Copyright Ó 2007 by the Genetics Society of America
DOI: 10.1534/genetics.107.079715
tint Maps to Mouse Chromosome 6 and May Interact With a Notochordal
Enhancer of Brachyury
Jiang I. Wu,*,1 M. A. Centilli,* Gabriela Vasquez,* Susan Young,*,2 Jonathan Scolnick,*,3
Larissa A. Durfee,* Jimmy L. Spearow,† Staci D. Schwantz,*,4
Gabriela Rennebeck*,5 and Karen Artzt*,6
*Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas,
Austin, Texas 78712-1064 and †Department of Neurobiology, Physiology and Behavior,
University of California, Davis, California 95616
Manuscript received July 31, 2007
Accepted for publication August 10, 2007
ABSTRACT
At the proximal part of mouse chromosome 17 there are three well-defined genes affecting the axis of
the embryo and consequently tail length: Brachyury, Brachyury the second, and the t-complex tail interaction
(T1, T2, and tct). The existence of T1 and tct in fact defines the classical ‘‘t-complex’’ that occupies 40 cM
of mouse chromosome 17. Their relationship to each other and various unlinked interacting genes has
been enigmatic. The tint gene was the first of the latter to be identified. We report here its genetic
mapping using a microsatellite scan together with outcrosses to Mus spretus and M. castaneous followed by a
subsequent testcross to T, T1, and T2 mutants. Surprisingly, tint interacts with T2 but not with T1. The
implications of our data suggest that T2 may be part of the T1 regulatory region through direct or indirect
participation of tint.
A
combination of classical genetics, gene cloning,
and experimental embryology has revealed that
neural tube defects in mice and, by implication, in humans are a developmentally heterogeneous group of
malformations ( Juriloff and Harris 2000; Copp et al.
2003). This heterogeneity and contributing environmental factors have been some of the reasons for the
sporadic nature of these conditions. Furthermore, an
expanding body of evidence indicates that neural tube
development is a multigenic process that may involve
several independently segregating genes (Estibeiro
et al. 1993; Copp 1994; Helwig et al. 1995; Greco et al.
1996; Doudney and Stanier 2005). The combination
of Brachyury (T ) and tct is one of the oldest and most
We dedicate this article to the memory of our dear colleague Lori
Flaherty (1946-2006).
1
Present address: Howard Hughes Medical Institute, Stanford University,
Stanford, CA 94305.
2
Present address: Genetics, Genomics and Development Division of the
Department of Molecular & Cell Biology, UC Berkeley, Berkeley, CA
94720.
3
Present address: Salk Institute, 10010 North Torrey Pines Rd., La Jolla,
CA 92037.
4
Present address: Department of Gene and Cell Medicine, Mount Sinai
School of Medicine, New York, NY 10029.
5
Present address: Greehey Children’s Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78225.
6
Corresponding author: Institute for Cellular and Molecular Biology,
University of Texas, 1 University Station, A4800, Austin, TX 78712-1064.
E-mail: [email protected]
Genetics 177: 1151–1161 (October 2007)
penetrant models for this developmental defect (Park
et al. 1989) but has been incompletely understood.
The t complex located on proximal third of mouse
chromosome 17 is characterized by four inversions that
prevent recombination between mutant and wild-type
chromosomes (Figure 1, striped bars). Every t-bearing
chromosome trapped in the wild in Europe and the
Americas always contains at least one recessive embryo
lethal (Silver 1981). In fact, in the days before mouse
knockout technology, most of the known recessive
embryo lethals in mice were mapped to the t-complex.
None of them are cloned yet because of their complicated genetics. The net effect of the inversions is that
t-haplotypes act like the balancer chromosomes of Drosophila locking up the entire region against recombination. However, exceptional recombination can occur
at the rate of 1/1000 because of inverted repeats. Other
remarkable features of t-haplotypes are male transmission ratio distortion making t’s look like a virus in terms
of population genetics. In fact, t-haplotypes are found
in 10% of wild mice (Ardlie and Silver 1996).
T is a deletion (see Table 1 for all genes mentioned)
that includes a semidominant mutation that has been
found in only non-t-carrying wild-type chromosomes. It
causes a short tail in heterozygotes and is lethal in
homozygotes. Affected embryos die shortly after gastrulation, presenting a complete loss of the posterior mesoderm due to primitive streak defects. Differentiation of
anterior portions of the notochord is also disrupted
followed by its complete absence in posterior portions
1152
J. I. Wu et al.
Figure 1.—Diagram of the proximal third of
chromosome 17. The striped chromosome represents inverted regions in t-haplotypes and the
solid circle, the centromere. Various markers
are shown: H-2, the mouse major histocompability complex; tf, tufted, a visible hair growth pattern
mutation; various lethals; and D17Mit170, a
marker near the distal end of the T deletion.
See text for explanation of T1, T2, and tct.
of the embryo (Gruneberg 1958; Yanagisawa et al.
1981).
In the mouse, studies have shown that, although the
Brachyury phenotype is dependent on the presence of a
mutation mapping to the T/t locus, its severity can be
profoundly affected by changes in genetic background
(Wittman and Hamburgh 1968; Mikova and Ivanyi
1974). This suggests that the Brachyury gene product
might interact with multiple factors during embryo development that can affect the variability at which the
‘‘T pathway’’ functions.
Finally, and most important for this study, every wild
trapped t-haplotype by definition carries a mutation
proximally known as tct (t-complex interaction factor) (see
Figure 1). tct as studied in exceptional recombinants
without a linked lethal has no phenotype on its own
even in homozygotes. The classical identification of a
t-haplotype is that in compound heterozygotes (T/tct),
it produces a tailless rather than a short-tailed mouse.
Thus, tct interacts with the classical T deletion.
Great effort has gone into the analysis of the relationship between T and tct ( Justice and Bode 1988). The
interaction between these two mutations suggests that
the lesions are in the same gene. However, genetic analysis of this phenomenon has been immensely complicated by the fact that tct is carried exclusively by all
t-haplotypes that contain multiple inversions (Howard
et al. 1990) that have historically made recombinational
analysis impossible. Over the years, the assessment remained that either T and tct were two alleles at a single
locus or mutations at two closely linked loci (Rennebeck
et al. 1995, 1998). tct is still not cloned.
The cloning of the T gene (Herrmann et al. 1990)
did not resolve the one vs. two gene question because
the t-haplotype copy of T has not been shown to have a
mutation.
The Brachyury gene, hereafter designated as T1 (Table
1), is the founding member of the T-box gene family
of transcriptional factors (Papaioannou 1997; Showell
et al. 2004). T1 is a classical transcriptional activator that
is localized to the nucleus, binds DNA, and functions cell
autonomously (Kispert and Herrmann 1993; Wilson
et al. 1993; Conlon et al. 1996). The N-terminal region of
the protein contains a rather large DNA-binding core
followed by an activation domain at the C terminus. The
finding of repressor domains together with activation
domains has generated some debate since the former has
not been conserved among vertebrate species (Kispert
et al. 1995). Brachyury homologs have been identified in
many different organisms: Xenopus laevis (Xbra), zebrafish
(ntl ), Halocynthia (As-T ), and Ciona (CiBra and CsBra)
(Smith et al. 1991; Schulte-Merker et al. 1994; Yasuo
et al. 1996; Corbo et al. 1997). Comparison of their
function has shown that Brachyury’s role in morphogenesis and mesodermal cell fate is evolutionarily conserved.
Curiously, subtle phenotypic differences among Brachyury
mutations in different species suggest that its regulation,
despite being considered conserved, requires different
molecular players (Halpern et al. 1993).
A second Brachyury gene, named ‘‘Brachyury the
second’’ (T2), was identified and cloned (Rennebeck et al.
1995). T2Bob is the only mutation of this gene identified so
far. It was found by the analysis of a transgenic insertion
that is known to map 12 kb upstream of the first T1 gene on
the same (1) DNA strand. The original T mutation has
been shown to be a deletion of about 200 kb with the T1
gene located approximately in the middle (Herrmann
et al. 1990) and is now known to delete both the T1 and
T2 genes. T1 and T2 do not compliment each other or tct.
The phenotype of T2/T2 is similar to T1 but is less affected
in all measurable ways. The embryological defect in T2Bob
starts more caudally than in T1 embryos and is mostly
limited to the notochord.
The genome sequencing in mice revealed that there
are no genes directly telomeric to T1; however, in addition to T2, there are several coding units centromeric
that could also be included in the original T deletion.
These genes include chemokine receptor 6 (Ccr6); brain
protein 44-like (Brp44l ); SFT2 domain containing 1 (Sft2d1),
a SNARE-like protein believed to be required for traffic
Ruvinsky (2002)
Agulnik (1998)
Agulnik (1999)
This article
Not cloned
QTL
QTL
Marker for distal end of Del(17)T
9: 32 cM
9: 57.695–57.696
15: 82.468–82.469
17: 8.54
Chr
Chr
Chr
Chr
Silver (1981)
This article
Abdelkhalek(2004)
Lyon (1956)
Harrison (2000)
Not cloned, only in t-haplotypes
Not cloned
Point mutation in the homeobox
Abnormal hair growth, not cloned
Transcription factor
17: 4.02 cM
6: 81.09–88.09
6: 85.38–85.39
17: 13.3 cM
2: 70.28–70.27
Chr
Chr
Chr
Chr
Chr
Rennebeck (1998)
Rennebeck (1999)
Transcription factor, hypermorph
Transgene insertion
Chr 17: 8.27–8.28
Chr 17: 8.23–8.26
Herrmann (1990)
Deletion .200 kb
Chr 17: 8.04–8.54
Brachyury the first
Brachyury the second
t complex tail interaction
t-interacting factor
Notochord homolog (Xenopus laevis)
tufted
trans-acting transcription factor 5
abnormal feet and tail
brachyury modifier 1
brachyury modifier 2
DNA segment, Chr 17, MIT 170
T1
T2
tct
tint
Noto
tf
Sp5
Aft
brm1
brm2
D17Mit170
Del(17)T, deletion on Chr 17,
Brachyury
T1Wis, Brachyury Wisconsin
T2Bob, Brother of Brachyury,
T2Tg(H2K)1Art
Only existing allele
Only existing allele
Nototc, truncate
None
Sp5tm1Rbe, targeted mutation 1,
Rosa Beddington
Only existing allele
QTL
QTL
Not relevant
Brachyury
T
Map position (Chr: Mb)
Allele symbol, name(s)
Gene name
Gene symbol
Summary of genes mentioned
TABLE 1
Notes
Reference
tint Maps to Mouse Chromosome 6
1153
through the Golgi complex (Banfield et al. 1995); and
two genes of unknown function, 4930506C21Rik and
9630019k15Rik. Although the T1 and T2 genes map physically close to one another, they are unrelated in sequence.
For decades, simple maintenance of exceptional
proximal tct stocks constituted an unheralded enhancer
screen. Homozygous tct/tct, without the presence of a
lethal, produces exclusively normal tailed progeny; however, the appearance of a single short-tailed offspring
led to the identification of a new mutation, tint (Artzt
et al. 1987). tint is unlinked to T as measured by independent segregation of the visible marker tufted (tf ).
It was the first non T/t complex, non-chromosome 17
tail interacting factor that by itself has no phenotype in
homozygotes but interacts with great specificity with
both the original T deletion and tct.
We report here the genetic mapping of tint. A microsatellite scan was used together with outcrosses to Mus
spretus and M. castaneous followed by a subsequent testcross to T, T1, and T2 mutants. Surprisingly, tint interacts with T2Bob but not with T1Wis. The implications of
such interaction suggest that T2 may be part of T1 regulation through indirect or direct participation of tint.
MATERIALS AND METHODS
Mice: The inbred strain homozygous for tint is designated
tint/Art (Artzt et al. 1987) and has been brother sister mated
since 1986 (more than F96 generations). However, this strain
had many genetic inputs including random bred and wild
mice and thus their microsatellite genotype was unknown.
tint/tint have normal tails because the gene is only evident
in compound with either T or tct from the t complex on
chromosome 17. To produce a heterozygous F1, tint/tint mice
were crossed to a ‘‘non inbred’’ subline of M. spretus (Spr/
Jls-RP), which took origin in Spain and was maintained by the
late Verne Chapman at Roswell Park until 1990 when it was
transferred to the JLS colony at the University of California
at Davis. The more commonly used SPRET/Ei strain was
obtained from the Jackson Laboratory and was used in later
experiments as a control for microsatellite mapping. Cast/Ei
also came from Jackson. BTBRTF/Art (hereafter referred to as
BTBR) was originated by L. C. Dunn and transferred to the
colony of the late D. Bennett and K. Artzt. In preliminary
microsatellite typing BTBR was found to be very similar, but
not identical, to tint/Art. truncate (tc) homozygotes were
kindly provided by Achim Gossler (Medizinische Hochschule,
Hannover, Germany). The Brachyury mutant (Twis) was a gift
from Virginia Pappaionnou (Columbia University, New York).
Genotyping: DNA samples were isolated from tail or ear
clippings using a protocol adapted from Sambrook et al.
(1989) and normalized to concentrations of 50 or 100 ng/ml.
Primer sets were synthesized by IDT (Coralville, IA) and
checked for polymorphisms. Briefly, primers were 59 endlabeled with 32P using T4 polynucleotide kinase (Invitrogen,
Frederick, MD) as follows: primer mix, 5 pmol final concentration; 53 forward reaction buffer, 1 ml; T4 polynucleotide
kinase (10 units/ml), 0.5 ml; 32P dATP (10 mCi/ml), 1 ml; water
to 10 ml. The reaction was incubated at 37° for 30 min and then
used for PCR analysis. PCR reactions were prepared according
to a protocol adapted from HotMaster Taq DNA polymerase
(Eppendorf AG, Hamburg, Germany). Specifically, a reaction
mix cocktail was prepared containing the following final
1154
J. I. Wu et al.
TABLE 2
Mapping crosses and tail phenotypes at birth
spretus cross
P
F1
Chr 17
Chr 6
1Ttint/1Ttint
1Ttint/1Tsp 1Tsp
int/int
int/sp
Progeny
Test cross
Chr 17
Chr 6
3
3
1Tsp 1Tsp/1Tsp 1Tsp
T/1Tsp 1Tsp
sp/sp
BT/BT
Phenotype
Expected (%)
Observed no.
1Ttint/1TBT
1TBT/1Tsp 1Tsp
1Ttint/1TBT
1TBT/1Tsp 1Tsp
T/1Tsp 1Tsp
T/1Tsp 1Tsp
sp/BT
sp/BT
int/BT
int/BT
sp/BT
int/BT
Normal tail
Normal tail
Normal tail
Normal tail
Normal tail
Short tail
T/1Ttint
T/1Ttint
sp/BT
int/BT
Short tail
Tailless
Total
Observed (%)
62.5
478
67
25
148
21
12.5
100
87
713
12
100
castaneus cross
Chr 17
P
F1
Ttint
Ttint
1 /1
1Ttint/1Tcas
Chr 6
int/int
int/cas
Progeny
Test cross
Ttint
TBT
3
3
Phenotype
1 /1
1TBT/1Tcas
1Ttint/1TBT
1TBT/1Tcas
T/1Tcas
cas/BT
cas/BT
int/BT
int/BT
cas/BT
Normal tail
Normal tail
Normal tail
Normal tail
Short tail
T/1Ttint
T/1Tcas
cas/BT
int/BT
Short tail
Tailless
T/1Ttint
int/BT
Tailless
Total
Chr 17
Chr 6
Tcas
cas/cas
BT/BT
Expected (%)
Observed no.
Tcas
1 /1
T/1TBT
Observed (%)
50
269
51.7
25
136
26.2
25
115
22.1
100
520
100
Gene symbols: 1Ttint, wild type of T from the tint strain; 1Tsp, wild type of T from spretus (for clarity, we assume
it is duplicated); 1TBT, wild type of T from BTBRTF; 1Tcas, wild type of T from castaneous; T, the original Bracyhury
deletion. The chromosome 6 column refers to the tint locus, mutant ‘‘int,’’ wild type from spretus ‘‘spret,’’ from
castaneous ‘‘cast,’’ or from BTBRTF ‘‘BT.’’
concentrations per reaction: 13 HotMaster Taq buffer with
Mg21, 2.5 mm Mg21; dNTP mix (Roche Diagnostics, Mannheim, Germany), 0.2 mm; 32P dATP-labeled primer mix, 0.08
mm; DNA template, 50 or 100 ng; HotMaster Taq DNA
polymerase, 2.5 units; water to 25 ml. PCR conditions were as
follows: 94°, 2 min; (94°, 20 sec; 56°, 20 sec; 65°, 45 sec) 35
cycles; 65°, 5 min. Optimal annealing temperatures for individual primer sets varied from 54.5° to 58.0°. A 20-ml aliquot
of the PCR product was loaded onto nondenaturing polyacrylamide gels containing 13 TBE and ranging from 6 to 10%
polyacrylamide to resolve the products. Gels were run at 40 V
overnight or 150 V for 3 hr, transferred to filter paper, dried,
and transferred to a Kodak (Rochester, NY) BioMax MS intensifying phosphor screen for 1–2 hr. The gels were scanned
using a Bio-Rad (Hercules, CA) molecular imager FX.
Our initial strategy in the genome-wide microsatellite scan was
to assay with markers that were spaced 30 cM apart. Later we
used the MB positions defined by the mouse genome mapping
project (Ensembl v.45, June 2007). All of the markers used are
listed in supplemental Table S1 at http://www.genetics.org/
supplemental/. When linkage was found, more closely spaced
markers were used to refine the tint map position. All recombination was tested in females.
RESULTS
Design of crosses: The design of the mapping crosses
for tint is shown in Table 2. We first outcrossed tint/Art
to spretus or castaneous and then test crossed the F1 to
the original Brachyury deletion for the presence of tint.
Of the testcross progeny, all normal tails were discarded
at birth since they segregate genotypes that are phenotypically indistinguishable and therefore are not
informative.
The remaining mice that receive T in the testcross
are of two genotypes with respect to tint and these are
expected in equal numbers. They segregate for 1tint vs.
Brachy
Brachy
Long Brachy
Brachy
Brachy
Brachy
Short Brachy
Short Brachy
Curved Brachy
Short Brachy
Short Brachy
Short Brachy
Short Brachy
Short Brachy
Short Brachy
Tailless stump
Tailless stump
Tailless stump
Tailless stump
Tailless stump
Tailless
Tailless
Tailless
Tailless
Tailless
Tailless
Tailless
Tailless
Visual
phenotype
Probe
48
46
39
37
24
24
14
10
6–9
4
2
2
2
2
2
1
1
1
1
1
0
0
0
0
0
0
0
0
84
80
68
65
42
42
24
17
13
7
4
4
4
4
4
2
2
2
2
2
0
0
0
0
0
0
0
0
C
C
C
C
C
I
I
I
I
I
I
I
I
I
NDb
I
I
I
I
I
I
I
I
ND
I
I
I
C
C
C
C
C
C
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
ND
I
I
I
I
I
I
I
C
C
C
C
C
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
ND
I
C
C
C
C
C
I
I
I
I
I
I
I
I
I
I
I
C
I
I
I
I
I
I
I
I
I
I
I
C
C
C
C
C
I
I
I
I
I
I
I
I
I
I
I
C
I
I
I
I
I
I
I
I
I
I
I
C
C
C
C
C
I
I
I
I
I
I
I
I
I
I
I
C
I
I
I
I
I
I
I
I
I
I
ND
C
C
C
C
C
I
I
I
I
I
I
I
I
I
I
I
C
I
I
I
I
I
I
I
I
I
I
I
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
C
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
I
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
Tail
D6mit386 D6mit22 D6mit323 D6mit40 D6mit263 D6mit131 D6mit365 D6mit104 D17mit170
81.94
85.09
86.87
88.08
88.95
90.32
94.07
110.93
8.54
length (mm)a % normal tail Mb position:
The two recombinants are 17 and 28; marker megabase positions Ensembl Dec 2006. I, tint; C, castaneous type.
a
At the age measured (2 weeks), normal tail length was 57 mm.
b
ND, not done.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Mouse
Results of ‘‘blind’’ typing in castaneous test cross
TABLE 3
tint Maps to Mouse Chromosome 6
1155
1156
J. I. Wu et al.
Figure 2.—All recombinants for chromosome 6.
M. spretus (S) on chromosome 17. For the markers close
to where tint resides, all the short-tail mice are S and
all the tailless mice are expected to be tint (I) type
(except for recombinants). For all other chromosomes,
the short-tail mice and tailless mice should consist of
an equal number of S and I types. A similar situation
applies to the M. castaneous cross where markers were
designated as (C) types.
There are two noteworthy considerations in the design of this cross. Since the tint parental strain and the
T/1 parent used for the test crosses are on similar
genetic backgrounds, we expect them to be nonpolymorphic with most microsatellite markers. Thus, for
most markers, an S- or C-specific PCR product was necessary. In the case where I and BTBRTF/Art were polymorphic, the I product could be used to type regardless
of the other band. Furthermore, it is important to
distinguish S and I type in most cases. The litter sizes
of these backcross mice averages 11–12 pups so that
normal tail mice can be culled at birth and there are still
reasonable numbers of test cross progeny to genotype.
Phenotypic overlap problems: In the spretus testcross,
an excess over the predicted number of normal tailed
were initially recorded: 63 vs. the 50% expected (Table 2).
Thus, it was apparent early on that either M. spretus had
a duplication of Brachyury (T1) and the closely linked
Brachyury the second (T2) makes twice as much product
or M. spretus contains dominant suppressors of short
tail. We tested this hypothesis by crossing T heterozygotes to M. spretus and found that only normal-tailed
offspring were produced (n ¼ 48), thus confirming one
of the possibilities above. M. castaneous crossed to T/1
produced short- and normal-tailed offspring in the expected proportions (50:50). Thus, we decided to map
tint in both M. spretus and M. castaneous crosses. In both
test crosses to T/1, there was a dearth of tailless progeny. For this, it was necessary to type each individual
mouse to be certain it was carrying the T deletion. We
ascertained a microsatellite maker (D17mit170) near
the distal deletion breakpoint that was polymorphic for
BTBRTF mice segregating for T or 1T. This marker
became a useful tool for genotyping T. However, after
eliminating all the noncarriers of T, there were still less
than the original expected numbers of tailless mice
born. It soon became clear that tint is on chromosome 6
(see below). In the castaneous cross where there was no
tint Maps to Mouse Chromosome 6
Figure 3.—LOD scores for both models. Top, original phenotype; bottom, reclassified phenotype based on typing for T
and the tint region of chromosome 6.
genetic complication of a possible duplication, we
retrospectively classified all mice on the basis of having
the T deletion and an I (tint) or C (M. castaneous) genotype for the relevant region of chromosome 6. We tested
the feasibility of this hypothesis in the M. castaneous cross
by blindly typing an additional 28 mice from the testcross whose tails had been measured. They were typed
using several markers for carrying T and being I or C for
the relevant region of chromosome 6. In every case but
two, which turned out to be recombinants, the measured tail length correlated with the genotype (Table 3).
However, it was evident that there is phenotypic overlap
in the tail length range of 42% of normal. These mice,
including the two recombinants, were then included with
the castaneous cross typing results (Figure 2). When we
ran pair wise recombination fractions and LOD scores on
both models using R/qtl (Li et al. 2006), it appeared that
the retroactive classification based on the presence of T
and chromosome 6 genotype seemed robust (Figure 3).
Tint maps to chromosome 6: The major component
of the tint and T interaction maps to chromosome 6 in
both crosses. With respect to markers on chromosome
6, we examined 161 mice from M. castaneous and 111
mice from the M. spretus cross. Recombinants in the
critical regions are shown in Figure 2. Only some of the
1157
probes could be used in both crosses because not all of
them were dually polymorphic. Except for recombinants, all the tailless mice were I and all short-tailed mice
were C or S for the relevant region. Both crosses are in
agreement and the smallest possible region is defined
by recombinants in the M. castaneous cross marked by
D6mit386 at 81.09 Mb and D6mit40 at 88.1 Mb. This is
a relatively large region and we were unable to divide
it further using microsatellites. Regardless of this area
being gene dense with .30 genes, we have not made an
attempt to further define the interval but rather opted
for the candidate gene approach.
tint is not Noto: The genetic map in this region
contained at least one probable candidate. The truncated
(tc) mutation has now been cloned and renamed Noto
(notochord homolog, Xenopus laevis) and resides on chromosome 6 at 85.39 Mb (Pavlova et al. 1998; Abdelkhalek
et al. 2004). The phenotype of tc is recessive and not
completely penetrant. Nototc causes an interruption of
the notochord at E9.5 leading to a short or absent tail.
We tested Nototc for interaction with both tint and the T
deletion and found no more than the expected number
of short tails, suggesting no interaction between the
2 genes (n ¼ 49 tint; n ¼ 86 for T; data not shown). In
addition, the sequence of the three coding exons of
Noto in tint/tint showed no significant differences in
BTBRTF/Art, M. spretus, or M. castaneous.
tint interacts with T2 and not with T1: Since T2 was
not identified at the time tint was discovered to interact
with the original T deletion, it seemed reasonable to retest
separately the interaction of tint with T1 and T2. This was
accomplished by crossing tint/tint to T1Wis and T2Bob independently. T1Wis is a hypermorph allele of T1 that causes
the carrier to have no tail at all (Herrmann 1991). Any
interaction with tint would therefore be expected to
have less than no tail or sacroccygeal spina bifida, usually
detected by a blood blister on the rump (Park et al. 1989).
Since some of these might not be viable, all pregnant
females were inspected for newborns twice daily. In cases
of severe spina bifida, there might even be a loss of T1Wis
embryos. T2Bob carriers consistently have a 50% tail length
on the BTNTTF/Art background; thus, any reduction
in tail length was easily recognized. Much to our surprise,
it was clear that tint interacted with T2 and not with T1
(Table 4). Whereas T1Wis maintains a relatively constant
ratio of normal tails to tailless offspring in all three crosses,
the T2Bob mutation shows a dramatic shortening of the
tail in successive crosses to tint. The proportion of tailless
mice rose from 0 in the control to 17% in the F1 cross
and to 33% in the backcross. Thus, remarkably, the real
interaction of tint is with T2 and not T1.
DISCUSSION
tint maps to chromosome 6 and interacts with T2:
tint maps between D6mit386 at 81.1 Mb and D6mit40 at
88.1 Mb on chromosome 6. This region contains .30
1158
J. I. Wu et al.
TABLE 4
tint interaction with T1Wis and T2Bob
Control: T1wis, 1/1 3 1/1, 1/1
Control: T2Bob, 1/1 3 1/1, 1/1
Phenotype
No.
%
No.
%
Normal tail
Short tail 50%
Short tail 25%
Stump ,10%
Tailless
Total
69
0
5
6
55
135
51.1
0
3.7
4.4
40.7
100
91
84
0
0
0
175
52.0
48.0
0
0
0
100
F1: T1wis, 1/1 3 tint/tint
F1: T2Bob, 1/1 3 tint/tint
Phenotype
No.
%
No.
%
Normal tail
Short tail 50%
Short tail 25%
Stump ,10%
Tailless
Total
150
0
0
0
136
286
52.4
0
0
0
47.6
100
168
1
98
23
62
352
47.7
0
27.8
6.5
17.6
100
BC1: T1wis, 1/tint 3 tint/tint
BC1: T2Bob, 1/tint 3 tint/tint
Phenotype
No.
%
No.
%
Normal tail
Short tail 50%
Short tail 25%
Stump ,10%
Tailless
Total
304
0
0
0
255
560
54.2
0
0
0
45.5
100
150
0
44
17
104
315
47.6
0
14
5.4
33
100
genes and among these Noto is the only particularly
striking candidate. Initially named truncate, Nototc shows
decreased tail length, truncated notochord, abnormal
somite, and sclerotome formation (Theiler 1959;
Abdelkhalek et al. 2004). While Nototc shows a defect
in axial development, we saw no interaction when
crossed to the T deletion. Furthermore, sequence of
noto’s three exons in tint, M. castaneous, and M. spretus
mice did not show any significant changes. Thus, Noto is
excluded as a candidate for tint.
We were surprised to discover tint’s interaction is with
T2 rather than with T1. The transgenic insertion in T2Bob
was thought not to affect the T1 gene, its defined
regulatory region, its expression in the primitive streak,
or in the few notochord cells present (Rennebeck et al.
1995). At E9.5, T2Bob embryos show quite a distinct
phenotype from T1Wis and Tembryos, being less affected
in all measurable ways. The embryological defect in
T2Bob starts more caudally than in T1 embryos and is
mostly limited to the notochord. Furthermore, the
presence of floor plate in T2Bob indicates that notochord
cells were present, at least in the initial process of the
notochord formation, but they were not maintained in
later stages. Therefore, the interaction of tint with T2
might modulate the development of axial or paraxial
mesoderm or both.
Two other genes and two QTL have been reported to
interact with T, but in all cases their interaction has been
tested against the original T deletion that deletes both
T1 and T2. In 2000, the laboratory of Rosa Beddington
identified the trans-acting transcription factor (Sp5) mapping to chromosome 2 at 70.2 Mb. It is similar to tint in
that it has no homozygous phenotype and genetically
interacts with the T deletion (Harrison et al. 2000).
Ruvinsky et al. (2002) showed that the dominant
mutation Abnormal feet and tail (Aft) located on chromosome 9 at 32 cM also interacts with the T deletion. In
this case, the compound heterozygote mice display
kinky tails and syndactyly in the hindlimbs. The two T
interacting QTL (brm1 and brm2) described by Agulnik
et al. (1998) are on chromosomes 9 and 15, respectively.
Thus, in all four cases cited above, it is not clear whether
these genes are interacting with T1 or T2. Perhaps an
important lesson learned from the results presented
here is that establishing genetic interactions using a
deletion, however small, is not an appropriate strategy.
Is the notochord enhancer of Brachyury embedded in
T2? Numerous studies have revealed that Activin A
(TGFb family) and basic FGF are the main inducers of
T1 expression (Smith et al. 1991; Amaya et al. 1993;
Labonne and Whitman 1994). Transgenic studies
in mice and manipulation experiments in frogs have
tint Maps to Mouse Chromosome 6
1159
Figure 4.—Conserved genomic sequences in mouse T2 are circled in red.
identified a minimal region (400 bp upstream of the
initiation site) of the T1 gene that is responsible for the
mesoderm and notochord expression in frogs. However, in mice, a larger region is probably required since
there was no rescue of the Brachyury homozygous
phenotype (Stott et al. 1993). This minimal region
contains an E-box and two canonical Lef1/Tcf binding
sites that, in mice, are responsible for partially rescuing
the Brachyury phenotype (Stott et al. 1993; Clements
et al. 1996). Differences among vertebrate species vouch
for the complexity of the regulation of Brachyury. Other
binding sites such as goosecoid, Mix1 (now named
Mixl1), and Otx2 that function as repressors of Xbra
expression have being identified in frogs (Latinkic
et al. 1997). Interestingly, these regulatory regions are
not present in the mouse promoter, suggesting that the
regulatory elements may vary between vertebrate species (Yamaguchi et al. 1999). Although most of these
studies show the regulation of Brachyury in the mesoderm, regulatory elements controlling its expression in
the notochord are not completely understood in mice.
Although T2 appears to be a bona fide gene in all
respects, it is possible that it is important solely in
rodents, since no conservation has been identified in
any other vertebrate. In contrast, genome comparisons
have shown that there are highly conserved sequences
between mice and humans that map to the distal end
of T2 (Figure 4). As mentioned above, the original
mutation T2Bob is a transgenic mutation that has inserted
200 kb between the last two exons of T2 followed by a
deletion of 3 kb of the intronic sequence (Rennebeck
et al. 1998).
1160
J. I. Wu et al.
We cannot exclude the possibility that T2 is an important gene in mice and rats, or that it is probably allelic
with tct since the tct copy of T2 has a significant amino
acid change at the carboxyl terminus (Rennebeck et al.
1998). However, taken together, our results lead us to
the following alternate hypothesis from our original interpretation of T2. It is possible that the prevailing defect in the T2Bob mutants is that its transgenic insertion at
the 39 end interferes with a previously unidentified T1
notochord enhancer. After the insertion that putative
enhancer would be separated from T1 by 200 kb instead of 12 kb or may have even been deleted with the 3-kb
intronic sequence.
While a full understanding of the genes involved
in the vertebrate neural tube formation is still evolving,
the impact of tint interaction with T2Bob can be further
appreciated by the identification of Brachyury’s notochord regulatory sequences.
This work was supported by National Institutes of Health Grant
HD10668 (to K.A.).
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Communicating editor: T. R. Magnuson