cell was measured using
ImageJ software (NIH).
We constructed general linear models in SYSTAT v. 8.0 (SAS, 1998) to test the
null hypothesis that there was no difference in the total number of sperm cells, the
proportion of live, dead, and undifferentiated sperm cells, and the proportion of motile
and nonmotile sperm between taxa and between the hCG treatment. An ANOVA was
used to test the hypothesis that there was no difference in the size of spermatozoa
between treatments.
58
3.3.3 Microarray Experiments
RNA was extracted from adult testis in Xenopus laevis (n = 4), hybrids of X.
laevis x. X. muelleri (n=4) and X. muelleri (n = 3). Adults were euthanized with MS-222
and testes were immediately removed, placed in RNA extraction solution, and
homogenized. RNA was recovered using GeneHunter and Ambion RiboPure total RNA
kits. Samples of RNA were checked for purity by examination of the 28S and 18S
ribosomal RNA bands from denaturing gel electrophoresis, by 260/280 ratios from
scans with a Nanodrop ND 1000 spectrophotometer, and by readouts of the Agilent
Bioanalyzer. Total RNA samples were prepared and hybridized to Affymetrix Xenopus
laevis GeneChip Genome Arrays at the University of Texas Southwestern Medical
Center Microarray Array Core Facility following standard Affymetrix protocols.
Affymetrix Microarray Analysis Suite (MAS) v.5.0 was used to scan and process each
microarray chip. The signals of quality control and poly(A) transcripts revealed that
hybridizations were of high quality in all chips. Quality control probe sets (i.e., spike in
and housekeeping genes) were removed in subsequent statistical analyses.
Hybridizing RNA from a heterospecific species to a microarray designed for a
related species can have a dramatic impact on the signal recovered from microarrays
(Nuzhdin et al., 2004; Gilad et al., 2005; Gilad and Borevitz, 2006). Consequently, we
directly assayed sequence divergence by hybridizing genomic DNA from Xenopus
laevis and X. muelleri each separately to the Affymetrix Xenopus laevis GeneChip
Genome Array. Genomic DNA was extracted from liver from eight individuals/species
(four male and four female) using a QIAGEN DNeasy kit. Genomic DNA was
59
quantified using a Nanodrop ND 1000 spectrophotometer and 2.5 µg/µl from each of
the eight individuals was pooled to produce 20 µg genomic DNA for subsequent
fragmentation. The 20 µg pooled genomic DNA was fragmented by a DNase I digestion
using 2.5 µl 10X One-Phor-All Buffer (Amersham Biosciences), 0.015 U/µg DNase I
(Amershame Biosciences), and 14.125 µl H2O to form a 25 µl reaction. Reactions were
incubated at 37°C for 10 minutes and the DNase I was inactivated by incubating the
reaction at 100°C for 10 minutes. Reactions were visualized on 2.5% agarose gels
stained with SYBR Green to confirm that fragments were in the 50-200 bp size range
necessary for hybridization to Affymetrix microarrays. Fragments were labeled on the
3’ termini and hybridized to the X. laevis Affymetrix Genome Array following standard
protocols recommended by Affymetrix.
We used an electronic mask to eliminate probes that behaved poorly due to
sequence divergence. To do so, we modified the Xspecies perl script (Version 1.1) in
Hammond et al. (2005) to incorporate information gained by comparing the ratio of
genomic hybridization intensity of X. muelleri to X. laevis for each probe pair. The
Xspecies perl script selects a probe-set when one or more PM probe-pair (s) meets a
user-specified criteria of hybridization intensity and creates a new probe definition file
(.cdf) that contains only those probe-pairs that meet the user-specified criteria. This, in
theory, should create a hybridization signal that interrogates gene expression in a less
biased manner compared to not eliminating these probes. To conservatively guard
against sequence divergence we set the lower ratio of hybridization intensity to 0.99 and
explored variation in the number of probes eliminated at a variety of upper thresholds
60
with the idea that higher intensity for X. muelleri is not as damaging compared to probes
that have a hybridization signal lower for X. muelleri when compared to X. laevis (Fig.
6). In examining the threshold variation on the number of probesets remaining, we
chose to analyze the .cdf generated at the 1.01 and 1.10 level. Both sets provided a
similar general pattern but the effect was more prominent for the less stringent
threshold. We report here the results generated using an upper threshold of 1.10 in
subsequent analyses of gene expression which resulted in the removal of 226,841
individual probes and provided 11,485 probesets/genes for further analysis.
3.3.4 Data Analysis
Using the chip definition files created from the Xspecies perl script analyses, we
conducted two separate comparisons to uncover patterns of differential expression
between Xenopus laevis and hybrids, X. muelleri compared to hybrids, and X. laevis
compared to X. muelleri. First, the Xenopus laevis and hybrid chips were normalized
using RMAexpress (Bolstad, 2005) using default parameters for background correction
and quantile normalization. These RMA normalized data were then imported into the R
statistical environment and tested for differences in expression between X. laevis and
hybrids for each of the 11,485 genes using a moderated t-statistic based on an empirical
Bayes method in the limma package found in Bioconductor (Smyth, 2004). The
TopTable function was then used to output the FDR-adjusted P-values for differential
expression. We normalized X. muelleri and hybrid chips together using RMA and
repeated the analyses to uncover differential expression between X. muelleri and
hybrids. Finally, we normalized X. laevis and X. muelleri chips together using RMA and
61
repeated the analysis to uncover genes misexpressed between the two species. This later
test was used to discover patterns of expression behavior in hybrids compared to
interspecies expression behavior.
3.4 Results
3.4.1 Sperm Abundance
One explanation for why male Xenopus laevis x X. muelleri are sterile could be
due to phenotypic defects associated with the process of spermatogenesis. The
production of amphibian sperm is a complex physiological process involving six key
stages of differentiation that include primary spermatogonia, secondary spermatogonia,
primary spermatocytes, secondary spermatocytes, and spermatids (Scheltinga and
Jamieson, 2003). Additionally, it is widely accepted that the process of spermatogenesis
is under hormonal control and that the injection of gonadotropins stimulates sperm
production (Pozzi et al., 2006; Rosemblit et al., 2006). If hybrid male Xenopus are
sterile due to phenotypic defects associated with spermatogenesis, we predict that
characteristics of sperm quality (i.e., abundance, motility, and morphology) should be
different in hybrids compared to the parental species. We tested this hypothesis by
injecting males with human chorionic gonadotropin hormone (hCG) to assay the effect
of hormone induced stimulation on spermatogenesis in hybrids and the two parental
species and then compared sperm characteristics of these injected males with uninjected
sexually mature males. Sperm abundance was quantified using a novel sperm assay that
allowed detection of live, dead, and undifferentiated sperm cells. Testes were
homogenized and then incubated with fluorescent dyes that intercalate with DNA of
62
sperm cells based on whether the cell is live or dead. Counts of live, dead, and
undifferentiated sperm cells were made using epifluorescence microscopy and we tested
the null hypothesis that sperm abundance was the same in hybrids compared to the two
parental species.
There was a dramatic difference in the abundance of sperm in Xenopus laevis
compared to hybrids (Fig. 3.1a-c). About 40 times more sperm cells were found in X.
laevis compared to hybrids (F1, 9 = 135.4; P=0.000) and there was no effect of the hCG
treatment on the total number of sperm cells ( F1, 9 = 0.001; P=0.978; Fig. 3.2). Only
one X. muelleri was available for analysis and the abundance of sperm for this injected
X. muelleri male (22,900 sperm/microliter) was comparable to the abundance of sperm
for injected X. laevis (Mean sperm = 26,833 sperm/microliter) but not comparable to
injected hybrids (Mean = 738 sperm/microliter).
Proportions of live and dead sperm cells revealed an effect of hCG treatment on
Xenopus laevis only. The proportion of live sperm cells was about 40% greater in
normal X. laevis compared to hybrids (F1, 8 = 7.13; P=0.028) and there was an affect of
hCG treatment on the proportion of live sperm cells (F1, 8 = 15.75; P=0.004). This hCG
treatment effect can be explained mainly by the X. laevis specific response to hCG
treatment (Taxa x Treatment interaction F1, 8 = 6.31; P=0.036) as the proportion of live
cells decreased by about 50% (because the proportion of dead cells increased from 30%
to 50% from the hCG treatment) in X. laevis but did not change in hybrids (Fig. 3.3).
The proportion of live and dead cells for X. muelleri (Live = 0.52; Dead = 0.48) was
higher
63
64
A
C
64
Figure 3.1. Visualization of live (green) and dead (red) sperm in sperm density assay using dual emission filter
for SYBR14 and propidium iodide. Xenopus laevis (A); hybrid (B); X. muelleri (C); and brightfield combined
with fluorescent image of undifferentiated cells of hybrids (D).
D
B
5
LOG10TOT
Log 10 Cells/microliter
6
4
3
TAXA
2
normal
injected
TREATMENT
hybrid
laevis
Figure 3.2. The number of total sperm cells in Xenopus laevis (black circles) and
hybrids (striped squares) in uninjected sexually mature males and hCG injected
sexually mature males. Error bars represent ± 1 standard error.
compared to injected X. laevis (Mean proportion Live X. laevis = 0.22; Mean proportion
Dead X. laevis = 0.78) but was closer to the mean proportion of live and dead cells for
normal X. laevis (Mean proportion Live = 0.69; Mean proportion Dead = 0.30).
There was a striking difference in the proportion of undifferentiated sperm cells
in Xenopus laevis compared to hybrids (Fig. 3.1b, d). In X. laevis there were practically
no undifferentiated sperm cells but in hybrids the majority (about 50%) of the sperm
suspension was comprised of undifferentiated sperm (F1, 8 = 46.073; P=0.000). The
hCG treatment had no effect on the number of undifferentiated sperm cells (F1, 8 =
0.013; P=0.911; Fig. 3.3). There was no significant number of undifferentiated sperm
cells for the sample of X. muelleri.
65
66
Proportion of Cells
0.0
0.0
normal
injected
TREATMENT
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
B
normal
injected
TREATMENT
66
Figure 3.3. Proportion of live (green circles), dead (red squares), and undifferentiated (black triangles) sperm
cells in Xenopus laevis (A) and hybrids (B) in normal/uninjected compared to hCG injected males. Error bars
represent ± 1 standard error.
PROPDEAD
PROPGGB
PROPLIVE
A
Proportion
of Cells
PROPDEAD
PROPGGB
PROPLIVE
Log 10 Sperm Cells
LOG10IMMOTIL
LOG10MOTILE
4
3
X. laevis
2
1
0
Hybrid
TAXA
normal
injected
TREAT
TREATMENT
hybrid
laevis
Figure 3.4. Numbers of motile (green circles) and immotile (red x’s) sperm in
Xenopus laevis and hybrids in normal/uninjected compared to hCG injected
males. Error bars represent ± 1 standard error.
3.4.2 Sperm Motility
Motility of sperm is important to successful fertilization in amphibian species.
Xenopus, like many amphibian species, have external fertilization and sperm become
motile and swim to fertilize eggs only after entering a lower osmolality environment
(Christensen et al., 2004). Hybrids produce a lower abundance of mature sperm and
while sterility may be explained in part by this lower abundance, sterility could be
complete if sperm in hybrids failed to become motile. We tested the null hypothesis that
67
sperm motility was the same in hybrids compared to the two parental species by
counting the number of motile and immotile sperm following activation. There were
about 70 times more motile sperm in X. laevis compared to hybrids (F1,
5
= 188.3;
P=0.000) and there was no effect of hCG treatment on the number of motile sperm (F 1,
5
= 0.258; P=0.633). Despite the large difference in number of motile sperm cells,
proportions of motile to immotile sperm were the same between X. laevis and hybrids
(F1, 5 = 0.570; P=0.484; Fig. 3.4). There was 12% motile and 88% immotile sperm out
of a total of 238 sperm cells observed in X. muelleri. This was close to the mean
percentage of sperm for X. laevis (Mean = 18% motile and Mean = 82% immotile).
3.4.3 Sperm Morphology
Abnormal sperm morphology is frequently associated with infertility (Pilder et
al., 1997) and misshapen or abnormal sperm morphology in hybrid males would suggest
an explanation for sterility in hybrids. We measured the area of sperm cells as a proxy
for size and tested the null hypothesis that the size of mature sperm was the same in
hybrids compared to the parental species.
Hybrid sperm were dramatically larger compared to the sperm of Xenopus
laevis and X. muelleri (F1, 51 = 74.0; P=0.000). Hybrid sperm were about 28 µm2 larger
than Xenopus laevis (Bonferroni corrected P=0.000) and 25 µm2 larger than X. muelleri
sperm (Bonferroni corrected P=0.000). The size of sperm did not differ between X.
laevis and X. muelleri (Bonferroni corrected P =0.513; Fig. 3.5).
68
laevis
hybrid
muelleri
SpermAREA
Area (µm2)
70
60
50
**
40
30
*
*
laevis
TAXA
muelleri
20
10
hybrid
Figure 3.5. Comparison of sperm area (µm2) in Xenopus laevis, X. muelleri, and
hybrids and representative brightfield images of sperm. Error bars represent ± 1
standard error and * denotes significance from a Bonferroni multiple
comparison test. Hybrids have larger sperm compared to X. laevis and X.
muelleri but sperm area does not differ between X. laevis and X. muelleri.
69
3.4.4 Gene Expression
Our analyses of sperm characteristics in hybrids compared to X. laevis and X.
muelleri revealed several phenotypic differences that may contribute to the sterility
pattern. We next analyzed the transcriptional pattern in hybrids and the two parental
species to explore the proximate mechanism of gene expression and its contribution to
the phenotype of hybrids. This approach allowed us to identify loci that may be
involved in generating the sterility phenotype and simultaneously examine the broad
pattern of gene expression in hybrids compared to the parental species.
We used Affymetrix Xenopus laevis Genome Arrays to generate a
transcriptional profile of gene expression in testes of hybrids, X. laevis, and X. muelleri.
The Affymetrix microarray is designed for X. laevis and it is widely accepted that
hybridizing RNA from a heterospecific species to a microarray designed for a related
species can have a dramatic impact on the signal recovered from microarrays (Nuzhdin
et al., 2004; Gilad et al., 2005; Gilad and Borevitz, 2006). To control for this effect we
directly assayed sequence divergence by hybridizing genomic DNA from Xenopus
laevis and X. muelleri each separately to the Affymetrix Xenopus laevis GeneChip®
Genome Array. By taking the ratio of hybridization intensity of X. muelleri/X. laevis for
each probe on the array we then screened out probes that did not hybridize properly due
to sequence divergence in X. muelleri. To be conservative we set the lower ratio of
hybridization intensity to 0.99 and explored variation in the number of probes
eliminated at a variety of upper thresholds with the idea that higher intensity for X.
70
muelleri is not as damaging compared to probes that have a hybridization signal lower
Number of probesets remaining
for X. muelleri when compared to X. laevis (Fig. 3.6). In examining the threshold
16000
14000
12000
10000
8000
6000
4000
2000
0
1
1.1
1.2
1.3
1.4
1.5
1.6
Upper threshold
Figure 3.6. Number of probesets remaining at various thresholds from
comparing the hybridization intensity of Xenopus muelleri vs. X. laevis.
variation on the number of probesets remaining, we chose to analyze the datasets
generated at the 1.01 and 1.10 ratio level. Both datasets provided a similar general
pattern but the effect was more prominent for the less stringent threshold. We report
here the results generated using an upper threshold of 1.10 in subsequent analyses of
gene expression which resulted in the removal of 226,841 individual probes and
provided 11,485 probesets/genes for further analysis. We tested the null hypothesis that
the expression level for each of the 11,485 transcripts/genes was the same in three
71
separate contrasts (hybrids vs. X. laevis; hybrids vs. X. muelleri; and X. laevis vs. X.
muelleri). Microarray analysis revealed an asymmetrical pattern of gene misexpression
between hybrids and the two parental species. Only 1.2% of genes (142/11,485) in our
sample of the transcriptome were misexpressed between Xenopus laevis and hybrids
whereas about 35% of genes (3,995/11,485) were misexpressed between X. muelleri
and hybrids (Fig. 3.7). There were more genes upregulated in hybrids relative to X.
laevis (92 vs. 50; G = 12.61; df=1; P<0.001) but there were more genes upregulated in
X. muelleri compared to hybrids (2,236 vs. 1,759; G = 57.1; df=1; P<0.001). The top
thirty most misexpressed transcripts in the two contrast tests were mainly dominated by
EST sequences with little functional information available but our analysis of gene
expression in the testis would suggest that these EST sequences have testis related
function. Among the annotated transcripts, many are known to have functions in the
process of spermatogenesis, spermiogenesis, or testis related functions or are involved
in regulating polymerase II transcription (Tables 3.1-3.4). There were 56 transcripts
recovered as differentially expressed in both contrasts (Table 3.5) and these transcripts
may have a more crucial role in the sterility pattern in hybrid males of Xenopus.
There was also a dramatic difference in expression between the two parental
species. About 60% of genes were differentially expressed between X. laevis and X.
muelleri (6956/11485). Of these differentially expressed transcripts, about 40%
(2824/6956) were upregulated in X. laevis relative to X. muelleri and 60% (4132/6956)
were upregulated in X. muelleri relative to X. laevis.
72
73
-log
2 P-value
-log2P-value
-6
-4
-2
0
2
4
6
-10
B
-8
-4
-2
0
Muelleri/Hybrid fold change (log2)
2
Muelleri/Hybrid Fold Change (log2)
-6
4
73
Figure 3.7. Volcano plots from FDR corrected t-tests of statistical significance (vertical axis) against magnitude of expression
change (horizontal axis), where each point corresponds to a gene/transcript. Expression change (fold-change) is defined as a
log2-transformed ratio of mean nonhybrid to mean hybrid expression level. (A) Xenopus laevis vs. Hybrids; (B) Xenpous
muelleri vs. Hybrids. The red line denotes FDR adjusted alpha 0.05. The horizontal deviation from 0 towards the right or left
reflects hybrid underexpression or overexpression, respectively.
Laevis/Hybrid Fold Change (log2)
Laevis/Hybrid
Fold Change (log2)
0
-8
0
-10
2
2
6
8
10
12
14
16
18
4
-log2Pvalue
4
6
8
10
12
14
16
18
A
-log2 P-value
6
74
BJ045215
BF072047
AF183394.1
BJ077199
BJ057383
CB560349
Xl.2583.1.S1_at
Xl.8438.1.A1_at
Xl.553.1.S1_at
Xl.13889.1.A1_at
Xl.4752.1.A1_at
Xl.10826.1.A1_at
BF025643
BG161445
Xl.15671.1.S1_at
Xl.11452.1.S1_at
Xl.2688.1.S1_at
BM261816
BJ056235
Xl.15612.1.A1_at
BF024995
BJ079143
Xl.24473.2.S1_at
Xl.23438.1.S1_at
BE679976
Xl.6988.1.A1_at
Xl.26339.1.A1_at
BI349938
Xl.901.1.S1_s_at
BJ090062
D42060.1
Xl.18999.1.A1_at
BF072385
BI449483
Xl.2955.1.A1_at
Xl.10407.1.A1_at
BJ091076
Xl.23530.1.S1_at
Xl.5907.1.A1_at
BJ090330
BC044077.1
Xl.9507.1.A1_at
BQ735473
BU912107
Xl.13044.1.S1_at
Xl.17907.1.A1_s_at
BJ097646
Xl.13064.1.A1_at
BF071889
BG486062
Xl.5076.1.S1_at
BG552599
BJ091208
Xl.14584.1.A1_at
Xl.11414.1.A1_at
BG021901
Xl.14330.2.S1_at
Xl.3637.1.S1_at
BF072333
GeneBank
ID
Xl.22458.1.A1_at
ProbeID
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
RALDH2
Pleiotrophic
factor
ESTs
MGC52668
ESTs
Glycoprotein
ESTs
Zinc finger
ESTs
ESTs
ESTs
Casein kinase I
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
Target Gene
LOC398494
MGC114836
MGC68790
LOC494660
MGC82200
MGC84465
ALDH1A2
MGC84594
gpm6a-A
zmat2
MGC82938
MGC81145
CKIe
MGC84983
LOC495201
LOC495423
MGC85135
Gene Symbol
74
Similar to heterogeneous nuclear ribonucleoprotein U
Moderately similar to sel-1 suppressor of lin-12 (Homo sapiens)
Catalytic activity
Weakly similar to GAP-associated tyrosine phosphoprotein p62
(H.sapiens)
Regulation of progression through cell cycle
Oxidoreductase activity
Weakly similar to and SOCS box-containing protein 2 (Homo
sapiens)
Protein disulfide oxidoreductase activity
Nucleic acid binding
Moderately similar to 5-AMP-activated protein kinase (H.sapiens)
Weakly similar to tubulin-specific chaperone B (H.sapiens)
Transcription factor TFIIE complex, RNA pol II transcription factor
Protein amino acid phosphorylation, nucleotide binding and kinase
activity
Weakly similar to nucleolar phosphoprotein Nopp34 (Homo
sapiens)
Protein amino acid phosphorylation, nucleotide binding and kinase
activity
Moderately similar to Myc-interacting zinc finger protein
(H.sapiens)
Description/Molecular Function
10.609
5.232
7.505
9.354
11.036
8.763
5.053
7.059
4.628
9.027
5.050
8.074
7.606
8.046
7.776
6.511
6.850
8.729
8.377
5.399
7.259
8.194
8.833
11.941
8.263
8.371
7.099
11.721
6.920
9.905
Mean
Laev.
0.404
0.538
0.136
0.471
0.549
0.223
0.445
0.500
0.547
0.353
0.656
0.332
0.397
0.136
0.518
0.388
0.471
0.604
0.292
0.539
0.251
0.286
0.600
0.382
0.492
0.546
0.839
0.426
0.367
1.226
SD Laev.
9.108
3.718
5.962
7.807
9.465
7.174
3.461
5.423
2.991
7.370
3.377
6.400
5.919
6.326
6.031
4.710
5.033
6.911
6.514
3.530
5.332
6.101
6.716
9.701
5.985
5.992
4.697
9.118
3.707
6.673
Mean
Hybrid
Table 3.1. Top 30 candidate transcripts upregulated in X. laevis and differentially expressed
between X. laevis and hybrid. Expression values are in log2 scale; SD=standard deviation of
expression values. P values are adjusted according to FDR moderated t-tests.
0.125
0.228
0.456
0.397
0.148
0.383
0.479
0.407
0.323
0.397
0.247
0.313
0.261
0.334
0.465
0.670
0.509
0.300
0.700
0.547
0.289
0.649
0.641
0.958
0.838
0.905
0.525
1.086
0.630
0.685
SD
Hybrid
L-H
1.501
1.514
1.543
1.547
1.571
1.590
1.592
1.636
1.636
1.658
1.673
1.674
1.688
1.720
1.745
1.801
1.817
1.818
1.863
1.869
1.926
2.093
2.117
2.240
2.278
2.379
2.403
2.604
3.213
3.232
0.0301
0.0432
0.0332
0.0454
0.0405
0.0269
0.0459
0.0408
0.0405
0.0320
0.0458
0.0248
0.0256
0.0151
0.0405
0.0455
0.0399
0.0384
0.0405
0.0405
0.0084
0.0269
0.0405
0.0459
0.0405
0.0408
0.0390
0.0405
0.0053
0.0393
P Value
75
CD361045
AF526274.1
BG020671
CB592496
BC043743.1
BJ091236
BJ099365
BJ041426
CD302009
AF549947.1
BQ730542
CB943601
BE027068
BQ398596
BM179219
Xl.21661.1.S1_at
Xl.22343.1.A1_at
Xl.6132.3.S1_a_at
Xl.8467.1.S1_at
Xl.2789.1.A1_at
Xl.14349.1.S1_at
Xl.12561.1.S1_at
Xl.8719.1.S1_at
Xl.5630.1.S1_x_at
Xl.20475.1.S1_at
Xl.24218.1.S1_at
Xl.11050.2.A1_at
Xl.19741.1.S1_at
Xl.16301.1.A1_at
AJ224125.1
Xl.252.1.S1_s_at
BC041496.1
BF612780
BG020931
AJ277826.1
Xl.3481.1.S1_at
Xl.25297.1.S1_at
Xl.11954.1.S1_at
Xl.23573.1.S1_at
D10259.1
BF071965
BJ056076
Xl.262.1.S1_at
Xl.3958.1.A1_at
Xl.16776.3.A1_at
Xl.26415.1.S1_at
GeneBank ID
L11294.1
BE026534
AF035016.1
AW766707
BC042271.1
CB561521
BU904046
ProbeID
Xl.831.1.S1_s_at
Xl.24869.1.A1_at
Xl.233.1.S1_s_at
Xl.8916.1.S1_at
Xl.21956.1.S1_at
Xl.24635.1.S1_at
Xl.24583.1.S1_at
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
XMeis1-3
dynein light chain 2
MGC131089
ESTs
ATPase
MGC114734
ESTs
Similar to thymine-DNA
glycosylase
T-box transcription
factor
ESTs
erg
ESTs
ESTs
PAK5 protein
Target Gene
neuropeptide Y
ESTs
synaptobrevin
ESTs
MGC53461
MGC86242
ESTs
Homeodomain for
Distal-less
ESTs
ESTs
PAK5
Cnef01
MGC80999
LOC495153
MGC132021
LOC398167
dynll2
atp1a3
Tbx1
TDG
erg-A; erg-B
75
Weakly similar to apolipoprotein C-I
precursor (H.sapiens)
Moderately similar to serinethreonine
protein phosphatase (H.sapiens)
ATPase activity, coupled to
transmembrane movement of ions
Weakly similar to putative lymphocyte
G0G1 switch protein 2 (H.sapiens)
Acyl-CoA binding
Homeodomain transcription factor
Microtubule motor activity
RNA binding
Protein amino acid phosphorylation
Transcription factor activity; sequencespecific DNA binding
Weakly similar to testis expressed protein
isoform (Mus musculus)
DNA repair; hydrolase activity, acting on
glycosyl bonds
Regulation of transcription, DNAdependent
Protein binding
Highly similar to ubiquitin-like 5 (Homo
sapiens)
MGC68565
distalless
Oxidoreductase activity
regulation of transcription, DNAdependent
Vesicle-mediated transport
Description/Molecular Function
Hormone activity
LOC398631
xsybi
Gene Symbol
NPY
8.974
7.291
5.052
2.843
4.109
7.953
5.561
7.985
6.624
3.535
5.046
5.016
4.817
5.736
3.862
6.598
3.272
3.683
4.007
6.897
4.221
7.515
6.005
2.954
4.874
3.417
3.883
6.978
4.239
3.000
Mean
Laev.
0.878
0.506
0.686
0.401
0.675
0.365
0.817
0.333
0.834
0.060
0.461
0.904
0.643
0.797
0.882
0.441
0.516
0.453
0.587
0.358
0.787
0.435
0.247
SD Laev.
0.464
1.235
0.336
1.180
0.330
0.890
0.287
11.080
9.389
7.137
4.927
6.347
10.174
7.764
10.176
8.788
5.677
7.288
7.264
7.096
8.007
6.142
8.887
5.570
6.290
6.433
9.269
7.189
10.445
8.865
6.966
8.633
7.016
7.225
10.026
7.280
6.016
Mean
Hybrid
0.402
0.231
0.245
0.408
0.795
0.292
0.446
0.238
0.303
0.909
0.168
0.510
0.283
0.586
0.321
0.151
0.872
1.104
0.562
0.326
0.731
0.471
0.358
0.907
0.781
1.506
0.387
0.178
0.691
0.579
SD
Hybrid
Table 3.2. Top 30 candidate transcripts upregulated in hybrids and differentially expressed
between X. laevis and hybrid. Expression values are in log2 scale; SD=standard deviation of
expression values. P values are adjusted according to FDR moderated t-tests.
-2.105
-2.098
-2.085
-2.084
-2.237
-2.221
-2.202
-2.191
-2.164
-2.142
-2.242
-2.248
-2.279
-2.271
-2.280
-2.289
-2.298
-2.607
-2.426
-2.373
-2.969
-2.929
-2.860
L-H
-4.013
-3.758
-3.600
-3.342
-3.048
-3.041
-3.016
0.0468
0.0152
0.0281
0.0160
0.0470
0.0067
0.0405
0.0053
0.0403
0.0405
0.0069
0.0460
0.0188
0.0405
0.0399
0.0056
0.0408
0.0418
0.0234
0.0053
0.0256
0.0053
0.0027
P Value
0.0053
0.0269
0.0383
0.0256
0.0016
0.0268
0.0053
76
BF072333
BJ098745
CA791375
CB561158
BQ736300
AF170275.1
M63872.1
BJ065331
BG022603
BG553598
AW148242
X65256.1
BG371327
BI347887
M63663.1
AB000736.1
BG811302
BG233631
BC046655.1
CB563855
BG885943
BM191733
AW159159
BQ400337
U38225.1
BJ097670
AF079559.1
U09769.1
BJ091236
BG730683
Xl.21527.1.S1_at
Xl.11477.1.S1_at
Xl.20548.1.A1_at
Xl.544.1.S1_at
Xl.801.1.A1_at
Xl.19995.1.S1_at
Xl.2956.1.S1_at
Xl.7311.1.A1_at
Xl.8231.1.A1_at
Xl.21454.1.A1_at
Xl.7631.1.A2_at
Xl.18472.1.A1_at
XlAffx.1.5.S1_at
Xl.25724.1.S1_at
Xl.17719.1.A1_at
Xl.13920.1.A1_at
Xl.23289.1.S1_at
Xl.24429.1.S1_at
Xl.18120.1.S1_at
Xl.14265.1.A1_at
Xl.8670.1.A1_at
Xl.19899.1.A1_at
Xl.1029.1.S1_at
Xl.8437.1.A1_at
Xl.298.1.S1_at
Xl.990.1.S1_at
Xl.2789.1.A1_at
Xl.9600.1.A1_at
GeneBank ID
Xl.22458.1.A1_at
Xl.4588.1.A1_at
ProbeID
ESTs
ESTs
ESTs
Homeobox protein
BIX1
Achaete-scute
homolog
ESTs
Glutamate
decarboxylase
ESTs
ESTs
Transmembrane
conductance
regulator
LOC398509
ESTs
X.borealis B1 protein
Myelin basic protein
ESTs
ESTs
MGC52879
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
Phosphoinositide-3kinase
ESTs
ESTs
Occludin
Goosecoid
Spectrin (alphafodrin)
Elastase
Target Gene
ash3b-A
Bix1
gad1-A
MGC52879
B1
Mbp
cftr-A
LOC398509
MGC82349
76
Transcription regulator activity
Weakly similar to putative lymphocyte
G0G1 switch protein 2 (H.sapiens)
Carboxy-lyase activity
Moderately similar to serinethreonine
kinase (Homo sapiens)
Regulation of transcription, DNAdependent
Moderately similar to bromodomain 2B
(Homo sapiens)
Transcription regulation
Structural constituent of myelin sheath
ATPase activity, coupled to
transmembrane movement of substances
Cytoskeleton formation
Serine-type endopeptidase activity
Moderately similar to DHHC1 protein
(Homo sapiens)
Regulation of transcription
LOC398133
goosecoid
sptan1-A
ela3b
Phosphatidylinositol 3-kinase activity
Ubiquitin-protein ligase activity
Description/Molecular Function
pik3ca-A
Ube2e2
Gene
Symbol
8.615
5.580
6.100
6.277
7.380
7.783
6.275
6.306
7.868
8.657
10.151
6.481
6.435
7.891
5.725
9.142
5.914
8.792
8.253
7.025
8.475
6.693
8.423
7.225
8.790
6.833
6.238
5.982
10.466
6.870
Mean Muell.
0.953
1.070
1.034
1.266
0.284
0.537
0.569
1.227
0.259
0.470
1.738
0.806
1.594
0.270
1.563
0.695
1.333
0.273
0.644
0.411
1.944
0.241
1.610
0.600
1.079
1.683
0.359
1.928
1.175
0.026
SD
Muell.
5.900
2.879
3.365
3.530
4.628
5.026
3.514
3.415
4.989
5.801
7.301
3.643
3.599
5.057
2.897
6.333
3.110
5.995
5.462
4.107
5.572
3.716
5.484
3.898
5.544
3.629
3.051
2.964
6.230
3.355
Mean
Hybrid
0.810
0.386
0.449
0.266
0.569
1.173
0.222
0.265
0.388
0.366
0.341
0.423
0.592
1.100
0.327
1.243
0.425
0.529
0.519
0.295
0.436
0.189
0.521
0.552
0.304
0.511
0.263
0.252
0.705
0.417
SD
Hybrid
Table 3.3. Top 30 candidate transcripts upregulated in X. muelleri and differentially
expressed between X. muelleri and hybrid. Expression values are in log2 scale; SD=standard
deviation of expression values. P values are adjusted according to FDR moderated t-tests.
M-H
2.716
2.701
2.736
2.747
2.752
2.757
2.760
2.891
2.879
2.856
2.849
2.839
2.836
2.834
2.828
2.809
2.805
2.796
2.791
2.918
2.903
2.977
2.940
3.326
3.246
3.204
3.187
3.018
4.236
3.515
0.0085
0.0054
0.0051
0.0070
0.0013
0.0113
0.0008
0.0053
0.0005
0.0007
0.0161
0.0023
0.0154
0.0072
0.0120
0.0139
0.0085
0.0010
0.0021
0.0005
0.0225
0.0002
0.0131
0.0009
0.0022
0.0107
0.0002
0.0175
0.0015
0.0002
P Value
77
BF049564
BJ085862
BC044001.1
BJ087266
CB592843
BJ076999
CD361360
L06232.1
BU908393
BM191868
Xl.24746.1.S1_at
Xl.2458.1.A1_at
Xl.2565.1.S1_at
Xl.5466.1.A1_at
Xl.5149.1.S1_at
Xl.16507.1.A1_at
Xl.3862.2.S1_x_at
Xl.7933.1.S1_at
Xl.7482.2.A1_x_at
Xl.2855.1.A1_at
BF615663
J03167.1
AW198888
S64727.1
BE678238
BJ051838
BJ082612
BQ385898
AW147826
BJ080015
BJ057196
BE677987
BG554560
BJ049960
BC041251.1
CD324878
Xl.3536.2.S1_x_at
Xl.702.1.S1_a_at
Xl.8046.1.S1_at
Xl.1079.1.S1_at
Xl.11981.1.S1_at
Xl.23752.2.S1_at
Xl.7947.1.A1_at
Xl.19459.1.S1_at
Xl.14065.1.A1_at
Xl.6585.1.S1_at
Xl.5160.1.S1_at
Xl.25536.1.A1_at
Xl.25863.1.A1_s_at
Xl.5904.1.S1_x_at
Xl.11443.1.S1_at
Xl.4839.1.S1_at
BC041270.1
BG552332
BC041228.1
BJ057240
Xl.21811.1.S1_at
Xl.3208.1.A1_at
Xl.2839.1.S1_at
Xl.24180.1.A1_at
GeneBank
ID
ProbeID
ESTs
Ferritin
ESTs
Ribosomal
protein S3a
ESTs
ESTs
ESTs
HIV-1 rev
binding protein 2
HMG-box
protein
ESTs
ESTs
ESTs
AP-3
H1 histone
cyclin B2
ESTs
ESTs
ESTs
Similar to alphaTubulin
ESTs
ESTs
ESTs
Translation
factor sui1
homolog
Beta-Tubulin
MGC53533
ESTs
Protein
translocation
complex
ESTs
GKAP42
ESTs
Target Gene
LOC495205
MGC114751
ap3s1
h1fx
HMG2L1
HRB2
MGC82659
rps3a
MGC68738
Ferritin
LOC495086
C-B2
LOC495200
sec61beta
MGC81070
77
Intracellular protein transport
Coated vesicle membrane
RNA binding
Regulation of transcription, DNAdependent
Structural constituent of ribosome
Signalosome complex
Highly similar to transcription
factor BTF3a (H.sapiens)
Regulation of progression through
cell cycle
Hydrogen-transporting ATP
synthase activity
Ferroxidase activity
Catalytic activity; ATP binding
Translation initiation factor activity
GTPase activity
Ubiquitin-protein ligase activity
Vesicle-mediated transport
LOC495063
gc20
betatub56d
MGC53533
MGC82859
GTPase activity
Histidine-tRNA ligase activity
Protein kinase
Exonuclease activity
Weakly similar to microsomal
glutathione S-transferase 3
(H.sapiens)
Description/Molecular Function
MGC53359
LOC445827
LOC496017
gkap42
MGC115620
Gene
Symbol
2.789
4.328
5.476
2.742
3.634
3.604
3.246
5.653
4.405
3.991
3.293
3.636
6.126
3.446
2.765
4.740
5.554
4.293
2.985
6.534
2.950
3.937
3.141
4.455
5.214
2.695
4.310
3.089
2.938
2.724
Mean
Muell.
0.072
0.821
0.623
0.331
0.689
0.924
0.734
1.594
0.715
1.137
1.216
1.006
0.938
1.086
0.540
0.800
0.611
1.057
0.562
0.488
0.847
1.076
0.963
1.418
0.317
0.547
0.808
0.635
0.502
0.378
SD
Muell.
8.738
10.255
11.380
8.621
9.485
9.444
9.214
11.717
10.469
10.009
9.276
9.797
12.213
9.516
8.955
10.999
11.866
10.557
9.407
12.946
9.295
10.276
10.331
11.379
12.032
9.318
11.573
10.287
11.273
10.532
Mean
Hybrid
1.408
0.499
0.253
0.566
0.549
0.334
0.420
0.457
0.323
0.847
0.295
0.477
0.183
0.449
0.548
0.582
0.229
0.181
0.387
0.258
0.433
0.234
0.571
0.232
0.095
0.159
0.254
0.512
0.361
0.236
SD
Hybrid
Table 3.4. Top 30 candidate transcripts upregulated in hybrids and differentially expressed
between X. muelleri and hybrid. Expression values are in log2 scale; SD=standard
deviation of expression values. P values are adjusted according to FDR moderated t-tests.
M-H
-5.950
-5.927
-5.904
-5.879
-5.851
-5.840
-5.968
-6.064
-6.064
-6.018
-5.983
-6.161
-6.087
-6.070
-6.190
-6.259
-6.312
-6.263
-6.423
-6.412
-6.345
-6.340
-7.190
-6.924
-6.818
-6.623
-7.263
-7.198
-8.336
-7.808
0.0006
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0005
0.0001
0.0004
0.0002
0.0002
0.0001
0.0002
0.0001
0.0001
0.0000
0.0001
0.0000
0.0000
0.0001
0.0001
0.0001
0.0002
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
P value
78
MGC82032
arpc1a
ESTs
MGC52668
RALDH2
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
nucleostemin
ESTs
actin related
protein
Xl.10826.1.A1_at
Xl.23530.1.S1_at
Xl.18999.1.A1_at
Xl.6988.1.A1_at
Xl.15612.1.A1_at
Xl.5907.1.A1_at
Xl.7749.1.A1_at
Xl.20487.1.A1_at
Xl.12917.1.A1_at
Xl.4873.1.S1_at
Xl.25940.1.S1_at
Xl.5178.2.A1_at
wu:fc55d07
MGC82200
ALDH1A2
MGC82938
LOC495423
ESTs
Xl.5076.1.S1_at
Gene Symbol
Target Gene
ESTs
Xl.19899.1.A1_at
Gene Symbol
Probeset ID
Target Gene
ESTs
ESTs
MGC82900
MGC82221
ESTs
ESTs
Protein, nucleotide, and GTP
binding; regulation of cell cycle
Adenosine kinase activity; purine
ribonucleoside salvage
Actin binding and cellular
morphogenesis
Description/Molecular Function
Nucleotide and ATP binding;
kinase activity
Moderately similar 5-AMPactivated protein kinase (Homo
sapiens)
Trafficking protein particle complex
6A (Xenopus tropicalis)
Oxidoreductase activity
Protein binding and transporter
activity
Similar to bromodomain, 2B
(Rattus norvegicus)
Description/Molecular Function
6.685
Zinc and metal ion binding and
oxidoreductase activity
78
10.780
8.972
11.053
7.059
9.354
8.501
8.135
8.494
9.027
8.074
8.046
8.377
7.099
Mean Laev.
5.026
Mean Laev.
9.905
7.259
7.799
8.974
7.013
Mean Laev.
4.007
5.561
Target of antiproliferative
antibody 1
Homeodomain transcription factor
LOC398167
cd81-b
Description/Molecular Function
Gene Symbol
Cnef01
CD81 antigen
Target Gene
ESTs
XMeis1-3
Probeset ID
Xl.22458.1.A1_at
Xl.13889.1.A1_at
Xl.10985.1.A1_at
Xl.24218.1.S1_at
Xl.8717.2.A1_at
Xl.18983.1.S1_at
Probeset ID
Xl.25297.1.S1_at
Xl.12561.1.S1_at
-1.433
-1.747
-2.105
-2.080
1.182
9.786
7.948
10.008
5.423
7.807
7.201
6.876
7.352
7.370
6.400
6.326
6.514
4.697
0.993
1.024
1.045
1.636
1.547
1.300
1.259
1.143
1.658
1.674
1.720
1.863
2.403
X. laevis vs. Hybrid Contrast
Mean Hybrid
L-H
3.844
X. laevis vs. Hybrid Contrast
Mean Hybrid
L–H
6.673
3.232
5.332
1.926
9.232
8.431
11.080
9.094
X. laevis vs. Hybrid Contrast
Mean Hybrid
L-H
6.433
-2.426
7.764
-2.202
0.0471
0.0454
0.0408
0.0408
0.0454
0.0269
0.0484
0.0405
0.0320
0.0248
0.0151
0.0405
0.0390
P Value
0.0484
P Value
0.0393
0.0084
0.0387
0.0405
0.0468
0.0348
P Value
0.0234
0.0405
8.936
8.068
10.873
8.765
-1.060
-2.606
-1.636
-1.333
3.514
2.760
4.319
3.749
6.970
3.847
4.021
4.252
2.972
4.940
4.850
4.680
3.757
3.945
3.285
9.489
7.561
9.719
4.949
7.413
6.777
6.434
6.935
6.953
6.327
5.879
6.082
4.298
-5.170
-3.812
-2.749
-1.102
-3.392
-2.525
-3.462
-1.995
-2.103
-1.647
-2.121
-2.136
-1.012
X. muelleri vs. Hybrid Contrast
Mean Muell.
Mean Hybrid
M-H
6.275
X. muelleri vs. Hybrid Contrast
Mean Muell.
Mean Hybrid
M-H
10.466
6.230
4.236
6.190
4.888
1.302
7.876
5.463
9.237
7.432
X. muelleri vs. Hybrid Contrast
Mean Muell.
Mean Hybrid
M-H
3.367
5.978
-2.611
5.194
7.367
-2.173
Table 3.5. Candidate loci (56 total) common to both contrasts (X. laevis vs
hybrids; X. muelleri vs. hybrids) divided according to corresponding expression
behavior (hybrid>(laevis = muelleri); hybrid<(laevis=muelleri); laevis>hybrid>muelleri;
laevis<hybrid<muelleri). Expression values are in log2 scale. P values are FDR
adjusted values.
0.0001
0.0001
0.0004
0.0402
0.0033
0.0168
0.0012
0.0302
0.0167
0.0049
0.0099
0.0079
0.0449
P Value
0.0008
P Value
0.0015
0.0332
0.0254
0.0015
0.0091
0.0248
P Value
0.0061
0.0208
79
erg
ESTs
Xl.252.1.S1_s_at
Xl.16427.1.A1_at
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
hermes
Xl.2331.1.A1_at
Xl.11050.2.A1_at
Xl.26364.1.A1_at
Xl.24104.1.A1_at
Xl.10225.1.A1_at
Xl.3975.1.A1_at
Xl.1555.1.A1_at
Xl.2895.1.A1_at
Xl.449.1.S1_at
Xl.525.1.S1_at
ESTs
forkhead box
D3
ESTs
Xl.8719.1.S1_at
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
ESTs
Xl.16776.3.A1_at
Xl.22032.1.A1_at
Xl.9401.1.A1_at
Xl.3958.1.A1_at
Xl.25538.1.A1_at
Xl.6844.1.A1_at
Xl.26410.1.A1_at
TDG
Xl.23573.1.S1_at
ESTs
ESTs
Xl.25810.1.A1_at
eIF4G protein
PAK5
Xl.11954.1.S1_at
Xl.14571.2.S1_at
ESTs
ESTs
ESTs
ESTs
ESTs
MGC53769
Xl.6034.1.A1_at
Xl.19741.1.S1_at
Xl.20475.1.S1_at
Xl.21956.1.S1_at
Xl.12866.1.S1_at
Xl.16399.1.S1_at
Xl.24133.1.S1_at
ESTs
ESTs
Xl.3481.1.S1_at
ESTs
Xl.2789.1.A1_at
Xl.8916.1.S1_at
Target Gene
Probeset ID
Table 3.5-continued.
LOC398092
LOC495973
MGC84386
MGC80999
foxd3-A
MGC83883
MGC84299
dynein light
chain 2
LOC495071
NAT1
erg-A; erg-B
MGC68565
LOC443658
PAK5 protein
MGC53461
G6pc
dkfzp434h2226
MGC82505
LOC495153
Gene Symbol
Nucleotide binding; nucleic acid
binding; RNA binding
Similar to hypothetical protein
FLJ14490 (Pan troglodytes)
Weakly similar to hypothetical
protein FLJ20125 (Homo sapiens)
Transcription factor activity;
regulation of transcription
Microtubule motor activity
Kinase activity
Syntaxin binding
Protein amino acid
phosphorylation
Protein, zinc, and metal ion
binding
DNA repair; hydrolase activity,
acting on glycosyl bonds
Protein binding
Transcription factor activity;
sequence-specific DNA binding
Similar to guanine nucleotide
exchange factor (1B384) (Gallus
gallus)
RNA binding; translation initiation
factor activity
Protein, zinc, and metal ion
binding
Glucose-6-phosphotase activity
Highly similar to ubiquitin-like 5
(Homo sapiens) (H.sapiens)
Lipid binding
Description/Molecular Function
Similar to putative lymphocyte
G0/G1 switch protein 2 (Gallus
gallus)
79
6.534
7.444
7.694
4.752
9.379
6.704
6.105
7.291
7.549
4.656
7.985
5.386
7.766
7.515
8.281
5.987
5.946
6.680
9.142
6.335
3.272
6.005
3.862
6.649
6.897
6.973
5.052
3.535
6.978
5.006
9.286
3.683
3.883
4.109
Mean Laev.
8.252
9.321
9.197
5.912
11.188
8.026
7.375
9.389
8.748
5.756
10.176
6.469
9.403
10.445
9.853
7.841
7.373
8.146
10.187
7.851
5.570
8.865
6.142
8.321
9.269
8.720
7.137
5.677
10.026
6.297
10.684
6.290
7.225
6.347
-1.718
-1.877
-1.503
-1.160
-1.809
-1.322
-1.270
-2.098
-1.199
-1.099
-2.191
-1.083
-1.637
-2.929
-1.571
-1.854
-1.426
-1.466
-1.044
-1.516
-2.298
-2.860
-2.280
-1.672
-2.373
-1.747
-2.085
-2.142
-3.048
-1.291
-1.398
-2.607
-3.342
-2.237
X. laevis vs. hybrid contrast
Mean Hybrid
L-H
0.0137
0.0053
0.0160
0.0393
0.0160
0.0323
0.0405
0.0152
0.0454
0.0471
0.0053
0.0455
0.0097
0.0053
0.0408
0.0405
0.0154
0.0344
0.0471
0.0405
0.0408
0.0027
0.0399
0.0188
0.0053
0.0344
0.0281
0.0405
0.0016
0.0384
0.0405
0.0418
0.0256
0.0470
P Value
8.522
10.027
9.869
6.464
12.105
8.802
8.118
10.294
9.672
6.677
11.184
7.758
10.541
11.593
10.969
8.816
8.316
9.426
11.538
9.109
6.808
10.213
7.423
9.755
10.759
10.434
8.731
7.117
11.611
7.640
12.243
7.988
9.004
8.615
Mean Muell.
7.646
9.016
8.870
5.501
10.989
7.642
6.961
9.077
8.402
5.391
9.898
6.294
9.118
10.186
9.570
7.448
6.974
7.812
9.909
7.458
5.128
8.526
5.679
7.979
8.962
8.373
6.708
5.234
9.739
5.833
10.438
5.853
6.801
5.900
0.876
1.011
0.999
0.963
1.116
1.160
1.157
1.217
1.269
1.285
1.285
1.464
1.423
1.407
1.399
1.368
1.342
1.615
1.630
1.651
1.680
1.688
1.744
1.776
1.797
2.061
2.023
1.884
1.872
1.807
1.805
2.135
2.202
2.716
X. muelleri vs. hybrid contrast
Mean Hybrid
M-H
0.0367
0.0189
0.0201
0.0289
0.0242
0.0225
0.0106
0.0108
0.0078
0.0079
0.0113
0.0047
0.0048
0.0159
0.0213
0.0236
0.0059
0.0116
0.0055
0.0111
0.0220
0.0108
0.0443
0.0040
0.0036
0.0054
0.0315
0.0221
0.0015
0.0037
0.0035
0.0286
0.0028
0.0085
P Value
Comparing the overlap in genes misexpressed in individual hybrid contrasts to the three
classes of expression behavior between X. laevis and X. muelleri (X. laevis > X.
muelleri; X. laevis < X. muelleri; X. laevis = X. muelleri) revealed that in general
hybrids have an intermediate level of expression compared to the two parental species
(Table 3.6). For example, of the 92 genes upregulated in hybrids relative to X. laevis;
92% were also upregulated in X. muelleri relative to X. laevis. Similarly, of the 1,759
genes that were upregulated in hybrids relative to X. muelleri, 92% were upregulated in
X. laevis compared to X. muelleri. These results suggest a general pattern of
intermediate expression in hybrids and are consistent with a semidominant model of
expression difference even despite the strong asymmetrical pattern of misexpression in
hybrids compared to the two parental species.
3.5 Discussion
Our analysis of sperm abundance, motility, and morphology in hybrids suggests
that the process and control of spermatogenesis is severely perturbed in hybrids.
Hybrids have about 40 times less total sperm compared to parental species. Mature
sperm in hybrids are capable of motility and the highly reduced abundance of sperm
suggests that these numbers may be insufficient to fertilize eggs in vivo. Additionally,
differential live/dead staining of sperm cells in hybrids showed that the majority of cells
consisted of undifferentiated sperm suggesting an overall pattern of arrested
development of spermatogenesis and/or spermiogenesis. Cells that have the
characteristic shape of sperm in hybrids are larger compared to X. laevis and X. muelleri
raising the possibility that these sperm cells have abnormal numbers of chromosomes.
80
81
0
85
7
92
L>M
L<M
L=M
Total
L<H
81
50
8
1
41
L>H
X. laevis vs. Hybrids
1759
132
1
1626
M<H
2236
206
2029
1
M>H
X. muelleri vs. Hybrids
Table 3.6. Comparison in the overlap of transcripts recovered as differentially expressed
from the two contrasts with hybrids (Xenopus laevis vs. hybrids and X. muelleri vs. hybrids)
and the interspecies contrast (Xenopus laevis vs. X. muelleri). The congruence between
patterns of expression behavior in hybrids compared to the interspecies comparison suggests
a model of semidominance where hybrids have an intermediate level of expression compared
to the two parental species.
X. laeivs vs.
X. muelleri
Kobel (1996) suggested that sperm cells of hybrids were aneuploid and our results may
be consistent with these observations. Future work focusing on chromosome content of
hybrid sperm would aid in determining the end result of meiosis in hybrids. Sperm
production in hybrids did not respond to hCG hormone treatment whereas the
proportion of dead cells increased dramatically in X. laevis. The dosage of hCG used in
the experiment is typical for stimulating reproduction in Xenopus but our results suggest
the possibility that the dose may be too high. hCG triggers the production of LH and
FSH which are involved in the differentiation of sperm (Pozzi et al., 2006; Rosemblit et
al., 2006). Overstimulation by excessive hCG could result in sperm death and may
explain the increased numbers of dead sperm in X. laevis but why hybrids fail to
respond to hCG treatment remains an intriguing question. If receptors for hCG are more
sensitive in hybrids, an abnormally high dose of hCG may cause these receptors to
become inactivated leading to a lack of response by the process of spermatogenesis.
Alternatively, hormone receptors in hybrids may fail to function properly due to the
hybrid genetic background which could lead to a lack of response to hCG treatment.
This later hypothesis would implicate a possible mechanism for the overall different
pattern of spermatogenesis observed in hybrids. Spermatogenesis, a process tightly
controlled by hormonal interactions with the hypothalamicpituitary-gonadal axis
(Scheltinga and Jamieson, 2003), may be misregulated due to improper interactions
with hormones and receptors. This hypothesis has received no investigation in studies
of reproductive isolation despite considerable attention directed to the process of
spermatogenesis as an explanation for Haldane’s rule (Wu and Davis, 1993; Wu et al.,
82
1996). The lack of response to hCG in hybrids suggests that hormonal regulation of
spermatogenesis may contribute to reproductive isolation in hybrids and support for this
hypothesis could be generated by assaying receptor function in hybrids compared to
parental species.
The genes misexpressed in hybrids may offer clues to the loci involved in the
sterility phenotype characteristic of hybrid males and therefore potential loci that
contribute to reproductive isolation between X. laevis and X. muelleri. Many of the most
misexpressed loci consist of EST targets and therefore currently there is little functional
information available. One EST of interest though has been identified to be the
transcription factor TFIIE complex which is a transcription factor important to the
function of RNA polymerase II transcription. This EST was downregulated 4 times in
hybrids relative to X. laevis suggesting that Pol II transcription may be impacted by the
lack of TFIIE in hybrids. Interestingly, this gene was not found to be misexpressed
compared to X. muelleri.
Many interesting genes related to spermatogenesis in other organisms appear in
the candidate gene lists. For example, type 2 retinaldehyde dehydrogenase (RALDH2)
was found to be 3.2 times lower in hybrids compared to X. laevis. This gene catalyzes
the important developmental modulator retinoic acid and is exclusively expressed in
mouse testis (Hsu et al., 2000). Casein kinase I (CKIe), a gene involved in protein
amino acid phosphoryalation through the utilization of ATP but not GTP was
downregulated 4.3 times in hybrids relative to X. laevis and a unique form of CKle is
expressed in rat testis (Graves et al., 1993).
83
The most misexpressed gene in hybrids relative to X. laevis was neuropeptide Y
(NPY). NPY is one of the most abundant and widespread nueropeptides in mammals and
has been suggested to play a role in controlling reproductive function. In particular,
NPY potentiates the release of LH and FSH in response to GnRH (Bauer-Dantoin et al.,
1992; Knox et al., 1995) and is modulated by testosterone. NPY is predominantly
synthesized in the hypothalamic arcuate nucleus but also expressed in the testes (mostly
in Leydig cells) of mouse and rat (Kanzaki et al., 1996; Terado et al., 2006).
Importantly, there is a strong association between testicular development and the
expression of NPY (Terado et al., 2006). NPY was upregulated 16 times in hybrids
relative to X. laevis and also was overexpressed in X. muelleri relative to X. laevis.
Other upregulated genes in hybrids relative to X. laevis and that have documented
expression patterns in the testes include PAK5 (p21 activated kinase 2 plays a role in
Sertoli-germ cell anchoring dynamics of rats; Siu et al., 2005) and dynll2 (dynein light
chain-1 was highly expressed in mouse and rat testis; Wang et al., 2005).
Examining genes misexpressed in hybrids compared to X. muelleri reveals that
there are also many interesting genes related to the function of the testis in other
organisms. Occludin which was upregulated about 9 times more in X. muelleri
compared to hybrids has an important role in the formation of tight junctions
surrounding Sertoli cells and seminiferous tubules in mammals (Gye, 2004) and
salamanders (Jin et al., 2007). Goosecoid is an important transcription factor during
early development of Xenopus and was found to be about 8 times overexpressed in X.
muelleri relative to hybrids. A related gene (goosecoid-like) is expressed primarily in
84
the brain and primoridial germ cells in mouse (Galili et al., 2000). B1, a X. borealis
specific gene included on the X. laevis microarray, was upregulated about 7 times in X.
muelleri compared to hybrids and plays an important role in the assembly of TFIIIA and
expression of 5S RNA by RNA polymerase III (Kaulen et al., 1991). Two tubulin
related genes (similar to alpha-tubulin and beta-tubulin) were overexpressed 140 and
85 times respectively in hybrids relative to X. muelleri. Beta tubulin 2 has a testis
restricted expression profile in Aedes aegypti and Drosophila (Nielsen et al., 2006;
Smith et al., 2007). Another gene of interest is cyclin B2, upregulated 70 times in
hybrids compared to X. muelleri. Cyclins have a demonstrated role in spermatogenesis
in the eel Anguilla japonica (Kajiura-Kobayashi et al., 2004) and mouse (Brandeis et
al., 1998) and play a role in cell cycle regulation particularly in response to cancerous
aberrations in germ cells (Diederichs et al., 2005). Ferritin was found to be upregulated
68 times in hybrids compared to X. muelleri. Recently mitochondrial ferritin has been
discovered to have a testis specific expression profile in mammals and flies (Missirlis et
al., 2006) leading to the possibility that we recovered a signal of this ferritin product in
Xenopus. Finally, HMG box protein, a transcriptional repressor that was upregulated
about 62 times in hybrids relative to X. muelleri, has been found to be expressed
strongly in mouse testis (Smith et al., 2004).
The gene expression data suggest two major patterns for expression behavior in
hybrids relative to the two parental species. First, for misexpressed genes in hybrids the
overwhelming preponderance of misexpressed genes follow a semi-dominant model of
expression behavior because hybrid expression is intermediate and/or additive
85
compared to expression differences between the two species (Table 3.6). Hybrids of
these two species of Xenopus then appear to follow a very different overall pattern of
misexpression compared to hybrids in other organisms. For example, hybrids of
Drosophila have distinctly nonadditive expression behavior because hybrids have more
misexpression compared to the genes misexpressed between the two parental species
(Michalak and Noor, 2003; Ranz et al., 2004). The same can be said for hybrids of
maize (Auger et al., 2005), Arabidopsis (Josefsson et al., 2006; Comai et al., 2003;
Wang et al., 2004; Wang et al., 2006), and Senecio (Hegarty et al., 2006). In the case of
Xenopus, more genes are misexpressed between species than for hybrids compared to
each parental species.
The second major pattern observed from the microarray results is that there was
a difference in the expression pattern of hybrids compared to the two parental species.
Surprisingly, a relatively few number of genes were misexpressed between X. laevis and
hybrids (142/11,485) while a substantial number of genes were misexpressed between
X. muelleri and hybrids (3,995/11,485). In effect, adult testes of hybrids have an
expression pattern very similar to X. laevis but substantially different compared to X.
muelleri. These results suggest that most genes in the hybrid genetic background follow
a dominant pattern of expression because the overall level of expression in hybrids is
equal to that of X. laevis only. This pattern of expression in hybrid testes differs from
analyses of proteins in hybrids of X. laevis and X. borealis (a related species to X.
muelleri). Both species specific proteins of X. laevis and X. borealis are expressed
86
in hybrid ovary (De Robertis and Black, 1979) and species-specific copies of several
allozyme loci appear to both be expressed in various developmental stages of tadpoles,
adult heart, and adult liver (Wall and Blackler, 1974). However, LDH isozyme in early
embryos resembled that of the maternal species (X. laevis victorianus) and this persisted
through metamorphosis (Vonwyl, 1983). These studies suggest that the allelic
dominance observed in the expression profile of hybrid testes is not distributed in other
tissue types but rather may be confined to the testes.
One interpretation of these results is that the collective molecular processes that
occur in adult frog testes (i.e. spermio- and spermatogenesis) are under the influence of
massive genomic imprinting and specifically, the maternal but not the paternal copies of
genes are expressed. This would provide an explanation for the similarity of hybrid
expression profiles to X. laevis and the dissimilarity compared to X. muelleri because
the mother of these hybrids is X. laevis and not X. muelleri. No evidence for imprinting
has been found for Xenopus but this hypothesis would require that imprinting may
occur in Xenopus and that maternal imprinting is the controller of the expression pattern
in whole frog testes (de la Casa-Esperón and Sapienza, 2003). One fruitful avenue to
explore this hypothesis would be to analyze the expression profiles from testes of
reciprocal cross hybrids (X. muelleri x X. laevis). If the expression profile becomes
more similar to X. muelleri then this would be evidence of strong maternal effects,
possible related to genomic imprinting, controlling the expression pattern in frog testes.
An alternative mechanism that may explain the dissimilar expression profiles is
that in hybrids the paternal copy of chromosomes is eliminated or quiescent at the
87
expression level. Hybrids then would express only the maternally contributed X. laevis
portion of the genome but this would require that the maternally contributed portion be
upregulated two fold to match the expression profile of X. laevis. The mechanisms
involved here are difficult to explain but one possibility may be that paralogous gene
copies, normally repressed, become expressed thereby providing expressed products to
match that of X. laevis. Genomic elimination of paternal copies does occur in insects
and frogs (Crouse, 1960; Brun et al., 1995; Mable and Bogart, 1995; Dobson and
Tanouye, 1998; Goday and Esteban, 2001). For example in the treefrog, gene silencing
and monoallelic dominance of one genome occurs in crosses of tetraploid Hyla
versicolor and diploid H. arborea (Mable and Bogart, 1995). In F1 triploid hybrids,
several allozyme loci had unexpected frequencies suggesting that in some cases the
paternal gene was silenced (i.e. Mpi-2) or that the maternal allele was silenced and the
single paternal allele had double expression (i.e. Got-1). The implication here is that the
foreign copy can be recognized and eliminated, and given the polyploid nature of
Xenoups a similar mechanism may be operating. Indeed, amphibians have remarkably
examples of genomic exclusion. One well documented example consists of the
hybridogenetic system of the Rana esculenta complex in Europe where the entire
paternal genome is eliminated during oogenesis (Vinogradov et al., 1990).
Bringing together two divergent genomes into a common genetic background
can induce a variety of genomic changes including genome instability, changes in
chromatin, and transcriptome shock (Osborn et al., 2003; Adams and Wendel, 2005;
Birchler et al., 2005; Chen and Ni, 2006; Hegarty et al., 2006). In particular, gene
88
silencing appears to be one major result of bringing together two divergent genomes
into a common genomic environment via hybridization. For example, interesting
patterns regarding rRNA gene expression have been established in interspecific hybrids
of Xenopus. Both Xenopus laevis and X. borealis (a species closely related to X.
muelleri) display two nucleoli per cell during embryogenesis. Hybrids between these
two species have one nucleolus in the majority of cells suggesting that the nucleolar
organizer is inactivated due to genomic incompatibility between the two species
(Blackler and Gecking, 1972). More specifically, in hybrids between Xenopus laevis
and X. borealis (in the cited papers mistakenly referred to as X. muelleri; Brown et al.,
1977), the rRNA of X. laevis is preferentially transcribed and X. borealis is repressed
(Honjo and Reeder, 1973). Repression of X. borealis rRNA is basically complete until
the swimming tadpole stage during which a low level of X. borealis rRNA is detectable.
In adults, the level of expression of the X. borealis copy ranges from zero to significant
amounts indicating the presence of much variation between individual hybrids. This
phenomenon, first called “differential amphiplasty” and currently termed “nucleolar
dominance”, is an epigenetic phenomena that is controlled at the level of transcription
and occurs in a wide variety of interspecific hybrids including the plant taxa Crepis
(Navashin, 1934; Wallace and Langridge, 1971), Salix (Wilkinson, 1944), Ribes (Keep,
1960; 1962), Solanum (Yeh and Peloquin, 1965), Hordeum (Kasha and Sadasivaiah,
1971; Lange and Jochemsen 1976; Nicoloff, 1979; Schubert and Kunzel, 1990),
Triticum (Crosby, 1957; Flavell et al., 1988; Thomposon and Flavell, 1988; Sardana et
al., 1993), Agropyron (Heneen, 1962), Brassica (Chen and Pikaard, 1997a; 1997b), and
89
Arabidopsis (Chen et al., 1998) as well as hybrids of Drosophila (Durica and Krider,
1977; 1978), and mammal somatic cell hybrids of mouse and human (Elicieri and
Green, 1969; Bramwell and Handmaker, 1971; Miller et al., 1976; Croce et al., 1977;
Soprano et al., 1979; Soprano and Baserga, 1980; Onishi et al., 1984). Nucleolar
dominance operates on tens of millions of base pairs of chromosomal DNA and this
large scale inactivation mechanism is only rivaled by X chromosome inactivation in
mammalian cells (Heard et al., 1997).
Can the mechanisms of nucleolar dominance, a pattern specifically related to the
RNA polymerase I transcription machinery of rRNA and not with RNA polymerase II
transcription of protein coding genes provide explanations for the asymmetrical
transcriptomic expression pattern observed in hybrids of Xenopus? The mechanisms
that discriminate maternal or paternal rRNA genes remain unclear but three hypotheses
have been proposed to account for the differential silencing of species specific alleles:
the species-specific transcription factor hypothesis, the enhancer-imbalance hypothesis,
and the chromatin imprinting hypothesis (Reeder, 1985; Pikaard, 2000; Viegas et al.,
2002).
3.5.1 Species-Specific Transcription Factor Hypothesis
The DNA sequences coding for ribosomal proteins (18S, 5.8S, and 25/26S) are
highly conserved but intergenic rDNA sequences are highly divergent amongst
eukaryotes (Appels and Dvorak, 1982; Dover and Flavell, 1984; Pikaard, 2000; Viegas
et al., 2002). According to the species-specific transcription factor hypothesis, because
of this divergent evolution rRNA gene transcription can occur only if co-evolved
90
species-specific transcription factors bind to corresponding regulatory sequences. When
genomes from two different species are brought together, there is a failure of rDNA
transcription because of a lack of appropriate specific transcription factors. Cell-free
transcription systems of mouse-human hybrid cell lines revealed that the promoter for
mouse or the promoter for human rRNA would not function in a cell-free extract made
from another species (Grummt et al., 1982; Miesfeld and Arnheim, 1984; Miesfeld et
al., 1984; Schnapp et al., 1991). In plants, the tomato rRNA gene promoter does not
work when transfected into Arabidopsis protoplasts and the tobacco rRNA gene
promoter does not work when placed into a bean cell-free extract (Fan et al., 1995;
Doelling and Pikaard, 1996). These findings suggest that transcription factors had coevolved with their corresponding regulatory DNA sequences because transcription
function was erased when forced into a heterospecific environment. One prediction of
this co-evolution model is that the amount of sequence divergence would be inversely
related to how effective the mechanisms of the species-specific transcription hypothesis
operate and this appears to be the case. When more closely related species are brought
together into a common genomic environment, rRNA gene promoters are fully
functional. For example, in Brassica napus, a hybrid derived from B. rapa and B.
oleracea, only B. rapa rRNA is transcribed reflecting a pattern of nucleolar dominance
but both rRNA genes were transcribed when transfected into protoplasts of the other
species (Frieman et al., 1999). The same pattern occurs in Arabidopsis suecica, a hybrid
of A. thaliana and A. arenosa; A. thaliana and A. arenosa rRNA genes are expressed in
both species and transfected A. thaliana rRNA is active when introduced into the hybrid
91
(Chen et al., 1998). These congeneric interspecific hybrids are more closely related
compared to the previous examples and here species specific transcription factors work
fine in a heterospecific background. In the case of Xenopus, it is possible at least for
some loci that transcription factors may fail to bind to their corresponding regulatory
targets causing a silencing of underdominant genes because the two species examined in
this study are substantially divergent. The estimated divergence time for the last
common ancestor of X. laevis and X. muelleri is 21-35 mya (Evans et al., 2004; 2005;
Chain and Evans 2006). However, this mechanism could operate on a genome wide
scale in adult testes only if a key transcription factor, or factors, derived from
maternally contributed X. laevis genes and involved in the regulation of X. muelleri
chromatin was sufficiently divergent to misregulate the proper expression of X. muelleri
alleles. Allele specific imprinting at the level of chromatin has been recently reported in
Arabidopsis (Gehring et al., 2006) and in the case of Xenopus evidence for this
hypothesis would be obtained by identifying chromatin remolding factors that operate
from the maternal genome to regulate paternal copy chromatin and/or paternal copy
transcription.
3.5.2 Enhancer Imbalance Hypothesis
The enhancer imbalance hypothesis proposes that differences in the amount of
intergenic sequences provide a competitive environment for the transcription of rRNA
genes. If particular intergenic sequences are more effective at controlling transcription
then these sequences will outcompete sequences contributed by the other species
creating an imbalance in the way that enhancers operate to sequester transcription
92
factors. In Xenopus, the repetitive DNA elements located upstream of the rRNA
promoter act as orientation and position independent enhancers of transcription (Busby
and Reeder, 1983; Labhart and Reeder, 1984; Pikaard and Reeder, 1988). In X. laevis,
each intergenic spacer contains a 60/81 bp repeat with a 42 bp core element that shares
90% sequence similarity with the gene promoter. In X. borealis, complete 60/81 bp
repeats are absent but the 42 bp core element is present suggesting that the 42 bp core
element controls the enhancer effect (Reeder, 1985; Boseley et al., 1979; Sollner-Webb
and Reeder, 1979; Moss, 1983). In hybrids, only the rRNA genes of X. laevis are
transcribed (Blackler and Gecking, 1972; Honjo and Reeder, 1973; Cassidy and
Blacker, 1974) and this appears to be due to the more numerous enhancer elements
located in X. laevis sequence compared to X. borealis which sequester a critical
transcription factor necessary for transcription (Reeder et al., 1983; Reeder, 1984;
Reeder and Roan, 1984). Co-injection of rRNA minigenes with complete spacers into
oocytes of X. laevis and X. borealis showed that X. laevis minigenes were preferentially
transcribed and this occurred even when injecting recombinant constructs that had
spacer and promoter regions swapped (Reeder and Roan, 1984). These experiments
support the notion that species specific differences in intergenic regions, and not gene
promoters, are responsible for the failure of the X. borealis allele to be transcribed in
hybrids.
The enhancer imbalance hypothesis stems directly from biochemical
interactions of transcription factors and the enhancer elements that regulate gene
expression and therefore follows models of cis-trans evolution that have recently been
93
proposed (Porter and Johnson, 2002; Landry et al., 2005). Differences in genomic
content between species then may be consistent with this model because differing
amounts of intergenic DNA and/or enhancers may cause problems with transcribing one
allele from one of the two species. Examining published C-values for Xenopus
(available on the Animal Genome Database-http://www.genomesize.com/), the genomic
content of Xenopus laevis and X. muelleri differs by about 430 Mb (Mean X. laevis
3231 Mb; Mean X. muelleri 3660 Mb; t= -2.76; df=1; P =0.02). By combining these
two genomes into the same genomic environment, different enhancer content resulting
from differences in genome size, may contribute to the patterns observed in gene
expression of X. laevis, X. muelleri, and their F1 hybrid.
3.5.3 Chromatin Imprinting Hypothesis
DNA methylation and covalent modifications of histone tails within
nucleosomes
(acetylation,
phosphorylation,
sumoylation,
ubiquination,
and
methylation) have an impact on chromatin structure, gene transcription, and epigenetic
information (Shilatifard, 2006). Indeed modifications to chromatin are applied and
removed in a highly specific fashion leading to the proposition of a histone code that is
read by chromatin-associated factors (Jenuwein and Allis, 2001). The chromatin
imprinting hypothesis proposes that changes in the regulation of chromatin state explain
the dominance seen in the preferential transcription of rRNA genes (Viegas et al.,
2002).
DNA
hypomethylation
and
histone
hyperacetylation
correlate
with
transcriptional activity and DNA hypermethylation and histone hypoacetylation
correlate with transcriptional silencing and therefore allele specific regulation of
94
chromatin state may render some gene copies to be transcribed while rendering others
to be silent (McStay, 2006).
There is a striking correlation between genome size and the direction of
silencing in interspecific hybrids. On average, the parental species with the smaller
genome is always dominant whereas under-dominant loci came from the smaller
genome of the two parental species (Viegas et al., 2002). The argument here is that
larger genomes have more heterochromatin and when combined into the same genomic
environment via hybridization this increased heterochromatin content changes the
spatial structure of the genome within the nucleus leading to preferential silencing of
one parental copy. In effect, the positional configuration of chromatin in the hybrid
nucleus is imbalanced and the genome with the larger content will be subjected to
heterochromatinization via chromatin remolding mechanisms (Richards and Elgin,
2002). As stated above, the genomic content differs between X. laevis and X. muelleri
and the smaller of the two genomes (X. laevis) is the species for which hybrids are most
similar at the expression level. Additionally, several genes involved in chromatin
maintenance are misexpressed in hybrids compared to X. muelleri but not in hybrids
compared to X. laevis (i.e. Ube2e2 that has ubiquitin-protein ligase activity and SET
which is involved in chromatin remolding).
Our analysis of the phenotype of hybrids revealed a dramatically lower
abundance of sperm in hybrids, increased numbers of undifferentiated sperm cells, a
lack of response to hCG treatment, larger sperm compared to both parental species and
even despite these phenotypic abnormalities, the presence of a few motile sperm.
95
Coupling these phenotypic data with microarray analyses provides loci that may lead to
the sterile phenotypic condition of hybrids. These data are important for identifying
genes germane to postzygotic reproductive isolation between species and we present the
first lists of these loci for Xenopus. At the broad gene expression level, more genes are
misexpressed between species compared to hybrids. This is different compared to gene
expression analyses of hybrids in other organisms where in general more misexpression
occurs in hybrids. Genes that are misexpressed in hybrids compared to each species
follow a semi-dominant model of expression behavior because most genes have an
intermediate level of expression in hybrids. Despite the semi-dominance of
misexpressed genes, there was strong pattern of dominance for the X. laevis genome
because hybrids had fewer genes misexpressed compared to X. laevis than compared to
X. muelleri. This pattern implies a silencing mechanism for the paternally inherited X.
muelleri alleles and has implications for the evolution of species differentiation at the
expression level (Malone et al., 2006). This silencing mechanism may be a natural
phenomenon related to amphibian testis function or alternatively could be a genuine
response to the hybrid genetic background. Most interesting is that this pattern of
silencing, reminiscent of the widespread phenomenon of nucleloar dominance, has also
been documented in Arabidopsis allotetraploid hybrids (Wang et al., 2006). We propose
three hypotheses derived from large scale silencing of Xenopus rRNA genes which
could explain the species-level dominance pattern. By doing so we focus much needed
attention on the molecular mechanisms that promote gene transcription and in particular
96
how these mechanisms relate to the expression basis of reproductive isolation in
Xenopus.
97
CHAPTER 4
TESTIS-DERIVED MICRORNA PROFILES OF AFRICAN CLAWED FROGS
(XENOPUS) AND THEIR STERILE HYBRIDS
4.1 Abstract
Gene regulation was long predicted to play a vital role in speciation and species
divergence. Only recently with the advent of new technologies, however, has it been
possible to address the question about the relative contributions of different mechanisms
of gene expression to the evolution of phenotypic diversity. Here we broaden the
question and ask whether microRNAs, a large class of small regulatory RNAs, play a
role in reproductive isolation between species by contributing to hybrid male sterility.
MicroRNAs from testes of African Clawed Frogs (Xenopus) were extracted and the
expression profiles of sterile hybrids were compared with males of a parental species.
Hybrid testes were largely microRNA-depleted relative to those of non-hybrids, and this
pattern was validated with quantitative RT-PCR. A number of candidate differential
microRNAs from this study have previously been described as testis-specific in the
mouse, suggesting that microRNA structural conservation may be associated with
functional retention. Predicted targets of the top three candidate microRNAs (miR-100,
miR-125b, and miR-202*) were surveyed and a substantial overlap between the target
Gene Ontology classes is demonstrated. The consequences of decreased microRNAs in
98
hybrid testes are also discussed in relation to other changes in the transcriptome, as well
as male sterility.
4.2 Introduction
Comparative genomics provides new tools for helping to understand how
phenotypic differences between species are encoded in their genomes and what genetic
processes underlie the formation of new species (speciation). It has long been
recognized that speciation often results in sterility and inviability of species hybrids but
only recently has it been possible to identify genes causing incompatibilities on the
hybrid genetic background and associated hybrid defects (Wittbrodt et al., 1989; Pilder
et al., 1993; Ting et al., 1998; Barbash et al., 2003; Presgraves et al., 2003; Masly et al.,
2006). The evolution of reproductive isolation between species has been formalized as
the so-called Dobzhansky-Muller model of incompatible epistatic interactions between
genes that have diverged in different gene pools (Dobzhansky, 1937; Muller, 1940).
Despite its attractiveness, this broad model has obvious limitations for generating
specific predictions related to the genetics of reproductive isolation. In particular, the
Dobzhansky-Muller model does not easily accommodate such widespread phenomena
as maternal effects, transposable element mobilization, genomic imprinting and other
epigenetic changes despite their importance to reproductive isolation (e.g., Vrana et al.,
1998; Liu and Wendel, 2000; Shan et al., 2005; Salmon et al., 2005; Josefsson et al.,
2006; Ungerer et al., 2006; Nasrallah et al., 2007). The merging of two distinct genomes
typically sets in motion extensive modifications of the genome and transcriptome,
creating cascades of novel expression patterns (Michalak and Noor, Wu et al., 2003;
99
Michalak and Noor, 2004; Ranz et al., 2004; Auger et al., 2005; Hegarty et al., 2006),
regulatory interactions and new phenotypic variation (Riddle and Birchler, 2003;
Adams and Wendel, 2005a; 2005b). To complete this picture, we examined the
contribution of microRNAs to hybrid transcriptome anomalies and male sterility.
MicroRNAs (miRNAs) are a large class of small (~22-nucleotide long) non-coding
ribonucleic acids that function as fundamental transcriptional and post-transcriptional
regulators in plants and animals (Ambros 2001; Bartel, 2004).
There are several reasons to expect that miRNAs and other small RNAs have a
critical role to play in creating postzygotic reproductive isolation. First, it is becoming
increasingly clear that there are barely any cellular or developmental functions
unaffected by miRNAs. For example, it has been estimated that known miRNAs make
up >1% of the human gene content and regulate >30% of all protein-coding genes (Krek
et al., 2005; Lewis et al., 2005; Xie et al., 2005). Second, there are abundant testisspecific miRNAs (Lagos-Quintana et al., 2002; Yu et al., 2005), repeat-associated small
interfering RNAs (rasiRNA; Aravin et al., 2003; 2004), and Piwi-interacting RNAs
(piRNA) involved with spermato- and spermiogenesis (Girard et al., 2006; Grivna et al.,
2006; Watanabe et al., 2006; Lau et al., 2006; Kim, 2007), which all provide candidate
causative agents of hybrid male sterility. Finally, given that miRNAs along with other
transcripts are abundant in maternal deposits (Tang et al., 2007) it is tempting to
speculate that species-specific miRNA deposits may account for cases of reproductive
isolation with maternal effects, when reciprocal crosses between the same two species
produce different patterns of hybrid defects.
100
African Clawed Frogs (Xenopus) provide a novel system for exploring the
importance of miRNAs to the problem of reproductive isolation through hybrid male
sterility. There are >20 Xenopus species and experiments with over 100 reciprocal
crosses between selected pairs of species have revealed that hybrid males and rarely
females are sterile (Kobel and Du Pasquier, 1975; Müller, 1977; Kobel et al., 1981;
Picker, 1985; Picker and de Villiers, 1989; Kobel, 1996a; 1996b; Kobel et al., 1996;
Picker et al., 1996; Fischer et al., 2000). Certain reciprocal crosses are more affected or
not possible, suggesting a contribution of maternal effects. Additionally, there is
evidence that hybridization in Xenopus is accompanied by a transcriptome shock and
preferential silencing of one of the parental genomes (Malone et al., 2007), thus
suggesting the activity of ample transcriptional repressors.
As spatial and temporal expression patterns of miRNAs can provide clues about
their possible functions, tissue specificity of miRNA signaling is currently an area of
active research. For example, miRNA profiling identified pancreas islet-specific
miRNA involved in insulin secretion (Poy et al., 2004) as well as brain-specific miRNA
regulating the size of dendritic spines-postsynaptic sites of excitatory synaptic
transmission (Schratt et al., 2006). Here we report significant changes to miRNA levels
between testes of sterile Xenopus hybrids (Xenopus laevis x X. muelleri) and testes of
the parental Xenopus laevis. The detected repertoire of miRNAs additionally suggests
that testis-specific miRNA pathways are conserved between as distant taxa as Xenopus
and the mouse. An analysis of the miRNA putative targets shows that a diversity of
101
gene activities could be affected and that there was substantial cross-targeting between
the top candidate miRNAs, at least at the level of the target Gene Ontology categories.
4.3 Materials and Methods
4.3.1 RNA Extraction
A total of 5 micrograms/sample of RNA was extracted from mature testis in
Xenopus laevis (n = 3) and sterile hybrids of X. laevis x. X. muelleri (n=3) using
Ambion MirVana miRNA isolation kit and the manufacture’s protocols. Adults were
euthanized with MS-222 and tissue was immediately extracted and homogenized in
lysis/binding buffer Samples of RNA were checked for quality using Nanodrop ND
1000 spectrophotometer and the Agilent 2100 Bioanalyzer. The origin of animals and
the patterns of hybrid sterility have been described elsewhere (Malone et al., 2006;
2007).
4.3.2 miRCURYTM LNA microRNA Arrays
The Exiqon (Vedbaek, Denmark) miRCURYTM LNA microRNA arrays and the
service to process the samples were used. This technology is based on the use of locked
nucleic acid (LNA)-modified oligonucleotides that exhibit improved thermostability in
nucleic acid duplexes (Castoldi et al., 2006). The arrays consist of control probes,
mismatch probes, and 1458 capture probes interrogating all known miRNAs as
annotated in miRBase Release 8.1 (Griffiths-Jones et al., 2006), including 7 cloned
Xenopus laevis (Watanabe et al., 2005) and 177 predicted Xenopus tropicalis miRNAs,
as well as 146 miRPlusTM miRNA sequences unavailable in miRBase. Thirty control
capture probes include 10 spike-in, 8 negative control, and 12 small nuclear RNAs
102
(snoRNA)-interrogating probes. The arrays are manufactured on a standard size glass
slide and organized as 8 sub-arrays in 4 replicates each with a spot size of 120 µm. For
labeling, the miRCURYTM LNA kit was used according to the Exiqon protocols. The six
biologically independent samples were labeled with Hy3 and Hy5 dyes and
competitively hybridized on three arrays, each contrasting hybrid with X. laevis
samples. Dyes were alternated between sample types on one of the arrays. The spike-ins
were labeled as normal miRNA types and hybridized to capture probes specifically
designed for these RNAs.
4.3.3 Microarray Data Processing
Slides were scanned using a Genepix 4000B laser scanner (Axon Instruments)
and microarray images were preprocessed using the Genepix Pro 4.0 software. Data
were normalized using the background subtraction and the global Lowess (LOcally
WEighted Scatterplot Smoothing) regression algorithm. Spike-in controls (10 different
RNA oligonucleotides) were used to assess the overall performance of hybridizations.
Signals from the Hy3 and Hy5 channels were highly correlated (R2 range between 0.972
and 0.992) indicating highly reproducible labeling and hybridization across all slides
(Fig. 3.1). The normalized expression values were log2-transfromed and implemented in
Cyber-T (Baldi and Long, 2001) and EDGE (Leek et al., 2005) for tests of differential
gene expression. To control the false discovery rate, a Bonferroni correction was
applied to the P-values generated by Cyber-T and q-values were estimated using EDGE.
Hierarchical clustering with Euclidean distances and heuristic ordering in Edge was
103
used to visualize the global pattern of miRNA expression in hybrids relative to X.
laevis.
Array 1
Array 2
100000
100000
2
2
Normalized Hy5 signals
R = 0.992
10000
1000
100
100
1000
10000
10000
1000
100
100
100000
Normalized Hy3 signals
1000
10000
Normalized Hy3 signals
Array 3
100000
2
R = 0.991
Normalized Hy5 signals
Normalized Hy5 signals
R = 0.972
10000
1000
100
100
1000
10000
100000
Normalized Hy3 signals
Figure 4.1. Hy5 vs Hy3 scatterplots for the spike-in controls. These diagrams
show that two different dyes used in spike-in controls produced similar
fluorescence signal. The black trendline represents X=Y.
104
100000
4.3.4 Quantitative Real-Time PCR Validation
Differentially expressed miRNAs were subject to quantitative RT-PCR
validation using TaqMan MicroRNA Assays from Applied Biosystems (ABI product
numbers 4373219, 4373148, 4373019, 4373160, 4373274). As there appears to be no
consensus regarding endogenous control selection for the miRNA relative
quantification yet, we chose to use hsa-miR-382 that according to our microarray data
was highly expressed at very similar levels in all samples. A total of 10 ng/sample of
RNA was extracted as described above from testes of 10 X. laevis and 10 hybrid males.
cDNA was produced using the ABI TaqMan MicroRNA Reverse Transcription Kit
(product # 4366596) and the manufacturer protocol. To test for genomic DNA and
reaction contamination, two types of negative controls were used for PCR: reverse
transcriptase-omitted products and blanks (DEPC-treated water). No amplification was
observed in any of the negative controls. The ABI 7300 RT PCR System and the
instrument default cycling conditions were used. Each RNA sample was assayed twice
for every miRNA in separate RT reactions. The threshold cycle (CT) ratios between the
target miRNA and the endogenous control were calculated, arcsin-transformed, and
one-way repeated measures ANOVA was used to test differences between male types
(X. laevis and hybrids) and determine the effect of technical replications.
4.3.5 miRNA Target Prediction
To identify potential binding genomic sites for candidate miRNA, the miRanda
algorithm provided by a Welcome Trust Sanger Institute server was used
(http://microrna.sanger.ac.uk/targets). The miRanda uses dynamic programming
105
alignment to identify highly complementary sites which are scored between 0 and 100,
where 0 represents no complementarity and 100 complete complementarity (John et al.,
2004). The algorithm incorporates a weighted scoring system that rewards
complementarity at the 5' end of the miRNA and the Vienna RNA folding routines to
estimate the target site thermodynamic stability (Hofacker, 2003; Enright et al., 2003).
Statistical overrepresentations of the target Gene Ontology (GO) categories were
examined with GoMiner (Zeeberg et al., 2003).
4.4 Results
4.4.1 Differential Expression
There was a global miRNA underexpression in hybrids compared with parental
X. laevis (Fig. 3.2). Both Cyber-T and Edge produced an identical order of the top
candidate miRNAs differentially expressed between the parental X. laevis and F1
hybrids (Supplementary Table 1). Three miRNAs remained significantly different at the
0.05 alpha-level after a Bonferroni correction (0.05/1458) was applied to the Cyber-T
results: xtr-miR-202*, hsa-miR-494, and dre-miR-202*, all underexpressed in hybrids
(Table 3.1). The Edge-generated-q-values for the top three and the next five best
candidates (according to their P-values) were all equal to 0.125. The fact that two of the
best candidate miRNAs, xtr-miR-202* from X. tropicalis and dre-miR-202* from
Danio rerio, have identical sequences is interpreted as an indication of high array
reproducibility. Out of the top ten candidate miRNAs, six were previously known in
Xenopus, three were known exclusively in mammals (miR-494, mmu-miR-489, and
mmu-miR-155), and one is from miRPlus. These mammalian miRNA sequences did not
106
Figure 4.2 Hierarchical clustering visualizing miRNA overerepresentation (red
color) in the parental X. laevis relative to hybrids. Only differentially expressed
(q-value < 0.2) miRNAs were included.
produce significant hits when blasted against the X. tropicalis genome (X. laevis and X.
muelleri genomes are unavailable).
4.4.2 Quantitative Real-Time PCR Analysis
As the false discovery rate estimated as q-values was relatively high, differential
expression of miR-202*, miR-494, miR-125b, and miR-100 was independently tested
107
Table 4.1. Top five candidate miRNAs differentially expressed
between hybrid and non-hybrid testes. Fold change is a mean ratio
between X. laevis and hybrid expression values.
miRNA
Fold change Edge P-value Cyber-T P-value
xtr-miR-202*
2.491
0.0004
9.776-6 *§
hsa-miR-494
2.436
0.0008
1.921-5 *
dre-miR-202*
2.392
0.0012
3.206-5 *§
xtr-miR-125b
2.179
0.0016
7.321-5 §
miRPlus 17927 2.117
0.0020
0.0025
* Significant at P < 0.05 after Bonferroni correction
§ Confirmed with quantitative RT-PCR
using larger RNA sampling and qRT-PCR. Amplification patterns of miR-202*, miR125b, and miR-100 confirmed results of the microarray analysis and showed consistent
miRNA underexpression in hybrids relative to X. laevis (Table 4.2). Expression of miR494 was low compared to the other three miRNAs and no statistically significant
differential expression between X. laevis and hybrids was observed. Although
reproducibility between the consecutive RT reactions was remarkably high, their
statistical effect was significant in all four assays (Table 4.2). Interaction factors from
the one-way repeated measures ANOVA were not significant (P>0.05).
4.4.3 Predicted Targets of Candidate miRNAs
MicroRNAs positively verified with qRT-PCR were analyzed with respect to
their putative targets with MiRanda (Table 4.3). A total of 23 GO categories related to
Biological Processes (BPGO) were overrepresented among putative targets of miR202* (Supplementary Table 2). These categories included eight chemotaxis-, six protein
processing- and two behavior-related processes. Only two Cell Component GO
(CCGO) categories were overrepresented in miR-202* targets: small ribosomal subunit
108
Table 4.2. ANOVA analysis of TaqMan RT-PCR results.
H Mean CT
-ratio
ANOVA
Effects
MS
Effect
MS
Error
Fdf(1, 8)
P-value
0.6032
Type (1)
Replicates (2)
(1) x (2)
Interaction
0.0065
0.0014
6.68-5
0.0006
0.0001
0.0001
11.3723
9.5812
0.4675
0.0100
0.0148
0.5134
miR-494
1.1480
1.1420
Type (1)
Replicates (2)
(1) x (2)
Interaction
0.0002
0.0012
1.74-5
0.0015
0.0005
0.0005
0.1175
2.5307
0.0365
0.7406
0.1503
0.8531
miR-125b
0.6844
0.7438
Type (1)
Replicates (2)
0.0176
0.0251
0.0007
0.0003
25.7364
92.0373
(1) x (2)
Interaction
0.0002
0.0002
0.6183
0.0010
1.16 x
10-5
0.4543
Type (1)
Replicates (2)
0.0104
0.0084
0.0008
0.0002
12.3913
52.7265
(1) x (2)
Interaction
6.91-5
0.0002
0.4335
L Mean
CT-ratio
miR-202*
0.5834
miR-100
0.7089
0.7544
0.0078
8.71 x
10-5
0.5288
and cell surface. There were 12 Molecular Function GO (MFGO) categories
overrepresented among the miR-202* targets, including six various acetyltransferase
activities. In miR-125b putative targets, 47 BPGO, 11 CCGO, and 23 MFGO categories
were overrepresented. A similar analysis of miR-100 showed that 19 BPGO, 8 CCGO,
and 15 MFGO target categories were overrepresented. There was a substantial overlap
(24%) between overrepresented BPGO categories of all three miRNA targets, including
chromatin assembly, cellular protein metabolism, protein localization and metabolism.
109
110
110
Beta-1,3-galactosyltransferase 5
ENSXETT000
00013476
pecr
Jumonji/ARID domain-containing protein
1A (Retinoblastoma-binding protein 2)
(RBBP-2)
Pecr-prov protein (fragment)
Heparan sulfate (Glucosamine) 3-Osulfotransferase
JARID1A
xtr-miR-125b
hs3st1
Egg005f01.1
zinc finger, AN1-type domain 2A
Hypothetical protein LOC496606
ap3s2
ENSXETT000
00058115
cAMP-dependent protein kinase type Ialpha regulatory subunit (Tissue- specific
extinguisher 1) (TSE1).
Sacsin
Hypothetical protein
Description
ENSXETT000
00039632
Gene
xtr-miR-202*
BC076989.1
17.442
17.5634
17.6511
18.0788
18.5336
18.5336
18.8403
20.142
20.2392
Score
-27.59
-22.72
-34.21
-27.24
-12.62
-18.46
-22.04
-19.5
-14.06
Energy
3.0-5
0.0004
0.0042
0.0023
0.0047
0.0047
0.0035
10-6
0.0009
P-value
UCAAUCCCAGAGUCCCU
:||||| | ||||||||
GGTTAGAGACTCAGGGA
GUGUUCAAUCCCAGAGUCCCU
|| :|| | ||| |||:|||:
CAGGAGGTCGGGCCTCGGGGG
UGUCCAAUUCCCAGAGUCCCU
|||
|::| ||:|||||||
ACATAATGGGAGTTTCAGGGA
AGUGUUCAAUCCCAGAGUCCCU
||| | |:||||||||||||
TCAGCACATGGGGTCTCAGGGA
UUCUCCAUAUACGUAUCCUU
:|| |||:| ||:|||||:|
GAGTGGTGTTTGTATAGGGA
UCCAUA-UA-CGUAUCCUUU
|||||| || |||||||||
AGGTATCATCTCATAGGAAA
UUUCUCCAUAUACGUAUCCUU
||:||| | | | :|||||||
AAGGAGCT-TCTTTATAGGAA
UUCUCCAUAUACGUAUCCUUU
|| | || ||| |||||||||
AAAATGTTTATACATAGGAAA
UUCUCCAUAUACGUAUCCUU
|:|||| ||| ||||||||
AGGAGGCATAACCATAGGAA
Alignment
Table 4.3. MIRANDA-predicted top candidate targets of miR-202*, miR-125b, and xtr-miR-100.
111
Cerebellin-2 precursor
CBLN2_HUM
AN
Hypothetical protein
TNeu066d14.1
Glucagon-like peptide 2 receptor
precursor
Uridine-cytidine kinase 2
UCK2
GLP2R
Protein cereblon
xtr-miR-100
CRBN_XENT
R
Table 4.3-continued.
111
16.6989
16.9971
17.0555
17.0573
18.0964
-27.64
-23.87
-21.39
-21.18
-25.93
0.0018
0.0055
0.0052
0.0003
0.0018
GUGUUCAAGC--CUAGAUGCCCAA
|| || |||| |:|:|::|||||
CAGAACTTCGCTGGTTTGTGGGTT
UGUUCAAGCCUAGAUGCCCAA
|:||||| | ||||| |||||
ATAAGTT-GTATCTAGGGGTT
GUUCAAGCCUAGAUGCCCAA
||| |||| :|:||||||||
CAA-TTCGAGTTTACGGGTT
GUGUUCAAGCCUAGAUGCCCAA
:||||| | | ||||||||||
TACAAG-TGGTCTCTACGGGTT
GUGUUCAAGCCUAGAUGCCCAa
||||| | :||||:||:||||
CACAACTCTGGATTTATGGGTg
A 19% overlap between overrepresented CCGO (including intracellular organelle part)
and no clear overlap between MFGO categories for these combined miRNA targets
were observed.
4.5 Discussion
Here we report the identification of a number of miRNAs depleted in testes of
sterile hybrids relative to fertile parentals in Xenopus. To our knowledge, this is first
miRNA profiling in adult Xenopus. Previously, a number of X. laevis miRNA were
isolated from various developmental stages ranging from oocytes to tadpoles (Watanabe
et al., 2005; Chen et al., 2006). Although miRBAse (Release 9.1) provides a total of 177
predicted miRNAs for Xenopus tropicalis, no data on their spatial and temporal
distribution have been available.
Sequences of a large proportion of miRNAs are highly conserved among taxa
and many are expressed in a tissue-specific manner (e.g., Lim et al., 2005; Aboobaker et
al., 2005; Chen and Rajewsky, 2007). Although this does not necessarily imply spatial
and temporal conservation of their expression, such overlap in expression between
various taxa has been demonstrated (Miska et al., 2004; Sempere et al., 2004; Ason et
al., 2006). For example, miR-10 regulates Hox genes in both Drosophila and
vertebrates (Sempere et al., 2006; Lemons and McGinnis, 2006). Hence, one may ask
whether the miRNAs that we observed to be differentially expressed between hybrid
and non-hybrid Xenopus males match the list of testis-specific miRNAs isolated from
the mouse (Lagos-Quintana et al., 2002; Yu et al., 2005). One of such testis-specific
miRNAs, miR-122a, for example, is known to target a reporter mRNA containing
112
sequences from the 3’-UTR of the Transition Protein 2 (Tnp2) mRNA (Yu et al., 2005).
Four species of transition proteins, including Tnp2, facilitate chromatin transformation
from the nucleosome structure to the nucleoprotamine structure during spermatid
differentiation in mammals. All genes involved in mammalian spermiogenesis, such as
Tnp2, are subject to translational repression for up to 7 days (Kleene, 2003), and small
RNAs are logical candidate silencers in this process.
Remarkably, one of our top three candidate miRNAs, miR-100, has been
characterized as testis-specific in mice (Yu et al., 2005). Its targets and a potential role
in spermatogenesis/spermiogenesis are unknown, but the miRanda-predicted best
candidate target of miR-100 in X. tropicalis is cereblon (CRBN; Table 2), in mammals
known to be enriched in brain and testes (Xin et al., 2007). CRBN encodes for an ATPdependent Lon protease that selectively degrades short-lived polypeptides and regulates
mitochondrial replication and transcription (Higgins et al., 2004). In brain, CRBN is
typically associated with the large conductance Ca2+-activated K+ (BK(Ca)) channels
and mutations in the CRBN gene result in long-term potentiation and learning
dysfunctions leading to mental retardation (Higgins et al., 2004; Yu et al., 2005; Xin et
al., 2007). The complementarity between miR-100 and its putative targets, including
CRBN, is not perfect but this seems to be typical of most animal miRNAs that unlike
siRNAs and plant miRNAs are characterized by an imperfect match to their mRNA
targets (Zamore et al., 2000; Valencia-Sanchez et al., 2006). The GO categorization of
the putative miR-100 targets in Xenopus suggests enrichment in genes related to
113
neurophysiological processes, sensory perception of light stimulus, exocytosis,
chromatin and nucleosome assembly, and protein localization.
Beside miR-100, seven other miRNAs from the Yu et al.’s (2005) list of the 29
mouse testis-specific miRNAs were also present in our list: miR-16, miR-143, miR-21,
miR-30b, miR-15b, miR-7c, miR-467 (Supplementary Table 1). This finding suggests a
considerable conservation of male germline miRNA pathways in vertebrates and
necessitates further explorations into the role of miRNA in male reproduction and
hybrid sterility. There is no evidence, however that the other top two candidate
miRNAs, miR-202* and miR-125b, are testis-specific in Xenopus or other vertebrates
(Lagos-Quintana et al., 2002; Chen et al., 2005).
Lee et al. (2005) have used a siRNA against the loop region of the mirR-125b
precursor to selectively down-regulate the mature miRNA in human cell lines. The
depletion of miR-125b resulted in a significant proliferation defect in PC-3 (prostate
cancer) cells. The miRanda-predicted targets of mirR-125b in Xenopus are enriched in
transferase and hydrolase/protease activities and associated with such biological
processes as proteolysis, protein complex, chromatin and nucleosome assembly, DNA
packaging, RNA export and localization. There was a substantial overlap between the
target GO categories of the three miRNAs, with the largest between miR-100 and miR125b (19%) and smallest between miR-100 and miR-202* (2%). This result suggests
the miRNA cross-targeting of such functions as chromatin assembly, nucleosome
assembly, and cellular protein metabolism.
114
In contrast to many hybrid anomalies in the expression of translated genes,
differential miRNAs were consistently underrepresented and virtually no significant
overrepresentations were observed in hybrids relative to the parental species. This may
reflect an overall miRNA underexpression due to hybrid genetic incompatibilities
between upstream components of the miRNA pathway. In animal cells, miRNAs are
transcribed from genes as primary miRNAs by RNA polymerase II, then cleaved by
Drosha, an RNase III enzyme, into hairpin-like precursor miRNA (Lee et al., 2003), and
eventually transported into the cytoplasm with the help of Exportin (Yi et al., 2003). In
the cytoplasm, pre-miRNAs are cut into double-stranded RNA duplexes by Dicer,
another RNase III enzyme, processed into mature miRNAs and incorporated into RISC
complex with other components for target recognition (Lee et al., 2002). Dysfunctions
at any of these levels are expected to result in a global underexpression of miRNAs
which in turn may lead to overexpression of their target genes on the hybrid genomic
background.
Could this prediction of uniform upregulation of the target genes due to
misregulation of the upstream components in the miRNA pathway explain the pattern of
other gene misexpressions in hybrids between X. laevis and X. muelleri? Given the lack
of direct experimental data on the true miRNA targets in these two Xenopus species, all
answers to this question will necessarily be speculative at this moment. Nevertheless,
Malone et al. (2007) have observed that 65% of all hybrid gene misexpressions relative
to the parental X. laevis were in fact overexpressions. Among all misexpressed genes,
only six out of 142 were predicted target genes of Xenopus miR-100, miR-125b or miR115
202*, and all six but one (a miR-100 target) were overexpressed in hybrids. This
tendency for enrichment in upregulations among putative miRNA targets was also
apparent in a comparison of hybrid males with the other parental species, X. muelleri:
44% of all misexpressions (n = 3,995) were represented by overexpressions in hybrids,
and 51% of the misexpressed putative miRNA targets (n = 180) were upregulated (Chi
square test, P = 0.085).
As there are examples of negative feedback loops that involve multiple miRNAs
and transcription factors (Johnston et al., 2005; Yoo et al., 2005; Fazi et al., 2005), it is
likely that the prediction of uniform upregulation of miRNA targets due to miRNA
misexpression is an oversimplification. A comprehensive understanding of the genetic
basis of hybrid defects and speciation will require further insights into how transcription
factors, miRNAs, signaling pathways and epigenetic regulators are wired together into
multicomponent regulatory networks.
116
CHAPTER 5
GENE EXPRESSION ANALYSIS OF THE OVARY OF FERTILE HYBRID
FEMALES OF X. LAEVIS AND X. MUELLERI
5.1 Abstract
Hybridizing any two species of Xenopus results in sterile hybrid males and
fertile hybrid females. Previous work has demonstrated a dramatic asymmetrical pattern
of misexpression in hybrid males compared to the two parental species with relatively
few genes misexpressed in comparisons of hybrids and the maternal species (X. laevis)
and dramatically more genes misexpressed in hybrids compared to the paternal species
(X. muelleri). In this work, we examine the gene expression pattern in hybrid females of
X. laevis x X. muelleri to determine if this asymmetrical pattern of expression also
occurs in hybrid females. Additionally, we also wanted to test the prediction that hybrid
females have less gene misexpression given the fact that hybrid females are fertile
whereas hybrid males are completely sterile. We find a similar pattern of asymmetry in
expression compared to males in that there were more genes misexpressed between
hybrids and X. muelleri compared to hybrids and X. laevis. We also found a dramatic
increase in the number of misexpressed genes with hybrid females having about 20
times more genes misexpressed compared to hybrid males and therefore the match
between phenotype and expression pattern is not supported. We discuss these intriguing
findings in the context of reproductive isolation and suggest that divergence in female
117
expression may be involved in sterility of hybrid males due to the spermatogenesis is
special component of faster male evolution.
5.2 Introduction
Frogs of the genus Xenopus provide a striking exception to the most widespread
generalization in evolutionary biology–Haldane’s rule. Xenopus have ZW sex
determination and Haldane’s rule would predict that hybrid females should suffer the
most dramatic effects of hybridization but contrary to expectation, F1 hybrid males are
completely sterile and hybrid females are fertile (Chapter 1; Kobel, 1996).
Analyses of spermatogenesis in hybrid males of X. laevis x X. muelleri have
shown that males have a dramatically lower abundance of motile sperm, increased
numbers of undifferentiated sperm cells, and larger mature sperm cells compared to
parental species. The gene expression pattern for hybrid males shows a striking
asymmetric pattern in that relatively few genes are misexpressed between hybrids and
the maternal species (X. laevis) whereas there are dramatically more genes
misexpressed between hybrid males and the paternal species, X. muelleri. These results
suggest intriguing mechanisms operating on the transcriptome in hybrid males of
Xenopus that may reflect strong maternal and/or species dominance effects (Malone et
al., 2007).
Hybrid females are just as fertile as conspecific species (Chapter 1) and given
the phenotype of hybrid females, a reasonable prediction would be that gene expression
should be similar to the two parental species. However, given the asymmetrical pattern
of expression operating in hybrid males, it is of interest to investigate the pattern of
118
gene expression in hybrid oogenesis, particularly since oogenesis in hybrids does not
seem to be affected by the hybrid genetic background compared to hybrid males.
In this study, we analyzed the gene expression pattern of adult ovary in hybrid
females of X. laevis x X. muelleri compared to the two parental species. Our analyses
reveal a pattern of asymmetrical gene expression like that in hybrid males but
surprisingly there is a dramatic increase in the number of genes misexpressed in hybrid
females compared to the two parental species relative to hybrid males. This increased
level of gene misexpression suggests that oogenesis can tolerate dramatically more
misexpression compared to spermatogenesis and points further evidence to the
spermatogenesis is special component of the faster male evolution hypothesis for
Haldane’s rule.
5.3 Materials and Methods
5.3.1 Microarray Experiments
RNA was extracted from adult ovary in Xenopus laevis (n = 4), hybrids of X.
laevis x. X. muelleri (n=2) and X. muelleri (n = 3). Origin of parents and methodology
for creating hybrids has been described elsewhere (Malone et al., 2006; Malone et al.,
2007). Adults were euthanized with MS-222 and 50 mg of ovary was dissected and
homogenized in RNA extraction solution using a hand held pestle. RNA was recovered
using GeneHunter and Ambion RiboPure total RNA kits. Samples of RNA were
checked for purity by examination of the 28S and 18S ribosomal RNA bands from
denaturing gel electrophoresis, by 260/280 ratios from scans with a Nanodrop ND 1000
spectrophotometer, and by readouts of the Agilent Bioanalyzer. Total RNA samples
119
were prepared and hybridized to Affymetrix Xenopus laevis GeneChip Genome Arrays
at the University of Texas Southwestern Medical Center Microarray Array Core Facility
following standard Affymetrix protocols. Affymetrix Microarray Analysis Suite (MAS)
v.5.0 was used to scan and process each microarray chip. The signals of quality control
and poly(A) transcripts revealed that hybridizations were of high quality in all chips.
Quality control probe sets (i.e., spike in and housekeeping genes) were removed in
subsequent statistical analyses. Hybridizing RNA from a heterospecific species to a
microarray designed for a related species can have a dramatic impact on the signal
recovered from microarrays (Nuzhdin et al., 2004; Gilad et al., 2006; Gilad and
Borevitz, 2006; but see Oshlack et al., 2007). To control for this effect, we used an
electronic mask generated from hybridizing genomic DNA from X. laevis and X.
muelleri onto the X. laevis microarray (Malone et al., 2007). This mask which screens
out probes that have significant sequence divergence in X. muelleri provides 11,485
probesets/genes for further analysis.
5.3.2 Data Analysis
We conducted three separate comparisons to uncover patterns of differential
expression between Xenopus laevis and hybrids, X. muelleri compared to hybrids, and
X. laevis compared to X. muelleri. First, the Xenopus laevis and hybrid chips were
normalized using RMAexpress (Bolstad, 2005) using default parameters for background
correction and quantile normalization. These RMA normalized data were then imported
into the R statistical environment and tested for differences in expression between X.
laevis and hybrids for each of the 11,485 genes using a moderated t-statistic based on an
120
empirical Bayes method in the limma package found in Bioconductor (Smyth, 2004).
The TopTable function was then used to output the FDR-adjusted P-values for
differential expression. We normalized X. muelleri and hybrid chips together using
RMA and repeated the analyses to uncover differential expression between X. muelleri
and hybrids. Finally, we normalized X. laevis and X. muelleri chips together using RMA
and repeated the analysis to uncover genes misexpressed between the two species. This
later test was used to discover patterns of expression behavior in hybrids compared to
gene expression behavior between the two conspecific species.
5.4 Results
There was a substantial amount of gene misexpression in hybrids compared to the two
parental species. About 14% (1,616/11,485) of genes were misexpressed in hybrid
females compared to females of X. laevis and 63% (7,279/11,485) of genes were
misexpressed between hybrids and X. muelleri (Fig. 5.1). The number of genes
upregulated in hybrids relative to X. laevis compared to the number of genes
upregulated in X. laevis relative to hybrids was the same (839 vs. 777; G = 2.38; df = 1;
P>0.05) but there were significantly more genes upregulated in X. muelleri compared to
hybrids (4,349 vs. 2,930; G = 139.2; df=1; P<0.0001). Many of the top 30 most
misexpressed genes for each class of gene expression behavior are EST sequences with
little functional information but our results imply that these EST sequences play a role
in oogenesis in Xenopus. Of the top 30 candidate genes with known function many have
a documented role in oogenesis in other organisms (Table 5.1-5.4). Comparing the two
lists of differentially expressed genes showed that about 68% (1105/1616) were
121
common to both X. laevis vs. hybrids and X. muelleri vs. hybrids. This common set of
differentially expressed genes suggests a set of genes that are uniquely expressed in
hybrids relative to the two parental species.
Gene expression between the two parental species was also dramatically
different. More than 76% (8,741/11,485) of genes were differentially expressed
between females of X. laevis and X. muelleri. Of these differentially expressed genes,
about 60% (5,203/8,741) were upregulated in X. muelleri relative to X. laevis (5,203 vs.
3,538; G = 159.5; df=1; P<0.0001). Comparing the overlap in genes misexpressed in
the two hybrid contrasts to the three classes of expression behavior between X. laevis
and X. muelleri (X. laevis > X. muelleri; X. laevis < X. muelleri; X. laevis = X. muelleri)
shows a general pattern of semidominance in expression behavior (Table 5.5). For
example, of the 839 genes upregulated in hybrids relative to X. laevis; 90% were
upregulated in X. muelleri relative to X. laevis. Similarly, of the 2,930 genes that were
upregulated in hybrids relative to X. muelleri, 91% were upregulated in X. laevis
compared to X. muelleri. These results suggest a general pattern of intermediate
expression in hybrids and are consistent with a semidominant model of expression
difference even despite the asymmetrical pattern of misexpression in hybrids compared
to the two parental species.
122
123
-2
0
2
4
6
8
Fold Change(L/H)
0
-4
0
-6
2
2
-8
4
6
8
10
12
4
6
8
10
12
14
-8
-6
B
-4
0
2
FoldChange(M/H)
-2
4
6
123
Figure 5.1. Volcano plots from FDR corrected t-tests of statistical significance (vertical axis) against magnitude of expression
change (horizontal axis), where each point corresponds to a gene/transcript. Expression change (fold-change) is defined as a
log2-transformed ratio of mean nonhybrid to mean hybrid expression level. (A) Xenopus laevis (L) vs. Hybrids (H); (B)
Xenpous muelleri (M) vs. Hybrids (H). The red line denotes FDR adjusted alpha 0.05. The horizontal deviation from 0
towards the right or left reflects hybrid underexpression or overexpression, respectively.
-log2Pvalue
14
A
-log2Pvalue
8
124
GenBankID
AW147858
BJ081331
BM180490
BG730898
CB562594
BJ079520
BM191810
BQ388097
BG023013
BJ078115
BG021901
BJ051966
BQ383513
BG514303
BM180520
BJ050593
BG408234
BG161783
BJ084136
BF025269
CB560349
AW632884
BG234472
BQ400343
BJ045456
AW633197
BJ048949
BI444111
BM179493
BJ057192
ProbeID
Xl.1473.1.A1_at
Xl.7484.1.A1_at
Xl.14874.1.A1_at
Xl.2557.1.S1_at
Xl.4581.1.A1_at
Xl.14122.1.A1_at
Xl.12012.2.A1_at
Xl.23885.1.S1_at
Xl.3628.1.A1_at
Xl.24509.1.A1_at
Xl.14330.2.S1_at
Xl.10705.1.A1_at
Xl.1973.1.S1_at
Xl.4825.1.A1_at
Xl.14885.1.A1_at
Xl.16512.1.A1_at
Xl.1448.2.S1_a_at
Xl.16106.1.A1_at
Xl.25509.1.A1_at
Xl.25610.1.A1_at
Xl.10826.1.A1_at
Xl.8907.1.S1_at
Xl.6727.1.A1_at
Xl.2780.1.S1_at
Xl.16426.1.A1_at
Xl.6421.1.A1_at
Xl.22941.1.A1_at
Xl.25368.2.A1_at
Xl.14796.1.A1_at
Xl.19146.3.A1_at
EST
EST
EST
EST
EST
proliferation2G4
EST
TIA1
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
EST
Target Gene
MGC69123
MGC82526
MGC81986
MGC83726
MGC68457
Pa2g4
LOC780754
tial1-a
MGC82938
LOC495116
LOC495259
MGC82215
LOC495457
MGC78898
MGC132176
MGC85135
LOC398314
LOC494727
MGC84382
Gene
Symbol
124
Moderately similar to A57088 nucleoporin-like
protein Rab;regulation of GTPase activity
Weakly similar to IFR1_HUMAN INTERFERONRELATED DEVELOPMENTAL REGULATOR
1(H.sapiens)
similar to thyroid hormone receptor associated
protein 3 (predicted) [Rattus norvegicus]
Moderately similar to AAKG_HUMAN 5-AMPactivated protein kinase, (H.sapiens)
Xenopus laevis, Similar to proliferation-associated
2G4
regulation of transcription, DNA-dependent
nucleic acid binding
Moderately similar to pallidin; pallid (mouse)
homolog, pallidin (Homo sapiens) (H.sapiens)
heme oxygenase (decyclizing) activity
nucleic acid binding
Weakly similar to A36368 transcription factor CBF,
CCAAT-binding - human (H.sapiens)
ubiquitin-protein ligase activity
Weakly similar to FW1A_HUMAN F-boxWD-repeat
protein
Description/Molecular Function
Weakly similar to hypothetical protein MGC3731
(Homo sapiens) (H.sapiens)
intracellular signaling cascade
Highly similar to T2D1_HUMAN TRANSCRIPTION
INITIATION FACTOR TFIID 250 KD SUBUNIT
Weakly similar to A39599 55K erythrocyte
membrane protein - human (H.sapiens)
Dihydrolipoamide acetyltransferase
6.21
7.10
7.88
6.79
6.95
7.28
6.41
6.58
8.68
8.37
7.16
7.59
8.24
6.09
8.67
7.41
10.16
8.79
7.03
7.39
7.76
7.18
9.04
8.63
10.12
7.34
9.51
7.35
10.18
7.33
Mean
Laevis
0.61
0.69
0.52
0.15
0.49
0.75
0.58
0.40
0.38
0.31
0.09
0.44
0.23
0.34
0.34
0.19
0.84
0.46
0.48
0.34
0.50
1.34
0.30
0.27
0.30
0.40
0.42
0.35
0.56
0.54
SD
Laevis
3.42
4.32
5.11
3.98
4.00
4.33
3.52
3.71
5.82
5.42
4.19
4.50
5.18
3.09
5.45
4.19
6.99
5.64
3.91
3.63
4.05
3.52
5.50
5.34
6.10
3.45
5.70
3.29
4.28
3.01
Mean
Hybrid
0.56
0.68
0.90
0.43
1.03
0.33
0.64
0.46
0.21
0.37
0.77
1.91
0.69
0.23
0.79
0.39
0.62
0.23
1.11
0.77
0.59
0.77
0.56
0.36
0.18
0.37
1.02
0.54
0.43
0.25
SD
Hybrid
Table 5.1. Top 30 candidate transcripts upregulated in X. laevis and differentially expressed
between females of X. laevis and hybrids. Expression values are in log2 scale; SD=standard
deviation of expression values. P values are adjusted according to FDR moderated t-tests.
2.79
2.78
2.78
2.81
2.95
2.95
2.89
2.87
2.86
2.95
2.98
3.10
3.05
3.00
3.22
3.22
3.17
3.15
3.12
3.76
3.71
3.66
3.53
3.29
4.02
3.88
3.81
4.06
5.90
4.32
L-H
0.0139
0.0182
0.0151
0.0041
0.0148
0.0149
0.0124
0.0063
0.0057
0.0047
0.0057
0.0325
0.0057
0.0044
0.0064
0.0041
0.0182
0.0057
0.0142
0.0057
0.0057
0.0325
0.0041
0.0041
0.0041
0.0041
0.0072
0.0041
0.0041
0.0041
adj.P.Val
125
BC044959.1
BJ084191
D78003.1
U07179.1
BI448285
AF170341.1
:AF170344.1
BC047974.1
BI478140
BG346438
BJ052360
BJ091634
L09728.1
Xl.19610.1.A1_at
Xl.75.1.S1_at
Xl.882.1.S1_at
Xl.17327.1.A1_at
Xl.747.1.S1_at
Xl.545.1.S1_at
Xl.23647.1.S1_at
Xl.19047.1.A1_at
Xl.9113.1.A1_at
Xl.16847.1.A1_at
Xl.13666.1.A1_at
Xl.721.1.S1_at
CB984321
BE491023
BG552091
BJ099673
BM180884
BJ091634
Xl.24699.1.S1_at
Xl.10600.1.S1_at
Xl.11324.1.A1_at
Xl.13010.1.A1_at
Xl.14915.1.A1_at
Xl.13666.1.A1_x_at
Xl.433.2.S1_at
BC045272.1
BC042271.1
Xl.8630.1.S1_s_at
Xl.21956.1.S1_at
BI940804
BJ084267
BC041496.1
BJ039322
Xl.23573.1.S1_at
Xl.16239.1.A1_at
Xl.24058.1.S1_at
Xl.16509.1.A1_at
BJ098608
Xl.24502.1.S1_at
CB560511
BJ075928
BI675584
Xl.19075.1.A1_at
Xl.24516.1.S1_at
Xl.13831.3.S1_at
GenBankID
BG552470
ProbeID
Xl.4605.1.A1_at
c4
Ldehydrogen
ase A
EST
galectin-1
metastasis
associated 1
cell death 2
Coatomerpro
tein
chimerin
EST
EST
transcription
factor DLL4
EST
EST
EST
neurotrophin
receptor B
EST
EST
EST
EST
EST
EST
EST
EST
MGC53990
MGC53461
EST
thymine-DNA
glycosylase
EST
EST
Target Gene
EST
Dlx2
copa
chn1
mta2
pdcd2
ldha
MGC68503
MGC64502
c4
trkb-b
MGC82121
LOC495356
MGC79012
MGC53990
MGC53461
TDG
GeneSymbol
MGC83384
125
regulation of transcription,
DNA-dependent
sugar binding
transcription factor activity,
regulation of transcription
Apoptosis
ER to Golgi vesiclemediated transport i
signal transduction
oxidoreductase activity
protein amino acid
phosphorylation
similar to Angiopoietin-1
receptor precursor (mTIE2)
endopeptidase inhibitor
activity i fourth component
of complement
Highly similar to histone
H2A.FZ variant, isoform
1(Homo sapiens)
Weakly similar to
Apolipoprotein E precursor
(H.sapiens); lipid binding
Highly similar to alpha
cardiac actin (H.sapiens)
Similar to serum-inducible
kinase; protein
serine/threonine kinase
act.
Weakly similar to HES2
(Hairy and enhancer of
split 2) (H.sapiens)
Weakly similar to forkhead
box P2; (Homo sapiens)
hydrolase activity, acting
on glycosyl bonds
Descriptoin
7.89
8.05
6.10
4.22
6.81
7.85
4.11
5.29
4.77
5.51
4.09
4.48
3.68
6.99
4.04
6.41
5.64
3.65
4.88
3.44
5.43
5.11
7.61
4.74
8.39
5.55
5.39
4.56
5.53
Mean
Laevis
3.41
0.58
0.35
0.76
0.74
0.77
0.51
0.36
0.94
0.49
0.21
0.47
0.05
0.40
0.28
0.35
0.60
0.45
0.36
0.94
0.46
0.46
0.30
0.80
0.36
0.22
1.16
1.36
0.70
1.29
SD
Laevis
0.39
10.04
10.28
8.32
6.44
8.97
10.14
6.34
7.60
7.07
7.80
6.41
6.81
6.01
9.40
6.44
8.93
8.05
6.51
7.68
6.23
8.22
7.72
10.17
8.01
11.57
9.22
8.66
8.65
9.67
Mean
Hybrid
7.63
0.35
0.01
0.12
0.36
0.54
0.13
0.40
0.06
0.19
0.55
0.00
0.36
0.00
0.09
1.00
1.00
0.17
1.79
0.25
0.11
1.89
0.35
0.37
0.75
0.00
0.15
0.46
0.00
0.27
SD
Hybrid
0.44
-2.15
-2.23
-2.22
-2.22
-2.16
-2.29
-2.23
-2.31
-2.29
-2.29
-2.32
-2.33
-2.33
-2.41
-2.40
-2.52
-2.41
-2.85
-2.80
-2.79
-2.79
-2.61
-2.57
-3.26
-3.18
-3.67
-3.27
-4.09
-4.14
L-H
-4.22
Table 5.2. Top 30 candidate transcripts upregulated in hybrids and differentially expressed
between females of X. laevis and hybrids. Expression values are in log2 scale; SD=standard
deviation of expression values. P values are adjusted according to FDR moderated t-tests.
0.0199
0.0071
0.0276
0.0279
0.0350
0.0124
0.0096
0.0387
0.0117
0.0075
0.0101
0.0043
0.0076
0.0057
0.0185
0.0244
0.0094
0.0327
0.0256
0.0064
0.0417
0.0057
0.0230
0.0062
0.0041
0.0210
0.0405
0.0060
0.0205
adj.P.Val
0.0041
126
BJ097640
BM179326
:X63425.1
BJ056161
Xl.7661.1.S1_at
Xl.2200.1.A1_at
Xl.1140.1.S1_s_at
Xl.15786.1.A1_at
BC041550.1
Xl.8049.1.S1_a_at
CB756768
BC045223.1
AW147985
CA988460
BM261049
Xl.6902.1.A1_at
Xl.25755.1.A1_at
Xl.7619.1.S1_a_at
Xl.23754.1.S1_at
Xl.23241.1.S1_at
BU904283
BG555239
Y17861.1
BJ097608
Xl.25283.1.S1_s_at
Xl.24302.1.A1_at
Xl.61.1.S1_s_at
Xl.15150.1.A1_at
CD324865
BG022283
BQ383420
BM261211
Xl.14298.1.A1_at
Xl.4311.1.A1_at
Xl.2546.1.S1_at
Xl.17949.1.S1_at
BE026658
Xl.25735.1.S1_at
AW782701
X63427.1
CD362680
AF256087.1
BF428365
Xl.3326.2.S1_a_at
Xl.24194.1.S1_at
XlAffx.1.12.S1_at
Xl.23898.1.A1_x_at
CB564916
BJ077543
Xl.17322.1.A1_a_at
Xl.8805.1.S1_s_at
BM191810
BC043865.1
AW764672
BI445766
Xl.12012.2.A1_at
Xl.7034.1.S1_at
Xl.5802.1.A1_x_at
Xl.5299.1.S1_at
Xl.6272.1.A1_at
GenBank ID
Probe ID
Bmp2
EST
EST
EST
EST
zf-e326
EST
EST
Psma2
EST
MGC83120
ribosomal
protein L4
Similar to VAMP
EST
EST
EST
LAP2
EST
EST
EST
EST
Bmp7
EST
Xcat 2
EST
EST
EST
LOC398646
EST
SEB-4
Target Gene
Nucleic acid binding
Weakly similar to myeloidlymphoid or mixed-lineage
leukemia 2; ALL1-related gene (Homo sapiens)
Bone morphogentic protein, ossification, growth factor
activity
Ribosome biogenesis and assembly
Xenopus borealis Xcat-2
membrane alanyl aminopeptidase activity
Weakly similar to GRF1_HUMAN G-rich sequence factor-1
(GRSF-1) (H.sapiens)
Moderately similar to MOB-LAK (Homo sapiens)
(H.sapiens)
seb4-a
Bmp2
MGC83224
LOC495305
LOC734179
zf-e326
LOC495016
Psma2
MGC68573
rpl-4
MGC83120
MGC53868
126
Weakly similar to MCA3_HUMAN Multisynthetase complex
auxiliary component p18 (H.sapiens)
Calcium ion binding
Bone morphogenetic protein 2; growth factor activity;
ossification
tRNA processing
Ribosomal protein L1; structural constituent of ribosome
Proteasome subunit XC3; ubiquitin-dependent protein
catabolism
cytochrome-c oxidase activity
Moderately similar to SYQ_HUMAN Glutaminyl-tRNA
synthetase(H.sapiens), glutamate-tRNA ligase activity,
protein biosynthesis
Intracellular signaling cascade
Highly similar to B-cell CLLlymphoma 11A (zinc finger
protein); (H.sapiens); nucleic acid binding
Similar to VAMP (vesicle-associated membrane protein)associated protein A, structural molecule activity
Highly similar to Calcium-binding protein p22 (Calciumbinding protein CHP) (H.sapiens); calcium ion binding
Lamina associated polypeptide 2; nuclear envelope
LAP2
MGC68575
Highly similar to RL2B_HUMAN 60S ribosomal protein
L23a (H.sapiens); structural constituent of ribosome
MGC85348
MGC68434
MGC68920
Xcat 2
MGC82089
LOC398646
Description/Molecular Function
Weakly similar to FW1A_HUMAN F-boxWD-repeat protein
1B (H.sapiens)
Similar to pantophysin, transporter activity
Gene Symbol
9.29
8.88
11.18
6.64
7.67
9.85
8.87
8.23
8.19
7.41
11.01
10.36
8.22
7.37
11.11
9.69
10.11
7.54
7.73
6.75
9.66
8.03
8.36
10.81
8.12
7.00
8.50
10.03
9.84
8.67
Mean
Muell.
0.18
0.19
0.14
0.02
0.18
0.09
0.30
0.37
0.25
0.23
0.19
0.23
0.28
0.47
0.08
0.04
0.12
0.32
0.29
0.24
0.23
0.53
0.60
0.09
0.12
0.09
0.22
0.23
0.32
0.44
SD
Muell.
5.79
5.40
7.64
3.13
4.09
6.28
5.32
4.68
4.58
3.83
7.37
6.65
4.47
3.60
7.22
5.83
6.28
3.75
3.82
2.86
5.68
3.94
4.27
6.79
4.12
2.90
3.16
5.60
5.46
4.47
Mean
Hybrid
0.07
0.02
0.40
0.11
0.30
0.41
0.01
0.37
0.10
0.21
1.79
0.28
0.43
0.04
0.18
0.05
0.21
0.22
0.46
0.37
0.47
0.43
1.21
0.43
0.11
0.14
0.55
0.29
0.27
1.24
SD
Hybrid
Table 5.3. Top 30 candidate transcripts upregulated in X. muelleri and differentially expressed
between females of X. muelleri and hybrids. Expression values are in log2 scale; SD=standard
deviation of expression values. P values are adjusted according to FDR moderated t-tests.
3.50
3.48
3.54
3.51
3.58
3.57
3.55
3.55
3.61
3.58
3.64
3.71
3.75
3.76
3.88
3.86
3.82
3.79
3.90
3.89
3.97
4.09
4.09
4.02
4.00
4.10
5.35
4.44
4.38
4.19
M-H
0.0002
0.0002
0.0002
0.0001
0.0002
0.0002
0.0002
0.0004
0.0002
0.0002
0.0059
0.0002
0.0003
0.0003
0.0001
0.0001
0.0001
0.0002
0.0003
0.0002
0.0002
0.0005
0.0019
0.0002
0.0001
0.0001
0.0002
0.0002
0.0002
0.0014
P value
127
BJ076394
:BQ398421
AY172320.1
CB561588
BF232270
BE026874
AW632842
BQ398301
U46131.1
BC041270.1
BE677987
AW147826
Xl.4504.1.A1_at
Xl.7045.1.S1_a_at
Xl.7252.1.S1_at
Xl.2565.3.S1_x_at
Xl.7837.1.A1_at
Xl.25809.1.A1_at
Xl.6605.1.A1_at
Xl.4170.2.A1_at
Xl.1014.1.S1_at
Xl.2839.1.S1_at
Xl.25536.1.A1_at
Xl.14065.1.A1_at
Xl.1018.1.A1_at
BC041216.1
BJ055271
AF184090.1
BM261081
U44950.1
Xl.3401.2.A2_at
Xl.23448.1.S1_at
Xl.2060.1.A1_x_at
Xl.576.1.S1_at
Xl.24785.1.S1_at
BG016692
Xl.14775.1.A1_at
Xl.3668.1.S1_at
BJ089477
BF615663
BM179359
AF450296.1
Xl.21357.2.S1_at
Xl.7151.1.S1_at
Xl.3536.2.S1_x_at
BJ045324
Xl.6585.1.S1_at
CD361360
BJ080015
Xl.4744.1.S1_at
Xl.3862.2.S1_x_at
BJ053357
BE491637
Xl.17345.1.A1_at
GenBank ID
X53745.1
BJ098891
BC041555.1
ProbeID
Xl.4276.1.S1_at
Xl.8319.1.S1_at
Xl.21809.1.S1_at
EST
EST
Cdc21 protein
Protein
translocation
complex
EST
EST
EST
LOC494857
EST
Germes
Similar to alphaTubulin at 84B
EST
SWI/SNF
EST
fatvg
EST
EST
EST
EST
Vitelline
envelope
glycoprotein
Translation
factor sui1
EST
XLCL2
Claudin7L1
Similar to HIV-1
rev binding
protein 2
EST
Target Gene
Cyclin A1
Herz03
MGC53900
sec61beta
cdc21
LOC494857
MGC132211
MGC80281
MGC53359
LOC398520
fatvg
MGC81067
smarce1
MGC68561
LOC495200
gc20
lzpb-a
LOC446970
LOC397879
MGC53400
HRB2
LOC495025
MGC115708
GeneSymbol
LOC397885
Herz03
MGC53900
127
Similar to protein translocation complex beta
Weakly similar to hypothetical protein MGC2577 (Homo
sapiens)
Cell differentiation
Xenopus laevis DNA replication initiator protein, DNA
replication initiation, regulation of transcription
Xenopus laevis, Similar to alpha-Tubulin at 84B, microtubulebased movement
Highly similar to hypothetical protein FLJ10900 (H.sapiens),
electron transport
Histidine catabolism
Weakly similar to ACRC protein; putative nuclear protein
(H.sapiens)
Weakly similar to CTF1_HUMAN Cardiotrophin-1 (CT-1)
(H.sapiens)
Translation initiation factor activity
Moderately similar to hypothetical protein FLJ10738
(H.sapiens); 3'-5' exonuclease activity
Highly similar to transcription factor BTF3a - (H.sapiens)
Similar to SWISNF, actin dependent regulator of chromatin,
regulation of transcription
Xenopus laevis vitelline envelope 37k glycoprotein xlZPB
RNA binding
Xenopus laevis cldn7L1 mRNA for Claudin7L1, structural
molecule activity
Weakly similar to POL2_MOUSE Retrovirus-related POL
polyprotein (M.musculus)
Xenopus laevis F-box protein (PXP17), meiosis
Similar to axotrophin; likely ortholog of mouse axotrophin
(H.sapiens), protein binding
Similar to calcium modulating ligand
Weakly similar to MIC2_HUMAN T-cell surface glycoprotein
E2 precursor (H.sapiens)
Moderately similar to ubiquitin thiolesterase (H.sapiens);
ubiquitin-dependent protein catabolism
Descriptoin/Molecular Function
Regulation of progression through cell cycle
4.36
3.61
5.91
4.14
2.94
4.26
3.59
4.21
4.44
2.67
3.60
4.11
3.75
3.61
4.94
4.41
4.69
4.19
3.29
4.10
3.72
3.81
4.46
3.90
3.22
4.78
3.33
Mean
Muell.
5.12
4.84
3.00
1.14
0.15
0.17
0.46
0.43
0.49
0.26
0.59
0.41
0.16
0.18
1.10
0.62
0.40
0.56
0.07
0.31
0.26
0.41
0.53
0.63
0.18
0.60
0.27
0.20
0.60
0.21
SD
Muell.
0.58
0.22
0.31
10.15
9.33
11.76
10.00
8.88
10.18
9.50
10.11
10.39
8.64
9.55
10.10
9.88
9.69
11.02
10.41
10.84
10.32
9.45
10.31
9.95
10.23
10.82
10.42
9.75
11.43
10.03
Mean
Hybrid
12.76
11.65
9.78
0.93
0.12
0.08
0.08
0.02
0.36
0.02
0.11
0.27
0.24
0.27
0.06
0.23
1.64
0.03
0.03
0.21
1.21
0.17
0.29
0.14
0.22
0.06
0.03
0.14
0.16
0.99
SD
Hybrid
0.10
0.03
0.23
Table 5.4. Top 30 candidate transcripts upregulated in hybrids and differentially expressed
between females of X. muelleri and hybrids. Expression values are in log2 scale; SD=standard
deviation of expression values. P values are adjusted according to FDR moderated t-tests.
-5.79
-5.72
-5.85
-5.86
-5.94
-5.92
-5.91
-5.90
-5.95
-5.97
-5.96
-5.99
-6.13
-6.08
-6.08
-5.99
-6.15
-6.13
-6.16
-6.22
-6.24
-6.43
-6.36
-6.51
-6.53
-6.65
-6.70
M-H
-7.64
-6.80
-6.79
0.0012
0.0001
0.0001
0.0002
0.0001
0.0002
0.0001
0.0002
0.0002
0.0001
0.0001
0.0007
0.0002
0.0008
0.0002
0.0001
0.0001
0.0004
0.0001
0.0002
0.0002
0.0001
0.0002
0.0001
0.0001
0.0002
0.0002
P value
0.0001
0.0001
0.0001
128
X. laeivs vs.
X. muelleri
753
62
839
L=M
Total
24
L<M
L>M
L<H
128
777
86
65
626
L>H
X. laevis vs. Hybrids
2930
267
9
2654
M<H
4349
328
3997
24
M>H
X. muelleri vs. Hybrids
Table 5.5. Comparison in the overlap of transcripts recovered as differentially
expressed from the two contrasts with hybrids (Xenopus laevis vs. hybrids and
X. muelleri vs. hybrids) and the interspecies contrast (Xenopus laevis vs. X. muelleri).
The congruence between patterns of expression behavior in hybrids compared to
the interspecies comparison suggests a model of semidominance where hybrids have
an intermediate level of expression compared to the two parental species.
5.5 Discussion
Our analysis of hybrid females relative to the two parental species provides key
insight into the process of oogenesis in hybrid females and the two parental species.
There is an asymmetrical pattern of gene misexpression with about 4.5 times more
genes differentially expressed between hybrids and Xenopus muelleri compared to X.
laevis. This result implies that strong maternal and/or species dominance effects act in
oogenesis and these are reflected in the hybrid transcriptome. Hybrid females have a
general pattern of semidominance in gene expression with the majority of genes being
expressed at intermediate levels compared to the two parental species. Finally, there is a
dramatic divergence in gene expression between the two parental species with more
than 76% of genes differentially expressed between X. laevis and X. muelleri. This
suggests that the process of oogenesis differs widely at the gene expression level
between these two species of Xenopus.
It is important to consider the methodology used to gather the samples of RNA
for this study. Samples of ovary (50 mg portions) were dissected and then homogenized
in RNA extraction solution. Therefore, we gathered a sample of ovary rather than the
entire ovary and this sample is a heterogeneous representation of oogenesis, rather than
a direct assessment of specific stages of oocyte development. Given the heterogeneous
nature of the tissue used to gather RNA, it is even more surprising that we found such
strong effects. Increased heterogeneity among samples would decrease the ability to
reject the null hypothesis that gene expression for a particular give is the same between
hybrids and conspecifics. Increased heterogeneity among samples would increase the
129
standard error and thereby decrease power to reject the null hypothesis. In fact though,
even despite the heterogeneous nature of the samples collected, we still reject a large
portion of null hypotheses suggesting that our microarray results represent real
biological effects, rather than statistical artifacts. Additionally, our results remain robust
even when using different normalization techniques (scaling and RMA) providing
further confidence that our results are not statistical artifacts (not shown).
The top 30 candidate genes for the contrasts between hybrids and the two
parental species provide many genes with known roles in mitosis, meiosis, and
oogenesis in general (Table 5.1-5.4). One EST, MGC132176, is predicted to have heme
oxygenase activity and this EST was upregulated 12 times in X. laevis relative to
hybrids. Heme oxygenase plays a role in regulating ovarian steriodgenesis in rats and
our results suggest this may be in the case in Xenopus (Alexandreanu and Lawson,
2003). Of the genes with known function, many have been documented to play a role in
oogenesis. For example, the proliferation associated protein PA2G4, which was
upregulated in X. laevis about 8 times higher than in hybrids, has been previously
isolated from Xenopus oocytes and is believed to play an important role in DNA
replication and cell cycle progression (Izuta et al., 2004). One EST that is similar to the
human transcription factor Hairy and enhancer of split 2, was upregulated 18 times in
hybrids relative to X. laevis, and is known to be regulated by reproductive hormones in
adult rat ovary (Yamada et al., 2004). Neurotrophin receptor B (Trkb-b) was
upregulated 5 times in hybrids relative to X. laevis and plays a critical role in ovulation,
steroid secretion, and follicular development in the ovary of rodents and humans
130
(Waraksa et al., 1995; Dissen et al., 1996a; 1996b; Mayerhofer et al., 1996; Seifer et al.,
2006).
Examining candidate gene lists for the Xenopus muelleri vs. hybrid comparison
also reveal many genes involved in oogenesis. For example two bone morphogenetic
proteins, Bmp7 and Bmp2, are 17 and 11 times respectively upregulated in X. muelleri
relative to hybrid females. Bone morphogenetic proteins are part of a class of proteins
involved in the development and patterning of the adult ovary (Knight and Glister,
2006; Von Schalburg et al., 2006). Xcat-2 was upregulated 17 times more in X. muelleri
relative to hybrids and is involved in the formation of germ plasm during stage I
oocytes of Xenopus (Kloc and Etkin, 1995; Zhou and King, 1996). Another gene of
interest is LAP2, upregulated 14 times higher in X. muelleri compared to hybrids and
specific isoforms of LAP2 are expressed exclusively in the ovary of anurans and
salamanders (Lang et al., 1999; del Pino et al., 2002; Brown et al., 2002). Cyclin A1, the
most divergently expressed gene, was upregulated nearly 200 times higher in hybrids
relative to X. muelleri and expressed the same in X. laevis and hybrids. Cyclin A1 plays
a major role in mammalian gametogenesis and meiosis (Kierszenbaum, 2006) and is a
partner of Cdk2, a key gene involved in the cell cycle both in mitosis and meiosis.
Female and male knockout Cdk2 mice are viable but both infertile (Berthet et al., 2003).
Curiously, disruption of Cyclin A1 expression results in male infertility but not female
infertility and specifically causes the developmental arrest of spermatogenesis during
meiosis I (Liu et al., 1998; Persson et al., 2005). A vitelline envelope glycoprotein, lzpba, was upregulated 75 times in hybrids relative to X. muelleri, and vitelline envelope
131
proteins have an obvious role in the formation of oocytes during amphibian oogenesis
(Kubo et al., 2000). Finally, Germes, a gene that localizes to the germ plasm during
early oogenesis in Xenopus was upregulated 62 times higher in hybrids compared to X.
muelleri (Berekelya et al., 2003).
Perhaps the surprising result of our analyses is that hybrid females are fertile yet
have a dramatic increase in gene misexpression compared to hybrid males which are
completely sterile (Malone et al., 2007). These results would seem to contradict what
we might intuitively predict; specifically that it seems reasonable to assume that normal
phenotypes should have greater similarity in expression profiles and perturbed
phenotypes should have greater divergence in expression. Hybrid males, which are
completely sterile, have only 56 genes uniquely misexpressed compared to both
parental species whereas hybrid females, which are fertile, have nearly 20 times more
genes misexpressed (1,105). However, these results are consistent with patterns of sexbiased gene expression in which female-biased genes were found to be more
divergently expressed between species compared to male-biased genes (Malone et al.,
2006) providing further evidence that this pattern represents real biological effects.
Thus, we are left with a question, how can the process of oogenesis tolerate such
dramatic differences in the level of gene expression, yet still produce fertile eggs,
whereas the process of spermatogenesis in hybrid males has relatively few genes
misexpressed yet results in complete sterility.
To date, there has been little exploration of this question because studies of gene
expression and reproductive isolation have focused on the sterility phenotype which
132
typically involves males. However hybrid females of Drosophila melanogaster and D.
simulans , which are sterile, have been analyzed and hybrids had a majority of genes
misexpressed compared to the two species (Ranz et al., 2004). Males of this
interspecific cross are inviable and therefore a comparison cannot be made to hybrid
males. Recent work has shown that critical genes involved in mammalian female
reproduction undergo rapid diversification due to retrotransposed genes in Mus
musculus and these results may provide a clue to the divergent expression pattern
occurring in females of Xenopus which could be a general pattern of female
reproduction (Evsikov et al., 2006).
Xenopus do not conform to a fundamental generalization in evolutionary
biology-Haldane’s rule (Chapter 1; Kobel, 1996). Patterns of sex-biased gene
expression and sex-reversal experiments suggest that the spermatogenesis is special
component of the faster male evolution hypothesis (Wu and Davis, 1993) is the best
explanation for sterile males in Xenopus even though females are the heterogametic sex
(Chapter 6). Given the divergent pattern of expression in hybrids and females between
species, we suggest the following scenario to explain why spermatogenesis can be a
special mechanism generating hybrid male sterility.
First, oogenesis relies on a staggering amount of gene expression with up to
45% of all mouse genes and 55% of all Drosophila genes expressed in the mature
oocyte (Evsikov et al., 2006; Tadros et al., 2007) and additionally this abundant
transcription results in maternally deposited RNAs and proteins which foster oocyte
growth and early development (Seydoux, 1996; Evsikov et al., 2006). In particular,
133
much of this RNA deposition functions to localize coding and non-coding RNAs
essential to germ cell development into a distinct subcellular domain that can be moved
into the vegetal cortex of the oocyte. Interestingly, RNAs localized in the germ plasm
may not be translated for years highlighting the importance of the germ plasm as a
storage unit for RNA and furthermore many of these stored RNAs are involved in
translational regulation of germ cell specific expression (MacArthur et al, 1999;
Houston and King, 2000a, 2000b; King et al., 2005).
We find dramatic differences in gene expression between females of two species
of Xenopus and many of the most dramatic differences have to do with Early/METRO
pathway (e.g. Germes, Fatvg, Cyclin A1) of germ plasm specification (King et al.,
2005). These dramatic, sometimes 200 times different, RNA abundance levels indicate
a major difference in the amount of key genes involved in germ plasm specification and
maternally loaded RNAs between species. This result would suggest that each species
has a divergent way of completing oogenesis with regard to gene expression.
During fertilization, sperm fertilize an egg and this starts the dramatic changes
that turn the mature oocyte into a functioning zygote (Stitzel and Seydoux, 2007). Each
sperm delivers a haploid genome along with mature RNA that initiates and directs
subsequent development (Ostermeier et al., 2004). The interaction between sperm and
the genome it delivers to the egg and stored RNA must coevolve in such a way to
ensure successful development. Consider now sperm from a different species, adapted
for fertilizing eggs of its conspecific species, that now successfully fertilizes an embryo
from a different species. As our data suggest, the paternal genome will now interact
134
with a radically different embryo with drastically different amounts of stored maternal
RNAs. We therefore suggest the possibility that disruption of spermatogenesis in adult
hybrid males occurs because of radically divergent expression in females during
oogenesis. Oocytes armed with pools of RNA adapted for one species, work in the
sense that they can be fertilized and develop but, the initial differences in maternally
stored RNAs generate subsequent dysfunctions in males because molecular interactions
that generate the adult testis and subsequent spermatogenesis are misregulated due to
the differences in maternally stored RNA populations. Spermatogenesis is special in the
sense that during early development key factors fail to interact properly to generate a
normally functioning testis.
Several genes from our microarray results suggest directions by which this
hypothesis could be tested. One example is Cyclin A1 which was upregulated about 200
times more in hybrids and 170 times more in X. laevis compared to X. muelleri.
Knocking out Cyclin A1 in mice causes completely sterility and the interaction between
Cyclin A1 and cdk2 is crucial for normal development (Liu et al., 1998; Kierszenbaum,
2006). Cyclin A1 is also known to be maternally deposited in Xenopus and regulates the
progression of the cell cycle and apoptosis (Carter et al., 2006). Our hypothesis suggests
that factors like Cyclin A1 which are loaded into embryos in drastically different
amounts may play a role in misdirecting the development of the hybrid testis.
Our work provides an important first glimpse into the expression pattern of
hybrid females and parental species. We find an asymmetrical expression pattern
similar to the pattern of expression in hybrid male Xenopus and allotetraploid
135
Arabidopsis (Malone et al., 2007; Wang et al., 2006). However, hybrid females have a
dramatic increase in the number of misexpressed genes compared to sterile males and
we suggest that this gene expression divergence plays a role in hybrid male sterility.
Our results call for attention as to how divergent expression in females plays a role in
reproductive isolation between species.
136
CHAPTER 6
A TEST OF THE FASTER MALE EVOLUTION HYPOTHESIS FOR HALDANE’S
RULE USING SEX REVERSED HYBRIDS OF XENOPUS
6.1 Abstract
One of the defining patterns of reproductive isolation is Haldane’s rule where
the heterogametic sex (XY or ZW) suffers the most dysfunctional effects of
interspecific hybridization. Xenopus have ZW sex determination yet in crosses across
all species there is no obvious inviability and hybrid males are completely sterile
whereas hybrid females are fertile; a pattern exactly opposite to Haldane’s rule. One
compelling hypothesis to explain this pattern is faster male evolution. Faster male
evolution states that spermatogenesis is a sensitive process that is easily perturbed in a
hybrid genetic background and/or sexual selection on male expressed genes drives them
to evolve faster than female expressed genes. A powerful experiment to test the faster
male evolution hypothesis is to create sexreversed individuals, that is, individuals that
have the genotype of one sex and the phenotype of the opposite sex. We used the novel
ability in Xenopus to create sexreversed hybrids combined with microarray expression
data to provide a powerful new test of faster male evolution. The phenotype of sexreversed individuals is exactly the same as normal individuals; hybrid males are sterile
and hybrid females are fertile irrespective of their genetic background. Gene expression
137
profiles match these adult phenotypes in that no genes were detected to be differentially
expressed. Our results suggest that faster male evolution and in particular, the sensitive
spermatogenesis component, is the most likely mechanism operating to produce
patterns of reproductive isolation in Xenopus.
6.2 Introduction
Haldane’s rule remains one of the few generalizations of evolutionary biology
(Haldane, 1922; Coyne and Orr, 2004). Generally, and across a variety of taxa (e.g.
Drosophila, birds, butterflies, mammals), the heterogametic sex (ZW or XY) suffers the
most dysfunctional effects of interspecific hybridization. The ubiquity of Haldane’s rule
is particularly significant because it implies that common mechanisms are responsible
for the process of isolating and maintaining distinct biological species. Understanding
Haldane’s rule then provides key insight into a fundamental problem in evolutionary
biology—the origin of new species.
Intensive study in the past two decades has nearly solved the general
mechanisms operating to produce Haldane’s rule (Wu and Davis, 1993; Wu et al., 1996;
Laurie, 1997; Orr, 1997; Coyne and Orr, 2004). What is needed now is an examination
of cases that consistently fail Haldane’s rule in order to evaluate the proposed
explanations.
Frogs of the genus Xenopus provide a model system to address this problem.
Female Xenopus have heterogametic sex chromosomes (ZW) and males are
homogametic (ZZ) yet in over 100 crosses from across clades of the more than 20
species, males are sterile and females are fertile (Kobel et al., 1981; Picker, 1985; Kobel
138
and Du Pasquier, 1986; Picker and de Villiers, 1989; Kobel, 1996; Kobel et al., 1996;
Picker et al., 1996; Fischer et al., 2000); a pattern exactly opposite of Haldane’s rule.
Perhaps most remarkable is the ancient age of the two main clades of Xenopus (53-64
mya) yet the absence of hybrid inviability (Kobel, 1996; Evans et al., 2004; 2005; Chain
and Evans, 2006).
Only one of the two leading hypotheses for Haldane’s rule can explain this
pattern. Dominance effects, which appear to be the most likely mechanism in other
organisms with ZW sex determination (birds and butterflies; Presgraves, 2002; Price
and Bouvier, 2002), cannot explain the case for Xenopus because the heterogametic sex,
in this case females, should be affected by all Z-linked recessive alleles that cause
hybrid dysfunction. This is not the case because males are sterile and females are fertile.
Faster male evolution is a compelling hypothesis to explain this pattern (Wu and
Davis, 1993; Wu et al., 1996). Fastermale evolution suggests that either 1)
spermatogenesis may be a sensitive process that is easily perturbed in hybrids or 2)
sexual selection on male-expressed genes drives them to evolve faster than femaleexpressed genes thereby leading to hybrid male defects. This would provide an
explanation for the sterility pattern in Xenopus and more generally, shed light on the
mechanisms of faster male evolution creating hybrid male sterility.
The ultimate test of faster-male evolution that would dissect sex chromosome
effects from physiological sex effects would be to create sex-reversed hybrids; genetic
males (Z1Z2-where subscripts denote the origin of chromosomes from either species 1
or species 2) that are phenotypically female and genetic females (Z1 W2) that are
139
phenotypically male. If genotypic females sex-reversed to males are fertile and
genotypic males converted to females are sterile, this would suggest sex chromosome
effects override the physiological phenotype providing little evidence for the
spermatogenesis is special component of faster male evolution. If genotypic females
sex-reversed to males are sterile, while genotypic males converted to females are fertile,
then this would suggest that male sterility is completely determined by the physiological
male phenotype. Such a result would be consistent with the faster male evolution
hypothesis, particularly the sensitive spermatogenesis component. The other two
possibilities, both sex-reversed males and females sterile or both fertile, would also
imply that sterility is independent of sex chromosomes and overwhelmed by,
respectively, physiological “maleness” or “femaleness”. Examining the ensuing fertility
pattern of sex-reversed compared to normal hybrids and coupling these results with
profiles of gene expression in the gonads provides a unique way to understand the
contribution of genes related to spermatogenesis on hybrid sterility.
6.3 Materials and Methods
6.3.1 Crosses and Sex-reversal Treatment
Crosses were made to produce clutches of F1 X. laevis x X. muelleri hybrids
used for the sex-reversal experiment. Female X. laevis were purchased from Xenopus
Express and were derived from the Cape region of South Africa. Male X. muelleri were
wild caught from Swaziland and provided by R. C. Tinsley. Pairs were injected with
hCG hormone and placed in 10 L of artificial pond water. Artificial pond water was
produced by creating one stock solution (A) with 1.50 M NaCl and 0.16 M CaCl2, and
140
another stock solution (B) of 0.03 M NaHCO3. To make 10 L of artificial pond water,
40 ml of stock solution A and 40 ml of stock solution B was combined with 10 L of
water. Fertilized eggs were separated and then raised to sexual maturity.
Portions of two clutches were subjected to sex-reversal treatment (Fig. 6.1).
Tadpoles were raised in 54 L of de-chlorinated water and to produce sex-reversed
females we added estradiol (50 µg/L) to the water during the third week of development
(Nieuwekoop and Faber tadpole stages 52-55; Nieuwkopp and Faber, 1956) and daily
until metamorphosis was complete (Chang and Witschi, 1956). Sex-reversed hybrid
males were produced by gonadal implantation using testis of X. laevis and following the
methods of Mikamo and Witschi (1963) and Watson and Kelley (1992).
Z2W1
Z1Z2
Sex Reversal
Z2W1
Z1Z2
Z2W1
Z1Z2
Sterile or Fertile?
All Female Phenotype
All Male Phenotype
Figure 6.1. Flowchart of one replicate of the sex-reversal experiment.
141
To distinguish ZW vs. ZZ genotypes in sex-reversed individuals we used a W
chromosome marker kindly supplied by Dr. Michiko Ito and based on Yoshimoto et al.
(2006). DNA was extracted from toe clips from adult sex-reversed frogs using the
QIAGEN DNAeasy kit. Multiplex PCR amplification with W and Z chromosome
specific primers was performed and provided unambiguous assignment of ZW or ZZ
individuals (Fig. 6.2). Thermocycling conditions consisted of 94°C for 5 minutes,
followed by 30 cycles of 94°C denaturation, 55°C annealing, and 72°C extension.
Figure 6.2. A sample of unambiguous genotype determination of sex-reversed
individuals. Double bands indicate the presence of W specific marker as
amplified from PCR reactions and indicated ZW genotype. Single band
represents Z chromosome marked and is amplified only in ZZ genotypes. λ is
100 bp DNA ladder. Neg=Negative control.
142
6.3.2 Test Crosses
We performed test crosses to determine the fertility pattern of sex-reversed
compared to normal hybrids. Samples of normal and sex-reversed individuals were
injected with hCG and then paired with an opposite sex X. laevis in 10 L of artificial
pond water. Samples of eggs were examined for fertilization efficiency and the total
number of fertilized and unfertilized embryos were counted to determine fertilization
success. For phenotypic female hybrids we also counted the total number of eggs
deposited after mating to produce an estimate of female fecundity. The number of
individuals that hatched in the sample of fertilized embryos and the number of
individuals that reached the swimming tadpole stage were also counted to produce
estimates of hatching success. Statistical analyses were performed using a one-way
ANOVA in R software between sexreversed and normal hybrids to determine
differences in the fertility pattern in sexreversed individuals.
6.3.3 RNA Extraction and Microarray Analysis
RNA was extracted from adult ovary (n=4; 2/hybrid clutch) and testis (n=3; two
individuals from one clutch and one individual from the second clutch) from sexreversed hybrids. For normal hybrids, we extracted ovary (n=4) and testis (n=3) from
sexually mature individuals produced from separate crosses that were not subject to sexreversal treatment. Adults were euthanized with MS-222 and gonads were immediately
removed, placed in RNA extraction solution, and homogenized. RNA was recovered
using Ambion RiboPure total RNA kits. Samples of RNA were checked for purity by
examination of the 28S and 18S ribosomal RNA bands from denaturing gel
143
electrophoresis, by 260/280 ratios from scans with a Nanodrop ND 1000
spectrophotometer, and by readouts of the Agilent Bioanalyzer. Total RNA samples
were prepared and hybridized to Affymetrix Xenopus laevis GeneChip Genome Arrays
at The University of Texas at Arlington following standard Affymetrix protocols.
Affymetrix GeneChip Operating Software (GCOS) was used to scan and process each
microarray chip. The signals of quality control and poly(A) transcripts revealed that
hybridizations were of high quality in all chips.
To control for the effect of sequence divergence we used the electronic mask
found in Malone et al. (2007). This mask, which is derived from hybridizing genomic
DNA from X. laevis and X. muelleri removes genes that have high sequence divergence
between species and provides 11,485 probesets/genes available for analysis.
Hierarchical clustering of expression data was performed using the made4
package in R software (Culhane et al., 2005). Separate comparisons were made for
phenotypic females and phenotypic males. First, microarray hybridizations of ZW and
ZZ individuals of a particular phenotypic sex were normalized using default parameters
in RMAexpress (Bolstad, 2005). These RMA normalized expression data were then
log2 transformed and each of the 11,485 genes were tested for differences in expression.
We used a moderated t-statistic based on an empirical Bayes method in the limma
package found in Bioconductor (Smyth, 2004). The TopTable function was then used to
output FDR-adjusted P-values for differential expression. Not a single gene was found
to be differentially expressed in either ZW vs. ZZ females or ZW vs. ZZ males.
144
6.4 Results
The proportion of fertilized eggs, and proportion of eggs that hatched into
tadpoles was similar in normal compared to sex-reversed females. There was also no
difference in the number of eggs laid during mating (Table 6.1). Normal hybrid males
(n=15) and sexreversed males (n=3) failed to fertilize any eggs and therefore both types
of males are completely sterile. These fertility data show that phenotypically, sexreversed males and sex-reversed females have the same phenotype as normal
individuals even despite having an inverse genetic background.
Table 6.1. Fertility results from test crosses of ZW and ZZ
individuals from the same phenotypic sex. ZW and ZZ females
have the same fertility characteristics and both ZW and ZZ
phenotypic males are completely sterile.
% Eggs
Fertilized and
Dividing
Phenotype
Phenotypic
Females
Total Number of
Eggs Deposited
Genotype
ZW
Mean
0.52
SD
0.27
Mean
0.51
SD
0.34
Mean
2088.7
SD
1390.9
ZZ
0.52
0.28
0.56
0.29
1388.3
1151.5
ANOVA (ZW vs. ZZ)
ZW
Phenotypic
Males
% of Fertilized
Eggs that Hatched
into Tadpoles
ZZ
F 1,15 = 0.0004;
P=0.995
0
0
-
145
F 1,14 = 0.1013;
P=0.756
-
F 1,14 = 1.22;
P=0.289
-
-
-
Hierarchical clustering of the expression data shows the striking similarity in
normal and sex-reversed individuals (Fig. 6.3). The expression profiles are so similar in
fact that no evidence of differential expression for any genes can be detected in
comparisons of normal and sex-reversed females or normal and sex-reversed males.
These expression data match the overall fertility pattern as sex-reversed individuals
have the same phenotype as their normal counterparts.
6.5 Discussion
These results suggest that W chromosome in Xenopus bears few if any
additional genes expressed in adult gonads and consists mainly of loci controlling the
master regulatory switch that determines physiological sex. Overriding this mechanism
PHENOTYPIC FEMALES
laevis x muelleri SR♂
laevis x muelleri ♂
laevis x muelleri ♂
laevis x muelleri SR♂
laevis x muelleri SR♂
laevis x muelleri ♂
laevis x muelleri SR♀
laevis x muelleri SR♀
laevis x muelleri ♀
laevis x muelleri ♀
laevis x muelleri ♀
laevis x muelleri ♀
laevis x muelleri SR♀
laevis x muelleri SR♀
in hybrids through sex-reversal treatment provides no change in phenotype or
PHENOTYPIC MALES
Figure 6.3. Hierarchical clustering of gene expression data for normal and sexreversed hybrids of Xenopus laevis x X. muelleri. FDR corrected moderated t
tests reveal no difference in gene expression between normal and sex-reversed
females and normal and sex-reversed males. ♀=normal female (ZW); ♂=
normal male (ZZ); SR ♀ = sex-reversed female (ZZ); SR ♂ = sex-reversed male
(ZW).
146
expression pattern in the adult gonad and therefore does little to contribute to Haldane’s
rule in these frogs.
The best explanation for why these organisms fail Haldane’s rule is the faster
male evolution hypothesis. First, there is a match between the fertility pattern and
expression profiles of normal and sex-reversed individuals. Hybrid males, both normal
and sexreversed, are sterile whereas both types of hybrid females are fertile.
Physiological maleness appears to be the most important predictor of sterility rather
than chromosomal background and additionally implies that gene dosage effects (Zhang
and Oliver, 2007) are not important to the physiological processes involved in
oogenesis and spermatogenesis (Hayes, 1998). Given the results from the sex-reversal
experiment it is difficult to separate the two components of faster male evolution, that is
whether sterility is about the sensitivity of spermatogenesis and/or sexual selection on
male expressed genes because both genetic backgrounds yield the same phenotype.
However, there are numerically more genes involved in spermatogenesis in both X.
laevis and X. muelleri (Malone et al., 2006) and these more numerous genes would be
consistent with the idea that spermatogenesis is more easily perturbed in hybrid males
compared to hybrid females. Interestingly though, the divergence in gene expression
between species is higher for female compared to male-expressed genes (Malone et al.,
2006), suggesting that female expressed genes diverge faster between species compared
to male expressed genes. This would be inconsistent with the idea that strong sexual
selection on male expressed genes causes them to divergence faster leading to hybrid
male
sterility.
Overall then the preponderance of evidence
147
suggests that
spermatogenesis is a sensitive process due to the more numerous genes involved and is
easily perturbed in a hybrid genetic background causing hybrid male, rather than hybrid
female sterility. The interesting role of female gene expression divergence and the fact
that males are sterile and females are fertile suggests that organisms with ZW sex
determination may posses intriguing mechanisms operating to produce patterns of
postzygotic reproductive isolation.
148
APPENDIX A
SUPPLEMENTAL TABLES FOR CHAPTER 1
149
150
A.
X. laevis RMA
1120
108
1244
43.03
1
87
971
33.6
U
Totals
%
16
883
M
F
M
F
23.4
676
455
90
131
U
2891
650
1211
1030
Totals
X. laevis Global Scaling
22.5
41.9
35.63
%
150
B.
%
Totals
U
M
F
31.1
900
154
35.4
1023
99
918
6
745
0
M
F
33.5
968
674
192
102
U
2891
927
1110
854
Totals
X. muelleri Global Scaling
Table A.1. The classification of male-biased (M), female-biased (F), and
unbiased (U) gene expression behavior based on LPE tests from Global Scaling and
RMA normalization for X. laevis (A) and X. muelleri (B). Note that the percentage of
each class of expression behavior is extremely similar between the two normalization methods.
X. muelleri RMA
32.1
38.4
29.5
%
151
392
2045
2437
84.3
146
308
454
15.7
N
Y
Total
%
151
Y
N
2891
2353
538
Total
81.4
18.6
%
Species Effect Global Scaling
Table A.2. The classification of genes that were expressed the same between
species (N) and those that were differentially expressed between species (Y) based on LPE
tests for the Global Scaling and RMA normalization datasets.
Species Effect RMA
152
24
155
222
432
14.9
69
99
54
290
10.0
>2 ≤ 3
<2
Total
%
30
68
>3 ≤ 4
>4
>3 ≤ 4
>4
152
25.6
735
538
126
47
25
>2 ≤ 3
49.6
1433
1238
162
24
9
<2
2891
2052
543
164
132
Total
Species Fold Change Global Scaling
71.0
18.8
5.7
4.6
%
Table A.3. The classification of genes that were expressed the same between
species (N) and those that were differentially expressed between species (Y) based on LPE
tests for the Global Scaling and RMA normalization datasets.
Species Fold Change RMA
153
286.97
Xl.26159.2.A1_at
Xl.7966.1.A1_at
273.07
135.31
Xl.2790.1.S1_at
Xl.2509.1.S1_a_at
149.67
Xl.23787.3.S1_at
298.17
147.53
Xl.18853.1.A1_at
315.28
46.34
Xl.12093.1.S1_at
Xl.22727.1.S1_a_
at
131.12
Xl.3444.1.S1_at
Xl.25717.1.A1_at
79.70
458.30
Xl.7046.1.S1_at
231.39
Xl.1169.1.S1_at
212.31
Xl.637.1.A1_at
33.54
366.78
Xl.3362.1.S1_at
459.43
174.84
Xl.12560.1.S1_at
Xl.12112.1.S1_at
146.71
Xl.5747.1.S1_at
Xl.12778.1.A1_at
237.63
374.12
395.59
333.82
Xl.25073.1.S1_at
Xl.220.1.S1_at
Xl.25553.1.S1_at
78.71
Xl.3257.1.S1_at
Xl.1095.1.S1_at
81.84
218.97
Xl.5846.10.S1_at
Mean
Muelleri
Xl.848.1.S1_at
Affymetrix
Probeset ID
1157.21
99.39
1233.06
91.55
548.48
767.70
587.40
212.41
647.95
114.43
370.37
1471.40
107.68
185.79
34.41
74.88
1319.04
938.66
3222.20
3341.75
3634.08
5669.93
691.68
4732.36
2119.83
Mean
Laevis
365.12
713.46
361.33
562.26
157.97
217.48
174.96
68.54
178.87
1012.39
110.38
342.63
1031.94
48.64
313.33
739.34
263.89
175.25
536.34
586.10
534.03
895.76
90.41
281.19
119.72
Mean
Muelleri
Male
1813.33
140.96
1899.17
106.28
837.35
1239.58
997.47
403.81
1075.14
166.63
685.17
2367.63
148.19
351.44
43.31
101.94
1997.06
1383.56
4751.43
6658.67
7030.15
11314.98
1351.34
5946.35
2633.36
Mean Laevis
Male
Male Biased in Both Species for Genes Differentially Expressed between Species
2.31
2.34
2.39
2.40
2.41
2.51
2.51
2.56
2.59
2.60
2.63
2.79
2.80
2.85
2.85
2.86
2.92
2.98
3.15
3.51
3.72
3.66
3.90
4.40
4.46
153
Abslog2
Sex Fold
Change
ESTs, Moderately similar to VATD_HUMAN Vacuolar ATP synthase subunit D (V-ATPase D
subunit) (Vacuolar proton pump D subunit) (V-ATPase 28 kDa accessory protein) (H.sapiens)
ESTs, Moderately similar to POR3_HUMAN Voltage-dependent anion-selective channel protein 3
(VDAC-3) (hVDAC3) (Outer mitochondrial membrane protein porin 3) (H.sapiens)
ESTs
ESTs, Weakly similar to NCR2_HUMAN Nuclear
receptor co-repressor 2 (N-CoR2) (Silencing mediator of retinoic acid and thyroid hormone receptor)
(SMRT) (SMRTe) (Thyroid-, retinoic-acid-receptor-associated co-repressor) (T3 receptor-associating
factor) (TRAC) (CTG26) (H.sapiens)
Xenopus laevis clone S10-57-E10 mRNA sequence
ESTs
ESTs, Moderately similar to AQHUP annexin V - human
ESTs, Weakly similar to hypothetical protein MGC16186 (Homo sapiens)
Xenopus laevis Xtr gene for tudor protein, complete cds.
ESTs, Weakly similar to hypothetical protein MGC3037 (Homo sapiens)
ESTs, Moderately similar to cytochrome c (Homo sapiens) \
X.laevis mRNA for elongation factor-1 delta
Xenopus laevis mRNA for G-protein alpha subunit (g(alpha)sx2
ESTs
Xenopus laevis early growth response protein mRNA, complete cds.
Xenopus laevis, Similar to troponin T3, skeletal, fast,
Xenopus laevis, Similar to succinate dehydrogenase complex, subunit B, iron sulfur (Ip),
Xenopus laevis, clone MGC:53076 IMAGE:5542078, mRNA, complete cds
X.laevis mRNA homologous to rat ribosomal protein S27
Sperm-specific basic nuclear protein 5 (Xenopus laevis, mRNA, 501 nt)
Xenopus laevis, similar to actin alpha
Weakly similar to X-prolyl aminopeptidase 2, membrane-bound; aminoacylproline aminopeptidase;
aminopeptidase P; X-prolyl aminopeptidase 2 (aminopeptidase P) (Homo sapiens)
Weakly similar to hypothetical protein FLJ13263 (Homo sapiens)
Xenopus laevis mRNA for sperm-specific basic nuclear protein
Xenopus laevis, Similar to ribosomal protein L5
Target Description
Table A.4. Top 25 candidate transcripts as recovered using RMA normalization
and LPE tests for different classes of sex and non sex-biased genes. Transcripts are ordered
from the highest to lowest absolute value of the log2 FC.
154
87.29
119.87
239.97
Xl.15148.2.A1_at
Xl.7723.1.S1_at
Xl.8175.1.S1_at
558.57
211.17
161.05
Xl.25936.1.A1_at
Xl.22643.3.S1_at
324.28
Xl.8327.1.S1_at
Xl.337.3.S1_at
226.74
Xl.2877.1.A1_at
Xl.16888.1.A1_at
67.55
124.42
Xl.23370.1.S1_at
116.32
225.35
Xl.25748.1.S1_at
Xl.6982.1.A1_at
196.22
Xl.8592.1.S1_at
Xl.25377.1.S1_at
315.55
Xl.489.1.S1_at
131.89
174.02
Xl.8388.1.S1_at
1063.08
208.23
Xl.1739.1.S1_at
Xl.8049.1.S1_a_at
323.70
Xl.13115.1.S1_at
Xl.26114.1.A1_at
220.64
434.55
Xl.24736.1.S1_at
128.57
181.21
Xl.12858.1.A1_at
172.25
Xl.715.2.A1_x_at
185.88
Xl.4515.1.S1_at
Mean
Muelleri
Female
Xl.7933.1.S1_at
Affymetrix
Probeset ID
277.10
111.07
104.36
182.44
677.16
177.80
337.21
259.60
78.88
128.34
950.78
114.23
120.51
271.89
248.09
339.04
178.92
223.74
388.83
502.57
226.13
161.97
144.37
155.22
207.87
Mean
Muelleri
Male
593.48
299.41
34.83
81.87
1480.90
507.66
905.68
578.97
222.22
289.25
258.20
426.60
40.28
795.38
649.32
1144.23
50.12
649.54
1182.57
1821.79
958.56
38.86
618.95
1817.13
2589.56
Mean Laevis
Female
716.15
308.67
36.43
72.40
1725.61
399.88
885.84
735.07
175.57
398.41
451.65
324.40
41.40
699.97
714.07
936.11
58.80
781.35
1153.70
1985.07
1033.98
34.24
725.12
1615.14
2915.18
Mean
Laevis
Male
Unbiased in Both Species for Genes Differentially Expressed between Species
Table A.4-continued.
1.38
1.38
1.38
1.39
1.42
1.45
1.45
1.47
1.48
1.51
1.53
1.58
1.59
1.63
1.67
1.69
1.69
1.75
1.76
2.06
2.16
2.26
2.33
3.36
3.83
Abslog2
Species
Fold
Change
154
ESTs, Moderately similar to dpy-30-like protein (Homo sapiens)
Xenopus laevis, clone MGC:53437 IMAGE:5571896, mRNA, complete cds.
ESTs, Weakly similar to F35D11.4.p (Caenorhabditis elegans) (C.elegans)
ESTs, Weakly similar to KIAA0494 gene product (Homo sapiens)
Xenopus laevis cytoplasmic myosin II regulatory light chain mRNA, complete cds
ESTs, Highly similar to O-linked GlcNAc transferase; O-GlcNAc transferase (uridine diphospho-Nacetylglucosamine:polypeptide beta-N-acetylglucosaminyl transferase); uridine diphospho-Nacetylglucosamine:polypeptide beta-N-acetylglucosaminyl transferase (Homo sapiens)
Xenopus laevis cDNA clone 5O8
ESTs, Moderately similar to TXNL_HUMAN Thioredoxin-like protein (32 kDa thioredoxin-related protein) (H.sapiens)
ESTs, Weakly similar to EAF1 protein (Homo sapiens)
Xenopus laevis, Similar to VAMP (vesicle-associated membrane protein)-associated protein A, 33kDa, clone MGC:53868
IMAGE:5542990, mRNA, complete cds
ESTs, Moderately similar to OM07_HUMAN Probable mitochondrial import receptor subunit TOM7
homolog(Translocase of outer membrane 7 kDa subunit homolog) (Protein AD-014) (H.sapiens)
ESTs
ESTs, Moderately similar to three-PDZ containing protein similar to C. elegans PAR3 (partiti; three-PDZ containing
protein similar to C. elegans PAR3 (partitioning defect); par-3 (partitioning defective 3, C.elegans) homolog (Homo
sapiens)
Xenopus laevis, clone IMAGE:5543627, mRNA
ESTs, Weakly similar to COXB_HUMAN Cytochrome c oxidase polypeptide Vb, mitochondrial precursor (H.sapiens)
Xenopus laevis, clone MGC:53388 IMAGE:5571478, mRNA,
Xenopus laevis exonuclease ExoI (EXOI) mRNA, complete cds.
Xenopus laevis, similar to Ubiquinol-cytochrome C reductase complex, clone MGC:53943 IMAGE:5570350, mRNA,
complete cds.
Xenopus laevis Xe-p9 mRNA, complete cds
Xenopus laevis glutathione s-transferase mRNA, complete cds
ESTs, Moderately similar to hypothetical protein MGC2594 (Homo sapiens)
ESTs, Weakly similar to hypothetical protein MGC12992 (Homo sapiens)
Xenopus laevis Rho GTPase Cdc42 mRNA, complete cds
Xenopus laevis, Similar to cold inducible RNA-binding protein, mRNA, complete cds.
Xenopus laevis beta-tubulin mRNA, complete cds.
Target Description
155
Xl.2618.1.A1_x_at
Xl.13786.1.S1_at
Xl.10313.1.A1_at
Xl.4246.1.S1_at
Xl.2622.1.S1_x_at
Xl.1171.1.A1_at
Xl.16654.3.S1_a_at
Xl.5692.1.S1_at
Xl.11560.1.S1_at
Xl.19047.1.A1_at
Xl.15278.1.A1_at
Xl.7748.1.S1_at
Xl.16292.1.A1_at
Xl.21811.1.S1_at
Xl.3708.1.S1_at
Xl.5092.1.A1_at
Xl.2480.1.A1_at
Xl.21883.1.S1_at
Xl.3447.1.S1_x_at
Xl.6650.1.S1_at
Xl.21865.1.S1_s_at
Xl.26325.1.S1_at
Xl.11087.1.A1_at
Xl.12077.1.S1_at
Xl.24281.1.A1_at
Affymetrix
Probeset ID
540.21
306.92
270.02
463.98
584.56
198.43
193.65
316.28
541.08
530.66
588.02
483.92
968.41
650.82
569.93
122.70
385.67
206.15
145.28
145.80
254.85
290.51
141.14
117.66
216.25
353.68
370.68
327.33
331.92
599.02
476.35
617.12
88.81
274.39
164.80
439.33
263.82
279.99
459.91
174.50
875.43
163.75
78.32
1225.92
439.48
170.80
664.16
1255.30
375.70
387.12
Mean
Laevis
Mean
Muelleri
202.00
397.30
122.72
1269.97
559.70
982.72
421.00
573.78
492.72
504.06
249.57
162.38
161.28
517.52
376.33
215.75
241.20
391.62
874.96
645.03
300.43
336.00
101.61
247.62
692.20
Mean
Muelleri
Male
326.54
642.98
203.37
758.30
965.73
1725.25
744.78
1023.36
894.56
918.48
492.29
327.71
328.55
1065.95
850.52
488.53
582.22
953.99
2204.65
1639.25
771.43
889.46
298.71
835.16
2454.58
Mean Laevis
Male
Male Biased in Both Species for Genes Expressed the Same between Species
Table A.4-continued.
155
0.69
0.69
0.73
0.74
0.79
0.81
0.82
0.83
0.86
0.87
0.98
1.01
1.03
1.04
1.18
1.18
1.27
1.28
1.33
1.35
1.36
1.40
1.56
1.75
1.83
Abslog2
Sex Fold
Change
ESTs, Moderately similar to hypothetical protein FLJ12571 (Homo sapiens)
Xenopus laevis, Similar to cysteinyl-tRNA synthetase, clone MGC:53887 IMAGE:5543220, mRNA, complete
cds.
ESTs, Moderately similar to BTB (POZ) domain containing 2; BTB (POZ) domain containing 2; hypothetical
protein FLJ20386 (Homo sapiens)
Xenopus laevis, clone MGC:53564 IMAGE:4681607, mRNA, complete cds.
Xenopus laevis, Similar to aldehyde dehydrogenase 9 family, member A1, clone MGC:52697
IMAGE:4682691, mRNA, complete cds.
X.laevis mRNA for elongation factor 1 delta.
Xenopus laevis mRNA for alpha subunit of casein kinase II
Xenopus laevis, Similar to aspartyl-tRNA synthetase, clone MGC:53970 IMAGE:5570927, mRNA, complete
cds.
ESTs, Highly similar to similar to mouse Glt3 or D. malanogaster transcription factor II; likely ortholog of
mouse gene trap locus 3 (Homo sapiens)
ESTs, Highly similar to G protein pathway suppressor 1; Arabidopsis FUS6COP11 homolog (Homo sapiens)
ESTs, Highly similar to COPA_HUMAN Coatomer alpha subunit (Alpha-coat protein) (Alpha-COP)
(HEPCOP) (HEP-COP) (Contains: Xenin (Xenopsin-related peptide); Proxenin) (H.sapiens)
Xenopus laevis karyopherin-beta 3 variant mRNA, complete cds.
Xenopus laevis, clone MGC:52799 IMAGE:4724842, mRNA, complete cds.
ESTs, Moderately similar to T13147 isocitrate dehydrogenase (NAD+) (EC 1.1.1.41) beta chain isoform B human (H.sapiens)
Xenopus laevis, Similar to protein kinase anchoring protein GKAP42, clone MGC:52736 IMAGE:4683888,
mRNA, complete cds.
ESTs, Weakly similar to B35098 MHC class III histocompatibility antigen HLA-B-associated protein 2
(imported) - human (H.sapiens)
Xenopus laevis, similar to 30 kDa protein, clone MGC:54006 IMAGE:5571444, mRNA, complete cds.
ESTs, Weakly similar to A44302 protein-glutamine gamma-glutamyltransferase (EC 2.3.2.13) 2, splice form 2
- human (H.sapiens)
ESTs
Xenopus laevis, clone MGC:53258 IMAGE:5569848, mRNA, complete cds. Unknown (protein for
MGC:53258)
Xenopus laevis, Similar to protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), alpha isoform,
clone MGC:53746 IMAGE:4930712, mRNA, complete cds.
Xenopus laevis, Similar to RIKEN cDNA 1200009K13 gene, clone MGC:53445 IMAGE:5571963, mRNA,
complete cds.
ESTs, Weakly similar to hypothetical protein FLJ10466 (Homo sapiens) (H.sapiens)
Xenopus laevis intraflagellar transport protein 20 mRNA, complete cds.
ESTs, Highly similar to HS9A_HUMAN Heat shock protein HSP 90-alpha (HSP 86) (H.sapiens)
Target Description
156
236.04
310.85
164.39
294.08
168.55
Xl.21978.1.S1_at
Xl.179.1.S1_at
Xl.25684.1.S1_at
Xl.4535.2.S1_at
Xl.22.1.S1_at
398.46
204.93
117.69
Xl.1164.1.S1_at
Xl.19268.1.A1_at
Xl.2801.1.A1_at
533.30
353.71
Xl.4725.1.S1_at
299.57
226.39
Xl.6295.1.S1_at
Xl.4138.2.S1_x_at
159.07
Xl.4048.1.S1_at
Xl.105.1.S1_at
64.09
Xl.12179.1.A1_at
301.32
441.73
Xl.8319.1.S1_at
197.63
692.05
Xl.702.2.S1_at
Xl.8304.1.S1_at
147.09
Xl.25660.1.S1_at
Xl.20985.1.A1_at
135.36
134.54
Xl.4641.1.S1_at
188.96
126.36
Xl.8066.1.A1_at
Xl.744.1.S1_at
162.14
Xl.449.1.S1_at
Xl.25590.1.A1_at
38.33
Mean
Muelleri
Xl.23471.1.S1_at
Affymetrix
Probeset ID
466.34
775.16
1912.68
3247.00
1573.02
998.00
1945.02
856.82
1539.59
829.12
47.37
1620.18
1886.00
1381.99
833.73
358.12
2924.74
4505.72
863.64
1651.00
981.47
1094.45
1165.88
1276.52
414.64
Mean
Laevis
134.30
222.17
475.39
578.45
372.38
268.99
365.72
227.69
326.61
216.49
420.83
264.29
458.87
245.77
176.16
70.73
543.77
781.34
162.72
205.12
169.15
156.31
163.85
175.88
40.19
Mean
Muelleri
Female
897.65
1487.43
3315.52
4129.39
2689.22
1960.83
2706.41
1692.57
2452.14
1626.43
54.64
2103.57
3718.13
2041.49
1483.98
655.27
5114.94
7555.12
1637.63
2226.68
1930.91
2147.69
2303.49
2490.22
808.03
Mean Laevis
Female
156
2.74
2.74
2.80
2.84
2.85
2.87
2.89
2.89
2.91
2.91
2.95
2.99
3.02
3.05
3.07
3.21
3.23
3.27
3.33
3.44
3.51
3.78
3.81
3.82
4.33
Abslog2
Sex Fold
Change
Female Biased Genes in Both Species for Genes Differentially Expressed between Species
Table A.4-continued.
ESTs, Weakly similar to hypothetical protein FLJ14464 (Homo sapiens)
ESTs
Xenopus laevis maternal mRNA D7
Xenopus laevis cytoplasmic beta actin mRNA, complete cds
Xenopus laevis mRNA for p33 ringo (ls27).
ESTs, Weakly similar to HSHUB1 histone H2B.1 - human (H.sapiens)
Frog phospholipase C beta type (PLC1) mRNA, complete cds.
Xenopus laevis cDNA clone 30F5-2.
Xenopus laevis Plx1 mRNA, complete cds
ESTs, Weakly similar to B40335 histone H1-3 (validated) - human (H.sapiens)
African clawed frog L-myc oncogene (xL-myc2) mRNA, complete cds
Xenopus laevis CArG-binding factor A mRNA,partial cds
Xenopus laevis cldn4L2 mRNA for Claudin4L2, complete cds.
Xenopus laevis, Similar to NHP2 non-histone chromosome protein 2-like 1 (S.
cerevisiae),
Xenopus CDC47-2p (CDC47-2) mRNA, complete cds.
ESTs
Xenopus laevis clone S10-21-F5 mRNA sequence
X.laevis cyclin B2 mRNA, complete cds
Xenopus laevis ornithine aminotransferase (OAT) mRNA, complete cds
Xenopus laevis, Similar to DEADH (Asp-Glu-Ala-AspHis) box polypeptide 5
(RNA helicase, 68kDa)
ESTs
X.laevis mRNA for alpha-tubulin.
ESTs, Weakly similar to H2B histone family, member E (Homo sapiens)
Xenopus laevis RRM-type RNA-binding protein hermes mRNA, complete cds.
ESTs, Moderately similar to solute carrier family 6 (neurotransmitter transporter),
member 14; amino acid transporter B0+ (Homo sapiens)
Target Description
157
307.37
634.15
541.62
82.51
81.31
441.25
676.23
234.27
479.87
103.65
141.07
315.35
452.16
349.63
Xl.4222.1.S1_at
Xl.1694.1.S1_at
Xl.6615.1.A1_at
Xl.5313.1.S1_a_at
Xl.1701.2.A1_at
Xl.21410.2.S1_at
Xl.5135.1.A1_at
Xl.10377.1.S1_at
Xl.15484.1.S1_at
Xl.13518.2.S1_at
Xl.13697.1.A1_at
Xl.24761.1.S1_at
Xl.6855.1.S1_at
Xl.23498.1.A1_x_at
102.93
1052.96
225.85
Xl.8013.1.S1_at
Xl.11208.1.S1_at
Xl.23930.1.S1_at
Xl.467.1.S1_at
153.61
418.86
Xl.695.1.S1_at
220.75
216.25
Xl.1066.1.A1_at
1504.66
499.59
Xl.5598.1.A1_at
Xl.24414.1.S1_at
147.26
Xl.9364.1.S2_at
761.95
Xl.611.1.S1_at
Mean
Muelleri
Xl.93.1.S1_at
Affymetrix
Probeset ID
251.51
701.79
114.96
518.17
168.23
1822.80
240.67
417.52
493.13
363.09
175.50
125.70
618.74
289.21
936.66
600.38
104.05
110.36
765.40
1029.76
464.05
321.57
940.59
282.03
2311.47
Mean
Laevis
265.96
1377.04
130.69
444.40
176.65
2113.78
233.82
394.18
495.99
343.50
163.46
113.76
563.15
271.70
950.37
531.72
89.56
99.99
673.41
876.75
346.62
239.12
721.24
161.85
1031.17
Mean
Muelleri
Female
406.95
881.27
206.38
702.59
284.55
3427.46
381.79
653.17
826.11
581.97
283.86
203.99
1027.85
503.58
1822.83
1031.52
174.20
196.99
1356.98
2005.90
825.09
578.24
1855.06
525.31
4571.11
Mean
Laevis
Female
Female Biased Genes in Both Species for Genes Expressed Same between Species
Table A.4-continued.
0.61
0.64
0.66
0.66
0.69
0.70
0.71
0.73
0.74
0.76
0.80
0.84
0.87
0.89
0.94
0.96
0.96
0.98
1.01
1.19
1.25
1.27
1.36
1.70
2.15
Xenopus laevis clone S10-13-D10 mRNA sequence
ESTs, Moderately similar to hypothetical protein MGC4293 (Homo sapiens)
Xenopus laevis, Similar to RNA-binding region (RNP1, RRM) containing 1, mRNA, complete cds.
ESTs, Weakly similar to hypothetical protein MGC14595 (Homo sapiens)
Xenopus laevis polyprotein, serine proteases and ovochymase regions, mRNA, complete cds.
ESTs, Weakly similar to hypothetical protein FLJ22692 (Homo sapiens)
Xenopus laevis contractile ring component anillin mRNA, complete
ESTs, Weakly similar to RR40_HUMAN Exosome complex exonuclease RRP40 (Ribosomal RNA
processing protein 40) (CGI-102) (H.sapiens)
ESTs, Weakly similar to T12688 hypothetical protein 63B12.2 - fruit fly (Drosophila melanogaster)
(D.melanogaster)
ESTs, Moderately similar to ROG_HUMAN Heterogeneous nuclear ribonucleoprotein G (hnRNP G)
Glycoprotein P43) (H.sapiens)
ESTs, Weakly similar to Y37D8A.22.p (Caenorhabditis elegans)
ESTs
ESTs, Weakly similar to DDH2_HUMAN NG,NG-dimethylarginine dimethylaminohydrolase 2
(Dimethylargininase 2) (Dimethylarginine dimethylaminohydrolase 2) (DDAHII) (G6a) (H.sapiens)
ESTs, Weakly similar to A43932 mucin 2 precursor, intestinal - human (fragments) (H.sapiens)
ESTs, Weakly similar to A36000 sperm-binding glycoprotein ZP3 precursor - human (H.sapiens)
Xenopus laevis cDNA clone 5G23
ESTs, Moderately similar to PAR-6 gamma protein; par-6 (partitioning defective 6, C.elegans) homolog
gamma (Homo sapiens)
ESTs, Weakly similar to hypothetical protein MGC2376 (Homo sapiens)
ESTs, Weakly similar to RTP801 (Homo sapiens)
Xenopus laevis Wee1A kinase (Xe-Wee1A) mRNA, complete cds.
Xenopus laevis wee1 homolog mRNA, complete cds.
Xenopus laevis homeobox protein mRNA, complete cds.
ESTs
Xenopus laevis egg envelope component ZPAX mRNA,
Xenopus laevis oocyte lectin mRNA, complete cds.
Target Description
157
Abslog2
Sex
Fold
Change
APPENDIX B
SUPPLEMENTAL TABLES FOR CHAPTER 4
158
159
17285
14287
17397
10929
17927
19581
14286
11203
19015
17308
19580
11208
1
2
3
4
5
6
7
8
9
10
11
12
13
14348
13485
17575
17327
17454
10995
17502
19004
3320
11067
17421
11020
14
15
16
17
18
19
20
21
22
23
24
25
11041
Gene
Name
Rank
159
xtr-miR-125a, dre-miR-125a, fru-miR-125a, tni-miR-125a
xtr-miR-22, hsa-miR-22, mmu-miR-22, rno-miR-22, age-miR-22lca-miR-22, lla-miR-22, mdo-miR-
xtr-let-7a-e
xtr-miR-338, hsa-miR-338, mmu-miR-338, rno-miR-338, dre-miR-338, fru-miR-338, mdo-miR-338,
tni-miR-338
xtr-let-7c, hsa-let-7c, mmu-let-7c, rno-let-7c, dre-let-7c, gga-let-7c, ssc-let-7c
xtr-miR-199a*, hsa-miR-199a*, mmu-miR-199a*, bta-miR-199a*, dre-miR-199*, gga-miR-199*
xtr-miR-30c, hsa-miR-30c, mmu-miR-30c, rno-miR-30c, bta-miR-30c, dre-miR-30c, gga-miR-30c,
lla-miR-30c, mne-miR-30c, ptr-miR-30c, ssc-miR-30c
fru-miR-30c, tni-miR-30c
hsa-miR-630
0.01371
0.012903
0.0125
0.012097
0.011694
0.009677
0.009274
0.008871
0.008468
0.007661
0.008065
Hy5
0.007258
0.006452
0.006048
0.004839
0.004435
0.004032
0.003226
0.002823
0.002419
0.002016
0.001613
0.00121
0.000806
0.000403
P-Value
cel-miR-246
xtr-miR-10a, hsa-miR-10a, mmu-miR-10a, rno-miR-10a, aga-miR-10, age-miR-10a, bmo-miR-10,
bta-miR-10a, dme-miR-10, dps-miR-10, dre-miR-10a, ggo-miR-10a, mdo-miR-10a, ppa-miR-10a,
sla-miR-10a,
mmu-miR-207, rno-miR-207
xtr-miR-29a-29c, hsa-miR-29c, mmu-miR-29c, rno-miR-29c, bta-miR-29c, dre-miR-29a, fru-miR29a, gga-miR-29a-29c, mdo-miR-29a, ssc-miR-29c, tni-miR-29a
xtr-miR-29d
xtr-let-7i, hsa-let-7i, mmu-let-7i, rno-let-7i, bta-let-7i, dre-let-7i, fru-let-7i, gga-let-7i, mdo-let-7i, tnilet-7i,
mmu-miR-155
xtr-miR-142-5p, hsa-miR-142-5p, mmu-miR-142-5p, rno-miR-142-5p, bta-miR-142, dre-miR-142a5p, fru-miR-142a-142b, gga-miR-142-5p, tni-miR-142a-142b,
mmu-miR-489
xtr-miR-100,hsa-miR-100
miRPlus
xtr-miR-125b
dre-miR-202*
hsa-miR-494, mmu-miR-494, rno-miR-494
xtr-miR-202*
Name
Table B.1. microRNA expression results for comparisons of hybrids and X. laevis.
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.136364
0.136364
0.136364
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
Q-Value
9386.316
29282.57
5013.872
37917.33
80228.44
11550.85
5169.025
5512.278
57807.66
8879.057
9895.099
5139.964
12588.08
9627.908
14373.38
10760.45
63629.56
4836.386
5997.848
12038.12
7015.857
15727.71
38189.58
121162.6
161937.4
Mean_expr
160
11270
17275
14301
17334
11039
17366
11246
17510
17565
17898
27
28
29
30
31
32
33
34
35
36
37
17748
11030
17622
17512
17310
17869
17489
17473
14289
17601
11200
17299
14432
13140
17952
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
10898
13520
26
160
cel-miR-260
xtr-miR-138, hsa-miR-138, mmu-miR-138, rno-miR-138, bta-miR-138, dre-miR-138, fru-miR-138,
gga-miR-138, mdo-miR-138, tni-miR-138
miRPlus
hcmv-miR-UL22A*
mmu-miR-129-3p, rno-miR-129*, dre-miR-129*
xtr-miR-20a*
mmu-miR-540, rno-miR-540
hcmv-miR-UL70-3p
0.028226
0.027823
0.027016
0.026613
0.025
0.024597
0.024194
0.02379
0.023387
0.022581
miRPlus
mmu-miR-710
0.022177
0.021371
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.020565
0.020968
0.148876
0.148876
0.020161
0.019758
ppt-miR536
mmu-miR-711
gga-miR-202
hsa_negative_control_5
xtr-let-7a, hsa-let-7a, rno-let-7a, bta-let-7a, cbr-let-7, cel-let-7, dre-let-7a, fru-let-7a, gga-let-7a-,
gga-let-7j, mdo-let-7a, tni-let-7a
xtr-miR-26, hsa-miR-26a, mmu-miR-26a, rno-miR-26a, bta-miR-26a, dre-miR-26a, fru-miR-26,
gga-miR-26a, ggo-miR-26a, lla-miR-26a, mml-miR-26a, mne-miR-26a, ppa-miR-26a, ppy-miR26a, ptr-miR-26a, ssc-miR-26a, tni-miR-26
0.148876
0.018952
0.019355
miRPlus
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.018548
0.018145
0.016935
0.016532
0.016129
0.015726
0.015323
0.014516
0.014113
hsa-miR-602
xtr-miR-30b, hsa-miR-30b, mmu-miR-30b, rno-miR-30b, age-miR-30b, bta-miR-30b, dre-miR-30b,
fru-miR-30b, gga-miR-30b, ggo-miR-30b, lla-miR-30b, mml-miR-30b, mne-miR-30b, ppa-miR-30b,
ptr-miR-30b, tni-miR-30b
mmu-miR-434-3p
ebv-miR-BART13
xtr-let-7e
hsa-miR-29a, mmu-miR-29a, rno-miR-29a, age-miR-29a, bta-miR-29a, ggo-miR-29a, lla-miR-29a,
mml-miR-29a, mne-miR-29a, ppa-miR-29a, ppy-miR-29a, ptr-miR-29a, sla-miR-29a
hsa-miR-361, mmu-miR-361, rno-miR-361, bta-miR-361
dre-let-7h, fru-let-7h, tni-let-7h
rno-miR-347
dme-miR-289, dps-miR-289
22, mml-miR-22, mne-miR-22, ppa-miR-22, ppy-miR-22, ptr-miR-22, sla-miR-22
Table B.1-continued.
29029.22
4043.305
5195.415
6805.873
4007.654
3988.789
10648.72
70493.45
60692.06
102971.3
6254.172
205713.8
16453.39
18525.37
24598.5
6490.522
9514.796
11117.45
83623.71
5300.038
78559.83
88976.99
46201.28
38406.83
4850.166
5997.169
26478.82
161
13447
17604
14327
17878
19605
14349
11221
17422
13137
10934
77
78
79
80
81
82
83
84
11132
76
17646
67
68
75
17441
66
17552
11007
65
74
17330
64
17943
17953
63
17700
11279
62
73
11003
61
72
17747
60
17335
17678
59
71
hsa-miR-500, mmu-miR-500
10964
58
17685
17749
57
69
ebv-miR-BHRF1-3
11135
56
161
hsa-miR-526c
xtr-miR-129, hsa-miR-129, mmu-miR-129-5p, rno-miR-129, dre-miR-129, fru-miR-129, mdo-miR129, tni-miR-129
mmu-miR-695
mmu-miR-300, rno-miR-300
cbr-miR-246
0.043548
0.043145
0.042742
0.042339
0.041532
0.040323
0.040726
hsa_SNORD6
0.039919
0.039516
0.039113
0.03871
0.038306
0.0375
0.037097
0.03629
0.035887
0.035484
0.034677
0.034274
bta-miR-193a*
cbr-miR-252, cel-miR-252
mghv-miR-M1-7-5p
osa-miR171g, sbi-miR171c, zma-miR171g
hsa-miR-617
mmu-miR-760
gga-miR-124b
xtr-let-7b
dre-miR-26b
hsa-miR-648
0.033871
0.033468
miRPlus
gga-miR-130b
xtr-miR-206, hsa-miR-206, mmu-miR-206, rno-miR-206, dre-miR-206, gga-miR-206, mdo-miR206, mne-miR-206, ppy-miR-206
0.032661
0.033065
U6-snRNA-2
0.032258
0.031452
0.031048
0.030645
0.030242
0.029839
0.029435
0.029032
hsa-miR-202
xtr-miR-202, dre-miR-202, fru-miR-202, tni-miR-202
xtr-miR-10c
xtr-miR-155, hsa-miR-155, dre-miR-155, gga-miR-155
hsa-let-7b, mmu-let-7b, rno-let-7b, dre-let-7b, fru-let-7b, gga-let-7b, mdo-let-7b, tni-let-7b
hsa-miR-503
ath-miR168a-, ath-miR168b, gma-miR168, ptc-miR168a-, ptc-miR168b
13357
mmu-miR-503, rno-miR-503
cel-miR-271
55
14385
14288
54
53
Table B.1-continued.
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
25906.84
23526.1
20249.28
23142.69
6679.008
18824.64
74562.22
89219.84
36848.96
25612.42
32444.54
47705.93
5782.466
12078.64
8827.738
22598.78
43901.45
15241.39
11241.6
46835.02
41030.28
164711.1
61556.83
65113.45
6654.195
9160.121
42350.13
49007.54
30228.14
55497.93
60890.86
162
10936
17859
17921
10923
17960
14439
91
92
93
94
95
96
13201
17904
14434
13511
17399
13480
11146
17930
17537
17876
14446
13445
14329
11014
17699
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
10928
98
10993
cel-miR-264
16528
90
97
miRPlus
14407
89
162
cbr-miR-231
xtr-miR-214, hsa-miR-214, mmu-miR-214, rno-miR-214, age-miR-214, bta-miR-214, dre-miR-214,
fru-miR-214, ggo-miR-214, mdo-miR-214, mml-miR-214, mne-miR-214, ppa-miR-214, ppy-miR214, ptr-miR-214, sla-miR-214, ssc-miR-214, tni-miR-214
xtr-miR-130c, dre-miR-130c, fru-miR-130, gga-miR-130a, tni-miR-130
osa-miR166k-, osa-miR166l, sbi-miR166e-, sbi-miR166g, zma-miR166k-, zma-miR166j
cel-miR-243
0.148876
0.148876
0.148876
0.056452
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.056855
0.056048
0.055645
0.055242
0.054032
0.054435
miRPlus
0.053629
miRPlus
mghv-miR-M1-3
0.052823
0.052419
0.052016
0.051613
0.05121
hsa-miR-513
dme-miR-3, dps-miR-3
mmu-miR-501*
cel-miR-230
xtr-miR-210, hsa-miR-210, mmu-miR-210, rno-miR-210, ame-miR-210, bta-miR-210, dme-miR210, dps-miR-210, dre-miR-210, fru-miR-210, tni-miR-210
0.050403
0.050806
miRPlus
0.05
0.049597
0.049194
sof-miR168b
hsa-miR-125a, mmu-miR-125a, rno-miR-125a, bta-miR-125a
hsa-miR-198, age-miR-198, ggo-miR-198, ppa-miR-198, ppy-miR-198, ptr-miR-198, sla-miR-198
0.148876
0.047984
0.04879
0.148876
0.047581
miRPlus
xtr-miR-107, hsa-miR-107, mmu-miR-107, rno-miR-107, dre-miR-107, fru-miR-107, gga-miR-107,
ggo-miR-107, lla-miR-107, mdo-miR-107, mml-miR-107, mne-miR-107, ppa-miR-107, ppy-miR107, ptr-miR-107, ssc-miR-107, tni-miR-107
0.148876
0.148876
0.046774
0.047177
0.148876
0.148876
0.148876
miRPlus
0.046371
0.045968
0.045161
0.148876
0.148876
0.148876
xtr-miR-130b, hsa-miR-130b, mmu-miR-130b, rno-miR-130b
mmu-miR-706
cel-miR-235
mmu-miR-17-3p
19600
88
0.044758
miRPlus
17894
0.044355
0.043952
mmu-miR-685
osa-miR164e
87
13408
17464
86
85
Table B.1-continued.
13608.24
38000.32
20155.5
13060.69
28445.97
13892.5
42257.26
15519.95
62715.89
25530.58
30310.09
28382.59
33907.84
11660.94
26783.3
7855.019
34405.79
31235.94
89037.69
6248.881
34836.07
24328.23
25239.88
21983.3
4195.99
20712.61
27446.03
128123.3
37596.13
163
17423
17856
13533
17698
17834
14433
13230
11054
14306
17471
17550
13395
17561
11181
17694
17596
11118
13211
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
17346
17574
10935
122
123
13207
13407
121
126
11228
120
125
17809
119
17629
hsa-miR-612
17877
118
124
xtr-miR-130a, hsa-miR-130a, mmu-miR-130a, rno-miR-130a, dre-miR-130a
17272
117
hsa-miR-485-3p, mmu-miR-485-3p
ptc-miR164f
gga-miR-302b*
163
ebv-miR-BART6-3p
hsa-miR-95, ggo-miR-95, lla-miR-95, ppa-miR-95, ppy-miR-95, ptr-miR-95, sla-miR-95, ssc-miR95
ath-miR156g
hsa-miR-638
hsa-miR-628
hsa-miR-381, mmu-miR-381, rno-miR-381
hsa-miR-320, mmu-miR-320, rno-miR-320, bta-miR-320
ptc-miR395a
0.070565
0.070161
0.069758
0.069355
0.068548
0.068145
0.067742
0.067339
0.066935
0.066532
0.066129
0.065726
0.065323
0.064919
miRPlus
cbr-miR-230
0.064516
0.064113
0.06371
0.062903
0.0625
0.062097
0.061694
0.06129
0.060484
0.060081
0.059677
dre-miR-130b
dme-miR-31a, dps-miR-31a
miRPlus
hsa-miR-584
hsa-miR-615, mmu-miR-615
ptc-miR159e
mmu-miR-700
osa-miR164c
mmu-miR-330, rno-miR-330
0.059274
0.058871
miRPlus
hsa-miR-769-3p
0.058468
0.058065
0.057661
0.057258
hsa-miR-551a
mmu-miR-689
17321
116
hsa-miR-637
xtr-miR-98, hsa-miR-98, mmu-miR-98, rno-miR-98, age-miR-98, bta-miR-98, ggo-miR-98, mmlmiR-98, ppa-miR-98, ppy-miR-98, ptr-miR-98
17354
11182
115
114
Table B.1-continued.
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
94796
9037.328
7548.984
40594.68
7896.366
6143.833
9573.782
56668.74
26343.98
21300.84
142388.2
44758.2
15036.45
11614.41
25532.39
10434.77
45494.96
38835.58
12216.41
68290.07
55271.05
149129.1
11326.55
32459.27
7322.945
60151.53
88976.99
24601.91
47432.52
9718.004
12797.73
164
17295
13239
17337
5730
17522
17896
17291
13443
17398
14402
17939
11235
17320
11214
162
163
164
165
166
167
168
169
170
171
172
173
174
5560
161
14373
155
156
14370
17751
154
160
17942
153
17830
7590
152
13128
13490
151
159
17280
150
158
11177
149
13450
hsa-miR-185, mmu-miR-185, rno-miR-185
osa-miR319a-, osa-miR319b, sbi-miR319, zma-miR319a-, zma-miR319c-, zma-miR319b-, zmamiR319d
14293
148
157
cel-miR-265
17521
147
mmu-miR-705
mmu-miR-291a-5p-291b-5p, rno-miR-291-5p
mmu-miR-351, rno-miR-351
hsa-miR-671, mmu-miR-671
cbr-miR-38
hsa-miR-634
164
, gma-miR156b, mtr-miR156, osa-miR156k, sbi-miR156d, zma-miR156j
mghv-miR-M1-4
bta-miR-21*
hsa-miR-658
hsa-miR-208, mmu-miR-208, rno-miR-208
dre-miR-10d*
ptc-miR399l
hsa-miR-583
cbr-miR-241
0.083871
0.083468
0.083065
0.082661
0.082258
0.081855
0.081452
0.081048
0.080645
0.080242
0.079839
0.079435
0.079032
0.078629
0.078226
0.076613
0.077823
miRPlus
0.07621
0.075403
0.075
0.074597
0.074194
0.07379
0.073387
0.072984
0.072581
0.072177
0.071774
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.148876
0.070968
0.071371
hsa-miR-432*
hsa-let-7e, mmu-let-7e, rno-let-7e
miRPlus
mmu-miR-707
dme-miR-184*
hsa-miR-527
xtr-miR-15c, hsa-miR-15b, mmu-miR-15b, rno-miR-15b, age-miR-15b, bta-miR-15b, dre-miR-15b,
gga-miR-15b, ggo-miR-15b, lla-miR-15b, mml-miR-15b, mne-miR-15b, ppa-miR-15b, ppy-miR15b, ptr-miR-15b
mmu-miR-546
mmu-miR-714
17050
146
hsa-miR-375, mmu-miR-375, dre-miR-375, fru-miR-375, gga-miR-375, mdo-miR-375, tni-miR-375
xtr-let-7g, dre-let-7j
11088
145
Table B.1-continued.
5862.47
11468.28
59941.26
26566.83
183874.2
14906.71
4573.419
47590.21
49801.82
13170.35
104892.2
14811.72
18779.57
10220.01
58211.1
6374.807
14834.28
6843.194
15485.67
51110.52
91440.51
10139.72
9018.552
12785.83
8113.832
6385.832
22799.97
12367.8
22559.61
5328.956
165
17839
17505
10978
11278
17551
13471
14401
10586
13366
178
179
180
181
182
183
184
185
186
187
11142
13326
17504
17632
17492
19018
17511
14291
11116
14316
17497
13208
17517
11264
13265
13241
11151
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
13370
17714
177
10234
17915
176
175
ptc-miR472b
hsa-miR-516-5p
165
osa-miR535, ppt-miR535a-, ppt-miR535b-, ppt-miR535c-, ppt-miR535d
rno-miR-327
mmu-miR-688
ptc-miR159f
dre-miR-457b
rno-miR-664
hsa-miR-452, mmu-miR-452
mmu-miR-542-5p, rno-miR-542-5p
mmu-miR-713
hsa-miR-346
sv40-miR-S1-5p
mmu-miR-691
mmu-miR-709
ath-miR391
hsa-miR-510
ath-miR169d-, ath-miR169e-, ath-miR169f-, ath-miR169g, ptc-miR169n
ath-miR390a-, ath-miR390b, osa-miR390, ppt-miR390a-, ppt-miR390b, ptc-miR390a-, ptcmiR390b-, ptc-miR390c-, ptc-miR390d
hsa-miR-518f*-526a
cel-miR-38
sbi-miR156e, zma-miR156k
hsa-miR-572
U6-snRNA-1
hsa-miR-654
xtr-miR-184, hsa-miR-184, mmu-miR-184, rno-miR-184, aga-miR-184, ame-miR-184, dme-miR184, dps-miR-184, dre-miR-184, fru-miR-184, gga-miR-184, mdo-miR-184, mne-miR-184, ppymiR-184, ptr-miR-184, ssc-miR-184, tni-miR-184
0.099597
0.099194
0.09879
0.098387
0.097984
0.097581
0.097177
0.096774
0.096371
0.095968
0.095565
0.095161
0.094758
0.093548
0.093145
0.091935
0.091532
0.091129
0.090323
0.089919
0.089516
0.089113
0.08871
0.088306
0.0875
0.087097
0.085484
0.085081
miRPlus
lla-miR-198
0.084274
0.084677
mml-miR-198, mne-miR-198
Table
Table
Table
B.1-continued.
B.1-continued.
B.1-continued.
0.148876
0.151348
0.151348
0.151348
0.151348
0.151348
0.151348
0.151348
0.151348
0.151348
0.151348
0.151348
0.151348
0.151348
0.151348
0.151348
0.150794
0.150794
0.150794
0.150538
0.150538
0.150538
0.150538
0.150538
0.150538
0.150538
0.150538
0.148876
0.148876
0.148876
38680.71
8815.028
4892.582
31253.6
84195.52
9158.292
70759.89
6455.31
7527.081
25008.82
17218.21
15836.03
11483.75
75590.48
47409.62
74726.7
26530.43
13145.75
18517.26
11195.51
21922.42
26993.61
35097.13
21204.92
25643.12
12138.22
14457.94
32079.58
36964.74
58308.69
166
17881
13206
17566
17832
17961
11008
11082
17573
13131
14456
17683
13424
224
225
226
227
228
229
230
231
232
233
234
13381
11124
17745
216
217
223
11220
215
222
13469
214
11056
13132
213
221
17933
212
17871
11145
211
17722
13474
210
220
11097
209
219
osa-miR395t
14352
208
17532
17465
207
218
dre-miR-17a*
14267
206
0.100403
xtr-miR-16a, dre-miR-16a, gga-miR-16lca-miR-16
osa-miR415
cbr-miR-245, cel-miR-245
hsa-miR-518c*
hsa-miR-625
hsa-miR-370, mmu-miR-370, rno-miR-370
166
miRPlus
xtr-miR-106-20a, hsa-miR-20a, mmu-miR-20a, rno-miR-20a, bta-miR-20a, dre-miR20a, fru-miR-20, gga-miR-20a, mdo-miR-20, tni-miR-20
miRPlus
ptc-miR156k
hsa-miR-629
0.151699
0.152778
0.152778
0.115323
0.152778
0.152778
0.152778
0.152778
0.152778
0.152778
0.114919
0.114516
0.114113
0.11371
0.113306
0.112903
0.1125
0.152655
0.152655
0.110887
0.11129
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152344
0.152174
0.151699
0.110081
0.109274
0.109677
miRPlus
0.108871
hsa-miR-492
hsa-miR-324-3p, mmu-miR-324-3p, rno-miR-324-3p
0.107661
0.108468
miRPlus
0.107258
0.106855
0.106452
0.106048
0.105645
dre-miR-16b, fru-miR-16, tni-miR-16
ebv-miR-BART5
mmu-miR-298, rno-miR-298
sbi-miR164c
0.105242
0.104435
miRPlus
hsa-miR-519e*
0.104032
0.103629
0.103226
0.102823
0.101613
0.100806
hsa-miR-512-5p
hsa-miR-382, mmu-miR-382, rno-miR-382
ath-miR319a-, ath-miR319b-, ath-miR319c, gma-miR319a-, gma-miR319b, mtr-miR319, pptmiR319a-, ppt-miR319b-, ppt-miR319c-, ppt-miR319d, ptc-miR319a-, ptc-miR319b-, ptc-miR319c, ptc-miR319d-, ptc-miR319e-, ptc-miR319f-, ptc-miR319g-, ptc-miR319h
cbr-miR-47, cel-miR-47
mmu-miR-678
xtr-miR-363-5p, hsa-miR-363*, rno-miR-363-5p
17836
205
miRPlus
Table B.1-continued.
74470.72
17875.89
13418.82
4433.172
39796.4
33026.11
10521.29
6498.637
6061.344
37922.79
7090.683
27148.86
133101.5
83847.45
7971.083
89219.84
14733.28
5221.424
30303.71
20793.89
147397.4
363675.9
16354.49
8777.921
32403.12
47991.02
8819.17
14380.22
11553.59
74470.72
167
17498
13441
17713
17288
13263
17864
13396
11175
17452
19007
13470
17284
11130
11227
17583
249
250
251
252
253
254
255
256
257
258
259
260
261
262
17448
13352
17605
243
244
248
13354
242
247
13420
241
13382
17322
240
246
rlcv-miR-rL1-11
10967
239
13449
17597
238
245
ath-miR163
xtr-miR-17-5p, hsa-miR-17-5p-106a, mmu-miR-17-5p, rno-miR-17, age-miR-17-5p-106a, bta-miR106-17-5p, dre-miR-17a, fru-miR-17, gga-miR-17-5p-106, ggo-miR-17-5p-106alca-miR-17-5p, llamiR-17-5p, mdo-miR-17-5p, mml-miR-17-5p-106a, mne-miR-17-5p-106a, ppa-miR
13348
237
mmu-miR-329, rno-miR-329
mmu-miR-720
hsa-miR-498
mghv-miR-M1-2
167
0.134677
0.134274
0.133871
0.133065
0.132661
0.132258
hsa_SNORD3@
sbi-miR171f
0.131452
0.131048
ppt-miR533a-, ppt-miR533b
hsa-miR-525
0.130645
0.128629
miRPlus
ath-miR156h
0.128226
0.127823
0.127419
0.126613
0.125403
0.159351
0.159351
0.159351
0.159266
0.159266
0.159266
0.15918
0.15918
0.15918
0.157609
0.157609
0.157609
0.157609
0.157609
0.156754
0.156754
0.155996
0.12379
0.125
0.155996
0.123387
0.155738
0.15535
0.121774
0.122581
0.15535
0.15535
0.155208
0.154812
0.154812
0.153481
0.153481
0.153192
0.121371
0.120968
0.120161
0.119355
0.118952
0.117339
0.116935
0.116129
osa-miR530
rlcv-miR-rL1-9
xtr-miR-16b
mtr-miR399b-, mtr-miR399d, osa-miR399i, sbi-miR399b, zma-miR399b-, zma-miR399d
hsa-miR-601
ath-miR161
osa-miR171h, sbi-miR171e, zma-miR171k-, zma-miR171h
osa-miR395a
ath-miR419
hsa-miR-659
mmu-miR-467b
hsa-miR-16, mmu-miR-16, rno-miR-16, age-miR-16, bta-miR-16, ggo-miR-16, lla-miR-16, mdomiR-16, mml-miR-16, mne-miR-16, ppa-miR-16, ppy-miR-16, ptr-miR-16, sla-miR-16
13460
236
osa-miR395u
osa-miR159a-, osa-miR159b, sbi-miR159, sof-miR159a-, sof-miR159b-, sof-miR159d, zmamiR159a-, zma-miR159b
ath-miR156a-, ath-miR156b-, ath-miR156c-, ath-miR156d-, ath-miR156e-, ath-miR156f, gmamiR156a, osa-miR156a-, osa-miR156b-, osa-miR156c-, osa-miR156d-, osa-miR156e-, osamiR156f-, osa-miR156g-, osa-miR156h-, osa-miR156i-, osa-miR156j, ppt-miR156a, ptc-miR
13384
235
Table B.1-continued.
44350.47
5509.019
206676.9
10854.89
24883.56
383136.3
6094.773
8020.16
30545.6
187649.9
7189.398
4730.816
11233.64
13373.66
10759.52
8904.504
75999.94
22235.87
26748.44
5143.477
5947.011
10453.66
92173.34
10779.43
5420.13
7715.207
16024.48
42252.15
168
17558
17863
14263
10907
17861
14264
16681
19017
17903
10905
10906
284
285
286
287
289
290
291
292
293
294
295
11216
13373
17376
275
276
10904
17950
274
282
17955
273
281
14257
272
11248
14421
271
280
13400
270
11086
1090
269
13229
19008
268
279
17515
267
278
17890
266
19594
mmu-miR-292-5p, rno-miR-292-5p
10899
265
277
hsa-miR-557
17309
264
0.147581
miRPlus
miRPlus
0.168548
0.170161
0.172177
spike_control_h
miRPlus
spike_control_c
168
0.166935
spike_control_b
spike_control_g
spike_control_e
miRPlus
mmu-miR-290, rno-miR-290
0.183065
0.182661
0.17621
0.174597
0.172984
0.165323
miRPlus
mmu-miR-721
0.164919
hsa-miR-663
0.160887
0.160081
spike_control_d
ath-miR395b-, ath-miR395c-, ath-miR395f
0.155242
0.153226
0.15121
0.150806
0.150403
xtr-miR-451, hsa-miR-451, mmu-miR-451, rno-miR-451, dre-miR-451, gga-miR-451
hsa-miR-373*
ptc-miR319i
hsa-miR-296, mmu-miR-296, rno-miR-296
0.146774
spike_control_i
0.15
0.145968
0.146371
cel-miR-240
0.144355
0.141129
spike_control_d
osa-miR156l
0.140323
hsa_SNORD2
0.192373
0.192373
0.186433
0.18536
0.184278
0.184052
0.182526
0.181858
0.180944
0.179825
0.179825
0.176862
0.176601
0.171875
0.170251
0.168615
0.168615
0.168615
0.168615
0.166971
0.166667
0.166667
0.166667
0.165741
0.162639
0.162313
0.162313
0.139919
0.162264
0.162313
0.13871
0.139516
0.161932
0.160171
miRPlus
hcmv-miR-UL148D
0.137903
0.135887
spike_control_j
hsa-miR-623
13177
263
xtr-miR-143, hsa-miR-143, mmu-miR-143, rno-miR-143, dre-miR-143, ggo-miR-143, lla-miR-143,
mdo-miR-143, ppa-miR-143, ppy-miR-143, ptr-miR-143
Table B.1-continued.
11592.19
22445.92
6994.291
104181.1
16164.3
12048.54
5222.445
62075.01
4609.721
7490.509
171292.2
63936.06
64226.01
53861.78
131045.3
81912.52
13572.57
190997.3
76641.17
121429.4
5988.368
292758.6
6571.821
7848.975
62075.01
11834.86
9014.187
168571.8
262852
135184.6
37853.55
169
0.483468
0.513306
mtr-miR395b
miRPlus
11234
17431
17375
13169
17386
13472
13372
13438
13205
17514
13198
17875
299
300
301
302
303
304
305
307
308
309
310
rlcv-miR-rL1-2
169
ath-miR447a-, ath-miR447b
ath-miR395a-, ath-miR395d-, ath-miR395e, ptc-miR395b-, ptc-miR395c-, ptc-miR395d-, ptcmiR395e-, ptc-miR395f-, ptc-miR395g-, ptc-miR395h-, ptc-miR395i-, ptc-miR395j
osa-miR395b-, osa-miR395d-, osa-miR395e-, osa-miR395g-, osa-miR395h-, osa-miR395i-, osamiR395j-, osa-miR395k-, osa-miR395l-, osa-miR395s-, osa-miR395m-, osa-miR395n-, osamiR395p-, osa-miR395q-, osa-miR395r, sbi-miR395b-, sbi-miR395a-, sbi-miR395c-, sbimtr-miR395a-, mtr-miR395c-, mtr-miR395d-, mtr-miR395e-, mtr-miR395f-, mtr-miR395h-, mtrmiR395i-, mtr-miR395j-, mtr-miR395k-, mtr-miR395l-, mtr-miR395m-, mtr-miR395n-, mtr-miR395o,
sbi-miR395f
hcmv-miR-US4
mmu-let-7d*, rno-let-7d*
mghv-miR-M1-8
hsa-miR-594
mmu-miR-350, rno-miR-350
0.310887
0.474194
0.451613
0.441532
0.375403
0.341532
0.513307
0.485032
0.477273
0.456026
0.448771
0.382813
0.349422
0.319123
0.270764
0.245
0.262903
0.245
0.237097
0.244128
0.225589
0.195524
0.236694
0.234677
0.216129
298
0.186694
3980
297
aga-miR-7, ame-miR-7, bmo-miR-7, bta-miR-7, dme-miR-7, dps-miR-7, dre-miR-7a, tni-miR-7
14262
296
spike_control_f
Table B.1-continued.
7679.302
26068.88
97608.06
51722.36
61313.43
22316.1
22728.45
29132.46
5516.829
15528.32
17674.23
7607.282
11460.86
5204.438
170
protein folding
response to protein
stimulus
nucleosome assembly
chromatin assembly or
disassembly
sensory perception of
light stimulus
neurophysiological
process
localization
chromatin assembly
biopolymer
glycosylation
vesicle docking
sulfate transport
protein localization
visual perception
sensory perception
dre-miR-100
Biol Proc
response to unfolded
protein
vesicle docking during
exocytosis
exocytosis
transport
protein complex
assembly
nucleosome
chromatin
0.0287
0.0085
170
0.0321
integral to Golgi
membrane
0.0049
double-stranded RNA binding
calpain activity
anion transporter activity
nucleic acid binding
sialyltransferase activity
S-methyltransferase activity
inorganic anion transporter activity
0.0033
0.0252
0.0429
0.0137
0.0151
0.027
0.0119
0.0283
0.0033
0.0101
nucleotide binding
helicase activity
calmodulin binding
hydrolase activity, acting on
carbon-nitrogen (but not peptide)
bonds, in cyclic amidines
deaminase activity
guanyl nucleotide binding
GTP binding
0.0422
0.0041
0.0151
0.0183
0.0237
0.0183
FMN binding
0.0321
0.0214
0.0267
0.027
0.0331
0.0454
0.0287
Molec Funct
P-value
Cell Comp
intrinsic to Golgi
membrane
intracellular
organelle part
chromosomal part
organelle part
spliceosome
complex
P-value
Table B.2. Predicted targets of three microRNAs and GO category analysis.
0.0008
0.0467
0.0186
0.0385
0.0321
0.0397
0.0214
0.0327
0.0191
0.0467
0.0397
0.0467
0.0482
0.0482
0.0164
P-value
171
envelope
0.0433
exopeptidase activity
metallopeptidase activity
metalloexopeptidase activity
0.0289
0.0334
0.0234
171
fucosyltransferase activity
0.0242
0.027
0.0463
0.0083
0.0334
0.0014
0.0008
0.0254
0.0012
membrane alanyl aminopeptidase
activity
aminopeptidase activity
endopeptidase activity
phosphoric ester hydrolase activity
0.0334
0.0175
0.0343
0.0343
0.0176
0.0408
inorganic diphosphatase activity
catalytic activity
0.0104
0.0433
0.0014
nucleosome assembly
chromatin assembly or
disassembly
establishment and/or
maintenance of chromatin
architecture
DNA packaging
regulation of cell cycle
pyrimidine nucleotide
biosynthesis
pyrimidine nucleotide
metabolism
glycerol metabolism
cellular protein metabolism
0.0193
0.0063
translation initiation factor binding
MAP kinase phosphatase activity
0.0318
0.0455
0.0014
0.0058
0.0078
0.0455
P-value
alanine aminopeptidase activity
0.0302
RNA localization
response to protein stimulus
0.0397
0.0162
0.0058
0.0063
hydrolase activity, acting on ester
bonds
hydrolase activity
transferase activity, transferring
nitrogenous groups
transferase activity, transferring
hexosyl groups
copper ion binding
phosphoric monoester hydrolase
activity
Molec Funct
0.0423
0.0037
0.0154
RNA export from nucleus
0.0059
0.0104
0.0024
protein complex assembly
mRNA export from nucleus
organelle envelope
mitochondrial
membrane
nuclear membrane
organelle inner
membrane
nuclear envelope
endomembrane
system
0.0088
0.0088
intracellular
organelle part
organelle part
0.0077
nucleocytoplasmic transport
chromosome organization
and biogenesis (sensu
Eukaryota)
proteolysis
protein amino acid N-linked
glycosylation
0.0108
0.0456
0.0019
0.0154
0.0162
P-value
Cell Comp
organelle
membrane
nuclear membrane
part
P-value
response to unfolded protein
dre-miR-125b
Biol Proc
Table B.2-continued.
172
cellular metabolism
negative regulation of
metabolism
cellular physiological
process
negative regulation of
biological process
establishment of RNA
localization
regulation of development
nuclear transport
nuclear export
RNA transport
nucleic acid transport
chromatin assembly
mRNA transport
protein splicing
negative regulation of
cellular metabolism
intein-mediated protein
splicing
biopolymer metabolism
regulation of progression
through cell cycle
macromolecule metabolism
nucleobase, nucleoside,
nucleotide and nucleic acid
transport
peptide transport
glycoprotein metabolism
cellular macromolecule
metabolism
primary metabolism
Table B.2-continued.
172
solute:cation symporter activity
0.0338
0.0037
0.0053
0.023
0.0176
0.0004
0.0104
0.0103
0.0267
0.0037
0.0318
0.0063
0.0024
0.0037
0.0037
0.0464
0.0104
0.0104
0.0439
0.025
0.0292
peptidase activity
solute:sodium symporter activity
eukaryotic initiation factor 4E
binding
0.0198
0.0096
0.0395
0.0104
0.0052
0.0395
173
Table B.2-continued.
polyol metabolism
metabolism
negative regulation of
translational initiation
physiological process
protein metabolism
0.0104
0.0129
0.0031
0.0334
0.0065
173
174
phosphoric ester hydrolase activity
motor activity
acyltransferase activity
UDP-glycosyltransferase activity
0.0056
0.0056
0.0056
0.0056
174
heat shock protein binding
0.0056
0.01
0.0056
0.0461
0.0092
0.0126
0.0149
0.0421
acid phosphatase activity
acetyltransferase activity
0.0245
0.0285
N-acyltransferase activity
0.0092
Molec Funct
transferase activity, transferring
groups other than amino-acyl groups
transferase activity, transferring acyl
groups
protein tyrosine phosphatase activity
0.0056
cell surface
0.0305
P-value
unfolded protein binding
0.0056
0.0209
chemotaxis
phosphate transport
establishment of
protein localization
induction of positive
chemotaxis
cellular protein
metabolism
cellular
macromolecule
metabolism
positive regulation of
positive chemotaxis
regulation of positive
chemotaxis
positive regulation of
chemotaxis
regulation of
chemotaxis
positive chemotaxis
regulation of body
fluids
coagulation
protein polymerization
response to external
stimulus
taxis
protein localization
locomotory behavior
Cell Comp
small ribosomal
subunit
0.0439
0.0439
P-value
protein transport
dre-miR-202*
Biol Proc
Table B.2-continued.
0.0228
0.0203
0.0447
0.0277
0.017
0.0305
0.0203
0.0302
0.0078
0.0015
0.0328
0.0228
P-value
175
behavior
hemostasis
blood coagulation
protein metabolism
Table B.2-continued.
0.0135
0.0126
0.0126
0.0247
175
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BIOGRAPHICAL INFORMATION
John H. Malone received a B.S. in Wildlife and Fishereies Sciences from Texas
A&M University and an M.S. in Zoology from the University of Oklahoma. He will be
a postdoctoral fellow at the National Institues of Health starting September 2007.
225