The Effects of Retinoic Acid on
Spermatogonial Stem Cell Differentiation In
Vitro
Sabrina Sicilia
Department of Medicine
Division of Experimental Medicine
McGill University
Montreal, Quebec, Canada
November 2013
A thesis submitted to McGill University in partial fulfillment of the
requirement of the degree of Masters of Science
Sabrina Sicilia © 2013
i
TABLE OF CONTENTS
Abstract ...................................................................................................... page VI
Résumé ..................................................................................................... page VIII
Acknowledgements ..................................................................................... page X
Contributions by author ........................................................................... page XI
Abbreviations ........................................................................................... page XII
Chapter 1: Introduction .............................................................................. page 1
The embryonic origin of male germ cells .......................................... page 1
Stages in the production of mature spermatozoa ............................... page 4
Spermatogonial stem cells ............................................................... page 10
Factors involved in the maintenance of SSCs ..................... page 11
Factors involved in the differentiation of SSCs .................. page 16
The role of retinoic acid in the differentiation of SSCs ................... page 19
Isolating SSCs – elucidating their specific markers ........................ page 23
Donor derived spermatogenesis in a recipient testis ....................... page 26
In vitro SSC culturing ...................................................................... page 27
Research hypothesis ........................................................................ page 30
Research objectives ......................................................................... page 31
Chapter 2: Materials and Methods .......................................................... page 33
Establishment of a Germ-Cell Cell Line ......................................... page 33
Immunocytochemistry against RARα – no treatment ..................... page 34
Cell Chain Counting ........................................................................ page 36
Cluster forming activity assay ......................................................... page 38
Spermatogonial Transplantation ...................................................... page 40
In vitro Apoptosis Assay ................................................................. page 43
Flow Cytometric Analysis of Apoptosis ......................................... page 44
ii
Microarray ....................................................................................... page 45
Quantitative real-time RT-PCR ....................................................... page 46
ICC against LRPAP1 and Stra8 – with treatment ........................... page 48
Chapter 3: Results ..................................................................................... page 49
Expression pattern of RARα ............................................................ page 49
Overview of cluster cells’ morphology post-treatment ................... page 51
Overview of cluster cells’ morphology post-treatment ....... page 51
Cell chain kinetics post-treatment ....................................... page 52
Quantification of SSCs after RA treatment ..................................... page 56
Cluster forming activity assay ............................................. page 56
Transplantation Assay ......................................................... page 58
Transplantation – cultured with feeders .................. page 58
Feeder-Free Transplantation .................................... page 60
Cell death after RA treatment .......................................................... page 62
Gene expression pattern after RA treatment ................................... page 67
Protein expression pattern after RA treatment ................................ page 72
STRA8 expression after treatment .......................... page 72
LRPAP1 expression after treatment ........................ page 73
Chapter 4: Discussion and Future Experiments ..................................... page 77
Annex .......................................................................................................... page 84
Annex 1: Microarray data – Genes upregulated .............................. page 84
Annex 2: Microarray data – Genes downregulated ......................... page 91
Annex 3: Response to Thesis Evaluator’s Comments ................... page 105
References ................................................................................................ page 108
iii
LIST OF FIGURES
Chapter 1: Introduction
Figure 1
Spermatogenesis .................................................................... page 7
Figure 2
Cellular bridges ..................................................................... page 8
Figure 3
Organization of the seminiferous tubule epithelium ........... page 12
Figure 4
Retinoic acid in the post natal testis .................................... page 20
Figure 5
Spermatogonia Markers ....................................................... page 26
Figure 6
In vitro germ cell cluster culture ......................................... page 28
Chapter 2: Materials and Methods
Figure 7
Cell chain counting ............................................................ page 37
Figure 8
Morphology of cell chains ................................................. page 38
Figure 9
Cluster forming activity assay ........................................... page 40
Figure 10
In vivo donor derived GFP colonies .................................. page 43
Chapter 3: Results
Figure 11
RARα expression pattern in cluster cells ............................ page 50
Figure 12
Cluster cells’ morphology after RA treatment .................... page 51
Figure 13
Kinetics of various cell chain formations ............................ page 54
Figure 14
Total number of cell chains formed post-treatment ............ page 56
Figure 15
Cluster forming activity assay post-treatment ..................... page 58
Figure 16
Transplantation assay with feeder culture conditions ......... page 60
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Figure 17
Transplantation assay with feeder-free culture .................... page 62
Figure 18
In vitro apoptosis 24 hours post-treatment .......................... page 65
Figure 19
In vitro apoptosis 48 hours post-treatment .......................... page 66
Figure 20
Quantification of cellular apoptosis post-treatment ............ page 67
Figure 21
STRA8 expression pattern post-treatment ........................... page 73
Figure 22
LRPAP1 expression pattern post-treatment ........................ page 75
Figure 23
LRPAP1 expression pattern of cell chain post-treatment .... page 76
Chapter 4: Discussion and Future Experiments
Figure 24
Study summary figure ......................................................... page 79
LIST OF TABLES
Chapter 2: Materials and Methods
Table 1
LRPAP1, RARα, and STRA8 concise ICC protocol .......... page 36
Table 2
qPCR forward and reverse primer sequence ....................... page 47
Chapter 3: Results
Table 3
Microarray and qPCR data .................................................. page 71
v
ABSTRACT
The fundamental biological functions of spermatogonial stem cells (SSCs) is to
support sperm generation and as such the propagation of genetic material to future
generations. Like other stem cells, SSCs are defined by their ability to self-renew
and also generate daughter cells which are committed to differentiation (J. M.
Oatley & Brinster, 2008). It has been shown that retinoic acid (RA) is
quintessential in spermatogenesis; its role is most clearly seen in its absence
which results in infertility (Snyder, Small, & Griswold, 2010). In vivo RA acts on
undifferentiated
spermatogonia
transitioning
them
into
differentiating
spermatogonia, consequently RA can serve as a key inducer of spermatogonial
differentiation in vitro (Zhou et al., 2008).
The objectives of my study were to assess the effects of RA on aggregates of
undifferentiated spermatogonia in vitro (clusters), elucidate genes implicated in
RA induced differentiation of SSCs and determine markers that coincide with the
transition for SSC commitment to differentiation. I hypothesize that RA induces
the differentiation of SSCs in vitro and further that SSCs can respond rapidly to
RA.
Results obtained support the hypothesis that RA rapidly induces SSC
differentiation in vitro. These results included: observation of distinctive cell
chain morphology in vitro post-treatment that is reflective of spermatogonia
differentiation, SSC quantification confirms a decrease SSCs post-treatment and
vi
gene expression analysis reveals an increase in spermatogonial differentiation
markers and a decrease of SSC specific markers following treatment. In addition
to providing insight into RA directed SSC differentiation, gene expression
analysis also revealed genes which were upregulated rapidly following RA
treatment. These genes included LRPAP1 and Stxbp5; two genes have not been
previously described in the context of SSC differentiation and may be novel
markers for early SSC differentiation. These results indicate that the SSC-based,
in vitro experimental paradigm that I used in this study provides an effective
platform to further dissect the process of early SSC differentiation and its
mechanisms.
vii
RÉSUMÉ
La fonction biologique fondamentale des spermatogonies (SSCs) est de soutenir
la production de sperme et la propagation du matériel génétique aux générations
futures. Comme les autres cellules souches, SSCs sont définies par leur capacité
de se renouveler et de générer des cellules filles ayant débuté leur différenciation
(J. M. Oatley & Brinster, 2008). Il a été démontré que l'acide rétinoïque (RA) est
essentiel à la spermatogenèse, car son absence cause une infertilité (Snyder et al.,
2010). RA peut agir sur les spermatogonies indifférenciées pour induire leur
différenciation et donc peut servir comme un inducteur clé de la différenciation
des spermatogonies in vitro (Zhou et al., 2008).
Les objectifs de mon étude étaient d'évaluer les effets de la RA sur les agrégats de
spermatogonies indifférenciées in vitro (clusters), d’élucider les gènes impliqués
dans la différenciation des SSCs induite par RA et de valider des marqueurs qui
permettraient de déterminer la transition de différenciation. Mon hypothèse est
que l’RA induit la différenciation des SSCs in vitro et aussi que les SSCs peuvent
répondre rapidement à l’RA. Ce qui peut suggérer que les SSCs existent comme
une population cellulaire hétérogène contenant une proportion de cellules déjà
prédisposée pour répondre à l'induction de la différenciation.
viii
Les résultats obtenus supportent l'hypothèse que l’RA induit rapidement la
différenciation in vitro des SSCs. Les résultats obtenus après le traitement
incluent: une morphologie distincte des chaînes de cellules, une perte de SSCs,
une augmentation des marqueurs de différenciation des spermatogonies et une
diminution des marqueurs spécifiques des SSCs. En plus de confirmer que l’RA
dirige la différenciation des SSCs, l'analyse globale de l'expression des gènes a
également démontré que certains ont une expression accrue après le traitement
avec l’RA. Ces gènes incluent LRPAP1 et Stxbp5; deux gènes qui n'ont pas été
décrits précédemment dans le cadre de la différenciation des SSCs et peuvent être
de nouveaux marqueurs montrant le début de la différenciation des SSCs.
ix
ACKNOWLEDGEMENTS
I would like to thank my supervisor Dr Makoto Nagano, for not only being a
wonderful mentor who am I grateful to have had the opportunity to learn from,
but for also having a lot of patience with me and my decision to begin my
Doctorate in Veterinary Medicine during the completion of my Masters.
Thank you to both, Xiangfan Zhang and Dr Jonathan Yeh, for being so willing to
answer my questions and help me with projects. You were both two of my most
precious resources throughout my Masters. Also, thank you to my thesis
committee members, Dr Daniel Bernard, Dr Daniel Dufort and Dr Geoffrey
Hendy for your guidance, critical questions and valuable suggestions throughout
the completion of my Masters.
Thank you to Sabrina Robert for taking the time to help me translate and correct
my ‘résumé’.
Finally I want to thank my fiancé, Domenic Ciarlillo, for his continual support
throughout my Masters and also for encouraging me to continue moving forward
those days when the completion of my Masters seemed nowhere in sight.
x
CONTRIBUTIONS BY AUTHORS
Transplantation was performed by Xiangfan Zhang, a laboratory assistant in The
Nagano Laboratory. Cell sorting was performed by McGill University’s Flow
Cytometry and Cell Sorting Facility. Microarray analysis was performed by
Genome Quebec. Quantitative PCR analysis was a joint effort between Dr
Jonathan Yeh and myself.
xi
ABBREVIATIONS
4',6-diamidino-2-phenylindole
DAPI
A aligned
Aal
A paired
Apr
A single
As
Basic fibroblast growth factor/Fibroblast growth factor 2
bFGF/FGF2
Blood-testes barrier
BTB
Bone morphogenetic protein
BMP
Bovine serum albumin
BSA
Cluster forming activity
CFA
Days post partum
DPP
Deleted in azoospermia-like
DAZL
Dimethyl sulfoxide
DMSO
Enhanced green fluorescent protein
EGFP
Fibroblast growth factor
FGF
Follicule stimulating hormone
FSH
GDNF-family receptor α1
GFRA1
Glial cell line-derived neutrotrophic factor
GDNF
Glucocorticoid-induced leucine zipper
GLIZ
Glyceraldehyde 3-phosphate dehydrogenase
GapDH
Green fluorescent protein
GFP
Haematopoietic stem cell
HSC
Helix-loop-helix
HLH
xii
Human embryonic stem
hES
Immunocytochemistry
ICC
Induced pluripotent stem
iPS
Inhibitor of DNA binding 4
ID4
LDL receptor-related protein associated protein 1
LRPAP1
Low-density lipoprotein
LDL
Neurogenin 3
Ngn3
Neutral buffered formalin
NBF
Spermatogenesis and oogenesis specific HLH 1
SOHLH1
Paraformaldehyde
PFA
Phosphate buffered saline
PBS
Primordial germ cell
PGC
Promyelocytic leukaemia zinc finger
PLZF
Quantitative real time RT-PCR
qPCR
Retinoic acid
RA
Retinoic acid receptor
RAR
Retinoid X receptor
RXR
Sal-like protein 4
SALL4
Spermatogonial stem cell
SSC
Stem cell
SC
Stem cell factor
SCF
Stimulated by retinoic acid gene 8
STRA8
Vitamin A deficient
VAD
xiii
CHAPTER 1
INTRODUCTION
The Embryonic Origin of Male Germ Cells
Murine gonads arise from a thickening of the coelomic epithelium but the germ
cells which populate the embryonic gonads, primordial germ cells (PGCs), arise
from another region within the developing embryo and will eventually migrate
into the gonad. For mice the formation of PGCs begins at about embryonic day
6.5, when the posterior proximal epiblast, under the influence of morphogens:
bone morphogenetic protein (BMP) 4 and BMP8b both which originate from the
extraembryonic ectoderm, will aid in generating a population of cells which
express fragilis, a transmembrane protein (Gilbert, 2006; Harikae, Miura, &
Kanai, 2013). At the center of the cluster of fragilis expressing are cells that also
express blimp1, a transcriptional inhibitor; it is this initial blimp1 expressing cell
population, consisting of 10 – 100 cells, which will give rise to PGCs (Gilbert,
2006).
At about embryonic day 9 the PGCs then begin their migration through the
hindgut towards the developing gonads and by embryonic day 11.5 the PGCs
begin to populate the gonads (Harikae et al., 2013). Upon reaching the gonads, the
PGC population is comprised of between 2500 – 5000 cells. The proliferation of
1
this cell population during the migration stage is influenced by paracrine factors
secreted by cells encountered en-route to the gonads; These factors include:
fibroblast growth factor (Fgf) 7 and stem cell factor (SCF); two factors whose
absence results in the apoptosis of the PGC population (Gilbert, 2006).
Two forms of SCF are known: a membrane-bound form and a secreted form
(Gilbert, 2006). SCF, in its soluble form, is primarily secreted by the cells that
line the migration route that the PGCs follow towards the gonads. Soluble SCF
contributes to the migration, survival, and maturation of PGCs, as well as the
colonization and development of the developing gonads (Gilbert, 2006; Høyer,
Byskov, & Møllgård, 2005; Stoop et al., 2008). SCF binds to its receptor, the cKit receptor tyrosine kinase. Upon binding of the ligand, c-Kit receptors
homodimerize, which allows for the activation of the intrinsic tyrosine kinase
activity of the receptor and also creates binding sites for a number of intracellular
signalling molecules, including those that are essential for germ cell development
(Høyer et al., 2005; Sette, Dolci, Geremia, & Rossi, 2000; Stoop et al., 2008). cKit expression is not limited to PGCs but is also present on cells of postnatal
testes: pre-meiotic and meiotic germ cells and Leydig cells (Høyer et al., 2005;
Stoop et al., 2008). The survival of PGCs is dependent on the secretion of antiapoptotic factors such as SCF which are produced only by cells along the PGCs’
migratory route; due to the localization of survival factors, PGCs that migrated to
an ectopic location undergo apoptosis (Pesce, Farrace, Piacentini, Dolci, & De
Felici, 1993). Following migration into the gonad, germ cell development
2
undergoes different patterns dependent on the sex of the individual. Briefly,
female PGCs undergo meiosis and become arrested in the meiotic prophase until
they are recruited for oocyte maturation, while male PGCs become mitotically
arrested until after birth (Gilbert, 2006).
At about the same time at which the PGCs begin to populate the gonads, the
gonads also undergo a state of organ sex determination, thereby resulting in the
formation of either ovaries or testes. It is principally the sex determining gene,
Sry, a transcription factor, that induces male sex determination (Harikae et al.,
2013). I will focus on male germ cell development from here on.
In the embryonic testes, the gonadal sex cords engulf the PGCs which are termed
gonocytes upon arrival in the testis (Gilbert, 2006; A. Kolasa, K. Misiakiewicz,
M. Marchlewicz, & B. Wiszniewska, 2012). In addition to the germ cell
population, the embryonic testes are also colonized by a supporting somatic cell
lineage, the precursors of Sertoli cells. These precursors undergo a migratory
process from the coelomic epithelium between embryonic day 10 – 11.5 (Harikae
et al., 2013). In mice Sry is secreted from embryonic day 10.5 to 12 by the
supporting cells of the undifferentiated gonads, which later become Sertoli cells
(Adams & McLaren, 2002). The precursors of the Sertoli cells differentiate to
mature Sertoli cells and constitute the seminiferous tubule epithelium (Gilbert,
2006).
3
Mature Sertoli cells in postnatal testes are in direct contact with the resident germ
cells. There is also the presence of tight junctions between neighboring Sertoli
cells which allows for the formation of the blood-testes barrier (BTB); this barrier
is vital in the establishment of a very controlled micro-environment critical for
germ cell development and maturation (Hasegawa & Saga, 2012).
After birth, testicular somatic cells continue to provide factors that are critical for
the proliferation and survival of the male germ cells (D. G. de Rooij & Russell,
2000). Once again, SCF, a factor which is expressed by Sertoli cells in the testes,
is found to be essential. In postnatal testes, the membrane bound form of SCF,
rather than the secreted form, exhibits prominent effects (Dirk G. De Rooij,
2009). Gonocytes migrate to the basement membrane of the seminiferous
epithelium soon after birth and start proliferating by 3 days post partum (dpp).
The gonocytes form type A single (As) spermatogonia, the most primitive type of
spermatogonia, at around 6 dpp (D. G. de Rooij & Russell, 2000; Gilbert, 2006;
Phillips, Gassei, & Orwig, 2010; Wu et al., 2009).
Stages in the Production of Mature Spermatozoa
Spermatogenesis is the progressive formation of mature spermatozoa from SSCs;
it is a tightly regulated progression of mitotic and meiotic events which are
4
defined by morphological characteristics and molecular processes. (Abutarbush,
2008; Hess & Franca, 2009; J. M. Oatley & Brinster, 2008). Spermatogenesis can
be simply defined by three distinct phases: spermatogonial proliferation, meiosis
of spermatocytes and spermiogenesis of haploid spermatids (J. M. Oatley &
Brinster, 2008; Phillips et al., 2010). In mice, the spermatogenesis takes about
34.5 days to complete, and from a single As spermatogonium 4096 spermatids
may arise (Gilbert, 2006; Agnieszka Kolasa, Kamila Misiakiewicz, Mariola
Marchlewicz, & Barbara Wiszniewska, 2012). This physiological process occurs
in a very coordinated manner. There are cyclic cellular changes over time at
diverse segments of the seminiferous tubules; germ cells proceed through their
differentiation, from spermatogonia to spermatocytes to spermatids finally
developing into spermatozoa in an organized fashion, moving from the basal
lamina to the lumen of the seminiferous tubules (Gilbert, 2006; Oakberg, 1956;
Phillips et al., 2010). This cyclic change, composed of multiple different stages,
can be observed histologically and is referred to as the “spermatogenic cycle”. In
the mouse, the spermatogenic cycle has 12 distinct stages (Phillips et al., 2010).
The first events in spermatogenesis that shows clear morphological differentiation
is the formation of Apr spermatogonia from As spermatogonia (Zhou et al., 2008).
Mitotic divisions progress resulting in the sequential formation of A-aligned (Aal)
spermatogonia, type A1 through to type A4 spermatogonia, intermediate
spermatogonia and type B spermatogonia, refer to Figure 1 (Zhou et al., 2008). As
shown in Figure 2, male germ cells are unique because the cells do not undergo
5
complete cytokinesis upon cell division and consequently the dividing cells will
remain connected by a cytoplasmic bridge thereby resulting in the formation of a
syncytia (Gilbert, 2006). The cytoplasmic bridges between adjacent cells are
about 1µm in diameter and allow for the passage of molecules, ions and mRNA,
all of which are important in the synchronous progression of germ cell
development (Gilbert, 2006; Phillips et al., 2010). As a result every mitotic
division of spermatogonia results in the progressive lengthening of the cell chain,
starting with Apr spermatogonia which consists of two linked cells
The spermatogonia resulting from mitotic divisions can be categorized,
morphologically, into different populations: As spermatogonia, the proliferating
cell population which includes Apr and Aal spermatogonia, and the differentiating
cell population of which type A1 - A4 spermatogonia, intermediate spermatogonia
and type B spermatogonia belong (Agnieszka Kolasa et al., 2012). Historically,
As, Apr and Aal spermatogonia are characterized as undifferentiated spermatogonia
whereas succeeding spermatogonia are termed differentiating spermatogonia, with
type A1 spermatogonia representing the first differentiated spermatogonia (D. G.
de Rooij & Russell, 2000). Spermatogonial proliferation, a process which lasts
approximately eight days, will end with the formation of primary spermatocytes,
which arise from type B spermatogonia (Gilbert, 2006; Phillips et al., 2010).
6
FIGURE 1| Spermatogenesis. Primary cell types formed through spermatogenesis.
7
Cytoplasmic Bridge
As Spermatogonium
Apr Spermatogonia
Aal Spermatogonia
FIGURE 2| The presence of a cytoplasmic bridge is first observed upon the
formation of Apr spermatogonia and will persist throughout the spermatogonia,
spermatocyte and spermatid cellular stages of spermatogenesis.
In the second stage of spermatogenesis, meiosis of spermatocytes, a primary
spermatocyte will undergo a first meiotic division resulting in the formation of a
secondary spermatocyte. A second meiotic division follows, resulting in the
formation of haploid spermatids (Abutarbush, 2008). It is during meiotic prophase
that homologous recombination takes place, thereby allowing for genetic
recombination and consequently genetic diversity (Mackey et al., 1997). The
successive meiotic divisions are completed in approximately 13 days, after which
spermiogenesis will occur (Gilbert, 2006).
8
Spermiogenesis is the final process of spermatogenesis and results in the
formation of mature spermatozoa as a consequence of the transformation of
spermatids (Abutarbush, 2008). During spermiogenesis, spermatids undergo
various modifications such as: the fabrication of an acrosomal vesicle, the
formation of a flagellum and flattening and condensation of the nucleus to
approximately 1/50th of its initial volume (Gilbert, 2006; Hess & Franca, 2009).
These modifications lead to the formation of a more elongated cell with the
capability of cellular motility and fertilization. After about 13.5 days
spermatogenesis will terminate with the release of the mature spermatozoa into
the lumen of the seminiferous tubules (Gilbert, 2006; Zhou et al., 2008).
Finally it is essential for male fertility that spermatozoa be available continuously
in sufficient quantity. This is possible due to the presence of the spermatogenic
wave. Germ cell differentiation does not occur simultaneously throughout the
seminiferous tubules but rather occurs only at some locations; different stages of
the spermatogenic cycle are distributed throughout various seminiferous tubule
segments, allowing for the production of spermatozoa on a cyclical basis along
the seminiferous tubules therefore resulting in the consistent production of mature
spermatozoa (Abutarbush, 2008).
9
Spermatogonial Stem Cells (SSCs)
SSCs, like other stem cells (SCs), are defined by their long-term ability to selfrenew and also generate daughter cells committed to differentiation, thereby
maintaining life-long sperm production (J. M. Oatley & Brinster, 2008).
Historically all AS spermatogonia were considered to be SSCs, however, it is now
known that SSCs represent only a fraction of AS spermatogonia, in turn AS
spermatogonia are likely comprised of progenitor cells as well. It is estimated that
SSCs likely account for less than 20% of the AS spermatogonia population (M. C.
Nagano & Yeh, 2013).
It is assumed that a symmetric division of SSCs results in either two SSCs or two
differentiated cells, while an asymmetric division leads to one SSC and one
committed cells (D. G. de Rooij & Russell, 2000). An asymmetric division is
generally considered to be the basic mechanism of the maintenance of the SC
pool, since no change in SC number after this cell division. However, the same
consequence can be reached if two different types of symmetric division occur in
a coordinate manner so that the number of SCs and committed cells can be
balanced within a given system (in the testis in this case). It is also assumed that
symmetric differentiating division results in the production of A pr spermatogonia
that is destined to differentiate into spermatozoa (Agnieszka Kolasa et al., 2012).
As such, the manner of SSC division is believed to be critical for consequent
10
functional outcome of daughter cells and for controlling SSC fate decision.
Nonetheless, the type of SSC division is not clearly defined and far from being
understood at present. In fact, the current understanding about the “SC state” and
“the process of SSC fate control” can be considered chaotic, given a recent report
that postulated that some Apr spermatogonia may maintain a ‘SC potential’ (Jan et
al., 2012; Yoshida, Nakagawa, Hara, & Kitadate, 2010). Since “SSC
differentiation” and “SSC self-renewal” are two sides of a coin, it is important to
understand when SSC activity is indeed lost. If the timing of this event is
understood, then SSCs could be more definitively defined.
Factors Involved in the Maintenance of Spermatogonial Stem Cells
SSCs reside in a specific place in the seminiferous epithelium; they are tethered to
laminin, a component of the basal lamina, via specific cell adhesion molecules
known as integrins, more specifically integrins α6 and integrins β1; integrins α6
and integrins β1 are both found on the surface of SSCs (Shinohara, Avarbock, &
Brinster, 1999; Yoshida et al., 2010). SSCs are surrounded by Sertoli cells and
peritubular myoid cells (Fig. 3). This environment is termed the “SSC niche” (J.
M. Oatley & Brinster, 2008), and will be further described in this section.
The testis is composed of somatic cells as well as germ cells, as depicted in Figure
3. There are three major types of testicular somatic cells: 1) peritubular myoid
cells, which provide structural support as well as growth factors to the resident
11
germ cells; 2) Leydig cells, which produce growth factors and testosterone; and 3)
Sertoli cells, which primarily contribute to the creation of a micro-environment in
addition to providing growth factors, support, and allow for the exchange nutrition
and metabolites with developing germ cells (Abutarbush, 2008; Hermo, Pelletier,
Cyr, & Smith, 2010; Hess & Franca, 2009; Jan et al., 2012; Johnson, Thompson
Jr, & Varner, 2008; Agnieszka Kolasa et al., 2012). Supported by the somatic cell
environment, SSCs allow for the generation of numerous types of germ cells in
the testis: spermatogonia, spermatocytes, spermatids and spermatozoa. Although
SSCs make up only 0.03% of the total mouse testis cell population, this small cell
proportion is vital in the propagation of genetic information from one generation
to the next (Phillips et al., 2010).
FIGURE 3| Structural organization of the seminiferous tubule epithelium. Note the
presence of various germ cell types including: spermatogonia, spermatocytes and
mature sperm, in addition to the localization of the Sertoli cells. Image source:
(Gilbert, 2006), figure 19.2.
12
The microenvironment in which SSCs reside is called a ‘niche’. The SSC niche
components are assumed to include Sertoli cells, Leydig cells, myoid cells, and
the basement membrane (Hermo et al., 2010). Sertoli cells, in general, play a
critical and direct role in the maintenance of spermatogenesis, as each of Sertoli
cell is responsible for supporting a finite number of germ cells. Sertoli cells are
also believed to play an important role in the maintenance and differentiation of
SSCs; they exert their influence on spermatogenesis and SSCs in part by secreting
various growth factors and mediating hormonal actions (Gilbert, 2006).
Follicle stimulating hormone (FSH), a hormone produced by the pituitary gland,
links the endocrine system to spermatogenesis (Abutarbush, 2008). FSH
stimulates the Sertoli cells to produce glial cell line-derived neurotrophic factor
(GDNF) a key regulator of SSC self-renewal (Kanatsu-Shinohara et al., 2003;
Meng et al., 2000). GDNF binds to its receptor, GDNF-family receptor α1
(GFRα1), localized on the membrane of germ cells. (Gilbert, 2006; Jan et al.,
2012).
Binding of GDNF to GFRα1 mediates the activation of the c-RET
receptor which, in turn activates downstream signaling cascades that favor SSC
self-renewal (Gilbert, 2006). This regulation was demonstrated a study which
showed that transgenic mice over-expressing GDNF had an accumulation of
undifferentiated spermatogonia whereas haploinsufficiency of GDNF results in a
depletion of germ cells over time (Jan et al., 2012). Moreover, GDNF has been
shown to be required to maintain and expand the SSC population in vitro
(Kanatsu-Shinohara et al., 2003). Furthermore, the expression of GFRα1 is
13
reported to be restricted mostly to As and Apr spermatogonia, suggesting that this
receptor may be used as a marker for undifferentiated spermatogonia (Yang, Wu,
& Qi, 2013).
Another factor which is predominantly expressed in As and Apr spermatogonia is
NANOS2 (Suzuki, Sada, Yoshida, & Saga, 2009; Yang et al., 2013). NANOS2 is
a RNA-binding protein and is known to be an important regulator in the
maintenance of SSCs (Lodish, 2008; Sada, Hasegawa, Pin, & Saga, 2012). Overexpression of nanos2
results
in
an accumulation of undifferentiated
spermatogonia whereas a knockout of nanos2 results in the depletion of
undifferentiated spermatogonia (Jan et al., 2012). Moreover, it has recently been
shown that NANOS2 acts downstream of GDNF to allow for the maintenance of
SSCs (Sada et al., 2012).
Promyelocytic leukaemia zinc finger (PLZF) protein belongs to the zing finger
protein family and is a transcriptional repressor (Fahnenstich et al., 2003; Jan et
al., 2012). PLZF exerts control over the cell cycle and has been shown to be
involved in the self-renewal and maintenance of SSCs (Costoya et al., 2004;
Fahnenstich et al., 2003). Its expression is restricted to undifferentiated
spermatogonia, i.e. As through Aal spermatogonia (Costoya et al., 2004). PLZF
stimulates the transcription of Redd1, which represses the action of mTorc1,
thereby promoting the expression of both GFRα1 and c-Ret. The increased
14
presence of both these receptors, GFRα1 and c-Ret, on undifferentiated
spermatogonia increases their receptiveness to GDNF which in turn allows for the
maintenance of the SSC pool (Jan et al., 2012). Once again the importance of
PLZF in SSC maintenance and self-renewal is most evident in its loss; loss of its
activity results in age-dependent SSC depletion, ultimately resulting in sterility
and testicular atrophy (Costoya et al., 2004).
Basic fibroblast growth factor (bFGF or FGF2), secreted by the Sertoli cells is
needed to SSC self-renewal and maintenance. FGF2 exerts a direct effect on SSCs
but also acts on Sertoli cells by regulating the expression of GDNF thereby also
indirectly affecting SSCs (A. Kolasa et al., 2012; Phillips et al., 2010).
Foxo1 has recently been revealed to be expressed in gonocytes as well as
undifferentiated spermatogonia. Foxo1 belongs to the larger family of Foxo
transcription factors. The expression of this family of transcription factors is
usually under the influence of growth factors; they exert an influence on cell
cycling as well as cell death. Foxo1 is predominantly regulated by PI3K signalling
and upon activation targets Ret expression. Upon ablated expression of Foxo1,
SSCs show a decreased ability to maintain their population (Goertz, Wu,
Gallardo, Hamra, & Castrillon, 2011).
15
It also appears that some factors may be reserved for a subset of SSCs. For
instance, the first wave of spermatogenesis appears to be initiated by neurogenin 3
(Ngn3)-negative SSCs whereas ensuing waves of spermatogenesis appears to be
mediated by Ngn3-expressing SSCs (Hess & Franca, 2009).
Inhibitor of DNA binding 4 (ID4) has been shown to be found solely in A s
spermatogonia. A decrease in ID4 expression reduces SSC maintenance, and
complete loss of ID4 results in progressive SSC depletion and eventual sterility
(Jan et al., 2012).
Factors Involved in the Differentiation of Spermatogonial Stem Cells
In order for spermatogenesis to occur various factors are required for the
transition from an undifferentiated spermatogonium to a differentiating
spermatogonium, Aal spermatogonia to A1 spermatogonia; this event in
spermatogenesis is characterized as a critical step of spermatogonial
differentiation (Hess & Franca, 2009). Key factors for this transition include: SCF
and retinoic acid (RA) among others (Hess & Franca, 2009; A. Kolasa et al.,
2012; Snyder et al., 2010; Zhou et al., 2008).
16
As described previously, the SCF–c-Kit signalling pathway is essential for PGC
migration. In postnatal testes this signalling pathway is critical for the survival
and proliferation of spermatogonia. c-Kit (receptor) is primarily expressed by
mitotically active spermatogonia and shows the highest expression in type A
spermatogonia (Sette et al., 2000). More specifically it has been noted that c-Kit
expression is highest in late Aal spermatogonia, right before its transition to A1
spermatogonia (Jan et al., 2012). Accordingly, SCF–c-Kit signaling is known to
assist the Aal – A1 transition. A point mutation in SCF results in the death of type
A spermatogonia at this transition point and consequently the loss of
spermatogenesis (Berruti, 2004; Dirk G. de Rooij, Okabe, & Nishimune, 1999).
As such, mice carrying this mutation are sterile due to a block in spermatogenesis
which is characterized by a decrease in spermatogonia proliferation and an
increase in spermatogonia apoptosis (Berruti, 2004). This suggests that not only is
c-Kit necessary for supporting the differentiation of spermatogonia but it is also
necessary for the proliferation of differentiating spermatogonia as well as their
survival (A. Kolasa et al., 2012).
Similar to c-Kit expression in the adult mouse testes, CYCLIN D2 is expressed at
the transition point between Aal and A1 spermatogonia and all the way through
until the formation of spermatocytes. The upregulation of CYCLIN D2 is
indicative of its subsequent need in the progression through meiosis I and
consequently the succession of differentiation (Jan et al., 2012; Zhou et al., 2008).
17
There also appears to be a redundancy with the presence of factors which were
essential for PGC survival. Like SCF and c-Kit, BMP4 appears to be implicated in
the differentiation of undifferentiated spermatogonia. BMP4 exerts this function
in part by promoting the expression of c-Kit (A. Kolasa et al., 2012).
Another factor which has been shown to be necessary for spermatogonia
differentiation is SOHLH1, Spermatogenesis and Oogenesis specific Helix-LoopHelix protein 1. SOHLH1’s expression is predominantly found in Aal through to
type B spermatogonia. It has been suggested that SOHLH1 exerts its influence on
spermatogonial differentiation by opposing self-renewal mechanisms. Moreover
impaired function of SOHLH1 results in infertility due to the inability of
spermatogonia to undergo the initial stages of differentiation (Jan et al., 2012).
Foxo1 was previously described as being important for maintenance of
undifferentiated spermatogonia via its modulation of Ret expression (Goertz et al.,
2011). Accordingly there are factors which are present in differentiating
spermatogonia which influence the function of Foxo1 thereby favoring the
differentiation of spermatogonia. One such factor is glucocorticoid-induced
leucine zipper (GLIZ). GLIZ inhibits the nuclear translocation of Foxo1 and in so
doing favors differentiation over SSC self-renewal (Ngo et al., 2013).
18
Interestingly some factors responsible for SSC maintenance may also influence
SSC differentiation; GDNF may be one such factor. Although GDNF plays a
predominant role in the maintenance of SSCs, the formation of Aal spermatogonia
in-vitro has been attributed to the an interaction between GDNF and
NEUREGULIN-1 (Jan et al., 2012). Consequently GDNF appears to aid in
maintaining a balance between the maintenance of the SSC pool and
spermatogonial differentiation.
As previously mentioned SSCs reside in a niche situated at the base of the
seminiferous epithelium. As such the SSC niche is in close proximity to blood
vessels (A. Kolasa et al., 2012). This association with the testicular vascular
network allows for factors found in the blood to influence SSC maintenance and
self-renewal as well as SSC differentiation; one such factor is vitamin A (Zhou et
al., 2008).
The Role of Retinoic Acid in the Differentiation of Spermatogonial Stem
Cells
RA is the active metabolite of vitamin A (Zhou et al., 2008). RA signalling has
been typically described as being a paracrine signalling system (Griswold,
Hogarth, Bowles, & Koopman, 2012; Raverdeau et al., 2012). In such a system a
cell in close proximity to a target cell will secrete a factor which will diffuse to
19
the neighboring target cell resulting in activation of signalling cascade in the
target cell (Lodish, 2008). Such is the case in the testis, as depicted in Figure 4.
The Sertoli cells not only serve as a storage site for vitamin A and its oxidized
metabolite RA but also catalyze this oxidization (Griswold et al., 2012). The
availability of RA is further modulated by the presence of enzymes required for
vitamin A’s oxidation, as well as the presence of RA degradation enzymes. RA
degradation enzymes primarily include CYP26 enzymes which belong to the
cytochrome P450 enzyme family (Griswold et al., 2012; Snyder et al., 2010).
FIGURE 4| In-vivo in the post-natal testis RA is produced by neighboring Sertoli
cells and diffuses to SSCs to allow for the initiation of differentiation by
transitioning from Aal to A1 spermatogonia and furthermore induces pre-meiotic
expression of STRA8 in order to allow differentiated spermatogonia. Image source:
(Griswold et al., 2012), figure 1.
In order for a cell to be responsive to RA it must possess the appropriate
receptors. RA is a ligand for a nuclear receptor composed of two subunits that
20
form a heterodimeric complex: the retinoic acid receptor (RAR) and the retinoid
X receptor (RXR); 3 isoforms (α, β and γ) are present for each subunit (Raverdeau
et al., 2012). Upon ligand-receptor binding, the complex then binds to RA
response elements (RARE) of target genes and modulates gene expression
(Griswold et al., 2012).
RA has been shown to be essential for spermatogenesis and its ability to induce
differentiation in various cell types has already been noted (Snyder et al., 2010).
Mice deprived of a dietary intake of vitamin A, also referred to as a vitamin A
deficient (VAD) diet, have an accumulation of undifferentiated spermatogonia
(Griswold et al., 2012; Zhou et al., 2008). The accumulation of undifferentiated
spermatogonia is attributed to a block in differentiation occurring at the Aal to A1
transition point (Griswold et al., 2012; Raverdeau et al., 2012). Such a block in
spermatogenesis evidently results in infertility. However, administration of RA
restores fertility by overcoming the Aal to A1 transition point block which results
in the re-initiation of spermatogenesis (Snyder et al., 2010; Vernet et al., 2006).
Recent studies have demonstrated that RA is capable of inducing differentiation
of undifferentiated spermatogonia in vitro in the absence of Sertoli cells. Such a
finding suggests that undifferentiated spermatogonia are capable of responding
directly to RA without an aid from Sertoli cells (Zhou et al., 2008).
21
Further, a mutation to the RARα, the major RAR isoform found in the mouse
testis, results in a similar phenotype to that seen in rodents fed with VAD diets.
The restoration of fertility in VAD mice by RA administration and the infertility
observed in RARα-/- mice collectively suggest that RA binds to RARα and
triggers downstream differentiation cascades essential for spermatogenesis
(Chung, Wang, & Wolgemuth, 2009).
The ability of germ cells to undergo meiosis I is also dependent on the
responsiveness of germ cells to RA and this receptiveness is mediated by a protein
called Deleted in azoospermia-like (DAZL). DAZL knockout mice also show a
phenotype similar to VAD mice: an inability of Aal spermatogonia to differentiate
into A1 spermatogonia (Schrans-Stassen, Saunders, Cooke, & de Rooij, 2001). RA
promotes expression of Stimulated by retinoic acid gene 8 (STRA8), a factor that
is necessary for the commitment of meiosis progression (Griswold et al., 2012;
Jan et al., 2012; Zhou et al., 2008). However, this response is not seen in DAZL-/mice suggesting that DAZL can mediate the responsiveness of germ cells to RA.
(Zhang et al., 2011).
STRA8 expression is first noted in testes at 10 dpp (Griswold et al., 2012). This
first incidence of STRA8 expression coincides with the first wave of
spermatogenesis and more precisely when meiosis I of the first spermatogenic
wave occurs (Griswold et al., 2012; Raverdeau et al., 2012). In the testes of Stra8null mice mature spermatozoa are not produced due to an inability to initiate
22
meiosis, however spermatogonia do undergo the Aal to A1 spermatogonia
differentiation (Griswold et al., 2012; Zhou et al., 2008). Nonetheless, given that
the high expression of STRA8 in A and B spermatogonia, this protein has been
recognized as a marker of spermatogonial differentiation (Zhou et al., 2008).
These observations suggest that RA is quintessential in progression of events
critical for spermatogenesis. In summary, RA is produced by neighboring Sertoli
cells in vivo, as illustrated in Figure 4, and diffuses to spermatogonia to allow for
the initiation of differentiation from Aal to A1 spermatogonia while it also induces
pre-meiotic expression of STRA8 to promote meiotic entry of spermatogonia
(Griswold et al., 2012). There is a study reporting that RA also reduces SSC
numbers in vitro (Dann et al., 2008), suggesting that this factor may play a
broader role in spermatogenic regulation. It is of note, however, that RA’s actions
on SSCs have not been investigated in detail. In contrast to secreted factors that
promote SSC self-renewal, those that stimulate SSC differentiation are not well
known. Therefore, the potential of RA to act as a SSC differentiation factor makes
it an ideal tool to dissect the mechanism of SSC fate control.
Isolating Spermatogonial Stem Cells – Elucidating Their Specific Markers
Obtaining a pure population of SSCs would be an invaluable resource for the
study of SSCs, but this remains unattainable to date. Numerous markers, both
intracellular markers and surface markers, as shown in Figure 5, have been
23
examined; these markers have led to an enriched population of SSCs but not a
pure population. Some markers are known to be expressed in other SC types, such
as haematopoietic stem cells (HSCs). Past studies also show that SSC markers
appear to be conserved between different species, for instance between humans
and mice (A. Kolasa et al., 2012).
Some SSC surface markers have been determined. Thy1 (CD90), a glycosyl
phosphatidylinositol anchored glycoprotein of the Ig superfamily, is a protein
whose expression has been reported in multiple SC types. Thy1 was initially
revealed to be expressed on HSC and has also been shown to be expressed on
SSCs. Sorting for Thy1+ germ cells results in obtaining a cell population highly
enriched for SSCs (Kubota, Avarbock, & Brinster, 2003). Additionally isolating
testicular germ cells that express integrin β1 and integrin α6, both cell surface
receptors for extracellular matrix components, also allows for SSC enrichment
(Shinohara et al., 1999).
GFRα1 has been shown to be expressed on the surface of SSCs, however GFRα1
expression is not exclusive to SSCs but is also expressed on the surface of As, Apr
and Aal spermatogonia (Phillips et al., 2010). Indeed, no molecules have been
found exclusively in SSCs thus far, which is a part reason that SSC purification
has not been achieved.
24
It has also been shown that the negative selection for particular markers is
effective for SSC enrichment; such is the case for c-Kit. Since c-Kit expression
appears in Aal spermatogonia and persists until early spermatocytes, selecting cKit - testicular germ cells is an effective method to enrich for SSCs (M. C. Nagano
& Yeh, 2013).
Other studies have further aided in uncovering the surface phenotype of SSCs and
thus far the surface phenotype of SSCs has been described as α6-integrin+, β1integrin+, THY1+, CD9+, GFRα1+, CDH1+, αv-integrin-, c-Kit -, MCH-I- and
CD45- (Phillips et al., 2010).
Certain intracellular proteins have also been shown to be markers for SSCs. Such
markers include PLZF and Nanos2. PLZF and Nanos2’s expression, like that of
GFRα1, are not exclusive to SSCs but rather are expressed throughout As to Aal
spermatogonia (A. Kolasa et al., 2012; Phillips et al., 2010; Yang et al., 2013).
Therefore selecting for PLZF or Nanos2 would allow only for a cell population
enriched in SSCs. ID4, a transcriptional repressor, represents an intracellular
molecule which is exclusive to As spermatogonia; however selecting testicular
germ cells which express this protein once again leads to an enriched but not pure
population of SSCs (M. J. Oatley, Kaucher, Racicot, & Oatley, 2011). Ngn3 is
another molecule which is expressed in SSCs; however it has been noted that only
a small subfraction of SSCs are Ngn3+. This observation of heterogeneity of Ngn3
25
amongst SSCs has led some to suggest that perhaps Ngn3 represents a molecule
expressed in SSCs which are ‘primed’ to differentiate (M. C. Nagano & Yeh,
2013).
FIGURE 5| Numerous markers, both intracellular markers and surface markers, have
been characterized as being either present or absent for stem cells, progenitor cells and
differentiating spermatogonia. Image source (Phillips et al., 2010), figure 3.
Donor Derived Spermatogenesis in Recipient Testes
Determining whether a cell is a SC depends on that cell’s ability to regenerate
tissue structure and function upon transplantation into a non-functional tissue of
the same origin. Upon transplantation, the donor SC is required to self-renew in
order to restore a SC population in the tissue and must also generate daughter
26
cells committed to the differentiation to restore tissue structure and function (J. M.
Oatley & Brinster, 2008). Nearly two decades ago, it was shown that the
transplantation of a testis cell suspension, derived from genetically labelled donor
mice, into the testes of infertile host mice results in the regeneration of donorderived spermatogenesis and fertility restoration. Each donor SSC generates a
distinct colony within the recipient’s seminiferous tubules (J. M. Oatley &
Brinster, 2008). Such a long term reconstruction of spermatogenesis demonstrates
the presence of SCs in the donor testis cell suspension. Consequently the
transplantation is an unequivocal assay to assess whether a given suspension of
testis cells contains SSCs.
The In Vitro Spermatogonial Stem Cell Culturing
Using the transplantation assay, Kubota et al. (2003) demonstrated that Thy1+
cells from a mouse testis cell suspension contained nearly all SSCs. However this
population did not exclusively contain SSCs but was enriched about 30 fold for
SSCs compared to unselected testes populations (Kubota et al., 2003). Using a
SSC-enriched cell population, it has become possible to maintain and expand SSC
in vitro (J. M. Oatley & Brinster, 2008). Culturing Thy1+ testis cells in serum-free
culture conditions with defined growth factors results in the formation of germ
cell aggregates termed ‘clusters’ (Figure 6).
27
A
B
FIGURE 6| In vitro germ cell cluster culture system using GFP germ cells derived
from B6/GFP mice. A, bright field image of germ cell clusters. B, Image of germ
cell clusters captured using fluorescence microscopy. A and B were taken at 10x
magnification.
The essential growth factors required in this culture system are: GDNF, a factor
necessary for SSC self-renewal, soluble Gfrα1, a factor that facilitates binding of
GDNF to a co-receptor expressed on the SSC surface, and bFGF, a factor which
improves SSC self-renewal when added in combination with GDNF (Kubota et
al., 2003; J. M. Oatley & Brinster, 2008; Phillips et al., 2010). Transplantation of
these clusters into infertile mouse testes leads to regeneration of donor-derived
spermatogenesis in the host testes, thereby conferring the presence of SSCs in the
clusters. It is of note that the clusters are not solely composed of SSCs and the
majority of cluster cells are undifferentiated spermatogonia (J. M. Oatley &
Brinster, 2008).
28
Based on the SSC culture system, Yeh, Zhang, and Nagano (2007) recently
established a short-term assay for a semi-quantitative detection of SSCs, termed
cluster forming activity (CFA) assay. The basis of the CFA assay is the
correlation between the number of germ cell clusters formed and the number of
SSCs present in culture. It was shown that the number of clusters formed in
culture correlates with SSC numbers, as measured by transplantation (Yeh et al.,
2007). The CFA assay overcomes obstacles posed by the transplantation assay
such as: the 8 weeks time lapse required for data collection, the requirement for
skilled micro-injection and need for immunocompatibility between the donorrecipient subjects (Yeh et al., 2007). The CFA assay functions as a short-term, 1
week to obtain results, semi-quantitative method to assess SSCs in vitro (Yeh et
al., 2007). However, this assay cannot replace the transplantation assay, because it
does not detect the cells’ ability to differentiate and to regenerate complete
spermatogenesis.
29
REASEARCH HYPOTHSIS
Like other SCs, SSCs are defined by their ability to self-renew and also generate
daughter cells which are committed to differentiation. Although information
regarding their self-renewal and maintenance continue to accumulate,
mechanisms regarding SSC differentiation remain to be elucidated. This lack of
knowledge regarding SSC differentiation is a critical problem for understanding
SSC biology. It is known that RA plays a quintessential role in initiating the
transition of undifferentiated to differentiating spermatogonia and consequently
RA can serve as a key inducer of spermatogonial differentiation in vitro. It has
also been suggested that the SSC population may not be a homogenous one, but
rather a heterogeneous one (Yang et al., 2013). This may suggest that a
heterogeneous SSC population may comprise of some cells that are more ‘primed’
to respond to certain environmental cues whereas others may be more prepared to
contribute to the maintenance of the SC pool.
Based on our current knowledge regarding SSCs, I hypothesize that RA induces
the differentiation of SSCs in vitro and that SSCs may respond rapidly to
differentiation induction implying the possible existence as a heterogeneous SSC
population as alluded to in literature.
30
RESEARCH OBJECTIVES
The objectives of my study are to assess the effects of RA on aggregates of
undifferentiated spermatogonia in vitro (clusters) and elucidate genes implicated
in RA induced differentiation of SSCs; toward determining markers that identify
the transition for SSC commitment to differentiation.
Aim 1: Explore the expression pattern of RARα in undifferentiated
spermatogonia, 'cluster cells'

Immunocytochemisty against RARα on cluster cells
Aim 2: Examine the morphological changes that cluster cells undergo when
treated with RA

Cell chain counting
Aim 3: Examine if RA induces differentiation of SSCs, examine the extent of
differentiation, and explore molecular pathways regulated by RA induced
differentiation

Quantify SSCs after RA treatment using in vitro and in vivo SSC
functional assays

Verify if cell death increases after RA treatment using flow cytometry
and in vitro staining
31

Conduct microarray to assess if global gene expression is altered after
treatment with RA

Assess the expression of SSC and differentiation markers after RA
treatment using quantitative PCR (qPCR) and immunostaining
32
CHAPTER 2
MATERIALS AND METHODS
Establishment of a Germ-Cell Cell Line
Donor mice were either the progeny of C57BL/6 (B6) females and B6.129S7Gtrosa26Sor (ROSA26) males (The Jackson Laboratory), designated B6/ROSA
mice, or the progeny of C57BL/6 (B6) females and C57BL/6-Tg (CAG-EGFP)
males (The Jackson Laboratory), designated B6/GFP mice. B6/ROSA mice
express Escherichia coli lacZ transgene in all cell types. B6/GFP mice express
enhanced GFP (EGFP) under the control of a chicken beta-actin promoter and
cytomegalovirus enhancer in all cell types except erythrocytes and hair.
The establishment of the cluster culture, whether from a B6/GFP, as illustrated in
Figure 6, or B6/ROSA mouse strain, was undertaken as outlined by Yeh et al.
(2007). Briefly, a single cell suspension was prepared from the testes of 6 – 8 ddp
pups enzymatically digesting the testes tissue using collagenase I and IV,
followed by further digestion with trypsin and DNase. The cell suspension was
then filtered through a 40 μM mesh. The filtered cell suspension was subsequently
incubated with biotinylated rat anti-mouse CD90.2, also called THY1.2 (BD
PharmingenTM). The cells were next washed and incubation with dynabeads M280
Streptavidin (Invitrogen). The positive cell fraction was then seeded onto a layer
33
of mitotically inactive STO feeders at a concentration 2.5 x 104 cells/cm2. 500 µL
of culture medium per cm2 with the following growth factor concentrations: 40
ng/mL GDNF, 300 ng/mL GFRα1 and 1 ng/mL bFGF, was added to each of the
wells (Yeh et al., 2007). Cell culture plates were incubated at 37°C. Medium with
growth factors was changed every 3 days and cells were sub-cultured onto STO
feeders every 6 to 7 days, following trypsin digestion. Once cell cultured appeared
healthy and clusters of cells began to form (≈ 6 cells per cluster), typically by
passages 4 to 6, growth factor concentrations were reduced to the following for
continued culture maintenance: 20 ng/mL GDNF, 75 ng/mL GFRα1 and 1 ng/mL
bFGF; this concentration of growth factors will be hence forth referred to as
reduced growth factors. All animal procedures were approved by the Institutional
Animal Care and Use Committee of McGill University.
Immunocytochemistry
Immunocytochemistry (ICC) Against RARα – No Treatment
Antibodies used are listed in Table 1. Germ cell culturing occurred as outlined in
establishment of a germ-cell cell line. Medium was changed on day 3 of culture
and cultures were fixed on day 6 of culture. Cells were fixed overnight at room
temperature with 5% neutral buffered formalin (NBF), which was prepared by
removing half the medium contained within the well, 80 μL, and adding an equal
34
volume of 10% NBF. After overnight fixation, the 5% NBF solution was removed
and the cells were further fixed for 30 minutes at room temperature with 10%
NBF. After fixation was complete the wells were washed twice with PBS. Cells
were then permeabilized with a 1:4 DMSO to methanol solution at room
temperature for 5 minutes. Permeablization was followed by two rinses with deionized water. Non-specific binding was then blocked at room temperature for 30
minutes with 10% normal goat serum in a 1% bovine serum albumin (BSA)
dissolved in PBS solution. The blocking solution was removed and a primary
antibody solution, prepared with 1:200 rabbit anti-RARα antibody (SantaCruz,
SC- 551) in a 1% BSA solution, was added to the designated wells. The primary
antibody was incubated at 4°C overnight. For a negative control, no primary
antibody was added, only 1% BSA was added to a well. After primary antibody
incubation, the wells were washed twice with PBS for 5 minutes per wash. The
wells were then incubated with a secondary antibody solution, prepared with
1:250 Cy3 goat anti-rabbit IgG (Jackson Immuno 711-165-152) in a 1% BSA
solution, at room-temperature for 2 hours. After secondary antibody incubation,
the wells were washed twice with PBS for 5 minutes per wash. The wells were
then incubated with a 1:100 4',6-diamidino-2-phenylindole (DAPI) in PBS
solution at room-temperature for 5 minutes, to allow for nuclear staining. Staining
of cluster
cells
was
visualized
with
fluorescence
microscopy (Leica
Microsystems, fluorescence microscope) and images were captured at various
magnifications.
35
TABLE 1| Immunocytochemistry blocking solution details, primary antibody details
and secondary antibody details, including dilution values and manufacture
information, for specified targets.
Cell Chain Counting
Established B6/GFP cluster lines were utilized because they express GFP
constitutively. B6/GFP germ cell cultures were established as above. On day 6 of
culture, germ cells were passaged onto a 0.32 cm2 flat bottom culture plate (96
wells), on which a layer of feeders was present, with 500 µL of culture medium
per cm2 and reduced growth factors; this time point was noted as day 0 of
experimental culture. Germ cells were treated with 0.01% dimethyl sulfoxide
(DMSO), the control, 0.07 μM or 0.7 μM RA on day 3 of culture. On culture day
3, fresh medium and reduced growth factors were supplied. For 5 consecutive
days (i.e., culture days 3 to 8) following treatment, the number of cell chains
36
present was counted (Figure 7). Cell chains, as shown in Figure 8, were
characterized as being either 2 cells, 3 cells or ≥ 4 cells. Cell chain counting
experiments were based on data obtained from biological triplicate experiments.
Further for each biological replicate 2 wells were prepared and the mean was
taken to represent the number of cell chains.
Cell Chain Counting
0.7μM RA or 0.07μM RA or 0.01% DMSO
Day: 0
Seed
Count
GFP Cells
3
Treat
4
5
6
7
8
Count Chains:
2 Cell Chain
3 Cell Chain
≥4 Cell Chain
FIGURE 7| Cell chain counting. Established B6/GFP cluster lines were utilized. On
day 6 of culture, germ cells were passaged on which a layer of feeders. Germ cells
were treated with on day 3 of culture. On culture day 3, fresh medium and reduced
growth factors were supplied. For 5 consecutive days (i.e., culture days 3 to 8)
following treatment, the number of cell chains present was counted. Cell chains
were characterized as being either 2 cells, 3 cells or ≥ 4 cells.
37
2 Cell Chain
3 Cell Chain
A
B
≥4 Cell Chain
C
D
E
F
FIGURE 8| Morphology of cell chains. Images taken 2 days post-treatment with the
use of fluorescence microscopy. A, linear 2 cell chain. B, linear 3 cell chain. C,
linear 4 cell chain. D, linear 6 cell chain. E and F, linear 8 cell chain. All images
were taken at 40x magnification
Cluster Forming Activity Assay
The cluster forming activity assay was followed the protocol established by Yeh
et al. (2007). In short, an established B6/GFP germ cell line was utilized. On day
6 of culture, germ cells were sub-cultured onto a 2 cm2 flat bottom culture plate
(24 well), which had a layer of STO feeders, at a concentration of 4 to 5 x 104
GFP+ cells per cm2 with 500 µL of culture medium and reduced growth factors;
38
this time point was noted as day 0 of culture. On day 3 fresh medium and reduced
growth factors were replaced along with the addition of either 0.7 μM RA or the
control, 0.01% DMSO. Germ cells were subsequently harvested via trypsinization
48 hours post-treatment. The number of GFP+ cells in each treatment group was
counted and the cells were sub-cultured onto a 0.32 cm2 flat bottom culture plates
(96 well) at a split rate equivalent to 1:2. The cells were cultured with 500 µL of
culture medium per cm2 and reduced growth factors. The culture medium was
changed on day 3 of culture and reduced growth factors were added. On day 6 of
culture, the number of clusters consisting of at least 6 cells was counted (Figure
9).
39
Cluster Forming Activity Assay
0.7μM RA or 0.01% DMSO
Day: 0
Seed
3
5
Treat
Passage
Count
GFP Cells
Day: 0
Seed
Count
GFP Cells
3
Change
Medium
6
Count
Clusters
FIGURE 9| CFA assay. An established B6/GFP germ cell line was utilized. On day
6 of culture, germ cells were sub-cultured onto a flat bottom culture plate which had
a layer of STO feeders; this time point was noted as day 0 of culture. On day 3 fresh
medium and reduced growth factors were replaced along with the addition of either
0.7 μM RA or the control, 0.01% DMSO. Germ cells were subsequently harvested
via trypsinization 48 hours post-treatment. Cells were sub-cultured onto a flat
bottom culture plates at a split rate equivalent to 1:2. The culture medium was
changed on day 3 of culture and reduced growth factors were added. On day 6 of
culture, the number of clusters consisting of at least 6 cells was counted.
Spermatogonial Transplantation
An established B6/GFP germ cell line was utilized. On day 6 of culture, germ
cells were passaged onto a 2 cm2 flat bottom culture plate (24 well), on which a
layer of STO feeders were present, at a concentration of 4 to 5 x 10 4 GFP+ cells
per cm2 with 500 µL of culture medium per cm2 and reduced growth factors; this
time point was noted as day 0 of culture. On day 3 of culture fresh medium and
40
reduced growth factors were supplied to the culture along with the addition of
either 0.7 μM RA or the control, 0.01% DMSO. Germ cells were subsequently
harvested via trypsinization either 4 hours, 12 hours, 24 hours or 48 hours posttreatment, Germ cells were washed twice with phosphate buffered saline (PBS)
and were resuspended in injection medium (constituents: 10% trypan blue, 10%
DNase and 80% basic medium) at a concentration of 0.5 x 106 GFP+ cells per mL.
Subsequently about 7 μL of the cell suspension was injected into the efferent
duct of 129/B6 mice. Endogenous spermatogenesis of recipient mouse was
abolished by prior administration of busulfan (a chemotherapeutic agent) at a dose
of 50 mg/kg at least 4 weeks before transplantation. Recipient mouse testes were
collected 8 to 12 weeks post-transplantation, weighed, the tunica surrounding the
testis was removed and the tubules were mechanically dispersed. GFP + colonies
were counted with the use of a dissecting microscope. Each GFP colony (Figure
10) measuring more than 0.1 mm in length and covering more than 50% of the
basal surface of a recipient tubule was counted (M. Nagano, Avarbock, &
Brinster, 1999). Transplantation data was based on data obtained from biological
triplicate experiments. Transplantation was done at 4 time points, 4, 12, 24, and
48 hours after RA treatment. A DMSO control group was prepared for each time
point, thus, a total of 8 groups (4 time points x 2 groups). The numbers of
recipient testes for RA-treated and control groups were 19 and 18, respectively, at
4 hours; 12 and 18, respectively at 12 hours; 15 and 19, respectively, at 24 hours;
and 16 and 17, respectively, at 48 hours.
41
For feeder-free cultures, an established B6/GFP germ cell line was also utilized.
On day 6 of culture, germ cells were passaged onto a 2 cm2 flat bottom culture
plate which was coated with laminin at a concentration of 20 μg/mL overnight at
4°C. Germ cells were seeded at a concentration of 4 to 5 x 104 GFP+ cells per cm2
with 500 µL of culture medium per cm2 and reduced growth factors; this time
point was noted as day 0 of culture. The volume of culture medium upon initial
seeding was reduced to 250 µL per cm2 and growth factor concentration was 40
ng/mL GDNF, 150 ng/mL GFRα1 and 2 ng/mL bFGF. On day 1 of culture, 250
µL per cm2 of fresh medium was added to each well along with the addition of
thither 0.7 μM RA or the control, 0.01% DMSO. The resulting culture conditions,
total culture medium volume per cm2 and growth factor concentration, were the
same as those used in other experiments preformed.
On day 3 of culture, 48
hours post-treatment, germ cells were harvested via trypsinization and the
experiment protocol proceeded as outlined for spermatogonial transplantation
with feeders. Feeder-free transplantation data were based on data obtained from
biological triplicate experiments, for RA treated group n = 19 recipient testes, for
control group n = 16.
42
A
B
FIGURE 10| In vivo donor derived colonies. A, bright field image of mouse testis.
B, recipient mouse testis with donor derived colonies. Donor mouse stain: B6/GFP.
Recipient mouse strain: 126/B6. Images were taken at 1.6 x magnification.
In vitro Apoptosis Assay
B6/GFP germ cell cultures were established as previously noted. On day 6 of
culture, germ cells were passaged onto a 0.32 cm2 flat bottom culture plates (96well culture plates) onto which a layer of feeders was present and germ cell
density was adjusted accordingly. 500 µL of culture medium per cm2 and reduced
growth factors, this time point was noted as day 0 of culture. On day 3 of culture
fresh medium and growth factors were supplied to the culture along with the
addition of either 0.7 μM RA or the control, 0.01% DMSO. Cultured cells were
fixed in culture at 24 hours and 48 hours post-treatment. Cells were fixed
overnight at room temperature with 5% (NBF), which was prepared by removing
half the medium contained within the well, 80 μL, and adding an equal volume of
43
10% NBF. After overnight fixation, the 5% NBF solution was removed and the
cells were further fixed for 30 minutes at room temperature with 10% NBF. After
fixation was complete the wells were washed twice with PBS and apoptotic cell
staining was performed using the Click-iT® TUNEL Alexa Fluor® Imaging Assay
(InvitrogenTM) kit; the kit’s outlined experimental protocol for cells grown in a
96-well microplate was followed from Step 7.5. A positive control well was
prepared as per the manufacturer’s instructions. Staining of cluster cells was
visualized with fluorescence microscopy and images were captured at 40x
magnification.
Flow Cytometric Analysis of Apoptosis
B6/GFP germ cell cultures were established as previously noted. On day 6 of
culture, germ cells were passaged onto a 2 cm2 flat bottom culture plate, onto
which a layer of feeders was present, with 500 µL of culture medium per cm2 and
reduced growth factors, this time point was noted as day 0 of culture. On day 3 of
culture fresh medium and growth factors were supplied to the culture along with
the addition of either 0.7 μM RA or the control, 0.01% DMSO. Germ cells were
harvested at the following time point: 4 hours, 12 hours, 24 hours and 48 hours
post-treatment. Once cells were harvested, they were washed twice with PBS and
were subjected to sorting using the fluorescence-activated cell sorting (FACS)
Aria (BD Bioscience) to isolate the GFP+ germ cell population. Following sorting
44
the isolated cells were subjected to fixation for 30 minutes at 4°C using a 1%
paraformaldehyde (PFA) solution at a concentration of 106 cells/mL. Subsequent
to fixation, cells were washed twice with PBS and resuspended in 70% ice cold
ethanol at a concentration of 106 cells/mL, after which cells were stored at -30°C
until analysis. Once 3 biological replicates were acquired, apoptosis and cell cycle
analysis continued using the APO-DIRECTTM Kit (BD PharmingenTM). Stained
samples were then subjected to flow cytometric analysis using the C6 Flow
Cytometer® (Accuri Cytometers). Appropriate gated populations were established
using positive and negative cell controls supplied in the APO-DIRECTTM Kit.
Analysis of apoptosis experiments were based on data obtained from biological
triplicate experiments. Further for each biological triplicate n = 2 or 3, therefore
for each experiment n = 6 or 9.
Microarray
B6/GFP germ cell cultures were established as described above. On day 6 of
culture, germ cells were passaged onto a 2 cm2 flat bottom culture plate, onto
which a layer of feeders was present, with 500 µL of culture medium per cm2 and
reduced growth factors, this time point was noted as day 0 of culture. On day 3 of
culture fresh medium and growth factors were supplied to the culture along with
the addition of either 0.7 μM RA or the control, 0.01% DMSO. Culture cells were
subsequently harvested via trypsinization either 4 hours or 24 hours post45
treatment. Once cells were harvested, they were washed twice with PBS and were
subjected to sorting using the FACS Aria (BD Bioscience) to isolate the GFP+
germ cell population. RNA extraction was performed on the isolated cell
population with the use of the PicoPureTM RNA Isolation kit (Arcturus, Applied
Biosystems) and RNase-Free DNase Set (Qiagen). Three biological replicates
were prepared at each time point. After extraction the RNA was sent to Genome
Quebec where microarray experiments were performed using the Illumina Mouse
6 Expression Bead Chip. Data acquired were analyzed using Flex Array. A list of
genes of interest was compiled using threshold of ± 1.5 fold expression difference
between the RA treated germ cells and the control population, and with a
significance of p ≤ 0.05. Microarray experimental data were based on data
obtained from biological triplicate experiments. Further for each biological
triplicate n = 2, therefore for each experiment n = 6.
Quantitative Real-Time RT-PCR
Germ cell culturing followed the same experimental set-up as that used for the
microarray analysis. However cultured cells were harvested at 4 hours and 12
hours post-treatment. Cells were harvested, subjected to sorting allowing for the
isolation of the GFP+ germ cell population and RNA extraction was performed as
outlined in the microarray experimental set-up. RNA quality was assessed after
extraction. Three biological replicates were collected at each time point. cDNA
46
was prepared using 0.5 μg of isolated RNA and TaqMan® Reverse Transcription
Reagents (Invitrogen). Quantitative real-time RT-PCR (qPCR) was then
performed using the Rotor-Gene (Qiagen), designated forward and reverse
primers (Table 2), and the QuantiTect ® SYBR® Green PCR Kit (Qiagen).
Glyceraldehyde 3-phosphate dehydrogenase (GapDH) expression was used as a
control (Dheda et al., 2004). qPCR experimental data were based on data obtained
from biological triplicate experiments. Further for each biological triplicate n = 2,
therefore for each experiment n = 6.
TABLE 2| Forward and reverse primer sequences, direction 5’ to 3’, of specific
target genes used for qPCR gene expression analysis
47
Immunocytochemistry Against LRPAP1 and Stra8 – With Treatment
Germ cell culturing and fixation occurred as outlined in vitro apoptosis analysis;
however the treatment point analyzed was 48 hours post-treatment. Staining then
proceeded as outline in ICC against RARα – no treatment.
ICC for low-density lipoprotein receptor-related protein-associated protein 1
(LRPAP1) (Epitomics, p30533) and STRA8 (Abcam, ab494015) occurred in the
same above outlined protocol, substituting the blocking solution, primary and
secondary antibodies and dilutions with the appropriate antibodies and dilutions
as noted in the following table. ICC for LRPAP1 occurred using the same above
outlined protocol, however the membrane permeabilization and de-ionized water
rinsing steps were omitted and the blocking solution, primary and secondary
antibodies and dilutions were substituted with the appropriate antibodies and
dilutions as noted in Table 1.
48
CHAPTER 3
RESULTS
Expression Pattern of Retinoic Receptor α in Cluster Cells
The major isoform of the RA receptor found in the mouse testis is RARα (Chung
et al., 2009). To evaluate whether cluster cells express RARα, ICC techniques
were applied to cluster cultures on day 3 of culture. As shown in Figure 11, RARα
expression was observed throughout clusters. Such an observation indicates that
in all cluster cells do express the most prevalent isoform of the RARs and can
respond to RA.
49
Cy3 (RARα)
GFP (Germ Cells)
A
B
C
GFP (Germ Cells)
DAPI (Nuclear Staining)
D
Cy3 (RARα)
E
F
G
H
I
J
FIGURE 11| In vitro germ cell culture system with RARα staining. All images were
taken with the use of fluorescent microscopy. Images were captured at variable
magnifications. Images A, B, E, F and G were taken at 10x magnification. Images C,
D, H, I and J were taken at 20x magnification. Images A, B, C and D served as
negative controls, to which no primary antibody against RARα was added. Images
A, C, E and H show GFP detection, germ cell clusters. Images F and I show DAPI
detection, cell nuclei. Images B, D, G and J show Cy3 detection, staining against
RARα.
50
Overview of Cluster Cells’ Morphology Post-treatment
Overview of Cluster Cells’ Morphology Post-treatment
After 2 days of treatment with 0.7 µM RA cluster cells have altered cluster
morphology to distinct cell chains as compared to the control treatment group
(Figure 12). The cell chain is a morphology typical of spermatogonial
differentiation (Hamer, Roepers-Gajadien, Gademan, Kal, & de Rooij, 2003). In
the treated culture cell clusters appeared smaller and cell chains are not only more
prevalent but are also longer than those observed in the control culture.
0.01% DMSO
0.7μM RA
A
B
FIGURE 12| GFP+ cluster cell culture 3 days post-treatment. Images were taken
with the use of fluorescence microscopy at 10x magnification. A, culture treated
with control, 0.01% DMSO. B, culture treated with 0.7 µM RA.
In accordance with data obtained prior to the commencement of this project
(unpublished data which was obtained in an undergraduate research project I
51
undertook in 2010 in the Nagano Laboratory at McGill University) I treated
cluster cultures with RA on day 3 in vitro rather than day 0, which would coincide
with cell seeding, because no statistical significant difference in the number of
cell chains was observed. Furthermore I found that treatment on day 3 led to an
increased proliferation of cluster cells which would allow more ample cell
recovery at the time of cell collection for further analyses described later.
Cell Chain Kinetics Post-treatment
Figure 13 shows that upon treatment of RA more cell chains were observed in the
initial days of treatment. When considering 2 cell chains (Figure 13 A) a
significant difference was noted in the number of cell chains between 0.07 µM
RA and 0.01% DMSO (control) groups on days 1 and 2 post-treatment. Starting
on day 3 post-treatment a decline in the number of 2 cell chains was observed for
both the 0.07 µM RA and the 0.7 µM RA treated culture however it is at this time
point that a peak in 3 cell chains was observed for the treatment groups. The peak
of 3 cell chains observed 3 days post-treatment was statistically significant
compared to the control group. These results suggest that there was a progression
of cell chain formation from 2 cell chains to 3 cell chains in accordance with the
progression of spermatogonial differentiation.
52
It is important to note that on day 1 post-treatment there is a statistical
significance in the difference of 3 cell chains and also ≥ 4 cell chains (Figures
11B and C) between the control and both 0.07 µM RA and 0.7 µM RA treated
cultures. A statistical significance was also noted in the number of ≥ 4 cell chains
day 2 post-treatment between the control and the 0.07 µM RA culture and the
control and the 0.7 µM RA culture.
In general, the data shows that a significant increase in chain formation occurs
soon after RA treatment, typically by day 2 in vitro, followed by an overall
decline of chain numbers, regardless of chain size. This may imply that a longer
culture without replenishment of media and growth factors is detrimental for cell
survival. This may particularly be the case for the cells committed to
differentiation, as our culture condition is optimized for SSCs, rather than their
daughter cells. Consequently the decrease in cell chains over time may be
attributed to an increased apoptosis of cells which are advancing through the early
stages of spermatogenesis.
53
0.01% DMSO
25
Number of Cell Chains Observed
B
Cell Chain Kinetics: 2 Cell Chain
0.07μM RA
0.7μM RA
c
20
a
b
d
15
10
a, b
c, d
5
Number of Cell Chains Observed
A
Cell Chain Kinetics: 3 Cell Chain
9
0.01% DMSO
8
0.07μM RA
0.7μM RA
7
6
c
5
4
a
b
d
3
c, d
2
1
a, b
0
0
Day 1
Day 2
Day 3
Day 4
Day 1
Day 5
Day 2
Day 3
Day 4
Day 5
Number of Days Post Treatment
Number of Days Post Treatment
Cell Chain Kinetics: ≥ 4 Cell Chain
C
Number of Cell Chains Observed
12
0.01% DMSO
0.07μM RA
10
0.7μM RA
c
8
a
b
d
6
4
2
a, b
c, d
0
Day 1
Day 2
Day 3
Day 4
Day 5
Number of Days Post Treatment
FIGURE 13| Cell chain kinetics post-treatment. . A, number of 2 cell chains
observed over a 5 days period following the specified treatment condition. B,
number of 3 cell chains observed over a 5 day period following the specified
treatment condition. C, number of ≥ 4 cell chains observed over a 5 day period
following the specified treatment condition. . a, b, c and d denote significance
between the defined groups. Data values ± SEM. Note, no significance was noted
between the variable RA doses.
In order to establish experimental paradigms for further analysis, the total number
of cell chains formed over time was examined. Figure 14 demonstrates that
throughout days 1 to 2 post-treatment, the number of total cell chains in RA
treated cultures is significantly greater as compared to control cultures. Moreover
54
there is a peak in the total number of cell chains for both 0.07 µM and 0.7 µM RA
treated cultures 2 days post-treatment; which is followed by a progressive decline
in the total number of cell chains.
It is imperative to note that no statistical significance was observed at any time
point post-treatment for the formation of 2 cell chains, 3 cells chains or ≥ 4 cell
chains between the 0.07 µM RA and 0.7 µM RA treated cultures. Consequently
seeing no dose dependence was noted between the two RA treatment
concentrations; thus all future experiments utilized a RA treatment concentration
of 0.7 µM of RA since it is this concentration which is most prevalent in the
literature.
In summary, the cell chain counting experiments showed that both RA doses
induced the formation of cell chains a morphological phenotype associated with
differentiation. In addition there is a progression through cell chain formation; at
the same time that the number of 2 cell chains begins to decline, the number of 3
cell chains increases.
55
FIGURE 14| Total number of cell chains observed over a period of 5 days
following the treatment of cluster cells on day 3 with either 0.01% DMSO, 0.07 µM
RA or 0.7 µ RA. a, b, c and d denote significance between the defined groups. Data
values ± SEM. Note, no significance was noted between the variable RA doses
Quantification of Spermatogonial Stem Cells After Retinoic Acid Treatment
Cluster Forming Activity Assay
Following results of the chain counting assay, a CFA assay was employed as a
short term in vitro assay to quantify the number of SSCs present in culture after
treatment with RA for 48 hours (Yeh et al., 2007). The results shown in Figure 15
reveals that the number of SSCs present in culture 48 hours post-treatment with
56
0.7 µM RA, a mean of 921 clusters, was smaller by 9 fold as compared to the
control culture, suggesting a significant
decline in SSC numbers after RA
treatment.
Although the CFA does have its advantages, as short term semi-quantitative SSCs
quantifications, one must be cautious when using this technique since this assay is
not based on spermatogenesis regeneration, the hallmark of SSC activity. Per se
the number of SSCs attributed to cluster counting may only account for 70% of
the actual number of SSCs present (Yeh et al., 2007). Thus, I proceeded to the
transplantation assay to more definitively examine the effects of RA on SSCs.
57
FIGURE 15| Cluster forming activity assay. Data values ± SEM .Quantification of
the number of SSCs observed per 5 x 105 GFP+ cells 48 hours post-treatment with
the specified treatment condition. A 9 fold reduction in the number of SSCs was
observed between the control group and the 0.7 µM RA treated group. * denotes
significance, p < 0.05.
Transplantation Assay
Transplantation – Cultured With Feeders
SC transplantation represents an unequivocal assay to establish SC numbers; as is
the case for spermatogonial transplantation. This assay allows one to
retrospectively asses SSC numbers based on the colony number of donor derived
spermatogenesis established in a recipient testis.
58
Spermatogonial transplantation was performed at different periods post-treatment
to define a time frame in which RA exerts its effects on SSCs. The data shown in
Figure 16 indicate that when cluster cells are treated for 4 hours with RA, the
number of SSCs increased by 1.4 fold as compared to the control group, although
this increase was found not to be statistically significant (p ≤ 0.22). When cluster
cells are treated with RA for 12 hours, the number of SSCs, as compared to the
companion control culture is reduced by 2.1 fold, which was statistically
significant (p ≤ 0.05). After a 48 hours RA treatment, the number of SSCs was
19.6 fold less than the number of SSCs estimated to be present in the companion
control culture. This reduction in SSCs in the treated culture was statistically
significant, p ≤ 0.05.
However I did not detect a significant decline in SSC numbers after 24 hours RA
treatment. This observation was perplexing. A possibility is that the degree of
decline might vary widely at the beginning of the decline in SSC number and the
transplantation assay may have failed to generate clear-cut data due to its
inherently large coefficient of variation.
Nonetheless, results show a declining trend in SSC numbers after RA from 4 to 48
hours, suggesting that RA exerts a negative impact on SSCs as quickly as 12
hours after treatment and that by 48 hours, there is a significant reduction in the
number of SSCs. These data confirm the results of the CFA assay. Caution is
59
necessary however, because the culture system used in the above experiments
included a feeder later; thus, it is possible that RA affected the function of feeder
cells, thereby indirectly reducing SCC numbers. I addressed this issue in the next
experiment.
FIGURE 16| Spermatogonial transplantation assay. Culture conditions for cluster
cells included the presence of STO feeders. Cultures were treated for the designated
time prior to collection and transplantation. a, b and c denote significance between
the defined groups. The following fold changes relative to the companion control
cultures, +1.4, -2.1, 1 and -19.6 were observed for the 4, 12, 24, and 48 hour treated
cultures respectively.
Feeder-Free Transplantation
In this experiment the transplantation assay was utilized in conjunction with a
feeder-free culture system to assess the direct effects of RA on SSCs in vitro.
60
Clusters were treated with 0.7 µM RA for 48 hours after which all cells were
collected and transplanted into recipient testes. Figure 17 demonstrates that RA
treatment for 48 hours reduced the number of SSCs by 5.9 fold as compared to the
control culture, 108 SSCs (n = 19) vs. 627 SSCs (n = 16) respectively. This
decrease of SSCs in the RA treated group was significant.
This observation not only supports the hypothesis that RA induces the
differentiation of SSCs in vitro but also shows that RA can act directly on SSCs to
induce their differentiation.
It is essential to note that all other experiments, such as gene expression profiling
post-treatment, ICC etc., were conducted in the presence of a feeder layer. This
experimental setup was utilized due to technical constraints posed by a feeder-free
system, such as increased cell detachment from the culture wells when
manipulating the culture plates and when medium changes were performed. The
presence of a layer of feeder cells allowed for ease of manipulation without
substantial cell detachment thereby creating a more ‘rugged’ culture system.
Moreover a decrease of cell detachment during experimental manipulation
contributed to an improved cell recovery during experiments; this was essential
when considerable numbers of cluster cells needed to be recovered, as was the
case when collecting cells for performing the microarray. The presence of a
feeder layer is also essential for the establishment and maintenance clusters.
61
FIGURE 17| Transplantation assay with feeder-free culture conditions.
Quantification of the number of SSCs per 5 x 105 GFP+ cells 48 hours posttreatment with the specified treatment condition. A 5.9 fold reduction was observed
in the 0.7µM RA treated group. * denotes significance, p < 0.05.
Cell Death After Retinoic Acid Treatment
My data described above suggest that morphological changes in clusters can be
observed within 24 hours of RA treatments while SSC numbers may start to
decline as early as 12 hours. However, it is possible that RA induced SSC
apoptosis, leading to SSC loss. Therefore I addressed this possibility in the next
experiment.
To help reveal which cell population was undergoing apoptosis an in vitro
apoptosis detection assay was utilized at 24 hours and 48 hours following
62
treatment. Figure 19, reveals a similar localization of apoptotic cells between the
control culture and the RA treated culture after 24 hours of treatment, refer to
panels F, I, L and O. Furthermore Figure 18, panels I and L show that apoptotic
cells appear to be localized at the periphery of clusters and not in cell chains. The
same observations were noted for cultures which were treated for 48 hours, refer
to Figure 19.
I applied the TUNEL assay at 4, 12, 24 and 48 hours post-treatment and measure
the degree of cell death with the use of flow cytometric analysis. The data showed
that there was no significant difference between the level of apoptosis in cultures
with 0.7 µM RA and the control culture from 4 to 24 hours. However a
statistically significant increase, 4.1 fold increase, was observed in the percentage
of apoptotic cells at 48 hours in RA treated culture, 10.6% (n = 6) compared to the
control culture (2.6%, n = 6) at p ≤ 0.05. The data demonstrate that cell death is
not detectable in cluster cells until 48 hours after RA treatment.
Even though there is an increase in the percentage of cluster cell undergoing
apoptosis following 48 hours of treatment with 0.7 µM RA the identity of the
apoptotic cells to be SSCs is unknown; given the limitation resulting from the
inability to identify SSCs, flow cytometric analysis for apoptosis only allowed for
the quantification of apoptosis amongst all GFP + cluster cells. This cell population
includes: SSCs, progenitor spermatogonia and differentiating spermatogonia.
63
Morphological differentiation represented by cell chains formation occurs by 24
hours post-treatment and SSC decline can occur as early as hours following
treatment and shows a time-dependent trend, yet, cell death becomes detectable
only after 48 hours. Moreover only a 4.1 fold increase in apoptosis was noted 48
hours following treatment with 0.7 µM RA, when, the 19.6 fold reduction in SSCs
in the RA treated culture was observed. These results collectively lead to a
conclusion that RA induced SSC differentiation, rather than SSC death is the
major cause of the decline to SSCs.
64
FIGURE 18| In vitro apoptosis analysis of cluster cells 24 hours post-treatment.
Treatment was administered on day 3 of culture. A – C denote positive controls, D –
L denote RA treated cultures and M – O denote control treated cultures. A, D, G, J
and M, GFP+ cells, cluster cells. B, E, H, K and N, staining against apoptotic cells.
C, F, I, L and O, merged image of cluster cells and apoptotic cells to allow for the
localization of apoptotic cells. In the merged panels apoptotic germ cells appear as a
orange/red colour. All images were captured with fluorescence microscopy at a
magnification of 20x.
65
FIGURE 19| In vitro apoptosis analysis of cluster cells 48 hours post-treatment.
Treatment was administered on day 3 of culture. A – C denote positive controls, D –
L denote RA treated cultures and M – O denote control treated cultures. A, D, G, J
and M, GFP+ cells, cluster cells. B, E, H, K and N, staining against apoptotic cells.
C, F, I, L and O, merged image of cluster cells and apoptotic cells to allow for the
localization of apoptotic cells. In the merged panels apoptotic germ cells appear as a
orange/red colour. All images were captured with fluorescence microscopy at a
magnification of 20x.
66
FIGURE 20| Quantification of apoptosis of cluster cells after 3 days of treatment.
The percentage of apoptotic cells was measured at the following time points posttreatment: 4, 12, 24 and 48 hours post-treatment. * denotes significance, p ≤ 0.05.
Gene Expression Pattern After Retinoic Acid Treatment
Data collected from the experiments thus far suggest that RA acts directly on
SSCs to induce their differentiation as readily as 12 hours following treatment
with 0.7 µM RA. To further confirm in vitro RA induced SSC differentiation and
to identifying novel markers of SSC and spermatogonia differentiation, I
conducted a global gene expression analysis. I selected two time points post RA.
The first time point, 4 hours post-treatment, was selected to detect any genes that
could reflect early events of SSC differentiation. The second time point, 24 hours
post-treatment, was chosen because by this time, morphological differentiation
67
clearly occurs while the trend of SSC decline begins, yet cell death does not
become evident.
Microarray data showed an upregulation of 50 genes and a downregulation of 16
genes 4 hours following treatment (data threshold: fold difference ± 1.5, p ≤ 0.05);
among those genes, certain genes of interest were identified (Table 3).
Microarray data indicate that well established markers of spermatogonial
differentiation, c-Kit and STRA8 were upregulated by over 2 fold already 4 hours
post-treatment. This result was further validated by qPCR at the time point. qPCR
validation showed an upregulation of 3.1 fold for c-Kit and 44.85 fold for STRA8
expression at 4 hours. The data indicate that these differentiation marker genes
were rapidly expressed upon RA administration and support the notion that
spermatogonial differentiation was indeed induced in this experimental paradigm.
Microarray data at 24 hours post-treatment showed an upregulation of 175 genes
and a downregulation of 327 genes (data threshold: fold difference ± 1.5, p ≤
0.05); data collected from the microarray showed a more extensive list of markers
which further confirmed that RA induces SSC differentiation. c-Kit and STRA8,
were upregulated 8.86 and 6.37 fold respectively. qPCR validation confirmed an
extensive upregulation of 8.05 fold for c-Kit and 462.40 fold for STRA8. In
addition to the upregulation of differentiation specific markers, a downregulation
of SSC markers was observed 24 hours post RA treatment via microarray
68
analysis. A downregulation of all of the following markers was observed: Nanos2,
Plzf, Redd1 and Ret, further suggesting that RA is inducing a loss of SSC specific
markers and more specifically a loss of SSCs. The decreased expression of
Nanos2, Plzf, Redd1 and Ret, was further validated by qPCR analysis where all
gene except Redd1 showed a significant decrease after 12 hours of RA treatment
compared to the control.
Interestingly microarray analysis and qPCR analysis showed a decrease in cell
cycling genes, Ccnd1 and Ccnd2. Ccnd1 and Ccnd2 code for CYCLIN D1 and
CYCLIN D2 respectively. It has been shown that Ccdn2 is upregulated at the Aal
to A1 transition point; consequently microarray and qPCR data may suggest that
although SSC differentiation has been induced by RA, the culture system being
used does not support further advancement in spermatogenesis. Ccnd3 was also
examined by qPCR analysis however this gene showed no expression change at
either time point post RA treatment.
Other genes which were upregulated 24 hours post-treatment included Stxbp5 and
LRPAP1. Stxbp5 plays a role in the fusion of endocytic vesicles (van Loon et al.,
2010). Previous SSC differentiation studies have not noted Stxbp5 as a possible
marker of SSC differentiation. qPCR data at 12 hours following treatment does
not show a significant change in Stxbp5 expression between the RA treated cells
and the control population. LRPAP1 encodes for a lipid metabolizing protein
69
(Pandey, Pradhan, & Mittal, 2008). Like Stxbp5, LRPAP1 has previously not been
demonstrated to potentially be a marker for SSC differentiation. Therefore further
examining the protein expression pattern of LRPAP1 may be informative since
qPCR data showed a significant increase in its expression 12 hours post-treatment
with RA.
Other genes which were also examined by qPCR despite showing no expression
change in the microarray analysis were Gfrα1, Sohlh1 and SALL4. Gfrα1 showed
no change in expression pattern post-treatment at either of the time point, 4 hours
or 12 hours post-treatment. SOHLH1’s expression is known to be predominantly
found in Aal through to type B spermatogonia and it has been suggested that
SOHLH1 opposes SSC self-renewal mechanisms. qPCR analysis of Sohlh1
expression post-treatment does not show an upregulation in RA treated cells, this
lack of upregulation may be attributed to the lack of Aal spermatogonia at the
given collection time. Finally SALL4, a member of a family of zinc finger
transcription factors, was shown to be upregulated significantly 4 hours post RA
treatment, 1.85 fold. Interestingly SALL4 has been previously noted to be a stem
cell marker whose expression is only momentarily increased in the first stages of
spermatogonial differentiation (Oikawa et al., 2013); results obtain through the
microarray analysis mirror SALL4’s known expression pattern.
70
Take together results from the microarray analysis and qPCR-based validation
support the hypothesis that RA induces the differentiation of SSCs in vitro; as the
data indicate the increased expression of differentiation specific genes and the
downregulation of SSC specific genes. Moreover, the data also revealed some
genes, such as LRPAP1 and SALL4, which may be interesting to explore further.
TABLE 3| Microarray analysis data and qPCR data after treatment. Data is
represented as the fold change in the 0.7 µM RA treated group compared to the
companion 0.01% DMSO control group. Times (4, 12 and 24 hours) indicated the
length of treatment prior to analysis. For microarray data, all data shown has
statistical significance, p ≤ 0.05 and a fold change of ± 1.5 was used as a baseline
when selecting for gene expression analysis. For qPCR data *denotes significance
between the treated group and the control group, p ≤ 0.05.
71
Protein Expression Pattern After Retinoic Acid Treatment
To explore the expression pattern of certain proteins post RA treatment ICC
techniques were employed. Cultures were treated for a period of 48 hours thereby
allowing the protein products of genes of interest to be produced.
STRA8 Expression After Treatment
STRA8 protein localization was explored to confirm that differentiation markers
observed with the use of gene expression analysis indeed coincided with cells
which showed a morphological phenotype attributed to spermatogonia
differentiation. As illustrated in Figure 21 C and D, STRA8 expression is highly
present in cell chain post-treatment with 0.7 µM RA. Cluster cells also appear to
express STRA8 expression (Fig. 21B) however expression would not appear to be
as pronounced as the expression observed in cluster chains, based on qualitative
assessment. These ICC results confirm that the cell chain morphology attributed
to differentiating spermatogonia coincides with cells which are expressing
differentiation markers.
72
A
B
C
D
FIGURE 21| In vitro stating for STRA8 48 hours following treatment. Green, GFP+
germ cells. Red, Cy3 staining, STRA8. Yellow, merged image resulting from germ
cells (GFP+) which express STRA8 (Cy3+). A, negative control. B, culture treated
with 0.01% DMSO. C and D cell chains present in culture after treatment with
0.7µM RA. Image captured with the use of fluorescence microscopy. Image
captured at 20 x magnification.
LRPAP1 Expression After Treatment
Gene expression analysis revealed the upregulation LRPAP1, a gene which was
previously unexplored in the context of spermatogonial differentiation. LRPAP1’s
expression was significantly upregulated as readily as 12 hours post-treatment
with 0.7 µM RA, refer to Table 3. To further characterize the expression of
LRPAP1 in cluster cells ICC techniques were used. The expression of LRPAP1
73
was explored 48 hours following treatment to allow protein formation as well as
transport to the cell membrane.
Staining for LRPAP1 reveals homogenous expression throughout clusters for both
the 0.01% DMSO treated cells as well as the 0.7 µM RA treated cells, refer to
Figure 22 L and Figure 22 F respectively. However the expression pattern for
LRPAP1 is interesting because it is lacking in cell chains, refer to Figure 22 I and
Figure 23 C. It appears that LRPAP1 is not expressed in differentiating
spermatogonia or more specifically its expression is lacking in longer cell chains.
Since LRPAP1’s expression is upregulated at the mRNA level as early as 12
hours (Table 3) and its expression at the protein level is seen only in clusters and
not in cell chains, this molecule may be a novel marker of early differentiation
process and be worth further examining in further studies.
74
FIGURE 22| LRPAP1 expression analysis 48 hours post-treatment. A – C, negative
control. D – I cluster cells treated with 0.7 µM RA. J – L cluster cells treated with
0.01% DMSO. A, D, G and J, GFP+ cells, cluster cells. B, E, H and K, DAPI,
nuclear staining. C, F, I and L, CY3 staining, LRPAP1. Images were captured with
florescence microscopy at 20x magnification.
75
FIGURE 23| LRPAP1 staining 48 hours following treatment with 0.7µM RA. A,
GFP+ cells represent cluster cells. B, CY3+ cells represent cells which express
LRPAP1. C, a merged and magnified image of panels A and B to help clearly
demonstrate the cells which express LRPAP1. Germ cells which express LRPAP1
appear yellow-orange. Images captures with the use of fluorescence microscopy at
20 x magnification.
76
CHAPTER 4
DISCUSSION AND FUTURE EXPERIMENTS
The field of SSC biology has seen numerous advances in the past years: SSC
culturing as well as in vitro expansion of SSCs are now possible, thanks to
discoveries of growth factors that promote SSC self-renewal. These events
followed the development of spermatogonial transplantation in 1994 that allowed
for the re-establishment of donor-derived spermatogenesis in recipient testes and
restoration of fertility in infertile mice (Brinster & Avarbock, 1994). Many years
ago the essential function of RA for cell differentiation in spermatogenesis was
shown. Thus, our knowledge has been accumulating about early stages of cell
proliferation and differentiation in spermatogenesis. Although the progresses in
SSC biology have been plentiful, many obstacles still remain. To date a pure
population of SSCs remains unattainable due to a lack of cell surface markers
required for its success. The mechanisms of action of RA in SSC differentiation
induction still remain largely unknown, and more importantly, the processes of
SSC commitment to differentiation are unidentified. I also emphasize important
questions that still remain to be answered – is there a specific point at which SSCs
become committed to differentiation, once committed to differentiation is this
decision reversible, and how do we detect the point of SSC commitment?
77
In this study I focused on demonstrating that RA acted directly on SSCs and
induced their differentiation. I revealed that SSCs commit to differentiation very
quickly after treatment with RA. I also found proteins which may serve as a
potential novel marker of early commitment to differentiation (Fig. 24). The
experimental paradigms used in my study had the following advantages that
allowed me to accomplish the outlined tasks. Firstly, in vitro commitment to
differentiation was readily assessed because of a unique characteristic of this
system; when spermatogonia become committed to differentiation they display a
distinctive morphological phenotype, cells that remain connected by cytoplasmic
bridges, referred to as cell chains. The in vitro approach allowed me to readily
follow the process of differentiation. In addition to assessing differentiation by
observing the number of cell chains, SSC transplantation served as an
unequivocal method to assess the changes in SSC numbers upon treatment with
RA.
78
FIGURE 24| Cluster cells were treated on day 3 of culture with 0.7 µM RA. Gene
expression analysis revealed the upregulation of differentiation specific markers and
the downregulation of SSC specific markers as rapidly as 4 hours following
treatment. At 12 hours post-treatment transplantation assay revealed a decrease in
SSC numbers and gene expression analysis showed an increase of differentiation
specific markers. 24 hours following treatment cell chains become a distinct feature
in germ cell cultures treated with RA and SSC specific markers are downregulated
while differentiation specific markers are upregulated. 48 hours following treatment
the total number of cell chains peak and the number of SSCs are substantially
decreased. Overall there appears to be a loss of SSCs with the length of treatment
and an increase in the number of germ cells undergoing differentiation.
Initial experiments undertaken (Fig. 11) confirmed that cluster cells in vitro
expressed RARα, suggesting that these cells have the appropriate receptor to
respond to RA stimulation and also showed that addition of RA to cluster cells in
culture results in a rapids formation, 24 hours following treatment, of cell chains.
It was also shown that this distinctive morphology is most prevalent 48 hours
following treatment,
suggesting that
SSCs are already committed to
79
differentiation by this time. The CFA assay further confirmed a significant
reduction in SSCs 48 hours post-treatment.
I then preformed the transplantation assay at various points following RA
treatment in anticipation of revealing SSC commitment over time after the
treatment. A RA induced effect on SSC numbers was noted as early as 12 hours
post-treatment and more drastically 48 hours following treatment (Fig. 16). It
should be noted that this experiment was also conducted under a feeder-free
condition demonstrating that RA directly acts on undifferentiated spermatogonia,
if not only on SSCs. It is therefore suggested that SSCs respond rapidly to RA and
the addition of RA in germ cells in vitro culture results in a significant decline in
the SSC population. This rapid response to differentiation induction suggests that
the SSC population may conceivably be a heterogeneous one; a cell population
composed of cells which are either primed for differentiation or self-renewal and
under particular conditions the presence of one population over the other may be
favored. Consequently, these results imply that the SSC state may be more fragile
or flexible than it has been expected, and we can readily manipulate the state.
My data also suggest that the reduction in SSC number is not the result of cell
death. Apoptosis analysis revealed that there was a significant increase in the
number of germ cells undergoing apoptosis only 48 hours following treatment, by
which time I observed evident changes in cell morphology and the initiation of
80
decline in SSC activity (Figs. 14, 16 and 20). It was interesting, however, that I
did not observe cell death in spermatogonial chain but in clusters (Figs. 18 and
19), as a total number of cell chains decline from day 2 to day 5 of culture (Fig.
14). The detection of apoptotic cells mainly in clusters may reflect that when the
cells in chains commit to cell death, they may rapidly come off the culture
substrate (a feeder layer throughout my experiments), making it difficult to detect
them.
It is noted that RA acts directly on SSCs for differentiation induction and SSCs
respond rapidly to the differentiation cue and undergo differentiation. Based on
these finding, I carried out global gene expression assays. Microarray data
revealed an upregulation of SSC differentiation markers, c-Kit and STRA8, and
also showed a downregulation of known SSC and undifferentiated spermatogonia
markers (Nanos2, PLZF, Redd1 and Ret), confirming SSC commitment to
differentiation and ongoing spermatogonial differentiation, following RA
treatment (Table 3).
The gene expression analysis also detected an upregulation of LRPAP1. Its
expression increased as quickly as 12 hours following treatment. This gene was
not previously described in the context of SSC differentiation and it may be
interesting if this gene is further explored to elucidate its role as a potential
marker for early SSC differentiation. I found that the expression of LRPAP1 was
81
localized to cells present in clusters and not to cell chains (Fig. 23). This is
unexpected and intriguing and may support the notion that LRPAP1 could be an
early commitment marker.
I propose the following experiments as future analysis of the possible involvement
of LRPAP1. Cluster cells will be FACS sorted for LRPAP1+ and LRPAP1- cells at
different time points following RA treatment, followed by transplantation assay to
asses SSC enrichment. I expect that SSCs are enriched in the LRPAP1 - fraction.
The results should also indicate the time-course changes in SSC numbers, which
will be informative to establish windows of SSC commitment process and may
possibly identify a time point where irreversible SSC differentiation takes place. It
could also be a powerful approach toward defining SSC differentiation process to
include sorting combinations with established SSC markers, such as Thy1
(renewal marker) and/or c-Kit (differentiation marker) together with LRPAP1.
For example, Thy1+/ c-Kit -/ LRPAP1- sorted cell fraction may be highly enriched
for SSCs, which can be assessed using spermatogonial transplantation. In addition
to investigating the role of LRPAP1 in SSC differentiation, conducting cell cycle
analysis of cluster cells following treatment with RA may reveal an alteration in
cell division potential of different cell fractions, taking into account the observed
decrease in Ccnd1 and Ccnd2 expression post-treatment and their association with
spermatogonial differentiation processes.
82
Although there is still a lot to decipher about SSC differentiation, the experiments
performed throughout my thesis confirmed the quintessential role of RA in the
induction of differentiation of SSCs and also revealed the possibility that SSCs
may exist in a fragile state where some SSCs are primed to respond rapidly to
differentiation cues. Furthermore the experiments performed confirmed that RA
can serve as an important tool for future experiments to aid in uncovering
important aspects about SSC differentiation.
83
ANNEX 1
MICROARRAY DATA
Genes Upregulated 24 Hours Post-Treatment with 0.7μM RA
(Threshold: fold increase ≥ 1.5, p ≤ 0.05)
KIT
Fold
Change
8.8560
REC8
6.9141
Nuclear
STRA8
6.3736
Cytoplasm
GBX2
4.1014
Nuclear
2310047D13RIK
4.0294
CYP26A1
3.8012
SCOTIN
2.7933
BLVRB
Target ID
Localization
Description
Membrane
Transmembrane tyrosine kinase receptor. Role in the maturation and differentiation of
spermatogonial stem cells
Human homolog of yeast Rec8, a meiosis-specific phosphoprotein involved in
recombination events
Stimulated by retinoic acid gene 8. Expression is induced by retinoic acid. Drived
gene expression in meiotic and post-meiotic germ cells.
Candidate control gene for cell pluripotency and differentiation in the embryo. In
undifferentiated embryonic stem cells but was downregulated in differentiated cell
populations
ER
membrane
ER
membrane
Cytochrome P450 retinoic acid-metabolizing enzyme, metabolizes retinoic acid into
several forms, thereby inactivating it
Scotin mRNA was induced coincident with apoptosis. Direct binding between p53 and
Scotin. Apoptosis induced by the p53/Scotin pathway is caspase dependent
2.6866
Cytoplasm
CLIC6
2.6572
Cyto + Mem.
TLE6
2.5084
Cytoplasm
The final step in heme metabolism in mammals is catalyzed by the cytosolic biliverdin
reductase enzymes A and B
Chloride intracellular channel protein 6. Has been shown to act with the dopamine
receptor 3
Significant similarity with the Drosophila groucho protein. X
RHOX10
2.5075
LLGL2
2.4321
D9ERTD280E
2.3611
GPRASP2
SEPT11
Cytoplasm
Lethal giant larvea homolog 2. plays a role in asymmetric cell division, epithelial cell
polarity, and cell migration
2.2945
Cytoplasm
G protien couple receptor associated sorting protein 2.
2.2890
Cytoplasm
ST6GAL1
2.2163
Golgi
membrane
SEPT11 belongs to the conserved septin family of filament-forming cytoskeletal
GTPases that are involved in a variety of cellular functions including cytokinesis and
vesicle trafficking
Relation to CDw75, a human leukocyte cell-surface antigen expressed in mature and
activated B cells but not in B cells at earlier stages of development or in plasma cells.
RGS10
2.2060
FAM171B
2.1525
LRPAP1
2.1296
Cell Surface
Low density lipoprotein receptor-related protein-associated protein 1. Studies
indicated that the molecule is present on the cell surface, forming a complex with the
heavy and light chains of the alpha-2-macroglobulin receptor
TCFCP2L1
2.1196
Nuclear
Transcription factor CP2-like. Transcriptional repressor. May suppress UBP1mediated transcriptional activation. Modulates the placental expression of CYP11A1
PLAT
2.0952
Secreted
Plasminogen activator. Serine protease. In the nervous system, PALT activity is
correlated with neurite outgrowth, neuronal migration, learning, and excitotoxic death.
Upregulates MMP9 (degrade the collagens of the extracellular matrix)
EGFP
2.0855
Cytoplasm
FAM115C
2.0431
Enhanced green fluorescent protein. In the cell line it is under the control of the
chicken actin promoter
Family with sequence similarity 115, member C.
PRTN3
2.0006
Extracellular
Proteinase 3. PR3 is a neutral serine protease that is able to cleave elastin.
LETMD1
1.9874
Outter Mito
memb.
LETM1 domain containing 1. Involved in tumorigenesis and may function as a
negative regulator of the p53/TP53
Regulator of G protein signalling 10. RGS proteins negatively regulate signaling
pathways involving 7-transmembrane receptors and heterotrimeric G proteins
Family with sequence similarty 171, member B.
84
NR6A1
1.9596
Nuclear
Nuclear receptor sub-family 6, group A, member 1. Aka Germ cell nuclear factor. This
gene encodes an orphan nuclear receptor which is a member of the nuclear hormone
receptor family. Its expression pattern suggests that it may be involved in neurogenesis
and germ cell development. The protein can homodimerize and bind DNA.
RPL3L
1.9440
Cytoplasm
Ribosomal protein L3 like. This gene has a tissue-specific pattern of expression, with
the highest levels of expression in skeletal muscle and heart. It is not currently known
whether the encoded protein is a functional ribosomal protein or whether it has
evolved a function that is independent of the ribosome.
LAMA5
1.9282
Basement
Memb.
Laminin alpha 5. Binding to cells via a high affinity receptor, laminin is thought to
mediate the attachment, migration and organization of cells into tissues during
embryonic development by interacting with other extracellular matrix components
AARD
1.9152
ANKRD39
1.8983
Ankyrin repear domain 39.
RNPEPL1
1.8704
HOXB5
1.8650
Arginyl aminopeptidase (aminopeptidase B)-like 1. Ubiquitous. Expressed at
relatively higher levels in heart and skeletal muscle
Homeobox B5. Encodes a nuclear protein with a homeobox DNA-binding domain.
The encoded protein functions as a sequence-specific transcription factor that is
involved in lung and gut development.Sequence-specific transcription factor which is
part of a developmental regulatory system that provides cells with specific positional
identities on the anterior-posterior axis.
9430080K19RIK
1.8504
RBP1
1.8498
DMRTC1C
1.8474
PFKFB4
Nuclear
Cytoplasm
Retinol binding protein 1. Carrier protein involved in the transport of retinol (vitamin
A alcohol) from the liver storage site to peripheral tissue.
1.8374
Cytoplasm
ZFP423
1.8334
Nuclear
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase. Synthesis and degradation of
fructose 2,6-bisphosphate
Zinc finger protein 423. It functions as a DNA-binding transcription factor by using
distinct zinc fingers in different signaling pathways. Thus, it is thought that this gene
may have multiple roles in signal transduction during development. Involved in
olfactory neurogenesis by participating in a developmental switch that regulates the
transition from differentiation to maturation in olfactory receptor neurons. Controls
proliferation and differentiation of neural precursors in cerebellar vermis formation
TXNDC13
1.8092
Membrane
Thioredoxin-related transmembrane protein 4.
GSTK1
1.8005
Peroxisome
Glutathione S-transferase kappa 1.This gene encodes a member of the kappa class of
the glutathione transferase superfamily of enzymes that function in cellular
detoxification. The encoded protein is localized to the peroxisome and catalyzes the
conjugation of glutathione to a wide range of hydrophobic substates facilitating the
removal of these compounds from cells.
SCARF2
1.8004
Membrane
Scavenger receptor class F, member 2. Probable adhesion protein, which mediates
homophilic and heterophilic interactions. In contrast to SCARF1, it poorly mediates
the binding and degradation of acetylated low density lipoprotein (Ac-LDL)
RBKS
1.7786
FCHO1
1.7759
H1FX
1.7644
GCAP27
1.7611
LOC100039276
1.7581
9430079M16RIK
1.7571
SLC27A2
Ribokinase. The ribokinase encoded by this gene belongs to the pfkB family of
carbohydrate kinases. It phosphorylates ribose to form ribose-5-phosphate in the
presence of ATP and magnesium as a first step in ribose metabolism
FCH domain only 1. Protein binding.
Nuclear
H1 histone family, member X. Histones H1 are necessary for the condensation of
nucleosome chains into higher order structures
1.7568
ER mem.
Perio mem.
Solute carrier family 27 (fatty acid transporter), member 2. Convert free long-chain
fatty acids into fatty acyl-CoA esters, and thereby play a key role in lipid biosynthesis
and fatty acid degradation. In vitro, also activates long- and branched-chain fatty acids
and may have additional roles in fatty acid metabolism. May be involved in
translocation of long-chain fatty acids (LFCA) across membranes
AXIN2
1.7549
Cytoplasm
COPZ2
1.7544
Cytoplasm
Axin 2. Inhibitor of the Wnt signaling pathway. Down-regulates beta-catenin.
Probably facilitate the phosphorylation of beta-catenin and APC by GSK3B
Coatomer protein complex, subunit zeta 2. The coatomer is a cytosolic protein
complex that binds to dilysine motifs and reversibly associates with Golgi nonclathrin-coated vesicles, which further mediate biosynthetic protein transport from the
ER, via the Golgi up to the trans Golgi network. Coatomer complex is required for
budding from Golgi membranes, and is essential for the retrograde Golgi-to-ER
transport of dilysine-tagged proteins
IQGAP3
1.7539
Cytoplasm
IQ motif containing GTPase activating protein 3.
B2M
1.7502
Plasma
Membrane
Beta-2-microglobulin. Component of the class I major histocompatibility complex
(MHC). Involved in the presentation of peptide antigens to the immune system.
85
RARB
1.7461
Nuclear
Retinoic acid receptor, beta. This gene encodes retinoic acid receptor beta, a member
of the thyroid-steroid hormone receptor superfamily of nuclear transcriptional
regulators. This receptor localizes to the cytoplasm and to subnuclear compartments. It
binds retinoic acid, the biologically active form of vitamin A which mediates cellular
signalling in embryonic morphogenesis, cell growth and differentiation. It is thought
that this protein limits growth of many cell types by regulating gene expression.
PDLIM4
1.7397
SGCB
1.7381
Plasma
Membrane
Sarcoglycan, beta (43kDa dystrophin-associated glycoprotein) Component of the
sarcoglycan complex, a subcomplex of the dystrophin-glycoprotein complex which
forms a link between the F-actin cytoskeleton and the extracellular matrix
COL22A1
1.7363
Secreted
CLGN
1.7340
GDF10
1.7221
ER
Membrane
Secreted
Collagen, type XXII, alpha 1. Acts as a cell adhesion ligand for skin epithelial cells
and fibroblasts
Calmegin. Calmegin is a testis-specific endoplasmic reticulum chaperone protein.
CLGN may play a role in spermatogeneisis and infertility.
Growth differentiation factor 10. The members of this family are regulators of cell
growth and differentiation in both embryonic and adult tissues. Studies in mice
suggest that the protein encoded by this gene plays a role in skeletal morphogenesis.
ABCA3
1.7168
Plasma
Membrane
ATP-binding cassette, sub-family A (ABC1), member 3. Plays an important role in the
formation of pulmonary surfactant, probably by transporting lipids such as cholesterol
LOC100045484
1.7121
EG630499
1.7096
FRAP1
1.7086
Plasma
Membrane
(In)
The protein encoded by this gene belongs to a family of phosphatidylinositol kinaserelated kinases. These kinases mediate cellular responses to stresses such as DNA
damage and nutrient deprivation. This protein acts as the target for the cell-cycle arrest
and immunosuppressive effects of the FKBP12-rapamycin complex
SERPINE2
1.7081
Secreted
Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1),
member 2. Serine protease inhibitor with activity toward thrombin, trypsin, and
urokinase. Promotes neurite extension by inhibiting thrombin. Binds heparin
NENF
1.6972
Secreted
Neuron derived neurotrophic factor. Displays neurotrophic activity and activates
phosphorylation of MAPK1/ERK2, MAPK3/ERK1 and AKT1/AKT in primary
cultured neurons. Does not have mitogenic activity in primary cultured astrocytes.
May play a role on neuronal differentiation and may have a transient effect on neural
cell proliferation in neural precursor cells. Neurotrophic activity is enhanced by
binding to heme
2410019G02RIK
1.6941
ZFP653
1.6924
Nuclear
Zinc finger protein 653. Transcriptional repressor
SEZ6
1.6891
Plasma
Membrane
DHX58
1.6753
Cytoplasm
EVPL
1.6738
Cytoplasm
NUDT18
1.6732
6230400G14RIK
1.6714
AGFG1
1.6651
Nucleus
OXCT1
1.6648
Mitoch.
Matrix
1700047I17RIK1
1.6631
2310014G06RIK
1.6570
RFX2
1.6489
RNASET2
1.6473
Secreted
EML3
1.6408
Cytoplasm
NGFRAP1
1.6397
GPX3
1.6392
1810020D17RIK
1.6361
PDZ and LIM domain 4. This gene encodes a protein which may be involved in bone
development. Mutations in this gene are associated with susceptibility to osteoporosis.
Nucleus
Seizure related 6 homolog (mouse). May play a role in cell-cell recognition and in
neuronal membrane signaling. Seems to be important for the achievement of the
necessary balance between dendrite elongation and branching during the elaboration
of a complex dendritic arbor
DEXH (Asp-Glu-X-His) box polypeptide 58. Participates in innate immune defense
against viruses.
envoplakin. Component of the cornified envelope of keratinocytes. May link the
cornified envelope to desmosomes and intermediate filaments
nudix (nucleoside diphosphate linked moiety X)-type motif 18. Probably mediates the
hydrolysis of some nucleoside diphosphate derivatives
ArfGAP with FG repeats 1. Required for vesicle docking or fusion during acrosome
biogenesis. Localized to cytoplasmic vessicles as well.
3-oxoacid CoA transferase 1. Key enzyme for ketone body catabolism. Transfers the
CoA moiety from succinate to acetoacetate.
regulatory factor X, 2 (influences HLA class II expression). It is a transcriptional
activator that can bind DNA as a monomer or as a heterodimer with other RFX family
members
ribonuclease T2.
echinoderm microtubule associated protein like 3. May modify the assembly dynamics
of microtubules, such that microtubules are slightly longer, but more dynamic
nerve growth factor receptor (TNFRSF16) associated protein 1.
Secreted
glutathione peroxidase 3 (plasma). Protects cells and enzymes from oxidative damage,
by catalyzing the reduction of hydrogen peroxide, lipid peroxides and organic
hydroperoxide, by glutathione
86
HGSNAT
1.6350
Membrane ?
ANKRD23
1.6347
Nucleus
CHCHD10
1.6288
Mitochondria
GIYD2
1.6276
Nucleus
ZMYM3
1.6266
Nucleus
AI448196
1.6237
Membrane
SLX1 structure-specific endonuclease subunit homolog B. Catalytic subunit of the
SLX1-SLX4 structure-specific endonuclease that resolves DNA secondary structures
generated during DNA repair and recombination. Has endonuclease activity towards
branched DNA substrates, introducing single-strand cuts in duplex DNA close to
junctions with ss-DNA
zinc finger, MYM-type 3. The encoded protein is a component of histone deacetylasecontaining
multiprotein complexes that function through modifying chromatin structure to keep
genes silent.
armadillo repeat containing, X-linked 4
LOC381629
1.6164
ARFGEF2
1.6133
Cytoplasm ?
ADP-ribosylation factor guanine nucleotide-exchange factor 2 (brefeldin A-inhibited)
2700038C09RIK
1.6128
HOXB4
1.6115
Nucleus
APEH
1.6101
Cytoplasm
SLC24A6
1.6083
Membrane
homeobox B4. Intracellular or ectopic expression of this protein expands
hematopoietic stem and progenitor cells in vivo and in vitro, making it a potential
candidate for therapeutic stem cell expansion.
N-acylaminoacyl-peptide hydrolase. This gene encodes the enzyme acylpeptide
hydrolase, which catalyzes the hydrolysis of the terminal acetylated amino acid
preferentially from small acetylated peptides. It can play an important role in
destroying oxidatively damaged proteins in living cells. Deletions of this gene locus
are found in various types of carcinomas, including small cell lung carcinoma and
renal cell carcinoma.
solute carrier family 24 (sodium/potassium/calcium exchanger), member 6. Transports
Ca(2+) in exchange for either Li(+) or Na(+),
9530020G05RIK
1.6031
CAPN5
1.6018
NRIP3
1.6013
A930005H10RIK
1.5995
LOC382163
1.5977
NUAK1
1.5937
COX6C
1.5911
Mito Inner
Mem.
IL11RA1
1.5900
Membrane
NLRP14
1.5886
Cytoplasm
PDE1B
1.5862
Cytoplasm
PRCP
1.5849
Lysosome
NACC2
1.5848
Nucleus
TMEM128
1.5847
Membrane
Transmembrane proteins 128.
KCNIP3
1.5843
Cyto & Cell
mem.
RND2
1.5833
Cytopla. Of
Vessic.
Kv channel interacting protein 3, calsenilin. This gene encodes a member of the family
of voltage-gated potassium (Kv) channel-interacting proteins, which belong to the
recoverin branch of the EF-hand superfamily.
Rho family GTPase 2. This gene encodes a member of the Rho GTPase family, whose
members play a key role in the regulation of actin cytoskeleton organization in
response to extracellular growth factors. This particular family member has been
implicated in the regulation of neuronal morphology and endosomal trafficking.
Cytoplasm ?
heparan-alpha-glucosaminide N-acetyltransferase. Lysosomal acetyltransferase that
acetylates the non-reducing terminal alpha-glucosamine residue of intralysosomal
heparin or heparan sulfate, converting it into a substrate for luminal alpha-N-acetyl
glucosaminidase
ankyrin repeat domain 23. May be involved in the energy metabolism. Could be a
molecular link between myofibrillar stretch-induced signaling pathways and muscle
gene expression
coiled-coil-helix-coiled-coil-helix domain containing 10
Calpain 5. Calpains are calcium-dependent cysteine proteases involved in signal
transduction in a variety of cellular processes.
nuclear receptor interacting protein 3.
NUAK family, SNF1-like kinase, 1. Involved in tolerance to glucose starvation.
Phosphorylates ATM. Suppresses Fas-induced apoptosis by phosphorylation of
CASP6, thus suppressing the activation of the caspase and the subsequent cleavage of
CFLAR
cytochrome c oxidase subunit Vic. Cytochrome c oxidase, the terminal enzyme of the
mitochondrial respiratory chain, catalyzes the electron transfer from reduced
cytochrome c to oxygen. This gene is up-regulated in prostate cancer cells
interleukin 11 receptor, alpha. The IL11/IL11RA/IL6ST complex may be involved in
the control of proliferation and/or differentiation of skeletogenic progenitor or other
mesenchymal cells.
NLR family, pyrin domain containing 14. May be involved in inflammation and
spermatogenesis. In the testis, expressed mainly in A dark spermatogonia, mid and late
spermatocytes and spermatids but not in mitotically active A pale and B
spermatogonia. In the testis, expressed mainly in A dark spermatogonia, mid and late
spermatocytes and spermatids but not in mitotically active A pale and B
spermatogonia
phosphodiesterase 1B, calmodulin-dependent. These enzymes are involved in many
signal transduction pathways and their functions include vascular smooth muscle
proliferation and contraction, cardiac contractility, platelet aggregation, hormone
secretion, immune cell activation, and they are involved in learning and memory
prolylcarboxypeptidase (angiotensinase C). The protein encoded by this gene is a
lysosomal prolylcarboxypeptidase, which cleaves C-terminal amino acids linked to
proline in peptides such as angiotension II, III and des-Arg9-bradykinin.
NACC family member 2, BEN and BTB (POZ) domain containing
87
A630053N20RIK
1.5817
ENTPD2
1.5784
Membrane
ectonucleoside triphosphate diphosphohydrolase 2. In the nervous system, could
hydrolyze ATP and other nucleotides to regulate purinergic neurotransmission.
RAB28
1.5775
Membrane
RAB28, member RAS oncogene family. The encoded protein may be involved in
regulating intracellular trafficking.
1700052O22RIK
1.5726
SMPD1
1.5720
Lysosome
sphingomyelin phosphodiesterase 1, acid lysosomal. The protein encoded by this gene
is a lysosomal acid sphingomyelinase that converts sphingomyelin to ceramide
LARP6
1.5709
Nucl & Cyto
APBB1
1.5686
AES
1.5657
JOSD2
1.5617
B230387C07RIK
1.5595
2900062L11RIK
1.5586
RPS6KA4
SH3GL2
NKX3-1
1.5572
MRAS
La ribonucleoprotein domain family, member 6.
Nucleus
amyloid beta (A4) precursor protein-binding, family B, member 1 (Fe65). It is an
adaptor protein localized in the nucleus.APP functions as a cytosolic anchoring site
that can prevent the gene product's nuclear translocation.
amino-terminal enhancer of split. Acts as dominant repressor towards other family
members. Inhibits NF-kappa-B-regulated gene expression. May be required for the
initiation and maintenance of the differentiated state
Josephin domain containing 2. May act as a deubiquitinating enzyme
1.5584
Nucleus
ribosomal protein S6 kinase, 90kDa, polypeptide 4
1.5580
Ctyo &
Membrane ?
Nucleus
SH3-domain GRB2-like 2. Implicated in synaptic vesicle endocytosis. May recruit
other proteins to membranes with high curvature
NH3 homeobox. The homeodomain-containing transcription factor NKX3-1 is a
putative prostate tumor suppressor that is expressed in a largely prostate-specific and
androgen-regulated manner. Loss of NKX3-1 protein expression is a common finding
in human prostate carcinomas and prostatic intraepithelial neoplasia
1.5560
Plasma
Membrane
HIST1H1C
1.5513
Nucleus
STAG3
1.5504
Nucleus
muscle RAS oncogene homolog. May serve as an important signal transducer for a
novel upstream stimuli in controlling cell proliferation. Weakly activates the MAP
kinase pathway
histone cluster 1, H1c. Histones H1 are necessary for the condensation of nucleosome
chains into higher order structures
stromal antigen 3. Meiosis specific component of cohesin complex. The cohesin
complex is required for the cohesion of sister chromatids after DNA replication
DOK4
1.5503
Membrane
2600009P04RIK
1.5485
2810423A18RIK
1.5484
ZNF512B
1.5476
BC044804
1.5405
2310021P13RIK
1.5405
LOC674135
1.5400
RNASET2B
1.5394
3110040M04RIK
1.5387
RAB11FIP4
Nucleus
docking protein 4. DOK4 functions in RET-mediated neurite outgrowth and plays a
positive
role in activation of the MAP kinase pathway (By similarity). Putative link with
downstream effectors of RET in neuronal differentiation.
zinc finger protein 512B
Extracellular
ribonuclease T2. This ribonuclease gene is a novel member of the Rh/T2/Sglycoprotein class of extracellular ribonucleases. It is a single copy gene that maps to
6q27, a region associated with human malignancies and chromosomal rearrangement.
1.5387
Endosome
DLG5
1.5362
Intracellular
TMEM9
1.5351
Lysosome
RAB11 family interacting protein 4 (class II). Acts as a regulator of endocytic traffic
by participating in membrane delivery. Required for the abcission step in cytokinesis,
possibly by acting as an 'address tag' delivering recycling endosome membranes to the
cleavage furrow during late cytokinesis.
discs, large homolog 5 (Drosophila). May play a role at the plasma membrane in the
maintenance of the structure of epithelial cells and in the transmission of extracellular
signals to the membrane and cytoskeleton
transmembrane protein 9. May be involved in intracellular transport
NELF
1.5338
Membrane
nasal embryonic LHRH factor. Couples NMDA receptor signaling to the nucleus.
Influences outgrowth of olfactory axons and migration of LHRH neurons
KHK
1.5337
Cytoplasm
ketohexokinase (fructokinase). This gene encodes ketohexokinase that catalyzes
conversion of fructose to fructose-1-phosphate. The product of this gene is the first
enzyme with a specialized pathway that catabolizes dietary fructose
1700023M03RIK
1.5333
TMEM41A
1.5308
Membrane
transmembrane protein 41A.
MIF4GD
1.5306
Nucleus
MIF4G domain containing. Functions in replication-dependent translation of histone
mRNAs which differ from other eukaryotic mRNAs in that they do not end with a
poly-A tail but a stem-loop. May participate in circularizing those mRNAs specifically
enhancing their translation
88
TBCEL
1.5301
Cytoplasm
TRH
1.5296
Sectreted
tubulin folding cofactor E-like. Acts as a regulator of tubulin stability
thyrotropin-releasing hormone. This hormone is responsible for the regulation and
release of thyroid-stimulating hormone, as well as prolactin. Deficiency of this
hormone has been associated with hypothalamic hypothyroidism.
ETHE1
1.5291
Cyto &
Nucleus
ZSWIM5
1.5287
ethylmalonic encephalopathy 1. Probably plays an important role in metabolic
homeostasis in mitochondria. May function as a nuclear-cytoplasmic shuttling protein
that binds transcription factor RELA/NFKB3 in the nucleus and exports it to the
cytoplasm. Suppresses p53-induced apoptosis by preventing nuclear localization of
RELA
zinc finger, SWIM-type containing 5
ATXN2
1.5284
Cytoplasm
ZFP282
1.5284
Nucleus
STXBP1
1.5279
Cytoplasm
HOXB6
1.5265
Nucleus
ANKRD56
1.5260
Membrane
ataxin 2. The autosomal dominant cerebellar ataxias (ADCA) are a heterogeneous
group of neurodegenerative disorders characterized by progressive degeneration of the
cerebellum, brain stem and spinal cord.
zinc finger protein 282. Binds to the U5 repressive element (U5RE) of the human T
cell leukemia virus type I long terminal repeat. It recognizes the 5'-TCCACCCC-3'
sequence as a core motif and exerts a strong repressive effect on HTLV-I LTRmediated expression
syntaxin binding protein 1. May participate in the regulation of synaptic vesicle
docking and fusion, possibly through interaction with GTP-binding proteins. Essential
for neurotransmission and binds syntaxin, a component of the synaptic vesicle fusion
machinery probably in a 1:1 ratio. Can interact with syntaxins 1, 2, and 3 but not
syntaxin 4. May play a role in determining the specificity of intracellular fusion
reactions
homeobox B6. Sequence-specific transcription factor which is part of a developmental
regulatory system that provides cells with specific positional identities on the anteriorposterior axis
ankyrin repeat domain 56.
SKIV2L
1.5250
Nucleus
superkiller viralicidic activity 2-like (S. cerevisiae). DEAD box proteins, characterized
by the conserved motif Asp-Glu-Ala-Asp (DEAD), are putative RNA helicases. They
are implicated in a number of cellular processes involving alteration of RNA
secondary structure such as translation initiation, nuclear and mitochondrial splicing,
and ribosome and spliceosome assembly. Based on their distribution patterns, some
members of this family are believed to be involved in embryogenesis,
spermatogenesis, and cellular growth and division.
SYRADB
1.5249
PPAP2C
1.5245
Membrane
phosphatidic acid phosphatase type 2C. Catalyzes the conversion of phosphatidic acid
(PA) to diacylglycerol (DG). In addition it hydrolyzes lysophosphatidic acid (LPA),
ceramide-1-phosphate (C-1-P) and sphingosine-1-phosphate (S-1-P).
MYO6
1.5229
Golgi,
nucleus &
Membrane
DUSP2
1.5180
Nucleus
myosin VI. This gene encodes a protein involved intracellular vesicle and organelle
transport, especially in the hair cell of the inner ear. Functions in a variety of
intracellular processes such as vesicular membrane trafficking and cell migration.
Required for the structural integrity of the Golgi apparatus via the p53-dependent prosurvival pathway.
dual specificity phosphatase 2. Regulates mitogenic signal transduction by
dephosphorylating both Thr and Tyr residues on MAP kinases ERK1 and ERK2. They
negatively regulate members of the mitogen-activated protein (MAP) kinase
superfamily (MAPK/ERK, SAPK/JNK, p38), which are associated with cellular
proliferation and differentiation.
LOC100047323
1.5167
1110012O05RIK
1.5156
IHPK1
1.5152
Cyto &
Nucleus
CNRIP1
1.5151
Cytoplasm ?
LOC100045864
1.5145
MIPEP
1.5141
Mito. Matrix
LIMA1
1.5138
Cytoplasm
LOC100044124
1.5133
SLC4A2
1.5127
LOC100045963
1.5126
ROGDI
1.5103
3632451O06RIK
1.5095
inositol hexakisphosphate kinase 1. Converts inositol hexakisphosphate (InsP6) to
diphosphoinositol pentakisphosphate (InsP7/PP-InsP5). Converts 1,3,4,5,6pentakisphosphate (InsP5) to PP-InsP4
cannabinoid receptor interacting protein 1. This gene encodes a G-protein coupled
receptor which interacts with the C-terminal tail of cannabinoid receptor 1. This
receptor plays a role in synaptic plasticity, analgesia, appetite, and neuroprotection.
mitochondrial intermediate peptidase.The product of this gene performs the final step
in processing a specific class of nuclear-encoded proteins targeted to the mitochondrial
matrix or inner membrane.
LIM domain and actin binding 1. Binds to actin monomers and filaments. Increases
the number and size of actin stress fibers and inhibits membrane ruffling. Inhibits actin
filament depolymerization. Bundles actin filaments, delays filament nucleation and
reduces formation of branched filaments
Membrane
solute carrier family 4, anion exchanger, member 2 (erythrocyte membrane protein
band 3-like 1). Plasma membrane anion exchange protein of wide distribution
Cytoplasm
rogdi homolog (Drosophila). May act as a positive regulator of cell proliferation
89
TMEM127
1.5088
Membrane
transmembrane protein 127. Controls cell proliferation acting as a negative regulator
of TOR signaling pathway mediated by mTORC1. May act as a tumor suppressor
CNPY2
1.5082
ER
canopy 2 homolog (zebrafish). Positive regulator of neurite outgrowth by stabilizing
myosin regulatory light chain (MRLC). It prevents MIR-mediated MRLC
ubiquitination and its subsequent proteasomal degradation
H2-Q8
1.5077
WHRN
1.5074
Cytoplasm
deafness, autosomal recessive 31. Necessary for elongation and maintenance of inner
and outer hair cell stereocilia in the organ of Corti in the inner ear
9030625A04RIK
1.5058
FA2H
1.5051
TPCN2
1.5028
ER
membrane
Lysosome
Mem.
fatty acid 2-hydroxylase. Required for alpha-hydroxylation of free fatty acids and the
formation of alpha-hydroxylated sphingolipids
two pore segment channel 2. Nicotinic acid adenine dinucleotide phosphate (NAADP)
receptor that may function as one of the major voltage-gated Ca(2+) channels (VDCC)
across the lysosomal membrane. May be involved in smooth muscle contraction
DNMT3B
1.5015
Nucleus
DNA (cytosine-5-)-methyltransferase 3 beta. CpG methylation is an epigenetic
modification that is important for embryonic development, imprinting, and Xchromosome inactivation. Studies in mice have demonstrated that DNA methylation is
required for mammalian development. This gene encodes a DNA methyltransferase
which is thought to function in de novo methylation, rather than maintenance
methylation.
90
ANNEX 2
MICROARRAY DATA
Genes Downregulated 24 Hours Post-Treatment with 0.7μM
RA (Threshold: fold decrease ≥ 1.5, p ≤ 0.05)
CDK4
0.6689
Cyto, Nuc,
Mem
cyclin-dependent kinase 4. The protein encoded by this gene is a member of the
Ser/Thr protein kinase family. This protein is highly similar to the gene products of
S. cerevisiae cdc28 and S. pombe cdc2. It is a catalytic subunit of the protein
kinase complex that is important for cell cycle G1 phase progression. The activity
of this kinase is restricted to the G1-S phase, which is controlled by the regulatory
subunits D-type cyclins and CDK inhibitor p16(INK4a). This kinase was shown to
be responsible for the phosphorylation of retinoblastoma gene product (Rb).
HMOX1
0.6684
ER
NRBP2
0.6684
Cytoplasm
RIN2
0.6683
Cytoplasm
BAG3
0.6675
Cytoplasm
T
0.6673
Nucleus
heme oxygenase (decycling) 1. Heme oxygenase cleaves the heme ring at the alpha
methene bridge to form biliverdin. Biliverdin is subsequently converted to bilirubin
by biliverdin reductase. Under physiological conditions, the activity of heme
oxygenase is highest in the spleen, where senescent erythrocytes are sequestrated
and destroyed
nuclear receptor binding protein 2. May regulate apoptosis of neural progenitor
cells during their differentiation
Ras and Rab interactor 2. as effector protein. May function as an upstream
activator and/or downstream effector for RAB5B in endocytic pathway. May
function as a guanine nucleotide exchange (GEF) of RAB5B, required for
activating the RAB5 proteins by exchanging bound GDP for free GTP
BCL2-associated athanogene 3. Inhibits the chaperone activity of HSP70/HSC70
by promoting substrate release. Has anti-apoptotic activity
T, brachyury homolog (mouse). Involved in the transcriptional regulation of genes
required for mesoderm formation and differentiation. Binds to a palindromic site
(called T site) and activates gene transcription when bound to such a site
FOXO6
0.6668
Cyto & Nucleus
C920006O11RIK
0.6666
HMGCL
0.6666
Mito. Matrix
KIF3C
0.6664
Cytoplasm
2900019M05RIK
0.6663
KRT24
0.6661
Cytoplasm ?
EFNA4
0.6661
Secreted
SPSB1
0.6661
Cytoplasm
splA/ryanodine receptor domain and SOCS box containing 1. Probable substrate
recognition component of a SCF-like ECS (Elongin BC-CUL2/5-SOCS-box
protein) E3 ubiquitin-protein ligase complex which mediates the ubiquitination and
subsequent proteasomal degradation of target proteins
1700054N08RIK
0.6654
GALNT2
0.6653
Golgi
UDP-N-acetyl-alpha-D-galactosamine:polypeptide
Nacetylgalactosaminyltransferase 2 (GalNAc-T2).Catalyzes the initial reaction in Olinked oligosaccharide biosynthesis, the transfer of an N-acetyl-D-galactosamine
residue to a serine or threonine residue on the protein receptor.
EIF1A
0.6647
Cytoplasm
eukaryotic translation initiation factor 1A, Y-linked. eems to be required for
maximal rate of protein biosynthesis. Enhances ribosome dissociation into subunits
and stabilizes the binding of the initiator Met-tRNA(I) to 40 S ribosomal subunits
1700013E18RIK
0.6646
COL1A1
0.6644
Secreted
collagen, type I, alpha 1. This gene encodes the pro-alpha1 chains of type I
collagen whose triple helix comprises two alpha1 chains and one alpha2 chain.
forkhead box O6. Transcriptional activator
3-hydroxymethyl-3-methylglutaryl-CoA lyase. Involved in the catabolism of
branched amino acids such as leucine
kinesin family member 3C. Microtubule-based anterograde translocator for
membranous organelles
keratin 24. This gene encodes a member of the type I (acidic) keratin family, which
belongs to the superfamily of intermediate filament (IF) proteins.
ephrin-A4. This gene encodes a member of the ephrin (EPH) family. The ephrins
and EPH-related receptors comprise the largest subfamily of receptor proteintyrosine kinases and have been implicated in mediating developmental events,
especially in the nervous system and in erythropoiesis.
91
ZSWIM4
0.6643
SLC40A1
0.6641
5930412G12RIK
0.6640
EPHA2
zinc finger, SWIM-type containing 4.
Membrane
solute carrier family 40 (iron-regulated transporter), member 1. May be involved in
iron export from duodenal epithelial cell and also in transfer of iron between
maternal and fetal circulation. Mediates iron efflux in the presence of a ferroxidase
0.6639
Membrane
EPH receptor A2. This gene belongs to the ephrin receptor subfamily of the
protein-tyrosine kinase family. EPH and EPH-related receptors have been
implicated in mediating developmental events, particularly in the nervous system.
CDH4
0.6636
Membrane
cadherin 4, type 1, R-cadherin (retinal). Cadherins are calcium dependent cell
adhesion proteins. They preferentially interact with themselves in a homophilic
manner in connecting cells; cadherins may thus contribute to the sorting of
heterogeneous cell types. May play an important role in retinal development
SCMH1
0.6624
Nucleus
WWC1
0.6606
Cytoplasm
sex comb on midleg homolog 1 (Drosophila). Component of the Polycomb group
(PcG) multiprotein PRC1 complex, a complex required to maintain the
transcriptionally repressive state of many genes, including Hox genes, throughout
development.
WW and C2 domain containing 1. The protein encoded by this gene is a
cytoplasmic phosphoprotein that interacts with PRKC-zeta and dynein light chain1. Alleles of this gene have been found that enhance memory in some individuals.
Three transcript variants encoding different isoforms have been found for this gene
CSNK1E
0.6593
Cytoplasm
NAB1
0.6593
Nucleus
LAPTM4A
0.6588
Membrane ?
AEBP1
0.6584
Cytoplasm
ARNTL
0.6573
Nucleus
B020017C02RIK
0.6562
2610016A17RIK
0.6559
ATPIF1
0.6558
Mitochondria
PCDH1
0.6556
Membrane
FOXJ2
0.6554
Nucleus
GJB2
0.6549
Membrane
SGK1
0.6546
Cyto & Nucleus
RPL22
0.6546
Cytoplasm
AK3L1
0.6543
Mitochondria
KANK3
0.6542
casein kinase 1, epsilon. The protein encoded by this gene is a serine/threonine
protein kinase and a member of the casein kinase I protein family, whose members
have been implicated in the control of cytoplasmic and nuclear processes,
including DNA replication and repair. The encoded protein is found in the
cytoplasm as a monomer and can phosphorylate a variety of proteins, including
itself.
NGFI-A binding protein 1 (EGR1 binding protein 1)Acts as a transcriptional
repressor for zinc finger transcription factors EGR1 and EGR2
lysosomal protein transmembrane 4 alpha. This gene encodes a protein that has
four predicted transmembrane domains. The function of this gene has not yet been
determined; however, studies in the mouse homolog suggest a role in the transport
of small molecules across endosomal and lysosomal membranes
AE binding protein 1. This protein seems to be activated by a novel mechanism,
whereby the direct binding of DNA enhances its protease activity. Adipocyteenhancer binding protein 1 may play a role in differentiated vascular smooth
muscle cells.
aryl hydrocarbon receptor nuclear translocator-like. The protein encoded by this
gene is a basic helix-loop-helix protein that forms a heterodimer with CLOCK.
This complex binds an E-box upstream of the PER1 gene, activating this gene and
possibly other circadian rhythym-associated genes. Three transcript variants
encoding two different isoforms have been found for this gene.
ATPase inhibitory factor 1. This gene encodes a mitochondrial ATPase inhibitor.
Alternative splicing occurs at this locus and three transcript variants encoding
distinct isoforms have been identified.
protocadherin 1. This gene belongs to the protocadherin subfamily within the
cadherin superfamily. The encoded protein is a membrane protein found at cell-cell
boundaries. It is involved in neural cell adhesion, suggesting a possible role in
neuronal development. The protein includes an extracelllular region, containing 7
cadherin-like domains, a transmembrane region and a C-terminal cytoplasmic
region. Cells expressing the protein showed cell aggregation activity. Alternative
splicing occurs in this gene
forkhead box J2. Transcriptional activator. Able to bind to two different type of
DNA binding sites.
gap junction protein, beta 2. They are continuously synthesized and degraded,
allowing
tissues
to
rapidly adapt to changing environmental conditions. Connexins play a key role in
many physiological processes including cardiac and smooth muscle contraction,
regulation of neuronal excitability, epithelial electrolyte transport and keratinocyte
differentiation
serum/glucocorticoid regulated kinase 1. This gene encodes a serine/threonine
protein kinase that plays an important role in cellular stress response. This kinase
activates certain potassium, sodium, and chloride channels, suggesting an
involvement in the regulation of processes such as cell survival, neuronal
excitability, and renal sodium excretion. High levels of expression of this gene may
contribute to conditions such as hypertension and diabetic nephropathy.
ribosomal protein L22. This gene encodes a cytoplasmic ribosomal protein that is a
component of the 60S subunit.
adenylate kinase 3. The protein encoded by this gene is a GTP:ATP
phosphotransferase that is found in the mitochondrial matrix.
KN motif and ankyrin repeat domains 3
92
ANKRD47
0.6539
FOXC2
0.6535
Nucleus
see above
DCTD
0.6534
Cytoplasm
STC2
0.6524
Secreted
IGF1R
0.6518
Membrane
CST3
0.6517
Secreted
cystatin C. As an inhibitor of cysteine proteinases, this protein is thought to serve
an important physiological role as a local regulator of this enzyme activity
ARHGAP21
0.6513
Golgi
Membrane
ASPHD2
0.6512
Membrane
Rho GTPase activating protein 21. Functions as a GTPase-activating protein
(GAP) for RHOA and CDC42. Downstream partner of ARF1 which may control
Golgi apparatus structure and function. Also required for CTNNA1 recruitment to
adherens junctions
aspartate beta-hydroxylase domain containing 2.
ABCA7
0.6510
Membrane
SLC2A3
0.6507
Membrane
CMTM7
0.6495
Membrane
A930002F06RIK
0.6487
TEX19.2
0.6484
CD82
0.6484
Membrane
INPPL1
0.6480
Cytoplasm &
Peripheral
Membrane
SPRED1
0.6473
Membrane
CLCF1
0.6472
Extracellular
MRPS6
0.6471
Mitochondria
EPHB1
0.6470
Membrane
IGF2BP3
0.6470
Nucleus & Cyto
forkhead box C2 (MFH-1, mesenchyme forkhead 1). This gene belongs to the
forkhead family of transcription factors which is characterized by a distinct DNAbinding forkhead domain. The specific function of this gene has not yet been
determined; however, it may play a role in the development of mesenchymal
tissues.
dCMP deaminase. Supplies the nucleotide substrate for thymidylate synthetase
stanniocalcin 2. This gene encodes a secreted, homodimeric glycoprotein that is
expressed in a wide variety of tissues and may have autocrine or paracrine
functions. The encoded protein has 10 of its 15 cysteine residues conserved among
stanniocalcin family members and is phosphorylated by casein kinase 2 exclusively
on its serine residues. Its C-terminus contains a cluster of histidine residues which
may interact with metal ions.
insulin-like growth factor 1 receptor. This receptor binds insulin-like growth factor
with a high affinity. It has tyrosine kinase activity. The insulin-like growth factor I
receptor plays a critical role in transformation events. Cleavage of the precursor
generates alpha and beta subunits. It is highly overexpressed in most malignant
tissues where it functions as an anti-apoptotic agent by enhancing cell survival.
ATP-binding cassette, sub-family A (ABC1), member 7. Plays a role in
phagocytosis by macrophages of apoptotic cells. Binds APOA1 and may function
in apolipoprotein-mediated phospholipid efflux from cells. May also mediate
cholesterol efflux. May regulate cellular ceramide homeostasis during
keratinocytes differentiation
solute carrier family 2 (facilitated glucose transporter), member 3. Facilitative
glucose transporter. Probably a neuronal glucose transporter
CKLF-like MARVEL transmembrane domain containing 7. This gene belongs to
the chemokine-like factor gene superfamily, a novel family that is similar to the
chemokine and transmembrane 4 superfamilies. This gene is one of several
chemokine-like factor genes located in a cluster on chromosome 3.
This metastasis suppressor gene product is a membrane glycoprotein that is a
member of the transmembrane 4 superfamily. Expression of this gene has been
shown to be downregulated in tumor progression of human cancers and can be
activated by p53 through a consensus binding sequence in the promoter. Its
expression and that of p53 are strongly correlated, and the loss of expression of
these two proteins is associated with poor survival for prostate cancer patients.
Two alternatively spliced transcript variants encoding distinct isoforms have been
found for this gene.
inositol polyphosphate phosphatase-like 1. May act by regulating AKT2, but not
AKT1,
phosphorylation
at
the
plasma
membrane. Part of a signaling pathway that regulates actin cytoskeleton
remodeling.
Required
for
the
maintenance
and
dynamic remodeling of actin structures as well as in endocytosis, having a major
impact
on
ligand-induced
EGFR
internalization and degradation. Regulates cell adhesion and cell spreading.
Required for HGF-mediated lamellipodium formation, cell scattering and
spreading.
sprouty-related, EVH1 domain containing 1. Tyrosine kinase substrate that inhibits
growth-factor-mediated activation of MAP kinase. Negatively regulates
hematopoiesis of bone marrow
cardiotrophin-like cytokine factor 1. Cytokine with B-cell stimulating capability.
Binds to and activates the ILST/gp130 receptor
mitochondrial ribosomal protein S6. This gene encodes a 28S subunit protein that
belongs to the ribosomal protein S6P family.
EPH receptor B1. Receptor for members of the ephrin-B family. Binds to ephrinB1, -B2 and -B3. Binding with the guidance cue ephrin-B2 at the optic chiasm
midline redirect ventrotemporal (VT) retinal ganglion cells (RGCs) axons
ipsilaterally. May be involved in cell-cell interactions in the nervous system
insulin-like growth factor 2 mRNA binding protein 3. RNA-binding protein that
act as a regulator of mRNA translation and stability. Binds to the 5'-UTR of the
insulin-like growth factor 2 (IGF2) mRNAs. Binds to sequences in the 3'-UTR of
CD44 mRNA
93
FGF9
0.6469
Secreted
fibroblast growth factor 9 (glia-activating factor). The protein encoded by this gene
is a member of the fibroblast growth factor (FGF) family. FGF family members
possess broad mitogenic and cell survival activities, and are involved in a variety
of biological processes, including embryonic development, cell growth,
morphogenesis, tissue repair, tumor growth and invasion. This protein was isolated
as a secreted factor that exhibits a growth-stimulating effect on cultured glial cells.
Mice lacking the homolog gene displayed a male-to-female sex reversal
phenotype, which suggested a role in testicular embryogenesis.
IRF2BP2
0.6453
Nucleus
interferon regulatory factor 2 binding protein 2. Acts as a transcriptional repressor.
Acts as a transcriptional corepressor in a IRF2-dependent manner. This repression
is not mediated at least in part by histone deacetylase activities
HELZ
0.6449
Nucleus
helicase with zinc finger. May act as an helicase that plays a role in RNA
metabolism in multiple tissues and organs within the developing embryo
AI450540
0.6447
B230327L12RIK
0.6445
SHISA2
0.6440
ER Membrane
PRR5
0.6440
shisa homolog 2 (Xenopus laevis). Plays an essential role in the maturation of
presomitic mesoderm cells by individual attenuation of both FGF and WNT
signaling (By similarity)
proline rich 5 (renal) This gene encodes a protein with a proline-rich domain. This
gene is located in a region of chromosome 22 reported to contain a tumor
suppressor gene that may be involved in breast and colorectal tumorigenesis. The
protein is a component of the mammalian target of rapamycin complex 2
(mTORC2), and it regulates platelet-derived growth factor (PDGF) receptor beta
expression and PDGF signaling to Akt and S6K1.
SESN1
0.6439
Nucleus
CRYGS
0.6432
sestrin 1. Involved in the reduction of peroxiredoxins. May also be regulator of
cellular growth
crystallin, gamma S. Crystallins are the dominant structural components of the
vertebrate eye lens
2900026A02RIK
0.6426
GP5
0.6421
Membrane
glycoprotein V. The GPIb-V-IX complex functions as the vWF receptor and
mediates vWF-dependent platelet adhesion to blood vessels. The adhesion of
platelets to injured vascular surfaces in the arterial circulation is a critical initiating
event in hemostasis
LOC100046770
0.6419
EGLN3
0.6418
Cyto & Nucleus
VWA2
0.6416
Secreted
egl nine homolog 3.May play a role in cell growth regulation in muscle cells and in
apoptosis in neuronal tissue. Promotes cell death through a caspase-dependent
mechanism
von Willebrand factor A domain containing 2
PLK2
0.6401
RAB31
0.6397
SCL0001487.1_50
0.6393
ICK
polo-like kinase 2. May play a role in the division of at least some cell types, such
as fibroblasts, and could function in embryogenesis, wound healing or neoplasia
Intracel.
Membrane
RAB31, member RAS oncogene family. Play essential roles in vesicle and granule
targeting
0.6391
Cyto & Nucleus
intestinal cell (MAK-like) kinase. This gene encodes an intestinal serine/threonine
kinase harboring a dual phosphorylation site found in mitogen-activating protein
(MAP) kinases. The protein localizes to the intestinal crypt region and is thought to
be important in intestinal epithelial cell proliferation and differentiation.
SMYD2
0.6389
Cytoplasm
ZFP36
0.6384
Nucleus
SET and MYND domain containing 2. Protein-lysine N-methyltransferase that
methylates both histones and non-histone proteins.
zinc finger protein 36, C3H type, homolog. Probable regulatory protein with a
novel zinc finger structure involved in regulating the response to growth factors.
KIF21A
0.6383
Cytoplasm
kinesin family member 21A. Microtubule-binding motor protein probably involved
in neuronal axonal transport. In vitro, has a plus-end directed motor activity
X99384
0.6378
ADM
0.6374
Secreted
SGPP2
0.6346
ER Membrane
adrenomedullin. Adrenomedullin, a hypotensive peptide found in human
pheochromocytoma, consists of 52 amino acids, has 1 intramolecular disulfide
bond, and shows a slight homology with the calcitonin gene-related peptide. It may
function as a hormone in circulation control because it is found in blood in a
considerable concentration.
sphingosine-1-phosphate phosphatase 2. Has specific phosphohydrolase activity
towards sphingoid base 1-phosphates. Has high phosphohydrolase activity against
dihydrosphingosine-1-phosphate and sphingosine-1-phosphate (S1P) in vitro. May
play a role in attenuating intracellular sphingosine 1-phosphate (S1P) signaling.
May play a role in pro-inflammatory signaling
RPAP3
0.6342
RSPH1
0.6340
BRWD3
0.6338
Cytoplasm
RNA polymerase II associated protein 3. This gene encodes an RNA polymerase
II-associated protein. The encoded protein may function in transcriptional
regulation and may also regulate apoptosis.
radial spoke head 1 homolog. May play an important role in male meiosis
bromodomain and WD repeat domain containing 3. It is thought to have a
chromatin-modifying function, and may thus play a role in transcription
94
RIPK3
0.6323
Cytoplasm
receptor-interacting serine-threonine kinase 3. It is a component of the tumor
necrosis factor (TNF) receptor-I signaling complex, and can induce apoptosis and
weakly activate the NF-kappaB transcription factor.
Rho GTPase activating protein 32. May be involved in the differentiation of
neuronal cells during the formation of neurite extensions. Involved in NMDA
receptor activity-dependent actin reorganization in dendritic spines. May mediate
cross-talks between Ras- and Rho-regulated signaling pathways in cell growth
regulation.
low density lipoprotein receptor. Binds LDL, the major cholesterol-carrying
lipoprotein of plasma, and transports it into cells by endocytosis. In order to be
internalized, the receptor-ligand complexes must first cluster into clathrin-coated
pits. In case of HIV-1 infection, functions as a receptor for extracellular Tat in
neurons, mediating its internalization in uninfected cells
B-cell CLL/lymphoma 6, member B. Acts as a sequence-specific transcriptional
repressor in association with BCL6.
insulin receptor substrate 2. May mediate the control of various cellular processes
by insulin
zinc finger and SCAN domain containing 5B. May be involved in transcriptional
regulation
lectin, galactoside-binding, soluble, 1. May regulate apoptosis, cell proliferation
and cell differentiation. Binds beta-galactoside and a wide array of complex
carbohydrates. Inhibits CD45 protein phosphatase activity and therefore the
dephosphorylation of Lyn kinase
F-box protein 6. Substrate-recognition component of some SCF (SKP1-CUL1-Fbox protein)-type E3 ubiquitin ligase complexes. Involved in endoplasmic
reticulum-associated degradation pathway (ERAD) for misfolded lumenal proteins
by recognizing and binding sugar chains on unfolded glycoproteins that are
retrotranlocated into the cytosol and promoting their ubiquitination and subsequent
degradation.
interleukin 17 receptor D. Feedback inhibitor of fibroblast growth factor mediated
Ras-MAPK signaling and ERK activation. May inhibit FGF-induced FGFR1
tyrosine phosphorylation. Regulates the nuclear ERK signaling pathway by
spatially blocking nuclear translocation of activated ERK without inhibiting
cytoplasmic phosphorylation of ERK. Mediates JNK activation and may be
involved in apoptosis
GRIT
0.6322
Membrane
etc…
LDLR
0.6320
Membrane
BCL6B
0.6320
Nucleus
IRS2
0.6318
Cytoplasm
ZSCAN5B
0.6315
Nucleus
LGALS1
0.6312
Secreted
FBXO6
0.6311
Cytoplasm
IL17RD
0.6307
Golgi
Membrane
LOC100047427
0.6305
6430550H21RIK
0.6297
LOC666559
0.6296
3110078M01RIK
0.6293
LOC382074
0.6287
PCP4L1
0.6285
SNAI1
0.6285
EG433229
0.6282
CYP51
0.6277
2610200G18RIK
0.6275
TMEM132C
0.6275
Membrane
IGF2BP1
0.6273
Cyto & Nucleus
MAP4K1
0.6271
insulin-like growth factor 2 mRNA binding protein 1. This gene encodes a member
of the insulin-like growth factor 2 mRNA-binding protein family.
mitogen-activated protein kinase kinase kinase kinase 1. May play a role in the
response to environmental stress. Appears to act upstream of the JUN N-terminal
pathway. May play a role in hematopoietic lineage decisions and growth regulation
LOC100047863
0.6258
P42POP
0.6252
Nucleus
Myb-related transcription factor, partner of profilin. Transcriptional repressor;
DNA-binding protein that specifically recognizes the core sequence 5'YAAC[GT]G-3'.
D15ERTD682E
0.6249
EG433016
0.6248
SETD1B
0.6248
Nucleus
FGL1
0.6246
Secreted
ARHGAP26
0.6240
Cell Junction
SET domain containing 1B. SET1B is a component of a histone methyltransferase
complex that produces trimethylated histone H3 at Lys4
fibrinogen-like 1. This protein is homologous to the carboxy terminus of the
fibrinogen beta- and gamma- subunits which contains the four conserved cysteines
of fibrinogens and fibrinogen related proteins. However, this protein lacks the
platelet-binding site, cross-linking region and a thrombin-sensitive site which are
necessary for fibrin clot formation.
Rho GTPase activating protein 26. The protein encoded by this gene is a GTPase
activating protein that binds to focal adhesion kinase and mediates the activity of
the GTP binding proteins RhoA and Cdc42. Defects in this gene are a cause of
juvenile myelomonocytic leukemi
Purkinje cell protein 4 like 1.
Cyto & Nucleus
snail homolog 1. The Drosophila embryonic protein snail is a zinc finger
transcriptional repressor which downregulates the expression of ectodermal genes
within the mesoderm. The nuclear protein encoded by this gene is structurally
similar to the Drosophila snail protein, and is also thought to be critical for
mesoderm formation in the developing embryo.
ER Membrane
cytochrome P450, family 51, subfamily A, polypeptide 1. This endoplasmic
reticulum protein participates in the synthesis of cholesterol by catalyzing the
removal of the 14alpha-methyl group from lanosterol.
transmembrane protein 132C.
95
PARP8
0.6233
Cytoplasm
poly (ADP-ribose) polymerase family, member 8. Poly (ADP-ribose) polymerase
(PARP) catalyzes the post-translational modification of proteins by the addition of
multiple ADP-ribose moieties. PARP transfers ADP-ribose from nicotinamide
dinucleotide (NAD) to glu/asp residues on the substrate protein, and also
polymerizes ADP-ribose to form long/branched chain polymers
C230098O21RIK
0.6232
EDG7
0.6226
Membrane
lysophosphatidic acid receptor 3. This gene encodes a member of the G proteincoupled receptor family, as well as the EDG family of proteins. This protein
functions as a cellular receptor for lysophosphatidic acid and mediates
lysophosphatidic acid-evoked calcium mobilization.
phosphofructokinase, platelet. FK catalyzes the irreversible conversion of fructose6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in
glycolysis.
dual specificity phosphatase 1. The expression of DUSP1 gene is induced in
human skin fibroblasts by oxidative/heat stress and growth factors. It specifies a
protein with structural features similar to members of the non-receptor-type
protein-tyrosine phosphatase family, and which has significant amino-acid
sequence similarity to a Tyr/Ser-protein phosphatase encoded by the late gene H1
of vaccinia virus. DUSP1 may play an important role in the human cellular
response to environmental stress as well as in the negative regulation of cellular
proliferation.
Rho guanine nucleotide exchange factor (GEF) 15. This gene encodes a protein
that functions as a specific guanine nucleotide exchange factor for RhoA. It also
interacts with ephrin A4 in vascular smooth muscle cells.
ankyrin 1, erythrocytic. Attaches integral membrane proteins to cytoskeletal
elements; binds to the erythrocyte membrane protein band 4.2, to Na-K ATPase, to
the lymphocyte membrane protein GP85, and to the cytoskeletal proteins fodrin,
tubulin, vimentin and desmin.
death-associated protein kinase 2. This gene encodes a protein that belongs to the
serine/threonine protein kinase family. This protein contains a N-terminal protein
kinase domain followed by a conserved calmodulin-binding domain with
significant similarity to that of death-associated protein kinase 1 (DAPK1), a
positive regulator of programmed cell death.
lectin, galactoside-binding, soluble, 7. Could be involved in cell-cell and/or cellmatrix interactions necessary for normal growth control. Pro-apoptotic protein that
functions intracellularly upstream of JNK activation and cytochrome c release
PFKP
0.6224
DUSP1
0.6218
Nucleus
ARHGEF15
0.6204
Cell Projection
ANK
0.6198
Cytoplasm
DAPK2
0.6189
Cytoplasm
LGALS7
0.6177
Cyto & Nucleus
NOTCH1
0.6173
Membrane
VAV2
0.6169
Cytoplasm
GBP3
0.6166
Membrane
TRIO
0.6163
Cytoplasm
MKIAA0282
0.6157
AXUD1
0.6157
LOC100040592
0.6145
WIF1
0.6140
HS3ST3B1
0.6119
Functions as a receptor for membrane-bound ligands Jagged1, Jagged2 and Delta1
to
regulate
cell-fate
determination. Upon ligand activation through the released notch intracellular
domain
(NICD)
it
forms
a
transcriptional activator complex with RBPJ/RBPSUH and activates genes of the
enhancer
of
split
locus.
Affects
the
implementation of differentiation, proliferation and apoptotic programs.
Guanine nucleotide exchange factor for the Rho family of Ras-related GTPases.
Plays
an
important
role
in
angiogenesis. Its recruitement by phosphorylated EPHA2 is critical for EFNA1induced
RAC1
GTPase
activation
and
vascular endothelial cell migration and assembly
This gene encodes a member of the guanylate-binding protein (GBP) family. GBPs
specifically
bind
guanine
nucleotides
(GMP, GDP, and GTP) and contain two of the three consensus motifs found in
typical GTP-binding proteins. The encoded protein interacts with a member of the
germinal center kinase family
triple functional domain (PTPRF interacting) Promotes the exchange of GDP by
GTP. Together with leukocyte antigen-related (LAR) protein, it could play a role in
coordinating cell-matrix and cytoskeletal rearrangements necessary for cell
migration and cell growth
Nucleus
cysteine-serine-rich nuclear protein 1. This gene encodes a protein that localizes to
the nucleus and expression of this gene is induced in response to elevated levels of
axin. The Wnt signalling pathway, which is negatively regulated by axin, is
important in axis formation in early development and impaired regulation of this
signalling pathway is often involved in tumors. A decreased level of expression of
this gene in tumors compared to the level of expression in their corresponding
normal tissues suggests that this gene product has a tumor suppressor function.
Binds to the consensus sequence 5'-AGAGTG-3' and has transcriptional activator
activity (By similarity). May have a tumor-suppressor function. May play a role in
apoptosis
Secreted
WNT inhibitory factor 1. The protein encoded by this gene functions to inhibit
WNT proteins, which are extracellular signaling molecules that play a role in
embryonic development. This protein contains a WNT inhibitory factor (WIF)
domain and five epidermal growth factor (EGF)-like domains, and is thought to be
involved in mesoderm segmentation. This gene functions as a tumor suppressor
gene, and has been found to be epigenetically silenced in various cancers
96
FRMD6
0.6119
STAT3
0.6118
Cyto &
Membrane
Cyto & Nucleus
FERM domain containing 6.
SOCS3
0.6117
Cytoplasm
suppressor of cytokine signaling 3. This gene encodes a member of the STATinduced STAT inhibitor (SSI), also known as suppressor of cytokine signaling
(SOCS), family. SSI family members are cytokine-inducible negative regulators of
cytokine signaling. The expression of this gene is induced by various cytokines,
including IL6, IL10, and interferon (IFN)-gamma. The protein encoded by this
gene can bind to JAK2 kinase, and inhibit the activity of JAK2 kinase.
POU3F1
0.6107
Nucleus
POU class 3 homeobox 1. Transcription factor that binds to the octamer motif (5'ATTTGCAT-3'). Thought to be involved in early embryogenesis and neurogenesis
F2R
0.6095
Membrane
coagulation factor II (thrombin) receptor. Coagulation factor II receptor is a 7transmembrane receptor involved in the regulation of thrombotic response.
Proteolytic cleavage leads to the activation of the receptor. F2R is a G-protein
coupled receptor family member.
4933432P15RIK
0.6089
ALDOA
0.6084
Cytosol ?
HS3ST3A1
0.6082
Golgi
Membrane
AGPAT4
0.6063
Membrane
SOX11
0.6060
Nucleus
aldolase A, fructose-bisphosphate. This gene product, Aldolase A (fructosebisphosphate aldolase) is a glycolytic enzyme that catalyzes the reversible
conversion of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and
dihydroxyacetone phosphate.
heparan sulfate (glucosamine) 3-O-sulfotransferase 3A1. Heparan sulfate
biosynthetic enzymes are key components in generating a myriad of distinct
heparan sulfate fine structures that carry out multiple biologic activities. The
enzyme encoded by this gene is a member of the heparan sulfate biosynthetic
enzyme family. It is a type II integral membrane protein and possesses heparan
sulfate glucosaminyl 3-O-sulfotransferase activity
1-acylglycerol-3-phosphate O-acyltransferase 4. This integral membrane protein
converts lysophosphatidic acid to phosphatidic acid, the second step in de novo
phospholipid biosynthesis.
SRY (sex determining region Y)-box 11. This intronless gene encodes a member
of the SOX (SRY-related HMG-box) family of transcription factors involved in the
regulation of embryonic development and in the determination of the cell fate. The
encoded protein may act as a transcriptional regulator after forming a protein
complex with other proteins.
BC023744
0.6059
SLC27A6
0.6055
Membrane
RPRML
0.6022
Membrane
PRRX2
0.6015
Nucleus
ADAMTS9
0.6012
Secreted
ANKRD37
0.5998
Nucleus
FDPS
0.5996
Cytoplasm
farnesyl diphosphate synthase. This gene encodes an enzyme that catalyzes the
production of geranyl pyrophosphate and farnesyl pyrophosphate from isopentenyl
pyrophosphate and dimethylallyl pyrophosphate. The resulting product, farnesyl
pyrophosphate, is a key intermediate in cholesterol and sterol biosynthesis, a
substrate for protein farnesylation and geranylgeranylation, and a ligand or agonist
for certain hormone receptors and growth receptors
MID1IP1
0.5994
Cyto &
Nucleus
MID1 interacting protein 1.Plays a role in the regulation of lipogenesis in liver.
Up-regulates ACACA enzyme activity. Required for efficient lipid biosynthesis,
including triacylglycerol, diacylglycerol and phospholipid. Involved in
stabilization of microtubules
8030402P03RIK
0.5990
GSTA3
0.5988
Cytoplasm
glutathione S-transferase alpha 3. These enzymes are involved in cellular defense
against toxic, carcinogenic, and pharmacologically active electrophilic compounds.
The enzyme encoded by this gene catalyzes the double bond isomerization of
precursors for progesterone and testosterone during the biosynthesis of steroid
hormones.
signal transducer and activator of transcription 3. In response to cytokines and
growth factors, STAT family members are phosphorylated by the receptor
associated kinases, and then form homo- or heterodimers that translocate to the cell
nucleus where they act as transcription activators. This protein is activated through
phosphorylation in response to various cytokines and growth factors including
IFNs, EGF, IL5, IL6, HGF, LIF and BMP2. This protein mediates the expression
of a variety of genes in response to cell stimuli, and thus plays a key role in many
cellular processes such as cell growth and apoptosis.
solute carrier family 27 (fatty acid transporter), member 6. FATPs are involved in
the
uptake
of
long-chain fatty acids and have unique expression patterns.
reprimo-like
paired related homeobox 2. Expression is localized to proliferating fetal fibroblasts
and the developing dermal layer, with downregulated expression in adult skin.
Increases in expression of this gene during fetal but not adult wound healing
suggest a possible role in mechanisms that control mammalian dermal regeneration
and prevent formation of scar response to wounding. The expression patterns
provide evidence consistent with a role in fetal skin development and a possible
role in cellularproliferation.
ADAM metallopeptidase with thrombospondin type 1 motif, 9. Members of the
ADAMTS family have been implicated in the cleavage of proteoglycans, the
control of organ shape during development, and the inhibition of angiogenesis.
ankyrin repeat domain 37.
97
BCAS1
0.5981
Cytoplasm
CYTH4
0.5980
Membrane
NIN
0.5979
Cytoplasm
1600021P15RIK
0.5976
NSBP1
0.5963
Nucleus
CAPG
0.5960
Cytoplasm
SCD2
0.5939
ER Membrane
HDAC7
0.5938
Cyto & Nucleus
PODXL
0.5931
Membrane
ITGB5
0.5924
Membrane
ARHGAP24
0.5924
Cytoplasm
CCND1
0.5917
Cyto & Nucleus
ID3
0.5894
Nucleus
PLEKHO2
0.5894
SOX4
0.5891
4930511J11RIK
0.5883
LDHA
breast carcinoma amplified sequence 1. This gene resides in a region at 20q13
which is amplified in a variety of tumor types and associated with more aggressive
tumor phenotypes.
cytohesin 4. he coiled-coil motif is involved in homodimerization, the Sec7 domain
contains guanine-nucleotide exchange protein (GEP) activity, and the PH domain
interacts with phospholipids and is responsible for association of PSCDs with
membranes. Members of this family appear to mediate the regulation of protein
sorting and membrane trafficking.
ninein (GSK3B interacting protein). This gene encodes one of the proteins
important for centrosomal function. This protein is important for positioning and
anchoring the microtubules minus-ends in epithelial cells. Localization of this
protein to the centrosome requires three leucine zippers in the central coiled-coil
domain
high-mobility group nucleosome binding domain 5. protein may function as a
nucleosomal binding and transcriptional activating protein.
capping protein (actin filament), gelsolin-like. The encoded protein reversibly
blocks the barbed ends of F-actin filaments in a Ca2+ and phosphoinositideregulated manner, but does not sever preformed actin filaments. By capping the
barbed ends of actin filaments, the encoded protein contributes to the control of
actin-based motility in non-muscle cells.
stearoyl-CoA desaturase 5. catalyzes the formation of monounsaturated fatty acids
from saturated fatty acids.
histone deacetylase 7. Histones play a critical role in transcriptional regulation, cell
cycle progression, and developmental events. Histone acetylation/deacetylation
alters chromosome structure and affects transcription factor access to DNA.
podocalyxin-like. This gene encodes a member of the sialomucin protein family.
The encoded protein was originally identified as an important component of
glomerular podocytes. Other biological activities of the encoded protein include:
binding in a membrane protein complex with Na+/H+ exchanger regulatory factor
to intracellular cytoskeletal elements, playing a role in hematopoetic cell
differentiation, and being expressed in vascular endothelium cells and binding to
L-selectin.
integrin, beta 5. Integrin alpha-V/beta-5 is a receptor for fibronectin.
Rho GTPase activating protein 24. ARHGAPs, such as ARHGAP24, encode
negative regulators of Rho GTPases (see ARHA; MIM 165390), which are
implicated in actin remodeling, cell polarity, and cell migration
cyclin D1. The protein encoded by this gene belongs to the highly conserved cyclin
family, whose members are characterized by a dramatic periodicity in protein
abundance throughout the cell cycle. Cyclins function as regulators of CDK
kinases. Different cyclins exhibit distinct expression and degradation patterns
which contribute to the temporal coordination of each mitotic event. This cyclin
forms a complex with and functions as a regulatory subunit of CDK4 or CDK6,
whose activity is required for cell cycle G1/S transition.
inhibitor of DNA binding 3. Members of the ID family of helix-loop-helix (HLH)
proteins lack a basic DNA-binding domain and inhibit transcription through
formation of nonfunctional dimers that are incapable of binding to DNA
pleckstrin homology domain containing, family O member 2.
Nucleus
SRY (sex determining region Y)-box 4. The encoded protein may act as a
transcriptional regulator after forming a protein complex with other proteins, such
as syndecan binding protein (syntenin). The protein may function in the apoptosis
pathway leading to cell death as well as to tumorigenesis and may mediate
downstream effects of parathyroid hormone (PTH) and PTH-related protein
(PTHrP) in bone development.
0.5873
Cytoplasm
KLF6
0.5869
Nucleus
TOB1
0.5855
Cytoplasm
lactate dehydrogenase A. The protein encoded by this gene catalyzes the
conversion of L-lactate and NAD to pyruvate and NADH in the final step of
anaerobic glycolysis. The protein is found predominantly in muscle tissue and
belongs to the lactate dehydrogenase family.
Kruppel-like factor 6. The zinc finger protein is a transcriptional activator, and
functions as a tumor suppressor. Multiple transcript variants encoding different
isoforms have been found for this gene, some of which are implicated in
carcinogenesis
transducer of ERBB2, 1. This gene encodes a member of the tob/btg1 family of
anti-proliferative proteins that have the potential to regulate cell growth. When
exogenously expressed, this protein supresses cell growth in tissue culture.
4833445A15RIK
0.5853
LOC100047934
0.5832
BNC1
0.5815
Nucleus
basonuclin 1. Likely to be a transcription factor specific for squamous epithelium
and for the constituent keratinocytes at a stage either prior to or at the very
beginning of terminal differentiation. May play a role in the differentiation of
spermatozoa and oocytes
98
MIDN
0.5813
Nucleus
midnolin. May be involved in regulation of genes related to neurogenesis in the
nucleolus
NPY
0.5806
Secreted
neuropeptide Y. This gene encodes a neuropeptide that is widely expressed in the
central nervous system and influences many physiological processes, including
cortical excitability, stress response, food intake, circadian rhythms, and
cardiovascular function. The neuropeptide functions through G protein-coupled
receptors to inhibit adenylyl cyclase, activate mitogen-activated protein kinase
(MAPK), regulate intracellular calcium levels, and activate potassium channels
SLC46A3
0.5805
Membrane
solute carrier family 46, member 3.
9530018I07RIK
0.5801
RRAGD
0.5789
Cytoplasm
Ras-related GTP binding D. RRAGD is a monomeric guanine nucleotide-binding
protein, or G protein. By binding GTP or GDP, small G proteins act as molecular
switches in numerous cell processes and signaling pathways.
9130211I03RIK
0.5780
BHLHB2
0.5778
Nucleus
IFITM2
0.5753
Cell Membrane
basic helix-loop-helix family, member e40. This gene encodes a basic helix-loophelix protein expressed in various tissues. Expression in the chondrocytes is
responsive to the addition of Bt2cAMP. The encoded protein is believed to be
involved in the control of cell differentiation.
interferon induced transmembrane protein 2. IFN-induced antiviral protein that
mediates cellular innate immunity to at least three major human pathogens, namely
influenza A H1N1 virus, West Nile virus (WNV), and dengue virus (WNV), by
inhibiting the early step(s) of replication. Induces cell cycle arrest and mediates
apoptosis by caspase activation and in p53-independent manner
CDKN1A
0.5736
Cyto & Nucleus
ASB9
0.5731
cyclin-dependent kinase inhibitor 1. The encoded protein binds to and inhibits the
activity
of
cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator of cell cycle
progression at G1. The expression of this gene is tightly controlled by the tumor
suppressor protein p53, through which this protein mediates the p53-dependent cell
cycle G1 phase arrest in response to a variety of stress stimuli. This protein can
interact with proliferating cell nuclear antigen (PCNA), a DNA polymerase
accessory factor, and plays a regulatory role in S phase DNA replication and DNA
damage repair.
ankyrin repeat and SOCS box containing 9. This gene encodes a member of the
ankyrin repeat and suppressor of cytokine signaling (SOCS) box protein family.
RASA3
0.5716
Membrane
RAS p21 protein activator 3. The protein encoded by this gene is member of the
GAP1 family of GTPase-activating proteins. The gene product stimulates the
GTPase activity of normal RAS p21 but not its oncogenic counterpart. Acting as a
suppressor of RAS function, the protein enhances the weak intrinsic GTPase
activity of RAS proteins resulting in the inactive GDP-bound form of RAS,
thereby allowing control of cellular proliferation and differentiation.
NDRG1
0.5713
Cyto & Nucleus
SERF1
0.5701
PCDH12
0.5699
N-myc downstream regulated 1. The protein encoded by this gene is a cytoplasmic
protein involved in stress responses, hormone responses, cell growth, and
differentiation.
small EDRK-rich factor 1A. The function of this protein is not known; however, it
bears low-level homology with the RNA-binding domain of matrin-cyclophilin, a
protein which colocalizes with small nuclear ribonucleoproteins (snRNPs) and the
SMN1 gene product.
protocadherin 12. The function of this cellular adhesion protein is undetermined
but mouse protocadherin 12 does not bind catenins and appears to have no affect
on cell migration or growth.
LOC381140
0.5697
PTRF
0.5693
LOC666036
0.5691
MEST
0.5689
2610028F08RIK
0.5681
PCBP4
Membrane
Membrane
polymerase I and transcript release factor. This gene encodes a protein that enables
the dissociation of paused ternary polymerase I transcription complexes from the 3'
end of pre-rRNA transcripts. This protein regulates rRNA transcription by
promoting the dissociation of transcription complexes and the reinitiation of
polymerase I on nascent rRNA transcripts.
ER Membrane
Mesoderm specific transcript homolog. This gene encodes a protein that is
preferentially expressed from the paternal allele.
0.5656
Cytoplasm
poly(rC) binding protein 4. This gene is induced by the p53 tumor suppressor, and
the encoded protein can suppress cell proliferation by inducing apoptosis and cell
cycle arrest in G(2)-M. This gene's protein is found in the cytoplasm, yet it lacks
the nuclear localization signals found in other subfamily members.
JAKMIP1
0.5648
Cytoplasm
SRF
0.5632
Nucleus
janus kinase and microtubule interacting protein 1. Associates with microtubules
and may play a role in the microtubule-dependent transport of the GABA-B
receptor. May play a role in JAK1 signaling and regulate microtubule cytoskeleton
rearrangements
serum response factor. This gene encodes a ubiquitous nuclear protein that
stimulates both cell proliferation and differentiation
99
MAP2K4
0.5613
Cytoplasm
mitogen-activated protein kinase kinase 4. This gene encodes a dual specificity
protein kinase that belongs to the Ser/Thr protein kinase family. This kinase is a
direct activator of MAP kinases in response to various environmental stresses or
mitogenic stimuli. It has been shown to activate MAPK8/JNK1, MAPK9/JNK2,
and MAPK14/p38, but not MAPK1/ERK2 or MAPK3/ERK3. This kinase is
phosphorylated, and thus activated by MAP3K1/MEKK.
ACSS1
0.5595
Mito Matrix
BATF
0.5590
Nucleus
acyl-CoA synthetase short-chain family member 1. It is primarily a cardiac enzyme
which produces acetyl-CoA mainly for the oxidation of acetate.
basic leucine zipper transcription factor, ATF-like. The protein encoded by this
gene is a nuclear basic leucine zipper protein that belongs to the AP-1/ATF
superfamily of transcription factors. The leucine zipper of this protein mediates
dimerization with members of the Jun family of proteins. This protein is thought to
be a negative regulator of AP-1/ATF transcriptional events.
SAMHD1
0.5590
Nucleus
GPC3
0.5584
Membrane
NDRL
0.5549
NPEPL1
0.5524
Cytoplasm
aminopeptidase-like 1. Probably catalyzes the removal of unsubstituted N-terminal
amino acids from various peptides
ZDHHC14
0.5517
Membrane
zinc finger, DHHC-type containing 14.
CCDC3
0.5471
Secreted
coiled-coil domain containing 3.
KLF7
0.5471
Nucleus
Kruppel-like factor 7. Transcriptional activator. Binds in vitro to the CACCC motif
of the beta-globin promoter and to the SP1 recognition sequence
CLIP2
0.5453
Cytoplasm
TNS3
0.5449
Cell Junction
CAP-GLY domain containing linker protein 2. The protein encoded by this gene
belongs to the family of cytoplasmic linker proteins, which have been proposed to
mediate the interaction between specific membranous organelles and microtubules.
This protein was found to associate with both microtubules and an organelle called
the dendritic lamellar body.
tensin 3. May play a role in actin remodeling. Involved in the dissociation of the
integrin-tensin-actin complex. EGF activates TNS4 and down-regulates TNS3
which results in capping the tail of ITGB1. Seems to be involved in mammary cell
migration. May be involved in cell migration and bone development
CD9
0.5440
Membrane
TENS1
0.5429
Cell Junction
GSTP1
0.5422
Cyto & Nucleus
ECH1
0.5419
Mitochondria
SMARCA2
0.5415
Nucleus
GBP2
0.5406
TAGLN2
0.5397
Cell Membrane
(IN)
Membrane
ABR
0.5387
Cytoplasm
IGH-6
0.5361
BC008150
0.5336
4930533K18RIK
0.5334
SCL0002785.1_49
0.5326
SAM domain and HD domain 1. This gene may play a role in regulation of the
innate immune response. The encoded protein is upregulated in response to viral
infection and may be involved in mediation of tumor necrosis factor-alpha
proinflammatory responses.
glypican 3. These proteins may play a role in the control of cell division and
growth regulation. The protein encoded by this gene can bind to and inhibit the
dipeptidyl peptidase activity of CD26, and it can induce apoptosis in certain cell
types
Tetraspanins are cell surface glycoproteins with four transmembrane domains that
form multimeric complexes with other cell surface proteins. The encoded protein
functions in many cellular processes including differentiation, adhesion, and signal
transduction, and expression of this gene plays a critical role in the suppression of
cancer
cell
motility
and
metastasis. Required from sperm-egg fusion.
tensin 3. May play a role in actin remodeling. Involved in the dissociation of the
integrin-tensin-actin complex. EGF activates TNS4 and down-regulates TNS3
which results in capping the tail of ITGB1. Seems to be involved in mammary cell
migration. May be involved in cell migration and bone development
glutathione S-transferase pi 1. This GST family member is a polymorphic gene
encoding active, functionally different GSTP1 variant proteins that are thought to
function in xenobiotic metabolism and play a role in susceptibility to cancer, and
other diseases.
enoyl CoA hydratase 1, peroxisomal. This gene encodes a member of the
hydratase/isomerase superfamily.
SWI/SNF related, matrix associated, actin dependent regulator of chromatin,
subfamily a, member 2. Members of this family have helicase and ATPase
activities and are thought to regulate transcription of certain genes by altering the
chromatin structure around those genes.
guanylate binding protein 2, interferon-inducible. Interferons are cytokines that
have antiviral effects and inhibit tumor cell proliferation.
transgelin 2. The protein encoded by this gene is a homolog of the protein
transgelin, which is one of the earliest markers of differentiated smooth muscle.
The function of this protein has not yet been determined.
active BCR-related gene. The protein encoded by this gene contains a GTPaseactivating protein domain, a domain found in members of the Rho family of GTPbinding proteins. Functional studies in mice determined that this protein plays a
role in vestibular morphogenesis, suggesting that Rho-related GTPases help
coordinate motor skills and balance.
100
PIF1
0.5326
Nucleus
ESRRB
0.5313
Nucleus
ETV5
0.5309
Nucleus
SCL0003799.1_2
0.5254
DDR1
0.5244
1200009O22RIK
0.5238
GABARAPL1
PIF1 5'-to-3' DNA helicase homolog. PIF1 is a 5-prime-to-3-prime DNA helicase
that negatively regulates telomerase, a reverse transcriptase that maintains telomere
length
estrogen-related receptor beta. This gene encodes a protein with similarity to the
estrogen receptor. Its function is unknown; however, a similar protein in mouse
plays an essential role in placental development.
ets variant 5. Binds to DNA sequences containing the consensus nucleotide core
sequence GGAA
Membrane
discoidin domain receptor tyrosine kinase 1. These kinases are involved in the
regulation of cell growth, differentiation and metabolism. The protein encoded by
this gene belongs to a subfamily of tyrosine kinase receptors with homology to
Dictyostelium discoideum protein discoidin I in their extracellular domain, and that
are activated by various types of collagen. Expression of this protein is restricted to
epithelial cells, particularly in the kidney, lung, gastrointestinal tract, and brain.
0.5229
Membrane
OPTN
0.5229
Cytoplasm
TUBB2B
0.5207
Cytoplasm
BARX1
0.5165
Nucleus
CHRD
0.5151
Secreted
GABA(A) receptor-associated protein like 1. Increases cell-surface expression of
kappa-type opioid receptor through facilitating anterograde intracellular trafficking
of the receptor
optineurin. Optineurin may play a role in normal-tension glaucoma and adult-onset
primary open angle glaucoma.
tubulin, beta 2B. The protein encoded by this gene is a beta isoform of tubulin,
which binds GTP and is a major component of microtubules.
BARX homeobox 1. This gene encodes a member of the Bar subclass of
homeobox transcription factors. Studies of the mouse and chick homolog suggest
the encoded protein may play a role in developing teeth and craniofacial
mesenchyme of neural crest origin. The protein may also be associated with
differentiation of stomach epithelia.
chordin. This gene encodes a secreted protein that dorsalizes early vertebrate
embryonic tissues by binding to ventralizing TGF-beta-like bone morphogenetic
proteins and sequestering them in latent complexes. The encoded protein may also
have roles in organogenesis and during adulthood.
DUSP7
0.5150
Cytoplasm
CMTM8
0.5143
Membrane
F2RL1
0.5142
Membrane
2810011L19RIK
0.5137
LOC214575
0.5098
PHGDH
0.5076
CCND2
0.5074
Nucleus
CDH15
0.5070
Membrane
SGK3
0.5063
Endosome
LPAR3
0.5032
Membrane
ELAVL2
0.5013
dual specificity phosphatase 7. Regulates the activity of the MAP kinase family in
response to changes in the cellular environment. MAPK activation cascades
mediate various physiologic processes, including cellular proliferation, apoptosis,
differentiation, and stress responses
CKLF-like MARVEL transmembrane domain containing 8. This gene is widely
expressed in many tissues, but the exact function of the encoded protein is
unknown
coagulation factor II (thrombin) receptor-like 1. Receptor for trypsin and trypsinlike enzymes coupled to G proteins that stimulate phosphoinositide hydrolysis.
May have a role in the regulation of vascular tone
phosphoglycerate dehydrogenase. 3-Phosphoglycerate dehydrogenase (PHGDH;
EC 1.1.1.95) catalyzes the transition of 3-phosphoglycerate into 3phosphohydroxypyruvate, which is the first and rate-limiting step in the
phosphorylated pathway of serine biosynthesis, using NAD+/NADH as a cofactor.
cyclin D2. This cyclin forms a complex with and functions as a regulatory subunit
of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. This
protein has been shown to interact with and be involved in the phosphorylation of
tumor suppressor protein Rb. Knockout studies of the homologous gene in mouse
suggest the essential roles of this gene in ovarian granulosa and germ cell
proliferation. High level expression of this gene was observed in ovarian and
testicular tumors.
cadherin 15, type 1, M-cadherin. This gene is a member of the cadherin
superfamily of genes, encoding calcium-dependent intercellular adhesion
glycoproteins.The protein is thought to be essential for the control of
morphogenetic processes, specifically myogenesis, and may provide a trigger for
terminal muscle cell differentiation.
serum/glucocorticoid regulated kinase family, member 3. This gene is a member of
the Ser/Thr protein kinase family and encodes a phosphoprotein with a PX (phox
homology) domain. The protein phosphorylates several target proteins and has a
role in neutral amino acid transport and activation of potassium and chloride
channels.
lysophosphatidic acid receptor 3. This gene encodes a member of the G proteincoupled receptor family, as well as the EDG family of proteins. This protein
functions as a cellular receptor for lysophosphatidic acid and mediates
lysophosphatidic acid-evoked
ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2. The protein
encoded by this gene is a neural-specific RNA-binding protein that is known to
bind to several 3' UTRs, including its own and also that of FOS and ID.
101
SPRY4
0.5007
Cytoplasm
sprouty homolog 4. Suppresses the insulin receptor and EGFR-transduced MAPK
signaling pathway, but does not inhibit MAPK activation by a constitutively active
mutant Ras. Probably impairs the formation of GTP-Ras
SIRPA
0.4992
Cell Surface
POLD4
0.4973
Nuclear
Protein tyrosine phosphatase non-receptor tye. The transmembrane protein SIRPalpha-1 is a substrate of activated RTKs and binds to SH2 domains
DNA polymerase delta complex is involved in DNA replication and repair
MAPK11
0.4902
Cytoplasm
PPFIBP2
0.4899
Transmembrane
HLX
0.4897
Nuclear
LHFPL2
0.4896
Membrane
6720422M22RIK
0.4889
FKBP11
0.4868
Membrane
MRC1
0.4859
Membrane
ISL1
0.4849
Nuclear
TWIST1
0.4818
Nuclear
OSBPL3
0.4816
Cytoplasm
TGFBR3
0.4798
Cyto + Mem.
PRICKLE1
0.4796
SLC9A3R1
0.4774
Nucl.
membrane
Cyto + Mem.
SP5
0.4766
Nuclear
NANOS3
0.4756
Cytoplasm
RSPO2
0.4751
Secreted
NRD1
0.4709
Cyto + Mem.
SOX13
0.4702
Nuclear TF
FST
0.4679
Secreted
EN1
0.4664
Nuclear
KLF2
0.4650
Nuclear
LOC234081
0.4642
Mitogen activated protein kinase 11. Mitogen-activated protein kinase (MAPK)
cascades represent one of the major signal systems used by eukaryotic cells to
transduce extracellular signals into cellular responses
Protein tyrosibe phosphatase receptor type, F polypeptide interacting protein
binding protein 2. Transmembrane protein-tyrosine phosphatases (PTPases)
Homeobox gene HB24. The HB24 gene encodes a diverged human homeodomain-containing protein known to be expressed in hematopoietic progenitors
and activated lymphocytes
Protein contains putative transmembrane domains
FKBP11 belongs to the FKBP family of peptidyl-prolyl cis/trans isomerases,
which catalyze the folding of proline-containing polypeptide
Macrophage manose receptor. C type.
Transcription factor. family of transcription factors that binds to the enhancer
region of the insulin gene. Neurod4 and Ngn2 actively participated with Isl1 and
Lhx3 to specify motor neuron subtype in embryonic chicken spinal cord and in P19
mouse stem cells.
TWIST1 belongs to the basic helix-loop-helix (bHLH) class of transcriptional
regulators that recognize a consensus DNA element called the E box. directly binds
2 independent HAT domains of acetyltransferases
Member of the OSBP family of intracellular lipid receptors
Transforming growth factor (TGF)-beta. Multifunctional cytokine that modulates
several tissue development and repair processes, including cell differentiation, cell
cycle progression, cellular migration, adhesion, and extracellular matrix
production.
PRICKLE1 interacts directly with REST. REST is a transcriptional repressor that
regulates gene expression throughout the body
Solute carrier family 9, member 3, regulator 1. Highly concentrated in the apical
aspect of polarized epithelial cells. Scaffold protein that connects plasma
membrane proteins with members of the ezrin/moesin/radixin family and thereby
helps to link them to the actin cytoskeleton and to regulate their surface expression.
Specificity protein 5. Transcriptional factor.
Nanos3 was expressed in male mouse gonads, in primordial germ cells (PGCs), in
bipotential gonads at embryonic day (E) 11.5, and in female gonads at E12.5.
Nanos3-null mice were viable and showed no apparent abnormalities, but the size
of ovaries and testes in Nanos3-null mice was greatly reduced. Morphologic
examination revealed only a few germ cells in the E12.5 genital ridge, and none
were found in adult gonads. PGCs were not subsequently maintained during
migration. Apoptosis did not appear to cause the depletion of PGCs.
R-spondins (RSPOs), such as RSPO2, are secreted proteins that regulate betacatenin signaling. human RSPO2 enhanced mouse Wnt3a (606359) signaling.
Overexpression of Rspo2 in Xenopus activated the Wnt/beta-catenin pathway
upstream of dishevelled.
N-arginine dibasic convertase. Nardilysin. A metalloendopeptidase that cleaves
peptide substrates at the N terminus of arginine residues in dibasic moieties, had
been previously purified from rat testis. During early mouse development, Nrd
convertase is expressed almost exclusively in neural tissues.
SRY box 13. Encodes a transcription factor characterized by a DNA-binding motif
known as the HMG (high mobility group) box. Expression in the adult mouse is
restricted to kidney and ovary. One mechanism of SOX13 function is the inhibition
of signaling by the developmentally important for Wnt/T cell factor (TCF)
pathway
Follistatin. Important regulator of activin and other members of the TGF-beta
superfamily. Highest expression in adult ovary, pituitary, and kidney, and in fetal
heart and liver. Direct inhibitor of activin and BMPs which regulate differentiation
of progenitor cell types, including hematopoietic cells. Sood et al.suggested that
follistatin may have a role in regulating stem cell renewal versus differentiation in
umbilical cord.
Engrailed1. In Drosophila, the 'engrailed' (en) homeobox protein plays an
important role during development in segmentation, where it is required for the
formation of posterior compartments
Trancrip. Factor. zinc finger transcription factors.
102
ANXA2
0.4608
Secreted
Annexin II. Annexin II, a major cellular substrate of the tyrosine kinase encoded
by the SRC oncogene, belongs to the annexin family of Ca(2+)-dependent
phospholipid- and membrane-binding proteins.
EPITHELIAL SPLICING REGULATORY PROTEIN 1.
RBM35A
0.4569
Nuclear
TRF
0.4556
Nuclear + cyto
HMHA1
0.4554
Cytosol
4833409N03RIK
0.4549
GYLTL1B
0.4542
PDGFB
CBR3
LOC100047261
0.4503
TIMP1
0.4499
BICC1
0.4457
AKR1B8
0.4443
ALDH1A3
0.4433
Cytoplasm
ALDEHYDE DEHYDROGENASE 1 FAMILY, MEMBER A3.
MTCH1
0.4423
MITOCHONDRIAL CARRIER HOMOLOG 1. May play a role in apoptosis.
ELL3
0.4415
Mito. Inner
Membrane
Nucleus
9130213B05RIK
0.4392
RET
0.4370
Membrane
FBXO32
0.4338
SERPINH1
0.4316
ER lumen
TMC6
0.4296
Transmembrane
RET protooncogene is one of the receptor tyrosine kinases, cell-surface molecules
that transduce signals for cell growth and differentiation.
An F-box motif is found in a family of proteins that function as one component of
the SCF (SKP1 (601434)-cullin (603134)-F-box) complex of Ub protein ligases
(E3s)
COLLAGEN-BINDING PROTEIN 2; glycoproteins that bind specifically to
collagen type I, collagen type IV, and gelatin.
Transmembrane channel like protein 6.
NAV1
0.4251
Cytoplasm
ZBTB16
0.4245
Nucleus
FOXF1A
0.4234
Nuclear
TMEM132E
0.4210
Transmembrane
SPG21
0.4194
Cyto + Mem.
NEUROG3
0.4141
Nuclear
LHX1
0.4138
Nuclear
S100A11
0.4095
Cyto + Nucl
RASL11A
0.4044
Nuclear
3100002J23RIK
0.3998
AW120700
0.3970
SPSB4
0.3910
Cytoplasm
FIGN
0.3900
Nuclear Matrix
CHRNA4
0.3870
Membrane
TELOMERIC REPEAT-BINDING FACTOR 1. TRF, had been found to associate
with double-stranded TTAGGG repeat arrays in vitro and to display strong
specificity for vertebrate telomere DNA
MINOR HISTOCOMPATIBILITY ANTIGEN HA-1.
GLYCOSYLTRANSFERASE-LIKE 1B
0.4528
Golgi
membrane
Secreted
0.4523
Cytoplasm
CARBONYL REDUCTASE 3. catalyzes the reduction of a large number of
biologically and pharmacologically active carbonyl compounds to their
corresponding alcohols
Secreted
Tissue inhibitor of metalloproteinase 1. TIMP is identical to the collagenase
inhibitor. Collagenase and related metalloproteinases are responsible for much of
the remodeling that occurs in connective tissue.
Bicaudal C homolog 1. This gene encodes an RNA-binding protein that is active in
regulating gene expression by modulating protein translation during embryonic
development. Mouse studies identified the corresponding protein to be under strict
control during cell differentiation and to be a maternally provided gene product.
PLATELET-DERIVED GROWTH FACTOR, BETA POLYPEPTIDE.
* Aldo-keto reductase family 1 member B15.
ELONGATION FACTOR, RNA POLYMERASE II, 3. can increase the catalytic
rate of transcription elongation by RNA polymerase II
RT-PCR ELISA detected moderate expression in heart, brain, lung, and ovary,
with lower expression in kidney, testis, and fetal brain
highest levels in undifferentiated, multipotential hematopoietic progenitor cells and
its expression declines as cells become more mature and committed to various
hematopoietic lineages
* Forkhead box F1.
* Transmembrane protein 123E.
ACIDIC CLUSTER PROTEIN. It binds to the hydrophobic C-terminal amino
acids of CD4 which are involved in repression of T cell activation.
The protein encoded by this gene is a basic helix-loop-helix (bHLH) transcription
factor involved in neurogenesis
structural characteristics of the LIM1 gene suggested that it encodes a
transcriptional regulatory protein involved in control of differentiation and
development of neural and lymphoid cells
S100 CALCIUM-BINDING PROTEIN A11. Immunohistochemical analysis of
human skin detected S100A11 in nuclei of differentiating cells in the suprabasal
layers, but not in nuclei of proliferating cells in the basal layer. Facilitates the
differentiation and the cornification of keratinocytes
Ras like family 11. Member of the small GTPase protein family with a high degree
of similarity to RAS. Northern blot analysis clearly detected 2 RASL11A
transcripts of 1.6 and 1.2 kb in bladder, prostate, testis, and colon, and lower
expression in thymus and leukocytes.
SPRY DOMAIN-CONTAINING SOCS BOX PROTEIN 4.
Figetin. Fign, which encodes a new member of the 'meiotic' or subfamily-7 group
of ATPases associated with diverse cellular activities. Facilitate a variety of
functions, including membrane fusion, proteolysis, peroxisome biogenesis,
endosome sorting, and meiotic spindle formation
CHOLINERGIC
RECEPTOR,
NEURONAL
NICOTINIC,
ALPHA
POLYPEPTIDE 4. The nicotinic acetylcholine receptors (nAChRs) are members
of a superfamily of ligand-gated ion channels that mediate fast signal transmission
at synapses.
103
EGR3
0.3868
Nuclear
Early Growth Response 3. transcriptional regulator. immediate-early growth
response gene which is induced by mitogenic stimulation.
GLUTATHIONE S-TRANSFERASE, ALPHA-4. GSTA4 is highly effective in
catalyzing 4-hydroxynonenal, an important product of peroxidative degradation of
arachidonic acid and a commonly used biomarker for oxidative damage in tissue
GSTA4
0.3859
Cytoplasm
ENC1
0.3695
Nucl. Mat. +
Cyto.
IRX1
0.3688
Nuclear
DDIT4
0.3566
Cytoplasm
EGR4
0.3525
Nuclear
PIP4K2A
0.3483
NEF3
0.3447
IER3
0.3413
Membrane
B3GNT1
0.3358
Golgi
membrane
SPHK1
0.3336
IFITM1
0.3151
Plasma
Membrane
SALL3
0.3131
Nuclear
ACTN2
0.3043
Cytoplasm
IGFBP5
0.3033
Secreted
LOC100045019
0.2918
IGFBP6
0.2851
Secreted
NANOS2
0.2830
Cytoplasm
PYGL
0.2741
EGR2
0.2460
Nuclear
PHLDA1
0.2352
Cytoplasmic
NEFM
0.2242
Pleckstrin Homology Like Domain Family A member 1. Overexpression of
TDAG51 elicited significant changes in cell morphology, decreased cell adhesion,
and promoted detachment-mediated apoptosis
Neurofilament 3.
TCL1
0.2043
Cyto + Nucl
Overexpression of TCL1 in human seminomas. TCL1 dysregulation could
contribute to the development of this germinal cell cancer as well as lymphoid
malignancies. TCL1 functions as a coactivator of the cell survival kinase AKT
MAGED1
0.1988
Cyto + Mem.
is restricted to tumor cells and testis.
Ectodermal neural cortex 1. p53 induced gene 10. ENC1, encodes an actin-binding
protein. ENC1 expression increased dramatically in a neuroblastoma cell line
undergoing retinoic acid-induced differentiation. Northern blot analysis of rat
tissues found high expression in brain, low expression in testis
Iroquois homeobox protein 1. Members of this family appear to play multiple roles
during pattern formation of vertebrate embryos. involved in several embryonic
developmental processes including anterior/posterior and dorsal/ventral patterning
of specific regions of the central nervous system, and regionalization of the otic
vesicle, branchial epithelium, and limbs.
DNA Damage inducible transcript 4. REDD1 is a transcriptional target of p53
induced following DNA damage. In differentiating primary human keratinocytes,
TP63 and REDD1 expression was coordinately downregulated. Promotes neuronal
cell death.
Early Growth Response 4. When eukaryotic cells are stimulated to undergo
mitogenesis or differentiation, the expression of a small subset of genes, termed
early response or immediate early genes, is rapidly activated. Activates the
transcription of target genes whose products are required for mitogenesis and
differentiation.
Phosphatidylinositol-5-phosphate 4-kinase, type II, alpha. the precursor to second
messengers of the phosphoinositide signal transduction pathways. Is thought to be
involved in the regulation of secretion, cell proliferation, differentiation, and
motility.
Neurofilament 3.
Immediate Early Response 3. Protects cells from apoptosis induced by FAS or
tumor necrosis factor-alpha.
BETA-1,3-N-ACETYLGLUCOSAMINYLTRANSFERASE.
SPHINGOSINE KINASE 1. novel lipid messenger with both intracellular and
extracellular functions. Intracellularly, it regulates proliferation and survival, and
extracellularly, it is a ligand for EDG1
Interferon Induced Trasnmembrane Protein. Exhibit antiproliferative and
differentiating activities that may confer on them potential as antitumor agents.
Demonstrated that fragilis, a member of the IFN-inducible transmembrane protein
family, marks the onset of germ cell competence. Those cells with the highest
expression of fragilis subsequently express 'stella' a murine gene that was detected
exclusively in lineage-restricted germ cells. Appear to modulate cell adhesion and
influence cell differentiation. Ifitm1 activity was required for primordial germ cell
transit.
SALL3 protein contains 4 double zinc finger (DZF) domains, each of which
contains sequences identical or closely related to the SAL box
Actinin alpha 2. Alpha-actinin is an actin-binding protein with multiple roles in
different cell types
INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 5. have been shown
to either inhibit or stimulate the growth promoting effects of the IGFs on cell
culture. They alter the interaction of IGFs with their cell surface receptors
INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 6.
Nanos2 expression was restricted to male mouse germ cells. Nanos2-null testes had
defects in spermatogenesis. no germ cells were detected. Apoptosis continued until
the germ cells had completely disappeared by 4 weeks of age. Nanos2 suppressed
meiosis in male mouse germ cells by preventing Stra8 expression, which was
required for premeiotic DNA replication, after Cyp26b1 downregulation.
Glycogen phosphorylase, liver form. Phosphorylase is an important allosteric
enzyme in carbohydrate metabolism. Enzymes from different sources differ in
their regulatory mechanisms and in their natural substrates
Early growth response 2.
104
ANNEX 3
In regards to comments made by the Masters’ thesis examiner:
Explain why: If LRPAP1 is only up-regulated after 12 hours post-treatment with
RA it is found only in clusters? It is proposed to be an early marker for
differentiation, so why is it seen in DMSO treated cultures?
An altered expression level can be considered a marker for a specific event, in this
case differentiation. Certain markers may be expressed in control cell population
however when the cell population receives a stimuli, for example RA, a particular
markers expression may be altered, increased or decreased. Consider HSCs, when
initially isolating HSC it was noted that an enriched HSC population was obtained
when sorting for the surface phenotype Thy-1low, whereas Thy-1high and Thy-1cell fractions did not contain HSC (Seita & Weissman, 2010; Spangrude,
Heimfeld, & Weissman, 1988).
An altered expression level relative to a base expression level may representative
of the expression pattern observed for LRPAP1 post-treatment with RA.
Untreated cluster cells as well as treated cluster cells appear to express LRPAP1
(Fig. 22F and L), however cell chains do not express LRPAP1 (Fig. 23C). Global
gene expression analysis and qPCR data shows that LRPAP1’s expression is
105
upregulated post-treatment (Table 3). Taking these results together, it could be
suggested that cells which express LRPAP1high may represent SSCs which have
recently committed to differentiation, where as LRPAP1 low or LRPAP1- cell
populations may represent cell fractions that are enriched from SSCs.
Explain why: There is background chain formation even in DMSO (e.g. Fig. 14).
DMSO itself is known to be an inducer in certain cell types. There is no mention
of this in the thesis.
The presence of cell chains in the control cell population (0.01% DMSO treated
cluster cells) may be attributed to spontaneous differentiation. When cluster cells
are maintained and/or expanded using standard culture conditions necessary for
cluster maintenance spontaneous chain formation is observed regularly;
suggesting that some cluster cells will differentiate under standard culture
conditions. This phenomenon has been observed in mouse embryonic stem cell
population as well as in mouse induced pluripotent stem (iPS) cells (Clark et al.,
2004; Li et al., 2013)
DMSO was used as a solvent in the production of the required RA concentration
in this study. A study assessing the effect of various DMSO concentrations,
0.01% (low dose), 0.1% (medium dose) and 1% (high dose), on the differentiation
106
potential human embryonic stem (hES) cells, stated the DMSO can cause cell
growth arrest and terminal differentiation of these cells but these consequences
were observed at DMSO concentrations of 1% and 2% (Pal, Mamidi, Das, &
Bhonde, 2012). During the experiments performed throughout this thesis a
cautionary concentration of 0.01% DMSO was used. Also as stated by Pal et al.
(2012) the action of DMSO may be variable between species, cell types,
developmental stages and concentrations.
107
REFERENCES
Abutarbush, Sameeh M. (2008). Saunders Comprehensive Veterinary Dictionary, 3rd ed.
The Canadian veterinary journal = La revue vétérinaire canadienne., 49(9), 906.
Adams, I. R., & McLaren, A. (2002). Sexually dimorphic development of mouse
primordial germ cells: switching from oogenesis to spermatogenesis.
Development, 129(5), 1155-1164.
Berruti, Giovanna. (2004). Germ Cell Differentiation Signaling Events, Male. In M.
Editor-in-Chief: Luciano (Ed.), Encyclopedia of Endocrine Diseases (pp. 146149). New York: Elsevier.
Brinster, R L, & Avarbock, M R. (1994). Germline transmission of donor haplotype
following spermatogonial transplantation. Proceedings of the National Academy
of Sciences, 91(24), 11303-11307.
Chung, S. S. W., Wang, X. Y., & Wolgemuth, D. J. (2009). Expression of retinoic acid
receptor alpha in the germline is essential for proper cellular association and
spermiogenesis during spermatogenesis. Development, 136(12), 2091-2100. doi:
10.1242/dev.020040
Clark, A. T., Bodnar, M. S., Fox, M., Rodriquez, R. T., Abeyta, M. J., Firpo, M. T., &
Pera, R. A. (2004). Spontaneous differentiation of germ cells from human
embryonic stem cells in vitro. Hum Mol Genet, 13(7), 727-739. doi:
10.1093/hmg/ddh088
Costoya, J. A., Hobbs, R. M., Barna, M., Cattoretti, G., Manova, K., Sukhwani, M., . . .
Pandolfi, P. P. (2004). Essential role of Plzf in maintenance of spermatogonial
stem cells. Nat Genet, 36(6), 653-659. doi: 10.1038/ng1367
Dann, C. T., Alvarado, A. L., Molyneux, L. A., Denard, B. S., Garbers, D. L., & Porteus,
M. H. (2008). Spermatogonial stem cell self-renewal requires OCT4, a factor
downregulated during retinoic acid-induced differentiation. Stem Cells, 26(11),
2928-2937. doi: 10.1634/stemcells.2008-0134
de Rooij, D. G., & Russell, L. D. (2000). All you wanted to know about spermatogonia
but were afraid to ask. J Androl, 21(6), 776-798.
De Rooij, Dirk G. (2009). The spermatogonial stem cell niche. Microscopy Research and
Technique, 72(8), 580-585. doi: 10.1002/jemt.20699
de Rooij, Dirk G., Okabe, Masaru, & Nishimune, Yoshitake. (1999). Arrest of
Spermatogonial Differentiation in jsd/jsd, Sl17H/Sl17H, and Cryptorchid Mice.
Biology of Reproduction, 61(3), 842-847. doi: 10.1095/biolreprod61.3.842
Dheda, K., Huggett, J. F., Bustin, S. A., Johnson, M. A., Rook, G., & Zumla, A. (2004).
Validation of housekeeping genes for normalizing RNA expression in real-time
PCR. Biotechniques, 37(1), 112-114, 116, 118-119.
Fahnenstich, Jasmin, Nandy, Andreas, Milde‐Langosch, Karin, Schneider‐Merck, Tanja,
Walther, Norbert, & Gellersen, Birgit. (2003). Promyelocytic leukaemia zinc
finger protein (PLZF) is a glucocorticoid‐ and progesterone‐induced transcription
factor in human endometrial stromal cells and myometrial smooth muscle cells.
Molecular Human Reproduction, 9(10), 611-623. doi: 10.1093/molehr/gag080
Gilbert, Scott F. (2006). Developmental biology. Sunderland, Mass.: Sinauer Associates
Publishers.
Goertz, Meredith J., Wu, Zhuoru, Gallardo, Teresa D., Hamra, F. Kent, & Castrillon,
Diego H. (2011). Foxo1 is required in mouse spermatogonial stem cells for their
108
maintenance and the initiation of spermatogenesis. The Journal of Clinical
Investigation, 121(9), 3456-3466. doi: 10.1172/JCI57984
Griswold, Michael D., Hogarth, Cathryn A., Bowles, Josephine, & Koopman, Peter.
(2012). Initiating Meiosis: The Case for Retinoic Acid. Biology of Reproduction,
86(2), 35, 31-37. doi: 10.1095/biolreprod.111.096610
Hamer, Geert, Roepers-Gajadien, Hermien L., Gademan, Iris S., Kal, Henk B., & de
Rooij, Dirk G. (2003). Intercellular bridges and apoptosis in clones of male germ
cells. International Journal of Andrology, 26(6), 348-353. doi: 10.1111/j.13652605.2003.00436.x
Harikae, Kyoko, Miura, Kento, & Kanai, Yoshiakira. (2013). Early gonadogenesis in
mammals: Significance of long and narrow gonadal structure. Developmental
Dynamics, 242(4), 330-338. doi: 10.1002/dvdy.23872
Hasegawa, Kazuteru, & Saga, Yumiko. (2012). Retinoic acid signaling in Sertoli cells
regulates organization of the blood-testis barrier through cyclical changes in gene
expression. Development, 139(23), 4347-4355. doi: 10.1242/dev.080119
Hermo, L., Pelletier, R. M., Cyr, D. G., & Smith, C. E. (2010). Surfing the wave, cycle,
life history, and genes/proteins expressed by testicular germ cells. Part 1:
background to spermatogenesis, spermatogonia, and spermatocytes. Microsc Res
Tech, 73(4), 241-278. doi: 10.1002/jemt.20783
Hess, RexA, & Franca, LuizRenato. (2009). Spermatogenesis and Cycle of the
Seminiferous Epithelium. In C. Y. Cheng (Ed.), Molecular Mechanisms in
Spermatogenesis (Vol. 636, pp. 1-15): Springer New York.
Høyer, Poul Erik, Byskov, Anne Grete, & Møllgård, Kjeld. (2005). Stem cell factor and
c-Kit in human primordial germ cells and fetal ovaries. Molecular and Cellular
Endocrinology, 234(1–2), 1-10. doi: http://dx.doi.org/10.1016/j.mce.2004.09.012
Jan, Sabrina Z., Hamer, Geert, Repping, Sjoerd, de Rooij, Dirk G., van Pelt, Ans M. M.,
& Vormer, Tinke L. (2012). Molecular control of rodent spermatogenesis.
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1822(12),
1838-1850. doi: http://dx.doi.org/10.1016/j.bbadis.2012.02.008
Johnson, Larry, Thompson Jr, Donald L., & Varner, Dickson D. (2008). Role of Sertoli
cell number and function on regulation of spermatogenesis. Animal Reproduction
Science, 105(1–2), 23-51. doi:
http://dx.doi.org/10.1016/j.anireprosci.2007.11.029
Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Ogura, A., Toyokuni, S., & Shinohara,
T. (2003). Restoration of fertility in infertile mice by transplantation of
cryopreserved male germline stem cells. Hum Reprod, 18(12), 2660-2667.
Kolasa, A., Misiakiewicz, K., Marchlewicz, M., & Wiszniewska, B. (2012). The
generation of spermatogonial stem cells and spermatogonia in mammals. Reprod
Biol, 12(1), 5-23.
Kolasa, Agnieszka, Misiakiewicz, Kamila, Marchlewicz, Mariola, & Wiszniewska,
Barbara. (2012). The generation of spermatogonial stem cells and spermatogonia
in mammals. Reproductive Biology, 12(1), 5-23. doi:
http://dx.doi.org/10.1016/S1642-431X(12)60074-6
Kubota, H., Avarbock, M. R., & Brinster, R. L. (2003). Spermatogonial stem cells share
some, but not all, phenotypic and functional characteristics with other stem cells.
Proceedings of the National Academy of Sciences of the United States of
America, 100(11), 6487-6492. doi: 10.1073/pnas.0631767100
Li, P., Hu, H., Yang, S., Tian, R., Zhang, Z., Zhang, W., . . . Li, Z. (2013). Differentiation
of induced pluripotent stem cells into male germ cells in vitro through embryoid
body formation and retinoic acid or testosterone induction. Biomed Res Int, 2013,
608728. doi: 10.1155/2013/608728
109
Lodish, Harvey F. (2008). Molecular cell biology. New York: W.H. Freeman.
Mackey, Z. B., Ramos, W., Levin, D. S., Walter, C. A., McCarrey, J. R., & Tomkinson,
A. E. (1997). An alternative splicing event which occurs in mouse pachytene
spermatocytes generates a form of DNA ligase III with distinct biochemical
properties that may function in meiotic recombination. Mol Cell Biol, 17(2), 989998.
Meng, X., Lindahl, M., Hyvonen, M. E., Parvinen, M., de Rooij, D. G., Hess, M. W., . . .
Sariola, H. (2000). Regulation of cell fate decision of undifferentiated
spermatogonia by GDNF. Science, 287(5457), 1489-1493.
Nagano, Makoto, Avarbock, Mary R., & Brinster, Ralph L. (1999). Pattern and Kinetics
of Mouse Donor Spermatogonial Stem Cell Colonization in Recipient Testes.
Biology of Reproduction, 60(6), 1429-1436. doi: 10.1095/biolreprod60.6.1429
Nagano, Makoto C., & Yeh, Jonathan R. (2013). Chapter Three - The Identity and Fate
Decision Control of Spermatogonial Stem Cells: Where Is the Point of No
Return? In M. W. Paul (Ed.), Current Topics in Developmental Biology (Vol.
Volume 102, pp. 61-95): Academic Press.
Ngo, Devi, Cheng, Qiang, O′Connor, Anne E., DeBoer, Kathleen D., Lo, Camden Y.,
Beaulieu, Elaine, . . . Morand, Eric F. (2013). Glucocorticoid-Induced Leucine
Zipper (GILZ) Regulates Testicular FOXO1 Activity and Spermatogonial Stem
Cell (SSC) Function. PLoS ONE, 8(3), e59149. doi:
10.1371/journal.pone.0059149
Oakberg, Eugene F. (1956). Duration of spermatogenesis in the mouse and timing of
stages of the cycle of the seminiferous epithelium. American Journal of Anatomy,
99(3), 507-516. doi: 10.1002/aja.1000990307
Oatley, J. M., & Brinster, R. L. (2008). Regulation of spermatogonial stem cell selfrenewal in mammals. Annu Rev Cell Dev Biol, 24, 263-286. doi:
10.1146/annurev.cellbio.24.110707.175355
Oatley, M. J., Kaucher, A. V., Racicot, K. E., & Oatley, J. M. (2011). Inhibitor of DNA
binding 4 is expressed selectively by single spermatogonia in the male germline
and regulates the self-renewal of spermatogonial stem cells in mice. Biol Reprod,
85(2), 347-356. doi: 10.1095/biolreprod.111.091330
Oikawa, T., Kamiya, A., Zeniya, M., Chikada, H., Hyuck, A. D., Yamazaki, Y., . . .
Nakauchi, H. (2013). Sal-like protein 4 (SALL4), a stem cell biomarker in liver
cancers. Hepatology, 57(4), 1469-1483. doi: 10.1002/hep.26159
Pal, Rajarshi, Mamidi, MuraliKrishna, Das, AnjanKumar, & Bhonde, Ramesh. (2012).
Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of
human embryonic stem cells. Archives of Toxicology, 86(4), 651-661. doi:
10.1007/s00204-011-0782-2
Pandey, P., Pradhan, S., & Mittal, B. (2008). LRP-associated protein gene (LRPAP1) and
susceptibility to degenerative dementia. Genes Brain Behav, 7(8), 943-950. doi:
10.1111/j.1601-183X.2008.00436.x
Pesce, M., Farrace, M.G., Piacentini, M., Dolci, S., & De Felici, M. (1993). Stem cell
factor and leukemia inhibitory factor promote primordial germ cell survival by
suppressing programmed cell death (apoptosis). Development, 118(4), 10891094.
Phillips, B. T., Gassei, K., & Orwig, K. E. (2010). Spermatogonial stem cell regulation
and spermatogenesis. Philos Trans R Soc Lond B Biol Sci, 365(1546), 16631678. doi: 10.1098/rstb.2010.0026
Raverdeau, Mathilde, Gely-Pernot, Aurore, Féret, Betty, Dennefeld, Christine, Benoit,
Gérard, Davidson, Irwin, . . . Ghyselinck, Norbert B. (2012). Retinoic acid
induces Sertoli cell paracrine signals for spermatogonia differentiation but cell
110
autonomously drives spermatocyte meiosis. Proceedings of the National
Academy of Sciences, 109(41), 16582-16587. doi: 10.1073/pnas.1214936109
Sada, Aiko, Hasegawa, Kazuteru, Pin, Pui Han, & Saga, Yumiko. (2012). NANOS2 Acts
Downstream of Glial Cell Line-Derived Neurotrophic Factor Signaling to
Suppress Differentiation of Spermatogonial Stem Cells. STEM CELLS, 30(2),
280-291. doi: 10.1002/stem.790
Schrans-Stassen, B. H., Saunders, P. T., Cooke, H. J., & de Rooij, D. G. (2001). Nature
of the spermatogenic arrest in Dazl -/- mice. Biol Reprod, 65(3), 771-776.
Seita, J., & Weissman, I. L. (2010). Hematopoietic stem cell: self-renewal versus
differentiation. Wiley Interdiscip Rev Syst Biol Med, 2(6), 640-653. doi:
10.1002/wsbm.86
Sette, C., Dolci, S., Geremia, R., & Rossi, P. (2000). The role of stem cell factor and of
alternative c-kit gene products in the establishment, maintenance and function of
germ cells. Int J Dev Biol, 44(6), 599-608.
Shinohara, T., Avarbock, M. R., & Brinster, R. L. (1999). beta1- and alpha6-integrin are
surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A,
96(10), 5504-5509.
Snyder, Elizabeth M., Small, Christopher, & Griswold, Michael D. (2010). Retinoic Acid
Availability Drives the Asynchronous Initiation of Spermatogonial
Differentiation in the Mouse. Biology of Reproduction, 83(5), 783-790. doi:
10.1095/biolreprod.110.085811
Spangrude, G. J., Heimfeld, S., & Weissman, I. L. (1988). Purification and
characterization of mouse hematopoietic stem cells. Science, 241(4861), 58-62.
Stoop, H., Honecker, F., van de Geijn, G. J. M., Gillis, A. J. M., Cools, M. C., de Boer,
M., . . . Looijenga, L. H. J. (2008). Stem cell factor as a novel diagnostic marker
for early malignant germ cells. The Journal of Pathology, 216(1), 43-54. doi:
10.1002/path.2378
Suzuki, H., Sada, A., Yoshida, S., & Saga, Y. (2009). The heterogeneity of
spermatogonia is revealed by their topology and expression of marker proteins
including the germ cell-specific proteins Nanos2 and Nanos3. Dev Biol, 336(2),
222-231. doi: 10.1016/j.ydbio.2009.10.002
van Loon, J. E., Leebeek, F. W., Deckers, J. W., Dippel, D. W., Poldermans, D.,
Strachan, D. P., . . . de Maat, M. P. (2010). Effect of genetic variations in
syntaxin-binding protein-5 and syntaxin-2 on von Willebrand factor
concentration and cardiovascular risk. Circ Cardiovasc Genet, 3(6), 507-512.
doi: 10.1161/circgenetics.110.957407
Vernet, N., Dennefeld, C., Rochette-Egly, C., Oulad-Abdelghani, M., Chambon, P.,
Ghyselinck, N. B., & Mark, M. (2006). Retinoic acid metabolism and signaling
pathways in the adult and developing mouse testis. Endocrinology, 147(1), 96110. doi: 10.1210/en.2005-0953
Wu, Xin, Schmidt, Jonathan A., Avarbock, Mary R., Tobias, John W., Carlson, Claire A.,
Kolon, Thomas F., . . . Brinster, Ralph L. (2009). Prepubertal human
spermatogonia and mouse gonocytes share conserved gene expression of
germline stem cell regulatory molecules. Proceedings of the National Academy of
Sciences, 106(51), 21672-21677. doi: 10.1073/pnas.0912432106
Yang, Lele, Wu, Wei, & Qi, Huayu. (2013). Gene expression profiling revealed specific
spermatogonial stem cell genes in mouse. genesis, 51(2), 83-96. doi:
10.1002/dvg.22358
Yeh, Jonathan R, Zhang, Xiangfan, & Nagano, Makoto C. (2007). Establishment of a
Short-Term In Vitro Assay for Mouse Spermatogonial Stem Cells. Biology of
Reproduction, 77(5), 897-904. doi: 10.1095/biolreprod.107.063057
111
Yoshida, Shosei, Nakagawa, Toshinori, Hara, Kenshiro, & Kitadate, Yu. (2010). O19.
Stem cell functionality in the mouse spermatogenesis. Differentiation, 80,
Supplement 1(0), S11. doi: http://dx.doi.org/10.1016/j.diff.2010.09.162
Zhang, L., Tang, J., Haines, C. J., Feng, H. L., Lai, L., Teng, X., & Han, Y. (2011). c-kit
and its related genes in spermatogonial differentiation. Spermatogenesis, 1(3),
186-194. doi: 10.4161/spmg.1.3.17760
Zhou, Q., Li, Y., Nie, R., Friel, P., Mitchell, D., Evanoff, R. M., . . . Griswold, M. D.
(2008). Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation
of murine gonocytes and spermatogonia induced by retinoic acid in vitro. Biol
Reprod, 78(3), 537-545. doi: 10.1095/biolreprod.107.064337
112