RETINOIC ACID ALPHA IN GERM CELLS IS IMPORTANT FOR MITOSIS

OF SPERMATOGONIA, SPERMATOGONIAL DIFFERENTIATION AND MEIOSIS

BY

SZE MING LAW

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY School of Molecular Biosciences

JULY 2013

©Copyright by SZE MING LAW

All Rights Reserved

© Copyright by SZE MING LAW, 2013 All Rights Reserved To the Faculty of Washington State University

The members of the Committee appointed to examine the dissertation of SZE MING

LAW find it satisfactory and recommend that it be accepted

______Kwan Hee Kim, Ph.D., Chair

______Derek McLean, Ph.D.

______Mary Hunzicker-Dunn, Ph.D.

______Wenfeng An, Ph.D.

ii ACKNOWLEDGEMENT

I wish to thank my advisor, Dr. Kwan Hee Kim, who gave me a chance to succeed and became the scientist I am today, and not only deeply cared about my professional development but also my personal growth. It has been such an inspiration to work under her supervision and guidance that I understand what it takes to be a good scientist. I would also like to thank my committee members, Drs. Derek McLean, Mary Hunzicker-Dunn and Wenfeng An for their positive criticism, helpful recommendations and constructive advice throughout my thesis work in the School of Molecular Biosciences.

I would like to express my gratitude to all the past and present members of the Kim lab, especially for Dr. Tim Doyle’s collaboration and helpful discussions on research topics, for

Jennifer Onken’s assistance and devoted work ethics to meet my animal needs for research, and for Natalie Peer, Zulema Garcia and Joseph Lawhead’s genuine effort to critically review my thesis.

Especially, I thank my husband, who has been my best friend for over ten years and has always been there for me to provide emotional support and taught me how much life can be more beautiful with a glass half full. Most importantly, I am grateful for my parents, who believed in me and supported me for all these years, extended their help as much as they can when I needed it the most, and urged me to do the best for myself in any difficult situations.

iii ALPHA IN GERM CELLS IS IMPORTANT FOR MITOSIS

OF SPERMATOGONIA, SPERMATOGONIAL DIFFERENTIATION AND MEIOSIS

ABSTRACT

By Sze Ming Law, Ph.D. Washington State University July 2013

Chair: Kwan Hee Kim

Spermatogenesis is governed by vitamin A, as shown by vitamin A deficient (VAD) testes, which lack advanced germ cells. Vitamin A signaling is mediated by retinoid receptors.

There are two families of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each with alpha, beta and gamma subtypes. Retinoic acid receptor alpha

(RARA), plays a significant role in the testis such that Rara-null males are infertile because of severe germ loss.

Striking similarities of the testicular phenotypes are detected between Rara-null and

VAD mice: severely degenerated testes, lack of germ cells, sloughing of mature spermatids, and infertility. To discern the molecular function of RARA in germ cells, Rara was conditionally deleted using stimulated by retinoic acid 8 (STRA8)-iCRE. With RARA function disabled in germ cells, morphological abnormalities detected in the testes included lack of germ cell organization, lack of lumen, sloughing cells, and vacuolization. Not surprisingly, germ-cell specific Rara conditional knockout mice (cKO) had a dramatic reduction in epididymal sperm number. Further analysis of cKO testes demonstrated decreased spermatogonial proliferation and differentiation, while meiotic defects such as reduced synapsis, synaptonemal fragmentation, and unrepaired double strand breaks were increased. Furthermore, functional spermatogonial

iv transplantation assays pointed to the possibility that RARA regulates spermatogonial stem cell colonization and proliferation, as shown by the reduction of donor-derived spermatogenesis from the cKO donor germ cells. The lack of RARA in the testes clearly shows quantifiable deficiencies during spermatogonial proliferation, differentiation, and meiosis.

Microarray expression studies of mRNAs from the enriched germ cells from wild type and cKO mice provided molecular evidence that RARA regulates spermatogonial differentiation at postnatal day 4 (P4) and meiosis at P8. Cell differentiation, cell adhesion, cell migration, and other pathways related to the early steps of spermatogonial differentiation were found to be functional categories significant in germ cells from P4. These were very distinct from synapsis, synaptonemal complex formation, and crossover formation related to meiosis, which were functional categories significant in germ cells from P8. In conjunction with phenotypic abnormalities, we provide evidence that RARA mediates retinoic acid function during spermatogonial proliferation, differentiation, and meiosis.

v TABLE OF CONTENTS

ACKNOWLEDGEMENT ...... iii

ABSTRACT ...... iv

INTRODUCTION ...... 1

INTRODUCTION ...... 2

The testis and spermatogenesis ...... 2

Vitamin A regulation and metabolism in rodent testis ...... 7

Vitamin A deficiency in mice and rats ...... 9

Retinoic acid receptors and retinoid X receptors ...... 9

Retinoic acid receptor alpha ...... 10

Spermatogonial stem cell transplantation assay ...... 12

Germ cell type-specific investigation of RARA function using Cre/LoxP system ...... 13

Summary of work ...... 15

REFERENCES ...... 19

CHAPTER TWO ...... 47

DISTINCT REQUIREMENT FOR RARA FUNCTION IN SPERMATOGONIAL

PROLIFERATION AND DIFFERENTIATION, AND DOUBLE STRAND BREAK

REPAIR ...... 47

ABSTRACT ...... 48

INTRODUCTION ...... 50

METHODS AND MATERIALS ...... 55

vi Generation of the Raraflox/flox and Rara conditional knockout mouse strains ...... 55

EYFP reporter animals ...... 56

Genotyping ...... 57

Testicular tissue collection and determination of testicular abnormalities ...... 58

Sperm count and fertility analysis ...... 58

Detection of mitosis (proliferation) by phosphohistone H3 (pHH3) immunofluorescence and

BrdU assay ...... 59

Detection of apoptosis by Terminal Deoxynucleotidyl transferase dUTP nick end labeling

(TUNEL) assay ...... 60

Antibodies, immunohistochemistry, and immunofluorescence ...... 61

Pachytene -spread ...... 62

Spermatogonial transplantation ...... 63

Post-transplantation recovery analysis ...... 64

Real-time RT PCR ...... 64

Statistical significance ...... 65

RESULTS ...... 66

Germ cell-specific deletion of RARA ...... 66

Lack of functional RARA leads to increased abnormal testicular tubules throughout

development ...... 67

Reduced number of tubules with the most advanced germ cell type ...... 67

Lack of RARA leads to decreased sperm count at P75 and P120 and a decreased fertility at

P120 ...... 68

Lack of RARA protein in germ cells leads to a decrease in mitosis of spermatogonia ...... 68

vii Lack of RARA leads to a decrease in the type B spermatogonia, meiotic germ cells, and

accumulation of undifferentiated spermatogonia ...... 69

No differences in apoptosis ...... 71

Retained unrepaired double strand breaks in Rara cKO pachytene spermatocytes ...... 71

RARA protein in germ cells is responsible for the colonization, initial proliferation and

differentiation of spermatogonia ...... 72

DISCUSSION ...... 74

ACKNOWLEDGEMENT ...... 82

CHAPTER THREE ...... 123

POTENTIAL TARGETS FOR RARA PROTEIN IN REGULATING THE

SPERMATOGONIAL DIFFERENTIATION AND MEIOSIS ...... 123

ABSTRACT ...... 124

INTRODUCTION ...... 126

MATERIALS AND METHODS ...... 128

Animals ...... 128

Germ cell enrichment ...... 128

RNA extraction ...... 129

Microarray processing and data analysis ...... 129

Real-time RT PCR ...... 130

Known Retinol (ROL), RA, RARA-regulated and retinoic acid response element

search ...... 131

RESULTS ...... 132

Microarray hybridization and GeneSpring analysis ...... 132

viii Retinol (ROL), RA and RARA regulated genes ...... 133

Functional clustering and pathway analysis ...... 135

Real time PCR of highly changing genes ...... 136

Rara regulation of double strand break (DSB) repair-related genes ...... 137

Rara regulates potential DMRT1 target genes ...... 137

DISCUSSION ...... 138

REFERENCE ...... 143

CHAPTER FOUR ...... 178

CONCLUSIONS AND FUTURE EXPERIMENTS ...... 178

CONCLUSION CHAPTER ...... 179

Action of retinoic acid (RA) and retinoic acid receptors on spermatogenesis ...... 179

Conclusions from the characterization of the germ cell-specific knockout mice and a

working model ...... 181

Conclusions from microarray experiments ...... 184

FUTURE EXPERIMENTS ...... 187

Stem cell function regulated by RARA ...... 187

Follow-up of transcriptome differentially regulated in the cKO vs. wild type germ cells . 188

REFERENCE ...... 191

APPENDIX ONE ...... 209

SUPPLEMENTAL MICROARRAY DATA ...... 209

ix LIST OF TABLES

CHAPTER TWO

Table 2-1. Genotyping primers...... 93

CHAPTER THREE

Table 3-1. Primers used for real time RT-PCR...... 167

Table 3-2. Summary of differentially regulated probe sets...... 168

Table 3-3. Top RARA regulated genes in germ cells at P4 and P8...... 169

Table 3-4. Canonical and non-canonical retinoic acid response elements (RAREs) in the proximal promoter region of genes that are regulated ± 2.0 fold in the P4 and P8 lists. Direct repeat of the RGKTCA motif separated by zero, one, two, three, four, five, six, seven, eight, nine and ten base pairs are listed...... 170

Table 3-5. Top ten Pathway Studio subnetworks for the P4 gene set...... 171

Table 3-6. Top ten Pathway Studio subnetworks for the P8 gene set...... 173

Table 3-7. Top ten Pathway Studio (GO) biological processes for the P4 gene set...... 174

Table 3-8. Top ten Pathway Studio Gene Ontology (GO) biological processes for the P8 gene set...... 175

Table 3-9. Genes whose gene products bind to DMRT1 in the P4 gene set...... 176

Table 3-10. Pathway Analysis of transcripts that are differentially regulated at P8 and related to

DNA repair...... 177

APPENDIX

Table A1-1. Total Pathway Studio subnetworks for P4 germ cell differentially regulated at ± 2.0 fold genes...... 210

x Table A1-2. Total Pathway Studio subnetworks for P8 germ cell differentially regulated at ± 2.0 fold genes...... 217

Table A1-3. Total Pathway Studio Gene Ontology (GO) for P4 germ cell differentially regulated at ± 2.0 fold genes...... 221

Table A1-4. Total Pathway Studio Gene Ontology (GO) for P8 germ cell differentially regulated at ± 2.0 fold genes...... 247

xi

LIST OF FIGURES

CHAPTER ONE

Figure 1-1. Testis and seminiferous tubules...... 33

Figure 1-2. Schematic of embryonic germ cell development...... 35

Figure 1-3. Schematic of postnatal spermatogenesis and RA action sites...... 37

Figure 1-4. Representation of the synaptonemal complex formation and chromosome behavior during meiotic prophase...... 39

Figure 1-5. Graphical representation of the pituitary-hypogonadal axis...... 41

Figure 1-6. Retinoic acid metabolism and mechanism of action of retinoid receptors...... 43

Figure 1-7. Representation of protein expression...... 45

CHAPTER TWO

Figure 2-1. Schematic drawings of generating Rara cKO animals...... 94

Figure 2-2. Expression of STRA8-iCRE during neonatal development...... 96

Figure 2-3. Tubular abnormalities in cKO mice across the developmental ages...... 98

Figure 2-4. Quantification of morphological abnormalities in the testes...... 102

Figure 2-5. Decrease in percentage of most advanced cell types...... 104

Figure 2-6. Epididymal sperm and male fertility analysis...... 106

Figure 2-7. Decrease in number of cells undergoing mitosis (proliferation)...... 108

Figure 2-8. Decrease in percentage of tubules with RHOX13-positive differentiating spermatogonia...... 110

Figure 2-9. Decrease in percentage of tubules with brightly stained γH2AX-positive leptotene and zygotene spermatocytes...... 112

Figure 2-10. Accumulation of undifferentiated spermatogonia at P10...... 114

xii Figure 2-11. TUNEL analysis to detect apoptosis...... 116

Figure 2-12. Decrease in percentage of pachytene spermatocytes with perfect synapsis and increase in percentage of double strand break (DSB) formation...... 118

Figure 2-13. Analyses of the recipients after spermatogonial stem cell transplantation...... 120

CHAPTER THREE

Figure 3-1. Germ cell enrichment with a step-step digestion...... 151

Figure 3-2. Real time PCR analysis of transcripts for somatic cell contamination in the enriched germ cell isolation procedure...... 153

Figure 3-3. Venn Diagram...... 155

Figure 3-4. Pathway analysis of common biological functions from transcripts that are differentially regulated at ± 3 fold or higher in P4 germ cells...... 157

Figure 3-5. Pathway analysis of common biological functions from transcripts that are differentially regulated at ± 3 fold or higher in P8 germ cells...... 159

Figure 3-6. Real time PCR verification of transcriptional changes in P4 germ cells...... 161

Figure 3-7. Real time PCR verification of transcriptional changes in P8 germ cells...... 163

Figure 3-8. Schematic drawing of transcripts in the P8 set that are involved in the DSB pathway.

...... 165

CHAPTER FOUR

Figure 4-1. The three commitment steps of spermatogenesis tightly regulated by RA and RARA.

...... 197

Figure 4-2. Regulation of RARA in the canonical transcription pathway, and RARA acting in non-canonical transcription and regulation pathways...... 199

xiii Figure 4-3. Proposed model of RARA regulation of spermatogonial mitosis, at the transition of undifferentiated spermatogonia to differentiating spermatogonia and meiosis...... 203

Figure 4-4. Co-regulator activity of RARA in the transcriptional activity of DMRT1...... 206

xiv CHAPTER ONE

INTRODUCTION

INTRODUCTION

The testis and spermatogenesis

The testis is a male specific reproductive organ, in which haploid gametes are produced with the purpose of passing on their genetic information to the next generation. A tough fibrous capsule (tunica) covers the testis and encloses two major compartments: the interstitial compartment (interstitium) and the seminiferous tubule compartment (Russell et al., 1990a)

(Fig.1-1). Blood and lymphatic vessels as well as Leydig cells are found in the interstitium

(Fig.1-1 B). Leydig cells are the major source of androgens for the testis including testosterone, estrogen, and other essential steroids for testicular function (Haider, 2004). The lymphatic endothelium, the myoid cells, and the basal lamina set the boundary of seminiferous tubules.

Both ends of the seminiferous tubules connect to the rete testis (Fig. 1-1 A). Myoid cells, along with the basal lamina, provide the structural support for Sertoli cells and germ cells in the seminiferous tubule (Fig.1-1 C). Within each tubule, germ cells form concentric circles with spermatogonia next to the basement membrane, followed by germ cells undergoing meiosis and haploid spermatid maturation in the adluminal compartment of the testis (Fig.1-1 B and C).

Along the diameter of the concentric circle, germ cells at different developmental stages associate with each other in a specific order, giving rise to the twelve, fourteen, and six stages of spermatogenic cycles in mice, rats, and humans, respectively (Russell et al., 1990b).

Germ cell formation begins in utero with primordial germ cells (PGCs) that form around embryonic day 6.25 (E6.25) and migrate from the proximal epiblast through the hindgut towards the genital ridge around E10.5 (Saitou and Yamaji, 2012; Tam and Snow, 1981) (Fig.1-2). From here, female (XX) and male (XY) PGCs enter two distinct pathways. While XX PGCs continue to proliferate until E13.5 and subsequently enter meiosis, XY PGCs are enclosed by testicular

2 cords, become prospermatogonia or gonocytes around E12.5 (McLaren, 1984) and are then

arrested at G0-like state around E13.5. Gonocytes remain quiescent around E16.5 until shortly after birth (Hilscher et al., 1974; Vergouwen et al., 1991).

Migrating from the center of the tubule towards the basement membrane, gonocytes differentiate to spermatogonial stem cells (SSCs) and resume mitosis to replenish the SSC population. They then produce undifferentiated spermatogonia by P3 (Sapsford, 1962; Oatley and Brinster, 2012). This is the beginning of the process known as spermatogenesis that produces numerous haploid spermatozoa. Spermatozoa are produced through three biological processes

(Fig.1-3): the renewal of spermatogonial stem cells and the production and expansion of progenitor spermatogonial cells by mitosis and differentiation; the reductive division of progenitor cells by meiosis during the spermatocyte stage (when DNA is recombined); and the maturation step called spermiogenesis that includes the chromatid compaction of haploid cells and flagellum formation (Russell et al., 1990a).

There are three types of spermatogonia: stem cell spermatogonia (Asingle (As)); proliferative undifferentiated spermatogonia (Apair (Apr); Aaligned (Aal)); and proliferative

differentiating spermatogonia. Differentiating spermatogonia arise from Aal spermatogonia that

differentiate into the type A1 differentiating spermatogonia without mitosis, followed by a

concomitant cell division in between each type to produce the type A2, A3, A4, Intermediate, and

B differentiating spermatogonia. Additionally, the type A1 differentiating spermatogonia can arise directly from gonocytes and kick start the first wave of spermatogenesis (Sapsford, 1962).

During the proliferative and differentiation phases of spermatogonia, mitotic divisions expand their population by 1000 fold. After meiosis, there is an additional four-fold expansion to provide a large number of sperm continuously over a long period of time (Russell et al., 1990a).

3 The most advanced differentiating spermatogonia (the type B spermatogonia) divide into preleptotene spermatocytes, which, with a round of DNA synthesis (4N DNA), prepare for meiosis. Spermatogonia and most preleptotene spermatocytes reside in the basal compartment, separated from the adluminal compartment by the Sertoli cell-Sertoli cell tight junctions (Fig.1-1

C). Preleptotene spermatocytes cross the tight junction to the adluminal side where meiosis begins. During meiosis, homologous recombine, genetic material is halved, and haploid spermatids are produced. Morphological changes occur as cells undergo prophase of the first meiotic division, and become leptotene, zygotene, pachytene and diplotene spermatocytes.

The presence of leptotene spermatocytes signals the initiation of meiotic prophase. At this stage, chromosomes condense, but remain unpaired (Fig.1-4 A). Chromosomes start to pair with their homologs in zygotene spermatocytes through the formation of synaptonemal complexes, a multi-protein structure, which can be used to identify the different meiotic prophases (Moses, 1968) (Fig.1-4 B). The pairing completes in pachytene spermatocytes.

Synaptonemal complexes dissipate and chromosomes separate from their partners at the diplotene stage. Then, Meiosis I occurs and secondary spermatocytes are formed via a reductive cell division. Round spermatids (haploid) are then rapidly produced in Meiosis II with another reductive cell division.

Round spermatids evolve into spermatozoa through several morphological changes without mitosis, known as spermiogenesis (Russell et al., 1990a). These morphological developments include flagellum emergence, acrosome development, nuclear condensation, and elimination of . Once the spermatozoa are produced, they are released from the Sertoli cells into the lumen of the seminiferous tubules, which is filled with liquid secreted by the Sertoli cells. From the lumen, the spermatozoa are moved into the rete testis through the contraction of

4 the peritubular myoid cells. From there, spermatozoa mature as they travel through the epididymis and mature sperm are stored in the caudal epididymis (Amann et al., 1993;Toshimori,

2003) (Fig.1-1 A)

Spermatogenesis in higher vertebrates is dependent on the function of Sertoli cells, as they provide a structural, nutritional, endocrine, and paracrine microenvironment to the germ cells (Griswold, 1995). Sertoli cell proliferation starts at E7 and ends around 2 weeks after birth, and is followed by the differentiation process at puberty. By this time, the number of Sertoli cells is set (Eskola et al., 1993) and this number defines the number of germ cells that can be supported in the adult testis (Orth et al., 1988).

Sertoli cells maintain the structural integrity of the seminiferous epithelium by attaching to the basement membrane and forming a polarized columnar shape that provides physical support and an interaction platform for the developing germ cells. Adjacent Sertoli cells compartmentalize the seminiferous epithelium through the formation of the junctional barrier

(Fig. 1-1 C), a specialized tight junction that separates the basal and adluminal compartments

(Weber et al., 1998). Disruption of the tight junctional complexes results in the disturbance of spermatogenesis (Cheng and Mruk, 2012). In addition to tight junctions, desmosomes and gap junctions hold adjacent Sertoli cells together (Gilula et al., 1976; Russell 1977a; Dym and

Fawcett, 1970; Russell and Peterson, 1985). Throughout the course of spermatogenesis, Sertoli cells remain in intimate contact with germ cells through adherens junctions, gap junctions, ectoplasmic specializations, and tubulobulbar complexes (Russell, 1977a; McGinley et al., 1979;

Russell, 1977b; Russell et al., 1988; Russell and Clermont, 1976; Russell and Malone, 1980;

Russell, 1979). With these junctional complexes in place, serum macromolecules are secluded from meiotic and post-meiotic germ cells that reside in the adluminal compartment. Instead,

5 Sertoli cells actively create the adluminal microenvironment by secreting nutrients and signaling molecules, to which the meiotic and post-meiotic germ cells are exposed. Sertoli cells also clean up defective germ cells by phagocytosis.

Testicular function is influenced both by endocrine (extra-testicular) and paracrine (intra- testicular) factors. Maintenance of normal spermatogenesis is dependent on the anterior pituitary hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are synthesized and secreted under the hypothalamic control of the gonadotropin-releasing hormone

(GnRH) (Fig.1-5). LH and FSH signal through luteinizing (LHR) and follicle- stimulating hormone receptor (FSHR), which are expressed by the Leydig and the Sertoli cells, respectively. Knocking out the LH receptor resulted in an arrest of spermatogenesis at the round spermatid stage (Zhang et al., 2001). Additionally, male mice without the FSH receptor had reduced testes size and testosterone levels (Krishnamurthy et al., 2000), essentially functioning at a quantitatively lowered spermatogenesis level. The paracrine regulation of spermatogenesis is provided by steroids, such as testosterone and estradiol secreted by Leydig cells, and by such as inhibin and activin synthesized by Sertoli cells (Cooke and Saunders, 2002) (Fig.1-5).

Receptor for testosterone (AR) has been deleted in various cell types, resulting in disruption of testicular function (Gendt and Verhoeven, 2012). Mutants for the estrogen receptors showed phenotypes of either infertility with ER-alpha or normal spermatogenesis with ER-beta (Eddy et al., 1996; Krege et al., 1998).

Interactions between Sertoli cells and germ cells through physical interaction and the secretion of signaling molecules are essential for the healthy progression of spermatogenesis

(Griswold et al., 1989; Jegou, 1991; Sylvester and Griswold, 1994; Wright et al 1989). Inhibin has a primary role of negatively regulating FSH secretion from the pituitary gland, whereas

6 activin has been proposed to affect germ cell maturation at the step when gonocytes differentiate

into spermatogonia (de Kretser et al., 2001). Another example of paracrine regulation is c-kit

receptor ligand, also known as Steel factor, synthesized by Sertoli cells. The lack of c-kit ligand

causes arrest of spermatogenesis due to blocking of the differentiation step of the Aal to the type

A1 (de Rooij, 2001; Loveland and Schlatt, 1997).

This intimate relationship between Sertoli cells and germ cells is organized so that germ cell development is precisely timed and spatially represented as the cycle of seminiferous epithelium (spermatogenic cycle) (Clermont, 1972). Defined association of germ cells, called stages, is morphologically distinct and can be identified in histological cross sections of the testis

(Russell et al., 1990b). There are twelve stages in the adult mouse. The types of germ cells found in the testis seem to be defined by the germ cells, not somatic cells (Franca et al., 1998), such that donor rat germ cells define the germ cell association patterns present in the mouse recipients after transplantation. At the same time, Sertoli cells regulate the microenvironment milieu for germ cell development as a function of the stage of the spermatogenic cycle (Parvinen, 1993).

Disruption of any factors that support the integrity of this germ cell organization would inevitably disturb spermatogenesis.

Vitamin A regulation and metabolism in rodent testis

In mammals, dietary vitamin A (retinoids) or retinol is required for the healthy development of spermatogenesis and overall animal health (Huang and Hembree, 1979; van Pelt and de Rooij, 1990a; Wolbach and Howe, 1925). Vitamin A cannot be synthesized de novo by animals and thus must be acquired through the diet (Fig.1-6). The equilibrium of retinoid molecules is regulated by two modes of action in the testis: 1) by the level of retinyl ester stored;

7 and 2) by the new synthesis or metabolism of retinoic acid. Vitamin A is reduced to retinyl ester, its storage form, by lecithin-retinol acyltransferase (LRAT) (Napoli, 1996). When required, retinol is secreted from the liver, bound to retinol binding protein (RBP or RBP4) and transthyretin (TTR) and transported through the circulatory system (Goodman, 1984). In the testis, peritubular myoid cells take up circulatory retinol and transfer it to cellular retinol binding protein (CRBP) (Blaner et al., 1987). Retinol is then bound by newly synthesized RBP in the myoid cells and secreted into the peritubular space as a complex, and then taken up by Sertoli cells (Davis and Ong, 1995). Stimulated by retinoic acid gene 6 (STRA6), a membrane bound receptor, binds to the RBP-retinol complex for retinol uptake (Kawaguchi et al., 2007) into

Sertoli cells. In Sertoli cells, retinol is metabolized into retinaldehyde by either alcohol dehydrogenase (ADH) or retinol dehydrogenase (RDH), and then into retinoic acid by retinaldehyde dehydrogenase (RALDH) (Ang et al., 1996; Russell et al., 1990a; Zhang et al.,

2001). Thereafter, retinoic acid transduces signals via activation of retinoid receptors. When no longer needed, cytochrome P450 (CYP26) enzymes metabolize retinoic acid through oxidation

(Hernandez et al., 2007; Uehara et al., 2007).

Because of the Sertoli cell-Sertoli cell tight junctions creating a blood-testis barrier

(Russell, 1977c), which restricts the transport of large protein molecules, retinol-RBP must be processed in Sertoli cells before crossing it or retinoic acid can be supplied to germ cells in the adluminal compartment. Alternatively, germ cells in the basal compartment may get retinol directly from the serum. LRAT is expressed in germ cells, and thus, it is possible that retinol is converted and stored as retinyl ester inside the germ cells (Sugimoto et al., 2012).

8 Vitamin A deficiency in mice and rats

In VAD rats, germ cell-depleted seminiferous tubules were found in males, and failure of

fertilization and implantation were seen in females (Wolbach and Howe, 1925; Evans, 1928).

The atrophic testes retained only Sertoli cells and spermatogonia in mice (van Pelt and de Rooij,

1990b), and a few preleptotene spermatocytes additionally in rats (Huang and Hembree, 1979).

This is due to the loss of associations between Sertoli cells and spermatids in rats, resulting in

sloughed germ cells seen in the epididymis (Unni et al., 1983). In addition, detailed investigation

indicated that early meiotic germ cells, pachytene spermatocytes, and multinucleated giant cells

became apoptotic in rats (Akmal et al., 1998). VAD mice and rats can regenerate

spermatogenesis in a synchronous manner after retinoid or repeated retinoic acid replenishment,

coupled with a retinoic acid-sufficient diet (Howell et al., 1963; Huang et al., 1983; Huang and

Hembree, 1979; Morales and Griswold, 1987; van Pelt and de Rooij, 1990b, 1991). Studies have

suggested that retinoic acid is critical for the initiation of meiosis (Akmal et al., 1997; Bowles et

al., 2006; Chung et al., 2004; Doyle et al., 2007; Koubova et al., 2006) and spermiogenesis

(Lufkin et al., 1993; Chung et al., 2005; Doyle et al., 2007) mediated through retinoic acid

receptors.

Retinoic acid receptors and retinoid X receptors

Retinol can be metabolized into two major types of retinoic acid (RA): 9-cis RA and all- trans-RA (Duester, 2000). They are ligands for two families of nuclear hormone receptors, the transcription factors retinoic acid receptor (RAR) and (RXR). Both 9-cis-RA and all-trans-RA can activate RAR, while RXRs are only activated if bound to 9-cis-RA

(Mangelsdorf, 1994). Each family has three functionally distinct subtypes: alpha, beta and

9 gamma (Chambon, 1996). RAR heterodimerizes with RXR, and as a heterodimeric complex,

binds to a DNA element called retinoic acid response element (RARE), with direct repeats of a

consensus sequence of PuG(G/T)TCA, separated by 1, 2 or 5 nucleotides (DR1, 2, 5) (Popperl

and Featherstone, 1993). Upon the activation of this heterodimer by RA, chromatin opens up and

becomes permissive for transcription of target genes (Bastien and Rochette-Egly, 2004). Genetic

knockouts of all six receptors have been generated and characterized. Only Rara knockout (KO)

male mice are infertile due to advanced germ cell loss, similar to that observed in vitamin A

deficient (VAD) mice, which lacks the ligand for all six receptors. Homozygous Rarb mutants

are growth-deficient, but fertile and have a normal longevity (Ghyselinck et al., 1997). Rarg

mutants are sterile not due to a defect in the testis, but instead it is thought to be a result of

squamous metaplasia of the seminal vesicles and prostate (Lohnes et al., 1993). Rxra mutants are embryonic lethal (Sucov et al., 1994). Rxrb mutant mice are sterile due to abnormal spermiogenesis and spermiation (release of spermatids). The number of spermatozoa is reduced as well as characterized by abnormal acrosomes and tails (Kastner et al., 1996). Lastly, Rxrg mutant mice are phenotypically normal (Krezel et al., 1996).

Retinoic acid receptor alpha

Cellular localization and developmental studies of Rara transcripts and RARA proteins in the rat testes indicated that both Sertoli and germ cells express this gene product starting from early postnatal ages (as early as P0 and P3~5 in gonocytes) (Akmal et al., 1997; Boulogne et al.,

1999; Cupp et al., 1999; Dufour and Kim, 1999) (Fig.1-7). The mouse Rara gene is conserved in the rat, human, chimpanzee, Rhesus monkey, dog, cow, chicken and zebrafish. An accumulation of the type A1 spermatogonia near the end of stage VIII, resulting from a block in

10 spermatogenesis, is a signature of VAD rats (Griswold et al., 1989). Rara mRNA level in the

VAD rats spiked within 30 minutes of retinol injection (Kim and Griswold, 1990). In addition,

the highest expression of Rara mRNA is between stage VIII and IX in the stage-synchronized

rats (Linder et al., 1991), corresponding remarkably well with the VAD biological block. Once

the protein is made and imported to the nucleus, it is degraded within two hours. The receptor to

be degraded is chaperoned by disulfide isomerase glucose-regulated protein 58 (GRp58) for

degradation in the endoplasmic reticulum (Zhu et al., 2010). This is important when interpreting

the effect of RA treatment beyond two hours, as it would be RARA-independent and could be a

result of accumulative toxicity. In addition, RARA nuclear localization and the subsequent

transcriptional activity are positively regulated by protein kinase C (PKC) (Braun et al., 2000)

and by small ubiquitin-like modifier-2 (SUMO-2) (Zhu et al., 2009). FSH binds its receptor and

activates cAMP and protein kinase A (PKA), which phosphorylates at PKA sites on RARA,

inhibiting the nuclear localization of RARA (Santos and Kim, 2010).

Rara-null animals have high neonatal mortality and exhibit male infertility phenotype.

The surviving males have depleted germ cells and vacuolization in the testis (Chung et al., 2004;

Doyle et al., 2007; Lufkin et al., 1993), partly due to apoptosis of germ cells in a stage-dependent

manner (Doyle et al., 2007). Careful examination of the testis of Rara-null mice at various stages

of development and the spermatogenic cycle has uncovered more detailed abnormalities. There

was a temporal arrest of preleptotene spermatocytes in the first three waves of spermatogenesis

and degenerating pachytene spermatocytes and spermatid arrest at steps 8 and 9 in the first wave of spermatogenesis. A delay in the onset of the second wave was also noted. Mature spermatids failed to align at the luminal epithelium. Disorganization of germ cells within the tubule occurred due to missing or decreased numbers of the predicted cell types, and a decrease in germ cell

11 proliferation (Chung et al., 2004; Doyle et al., 2007). In addition, apoptotic elongating

spermatids are frequently found in 9-week old mutant testes (Chung et al., 2005).

Sertoli cell-specific Rara conditional knockout (cKO) was generated by Vernet and

colleagues and showed there was age-dependent testicular degeneration with vacuolization

(Vernet et al., 2006), similarily seen in Rara-null testes (Chung et al., 2004; Doyle et al., 2007).

In addition, there was a delay in the first wave of spermatogenesis, with fewer percent tubules

expressing STRA8 at P5, fewer percent tubules with leptotene spermatocytes, and fewer percent

tubules with pachytene and post-meiotic germ cells at P20 (Vernet et al., 2006). There were

more apoptotic round spermatids, before the time of apoptotic elongating spermatids in the Rara-

null testes (Chung et al., 2005).

Given the aforementioned expression pattern of Rara transcript and protein, combined

with the phenotypic disruptions of Rara-null mice, it is clear that Rara is crucial around the time of the second wave, meiotic initiation, and at the time of elongating spermatid formation. The delay in the second wave is thought to be due to decreased spermatogonial proliferation and differentiation. More specifically, it may be due to inefficient conversion of undifferentiated spermatogonia to differentiated spermatogonia.

Spermatogonial stem cell transplantation assay

Mammalian spermatogenesis over a lifetime is supported by a limited number of stem cells, termed the spermatogonial stem cells (SSCs). Although SSCs are quiescent and less prone to cellular stress, once they are activated to divide, any damage can be amplified and lead to reduced or lack of spermatogenesis. Healthy spermatogenesis depends on the support of Sertoli cells and cross talk between Sertoli cells and germ cells.

12 The transplantation of SSCs from one mouse into the testes of a recipient mouse was

pioneered by Brinster and colleagues (Brinster and Zimmermann, 1994). This functional assay

allows one to study the quality of stem cells and the intricate relationship between germ cells and

Sertoli cells. When donor spermatogonial cells are enzymatically isolated and injected into recipient testes, which are infertile by either or cytotoxic chemicals, donor-derived spermatogenesis can be obtained from SSCs of the donor germ cells (Brinster and Zimmermann,

1994). SSC transplantation has been applied in many animal models and is now considered the gold standard for SSC functional assay in the testis (Boettger-Tong et al., 2000; Johnston et al.,

2001; Mahato et al., 2000; Rilianawati et al., 2003).

To determine the quality of stem cells in Rara-null cKO testes, germ cells were enzymatically digested, enriched, and transplanted into germ cell-depleted testes of W/Wv mice

(Doyle et al., 2007). Rara-null germ cells rarely colonized and regenerated, leading to the conclusion that RARA protein in germ cells is essential for regulating early spermatogonial proliferation and differentiation. In the complementary experiment, in which wild type germ cells of Rosa26 mice were injected into Rara-null testes, germ cells were able to proliferate and

differentiate, but the efficiency of spermatogenesis was low, as shown by the dramatically

decreased number of spermatocytes and spermatids (Doyle et al., 2007). Therefore, it was

concluded that RARA in Sertoli cells was crucial for supporting efficient meiosis and

spermiogenesis.

Germ cell type-specific investigation of RARA function using Cre/LoxP system

Because of the pleiotropic expression of RARA in the seminiferous tubule at different

stages of germ cell development and in various cell types, it is necessary to tease apart the gene

13 function using spermatogonial germ cell functional assay and cell type-specific conditional knockout approaches.

The Cre-LoxP site-specific recombination method has been widely used as an elegant system to study gene function in a cell type and in a developmental stage-specific manner.

Cyclization recombination (CRE) is an enzyme originated from bacteriophage P1. This 34- kilodalton enzyme is a site-specific DNA recombinase that recognizes a 34- consensus loxP ( of X-over of P1) site. When two loxP sites are placed at regions flanking a target sequence (flox target sequence), in this case, a common region of two main Rara isoforms, the two loxP sites are recombined and the target sequence is cut out in the presence of CRE recombinase. Cell type-specific promoters regulate Cre expression at desired developmental stages (Sauer, 1998).

Sertoli cell-specific Rara knockout mice were generated (Vernet et al., 2006a). In these

Sertoli cell-specific Rara mutants, spermatogenesis was reduced in an age-dependent manner.

There was an increase in the number of apoptotic cells in 9-week old mutants. In addition, a delay in the progression of the prepubertal wave of spermatogenesis occurred. In other words, there was a decreased efficiency of spermatogenesis. To study the function of Rara specifically in germ cells, promoters of Stimulated by retinoic acid gene 8 (Stra8) and Neurogenin 3 (Ngn3) were engineered to drive the expression of Cre. When mated to floxed Rara mice, germ cell- specific Rara conditional knockout animals were generated. After two generations of matings, both of floxed Rara were deleted. Stra8-iCre mRNA is expressed at P3 (Sadate-Ngatchou et al., 2008), similar to the expression pattern of Stra8 mRNA. Stra8 mRNA has the highest expression in the type A and type B spermatogonia, detectable starting at P3 and peaking at P10, when meiosis is initiated in the testis (Oulad-Abdelghani et al., 1996; Shima et al., 2004). In

14 ovaries, meiotic initiation starts at about E13.5 (McLaren and Southee, 1997), and Stra8 mRNA has the highest expression at E14.5 (Shima et al., 2004). On the other hand, Ngn3 and Ngn3-Cre mRNA starts to express around P3, in a subset of undifferentiated spermatogonia (Yoshida et al.,

2006; Yoshida et al., 2004). Both Stra8 and Ngn3 promoter driven-Cre recombinases deleted the floxed region of Rara and allowed the study of this gene function in germ cells, which is the goal of the current thesis study, illustrated in the next chapter.

Summary of work

All-trans retinoic acid (RA), a biologically active form of dietary vitamin A (retinoids), is important for mammalian spermatogenesis. Retinoic acid receptor (RAR) is a RA-dependent , mediating retinoid signaling. Retinoic acid receptor alpha (RARA) is crucial in the development of testicular germ cells, shown by Rara-null testes. Both vitamin A deficient

(VAD) and Rara-null testes are abnormal. Both vitamin A deficient (VAD) and Rara-null males are infertile due to depletion of germ cells. Phenotypic abnormalities of severely degenerated testes include: lack of advanced germ cells, sloughing of germ cells, and vacuole formation in the seminiferous epithelium.

Rara was conditionally deleted in germ cells using STRA8-iCRE (codon improved CRE) to disable RARA function to discern the functional phenotype and expression targets of this transcription factor specifically in germ cells. STRA8-iCRE was activated at postnatal day 1 (P1) in 100% of the animals (N=7), as shown by mating with reporter mice. Rara mRNA levels were consistently reduced in enriched germ cells from Rara cKO mouse at P4, 8 and 25, which were tested using quantitative real time RT-PCR.

15 With RARA function disabled, there was a 42% and 74% decrease in epididymal sperm

count at P75 and P120, respectively. Detectable morphological abnormality of vacuole formation

is apparent in the Rara cKO testes in P10 animals. Significant morphological abnormality of

disorganized germ cell layers was scored starting at P25, which worsened throughout development. By P365 (1 year), 50% of the seminiferous tubules were abnormal in the Rara mutant testes. These abnormalities included lack of germ cell organization, lack of lumen, vacuole formation, and sloughing of cells. There was more than a two-fold decrease in the most advanced germ cell types at P15, P20 and P30. Further investigation using mitosis markers, phosphohistone H3 (pHH3) and a synthetic nucleotide 5-bromo-2’deoxyuridine (BrdU), showed a decrease in proliferation of germ cells starting at P4. The expression pattern of a differentiating

spermatogonial marker, Reproductive gene 13 (RHOX13), showed a decrease starting

at P6. Analysis of pachytene chromosome spread using meiosis markers, SYCP3 and γH2AX,

suggested that RARA has a pivotal role in the double strand break (DSB) repair pathway.

Global transcriptome expression profiling of wild type and Rara cKO germ cells

highlighted and confirmed the role of RARA in cell differentiation and meiosis. Functional

pathway analysis algorithms identified cell differentiation, cell adhesion and cell migration as the

top three biologically functional processes that are significant at P4. As germ cells develop into

leptotene spermatocytes at P8, drastically different functions emerged. The top three biologically

functional processes significant at P8 are meiosis, spermatogenesis, and synaptonemal complex

assembly.

Detailed analysis of the mis-regulated transcripts revealed that some of them are primary

targets of RA (transcripts with RAREs), and some of them are secondary targets. Among the

secondary targets at P4, the highest up-regulated transcript is phosphatidylinositol 4-kinase type

16 2 beta (Pi4k2b, 10.2 fold in the cKO), which is involved in a kinase cascade, a non-genomic

effect of RARA summarized recently (Al Tanoury et al., 2013). In addition, the greatest down-

regulated transcript is nuclear distribution gene E-like homolog 1, A. nidulans (Ndel1, -7.5 fold in cKO), which is involved in polarity formation and organization during neurogenesis. As gonocytes differentiate and move from the center of the seminiferous tubule to the basement membrane, it may be important to rearrange the microtubule network and set up

polarity, an important aspect of asymmetric stem cell division (Knoblich, 2008). Interestingly,

proteins from 15 transcripts are known to physically bind to DMRT1 (Murphy et al., 2010), a

primary sex-determining gene in vertebrates, suggesting the potential role of RARA regulating

the downstream target of DMRT1 at P4. DMRT1 has been shown to restrict RA responsiveness

(Matson et al., 2010) and RARA may be responsible for this action. DMRT1 was shown to be

up-regulated by FSH (Chen and Heckert, 2001), it appears that FSH negatively regulates RARA

directly or indirectly through activating DMRT-1, a potential mechanism where FSH inhibits the

transcriptional activity of RARA, asides from phosphorylation of the PKA sites (Santos and

Kim, 2010).

Proteins from many of the differentially regulated transcripts at P8 are engaged in the

double strand break repair pathway during meiosis, which correlates to the phenotypic

abnormalities of Rara cKO germ cells at P8. The highest down-regulated transcript is essential

meiotic endonuclease 1 homolog 2, S. pombe (Eme2, -26.2 fold in cKO). The protein product of

this transcript forms a complex with MUS81, a structure-specific DNA endonuclease that

cleaves substrates such as 3’-flap structures, a product of DNA repair (Marti and Fleck, 2004).

Glial fibrillary acidic protein (Gfap, 10.3 fold in cKO) is the top up-regulated transcript and a

class-III . Intermediate filament has been shown to control the germ cell

17 nuclear activity in ciliate protozoa (Numata et al., 1985). Genes on the X and Y-chromosome have predominant roles in pre-meiotic stages of mammalian spermatogenesis (Maclean et al.,

2005; Wang et al., 2001). Remarkably, 9.2% of the transcripts are X or Y-linked in the list of differentially regulated transcripts that are ± 2.0 fold or greater, which is in accordance with the role of retinoic acid in meiotic activities (Griswold et al., 2012).

18 REFERENCES

Akmal, K.M., Dufour, J.M., Kim, K.H., 1997. Retinoic acid receptor alpha gene expression in

the rat testis: potential role during the prophase of meiosis and in the transition from

round to elongating spermatids. Biology of reproduction 56, 549-556.

Akmal, K.M., Dufour, J.M., Vo, M., Higginson, S., Kim, K.H., 1998. Ligand-dependent

regulation of retinoic acid receptor alpha in rat testis: in vivo response to depletion and

repletion of vitamin A. Endocrinology 139, 1239-1248.

Al Tanoury, Z., Piskunov, A., Rochette-Egly, C., 2013. Vitamin A and retinoid signaling:

genomic and non-genomic effects. Journal of lipid research.

Amann, R.P., Hammerstedt, R.H., Veeramachaneni, D.N., 1993. The epididymis and sperm

maturation: a perspective. Reprod Fertil Dev 5, 361-81.

Ang, H.L., Deltour, L., Hayamizu, T.F., Zgombic-Knight, M., Duester, G., 1996. Retinoic acid

synthesis in mouse embryos during gastrulation and craniofacial development linked to

class IV alcohol dehydrogenase gene expression. The Journal of biological chemistry

271, 9526-9534.

Bastien, J., Rochette-Egly, C., 2004. Nuclear retinoid receptors and the transcription of retinoid-

target genes. Gene 328, 1-16.

Blaner, W.S., Galdieri, M., Goodman, D.S., 1987. Distribution and levels of cellular retinol- and

cellular retinoic acid-binding protein in various types of rat testis cells. Biology of

reproduction 36, 130-137.

Boettger-Tong, H.L., Johnston, D.S., Russell, L.D., Griswold, M.D., Bishop, C.E., 2000.

Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of

supporting transplanted spermatogenesis. Biology of reproduction 63, 1185-1191.

19 Boulogne, B., Levacher, C., Durand, P., Habert, R., 1999. Retinoic acid receptors and retinoid X

receptors in the rat testis during fetal and postnatal development: immunolocalization and

implication in the control of the number of gonocytes. Biology of reproduction 61, 1548-

1557.

Bowles, J., Knight, D., Smith, C., Wilhelm, D., Richman, J., Mamiya, S., Yashiro, K.,

Chawengsaksophak, K., Wilson, M.J., Rossant, J., Hamada, H., Koopman, P., 2006.

Retinoid signaling determines germ cell fate in mice. Science 312, 596-600.

Braun, K.W., Tribley, W.A., Griswold, M.D., Kim, K.H., 2000. Follicle-stimulating hormone

inhibits all-trans-retinoic acid-induced retinoic acid receptor alpha nuclear localization

and transcriptional activation in mouse Sertoli cell lines. The Journal of biological

chemistry 275, 4145-4151.

Brinster, R.L., Zimmermann, J.W., 1994. Spermatogenesis following male germ-cell

transplantation. Proc Natl Acad Sci U S A 91, 11298-11302.

Chambon, P., 1996. A decade of molecular biology of retinoic acid receptors. FASEB J. 10, 940-

954.

Chen, J.K., Heckert, L.L., 2001. Dmrt1 expression is regulated by follicle-stimulating hormone

and phorbol esters in postnatal Sertoli cells. Endocrinology 142, 1167-1178.

Cheng, C.Y., Mruk, D.D., 2012. The blood-testis barrier and its implications for male

contraception. Pharmacol Rev 64, 16-64.

Chung, S.S., Sung, W., Wang, X., Wolgemuth, D.J., 2004. Retinoic acid receptor alpha is

required for synchronization of spermatogenic cycles and its absence results in

progressive breakdown of the spermatogenic process. Dev Dyn 230, 754-766.

20 Chung, S.S., Wang, X., Wolgemuth, D.J., 2005. Male sterility in mice lacking retinoic acid

receptor alpha involves specific abnormalities in spermiogenesis. Differentiation 73, 188-

198.

Chung, S.S., Wang, X., Wolgemuth, D.J., 2009. Expression of retinoic acid receptor alpha in the

germline is essential for proper cellular association and spermiogenesis during

spermatogenesis. Dev 136, 2091-2100.

Clermont, Y., 1972. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and

spermatogonial renewal. Physiological reviews 52, 198-236.

Cooke, H.J., Saunders, P.T., 2002. Mouse models of male infertility. Nature reviews. Genetics 3,

790-801.

Cupp, A.S., Dufour, J.M., Kim, G., Skinner, M.K., Kim, K.H., 1999. Action of retinoids on

embryonic and early postnatal testis development. Endocrinology 140, 2343-2352.

Davis, J.T., Ong, D.E., 1995. Retinol processing by the peritubular cell from rat testis. Biology

of reproduction 52, 356-364.

De Gendt, K., Verhoeven, G., 2012. Tissue- and cell-specific functions of the

revealed through conditional knockout models in mice. Molecular and cellular

endocrinology 352, 13-25. de Kretser, D.M., Loveland, K.L., Meehan, T., O'Bryan, M.K., Phillips, D.J., Wreford, N.G.,

2001. Inhibins, activins and follistatin: actions on the testis. Molecular and cellular

endocrinology 180, 87-92. de Rooij, D.G., 2001. Proliferation and differentiation of spermatogonial stem cells.

Reproduction 121, 347-354.

21 Doyle, T.J., Braun, K.W., McLean, D.J., Wright, R.W., Griswold, M.D., Kim, K.H., 2007.

Potential Functions of Retinoic Acid Receptor A in Sertoli Cells and Germ Cells During

Spermatogenesis. Annals of the New York Academy of Sciences 1120, 114-130.

Duester, G., 2000. Families of retinoid dehydrogenases regulating vitamin A function:

production of visual pigment and retinoic acid. European journal of biochemistry / FEBS

267, 4315-4324.

Dym, M., Fawcett, D.W., 1970. The blood-testis barrier in the rat and the physiological

compartmentation of the seminiferous epithelium. Biology of reproduction 3, 308-326.

Dufour, J.M., Kim, K.H., 1999. Cellular and subcellular localization of six retinoid receptors in

rat testis during postnatal development: identification of potential heterodimeric

receptors. Biology of reproduction 61, 1300-1308.

Eddy, E.M., Washburn, T.F., Bunch, D.O., Goulding, E.H., Gladen, B.C., Lubahn, D.B., Korach,

K.S., 1996. Targeted disruption of the gene in male mice causes

alteration of spermatogenesis and infertility. Endocrinology 137, 4796-4805.

Eskola, V., Nikula, H., Huhtaniemi, I., 1993. Age-related variation of follicle-stimulating

hormone-stimulated cAMP production, protein kinase C activity and their interactions in

the rat testis. Molecular and cellular endocrinology 93, 143-148.

Evans, H.M., 1928. The effects of inadequate vitamin A on the sexual physiology of the female.

J. Biol. Chem. 77, 651-654.

Franca, L.R., Ogawa, T., Avarbock, M.R., Brinster, R.L., Russell, L.D., 1998. Germ cell

genotype controls cell cycle during spermatogenesis in the rat. Biology of reproduction

59, 1371-1377.

22 Ghyselinck, N.B., Dupe, V., Dierich, A., Messaddeq, N., Garnier, J.M., Rochette-Egly, C.,

Chambon, P., Mark, M., 1997. Role of the retinoic acid receptor beta (RARbeta) during

mouse development. The International journal of developmental biology 41, 425-447.

Gilula, N.B., Fawcett, D.W., Aoki, A., 1976. The Sertoli cell occluding junctions and gap

junctions in mature and developing mammalian testis. Developmental biology 50, 142-

168.

Goodman, D.S., 1984. Overview of current knowledge of metabolism of vitamin A and

carotenoids. Journal of the National Cancer Institute 73, 1375-1379.

Griswold, M.D., 1995. Interactions between germ cells and Sertoli cells in the testis. Biology of

reproduction 52, 211-216.

Griswold, M.D., Bishop, P.D., Kim, K.H., Ping, R., Siiteri, J.E., Morales, C., 1989. Function of

vitamin A in normal and synchronized seminiferous tubules. Annals of the New York

Academy of Sciences 564, 154-172.

Griswold, M.D., Hogarth, C.A., Bowles, J., Koopman, P., 2012. Initiating meiosis: the case for

retinoic acid. Biology of reproduction 86, 35.

Haider, S.G., 2004. Cell biology of Leydig cells in the testis. International review of cytology

233, 181-241.

Hernandez, R.E., Putzke, A.P., Myers, J.P., Margaretha, L., Moens, C.B., 2007. Cyp26 enzymes

generate the retinoic acid response pattern necessary for hindbrain development.

Development 134, 177-187.

Hilscher, B., Hilscher, W., Bulthoff-Ohnolz, B., Kramer, U., Birke, A., Pelzer, H., Gauss, G.,

1974. Kinetics of gametogenesis. I. Comparative histological and autoradiographic

23 studies of oocytes and transitional prospermatogonia during oogenesis and

prespermatogenesis. Cell and tissue research 154, 443-470.

Howell, J.M., Thompson, J.N., Pitt, G.A., 1963. Histology of the lesions produced in the

reproductive tract of animals fed a diet deficient in vitamin A alcohol but containing

vitamin A acid. I. The male rat. Journal of reproduction and fertility 5, 159-167.

Huang, H.F., Hembree, W.C., 1979. Spermatogenic response to vitamin A in vitamin A deficient

rats. Biology of reproduction 21, 891-904.

Huang, H.F., Dyrenfurth, I., Hembree, W.C., 1983. Endocrine changes associated with germ cell

loss during vitamin A-induced recovery of spermatogenesis. Endocrinology 112, 1163-

1171.

Jegou, B., 1991. Spermatids are regulators of Sertoli cell function. Annals of the New York

Academy of Sciences 637, 340-353.

Johnston, D.S., Russell, L.D., Friel, P.J., Griswold, M.D., 2001. Murine germ cells do not require

functional androgen receptors to complete spermatogenesis following spermatogonial

stem cell transplantation. Endocrinology 142, 2405-2408.

Kastner, P., Mark, M., Leid, M., Gansmuller, A., Chin, W., Grondona, J.M., Decimo, D., Krezel,

W., Dierich, A., Chambon, P., 1996. Abnormal spermatogenesis in RXR beta mutant

mice. Genes Dev 10, 80-92.

Kawaguchi, R., Yu, J., Honda, J., Hu, J., Whitelegge, J., Ping, P., Wiita, P., Bok, D., Sun, H.,

2007. A membrane receptor for retinol binding protein mediates cellular uptake of

vitamin A. Science 315, 820-825.

Kim, K.H., Griswold, M.D., 1990. The regulation of retinoic acid receptor mRNA levels during

spermatogenesis. Mol Endocrinol 4, 1679-1688.

24 Knoblich, J.A., 2008. Mechanisms of asymmetric stem cell division. Cell 132, 583-597.

Koubova, J., Menke, D.B., Zhou, Q., Capel, B., Griswold, M.D., Page, D.C., 2006. Retinoic acid

regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci U S A 103,

2474-2479.

Krege, J.H., Hodgin, J.B., Couse, J.F., Enmark, E., Warner, M., Mahler, J.F., Sar, M., Korach,

K.S., Gustafsson, J.A., Smithies, O., 1998. Generation and reproductive phenotypes of

mice lacking . Proceedings of the National Academy of Sciences of

the United States of America 95, 15677-15682.

Krezel, W., Dupe, V., Mark, M., Dierich, A., Kastner, P., Chambon, P., 1996. RXR gamma null

mice are apparently normal and compound RXR alpha +/-/RXR beta -/-/RXR gamma -/-

mutant mice are viable. Proc Natl Acad Sci USA 93, 9010-9014.

Krishnamurthy, H., Danilovich, N., Morales, C.R., Sairam, M.R., 2000. Qualitative and

quantitative decline in spermatogenesis of the follicle-stimulating hormone receptor

knockout (FORKO) mouse. Biology of reproduction 62, 1146-1159.

Linder, C.C., Heckert, L.L., Roberts, K.P., Kim, K.H., Griswold, M.D., 1991. Expression of

receptors during the cycle of the seminiferous epithelium. Annals of the New York

Academy of Sciences 637, 313-321.

Lohnes, D., Kastner, P., Dierich, A., Mark, M., LeMeur, M., Chambon, P., 1993. Function of

retinoic acid receptor gamma in the mouse. Cell 73, 643-658.

Loveland, K.L., Schlatt, S., 1997. Stem cell factor and c-kit in the mammalian testis: lessons

originating from Mother Nature's gene knockouts. The Journal of endocrinology 153,

337-344.

25 Lufkin, T., Lohnes, D., Mark, M., Dierich, A., Gorry, P., Gaub, M.P., LeMeur, M., Chambon, P.,

1993. High postnatal lethality and testis degeneration in retinoic acid receptor alpha

mutant mice. Proc Natl Acad Sci USA 90, 7225-7229.

Maclean, J.A., 2nd, Chen, M.A., Wayne, C.M., Bruce, S.R., Rao, M., Meistrich, M.L., Macleod,

C., Wilkinson, M.F., 2005. Rhox: a new homeobox gene cluster. Cell 120, 369-382

Mahato, D., Goulding, E.H., Korach, K.S., Eddy, E.M., 2000. Spermatogenic cells do not require

estrogen receptor-alpha for development or function. Endocrinology 141, 1273-1276.

Mangelsdorf, D.J., 1994. Vitamin A receptors. Nutrition reviews 52, S32-44.

Marti, T.M., Fleck, O., 2004. DNA repair nucleases. Cellular and molecular life sciences :

CMLS 61, 336-354.

Matson, C.K., Murphy, M.W., Griswold, M.D., Yoshida, S., Bardwell, V.J., Zarkower, D., 2010.

The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls

the mitosis versus meiosis decision in male germ cells. Developmental cell 19, 612-624.

McGinley, D.M., Posalaky, Z., Porvaznik, M., Russell, L., 1979. Gap junctions between Sertoli

and germ cells of rat seminiferous tubules. Tissue & cell 11, 741-754.

McLaren, A., 1984. Meiosis and differentiation of mouse germ cells. Symposia of the Society for

Experimental Biology 38, 7-23.

McLaren, A., Southee, D., 1997. Entry of mouse embryonic germ cells into meiosis.

Developmental biology 187, 107-113.

Morales, C., Griswold, M.D., 1987. Retinol-induced stage synchronization in seminiferous

tubules of the rat. Endocrinology 121, 432-434.

Moses, M.J., 1968. Synaptinemal complex. Annu. Rev. Genet. 2, 363-412.

26 Murphy, M.W., Sarver, A.L., Rice, D., Hatzi, K., Ye, K., Melnick, A., Heckert, L.L., Zarkower,

D., Bardwell, V.J., 2010. Genome-wide analysis of DNA binding and transcriptional

regulation by the mammalian Doublesex homolog DMRT1 in the juvenile testis. Proc

Natl Acad Sci U S A 107, 13360-13365.

Napoli, J.L., 1996. Retinoic acid biosynthesis and metabolism. FASEB journal : official

publication of the Federation of American Societies for Experimental Biology 10, 993-

1001.

Numata, O., Sugai, T., Watanabe, Y., 1985. Control of germ cell nuclear behaviour at

fertilization by Tetrahymena intermediate filament protein. Nature 314, 192-194.

Oatley, J.M., Brinster, R.L., 2012. The germline stem cell niche unit in mammalian testes.

Physiological reviews 92, 577-595.

Orth, J.M., Gunsalus, G.L., Lamperti, A.A., 1988. Evidence from Sertoli cell-depleted rats

indicates that spermatid number in adults depends on numbers of Sertoli cells produced

during perinatal development. Endocrinology 122, 787-794

Oulad-Abdelghani, M., Bouillet, P., Decimo, D., Gansmuller, A., Heyberger, S., Dolle, P.,

Bronner, S., Lutz, Y., Chambon, P., 1996. Characterization of a premeiotic germ cell-

specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene. The

Journal of cell biology 135, 469-477.

Parvinen, M., 1993. Cyclic functions of Sertoli cells. In: Russell, L.D., Griswold, M.D., (eds)

The Sertoli cell. Cache River Press, Clearwater, Florida, 331-347.

Popperl, H., Featherstone, M.S., 1993. Identification of a retinoic acid response element

upstream of the murine Hox-4.2 gene. Molecular and cellular biology 13, 257-265.

27 Rilianawati, Speed, R., Taggart, M., Cooke, H.J., 2003. Spermatogenesis in testes of Dazl null

mice after transplantation of wild-type germ cells. Reproduction 126, 599-604.

Russell, L., Clermont, Y., 1976. Anchoring device between Sertoli cells and late spermatids in

rat seminiferous tubules. The Anatomical record 185, 259-278.

Russell, L., 1977a. Desmosome-like junctions between Sertoli and germ cells in the rat testis.

The American journal of anatomy 148, 301-312.

Russell, L., 1977b. Observations on rat sertoli ectoplasmic ('junctional') specializations in their

association with germ cells of the rat testis. Tissue and Cell 9, 475-498.

Russell, L., 1977c. Movement of spermatocytes from the basal to the adluminal compartment of

the rat testis. The American journal of anatomy 148, 313-328.

Russell, L.D., 1979. Spermatid-Sertoli tubulobulbar complexes as devices for elimination of

cytoplasm from the head region late spermatids of the rat. The Anatomical record 194,

233-246.

Russell, L.D., Malone, J.P., 1980. A study of Sertoli-spermatid tubulobulbar complexes in

selected mammals. Tissue & cell 12, 263-285.

Russell, L.D., Peterson, R.N., 1985. Sertoli cell junctions: morphological and functional

correlates. International review of cytology 94, 177-211.

Russell, L.D., Goh, J.C., Rashed, R.M., Vogl, A.W., 1988. The consequences of disruption

at Sertoli ectoplasmic specialization sites facing spermatids after in vivo exposure of rat

testis to cytochalasin D. Biology of reproduction 39, 105-118.

Russell, L.D., Ettlin, E.R., Hikim, A.P.S., Clegg, E.D., 1990a. Mammalian Spermatogenesis,

p.1-38. In Russell, L.D., Ettlin, E.R., Hikim, A.P.S., Clegg, E.D., (ed.), Histological and

Histopathological Evaluation of the Testis. Cache River Press, Clearwater.

28 Russell, L.D., Ettlin, E.R., Hikim, A.P.S., Clegg, E.D., 1990b. The Classification and Timing of

Spermatogenesis, p.41-56. In Russell, L.D., Ettlin, E.R., Hikim, A.P.S., Clegg, E.D.,

(ed.), Histological and Histopathological Evaluation of the Testis. Cache River Press,

Clearwater.

Sadate-Ngatchou, P.I., Payne, C.J., Dearth, A.T., Braun, R.E., 2008. Cre recombinase activity

specific to postnatal, premeiotic male germ cells in transgenic mice. Genesis 46, 738-742.

Saitou, M., Yamaji, M., 2012. Primordial germ cells in mice. Cold Spring Harbor perspectives in

biology 4.

Santos, N.C., Kim, K.H., 2010. Activity of retinoic acid receptor-alpha is directly regulated at its

protein kinase A sites in response to follicle-stimulating hormone signaling.

Endocrinology 151, 2361-2372.

Sapsford, C.S., 1962. Changes in the cells of the sex cords and seminiferous tubules during the

development of the testis of the rat and mouse. Aust J Zool 10, 178-193.

Sauer, B., 1998. Inducible gene targeting in mice using the Cre/lox system. Methods 14, 381-

392.

Shima, J.E., McLean, D.J., McCarrey, J.R., Griswold, M.D., 2004. The murine testicular

transcriptome: characterizing gene expression in the testis during the progression of

spermatogenesis. Biology of reproduction 71, 319-330.

Sucov, H.M., Dyson, E., Gumeringer, C.L., Price, J., Chien, K.R., Evans, R.M., 1994. RXR

alpha mutant mice establish a genetic basis for vitamin A signaling in heart

morphogenesis. Genes Dev 8, 1007-1018.

29 Sugimoto, R., Nabeshima, Y., Yoshida, S., 2012. Retinoic acid metabolism links the periodical

differentiation of germ cells with the cycle of Sertoli cells in mouse seminiferous

epithelium. Mechanisms of development 128, 610-624.

Sylvester, S.R., Griswold, M.D., 1994. The testicular iron shuttle: a "nurse" function of the

Sertoli cells. J Androl 15, 381-385.

Tam, P.P., Snow, M.H., 1981. Proliferation and migration of primordial germ cells during

compensatory growth in mouse embryos. Journal of embryology and experimental

morphology 64, 133-147.

Toshimori, K., 2003. Biology of spermatozoa maturation: an overview with an introduction to

this issue. Microscopy research and technique 61, 1-6.

Uehara, M., Yashiro, K., Mamiya, S., Nishino, J., Chambon, P., Dolle, P., Sakai, Y., 2007.

CYP26A1 and CYP26C1 cooperatively regulate anterior-posterior patterning of the

developing brain and the production of migratory cranial neural crest cells in the mouse.

Developmental biology 302, 399-411.

Unni, E., Rao, M.R., Ganguly, J., 1983. Histological & ultrastructural studies on the effect of

vitamin A depletion & subsequent repletion with vitamin A on germ cells & Sertoli cells

in rat testis. Indian J Exp Biol 21, 180-192. van Pelt, A.M., de Rooij, D.G., 1990a. The origin of the synchronization of the seminiferous

epithelium in vitamin A-deficient rats after vitamin A replacement. Biology of

reproduction 42, 677-682. van Pelt, A.M., de Rooij, D.G., 1990b. Synchronization of the seminiferous epithelium after

vitamin A replacement in vitamin A-deficient mice. Biology of reproduction 43, 363-367.

30 van Pelt, A.M., de Rooij, D.G., 1991. Retinoic acid is able to reinitiate spermatogenesis in

vitamin A-deficient rats and high replicate doses support the full development of

spermatogenic cells. Endocrinology 128, 697-704.

Vergouwen, R.P., Jacobs, S.G., Huiskamp, R., Davids, J.A., de Rooij, D.G., 1991. Proliferative

activity of gonocytes, Sertoli cells and interstitial cells during testicular development in

mice. Journal of reproduction and fertility 93, 233-243.

Vernet, N., Dennefeld, C., Guillou, F., Chambon, P., Ghyselinck, N.B., Mark, M., 2006.

Prepubertal testis development relies on retinoic acid but not rexinoid receptors in Sertoli

cells. Embo J 25, 5816-5825.

Wang, P.J., McCarrey, J.R., Yang, F., Page, D.C., 2001. An abundance of X-linked genes

expressed in spermatogonia. Nature genetics 27, 422-426.

Weber, J.E., Turner, T.T., Tung, K.S.K., Russell, L.D., 1988. Effect of cytochalasin D on the

integrity of the Sertoli cell (blood-testis) barrier. Am. J. Anat. 182, 130-147.

Wolbach, S.B., Howe, P.R., 1925. Tissue changes following deprivation of fat-soluble A

vitamin. J Exp Med 42, 753-777.

Wright, W.W., Zabludoff, S.D., Erickson-Lawrence, M., Karzai, A.W., 1989. Germ cell-Sertoli

cell interactions. Studies of cyclic protein-2 in the seminiferous tubule. Annals of the

New York Academy of Sciences 564, 173-185.

Yoshida, S., Sukeno, M., Nakagawa, T., Ohbo, K., Nagamatsu, G., Suda, T., Nabeshima, Y.,

2006. The first round of mouse spermatogenesis is a distinctive program that lacks the

self-renewing spermatogonia stage. Development 133, 1495-1505.

31 Yoshida, S., Takakura, A., Ohbo, K., Abe, K., Wakabayashi, J., Yamamoto, M., Suda, T.,

Nabeshima, Y., 2004. Neurogenin3 delineates the earliest stages of spermatogenesis in

the mouse testis. Developmental biology 269, 447-458.

Zhang, M., Chen, W., Smith, S.M., Napoli, J.L., 2001. Molecular characterization of a mouse

short chain dehydrogenase/reductase active with all-trans-retinol in intact cells, mRDH1.

The Journal of biological chemistry 276, 44083-44090.

Zhang, F.P., Poutanen, M., Wilbertz, J., Huhtaniemi, I., 2001. Normal prenatal but arrested

postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice.

Molecular endocrinology 15, 172-183.

Zhu, L., Santos, N.C., Kim, K.H., 2009. Small ubiquitin-like modifier-2 modification of retinoic

acid receptor-alpha regulates its subcellular localization and transcriptional activity.

Endocrinology 150, 5586-5595.

Zhu, L., Santos, N.C., Kim, K.H., 2010. Disulfide isomerase glucose-regulated protein 58 is

required for the nuclear localization and degradation of retinoic acid receptor alpha.

Reproduction 139, 717-731.

32 Figure 1-1. Testis and seminiferous tubules.

33 Figure 1-1. Testis and seminiferous tubules.

(A) Testis longitudinal-section view, showing the location of vas deferens, epididymis, efferent ducts, testis and rete testis. Elongated spermatids are produced in the testis and then travel to the epididymis through the rete testis. (B) Histological cross-section of one seminiferous tubule, showing the germ cell organization and the interstitium compartment. (C) Schematic representation of one section of the seminiferous tubule, showing germ cells at different stages of maturation, supported by two neighboring Sertoli cells. Polarized Sertoli cells are columnar shaped and associate with the germ cells from the basal to the adluminal compartment. The

Sertoli cell tight junction barrier (arrow) define the two compartments: stem cells and pre- meiotic cells (spermatogonia) are found on the basal side of the junction, whereas the meiotic

(spermatocytes) and the post-meiotic (round and elongating spermatids) cells are found organized in strict order of maturation towards the lumen.

34 Figure 1-2. Schematic of embryonic germ cell development.

35 Figure 1-2. Schematic of embryonic germ cell development.

In the mouse, germ cell competence is induced in the proximal epiblast cells around embryonic day 6.25~6.5 (E6.25~6.5). These epiblast cells are not lineage restricted and give rise to primordial germ cells (PGCs) and somatic cells. PGCs are thought to express PRDM1 and

PRDM14, two proteins that could regulate the specificity and development of PGCs. PGCs then migrate to the gonadal precursor, and then differentiate either toward the spermatogenic (male) or oogenic (female) pathway. Once PGCs become the male germ cells or gonocytes, they are arrested at the G0-like state until shortly after birth.

36 Figure 1-3. Schematic of postnatal spermatogenesis and RA action sites.

37 Figure 1-3. Schematic of postnatal spermatogenesis and RA action sites.

Gonocytes give rise to spermatogonial stem cells soon after birth. Stem cells can self-renew or commit to become undifferentiated spermatogonia, differentiating spermatogonia, and then spermatocytes through meiosis, and spermatids through spermiogenesis. Among many factors that have been known to affect the spermatogenic process, retinoic acid is required at the commitment steps throughout spermatogenesis, denoted by red arrows.

38 Figure 1-4. Representation of the synaptonemal complex formation and chromosome behavior during meiotic prophase.

39 Figure 1-4. Representation of the synaptonemal complex formation and chromosome behavior during meiotic prophase.

(A) Chromosome spread for leptotene, zygotene, pachytene, and diplotene spermatocytes.

Chromosome behavior is traced by immunostaining of a component (synaptonemal complex 3,

SYCP3) of the synaptonemal complex on chromosome spreads. (B) Synaptonemal complex is a multi-protein structure that forms between two homologous chromosomes during meiosis. It is a tripartite structure consisting of two parallel lateral regions and a central element, each with a group of protein components.

40 Figure 1-5. Graphical representation of the pituitary-hypogonadal axis.

41 Figure 1-5. Schematic representation of the pituitary-hypogonadal axis.

Both endocrine and paracrine signals orchestrate the process of spermatogenesis. Gonadotropin releasing hormone (GnRH), the master regulator that is secreted from the hypothalamus, stimulates the production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary gland. FSH acts on Sertoli cells, while LH acts on the Leydig cells.

Sertoli cell is the only cell type that expresses receptors for FSH. Signals from the gonadotropins exert directly on the Sertoli cells, which regulate spermatogenesis. Testosterone, inhibin and follistatin provide negative feedbacks, whereas activin provides positive feedbacks in the regulation of the axis.

42 Figure 1-6. Retinoic acid metabolism and mechanism of action of retinoid receptors.

43 Figure 1-6. Retinoic acid metabolism and mechanism of action of retinoid receptors.

Dietary vitamin A (retinol) exerts its biological effect via enzymes that convert the alcohol first to retinaldehyde by retinol dehydrogenase or alcohol dehydrogenase (RDH/ADH), and then to retinoic acid (RA) by retinaldehyde dehydrogenase (RALDH). Alternatively, retinol can be converted to retinyl ester for storage by lecithin: retinol acyltransferase or acyl-CoA: retinol acyltransferase (LRAT/ARAT). In the nucleus of the RA-target tissue, retinoid receptors (RARs and RXRs) can bind a regulatory DNA element (retinoic acid response element, RARE) in the presence of the ligand (RA), recruit co-activators (CoA) along with other transcriptional complexes and initiate the transcription of RA-regulated genes.

44 Figure 1-7. Representation of retinoid receptor protein expression in adults.

45 Figure 1-7. Representation of retinoid receptor protein expression in adults.

(A) The expression of all six retinoid receptors is indicated with the red color fillings for Sertoli

cell and germ cells. RARG and RXRB are only expressed in adult Sertoli cells, and not earlier.

(B) The biological effect of retinoic acid is mediated through RARA partnering with RXRG in germ cells, and RXRA in Sertoli cells (Dufour and Kim, 1999). (C) In adult mouse testes, RARA is localized in the nuclei of Sertoli cells, spermatogonia, preleptotene and pachytene spermatocytes, and round and elongating spermatids.

46 CHAPTER TWO

DISTINCT REQUIREMENT FOR RARA FUNCTION IN SPERMATOGONIAL

PROLIFERATION AND DIFFERENTIATION, AND DOUBLE STRAND BREAK

REPAIR

Sze Ming Law-Nagaokaa,d, Natalie R. Peera,e, Kylie J. Nelsona, Hui Lia, Derek McLeanb, Brenda

Murdocha, William D. Willisc, Eugenia H. Gouldingc, E. Mitch Eddyc, Kwan Hee Kima,f,g

aSchool of Molecular Biosciences, Center for Reproductive Biology, Washington State

University, Pullman, WA.

bDepartment of Animal Sciences, Center for Reproductive Biology, Washington State

University, Pullman, WA.

cLaboratory of Reproductive and Developmental Toxicology, Gamete Biology Section, National

Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC.

dSupported by NIH grant T32GM083864 (to S.M.L) from the National Institute of General

Medical Sciences (NIGMS).

eSupported by NIH grant T32GM008336 (to N.R.P) from the National Institute of General

Medical Sciences (NIGMS).

fSupported in part by NIH grant HD44569 (to K.H.K) from National Inistitute of Child Health

and Human Development (NICHD).

gCorrespondence: Kwan Hee Kim, School of Molecular Biosciences, 1715 NE S. Fairway Rd.,

Washington State University, Pullman, WA 99164. E-mail: [email protected]

Note: This work will yield a primary authorship publication. Manuscript is in preparation.

47 ABSTRACT

All-trans retinoic acid (RA), a biologically active form of dietary vitamin A (retinol), is

important for mammalian spermatogenesis. Retinoic acid receptor alpha (RARA) is a RA-

dependent transcription factor, mediating retinoid signaling, expressed both in Sertoli cells and

germ cells. RARA is crucial in the development of testicular germ cells. Rara-null mice are

characterized by high neonatal mortality, and adult male infertility due to severely degenerated

testes. The degenerated testes lack advanced germ cells, exhibit sloughing of spermatids, and

have vacuole formations in the seminiferous epithelium. To discern the functional role of this

transcription factor in germ cells, Rara was conditionally deleted (Rara cKO) specifically in

germ cells using stimulated by retinoic acid 8-codon improved Cre (STRA8-iCRE). STRA8-

iCRE was activated at postnatal day 1 (P1) in 100% of the EYFP reporter animals (n = 7). Rara mRNA was consistently reduced in enriched germ cells from Rara cKO mice at P4 and P8.

Morphological abnormalities of the Rara cKO testes were detected starting at P10 and P15, both with vacuole formations and disorganization of germ cells, which worsened as animals aged. At

P15, 20 and 30, there was more than a two-fold decrease in the most advanced germ cell types, and by P120, there was a 74% decrease in the epididymal sperm number. By P365, over 60% of the seminiferous tubules were abnormal in the Rara cKO testes. The abnormalities included germ cell disorganization, lack of lumen, large vacuole formation, and massive germ cell sloughing. Immunohistochemical investigation with antibodies to detect mitosis showed a decrease in the proliferation of germ cells at P4. An antibody against reproductive homeobox

gene 13 (RHOX13), a differentiating spermatogonial marker, showed a decrease in

differentiating B spermatogonia at P6. Analysis of pachytene spermatocyte chromosomal spreads

using antibodies to meiosis markers, synaptonemal complex protein 3 (SYCP3) and Phospho-

48 histone H2A.X (Ser139) (γH2AX), suggested that RARA has a pivotal role in the double strand break (DSB) repair pathway. In conclusion, RARA in male germ cells is required for maintaining the integrity of germ cell layers in the seminiferous epithelium, ensuring quantitatively normal spermatogonial proliferation and differentiation, and proper repair of DSBs during meiosis.

49 INTRODUCTION

Testicular germ cells are produced in the seminiferous tubules. Here, germ cells at

various developmental stages with so-called “nurse” Sertoli cells are arranged in concentric

layers. Over the course of germ cell development, Sertoli cells provide a supportive

microenvironment by the secretion of signaling molecules and nutritional factors, as well as by

establishing the three-dimensional architectural integrity required for germ cell development and

organization (Griswold, 1995). The production of germ cells, or spermatogenesis, in mammals is

comprised of three continuous phases in the testis: (1) the mitosis of spermatogonial stem cells

(SSCs) and undifferentiated and differentiating spermatogonia; (2) the meiotic divisions of

spermatocytes; and (3) the spermiogenesis for haploid spermatid maturation (Russell et al.,

1990a). SSCs and spermatogonia are positioned next to the basement membrane. As these germ

cells develop, they migrate toward the lumen of the seminiferous tubule. Elongated spermatids

are then released into the lumen of the seminiferous tubule and travel to the epididymis.

Functional sperm are produced as the sperm transits through the epididymis, where they are then

stored in the caudal epididymis.

Gonocytes or prospermatogonia in the center of the seminiferous cord migrate to the

basement membrane area and differentiate into spermatogonial stem cells (SSCs), which undergo

asymmetric mitotic division around postnatal day 3 (P3) (Sapsford, 1962; Oatley and Brinster,

2012). SSCs undergo mitosis to replenish the stem cell pool represented by the A single (As)

spermatogonia, and become A pair (Apr) and A aligned (Aal) spermatogonia. As, Apr, and Aal are considered the undifferentiated spermatogonia. Aal then continue their development into

differentiating spermatogonia, which include A1, A2, A3, A4, intermediate, and B spermatogonia, eventually expanding the number of spermatogonia 1000 fold. The type B spermatogonia divide

50 by the last round of mitosis to become preleptotene spermatocytes. Preleptotene spermatocytes

duplicate DNA and enter meiosis around P8, generating a series of primary spermatocytes.

Secondary spermatocytes and round spermatids can be seen around P20 and elongated

spermatids from the first wave of spermatogenesis are observed around P35 (Drumond et al.,

2011). To ensure non-stop production of gametes, a new wave of spermatogenesis begins every

8.6 days in mice (Russell et al., 1990b). Consequently, four to five layers of germ cells with

specific germ cell associations can be seen in a cross-section of adult testes. These associations

are divided into 12 time periods in mice, known as the 12 stages of the spermatogenic cycle

(Russell et al., 1990b).

Among many factors found to be essential in the process of spermatogenesis, dietary

retinol (vitamin A) or retinoids have been shown to be required for the healthy development of

spermatogenesis in rodents (Huang and Hembree, 1979; van Pelt and de Rooij, 1990a; Wolbach

and Howe, 1925). Vitamin A deficient (VAD) male mice and rats are infertile. Without vitamin

A in the daily diet, testes are devoid of most germ cells, except for the type A1 spermatogonia in

mice and a few additional preleptotene spermatocytes in rats (van Pelt and de Rooij, 1990b).

Retinoic acid, the acid form of vitamin A, is known to be the active signaling molecule among

retinoids and is known to affect the commitment step from Aal differentiation to A1 (van Pelt and de Rooij, 1990a; van Pelt and de Rooij, 1990b; Griswold et al., 1989; Zhou et al., 2008).

RA or RARA-specific agonist treatment of embryonic 14.5 rat ovary organ culture, accelerating meiotic entry implied the role of RA in meiosis (Livera et al., 2001). The ability of embryonic testis to enter meiosis after exogenous RA stimulation, as determined by meiosis markers expression and morphological examination pointed to the involvement of RA in meiotic entry regulation (Bowles et al., 2006; Koubova et al., 2006; MacLean et al., 2007; Trautmann et

51 al., 2008). Stimulated by retinoic acid gene 8 (Stra8) is indispensable for the entry and

progression of meiosis in the adult testis (Anderson et al., 2008; Mark et al., 2008), and thus has

been used as a marker for meiotic initiation. Combined with the observation that Stra8-null

embryonic ovaries cannot resume meiosis after exogenous RA treatment, RA has a established

role in regulating the entry of meiosis by inducing STRA8 expression (Koubova et al., 2006).

RA also plays a role at the transition from round spermatids to elongated spermatids (Huang and

Marshall, 1983; Akmal et al., 1997).

Retinoic acid signaling is mediated by two families of ligand-dependent transcription

factors: the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each with alpha,

beta and gamma subtypes (Chambon, 1996). Retinoid receptors are nuclear receptors, like the

estrogen and androgen receptors. One RAR binds one RXR to form a heterodimer in the nucleus.

In the presence of retinoic acid, the ligand for retinoid receptors, the heterodimeric receptor

complex recruits transcriptional co-activators and binds to a retinoic acid response element

(RARE), located upstream of a target gene, and regulates transcription (Bastien and Rochette-

Egly, 2004).

Rara-null male mice are infertile due to dramatic loss of germ cells (Chung et al., 2004;

Doyle et al., 2007; Lufkin et al., 1993). Further examination of the testes of Rara-null mice at

various stages of development has uncovered apoptosis of early meiotic germ cells in six-month-

old males (Doyle et al., 2007), as well as apoptosis of elongated spermatids in nine-week-old males (Chung et al., 2005). There were preleptotene spermatocytes arrested temporarily during three spermatogenic waves of spermatogenesis, increased number of degenerating pachytene spermatocytes compared to wild type mice during the first wave of spermatogenesis, spermatids temporarily arrested at steps 8 and 9 during the first wave of spermatogenesis, and a delay in the

52 onset of the second wave. The aforementioned phenotypes manifest as disorganization of germ

cell layers in the tubules, creating missing layers of germ cells or decreased numbers of predicted

cell types, causing a multi-level decrease in germ cell development (Chung et al., 2004; Doyle et

al., 2007). All the phenotypes point to the fact that RARA is required for meiosis and in germ

cell layer organization.

Cellular localization and developmental studies of Rara transcripts and RARA proteins in

the testis indicated that germ cells express the gene product starting from early postnatal ages, as

early as P0 and P3-5 in gonocytes (Akmal et al., 1997; Boulogne et al., 1999; Cupp et al., 1999;

Dufour and Kim, 1999), and highly express it in preleptotene and early meiotic spermatocytes at

P10 and P15 (Akmal et al., 1997). RA and other signaling molecules tightly regulate Rara

mRNA and proteins. As early as seven weeks during a VAD diet, Rara transcript and protein

levels declined to the low level observed in VAD rats (Akmal et al., 1998). Conversely, the level

of Rara mRNA in the VAD rats spiked within 30 minutes of retinol injection (Kim and

Griswold, 1990). In addition, the highest expression of Rara mRNA is between stage VIII and

IX in the stage-synchronized rats (Linder et al., 1991). The function of RARA protein is highly

regulated via nuclear localization and degradation levels. The protein is regulated by

posttranslational modifications acting directly on the receptor. RARA is modified by protein

kinase C (PKC), small ubiquitin-like modifier-2 (SUMO-2) and protein kinase A (PKA) on

specific sites of the receptor. These modifications regulate the nuclear localization and degradation of the receptor (Braun et al., 2002; Zhu et al., 2009; Santos and Kim, 2010). For example, PKA phosphorylates RARA on two of its PKA sites, leading to the inhibition of RARA nuclear localization (Santos and Kim, 2010). Additionally, disulfide isomerase glucose- regulated protein 58 (GRp58) chaperones RARA to the endoplasmic reticulum (ER) for

53 degradation by the ER-associated degradation (ERAD)-like system within two hours after RA

induction of RARA nuclear localization (Zhu et al., 2010).

Because of the pleiotropic expression of RARA in the seminiferous tubule, that both

Sertoli cells and germ cells express RARA, and the cross talk between germ cells and Sertoli

cells, it is necessary to tease out the gene function using cell type-specific conditional knockouts.

Vernet and colleagues have generated Sertoli cell-specific Rara knockout (Rara cKO) mice

(Vernet et al., 2006). In these Sertoli cell-specific Rara cKO, spermatogenesis was reduced in an

age-dependent manner. There was an increase in the number of apoptotic round spermatids in the

testes from nine-week-old mutants. In addition, there was a delay in the progression of the first wave of spermatogenesis, leading to decreased efficiency of spermatogenesis.

The phenotypes of germ-cell specific Rara cKO are of interest in the current study. Rara

was conditionally deleted in germ cells using STRA8-iCRE, which is expressed from Stra8-iCre.

Stra8-iCre has the 1.4 kb Stra8 promoter in front of a codon improved Cre recombinase (iCre)

sequence (Sadate-Ngatchou et al., 2008). We found that Rara in germ cells is critical for (1) the

mitosis of spermatogonia, as represented by decreased immunostaining with the mitotic markers,

phosphohistone H3 (pHH3) and 5-bromo-2’-deoxyuridine (BrdU), (2) the differentiation of

spermatogonia, indicated by decreased reproductive homeobox gene 13 (RHOX13) expression,

and (3) the meiotic process, specifically for the normal formation of the synaptonemal complex

and synapsis, and proper repair of double strand breaks (DSBs).

54 METHODS AND MATERIALS

Generation of the Raraflox/flox and Rara conditional knockout mouse strains

Rara was isolated from a mouse BAC clone (Invitrogen, Palo Alto, CA). Nucleotide numbers in the Rara genomic sequence are those from the Ensembl Gene ID

ENSMUSG00000037992. The genomic location is 98608221-98646663. The Rara DNA fragment containing exon 4 (nucleotides 98637229-98638979) was amplified by PCR using the mouse BAC clone 331 as a template, which was then ligated into the NheI site of pGkNeo(A) to produce the construct pGkE4Neo(A). The 3’-homologous region of Rara (nucleotides

98639103-98639132) was amplified by PCR using the mouse BAC clone 331 as a template and then ligated into the EcoRI site of pGkE4Neo(A) to produce pGk3’E4Neo(A). The 5’- homologous region of Rara (nucleotides 98634970-98634997) was amplified by PCR using the mouse BAC clone 331 as a template and then ligated into the BamHI and HindIII sites of pTK-

KS (+) (SE) to produce pGk3’E4Neo(A)5’. After digestion with BamHI and NotI, the DNA restriction fragment containing the 5’- homologous region of Rara along with thymidine kinase

(tk) was ligated into the NotI-BamHI site of pGk3’E4Neo(A)5’ to produce the Rara gene- targeting construct pGk3’E4Neo(A)5’TK. Consequently, exon 4 of Rara was placed in a sequence flanked by two loxP sites (Fig. 2-1 A) and the resulting floxed Rara construct was verified by DNA sequencing.

Recombinant ES cells containing floxed Rara construct were injected into blastocysts, which were transferred into pseudopregnant females. After selection, backcrossing and genotyping, a floxed Rara mouse in C57BL/6 genetic background was generated. Exon 4 (within the B domain) is common to the two main Rara isoforms. Thus, the excision of exon 4 by CRE

55 recombinase, which creates an in-frame deletion of 3489 base pairs, is predicted to disrupt

RARA function. A floxed Rara mouse was mated to a CMV-Cre mouse to verify the deletion.

Floxed Rara females (C57BL/6 genetic background) were mated to Stra8-iCre males

(FVB genetic background) (Jackson Laboratories, Jax stock #008208) to generate F1 generation

heterozygotes, Raraflox/+; cre+ (Fig. 2-1 C). Between the ages of P45 and P60, the F1 males were bred to homozygous floxed Rara females to generate F2 pups. In the F2 generation, only pups with flox/-; cre+ tail genotype were used for conditional Rara knockout sample collection.

Always using males carrying the iCre transgene and mating to flox female mice for two generations is necessary to ensure the efficient and clean deletion of Rara. Wild type control animals were generated in a similar fashion with the following exception: homozygous flox females were substituted with C57BL/6J (Rara+/+; cre-) females, so that wild type has the same genetic composition as the cKO mice, but has wild type Rara genes. Care and maintenance of animals was carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee of Washington State University, following the NIH guidelines.

EYFP reporter animals

B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J ((enhanced yellow fluorescent protein (EYFP) reporter) females (Jackson Laboratories, Jax stock #006148) were mated to Stra8-iCre males to generate Stra8-iCre-Eyfp F1 pups. Within the Eyfp reporter gene, there is a stop codon flanked by two loxP sites (Fig. 2-2). The stop codon is deleted when STRA8-iCRE recombines the two loxP sites, generating Eyfp; Stra8-iCre reporter F1 mice. Fresh tubules were examined for EYFP protein expression. Testes cross-sections from P0, P1, P3, P4, and P5 were immunostained for

GFP (Abcam, Cambridge, MA, cat # ab290) for the report of STRA8-iCRE activity. Then, by

56 mating the F1 male pups back to wild type females to generate Stra8-iCre-Eyfp F2 pups, the recombination efficiency of STRA8-iCRE was determined. Recombination efficiency (or penetrance) was calculated by the number of F2 generation pups, which express EYFP protein, divided by the number of pups that inherited the Eyfp (Fig. 2-2).

Genotyping

Tail snips of 0.5 cm were cut and digested using Viagen Direct Lysis Reagent (Ear or

Tail) (Viagen, Los Angeles, CA) with 0.4 mg/ml Proteinase K (Invitrogen, Carlsbad, CA) at

55°C on a heat block, until no visible tissue is left (about 4~6 hrs). After the digestion, Proteinase

K was deactivated at 85°C for 45 min. Crude lysate of 0.5~1 µl was then used directly for PCR reactions. Typical PCR reaction is composed of 1X GoTaq PCR buffer (Promega, Fitchburg,

WI), 0.25 mM dNTP, 0.5 µM of forward and reverse primer, and 0.25 U of GoTag (Promega

Corp., Madison, WI, cat # M300B) in a 50 µl reaction mix. All PCRs were run using the following program: 1 cycle at 94°C for 2 min; 35 cycles at 94°C for 30 sec, 60°C for 30 sec and

72°C for 1 min; and 1 cycle at 72°C for 7 min; hold temperature at 22°C. Primers for genotyping the parental, F1 and F2 animals are listed in Table 2-1. Genotyping was repeated for confirmation using a second pair of nested primers for each allele. W and W1 were used to detect the presence of the Rara wild type allele, F and F1 were used to detect the presence of the flox

Rara allele, Δ and Δ2 were used to detect the presence of Rara allele deletion, and M1 and M3 were used to detect the presence of Stra8-iCre transgene (Table 2-1).

57 Testicular tissue collection and determination of testicular abnormalities

Male mice were humanely euthanized at P1, P4, P5, P6, P8, P10, P11, P13, P15, P20,

P25, P30, P45, P61, P75, P120, P180, and P365. Testes were collected, fixed in Bouin’s solution for 30 min to 6 hrs, dependent on the size of the testis, and embedded in paraffin. Serial sections of four-µm thickness were cut and mounted on poly-L-lysine slides. To examine testicular morphology, sections were deparaffinized and hydrated through xylene and graded ethanol series, stained with hematoxylin and eosin, and examined using a Leitz DMRB microscope

(Leica, Wetzlar, Germany). Digital images were obtained using a Leica DFC 310 FX digital camera (Leica). To determine the percentage of abnormal tubules present in the testes, the number of abnormal tubules with vacuoles, sloughing of germ cells, lack of lumen, or disorganization of germ cell layers were counted in testicular sections, divided by the total number of tubules, and multiplied by 100. To obtain comprehensive sampling across the entire testis, a minimum of three sections spaced 50 µm apart were analyzed for each animal. This was repeated for three to six animals (n = 3-6).

Sperm count and fertility analysis

Sperm were collected from the caudal epididymis of mice at P75, P120, and P180. The excised epididymides were cut into five pieces, placed in 1X PBS, pre-warmed to 37°C for 5 min, and pipetted up and down using a P-1000 pipettor with large orifice tips to facilitate the sperm release. Fresh sperm were placed onto a hemacytometer and counted under an inverted phase-contrast Olympus IMT-2 inverted microscope (Olympus, Center Valley, PA). Each chamber was counted twice and averaged. Sperm clumps and free tails were not counted. The sperm counts were repeated for six to eleven animals (n = 6-11).

58 For fertility analysis, males at P75, P120 and P180 were mated to C57BL/6 wild type females between P58-78. The pairs were kept together for one week, and vaginal plug was confirmed before separation. Number and sex of live pups at P0 were recorded for multiple animals (n = 5-8). More animals are required to be tested for the fertility analyses.

Detection of mitosis (proliferation) by phosphohistone H3 (pHH3) immunofluorescence and

BrdU assay

Testicular sections from P4, P6, P8, P10, and P15 (n ≥ 3) were fixed in Bouin’s solution for one to two hours. After paraffin embedding, serial sections of four-µm thickness were cut and mounted on poly-L-lysine slides. After deparaffinization and hydration, tissue sections were microwaved with 0.01 M sodium citrate solution, permeabilized with 0.2% Triton X-100 in PBS for 45 min, blocked with 10% goat serum, and incubated overnight with rabbit polyclonal anti- pHH3 antibody at 1:1500 dilution (Millipore, Billerica, MA, cat # 06-570) to detect cells at mitotic prophase in the testis from animals at P4, P6, P8, P10, and P15. The slides were further processed by washing in PBS, incubated with biotinylated anti-rabbit IgG at 1:300 dilution

(Vector Laboratories, Burlingame, CA, cat # BA-1000), and then incubated with streptavidin conjugated to Alexafluor® 555 at 1:500 dilution (Life Technologies, Carlsbad, CA, cat #

S21381) to detect the primary antibody. 4’,6-diamidino-2-phenylindole (DAPI) at 1:10,000 dilution (Life Technologies, D1306) was used to counterstain the DNA. Sections were mounted with VectaShield mounting reagent (Vector Laboratories, cat # H-1000) and examined by fluorescent microscopy (Leitz DMRB microscope, Leica, Wetzlar, Germany) with pictures taken by Leica DFC 210 FX digital camera (Leica). Tubules with brightly stained cells undergoing mitotic prophase were counted as well as the total tubules in a given area to determine the

59 percentage of tubules with mitotic cells for at least three animals (n = 3). Three to 18 areas were counted, depending on the size of tissue.

5-bromo-2’-deoxyuridine and 5-fluoro-2’-deoxyuridine (BrdU) labeling reagent at 3 mg/ml in PBS (Life Technologies, Cat # 00-0103) was delivered to animals at P10 and P15 by intraperitoneal injections at a dose of 1 ml per 100 grams of body weight. Animals were killed 4 hours after the labeling procedure and both testes were surgically removed. One testis was fixed in Bouin’s solution and the other fixed in 4% paraformaldehyde mixed with 0.25% gutaraldehyde. Tissue was then embedded in paraffin, cut to four-µm thick sections and adhered to poly-L-lysine slides. After deparaffinization and hydration, tissue sections were microwaved with 0.01 M sodium citrate solution and stained with biotinylated anti-BrdU antibody following the manufacturer’s protocol (Life Technologies, cat # 93-3943). Sections were examined using a

Leitz DMRB microscope (Leica) and digital images were obtained using a Leica DFC 310 FX digital camera (Leica). The proliferation rate was determined by the number of BrdU-positive cells per round tubule for at least three animals (n = 3). 11-59 tubules were counted, depending on the size of tissue.

Detection of apoptosis by Terminal Deoxynucleotidyl transferase dUTP nick end labeling

(TUNEL) assay

Testicular sections from P10, P13, P15, P19, P25 and P75 (n = 3; except P25; n = 2) were fixed in 4% paraformaldehyde with 0.25% glutaraldehyde (GA). After paraffin embedding, serial sections of four-µm-thickness were cut and mounted on poly-L-lysine slides. To perform the

TUNEL assay, sections were deparaffinized and hydrated through xylene and graded ethanol series, and processed for the detection of apoptotic cells by TUNEL assay according to the

60 manufacturer’s instructions (Promega Corp., cat # G3250). Terminal deoxynucleotidyl transferase (TdT) mediates incorporation of fluorescein 12-dUTP at the 3’ hydroxyl end of fragmented DNA found in apoptotic cells. For a positive control, tissue sections were pretreated with deoxyribonuclease I. For a negative control, tissue sections were not incubated with TdT.

Sections were examined using a Leitz DMRB microscope (Leica) and digital images were obtained by using a Leica DFC 310 FX digital camera (Leica). Tubules with three or more apoptotic cells (representing apoptotic tubules) as well as the total number of tubules were scored for quantification.

Antibodies, immunohistochemistry, and immunofluorescence

For immunohistochemistry, antigens on deparaffinized and hydrated sections were heat- retrieved using 0.01 M sodium citrate solution. Tissue sections were blocked with 10% goat serum. Sections were then incubated overnight with rabbit anti-DDX4 antibody (1:500, Abcam, cat # ab13840), rabbit anti-STRA8 full-length peptide antibody (1:2500 dilution; gift from Dr.

Michael Griswold), or rabbit anti-RHOX13 peptide antibody (1:1200 dilution; gift from Dr.

Christopher Geyer). Sections were then washed in PBS, and incubated with biotinylated anti- rabbit IgG (1:300 dilution; Vector Laboratories, cat # BA-1000), followed by streptavidin- peroxidase (Vector Laboratories, cat # SA-5704) reaction and incubation with 3,3’- diaminobenzidine tetrahydrochloride (DAB) (Life Technologies, cat # 002020), and counter- stained with hematoxylin. For immunofluorescence, after microwaving with 0.01 M sodium citrate solution, the tissue sections were further permeabilized with 0.2% Triton X-100 in PBS for 45 min. Tissue sections were blocked with 10% goat serum, incubated overnight with rabbit polyclonal anti-DMRT1 antibody (1:400 dilution; gift from Dr. David Zarkower), rabbit

61 polyclonal anti-DDX4 antibody, or rabbit polyclonal anti-pHH3 antibody. Following PBS wash, tissue sections were either incubated with biotinylated anti-rabbit IgG (1:300 dilution; Vector

Laboratories), followed by streptavidin conjugated to Alexafluor-555 (1:500 dilution; Life

Technologies), or followed by anti-rabbit IgG conjugated to Alexafluor-555 secondary antibody

(1:1000 dilution; Life Technologies). The mouse monoclonal anti-γH2AX peptide antibody

(1:750 dilution; Millipore Corporation, cat # 05-636) incubation was followed by the use of

Mouse on Mouse Basic Kit following the manufacturor’s instruction (Vector Laboratories, cat #

BMK-2202). Tissue sections were labeled with 4’,6-diamidino-2-phenylindole (DAPI at

1:10,000 dilution; Life Technologies). Coverslips were mounted with VectaShield reagent

(Vector Laboratories). Sections were examined by fluorescence microscopy (Leitz DMRB,

Leica).

Pachytene chromosome-spread

Percentage of defective synaptonemal complexes was scored for pachytene meiotic spreads from mice at P15 using anti-SYCP3 (Santa Cruz Biotechnology, cat # sc-33874) and anti-γH2AX antibodies (Millipore cat # 07-164). Anti-SYCP3 immnostains the axial elements of the synaptonemal complex (SC), and anti-γH2AX marks DSB sites. The defects scored are SC fragmentation, autosomal γH2AX foci, partial asynapsis, forks, gaps and pair association. A pachytene chromosomal spread is considered to have a perfect synapsis if there are no defects such as forks/bubbles/gaps, asynapsis, partial asynapsis, SC association, SC fragmentation, non- homologous pairing, and synaptic failure. If there are more than three defect categories in the same cell, then it is possible the cell is 1) not at the right stage or 2) there are other technical

62 issues that might have caused the defects. Then, this cell is not scored. Total of 150 cells were

score for WT and 150 for cKO (n = 3).

Spermatogonial transplantation

Testes from Rara wild type (P8 and P16) and cKO male mice (P8, P11 amd P25) in the ¾

C57BL/6 and ¼ FVB genetic background provided the donor germ cells for spermatogonial

transplantation. The testes were detunicated and interstitial cells were first digested with 0.01 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO, cat # C2014). Isolated tubules were then further digested with 0.05% trypsin (Life Technologies, cat # 15090-046), 1 mg/ml collagenase,

2 µg/ml DNase I (Sigma, DN25), and 1.5 mg/ml hyaluronidase (Sigma, cat # H3757) to remove basement membranes and Sertoli cells. Single-cell suspensions of enriched germ cells were

obtained. Total germ cell number was determined using a hemacytometer under an inverted

phase-contrast Olympus IMT-2 inverted microscope (Olympus). Germ cell preparation will be

analyzed to determine the percent of cells that are germ cells. Enriched germ cells were then

resuspended at a concentration of 107 cells/ml in minimal essential media α with 0.03% trypan

blue dye (Life Technologies). Germ cells from one donor animal were transplanted into at least

two busulfan-treated ROSA26 recipients. One testis of the recipient was used for transplantation

and the contralateral testis from the same recipient animal served as a sham testis. A micro-glass

needle containing 7 µl (70,000 cells) of the donor germ cell suspension was inserted into a small

incision site close to the efferent ducts, connecting to the rete testes, where donor germ cells

enter the seminiferous tubules. After the surgery, the animals were allowed to recover for 70

days, by which time, all stages of germ cells including meiotic and post-meiotic germ cells are

63 represented in the recipient tubules injected with control stem cells from the wild type animals.

This procedure was repeated for at least three donor animals (n = 3).

Post-transplantation recovery analysis

The recipient testicular tissues were detunicated and fixed in 4% paraformaldehyde solution (pH 7.2) for one hour and stained with 1 mg/ml X-gal to detect the expression of beta- galactosidase before embedding in paraffin. After paraffin embedding, four-µm thick sections were cut and mounted on poly-L-lysine slides. To determine the donor-derived germ cell recovery, ten sections sliced 50 µm apart per testis from at least three animals were counter- stained with nuclear fast red to stain DNA. The sections were examined using a Leitz DMRB microscope and digital images were obtained using a Leica DFC 310 FX digital camera. Blue colored cells are endogenous germ cells and somatic cells, whereas the pink colored cells are the donor-derived germ cells and some endogenous somatic cells at the basement membrane and interstitial space. The tubules containing two or more layers of donor-derived pink-colored germ cells (round or elongated spermatids) were counted to quantify the donor-derived spermatogenesis. To further characterize the germ cell types present in the recovered tubules, immunohistochemistry or immunofluorescence was performed using antibodies for DDX4,

γH2AX, STRA8, and DMRT1.

Real-time RT PCR

Real time PCR was performed using forward and reverse primers flanking the floxed region of Rara to detect the presence of Rara mRNA in enriched germ cells from animals at P4 and P8 (n = 3, each with a technical duplicate). Real-time PCR primers were designed using

64 Primer Express version 2.0 (Applied Biosystems Technology, Foster City, CA). Forward primer for the detection of Rara mRNA was 5’ GAC AAA TCA TCC GGC TAC CAC TAT 3’,

Reverse primer was 5’ TTG TTT CGA TCG TTT CGC AC 3’. Briefly, cDNA was synthesized from 500 ng of RNA using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) and used as the template for real time PCR assay with a 7500 Fast real time PCR system

(Applied Biosystems Technology). Typically, a 25-µl reaction mixture contained 12.5 µl of 2X

Power SYBR Green PCR Master Mix (Applied Biosystems Technology), 500 nM of forward and reverse primers, 5 µl of diluted cDNA (1:20 dilution), and distilled water. The PCR cycles are as follows: 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, and 40 cycles at 95°C for

15 sec and 60°C for 1 min. The expression level of Rara mRNA was evaluated using the 2-(ΔΔCt) method (Schmittgen et al., 2008). Cycle threshold values (Ct) for Rara and the ribosomal S2 protein (Rps2) or glyceraldehyde-3-phophate dehydrogenase (Gapdh) gene, two housekeeping genes, were determined using Prism SDS version 1.1 software (Applied Biosystems

Technology). Ct values for Rara were normalized to those of either Rps2 or Gapdh values in each sample, and the fold change for Rara was calculated relative to the level in the reference sample. Examination of the dissociation curve indicated that there was only a single PCR product for each primer set. Amplification plots were logarithmic and similar to each other, indicating that PCR efficiencies were equivalent for primer sets and close to 1.

Statistical significance

Statistical significance was determined by one-way ANOVA, followed by pairwise comparisons of the means using the Student’s t method from JMP, version 10 software (SAS

Institute Inc., Cary, NC). The p ≤ 0.05 was considered significant.

65 RESULTS

Germ cell-specific deletion of RARA protein

To investigate the role of Rara within the postnatal male germ cell lineage, a transgenic mouse was generated bearing a modified Rara gene. A common region for the two main isoforms of Rara, exon 4, was engineered between two loxP sites (Fig. 2-1 A). Exon 4 is located within the B region that is part of the ligand-independent transcriptional activation function-1

(AF-1) domain (Rochette-Egly et al., 2000) of mouse Rara gene (Fig. 2-1 B). It binds cell- specific factors or co-activators. The transcriptional activity of RARs depends on the activation domains, AF-1 and AF-2 (Bour et al., 2005) (Fig. 2-1 B). Excision of the exon 4 between two loxP sites by CRE recombinase creates an in-frame deletion, and renders the transcriptional activity of RARA non-functional. By mating with transgenic mice bearing the Stra8-iCre transgene (Sadate-Ngatchou et al., 2008), exon 4 is deleted in both alleles after two generations of paternal mating (Fig. 2-1 C).

In the current study, by mating Stra8-iCre male to EYFP reporter female (Fig. 2-2 A), it was determined that STRA8-iCRE may function as early as P1 in a subset of gonocytes (Fig. 2-2

D) in seven out of seven transgenic mice (100%) as well as in 100% of animals tested at P2, P3,

P4, and P5 (Fig. 2-2 H). Moreover, the recombination efficiency (or penetrance) of CRE recombinase was 95.5%, calculated by the number F2 generation pups that express EYFP protein

(n = 21), divided by the number of pups with Eyfp allele (n = 22) (Fig. 2-2 B).

Consistently, the level of Rara mRNA from enriched germ cells in the Rara cKO testes from P4 (p =0.0002) and P8 (p = 0.0076) mice was reduced significantly (n = 3), as detected by real time RT-PCR (Fig. 2-2 I). The enriched germ cells will be characterized to determine the

66 percent contamination of cells. Alternatively, deletion of the Rara exon 4 will be quantitated in epididymal sperm DNA, to determine the percent of sperm with RARA function abolished.

Lack of functional RARA leads to increased abnormal testicular tubules throughout development

We investigated the testicular morphology throughout development in the germ cell- specific Rara cKO strain. Testicular abnormalities started to appear at P10 (Fig. 2-3 J) and P15

(Fig. 2-3 L), and became more severe as animals aged. The difference in the percent of abnormal tubules between the wild type and cKO mice was statistically significant starting at P25 (Fig. 2-3

N, Fig. 2-4). The abnormalities included lack of germ cell organization, various-sized vacuoles, lack of lumen in adults, and massive sloughing of germ cells (Fig. 2-3). There were, on average, two to five times more abnormalities in the cKO testes than their wild type counterparts (Fig. 2-

4), and as high as 60% in P365 cKO mice (Fig. 2-3 X, Fig. 2-4), suggesting malfunctioning of spermatogenesis in the cKO mice and worsening with advancing ages.

Reduced number of tubules with the most advanced germ cell type

Testicular cross-sections of wild type and cKO mice were scored for the relative number of most advanced germ cell types at P15, P20, and P30, when large pachytene spermatocytes

(Fig. 2-5 A and B), round spermatids (Fig. 2-5 C and D), and elongated spermatids (Fig. 2-5 E and F) are the most advanced germ cell types, respectively. In the testes of P15 animals, 39.8%

(± 3.6%) of the round tubules contained large pachytene spermatocytes in the cKO testes (Fig. 2-

5 B), compared to 70.8% (± 1.7%) in the wild type testes (Fig. 2-5 G, n = 3, p = 0.0001). The decline in the most advanced germ cell types is persistent at P20 and P30, where round spermatids and step 8-10 elongating spermatids were the most advanced cell types, respectively

67 (Fig. 2-5 C-F, n = 3, p = 0.0001 and 0.0006). These quantifiable abnormalities and the reduction

in spermatogenesis suggest that RARA maintains efficiency of spermatogenesis in the first wave

of spermatogenesis.

Lack of RARA leads to decreased sperm count at P75 and P120 and a decreased fertility at

P120

With the aforementioned abnormalities, it was not surprising that Rara cKO males at P75

and P120 had a 42% and 74% decrease in epididymal sperm count, respectively (Fig. 2-6 A n =

6~11, p = 0.0051 and 0.0002), and an increased presence of round spermatids in the epididymis

at P75 and P120 (data not shown). Although the animals are fertile at P75 and P120 (Fig. 2-6 B,

n = 5~8), the number of pups per litter was reduced for P120 cKO males mating with wild type

females. More replicate fertility tests will be conducted with males at P120 and males at P180

(they are ageing currently) to determine if there is an age-dependent fertility decline.

Lack of RARA protein in germ cells leads to a decrease in mitosis of spermatogonia

Starting around P3 during neonatal development, SSCs undergo asymmetric mitotic

divisions, accomplishing self-renewal of SSCs and producing undifferentiated spermatogonia.

Then, undifferentiated spermatogonia and the next advanced germ cell class, differentiating

spermatogonia, undergo mitosis to amplify the spermatogonial population by about 1000 fold.

One hallmark of mitosis is the phosphorylation of histone H3 at serine 10, which can be detected

by anti-phosphohistone H3 (pHH3) antibody. To investigate if RARA in germ cells has a role in

mitosis during spermatogonial proliferation and differentiation, pHH3 antibody was used to

detect mitotic cells (Fig. 2-7 A-H). There were 3.4 (± 0.5) pHH3-positive cells per 18.8 µm2 in the Rara cKO testis cross-sections compared to 9 (± 1.8) pHH3-positive cells per 18.8 µm2 in the

68 wild type at P4. This was a significant decrease in mitosis of spermatogonia at p = 0.0008 (Fig.

2-7 M, n = 3). The decrease in pHH3 staining persisted at P6 (cKO has 4.5 ± 0.6 cells per 18.8

µm2 compared to the wild type which has 7.4 ±0.8 cells per 18.8 µm2, n = 3, p = 0.0664) and at

P8 (cKO has 8.9 ± 0.9 cells per 18.8 µm2, compared to the wild type which has 11.0 ± 1.3 cells per 18.8 µm2, n = 3, p = 0.0024) (Fig. 2-7 M).

Additionally, 5-bromo-2’-deoxyuridine (BrdU) was injected into the wild type and Rara

cKO animals intraperitoneally at P10 and P15 to trace dividing cells that take up this synthetic

thymidine analog during DNA replication. Immunohistochemistry with anti-BrdU antibody was

used to detect the incorporation of the analogs (Fig. 2-7 I-L). There were 15.5 (± 1.4) BrdU-

positive cells per round tubule in the cKO testes compared to 28.0 (± 2.5) BrdU-positive cells per

round tubule in the wild type at P10 (n = 3, p = 0.11). There were 10.9 (± 0.3) BrdU-positive

cells per round tubule in the cKO testes, and 38.1 (± 16.1) BrdU-positive cells per round tubule

in the wild type testes at P15 (Fig. 2-7 N, n = 3, p = 0.11).

Lack of RARA leads to a decrease in the type B spermatogonia, meiotic germ cells, and

accumulation of undifferentiated spermatogonia

To determine the relative number of differentiating spermatogonia, we used a protein

marker, reproductive homeobox gene 13 (RHOX13) that is expressed in the intermediate and

type B spermatogonia. There was a greater percentage of tubule cross-sections with one or more

layers of germ cells staining brightly with the antibody against RHOX13 in the wild type (36.9%

± 5.0%) compared to the cKO (18.3% ± 3.2%) at P6 (Fig. 2-8 A, B and E) (n = 3, p = 0.0106),

and in the wild type (10.5% ± 3.1%) compared to the cKO (2.8% ± 1.2%) at P8 (Fig. 2-8 C, D

and E) (n = 3, p = 0.0475). These data suggest a decrease in the number of RHOX13-expressing

69 differentiated spermatogonial population due to either the reduction in mitosis of spermatogonia, or delay in RHOX13 expression.

To determine whether differentiated spermatogonia enter into meiosis normally, we used an antibody against phosphor-histone H2AX (Ser139) (γH2AX) to detect leptotene and zygotene spermatocytes. Similar to the delay observed for entering into a differentiating state for spermatogonia in cKO testes, this inefficiency persisted around P11, with fewer percentages of tubules brightly stained with γH2AX in cKO testes (compared Fig. 2-9 D to C, Fig. 2-9 E) (n = 3, p = 0.0001). This indicates a reduction into meiotic entry for leptotene and zygotene spermatocytes.

Furthermore, to determine whether the spermatogonia that failed to differentiate remained undifferentiated, immunostaining for the DM domain proteins double sex- and mab-3- related transcription factor 1 (DMRT1) was incubated with testicular sections from animals at

P10 (Fig. 2-10). DMRT1 has diverse and essential roles in the development of the mouse testis

(Raymond et al., 2000) and expresses highly in undifferentiated spermatogonia and at a lower level in differentiating spermatogonia and Sertoli cells (Matson et al., 2010). There were an increased number of DMRT-1-positive, brightly immunostained undifferentiated spermatogonia per tubule in the cKO (13.1 ± 1.6) compared to the WT (8.0 ± 0.4) at P10 (Compare Fig. 2-10 B to A, Fig. 2-10 C) (n = 3, p =0.0352). These results indicate an accumulation of undifferentiated spermatogonia at P10, confirming blocked spermatogonial differentiation regulated by RARA in germ cells. This is consistent with inefficiencies in progressing through the differentiation phase of spermatogonia and entry into meiosis.

70 No differences in apoptosis

To determine if this reduction in germ cell numbers was due to apoptosis, we performed

TUNEL assays for the testicular sections from mice at prepubertal ages (P10, P15, P19, or P25)

(Fig. 2-11). An increase in apoptosis in testes of both the cKO and the wild type was seen at P15, but there were no differences in apoptosis in the testicular sections between the wild type and cKO mice. Preliminary analysis of testes from P75 male mice showed no increase in apoptosis during the steady state of spermatogenesis in adults (n = 3) (data not shown). Further examination of apoptosis on more testicular sections at neonatal ages before P10 and at P120 and

P180 are necessary.

Retained unrepaired double strand breaks in Rara cKO pachytene spermatocytes

To understand how RARA is important during meiosis, the phenotypes of meiotic spermatocytes were examined using chromosome-spread techniques. During meiotic prophase, four phases of chromosomal rearrangement in leptotene, zygotene, pachytene, and diplotene spermatocytes orchestrate the step-wise and organized genetic recombination, chromosome pairing and synapsis (Moses, 1968). At the leptotene stage, chromosomes condense, but remain unpaired. Chromosomes start to pair with their homologs in zygotene spermatocytes through the formation of synaptonemal complexes, a multi-protein structure that holds homologous chromosomes together during meiotic prophase. In pachytene spermatocytes, chromosome spread shows the presence of 19 completely synapsed autosomal synaptonemal complexes and the partially synapsed pseudo-autosomal region (PAR) of the X and Y chromosome.

Synaptonemal complexes dissipate and chromosomes separate from their partners at the diplotene stage. Synaptonemal complex 3 (SYCP3) is a lateral element of synaptonemal

71 complexes, which was used to identify the synaptonemal complexes in this study. Meiotic recombination is initiated with the generation of programmed DNA double-strand breaks (DSBs) by endonuclease sporulation-specific 11 (SPO11) in leptotene spermatocytes. Phosphorylation of the γH2AX marks the DSB sites. By the pachytene spermatocyte stage, γH2AX DSB signal disappears from the fully paired autosomes and uniquely marks the sex chromosomes

(Mahadevaiah et al., 2001; Blanco-Rodriguez, 2012).

Compared to wild type testes at P15, there was less perfectly synapsed DNA in pachytene chromosomal spreads, indicated by SYCP3 staining, and more fragmented synaptonemal complexes detected in cKO testes (Fig. 2-12 A versus B (Circled), and C). Additionally, 28% of the Rara cKO pachytene chromosome-spreads retained autosomal γH2AX signals in the pachytene chromosome-spreads suggesting the presence of unrepaired DSBs in pachytene chromosomes (Fig. 2-12 B (Arrow) and C). Thus RARA appears to play a role in supporting precise synaptonemal complex formation, less damage to chromosomes, and resolution of double strand breaks during meiosis.

RARA protein in germ cells is responsible for the colonization, initial proliferation and differentiation of spermatogonia

To determine the quality of spermatogonial stem cells (SCCs), we performed spermatogonial stem cell transplantation. Previously, it has been shown that stem cells from

Rara-null male mice at 6 months of age did not colonize the germ cell-depleted W/Wv recipient testes (Doyle et al., 2007). This previous study suggested that the role of RARA in stem cells is to control spermatogonial colonization, proliferation and differentiation. Because of the cross talk between Sertoli cells and germ cells (Griswold, 1995), spermatogonial stem cells from Rara-

72 null mice could be different from spermatogonial stem cells from Rara cKO mice. In Rara-null mice, Sertoli cells have the mutant Rara alleles, whereas in germ cell-specific Rara cKO mice,

Sertoli cells have the wild type Rara alleles. Germ cells from Rara cKO male mice were obtained by enzymatic digestion, as described in Materials and Methods (Fig. 2-13 A).

Three donors’ testes from animals in ¾ C57BL/6 and ¼ FVB genetic background (wild type donors are P8 and P16, cKO donors were P8, P11 and P25) were used to isolate spermatogonial stem cells and 70,000 cells were injected into each recipient testis that were chemically-treated (busulfan) to remove endogenous germ cells (n = 2 for wild type and n = 3 for cKO). After 70 days post-transplantation, recipient testes were collected, fixed and stained for beta-galactosidase expression using X-gal. Cross-sections of the recipient testes were counter- stained with nuclear fast red to visualize donor-derived spermatogenesis, which does not have beta-galactosidase expression (Fig. 2-13 B-E). Endogenous germ cells regenerated appear blue, whereas the donor-derived germ cells are pink. The entire testis was examined for colonization

(6-10 cross-sections, 50 µm apart). On average, there were 7.8% ± 1.1% tubules with donor- derived colonization by wild type germ cells, while only 4.1% ±1.6% tubules with donor-derived colonization by cKO germ cells (p = 0.07). When the ages of the donor animals were taken into account, there seemed to be an age-effect on the number or the efficiency of stem cell colonization for cKO animals (Fig. 2-13 F). Additional experiments are required to address this issue further. Nonetheless, it is clear that there is a reduction in the competence of Rara cKO spermatogonial stem cells. They have either lost stem cell activity in colonization, proliferation and differentiation or alternatively, they have lost the ability to maintain stem cell number as animals age.

73 To determine if donor stem cells can indeed colonize but fail to proliferate and

differentiate, cross-sections of recipient testes will be immunostained for DMRT1 to determine

the number of brightly staining undifferentiated spermatogonia. These are on-going experiments.

DISCUSSION

Ablation of Rara specifically in germ cells allowed for the uncoupling of its regulatory

activity from the similar activities in the somatic cell environment. By mating Stra8-iCre

transgenic mice to EYFP reporter mice, iCRE recombinase was shown to be active as early as P1

(Fig. 2-2 D). Additionally, the iCRE recombination efficiency (penetrance) was 95.5%, similar

to a previous report of 97.7% recombination efficiency when Tg(ACTB-Bgeo/GFP)21Lbe

reporter animals were used (Sadate-Ngatchout et al., 2008). These data indicate that Rara may be

excised in germ cells of Rara cKO mice as early as P1 using STRA8-iCRE with a high

penetrance. If this occurs, Rara would be excised in mitotically quiescent gonocytes present in

the center of seminiferous cord at P1 (Drumond et al, 2011). Excision observed in our

experiment occurred earlier than expected according to the literature. We make sure to always

use male mice bearing the Stra8-iCre transgene. Previously, Stra8-iCre mRNA transcripts were

shown to be expressed in the testis at P3 (Sadate-Ngatchou et al., 2008), with the expectation that

the excision would occur later when CRE proteins are expressed. In two other studies, the

excision was shown to be active at P3 and P4. The iCRE activity was detected in the testes from

P3 or P4 mice when Rosa26mTmG male mice were bred to female Stra8-iCre-Dicerlox/lox and

Stra8-iCre-Droshalox/lox, and when Rosa26mTmG female mice were bred to male Stra8-iCre mice (Wu et al., 2012; Bao et al., 2013). If excision occurs at P3, Rara would be deleted in gonocytes that are migrating to the basement membranes to form spermatogonial stem cells and

74 resuming mitosis. In either situation, Rara was being excised fairly early in the germ cell development, in gonocytes, by STRA8-iCRE.

The removal of Rara by STRA8-iCRE revealed a quantifiable reduction in spermatogenesis throughout germ cell development (Fig. 2-3, 2-4, 2-5) and reduced fertility as animals aged (Fig. 2-6 B). Multiple testicular abnormalities were revealed around P10 and P15

(Fig. 2-3 and 2-4) and they became more severe as animals aged (P20-P365). Generally, the lack of RARA function in germ cells resulted in germ cell disorganization, as results of germ cell sloughing, with whole layers of germ cells being sloughed occasionally, blocking the lumen, showing varying sizes of vacuoles to the loss of single or multiple layers of germ cells in the seminiferous epithelium.

There was also a significant decline in the numbers of the most advanced germ cell types at P15, 20, and P30 (large pachytene spermatocytes at P15, round spermatids at P20, and elongated spermatids at P30) during the first wave of spermatogenesis (Fig. 2-5). By scoring for the most advanced germ cell types, we can avoid the dilution effect from germ cells in the subsequent developmental stages. Implication was that lack of RARA might cause a delay in spermatogenesis. At adult stages, analysis of sperm counts revealed age-dependent decreases of

42% and 74% in epididymal sperm number in the cKO mice at P75 and P120 compared to the wild type mice, respectively (Fig. 2-6 A). Fertility at P120 was lower in the cKO mice than in the wild type mice. Thus, the morphological data, altogether with sperm count and fertility tests showed a reduction in spermatogenesis across development and aging. These results suggest that

RARA has a role in the maintenance of the efficiency of spermatogenesis at the meiosis stage throughout development and aging.

75 By examining animals at various neonatal ages in detail, we found a significant reduction

in the number of spermatogonia undergoing mitosis at P4. Notable decreases in mitosis persisted

at P6, P8, P10 and P15 (Fig. 2-7). Decreases in mitosis are occurring in the undifferentiated and

type A1 to A4 series of differentiating spermatogonia, which are found in the testis of animals at

P4 (Drumond et al., 2011). P4 is also when differentiation of Aal undifferentiated spermatogonia

to the type A1 differentiating spermatogonia occurs. Therefore, the implication of the P4 results is that RARA in germ cells regulates the mitotic division of both undifferentiated and early, differentiating spermatogonia.

Additionally, there was a concomitant, significant decline in the number of intermediate and type B spermatogonia expressing RHOX13 in the cKO testes from P6 animals, when intermediate and type B spermatogonia are the most advanced cell types (Fig. 2-8). These data suggest RARA has a role in the differentiation of spermatogonia. RHOX13 is an important reporter molecule of RA signaling. The translation of Rhox13 mRNA has been demonstrated to coincide with the initiation of retinoic acid signaling in both male and female gonads (Geyer et al., 2012). Furthermore, testes of Rara cKO mice around P10 had increased numbers of undifferentiated spermatogonia compared to the wild type counterpart (Fig. 2-10), as well as a drastic reduction in percentage of tubules with most advanced early meiotic spermatocytes at

P11 (Fig. 2-9). This is in agreement with a study where neonates were made VAD in the background of an Lrat-null mutation (Li et al., 2011) and showed that germ cells failing to enter meiosis were associated with an increased number of undifferentiated spermatogonia. Thus, our data confirm that lack of RARA function in germ cells causes the inefficiency of spermatogenesis that starts in mitosis and differentiation of spermatogonia and persists through the entry into meiosis. These results are in agreement with the large volume of literature that

76 reports the requirement of the ligand retinoic acid for the differentiation of Aal spermatogonia to the type A1 spermatogonia and the initiation of meiosis, written in a review (Griswold et al.,

2012). The data shown in this study add further information to this literature that the receptor

RARA in germ cells participates in the RA signaling mechanism required for spermatogonial differentiation and the subsequent initiation of meiosis.

This inefficiency in spermatogenesis has also been reported for the Rara-null animals

(Doyle et al., 2007; Chung et al., 2004), and for Sertoli cell-specific Rara cKO mice (Vernet et al., 2006). In both cell-specific Rara mutants, spermatogenesis is reduced in an age-dependent manner, a disorganization of germ cells, and a delay in the progression of spermatogenesis in the first wave. However, a delay in spermatogenesis implies that sperm production will catch up later in the development. Testicular morphology of age-dependent degeneration indicates it is not a delay, but a decrease in spermatogenic output. Under these circumstances, we must consider that one of the roles of RARA in germ cells is to coordinate RA signaling via RARA with the

Sertoli cell. In other words, germ cells may require RARA to maintain their cellular association with Sertoli cells, an effect that requires a RARA-mediated signaling feedback from Sertoli cells.

For example, sloughing of germ cells has been a common phenotype for Rara-null and cell- specific Rara cKO mice. Sloughing may occur when there is a breakage of the intercellular bridge from germ cells of the same kind or when germ cells detach from Sertoli cells (Chapin,

1988). This phenotype may arise from mis-regulation of junctional protein interactions between

Sertoli cells and germ cells. Cooperation between these two cell types is important for the maintenance of the three-dimensional cell polarity required for proper organization of germ cells along the Sertoli cell membrane. Thus, it is not surprising that some of the phenotypes are shared between the Sertoli cell-specific (Vernet et al., 2006) and germ cell-specific Rara cKO mice.

77 However, we were surprised to find that the number of tubules with three or more

apoptotic cells in cKO testes from animals at P10, P13, P15, P19, P25 (Fig. 2-11), and P75 did

not differ from wild type testes. The increased abnormalities and the decreased number of germ

cells at various stages of development were not reflected in the apoptotic rate. This is unlike the

testes from Rara-null mice and Sertoli cell-specific Rara cKO (Vernet et al, 2006), in which

early meiotic spermatocytes, round and elongating spermatids showed apoptotic activities (Doyle

et al., 2007; Chung et al., 2005; Vernet et al., 2006). It is possible that apoptotic germ cells in

germ cell-specific Rara cKO mice are efficiently cleaned by the phagocytic mechanisms of the

wild type Sertoli cells. This scenario also indicates that RARA in Sertoli cells may be linked to

the mechanism of phagocytosis. Alternatively, RARA in Sertoli cells may protect germ cells

from apoptosis and both Rara-null mice and Sertoli cell-specific Rara cKO mice with Rara

mutant alleles in Sertoli cells could not protect germ cells from apoptosis. Previously, retinoic acid has been shown to up-regulate collagen 8A2, an extracellular matrix protein, which enhanced cell adhesion and protected hepatocarcinoma cells from undergoing apoptosis (Wang et al., 2013)

We report for a new finding that RARA in germ cells may have effects on the genomic integrity during meiosis. Chromosome spreads were used to study the meiotic process and scored for abnormalities (Fig. 2-12). The ineffectiveness of meiosis was showcased by the decreased percentage of pachytene spermatocytes with perfect synapsis and the increased percentage of synaptonemal complex fragmentation. The increase in SC fragmentation was also observed in another model organism. Atrad51, a homolog of RAD51 in Arabidopsis, when deleted by targeted recombination, has extensive chromosome fragmentation (Pradillo et al., 2012).

RAD51 may be regulated by RARA, similar to the role of estrogen receptor and RARA observed

78 to regulate breast cancer susceptibility gene BRCA1-mediated DNA repair in conjunction with co-activator CREB-binding protein (CBP) in breast cancer cell lines (Crowe and Lee, 2006)

Besides the normal sex body localization of γH2AX in pachytene spermatocytes, additional γH2AX foci, indicative of DSBs, were retained on the autosomal SCs from cKO mice.

There were two types: either small foci (S-foci) appeared along the synaptonemal complex (SC); or large foci (L-foci) appeared perpendicular to the SC, just like the chromatin loops. The difference in their appearance may indicate two distinct pathways of DSB formation. The S-foci mark sites of SPO11-dependent DSBs undergoing repair, while the L-foci indicate the SPO11- independent DSB events as well as the unrepaired SPO11-induced DSBs, shown by Spo11-null spermatocytes (Chicheportiche et al., 2007).

S- or L- foci indicate defective DSB repair or synaptic failure, seen similarly in azoospermic men with meiotic DSB defects (Sciurano et al., 2006), as well as in mice carrying such as Dmc1, Msh5, Atm, Sycp1, or Hormad2 (Barchi et al., 2005; de Vries et al.,

2005; Kogo et al., 2012). These genes code for proteins that participate in the DSB repair pathway, or are involved in SC assembly. Alternatively, these L-foci could be uncharacterized chromatin structural modifications (Chicheportiche et al., 2007). Therefore, unrepaired DSBs or uncharacterized chromatin structural modifications illustrate defective primary spermatocytes during meiotic prophase in Rara cKO testes.

In earlier studies, donor SSCs were enzymatically collected from testes of six month-old

Rara-null mice (C57BL/6), transplanted into W/Wv recipients, and after 70 days of recovery, no spermatogonial stem cells colonization and proliferation was observed (Doyle et al., 2007). It was concluded that RARA in germ cells could play a role in the determination of spermatogonial stem cell competence (Doyle et al., 2007). On the other hand, when donor germ cells were

79 enzymatically collected from animals (a mix 129/C57BL/6 genetic background, crossed to Actb-

EGFP mice) between the ages of P5 and P60, and injected into busulfan-treated NCr nude mice, the percentage of the repopulated tubules was not different (~12%) between the germ cells from the wild type and Rara-null mice (Chung et al., 2009). However, because of the wide range of donor ages, and the wide range of repopulation percentages, it is difficult to decipher whether there was an age effect on stem cell ability to regenerate spermatogenesis.

In this study, stem cell function of Rara-depleted stem cells from younger ages (P8 to

P25) was evaluated using the spermatogonial stem cell (SSC) transplantation technique. On average, cKO germ cell donor-derived spermatogenesis was about 50% of that found with the wild type germ cells (Fig. 2-13). This decreased donor germ cell-derived spermatogenesis implies that the stem cell competence was compromised, which could be due to a decreased ability of stem cells to colonize, proliferate and differentiate. Alternatively, this decreased competence could be due to a lower number of stem cells present in the Rara cKO testes.

Additionally, preliminary results suggested that donor animal ages are important. There was no difference in the regeneration rate when SSCs came from wild type donors at P8 or P16 (n = 1 for each age, n = 2 recipient for P8). However, the regeneration rate decreased with advancing ages when SSCs came from the cKO donors at P8, P11, and P25 (n = 1 for each age). The regeneration rate of SSCs from cKO donors was similar to that of the wild type donor SSCs at

P8, but decreased seven fold at P11 and decreased to virtually none at P25. It appears that the transplantable stem cell reserve or the quality of stem cells decreases over increasing ages in the cKO animals, while remaining unchanged in the wild type animals. It is possible that some events occurred during germ cell development between P8 and P11 that has affected either the quantity or quality of stem cells.

80 Other investigators have observed, similar to our results, a reduction in the stem cell reserve over time in GDNF-overexpressing mice (Meng et al., 2000). In another study, stem cell activity was decreased over increasing ages when stem cells overexpressed WNT4 (Boyer et al.,

2012). Whether similar phenomena happen with Rara cKO stem cells remains unknown and further replicate studies are needed to address this property of RARA in controlling the age- dependent stem cell reserve or the quality.

In conclusion, this study identified the distinctive roles of RARA in the germ line lineage, as well as similar roles of RARA as observed for Sertoli cells. We found that deletion of this

RARA transcription factor from germ cells caused a quantifiable decline in spermatogenesis, decreased mitosis, a block in spermatogonial differentiation, increased chromosomal fragmentation, and decreased resolution of double strand breaks during meiosis.

81 ACKNOWLEDGEMENT

We thank Jennifer Onken and Erika Larsonberg for managing the mouse colonies and genotyping and Jennifer Onken for fertility tests. We are grateful to Drs. Michael Griswold,

David Zarkower, Christopher Geyer and Jim Pru for their generous gifts of the STRA8, DMRT1 and RHOX13 antibodies, and EYFP mice respectively. We also wish to thank laboratory members for critically reading this manuscript. This research was supported by a grant from

NICHD to KHK and a grant from NIGMS to SML and NRP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIGMS.

82 REFERENCE

Akmal, K.M., Dufour, J.M., Kim, K.H., 1997. Retinoic acid receptor alpha gene expression in

the rat testis: potential role during the prophase of meiosis and in the transition from

round to elongating spermatids. Biology of reproduction 56, 549-556.

Akmal, K.M., Dufour, J.M., Vo, M., Higginson, S., Kim, K.H., 1998. Ligand-dependent

regulation of retinoic acid receptor alpha in rat testis: in vivo response to depletion and

repletion of vitamin A. Endocrinology 139, 1239-1248.

Anderson, J.A., Gilliland, W.D., Langley, C.H., 2009. Molecular population genetics and

evolution of Drosophila meiosis genes. Genetics 181, 177-185.

Bao, J., Ma, H.Y., Schuster, A., Lin, Y.M., Yan, W., 2013. Incomplete cre-mediated excision

leads to phenotypic differences between Stra8-iCre; Mov10l1 and Stra8-iCre; Mov10l1

mice. Genesis, 1-10.

Barchi, M., Mahadevaiah, S., Di Giacomo, M., Baudat, F., de Rooij, D.G., Burgoyne, P.S., Jasin,

M., Keeney, S., 2005. Surveillance of different recombination defects in mouse

spermatocytes yields distinct responses despite elimination at an identical developmental

stage. Molecular and cellular biology 25, 7203-7215.

Bastien, J., Rochette-Egly, C., 2004. Nuclear retinoid receptors and the transcription of retinoid-

target genes. Gene 328, 1-16.

Blanco-Rodriguez, J., 2012. Programmed phosphorylation of histone H2AX precedes a phase of

DNA double-strand break-independent synapsis in mouse meiosis. Reproduction 144,

699-712.

Boulogne, B., Levacher, C., Durand, P., Habert, R., 1999. Retinoic acid receptors and retinoid X

receptors in the rat testis during fetal and postnatal development: immunolocalization and

83 implication in the control of the number of gonocytes. Biology of reproduction 61, 1548-

1557.

Bour, G., Gaillard, E., Bruck, N., Lalevee, S., Plassat, J.L., Busso, D., Samama, J.P., Rochette-

Egly, C., 2005. Cyclin H binding to the RARalpha activation function (AF)-2 domain

directs phosphorylation of the AF-1 domain by cyclin-dependent kinase 7. Proceedings of

the National Academy of Sciences of the United States of America 102, 16608-16613.

Bowles, J., Knight, D., Smith, C., Wilhelm, D., Richman, J., Mamiya, S., Yashiro, K.,

Chawengsaksophak, K., Wilson, M.J., Rossant, J., Hamada, H., Koopman, P., 2006.

Retinoid signaling determines germ cell fate in mice. Science 312, 596-600.

Boyer, A., Yeh, J.R., Zhang, X., Paquet, M., Gaudin, A., Nagano, M.C., Boerboom, D., 2012.

CTNNB1 signaling in sertoli cells downregulates spermatogonial stem cell activity via

WNT4. PloS one 7, e29764.

Braun, K.W., Tribley, W.A., Griswold, M.D., Kim, K.H., 2000. Follicle-stimulating hormone

inhibits all-trans-retinoic acid-induced retinoic acid receptor alpha nuclear localization

and transcriptional activation in mouse Sertoli cell lines. The Journal of biological

chemistry 275, 4145-4151.

Chambon, P., 1996. A decade of molecular biology of retinoic acid receptors. FASEB J 10, 940-

954.

Chapin, R.E., 1988. Morphological evaluation of seminiferous epithelium of the testis. In Ch.7:

Physiology and Toxicology of Male Reproduction. Lamb, L.C., Foster, P.M., (eds.)

Academic Press, San Diego, 155-178.

84 Chicheportiche, A., Bernardino-Sgherri, J., de Massy, B., Dutrillaux, B., 2007. Characterization

of Spo11-dependent and independent phospho-H2AX foci during meiotic prophase I in

the male mouse. Journal of cell science 120, 1733-1742.

Chung, S.S., Sung, W., Wang, X., Wolgemuth, D.J., 2004. Retinoic acid receptor alpha is

required for synchronization of spermatogenic cycles and its absence results in

progressive breakdown of the spermatogenic process. Dev Dyn 230, 754-766.

Chung, S.S., Wang, X., Wolgemuth, D.J., 2005. Male sterility in mice lacking retinoic acid

receptor alpha involves specific abnormalities in spermiogenesis. Differentiation 73, 188-

198.

Chung, S.S., Wang, X., 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, 2091-2100.

Clagett-Dame, M., Knutson, D., 2011. Vitamin A in reproduction and development. Nutrients 3,

385-428.

Cooke, H.J., Saunders, P.T., 2002. Mouse models of male infertility. Nature reviews. Genetics 3,

790-801.

Crowe, D.L., Lee, M.K., 2006. New role for nuclear hormone receptors and coactivators in

regulation of BRCA1-mediated DNA repair in breast cancer cell lines. Breast cancer

research : BCR 8, R1.

Cupp, A.S., Dufour, J.M., Kim, G., Skinner, M.K., Kim, K.H., 1999. Action of retinoids on

embryonic and early postnatal testis development. Endocrinology 140, 2343-2352. de Vries, F.A., de Boer, E., van den Bosch, M., Baarends, W.M., Ooms, M., Yuan, L., Liu, J.G.,

van Zeeland, A.A., Heyting, C., Pastink, A., 2005. Mouse Sycp1 functions in

85 synaptonemal complex assembly, meiotic recombination, and XY body formation. Genes

& development 19, 1376-1389.

Doyle, T.J., Braun, K.W., McLean, D.J., Wright, R.W., Griswold, M.D., Kim, K.H., 2007.

Potential Functions of Retinoic Acid Receptor A in Sertoli Cells and Germ Cells During

Spermatogenesis. Annals of the New York Academy of Sciences 1120, 114-130.

Drumond, A.L., Meistrich, M.L., Chiarini-Garcia, H., 2011. Spermatogonial morphology and

kinetics during testis development in mice: a high-resolution light microscopy approach.

Reproduction 142, 145-155.

Dufour, J.M., Kim, K.H., 1999. Cellular and subcellular localization of six retinoid receptors in

rat testis during postnatal development: identification of potential heterodimeric

receptors. Biology of reproduction 61, 1300-1308.

Geyer, C.B., Saba, R., Kato, Y., Anderson, A.J., Chappell, V.K., Saga, Y., Eddy, E.M., 2012.

Rhox13 is translated in premeiotic germ cells in male and female mice and is regulated

by NANOS2 in the male. Biology of reproduction 86, 127.

Griswold, M.D., Bishop, P.D., Kim, K.H., Ping, R., Siiteri, J.E., Morales, C., 1989. Function of

vitamin A in normal and synchronized seminiferous tubules. Annals of the New York

Academy of Sciences 564, 154-172.

Griswold, M.D., 1995. Interactions between germ cells and Sertoli cells in the testis. Biology of

reproduction 52, 211-216.

Griswold, M.D., Hogarth, C.A., Bowles, J., Koopman, P., 2012. Initiating meiosis: the case for

retinoic acid. Biology of reproduction 86, 35.

Huang, H.F., Hembree, W.C., 1979. Spermatogenic response to vitamin A in vitamin A deficient

rats. Biology of reproduction 21, 891-904.

86 Huang, H.F., Marshall, G.R., 1983. Failure of spermatid release under various vitamin A states -

an indication of delayed spermiation. Biology of reproduction 28, 1163-1172.

Kim, K.H., Griswold, M.D., 1990. The regulation of retinoic acid receptor mRNA levels during

spermatogenesis. Mol Endocrinol 4, 1679-1688.

Kogo, H., Tsutsumi, M., Inagaki, H., Ohye, T., Kiyonari, H., Kurahashi, H., 2012. HORMAD2

is essential for synapsis surveillance during meiotic prophase via the recruitment of ATR

activity. Genes to cells : devoted to molecular & cellular mechanisms 17, 897-912.

Koubova, J., Menke, D.B., Zhou, Q., Capel, B., Griswold, M.D., Page, D.C., 2006. Retinoic acid

regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci U S A 103,

2474-2479.

Krishnamurthy, H., Danilovich, N., Morales, C.R., Sairam, M.R., 2000. Qualitative and

quantitative decline in spermatogenesis of the follicle-stimulating hormone receptor

knockout (FORKO) mouse. Biology of reproduction 62, 1146-1159.

Li, H., Palczewski, K., Baehr, W., Clagett-Dame, M., 2011. Vitamin A deficiency results in

meiotic failure and accumulation of undifferentiated spermatogonia in prepubertal mouse

testis. Biology of reproduction 84, 336-341.

Linder, C.C., Heckert, L.L., Roberts, K.P., Kim, K.H., Griswold, M.D., 1991. Expression of

receptors during the cycle of the seminiferous epithelium. Annals of the New York

Academy of Sciences 637, 313-321.

Livera, G., Rouiller-Fabre, V., Habert, R., 2001. Retinoid receptors involved in the effects of

retinoic acid on rat testis development.

87 Lufkin, T., Lohnes, D., Mark, M., Dierich, A., Gorry, P., Gaub, M.P., LeMeur, M., Chambon, P.,

1993. High postnatal lethality and testis degeneration in retinoic acid receptor alpha

mutant mice. Proc Natl Acad Sci USA 90, 7225-7229.

MacLean, G., Li, H., Metzger, D., Chambon, P., Petkovich, M., 2007. Apoptotic extinction of

germ cells in testes of Cyp26b1 knockout mice. Endocrinology 148, 4560-4567.

Mahadevaiah, S.K., Turner, J.M., Baudat, F., Rogakou, E.P., de Boer, P., Blanco-Rodriguez, J.,

Jasin, M., Keeney, S., Bonner, W.M., Burgoyne, P.S., 2001. Recombinational DNA

double-strand breaks in mice precede synapsis. Nature genetics 27, 271-276.

Mark, M., Jacobs, H., Oulad-Abdelghani, M., Dennefeld, C., Feret, B., Vernet, N., Codreanu,

C.A., Chambon, P., Ghyselinck, N.B., 2008. Stra8-deficient spermatocytes initiate, but

fail to complete, meiosis and undergo premature chromosome condensation. J Cell Sci

121, 3233-3242.

Matson, C.K., Murphy, M.W., Griswold, M.D., Yoshida, S., Bardwell, V.J., Zarkower, D., 2010.

The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls

the mitosis versus meiosis decision in male germ cells. Developmental cell 19, 612-624.

Meng, X., Lindahl, M., Hyvonen, M.E., Parvinen, M., de Rooij, D.G., Hess, M.W., Raatikainen-

Ahokas, A., Sainio, K., Rauvala, H., Lakso, M., Pichel, J.G., Westphal, H., Saarma, M.,

Sariola, H., 2000. Regulation of cell fate decision of undifferentiated spermatogonia by

GDNF. Science 287, 1489-1493.

Mose, M.J., 1968. The synaptinemal complex. Annu Rev Genet 2, 363-412.

Oatley, J.M., Brinster, R.L., 2012. The germline stem cell niche unit in mammalian testes.

Physiological reviews 92, 577-595.

88 Pelt, A.M.M.V., Rooij, D.G.D., 1990. Synchronization of the Seminiferous Epithelium after

Vitamin A Replacement in Vitamin A-Deficient Mice. Biology of Reproduction 43, 363-

367.

Pradillo, M., Lopez, E., Linacero, R., Romero, C., Cunado, N., Sanchez-Moran, E., Santos, J.L.,

2012. Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in

plant meiotic recombination. The Plant journal : for cell and molecular biology 69, 921-

933.

Rochette-Egly, C., Plassat, J.L., Taneja, R., Chambon, P., 2000. The AF-1 and AF-2 activating

domains of retinoic acid receptor-alpha (RARalpha) and their phosphorylation are

differentially involved in parietal endodermal differentiation of F9 cells and retinoid-

induced expression of target genes. Molecular endocrinology 14, 1398-1410.

Russell, L.D., Ettlin, E.R., Hikim, A.P.S., Clegg, E.D., 1990a. Mammalian Spermatogenesis,

p.1-38. In Russell, L.D., Ettlin, E.R., Hikim, A.P.S., Clegg, E.D., (ed.), Histological and

Histopathological Evaluation of the Testis. Cache River Press, Clearwater.

Russell, L.D., Ettlin, E.R., Hikim, A.P.S., Clegg, E.D., 1990b. The Classification and Timing of

Spermatogenesis, p.41-56. In Russell, L.D., Ettlin, E.R., Hikim, A.P.S., Clegg, E.D.,

(ed.), Histological and Histopathological Evaluation of the Testis. Cache River Press,

Clearwater.

Sadate-Ngatchou, P.I., Payne, C.J., Dearth, A.T., Braun, R.E., 2008. Cre recombinase activity

specific to postnatal, premeiotic male germ cells in transgenic mice. Genesis 46, 738-742.

Santos, N.C., Kim, K.H., 2010. Activity of retinoic acid receptor-alpha is directly regulated at its

protein kinase A sites in response to follicle-stimulating hormone signaling.

Endocrinology 151, 2361-2372.

89 Sapsford, C.S., 1962. Changes in the cells of the sex cords and seminiferous tubules during the

development of the testis of the rat and mouse. Aust J Zool 10, 178-193.

Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative CT

method. Nat Protoc 3, 1101-1108.

Sciurano, R.B., Rahn, M.I., Pigozzi, M.I., Olmedo, S.B., Solari, A.J., 2006. An azoospermic man

with a double-strand DNA break-processing deficiency in the spermatocyte nuclei: case

report. Human reproduction 21, 1194-1203.

Shima, J.E., McLean, D.J., McCarrey, J.R., Griswold, M.D., 2004. The murine testicular

transcriptome: characterizing gene expression in the testis during the progression of

spermatogenesis. Biology of reproduction 71, 319-330.

Small, C.L., Shima, J.E., Uzumcu, M., Skinner, M.K., Griswold, M.D., 2005. Profiling gene

expression during the differentiation and development of the murine embryonic gonad.

Biology of reproduction 72, 492-501.

Thomson, J.N., Howell, J.M., Pitt, G.A.J., 1964. Vitamin A and reproduction in rats. Proc R Soc

Lond (Biol) 159, 510-535.

Trautmann, E., Guerquin, M.J., Duquenne, C., Lahaye, J.B., Habert, R., Livera, G., 2008.

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes. Cell cycle 7, 656-

664.

Uehara, M., Yashiro, K., Takaoka, K., Yamamoto, M., Hamada, H., 2009. Removal of maternal

retinoic acid by embryonic CYP26 is required for correct Nodal expression during early

embryonic patterning. Genes Dev 23, 1689-1698.

90 Van Pelt, A.M., De Rooij, D.G., 1990a. The origin of the synchronization of the seminiferous

epithelium in vitamin A-deficient rats after vitamin A replacement. Biology of

reproduction 42, 677-682.

van Pelt, A.M., de Rooij, D.G., 1990b. Synchronization of the seminiferous epithelium after

vitamin A replacement in vitamin A-deficient mice. Biology of reproduction 43, 363-367. van Pelt, A.M., de Rooij, D.G., 1991. Retinoic acid is able to reinitiate spermatogenesis in

vitamin A-deficient rats and high replicate doses support the full development of

spermatogenic cells. Endocrinology 128, 697-704.

Vernet, N., Dennefeld, C., Guillou, F., Chambon, P., Ghyselinck, N.B., Mark, M., 2006.

Prepubertal testis development relies on retinoic acid but not rexinoid receptors in Sertoli

cells. Embo J 25, 5816-5825.

Wang, W., Xu, G., Ding, C.L., Zhao, L.J., Zhao, P., Ren, H., Qi, Z.T., 2013. All-trans retinoic

acid protects hepatocellular carcinoma cells against serum-starvation-induced cell death

by upregulating collagen 8A2. The FEBS journal 280, 1308-1319.

Wolbach, S.B., Howe, P.R., 1925. Tissue changes following deprivation of fat-soluble A

vitamin. J Exp Med 42, 753-777.

Wu, Q., Song, R., Ortogero, N., Zheng, H., Evanoff, R., Small, C.L., Griswold, M.D.,

Namekawa, S.H., Royo, H., Turner, J.M., Yan, W., 2012. The RNase III enzyme

DROSHA is essential for microRNA production and spermatogenesis. The Journal of

biological chemistry 287, 25173-25190.

Zhou, Q., Nie, R., Li, Y., Friel, P., Mitchell, D., Hess, R.A., Small, C., Griswold, M.D., 2008.

Expression of stimulated by retinoic acid gene 8 (Stra8) in spermatogenic cells induced

91 by retinoic acid: an in vivo study in vitamin A-sufficient postnatal murine testes. Biology

of reproduction 79, 35-42.

Zhu, L., Santos, N.C., Kim, K.H., 2009. Small ubiquitin-like modifier-2 modification of retinoic

acid receptor-alpha regulates its subcellular localization and transcriptional activity.

Endocrinology 150, 5586-5595.

Zhu, L., Santos, N.C., Kim, K.H., 2010. Disulfide isomerase glucose-regulated protein 58 is

required for the nuclear localization and degradation of retinoic acid receptor alpha.

Reproduction 139, 717-731.

92 Table 2-1. Genotyping primers.

Strain Primer Primer Primer Sequence Amplified PCR Pair Name Fragment Size Rara wild type W VZ200 TCC GAC TTG CGA CTC CCT CTA CTC A 554 bp TD200 TTA ACT CCT CCT GAA CTT TGG GAA G W1 6799* GTG CCC TTC CCT CCA TCT TCC TTA 589 bp 7365 GTC CTC CTC CGA CTT GCG ACT CC Floxed Rara F 6799* GTG CCC TTC CCT CCA TCT TCC TTA 575 bp 7352 CAT CGC CTT CTA TCG CCT TCT T F1 6799* GTG CCC TTC CCT CCA TCT TCC TTA 1017 bp 7794 ACC TTG CTC CTG CCG AGA AAG T Deleted Rara Δ 6799* GTG CCC TTC CCT CCA TCT TCC TTA 487 bp 7263 GCC CTG CGA CAC TCA CAC TCC TT Δ2 6799* GTG CCC TTC CCT CCA TCT TCC TTA 406 bp Δ2 CGG GTC ACC TTG TTG ATG ATG CA Stra8-icre M1 Stra8-2F GTG CAA GCT GAA CAA CAG GA 865 bp Stra8-R GTC CCC ATC CTC GAG CAG CCT C M3 Stra8-3F TGC CCA AGA AGA AGA GGA AA 403 bp Stra8-2R CCA GCA TCC ACA TTC TCC TT

* Common primer

93 Figure 2-1. Schematic drawings of generating Rara cKO animals.

94 Figure 2-1. Schematic drawings of generating Rara cKO animals.

Homologous recombination in embryonic stem cells (ESCs) replaced the wild type Rara gene with the targeting vector shown in (A). The Rara gene-targeting vector has exon 4 (E4) of Rara, engineered behind a neomycin cassette for positive selection by G-418, flanked by two loxP sites. A thymidine kinase gene was placed in front of the 5’ Rara homology region, allowing for negative selection by ganciclovir. ESCs with the targeting vector were injected into the blastocoel cavity of 4.5-day blastocysts, which were then surgically transferred into a pseudopregnant female. After multiple generations of selection, the Rara cKO mouse line was generated. (B) Protein domains of Rara. The B region is a part of the ligand-independent transcriptional activation function-1 (AF-1) domain (Rochette-Egly et al., 2000), known to bind cell-specific factors or co-activators, which is deleted upon CRE recombination. (C)

Representation of E4 (within the B domain) deletion after STRA8-iCRE recombination activity.

N: Not I, X: XhoI, E: EcoRI, B: BamHI, Nh: Nhe I, Nd: Nde I, C: Cla I, TK: thymidine kinase,

DBD: DNA binding domain, NLS: nuclear localization signal, LBD: ligand binding domain.

95 Figure 2-2. Expression of STRA8-iCRE during neonatal development.

96 Figure 2-2. Expression of STRA8-iCRE during neonatal development.

(A) Genetic construct showing EYFP reporter gene. Stop codon is deleted when STRA8-iCRE recombines the 2 loxP sites (white triangles), generating EYFP; STRA8-iCRE reporter F1 mice.

(B) By mating the F1 male pups back to wild type females, the recombination efficiency of

STRA8-iCRE was determined to be 95.5%. Testes cross-sections from P0 (C, false colored), P1

(D), P3 (E), P4 (F), and P5 (G) animals, immunostained for GFP. (H) Percentages of animals with EYFP expression in the testis, indicative of STRA8-iCRE activity. The number of animals used (N) are shown. (I) The level of Rara mRNA from isolated, enriched germ cells of cKO mice at P4 and P8 by real time RT-PCR assays (n = 3, *** p = 0.0002 and 0.0076).

97 Figure 2-3. Tubular abnormalities in cKO mice across the developmental ages.

98 Figure 2-3. Tubular abnormalities in cKO mice across the developmental ages.

99 Figure 2-3. Tubular abnormalities in cKO mice across the developmental ages.

100 Figure 2-3. Tubular abnormalities in cKO mice across the developmental ages.

Testes were collected from male mice at P1, P4, P5, P6, P8, P10, P11, P13, P15, P20, P25, P30,

P45, P61, P75, P120, P180, and P365, fixed in Bouin’s solution, embedded in paraffin, cut in four-µm thickness serial sections, and mounted on poly-L-lysine slides. To examine testicular morphology, sections were deparaffinized and hydrated, stained with hematoxylin and eosin, and examined using a microscope. Testes of Rara cKO male mice exhibited abnormalities compared to their wild type counterparts starting at P10 and P15 and they became worse as animals aged

(A-X). These micrographs represent three to six animals (n = 3-6).

101 Figure 2-4. Quantification of morphological abnormalities in the testes.

* * * * * *

102 Figure 2-4. Quantification of morphological abnormalities in the testes.

Morphological abnormalities were scored by counting tubules with abnormalities at P15, P25,

P45, P61, P75, P120, and P365, which included the presence of various sized vacuoles, sloughing of germ cells, lack of lumen, and disorganization of germ cell layers. The numbers from above were divided by the total number of tubules in the same area of the testicular section to obtain percentage of abnormal tubules. To obtain comprehensive sampling across the entire testis, a minimum of three sections spaced 50 µm apart were analyzed (n = 3~6). * denotes statistical significance at p ≤ 0.05.

103 Figure 2-5. Decrease in percentage of most advanced cell types.

104 Figure 2-5. Decrease in percentage of most advanced cell types.

(A-F) The number of tubules with the most advanced germ cell types were counted and scored using hematoxylin and eosin-stained testicular cross-sections. The most advanced germ cell types are late pachytene spermatocytes (A and B), round spermatids (C and D), and step 8-9 elongating spermatids (E and F) at P15, P20 and P30, respectively. (G) At all ages examined, there was a decrease in the percentage of tubules with advanced germ cell types (n = 3, *** p =

0.0001, 0.0001 and 0.0006).

105 Figure 2-6. Epididymal sperm and male fertility analysis.

106 Figure 2-6. Epididymal sperm and male fertility analysis.

(A) Sperm from the caudal epididymides were collected from mice at P75 and P120 and counted using a hemacytometer. Compared to the sperm number from the wild type mice, there was a 42% and 58% decrease in epididymal sperm number in cKO mice at P75 and

P120 (P180 data is not available yet; animals are aging). The sperm counts were repeated for six to eleven animals (n = 6~11). Differences in values between wild type and cKO are significant at p = 0.0051 and p = 0.0002 for P75 and P120, respectively. (B) Males at

P75 and P120 were mated to C57BL/6 wild type females between the ages of P58-78.

The pairs were kept together for one week before separating. Number and sex of live pups at P0 were recorded for multiple animals (n = 2~8). Differences in values between wild type and cKO are not significant at p ≤ 0.05. We note, however, that values for P120 are lower and more replicates are on going. P180 fertility tests will start as soon as males are 180 days old.

107 Figure 2-7. Decrease in number of cells undergoing mitosis (proliferation).

108 Figure 2-7. Decrease in number of cells undergoing mitosis (proliferation).

(A-H) Proliferation rates of young pups were represented and scored from testis cross-sections for pHH3 at P4 (A and B), P6 (C and D), P8 (E and F) and P10 (G and H). Phosphohistone H3

(pHH3) marks cells at mitotic prophase. (M) Number of pHH3-positive cells per 18.8 µm2 was counted. There was a significant decrease in the number of cells stained brightly for pHH3 at

P4, P6 and P8 (N = 3, *** p = 0.0008, 0.0084 and 0.0024). (I-L) Male pre-pubertal mice at P10

(I and J) and P15 (K and L) were subjected to intraperitoneal injection of BrdU four hrs prior to sacrifice, and testes cross-sections were immunostained with anti-BrdU antibody. (N) Number of

BrdU-positive cells per tubule was scored. There was reduced BrdU-positive cells/tubule, although not significant at p ≤ 0.05 (n = 3, p = 0.11).

109 Figure 2-8. Decrease in percentage of tubules with RHOX13-positive differentiating spermatogonia.

110 Figure 2-8. Decrease in percentage of tubules with RHOX13-positive differentiating spermatogonia.

(A-D) Percentage of tubules with RHOX13-positive differentiating spermatogonia was scored, using testicular sections immunostained with anti-RHOX13 antibody from mice at P4, P6 (A and

B), P8 (C and D), P10 and P15. RHOX13 is highly expressed in intermediate and type B spermatogonia. There was a decrease in the percentage of tubules with RHOX13-positive differentiating spermatogonia at P6 (E) (n = 3-4, p = 0.0106 and 0.0475).

111 Figure 2-9. Decrease in percentage of tubules with brightly stained γH2AX-positive leptotene and zygotene spermatocytes.

112 Figure 2-9. Decrease in percentage of tubules with brightly stained γH2AX-positive leptotene and zygotene spermatocytes.

(A-D) Testicular cross sections were immunostained for γH2AX, which stains brightly for leptotene and zygotene spermatocytes with double strand breaks (DSBs) in prophase of meiosis at P10 (A and B) and P11 (C). (E) Quantification of percentage of tubules with the brightly stained cells at P10, P11, 13, 15, 20 and 25. There was a significantly lower percentage of tubules with leptotene and zygotene spermatocytes in the cKO cross sections at P11. (n = 3, *** p = 0.0001 and ** p = 0.0016).

113 Figure 2-10. Accumulation of undifferentiated spermatogonia at P10.

114 Figure 2-10. Accumulation of undifferentiated spermatogonia at P10.

(A and B) Testicular cross sections were immunostained for DMRT1 and the number of undifferentiated spermatogonia per tubule immunostained brightly with DMRT1 was scored.

DMRT1 only expresses in the gonad. Antibody against DMRT1 immunostains Sertoli cells and differentiating spermatogonia lightly, and undifferentiated spermatogonia brightly. There were significantly more brightly immunostained, undifferentiated spermatogonia per tubule in the cKO testicular cross-sections compared to the wild type animals at P10. (C) (n = 3, p ≤ 0.05).

115 Figure 2-11. TUNEL analysis to detect apoptosis.

116 Figure 2-11. TUNEL analysis to detect apoptosis.

Percentage of tubules with three or more apoptotic cells was scored in testicular sections.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to detect

DNA fragmentation by labeling the terminal ends of nucleic acids. There was no change in the percentage of tubules with three or more apoptotic cells at P10, 15, 19 and 25 (n = 3 for P10,

P15, and P19; n = 2 for P25).

117 Figure 2-12. Decrease in percentage of pachytene spermatocytes with perfect synapsis and increase in percentage of double strand break (DSB) formation.

118 Figure 2-12. Decrease in percentage of pachytene spermatocytes with perfect synapsis and increase in percentage of double strand break (DSB) formation.

(A-B) Percentage of defective chromosomes was scored for pachytene meiotic spreads from mice at P15 using anti-SYCP3 and anti-γH2AX antibodies. Anti-SYCP3 immunostains the axial elements of the synaptonemal complex (SC), and anti-γH2AX marks DSB sites. (C) The defects scored are SC fragmentation (1), autosomal γH2AX foci (3), partial asynapsis (4), forks and gaps

(5) and pair association (6). A pachytene chromosomal spread has perfect synapsis (2) if there are no aforementioned defects. There was a significant decrease in the percentage of pachytene spermatocytes with perfect synapsis, and increased percent cells with DSBs (n = 3, 150 cells, p ≤

0.05).

119 Figure 2-13. Analyses of the recipients after spermatogonial stem cell transplantation.

120 Figure 2-13. Analyses of the recipients after spermatogonial stem cell transplantation.

121 Figure 2-13. Analyses of the recipients after spermatogonial stem cell transplantation.

(A) Graphical representation of the transplantation experiment. Donor testes of WT and cKO are from ¾ C57Bl/6 and ¼ FVB genetic background. Testes were detunicated and enzymatically digested in two steps, where interstitial cells were digested and removed in the first step, and the tubular basement membranes were digested in the second step. Recipient testes are from busulfan-treated ROSA mice. One testis was injected with donor germ cells, and the other testis served as a sham from each recipient animal. (B-E) Pictures are a representation of the testicular cross-sections of testes from recipient mice injected with WT germ cells (B and D) and cKO germ cells (C and E). Seminiferous tubules were stained with X-gal, which gave the blue color to the endogenous germ cells and somatic cells, whereas counter-staining with nuclear fast red gave the pink color and identified the donor re-derived germ cells and some endogenous somatic cells at the basement membrane and interstitial space. (F) The number of tubules with more than one layer of pink-color donor-derived germ cells was scored. Quantification of percentage of tubules with donor derived germ cells is shown (n = 2 with 20 sections 50µm apart for the wild type mice and n = 3 with 22 sections 50µm apart for cKO mice). WT-1 and WT-3 have donor germ cells from animals at P16 and P8, respectively. cKO-1, cKO-2, and cKO-3 have donor germ cells from animals at P25, P11, and P8, respectively.

122 CHAPTER THREE

POTENTIAL TARGETS FOR RARA PROTEIN IN REGULATING THE

SPERMATOGONIAL DIFFERENTIATION AND MEIOSIS

Sze Ming Law-Nagaokaa,b, Natalie R. Peera,c, Timothy J. Doylea, Kwan Hee Kima,d,e

aSchool of Molecular BioSciences, Center for Reproductive Biology, Washington State

University, Pullman, WA.

bSupported by NIH grant T32GM083864 (to S.M.L) from the National Institute of General

Medical Sciences (NIGMS).

cSupported by NIH grant T32GM008336 (to N.R.P) from the National Institute of General

Medical Sciences (NIGMS).

dSupported in part by NIH grant HD44569 (to K.H.K) from National Inistitute of Child Health

and Human Development (NICHD).

eCorrespondence: Kwan Hee Kim, School of Molecular BioSciences, 1715 NE S. Fairway Rd.,

Washington State University, Pullman, WA 99164. E-mail: [email protected]

Note: This work will yield a primary authorship publication. Manuscript is in preparation.

123 ABSTRACT

Retinoic acid receptor alpha (RARA) is a transcription factor known to regulate spermatogonial differentiation and meiosis, yet molecular players that control these functions are currently not yet well understood. We used transcriptome profiling to identify specific genes whose expressions were altered in germ cells without RARA function. Gene expression in enriched testicular germ cells was compared between wild type and germ cell-specific Rara conditional knockout (cKO) mice. We identified 322 and 407 probes that were regulated differentially at ± 2.0 fold or greater for postnatal day 4 (P4) and P8, respectively. Functional cluster analyses revealed two distinct sets of functions for the P4 and P8 sets of probes. These functions correspond remarkably well with biologically known functions of RARA and retinoic acid ligand (RA) in the testis. The six top functional categories for the P4 set were cell differentiation, cell adhesion, cell migration, pregnancy, contraction, and cell proliferation. For the P8 set, the top five functional categories were synapsis, male meiosis, spermatogenesis, synaptonemal complex, and crossover. Further analyses of the P4 set revealed 15 transcripts whose gene promoters physically interact with DMRT1, a protein known to play a role in sex determination, promote spermatogonial differentiation, and suppress meiosis. It is known that

DMRT1 down-regulates RA-dependent transcriptional activity by directly binding to the proximal promoter of RA-responsive genes. The 15 transcripts may be important for the regulation of RARA transcriptional activity and warrant further investigation to determine their roles in spermatogonial differentiation and meiosis. Analyses of the P8 set uncovered a number of gene products that participate in double strand break repair pathways during meiosis, one of which is EME2, a binding partner for MUS81. These two proteins form a complex, involved in resolving interference-independent crossovers (COs), possibly by cleaving recombination

124 intermediates such as D-loop substrates and nicked or intact Holliday junctions found during meiotic prophase. We have created unique databases from germ cells without RARA function that can provide the basis for mining gene clusters that may play critical roles in spermatogonial proliferation and differentiation and in meiosis in the mouse.

125 INTRODUCTION

RARA is one of six retinoid receptors mediating retinoic acid (RA) function (Chambon,

1996). There are two families of retinoid receptors, retinoic acid receptors (RARs) and retinoid X

receptors (RXRs). Rara-null male mice are infertile due to germ cell loss (Lufkin et al., 1993),

similar to vitamin A deficient (VAD) mice (Huang and Hembree, 1979; van Pelt and de Rooij,

1990; Wolbach and Howe, 1925). RARA in germ cells was found to be important in

spermatogonial proliferation and differentiation, as well as in resolving DNA double strand breaks during meiotic prophase (Chapter 2).

Three continuous mitotic, meiotic, and post-meiotic phases, each with multiple stages of germ cell development make up the process of spermatogenesis. Primordial germ cells become

prospermatogonia or gonocytes in embryos, and develop into spermatogonial stem cells (SSCs)

around postnatal day 3 (P3). Complex cellular signals direct SSCs to undergo mitosis to either

renew the stem cell pool, or divide into Apair and Aal undifferentiated spermatogonia. Aal cells can then differentiate into type A1, A2, A3, A4, intermediate and B differentiating spermatogonia, expanding the number of spermatogonia 1000 fold. Type B spermatogonia divide by the last round of mitosis to become preleptotene spermatocytes. Preleptotene spermatocytes duplicate

DNA and enter meiosis, generating a series of primary spermatocytes (leptotene, zygotene, pachytene and diplotene) and secondary spermatocytes (Russell et al., 1990). Secondary spermatocytes undergo a reductive division to produce haploid round spermatids, which mature to elongated spermatids by dramatic morphological changes known as spermiogenesis. It is also known that gonocytes can differentiate directly to the type A1 to jump start the first wave of

spermatogenesis (Yoshida et al., 2006).

It has been known that RA directs the expression of transcripts by activating the ligand-

126 dependent transcription factors, RARs and RXRs (Bastien and Rochette-Egly, 2004; Chambon,

1996). In response to RA signaling, RARs heterodimerize with RXRs and occupy characteristic

RA response elements (RAREs) located in the 5’ promoter region of target genes (Bastien and

Rochette-Egly, 2004). Occupancy of RAR/RXR heterodimers at cognate response elements

commonly determines the transcriptional responsiveness. RAREs are composed of two direct repeats of a core hexameric motif RGKTCA (coding in accordance to the IUPAC convention:

R=A/G; K=G/T). The classic RARE is a direct repeat of this motif, separated by 5 base pairs

(bp), also known as DR5. RAR/RXR can also bind to direct repeats separated by 2 bp (DR2), or

1 bp (DR1) (Bastien and Rochette-Egly, 2004; Cotnoir-White et al., 2011). Some of the well-

characterized genes with classic RAREs are involved in RA metabolism (Cyp26A1) (Loudig et

al., 2005), in RA signaling (Rara2, Rarb2, Rarg2) (de The et al., 1990; Lehmann et al., 1992;

Leroy et al., 1991), and in organismal development (Hoxa1, Hoxa4, Hoxb1) (Doerksen et al.,

1996; Huang et al., 1998; Langston and Gudas, 1992). Some RA-regulated genes do not have

RAREs (Lalevee et al., 2011), but are indirectly regulated by RARE-containing gene products.

To better understand the RA regulation network in the testis, we have profiled the

transcriptomes extracted from germ cells of wild type and germ cell-specific Rara conditional

knockouts (cKOs). Germ cells were isolated from two key developmental time points, postnatal

day 4 and 8 (P4 and P8), when spermatogonial differentiation and meiotic initiation occur in the

testis, respectively. Differentiation from Aal to A1-4 spermatogonia and mitosis are carried out around the time of P4, with the most advanced cell type being A4 spermatogonia (Drumond et

al., 2011). Meiosis is initiated and early meiotic spermatocytes are found around the time of P8

with leptotene spermatocytes being the most advanced germ cells (Drumond et al., 2011). Our

results suggest RARA mediates RA signaling to orchestrate spermatogonial differentiation and

127 meiosis of primary spermatocytes.

MATERIALS AND METHODS

Animals

Floxed Rara females (C57BL/6 genetic background) were mated to Stra8-iCre males

(FVB genetic background) (Jackson Laboratories, Jax stock #008208) to generate F1 generation

heterozygotes, Raraflox/+; cre+ (Fig. 2-1 C). F1 males between the ages of P45 and P60 were bred to homozygous floxed Rara females to generate F2 pups. In the F2 generation, only pups with flox/-; cre+ tail genotype were used for conditional Rara knockout sample collections. Wild type control animals were generated in a similar fashion with the following exception: homozygous floxed females were substituted with C57BL/6J (Rara+/+; cre-) females. Care and maintenance of animals were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee of Washington State University, following the NIH guidelines.

Germ cell enrichment

Testes from wild type and germ cell-specific Rara cKO neonates (both with ¾ C57BL/6 and ¼ FVB genetic background) at postnatal day 4 and 8 (P4 and P8) were surgically removed, detunicated, and digested in 1X Hank’s balanced salt solution (HBSS, pH 7.4), containing

0.01mg/ml collagenase at 37°C until tubules were free-flowing. Tubules were then settled by gravity, and supernatant containing interstitial cells was removed. Following two HBSS washes, tubules were subjected to a second digestion with 1 mg/ml collagenase, 2 µg/ml DNase, 1.5 mg/ml hyaluronidase, and 0.5 mg/ml trypsin in HBSS at 37°C. Fetal bovine serum was used to stop the trypsin enzymatic reaction and cells were then passed through a 40 µm-mesh sieve

128 screen before collection by centrifugation. The percentage of germ cells was determined by smearing the final suspension onto a slide with CytoSpin at 800 rpm for 6 min and immunostaining with germ cell nuclear antigen (GCNA) antibody. More than 200 cells were counted. The average percentage of germ cells in three separate germ cell preparations was determined.

RNA extraction

Total RNA was extracted from the enriched germ cell populations by using either

QIAGEN miRNeasy Mini Kit (cat# 217004, QIAGEN Inc., Valencia CA), or QIAGEN RNeasy

Mini Kit (cat# 74104 QIAGEN Inc., Valencia CA) following the manufacturer’s protocol. The quality of RNA was analyzed using 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara,

CA) and Nanodrop 2000c (Thermo Fisher Scientific Inc.Waltham, MA).

Microarray processing and data analysis

Extracted RNA was used to generate cRNA targets for microarray analysis. 500 ng of extracted total RNAs was first reverse transcribed into the first strand of cDNA using a T7-Oligo

(dT) Promoter Primer. The second strand of cDNA was synthesized by RNase H-mediated reaction. After purification, this double-stranded cDNA was used as templates for in vitro transcription (IVT) to generate complimentary RNA (cRNA) by T7 RNA Polymerase through incorporation of biotinylated nucleotides analog/ribonucleotide mix. Cleaned and fragmented biotinylated cRNA targets were then hybridized to the Affymetrix mouse genome 430.2 expression arrays overnight along with hybridization controls (Affymetrix Inc., Santa Clara,

CA), washed, incubated with Streptavidin-phycoerythrin, stained with biotinylated anti-

129 streptavidin antibody and scanned using Affymetrix GeneChip® scanner 3000. Transcriptome profiling (1 chip per RNA sample) was performed. The hybridization raw signals were acquired and normalized by either Affymetrix GeneChip® Operating Software (GCOS), or GeneChip®

Command Console® Software (AGCC). Then, they were exported to GeneSpring GX 12.5

(Agilent Technologies, Santa Clara, CA). The raw intensity data sets were normalized following the default setting of GeneSpring GX 12.5 (Agilent Technologies), in which data was subjected to Robust Multi-array Average (RMA) summarization, which includes background correction, normalization, and summarization, and baseline transformation to find the median of samples.

The normalized data was then filtered to include all genes with at least one sample having a raw signal ≥ 50. Moderated t-tests were performed to compare all RNA samples with asymptotic p- value computation. No multiple testing correction method was used. Only transcripts with a p- value ≤ 0.05 were considered in subsequent analyses. Pathway Studio 9.0 (Elsevier, Inc.

Rockville, MD) was used for the analysis of gene ontology clusters and subnetworks of cell processes.

Real-time RT PCR

Real-time PCR primers (Table 3-1) were designed using Primer Express version 2.0

(Applied Biosystems Technology, Foster City, CA). 500 ng of RNA was used to synthesize cDNA templates by using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA).

Real time PCR assay was performed with a 7500 Fast real time PCR system (Applied

Biosystems Technology). 1X Power SYBR Green PCR Master Mix (Applied Biosystems

Technology), 500 nM of forward and reverse primers, 5 µl of diluted cDNA (1:10 or 1:20), and distilled water were mixed in the reaction mix. The PCR cycles were as follows: 1 cycle at 50°C

130 for 2 min, 1 cycle at 95°C for 10 min, and 40 cycles at 95°C for 15 sec and 60°C for 1 min. All

real time PCR was conducted in technical duplicates with three biological samples. The

expression level was evaluated using the 2-(ΔΔCt) and the 2-ΔCt method (25). Cycle threshold values

(Ct) for the housekeeping genes (the ribosomal S2 protein (Rps2) or glyceraldehyde-3-phophate

dehydrogenase (Gapdh) gene) and the gene of interest were determined using Prism SDS version

1.1 software (Applied Biosystems Technology). Ct values for the gene of interest were

normalized to those of either Rps2 or Gapdh values in each sample, and then the fold change and

abundance for the gene of interest was calculated relative to the level in the reference sample. A

single PCR product for each primer set was observed by examining the dissociation curve. PCR

efficiencies were similar for each primer set based on the amplification plots of the genes of

interest compared to the endogenous controls. One-way ANOVA was performed, followed by

pairwise comparisons of the means at a p value of < 0.05, using JMP version 10 software (SAS

institute Inc. Cary, NC).

Known Retinol (ROL), RA, RARA-regulated genes and retinoic acid response element search

The number of genes regulated by retinol (ROL), RA, and RARA were extracted from

Pathway Studio 9.0 Program. These ROL-, RA-, RARA- regulated gene sequences on the P4 and

P8 lists were used to identify the retinoic acid response element (RARE) in their promoters using

an online platform: Transcriptional Regulatory Element Database (TRED). 2000 base pairs of

the proximal promoter from genes of interest were extracted from the database created between

the European Bioinformatics Institute and the Wellcome Trust Sanger Institute, Ensembl

(www.ensembl.org). The promoter sequence was inserted into the TRED searchable database for

the direct repeat of the core hexameric motif RGKTCA (coding is according to the IUPAC

131 convention: R=A/G; K=G/T), separated by one, two, or five nucleotides in between two direct repeats of the hexameric core (DR1, DR2, DR5). Additional repeats (DR0-DR10) were also included in the analysis, as they may provide valuable information in evaluating non-canonical

RAREs.

RESULTS

Microarray hybridization and GeneSpring analysis

Through a two-step enzymatic digestion, enriched germ cells were collected from testes of WT and cKO animals at P4 and P8 (Fig. 3-1). Total RNA was extracted from three separate sets of enriched germ cells of wild type (WT) and Rara cKO pups (Fig. 3-1). To confirm that enriched germ cells collected from WT and cKO have similar somatic cell contamination, real time PCR was performed for Sertoli cell marker Gata4, Leydig cell marker Hsd3b1, and germ cell marker Stra8 (Fig. 3-2). There was no change in the relative amount of Gata4 and Hsd3b1, indicating that the contamination levels of Sertoli cells and Leydig cells were similar between wild type and cKO germ cells. Stra8 was shown to decrease by 1.6 and 3.9 fold in the cKO at P4 and P8, respectively, indicating that germ cells are enriched. Real time PCR was also performed to confirm the decrease in Rara transcripts (Fig. 3-2). There was a decrease of 4.7 and 2.8 fold in the cKO at P4 and P8, respectively, indicating that there is a decreased level of Rara transcripts in the enriched germ cells from cKO mice.

After confirming the quality of the germ cells and that there was a decreased level of

Rara transcripts from enriched germ cells, the RNA extracted were used to generate cRNA targets for microarray analysis. These targets were hybridized to the Affymetrix mouse genome

430.2 expression arrays (Affymetrix Inc., Santa Clara, CA). The hybridization raw signals were

132 acquired and normalized by Affymetrix GeneChip® Operating Software (GCOS), or

GeneChip® Command Console® Software (AGCC) and then exported to GeneSpring GX 12.5

(Agilent Technologies, Santa Clara, CA) for further normalization and filtering. A probe set that had at least one sample with a raw signal ≥ 50 and statistical significance of p ≤ 0.05 were considered in later analyses (Table 3-2). Of the 3279 and 4814 significantly changed probe sets,

322 and 407 were changed ± 2 fold or greater, 32 and 94 were changed ± 3 fold by the lack of

Rara, compared to wild type at P4 and P8, respectively (Table 3-2).

In the P4 gene set, there were a total of 322 probes (283 annotated unique genes) differentially regulated at ± 2.0 fold or greater, comparing cKO to the WT values. A complete list of the 322 probes is found in Appendix Table A1-5. There are 64 down-regulated probes and

258 up-regulated probes in germ cells from Rara cKO mice, showing the distinct dichotomy of expression, with over four times more up-regulated probes than down-regulated probes. In the P8 gene set, there were a total of 407 probes (360 annotated unique genes) differentially regulated at

± 2.0 fold or greater, comparing cKO to the WT values. A complete list of 407 probes is found in Appendix Table A1-6. Out of the 407 probes, 78 are up-regulated probes and 329 are down- regulated in germ cells from Rara cKO mice. There are over four times more down-regulated probes than up-regulated probes, opposite of the P4 set.

Retinol (ROL), RA and RARA regulated genes

With ROL, RA, and RARA designated as the regulators, the search against the entire database of Pathway Studio Program yielded 530 genes known to be regulated by ROL, RA, or

RARA, of which 93 (33% of 283 annotated unique genes) were found in the P4 gene set and 73

(20% of 360 annotated unique transcripts) were found in the P8 gene set (Fig. 3-3). Then, the

133 ROL, RA, and RARA P4 and P8 lists were used with Transcriptional Regulatory Element

Database (TRED) program to identify those genes that have canonical (DR-1, DR-2, DR-5) and non-canonical (DR with 0 to 10 spacing, except for DR-1, DR-2, and DR-5) retinoic acid response elements (RAREs) in their proximal promoters. As a positive control, Cyp26a1 was analyzed and found to have DR-5 as previously known (Loudig et al., 2000). As negative controls, Gata1, Pten, Dmrt1, and Sgp1 were analyzed and were found to contain no RAREs, also as expected.

Only three genes from the P4 gene set and ten genes from the P8 gene set were identified to have canonical RAREs (DR-1, DR-2, DR-5); these have the potential to be directly regulated by RARA (Table 3-4). For the P4 gene set, the genes with canonical RAREs are Lhx9, LIM homeobox protein 9, Kif18b, family member 18B, and Plekhb1 for the P4 gene set. For the P8 gene set, the ten genes with canonical RAREs are Ubiquitin-conjugating enzyme E2C

(Ube2c), X-linked lymphocyte-regulated 5A/B/C (Xlr5a/Xlr5b/Xlr5c), (cadherin associated protein) delta 2 (Ctnnd2), Peptidylprolyl isomerase (cyclophilin)-like 6 (Ppil6),

SEC14-like 2 (S. cerevisiae) (Sec14l2), Dispatched homolog 1 (Drosophila) (Disp1), Pleckstrin homology domain containing, family B (evectins) member 1 (Plekhb1), Activating transcription factor 7 interacting protein 2 (Atf7ip2), REC8 homolog (yeast) (Rec8), and testis expressed 101

(Tex101). The fold changes and RAREs are listed in Table 3-4. Other genes are regulated by ligands ROL and RA, but not directly regulated by RARA (Leid et al., 1992; Umesono et al.,

1991).

134 Functional clustering and pathway analysis

To understand the functions of genes at these two early postnatal developmental time points of P4 and P8, the complete list of genes regulated ± 2.0 fold for the two time points were imported into Pathway Studio 9.0 (Elsevier, Inc. Rockville, MD) to find subnetworks of cell processes and Gene Ontology (GO) clusters. At P4, when spermatogonia actively expand their population by continuous mitosis and differentiation, functional clusters associated with these cell processes were cell differentiation, cell adhesion, and cell migration (Pathway Studio 9.0)

(Table 3-5). A complete list can be found in Table A1-1. Multicellular organismal development, , and cell differentiation were the top three GO biological process clusters for this group of genes (Table 3-7) and the complete list of GO clusters are found in Table A1-3. To better understand biological functions, the highly regulated genes that are changed ± 3.0 fold or higher were put into Pathway Studio. The results for the ± 3.0 fold or higher changing genes and their biological functions are shown (Fig. 3-4). The biological functions are, for example, cell cycle, cell migration, cell differentiation, G1/S transition, actin organization, and mitotic entry.

All the aforementioned processes are centered on cell differentiation, consistent with RA’s known involvement in the differentiation processes in multiple cell types (reviewed in Clagett-

Dame and Knutson, 2011).

Actively transcribed transcripts at P8 were found to be involved in meiosis, spermatogenesis, and synaptonemal complex assembly, according to the subnetwork cell processes (Table 3-6; complete list on Table A1-2) and synapsis, male meiosis and spermatogenesis, according to the Gene Ontology biological processes (Table 3-8, complete list on Table A1-4). Some of the highly regulated genes were involved in meiosis-related processes

135 (Fig. 3-5), for example, premeiotic DNA synthesis, pachytene checkpoint, synaptonemal complex formation, DNA repair, meiosis, and synaptonemal complex assembly.

Real time PCR of highly changing genes

The top ten up-regulated and ten down-regulated RARA-regulated genes with 2.0 fold or greater change when comparing the cKO to the WT values are listed (Table 3-3) for both the P4 and P8 gene sets. Real time PCR was performed for some highly changing genes from the P4 and P8 gene sets to confirm the transcriptional changes on microarray. The selected genes for the

P4 gene sets are Xaf1, XIAP associated factor 1, Gfap, glial fibrillary acidic protein, Tm4sf5, transmembrane 4 superfamily member 5, Pnmt, phenylethanolamine-N-methyltransferase,

Kcnc4, potassium voltage gated channel, Shaw-related subfamily, member 4, Coro1a, coronin, actin binding protein 1A, Tfap2c, transcription factor AP-2, gamma, and Lepr, leptin receptor.

Fold changes from the real time PCR were similar to the transcriptional changes observed from the microarray results (Fig. 3-6).

The selected genes for the P8 gene set are Eme2, essential meiotic endonuclease 1 homolog 2 (S. pombe), Dmc1, DMC1 dosage suppressor of mck1 homolog, meiosis-specific homologous recombination (yeast), Epcam, epithelial cell adhesion molecule, Rhox13, reproductive homeobox 13, Tex101, testis expressed gene 101, Smc1b, structure maintenance of chromosomes 1B, Prss50, protease, serine, 50, Tex11, testis expressed gene 11, Ly6k, lymphocyte antigen 6 complex, locus K, Taf7l, TAF7-like RNA polymerase II, TATA box binding protein (TBP)-associated factor, Sycp1, synaptonemal complex protein 1, and Spp1, secreted phosphoprotein 1. Fold changes from real time PCR were similar to the transcriptional changes observed from the microarray results (Fig. 3-7).

136 Rara regulation of double strand break (DSB) repair-related genes

RARA has been indicated to be involved in the resolution of DSBs (Chapter 2). During

meiosis, precisely controlled steps of DNA recombination between homologous chromosomes

are carried out by an array of proteins (Bolcun-Filas and Schimenti, 2012; Amunugama and

Fishel, 2012). A large group of these proteins are found differentially regulated in the cKO germ

cells from animals at P8 (Fig. 3-8). For example, PRDM9, PR domain protein 9, is

thought to be a major determinant of meiotic recombination hotspots in humans and mice

(Baudat et al., 2009). SPO11, Spo11 meiotic protein covalently bound to DSB homolog (S.

cerevisiae), is thought to mediate DNA cleavage and form DSBs that initiate meiotic

recombination. TEX11, testis expressed 11, binds to the DSB sites. TEX15, testis expressed 15,

BRCA1, breast cancer 1 and BRCA2, breast cancer 2, are three proteins that play central roles in

DNA repair (Yang et al., 2008). EME2, essential meiotic endonuclease 1 homolog 2 (S. pombe),

in a complex with MUS81, acts as a DNA structure-specific 3'-flap endonuclease. There are also

groups of cohesin proteins, synaptonemal complex proteins, and synaptic checkpoint proteins

that are differentially regulated (Fig. 3-8).

Rara regulates potential DMRT1 target genes

The DM domain proteins Double sex- and MAB-3-related transcription factor 1

(DMRT1) acts as primary sex-determining gene in vertebrate clades including fish, amphibians,

and probably birds (Matsuda et al., 2002; Smith et al., 2009; Yoshimoto et al., 2008). It has

diverse and essential roles in the development of the mouse testis (Raymond et al., 2000). 15

transcripts differentially regulated in the Rara germ cell-specific deleted testes that have promoters binding to DMRT1 (Murphy et al., 2010) (Table 3-9). Their corresponding genes were

137 Bcat1, branched chain aminotransferase 1, cytosolic, Kcnc4, potassium voltage gated channel,

Shaw-related subfamily, member 4, Prrx1, pair related homeobox 1, Adamts20, A disintegrin- like and metallopeptidase (reprolysin type) with thrombospondin type I motif, 20, Acta2, actin, alpha2, smooth muscle, aorta, Bnc2, basonuclin 2, Col4a4, collagen, type IV, alpha 4, Aff3,

AF4/FMR2 family, member 3, Igfbp6, Insulin-Like growth factor binding protein 6, Col4a3, collagen, Type IV, alpha 3, Icosl, Icos ligand, Cldnd1, claudin domain containing 1, Spp1, secreted phophoprotein 1, Ppm1e, protein phophatase 1E (PP2C domain containing) and Nfia, /A.

DISCUSSION

RARA mediates the effect of RA, a major paracrine factor that regulates spermatogonial differentiation and meiosis. The present study profiled the transcriptome changes of the enriched germ cell-specific Rara cKO germ cells compared to the WT germ cells to discover target genes of RARA. In the absence of RARA, the expression of target genes is expected to be mis- regulated. Pathway analysis provided insights into the two functional clusters regulated by

RARA for the P4 (Table 3-5, Table 3-7, A1-1, and A1-3) and P8 gene sets (Table 3-6, 3-8, A1-2, and A1-4). Top six functional clusters were cell differentiation, cell adhesion, cell migration, pregnancy, contraction, and cell proliferation for the P4 gene set. These functional clusters fit well with the type of germ cells present in P4 seminiferous epithelium, including gonocytes, undifferentiated and differentiated spermatogonia, which are undergoing mitosis. On the other hand, the top five functional clusters were synapsis, male meiosis, spermatogenesis, synaptonemal complex, and crossover for the P8 gene set. During meiosis, germ cells are geared up to go through the formation and repair of DSBs, chromosomal synapsis, and recombination.

138 These demonstrate that RARA has the potential to regulate genes involved in spermatogonial

differentiation, migration, and proliferation at P4 and meiosis, synapsis, and crossover during

recombination at P8.

In addition, genes on the X and Y-chromosomes have a predominant role in pre-meiotic

stages of mammalian spermatogenesis (Maclean et al., 2005; Wang et al., 2001). Interestingly,

among the 360 annotated unique transcripts from the P8 gene set that are differentially regulated

at ± 2.0 fold or greater, 7.6% (1 on Y chromosome, 32 on X chromosome) are X or Y-linked. At

least 8.1% of them are germ cell specific, compared with the top 50 changing genes differentially

expressed in E14 wild type 129 gonads and W/Wv gonads (Rolland et al., 2011).

We examined if the genes identified in the P4 and P8 lists have RAREs and if they could be regulated directly by RARA. 93 and 73 unique transcripts were found to be regulated by retinol (ROL), RA, and RARA at P4 and P8, respectively (Fig. 3-3). However, only a small portion of transcripts have canonical RAREs (Table 3-4), suggesting that most of the differentially regulated genes are secondary or tertiary targets of RARA. Further investigations are warranted to investigate the role of the three genes with canonical RAREs during spermatogonial migration, mitosis, and differentiation and ten genes with canonical RAREs during meiosis, crossover, and synapsis.

The top three genes up-regulated at P4, when RARA function is deleted, were

Phosphatidylinositol 4-kinase type 2 beta (Pi4k2b), XIAP associated factor 1 (Xaf1), and Ctr9,

Paf1/RNA polymerase II complex component, homolog (S. cerevisiae) (Table 3-3). Together,

PI4K2A and the type III PI4Ks (PIK4CA and PIK4CB) contribute to the overall PI4-kinase activity of the cell and are involved in the regulation of vesicular trafficking, which is especially significant for the plasma membrane as well as the endosomal and Golgi compartments. XAF1

139 may act to negatively regulate the inhibitor of apoptosis protein (IAP) family. During the

differentiation of undifferentiated spermatogonia to differentiating spermatogonia (A1-A4), apoptosis of A2-A4 spermatogonia has been shown to occur (de Rooij, 2001). CTR9 is a

component of the Platelet-activating factor acetylhydrolase 1 (PAF1 complex, PAF1C). The

transcription of Hox and Wnt target genes requires PAF1C. This complex is involved in the

methylation of histone H3 lysine 3 (H3K4me3).

The top three genes down-regulated in germ cells of Rara cKO mice at P4 were Nuclear

distribution gene E-like homolog 1 (A. nidulans) (Ndel1), RIKEN cDNA 2610507I01 gene

(2610507I01Rik), and Solute carrier family 25 (mitochondrial carrier, peroxisomal membrane

protein), member 17 (Slc25a17) (Table 3-3). NDEL1 is required to organize the cellular

microtubule array and anchor at the centrosome. In a complex with Platelet-

activating factor acetylhydrolase 1b, regulatory subunit 1 (LIS1), NDEL1 is involved in polarity

formation and microtubule organization during neurogenesis (Yamada M et al., 2010). The

complete loss of Ndel1 results in neuronal migration defects. This gene may be important for

spermatogonial stem cell migration and movement of germ cells along the membranes of Sertoli

cells. SLC25A17 is a peroxisomal transporter, catalyzing coenzyme A and other cofactors from

the cytosol into the peroxisomal matrix by a counter-exchange mechanism.

During meiosis, germ cells progress through the formation and repair of DSBs,

chromosomal synapsis and recombination. The top three up-regulated genes in germ cells of

Rara cKO mice at P8 were Glial fibrillary protein (Gfap), XIAP associated factor 1 (Xaf1), and

Potassium voltage gated channel, Shaw-related subfamily, member 4 (Kcnc4) (Table 3-3). GFAP

is a class-III intermediate filament, which acts as a marker to distinguish astrocytes from other

glial cells. XAF1 may act to negatively regulate the inhibitor of apoptosis protein (IAP) family,

140 shared with the P4 data set. KCNC4 exerts the effect of the voltage-dependent potassium ion permeability of excitable membranes. The top three down-regulated genes in germ cells of Rara cKO mice were Essential meiotic endonuclease 1 homolog 2 (S. pombe) (Eme2), Zinc finger,

DHHC domain containing 14 (Zdhhc14), and Protease, serine 50 (Prss50) (Table 3-3). Essential meiotic endonuclease 1 homolog 2 (EME2) forms a complex with MUS81 and acts as a DNA endonuclease that has high efficiency in cleaving 3'-flap structures (Fig. 3-8). ZDHHC14 has no known functions, while PRSS50 has threonine endopeptidase activity.

A recent study by our laboratory showed an unrepaired DSB phenotype by immunostaining for γH2AX in Rara cKO pachytene spermatocytes at P15 (Chapter 2). It is highly possible that EME2 is involved in the DSB repair pathway (Fig. 3-8), along with all the proteins known to be critical players during the meiotic recombination. EME2 interacts with

MUS81 endonuclease homolog (S. cerevisiae) to form a DNA structure-specific endonuclease

(xeroderma pigmentosa complementatin group F, also known as ERCC4, XPF-type), which cleaves substrates such as 3'-flap structures (Ciccia et al., 2008). It is involved in the replication fork during DNA synthesis, which is a process preceding meiosis. Interestingly, Mus81-/- has defects in the homologous recombination pathway. It collaborates with Flap structure-specific endonuclease 1 (FEN1), by interacting with MUS81 (Shin et al., 2012). A decrease in Eme2 expression caused by the loss of RARA suggests that EME2 is a target of RARA. It is interesting that when P2 testes were treated in organ culture with WIN18,446, a compound that inhibits the conversion of ROL to RA, Eme2 was down regulated 96.4 fold (Hogarth et al., 2011). However,

Eme2 has no RAREs (TRED).

Furthermore, 15 genes from the P4 gene set code for proteins that bind DMRT1 (Table 3-

9). Cell type-specific gene targeting unraveled the role of DMRT1 in germ cell differentiation,

141 proliferation, migration, and pluripotency of germ cells (Kim et al., 2007; Krezel et al., 1996).

DMRT1 is expressed in undifferentiated spermatogonia, premeiotic germ cells and Sertoli cells.

DM-domain proteins bind as homodimers or heterodimers with other DM-domain proteins to similar consensus elements in vitro and in vivo (Erdman et al., 1996; Murphy et al., 2010;

Murphy et al., 2007; Yi and Zarkower, 1999).

To better understand how DMRT1 directs mouse testicular development and function,

Murphy and colleagues performed an analysis with chromatin immunoprecipitation and microarray (ChIP-chip) (Murphy et al., 2010). Using P9 testicular mRNA from wild type and germ cell-specific Dmrt1 deleted testes, they identified DMRT1-bound genes that are abnormally regulated (Murphy et al., 2010). One of these candidate transcripts is Branched chain aminotransferase 1, cytosolic (Bcat1), which is germ cell specific and up-regulated 3.7 fold in cKO germ cells. BCAT1 catalyzes the first reaction in the catabolism of the essential branched chain amino acids leucine, isoleucine, and valine, as well as taking part in the pantothenate and

CoA biosynthesis pathways. It is a direct target of c- (Eden et al., 1996), and may be involved in the G1/S transition of the cell cycle (Schuldiner et al., 1996). Furthermore, Bcat1 is up-regulated upon Dmrt1 deletion (Murphy et al., 2010). Importantly, DMRT1 was shown to be an interacting partner with RARA (Zhu et al., 2010), suggesting DMRT1 and RARA may be co- regulated during spermatogonial differentiation and meiosis.

The present transcriptome analysis suggests that RARA mediates RA signal to mainly orchestrate cell differentiation and meiosis at two different developmental stages of germ cells,

P4 and P8, respectively. The direct and indirect target genes identified in the study provide a valuable knowledge base for the investigation of the mechanism of RA action mediated through

RARA.

142 REFERENCE

Al Tanoury, Z., Piskunov, A., Rochette-Egly, C., 2013. Vitamin A and retinoid signaling:

genomic and non-genomic effects. Journal of lipid research.

Adelman, C.A., Petrini, J.H., 2008. ZIP4H (TEX11) deficiency in the mouse impairs meiotic

double strand break repair and the regulation of crossing over. PLoS genetics 4,

e1000042.

Amunugama, R., Fishel, R., 2012. Homologous recombination in eukaryotes. Progress in

molecular biology and translational science 110, 155-206.

Bastien, J., Rochette-Egly, C., 2004. Nuclear retinoid receptors and the transcription of retinoid-

target genes. Gene 328, 1-16.

Baudat, F., Buard, J., Grey, C., Fledel-Alon, A., Ober, C., Przeworski, M., Coop, G., de Massy,

B., 2010. PRDM9 is a major determinant of meiotic recombination hotspots in humans

and mice. Science 327, 836-840.

Bolcun-Filas, E., Schimenti, J.C., 2012. Genetics of meiosis and recombination in mice.

International review of cell and molecular biology 298, 179-227.

Chambon, P., 1996. A decade of molecular biology of retinoic acid receptors. FASEB J 10, 940-

954.

Chen, J., Silver, D.P., Walpita, D., Cantor, S.B., Gazdar, A.F., Tomlinson, G., Couch, F.J.,

Weber, B.L., Ashley, T., Livingston, D.M., Scully, R., 1998. Stable interaction between

the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic

cells. Molecular cell 2, 317-328.

Ciccia, A., McDonald, N., West, S.C., 2008. Structural and functional relationships of the

143 XPF/MUS81 family of proteins. Annual review of biochemistry 77, 259-287.

Clagett-Dame, M., Knutson, D., 2011. Vitamin A in reproduction and development. Nutrients 3,

385-428.

Cloud, V., Chan, Y.L., Grubb, J., Budke, B., Bishop, D.K., 2012. Rad51 is an accessory factor

for Dmc1-mediated joint molecule formation during meiosis. Science 337, 1222-1225.

Cotnoir-White, D., Laperriere, D., Mader, S., 2011. Evolution of the repertoire of nuclear

receptor binding sites in genomes. Molecular and cellular endocrinology 334, 76-82. de Rooij, D.G., 2001. Proliferation and differentiation of spermatogonial stem cells.

Reproduction 121, 347-354. de The, H., Vivanco-Ruiz, M.M., Tiollais, P., Stunnenberg, H., Dejean, A., 1990. Identification

of a retinoic acid responsive element in the retinoic acid receptor beta gene. Nature 343,

177-180.

Doerksen, L.F., Bhattacharya, A., Kannan, P., Pratt, D., Tainsky, M.A., 1996. Functional

interaction between a RARE and an AP-2 binding site in the regulation of the human

HOX A4 gene promoter. Nucleic acids research 24, 2849-2856.

Drumond, A.L., Meistrich, M.L., Chiarini-Garcia, H., 2011. Spermatogonial morphology and

kinetics during testis development in mice: a high-resolution light microscopy approach.

Reproduction 142, 145-155.

Eden, A., Simchen, G., Benvenisty, N., 1996. Two yeast homologs of ECA39, a target for c-Myc

regulation, code for cytosolic and mitochondrial branched-chain amino acid

aminotransferases. The Journal of biological chemistry 271, 20242-20245.

144 Erdman, S.E., Chen, H.J., Burtis, K.C., 1996. Functional and genetic characterization of the

oligomerization and DNA binding properties of the Drosophila doublesex proteins.

Genetics 144, 1639-1652.

Griswold, M.D., Hogarth, C.A., Bowles, J., Koopman, P., 2012. Initiating meiosis: the case for

retinoic acid. Biology of reproduction 86, 35.

Hawley, R.S., Friend, S.H., 1996. Strange bedfellows in even stranger places: the role of ATM in

meiotic cells, lymphocytes, tumors, and its functional links to . Genes & development

10, 2383-2388.

Hogarth, C.A., Evanoff, R., Snyder, E., Kent, T., Mitchell, D., Small, C., Amory, J.K., Griswold,

M.D., 2011. Suppression of Stra8 expression in the mouse gonad by WIN 18,446.

Biology of reproduction 84, 957-965.

Huang, D., Chen, S.W., Langston, A.W., Gudas, L.J., 1998. A conserved retinoic acid responsive

element in the murine Hoxb-1 gene is required for expression in the developing gut.

Development 125, 3235-3246.

Huang, H.F., Hembree, W.C., 1979. Spermatogenic response to vitamin A in vitamin A deficient

rats. Biology of reproduction 21, 891-904.

Kim, S., Bardwell, V.J., Zarkower, D., 2007. Cell type-autonomous and non-autonomous

requirements for Dmrt1 in postnatal testis differentiation. Developmental biology 307,

314-327.

Krezel, W., Dupe, V., Mark, M., Dierich, A., Kastner, P., Chambon, P., 1996. RXR gamma null

mice are apparently normal and compound RXR alpha +/-/RXR beta -/-/RXR gamma -/-

mutant mice are viable. Proc Natl Acad Sci USA 93, 9010-9014.

145 Lalevee, S., Anno, Y.N., Chatagnon, A., Samarut, E., Poch, O., Laudet, V., Benoit, G.,

Lecompte, O., Rochette-Egly, C., 2011. Genome-wide in silico identification of new

conserved and functional retinoic acid receptor response elements (direct repeats

separated by 5 bp). The Journal of biological chemistry 286, 33322-33334.

Langston, A.W., Gudas, L.J., 1992. Identification of a retinoic acid responsive enhancer 3' of the

murine homeobox gene Hox-1.6. Mechanisms of development 38, 217-227.

Lauberth, S.M., Nakayama, T., Wu, X., Ferris, A.L., Tang, Z., Hughes, S.H., Roeder, R.G.,

2013. H3K4me3 interactions with TAF3 regulate preinitiation complex assembly and

selective gene activation. Cell 152, 1021-1036.

Lehmann, J.M., Zhang, X.K., Pfahl, M., 1992. RAR gamma 2 expression is regulated through a

retinoic acid response element embedded in Sp1 sites. Molecular and cellular biology 12,

2976-2985.

Leid, M., Kastner, P., Chambon, P., 1992. Multiplicity generates diversity in the retinoic acid

signalling pathways. Trends in biochemical sciences 17, 427-433.

Leroy, P., Nakshatri, H., Chambon, P., 1991. Mouse retinoic acid receptor alpha 2 isoform is

transcribed from a promoter that contains a retinoic acid response element. Proc Natl

Acad Sci U S A 88, 10138-10142.

Loudig, O., Maclean, G.A., Dore, N.L., Luu, L., Petkovich, M., 2005. Transcriptional co-

operativity between distant retinoic acid response elements in regulation of Cyp26A1

inducibility. The Biochemical journal 392, 241-248.

Lufkin, T., Lohnes, D., Mark, M., Dierich, A., Gorry, P., Gaub, M.P., LeMeur, M., Chambon, P.,

1993. High postnatal lethality and testis degeneration in retinoic acid receptor alpha

mutant mice. Proc Natl Acad Sci USA 90, 7225-7229.

146 Maclean, J.A., 2nd, Chen, M.A., Wayne, C.M., Bruce, S.R., Rao, M., Meistrich, M.L., Macleod,

C., Wilkinson, M.F., 2005. Rhox: a new homeobox gene cluster. Cell 120, 369-382.

MacQueen, A.J., Hochwagen, A., 2011. Checkpoint mechanisms: the puppet masters of meiotic

prophase. Trends in cell biology 21, 393-400.

Matsuda, M., Nagahama, Y., Shinomiya, A., Sato, T., Matsuda, C., Kobayashi, T., Morrey, C.E.,

Shibata, N., Asakawa, S., Shimizu, N., Hori, H., Hamaguchi, S., Sakaizumi, M., 2002.

DMY is a Y-specific DM-domain gene required for male development in the medaka

fish. Nature 417, 559-563.

Murphy, M.W., Sarver, A.L., Rice, D., Hatzi, K., Ye, K., Melnick, A., Heckert, L.L., Zarkower,

D., Bardwell, V.J., 2010. Genome-wide analysis of DNA binding and transcriptional

regulation by the mammalian Doublesex homolog DMRT1 in the juvenile testis. Proc

Natl Acad Sci U S A 107, 13360-13365.

Murphy, M.W., Zarkower, D., Bardwell, V.J., 2007. Vertebrate DM domain proteins bind

similar DNA sequences and can heterodimerize on DNA. BMC molecular biology 8, 58.

Parvanov, E.D., Petkov, P.M., Paigen, K., 2010. Prdm9 controls activation of mammalian

recombination hotspots. Science 327, 835.

Phillips, B.T., Gassei, K., Orwig, K.E., 2010. Spermatogonial stem cell regulation and

spermatogenesis. Philosophical transactions of the Royal Society of London. Series B,

Biological sciences 365, 1663-1678.

Raymond, C.S., Murphy, M.W., O'Sullivan, M.G., Bardwell, V.J., Zarkower, D., 2000. Dmrt1, a

gene related to worm and fly sexual regulators, is required for mammalian testis

differentiation. Genes Dev 14, 2587-2595.

147 Rolland, A.D., Lehmann, K.P., Johnson, K.J., Gaido, K.W., Koopman, P., 2011. Uncovering

gene regulatory networks during mouse fetal germ cell development. Biology of

reproduction 84, 790-800.

Romanienko, P.J., Camerini-Otero, R.D., 2000. The mouse Spo11 gene is required for meiotic

chromosome synapsis. Molecular cell 6, 975-987.

Russell, L.D., Ettlin, E.R., Hikim, A.P.S., Clegg, E.D., 1990. Mammalian Spermatogenesis, p.1-

38. In Russell, L.D., Ettlin, E.R., Hikim, A.P.S., Clegg, E.D., (ed.), Histological and

Histopathological Evaluation of the Testis. Cache River Press, Clearwater.

Schuldiner, O., Eden, A., Ben-Yosef, T., Yanuka, O., Smchen, G., Benvenisty, N., 1996.

ECA39, a conserved gene regulated by c-Myc in mice, is involved in G1/S cell cycle

regulation in yeast. Proc Natl Acad Sci U S A 93, 7143-7148.

Shin, Y.K., Amangyeld, T., Nguyen, T.A., Munashingha, P.R., Seo, Y.S., 2012. Human MUS81

complexes stimulate flap endonuclease 1. The FEBS journal 279, 2412-2430.

Smith, C.A., Roeszler, K.N., Ohnesorg, T., Cummins, D.M., Farlie, P.G., Doran, T.J., Sinclair,

A.H., 2009. The avian Z-linked gene DMRT1 is required for male sex determination in

the chicken. Nature 461, 267-271.

Umesono, K., Murakami, K.K., Thompson, C.C., Evans, R.M., 1991. Direct repeats as selective

response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell

65, 1255-1266. van Pelt, A.M.M.V., de Rooij, D.G.D., 1990. Synchronization of the Seminiferous Epithelium

after Vitamin A Replacement in Vitamin A-Deficient Mice. Biology of Reproduction 43,

363-367.

148 Wang, P.J., McCarrey, J.R., Yang, F., Page, D.C., 2001. An abundance of X-linked genes

expressed in spermatogonia. Nature genetics 27, 422-426.

Williams, R.S., Williams, J.S., Tainer, J.A., 2007. Mre11-Rad50-Nbs1 is a keystone complex

connecting DNA repair machinery, double-strand break signaling, and the chromatin

template. Biochemistry and cell biology = Biochimie et biologie cellulaire 85, 509-520.

Wolbach, S.B., Howe, P.R., 1925. Tissue changes following deprivation of fat-soluble A

vitamin. J Exp Med 42, 753-777.

Yamada, M., Hirotsune, S., Wynshaw-Boris, A., 2010. The essential role of LIS1, NDEL1 and

Aurora-A in polarity formation and microtubule organization during neurogensis. Cell

adhesion & migration 4, 180-184.

Yang, F., Eckardt, S., Leu, N.A., McLaughlin, K.J., Wang, P.J., 2008. Mouse TEX15 is essential

for DNA double-strand break repair and chromosomal synapsis during male meiosis. The

Journal of cell biology 180, 673-679.

Yi, W., Zarkower, D., 1999. Similarity of DNA binding and transcriptional regulation by

Caenorhabditis elegans MAB-3 and DSX suggests conservation

of sex determining mechanisms. Development 126, 873-881.

Yoshida, S., Sukeno, M., Nakagawa, T., Ohbo, K., Nagamatsu, G., Suda, T., Nabeshima, Y.,

2006. The first round of mouse spermatogenesis is a distinctive program that lacks the

self-renewing spermatogonia stage. Development 133, 1495-1505.

Yoshimoto, S., Okada, E., Umemoto, H., Tamura, K., Uno, Y., Nishida-Umehara, C., Matsuda,

Y., Takamatsu, N., Shiba, T., Ito, M., 2008. A W-linked DM-domain gene, DM-W,

participates in primary ovary development in Xenopus laevis. Proc Natl Acad Sci U S A

105, 2469-2474.

149 Zhu, L., Santos, N.C., Kim, K.H., 2010. Disulfide isomerase glucose-regulated protein 58 is

required for the nuclear localization and degradation of retinoic acid receptor alpha.

Reproduction 139, 717-731.

150 Figure 3-1. Germ cell enrichment with a two-step digestion.

151 Figure 3-1. Germ cell enrichment with two-step digestion.

Testes from WT and cKO mice in the ¾ C57Bl/6 and ¼ FVB genetic background were detunicated and first digested in HBSS (pH 7.4) containing 0.01 mg/ml collagenase at 37°C until tubules were free-flowing. Then loose tubules were washed with HBSS twice to remove the interstitial cells before they were digested further with 1 mg/ml collagenase, 2 µg/ml DNaseI, 1.5 mg/ml hyaluronidase, and 0.5 mg/ml trypsin (0.05%). Once single cell suspension was obtained,

FBS was used to stop the trypsin activity. Cell suspension was passed through a 40 µm-mesh cell strainer before being spun down, washed and RNA extracted for microarray hybridization (n =3).

152 Figure 3-2. Real time PCR analysis of transcripts for somatic cell contamination in the enriched germ cell isolation procedure.

153 Figure 3-2. Real time PCR analysis of transcripts for somatic cell contamination in the enriched germ cell isolation procedure.

Gata4 was used as the Sertoli cell marker. Hsd3b1 was used as the Leydig cell marker.

Both Gata4 and Hsd3b1 in the two time points were not significantly different, suggesting the level of somatic cell contamination is about the same between WT and cKO. Stra8 was used as the germ cell marker. Stra8 expression was decreased by 1.6 and 3.9 fold in the cKO at P4 and

P8, respectively. Rara expression was decreased by 4.7 and 2.8 fold in the cKO at P4 and P8, respectively. The Y-axis denotes the relative fold change expressed with ± SEM of the transcript level, in relation to that of the WT. Real time PCR was performed in technical duplicates and biological triplicates. Asterisks denote p values, indicated below on each graph.

154 Figure 3-3. Venn Diagram.

155 Figure 3-3. Venn diagram.

The number of unique genes regulated by retinoic acid (RA), retinol (ROL) or RARA in P4 and

P8 germ cells that are differentially regulated ± 2.0 fold. Using Pathway Studio 9.0, the total number of genes regulated by RA, ROL and RARA was determined to be 530. The total number of unique transcripts that are differentially regulated ±2.0 fold is 283 for P4 and 360 for P8.

Among the 530 genes, 93 were found in the P4 list, and 73 were found in the P8 list. Between P4 and P8 gene sets, there are 80 genes overlapping.

156 Figure 3-4. Pathway analysis of common biological functions from transcripts differentially regulated at ± 3 fold or higher in P4 germ cells.

157 Figure 3-4. Pathway analysis of common biological functions from transcripts differentially regulated at ± 3 fold or higher in P4 germ cells.

Red color denotes up-regulated transcripts or pathways, whereas green color denotes down- regulated transcripts. Numbers represent the fold changes of each gene. Dotted arrows represent a regulation relationship, with the plus symbol identifying a positive regulation.

158 Figure 3-5. Pathway analysis of common biological functions from transcripts differentially regulated at ± 3 fold or higher in P8 germ cells.

159 Figure 3-5. Pathway analysis of common biological functions from transcripts differentially regulated at ± 3 fold or higher in P8 germ cells.

Green color denotes down-regulated transcripts. Numbers represent fold changes for each gene.

A regulation relationship is indicated by a dotted arrow, and a plus symbol means a positive regulation.

160 Figure 3-6. Real time PCR verification of transcriptional changes in P4 germ cells.

161 Figure 3-6. Real time PCR verification of transcriptional changes in P4 germ cells.

Real time PCR of Xaf1, Gfap, Tm4sf5, Pnmt, Kcnc4, Coro1a, Tfap2c and Lepr from the

P4 list. The Y-axis denotes the relative fold change expressed with ± SEM of the transcript level, in relation to that of the WT. Real time PCR was performed in technical duplicates and biological triplicates, * p < 0.05. All the relative fold changes confirmed the changes in the microarray.

162 Figure 3-7. Real time PCR verification of transcriptional changes in P8 germ cells.

163 Figure 3-7. Real time PCR verification of transcriptional changes in P8 germ cells.

Real time PCR of Eme2, Dmc1, Epcam, Rhox13, Tex101, Smc1b, Prss50, Tex11, Ly6k,

Taf7l, Sycp1, and Spp1. The Y-axis denotes the relative fold change expressed with ± SEM of the transcript level, in relation to that of the WT. Real time PCR was performed in technical duplicates and biological triplicates, * p < 0.05, and confirming the changes in the microarray.

164 Figure 3-8. Schematic drawing of transcripts in the P8 set that are involved in the DSB pathway.

165 Figure 3-8. Schematic drawing of transcripts in the P8 set that are involved in the DSB

pathway.

During the prophase of meiosis, homologous chromosomes pair and undergo

recombination, preferentially at sites that are termed hotspots. PRDM9 (Parvanov et al., 2010)

was shown to activate the recombination hotspots. SPO11 (Romanienko and Camerini-Otero,

2000), which belongs to the topoisomerase family, creates double strand breaks (DSBs) that are

required for recombination. TEX11 (Adelman and Petrini, 2008), the MRE11/RAD50/NBS1

complex (Williams et al., 2007), and ATM binds to the DSB sites. H2AX phosphorylated by

ATM, become γH2AX, and marks the double strand breaks (Hawley and Friend, 1996). TEX15

(Yang et al., 2008), BRCA1 and BRCA2 (Chen et al., 1998), and the cohesion complex along

with the SC proteins that hold the homologous chromosomes together were found to be involved

in the DSBs repair/recombination pathway (Bolcun-Filas and Schimenti, 2012). The proteins involved in the synapsis checkpoint ensure the proper progression of meiosis (MacQueen and

Hochwagen, 2011). RAD51 and DMC1 are two single stranded DNA binding proteins that mediate the D-loop formation during the repair (Cloud et al., 2012). The MUS81-EME1 or

MUS81-EME2 endonuclease complex resolves the joint molecules and results in interference- independent crossovers (Shin et al., 2012).

166 Table 3-1. Primers used for real time RT-PCR.

P4

Gene Name Forward Primer Reverse Primer Xaf1 TCCCAACTGCTAAGAAAGTCATCA AGGTGAGCGGTTATAGCAAAGC Tm4sf5 CCTGGTGGTCGCATCAAGT CACACCAAAGGTCGCATTCA Tfap2c GCTGAACCGGAAGAGAATGG GGCTTAGAGGTCCAGTCCCAAT Pnmt GAGCCTTTGACTGGAGTGTGTATAGT GCTCGAAGCTGGCGTTCTT Gfap GCACCTTCTCTGAGGCTGATTG GGACAACTGAGCGGACACTGT Lepr GGATATACTTGCCATGGTGAAGAA ACGGTGTTTTAAGCTTGAAAGGA Kcnc4 CGAGAGGAGACCAGTTGCAAT TCACCCGGCCTCTGCTATT Coro1a TGCCATGACAGTGCCTAGAAAG TCCTGCAGTGGGCGGATA

P8

Gene Name Forward Primer Reverse Primer Dmc1 ACTGCAGATCCAGGAGCAACTAT GAGCCAGAATGTGTCCACCAA Eme2 CCAGCAAGTCAGTAGCCACCTA TGGAGTCTCTGAGCACAAGGTAGA Epcam AGGAAGAAATCAGCAAAATATGAGAAG GCACGGCTAGGCATTAAGCT Rhox13 CAGGTCGAGGAAATGGAAAGC TTGCAAGCTCTCCCCTTGTAA Smc1b GTGGCTGCTCTGGCTCTTCT TCCAGGGCTGCATCTACTTCA Spp1 GATTTGCTTTTGCCTGTTTGG AGCTGCCAGAATCAGTCACTTTC Sycp1 CAGGAGGCAGCATGGAGAAG TCACGGCGGACACTTGACT Taf7l CCACCATGCGGTTGTTCAA CGATAACACAAGGCAGATTAACCA Tex11 GAAAGGCATCTTCCATGTGCTT GACGCACATAGATGCTTTCTTGA Tex101 TGAAGGAGACCTGTAGTTACCAGTCA TCTGAGAGGCCCCGATTTC Ly6k CTCGTAGTTCTGGGCTTACAGTTG TCTGCGCCTCACACACATG Prss50 TCTGTGCTGGCATCCTTATCG TGGTTCTGGCTCAAGCAATG

167 Table 3-2. Summary of differentially regulated probe sets.

Cut-off P4 P8 Total 45101 45101 # of probes* of signal 50 with p value < 0.05 3297 4814 # of probes of signal 50 with p value < 0.05 and changed ± 1.5 fold or greater 1636 1665 # of probes of signal 50 with p value < 0.05 and changed ± 2.0 fold or greater 322 407 # of probes of signal 50 with p value < 0.05 and changed ± 3.0 fold or greater 32 94

* Multiple probe sets were used to represent one transcript. There are over 45,000 probe sets representing over 39,000 transcripts and variants on the Mouse 430.2 microarray.

168 Table 3-3. Top RARA regulated genes in germ cells at P4 and P8.

P4

Gene Name Fold Change Gene Symbol Gene Title 1420411_a_at 10.200 Pi4k2b Phosphatidylinositol 4-Kinase Type 2 Beta 1443621_at 8.008 Xaf1 XIAP Associated Factor 1 1422679_s_at 6.434 Ctr9 Ctr9, Paf1/RNA Polymerase II Complex Component, Homolog (S. Cerevisiae) 1426509_s_at 5.798 Gfap Glial Fibrillary Acidic Protein 1424445_at 5.098 Tm4sf5 Transmembrane 4 Superfamily Member 5 1450606_at 3.740 Pnmt Phenylethanolamine-N-Methyltransferase 1437176_at 3.708 Nlrc5 NLR Family, CARD Domain Containing 5 1450871_a_at 3.696 Bcat1 Branched Chain Aminotransferase 1, Cytosolic 1417150_at 3.546 Slc6a4 Solute Carrier Family 6 (Neurotransmitter Transporter, Serotonin), Member 4 1418726_a_at 3.506 Tnnt2 T2, Cardiac 1440342_at -2.612 G530011O06Rik RIKEN Cdna G530011O06 Gene 1455374_at -3.024 Kcnj3 Potassium Inwardly-Rectifying Channel, Subfamily J, Member 3 1416658_at -3.262 Frzb Frizzled-Related Protein 1453540_at -3.296 5430404G13Rik RIKEN Cdna 5430404G13 Gene 1449827_at -3.432 Acan Aggrecan 1429074_at -4.477 1700026D08Rik RIKEN Cdna 1700026D08 Gene 1456156_at -4.630 Lepr Leptin Receptor 1424912_at -5.061 Slc25a17 Solute Carrier Family 25 (Mitochondrial Carrier, Peroxisomal Membrane Protein), Member 17 1453749_at -6.357 2610507I01Rik RIKEN Cdna 2610507I01 Gene 1424893_at -7.524 Ndel1 Nuclear Distribution Gene E-Like Homolog 1 (A. Nidulans)

P8

Gene Name Fold Change Gene Symbol Gene Title 1426509_s_at 10.324 Gfap Glial Fibrillary Acidic Protein 1443621_at 9.042 Xaf1 XIAP Associated Factor 1 1425090_s_at 6.710 Kcnc4 Potassium Voltage Gated Channel, Shaw-Related Subfamily, Member 4 1460285_at 6.539 Itga9 Integrin Alpha 9 1425530_a_at 5.082 Stx3 Syntaxin 3 1420549_at 4.181 Gbp1 Guanylate Binding Protein 1 1448265_x_at 3.796 Mpzl2 Myelin Protein Zero-Like 2 1444677_at 3.460 C77673 Expressed Sequence C77673 1424045_at 3.261 5730437N04Rik RIKEN Cdna 5730437N04 Gene 1449254_at 3.191 Spp1 Secreted Phosphoprotein 1 1453544_at -5.842 Dmrtc1c /// Dmrtc1c2 Dmrt-Like Family C1c /// Dmrt-Like Family C1c2 1457100_at -5.861 AW552889 Expressed Sequence AW552889 1429035_at -6.393 Dpep3 Dipeptidase 3 1453331_at -6.417 1700013H16Rik RIKEN Cdna 1700013H16 Gene 1455739_at -6.776 Gm4980 Predicted Gene 4980 1441886_at -6.816 Ccdc79 Coiled-Coil Domain Containing 79 1438975_x_at -7.067 Zdhhc14 Zinc Finger, DHHC Domain Containing 14 1439739_at -7.326 Prss50 Protease, Serine, 50 1437614_x_at -8.765 Zdhhc14 Zinc Finger, DHHC Domain Containing 14 1460628_at -26.598 Eme2 Essential Meiotic Endonuclease 1 Homolog 2 (S. Pombe)

169 Table 3-4. Canonical and non-canonical retinoic acid response elements (RAREs) in the proximal promoter region of genes that are regulated ± 2.0 fold in the P4 and P8 lists.

Direct repeat of the RGKTCA motif separated by zero, one, two, three, four, five, six, seven, eight, nine and ten base pairs are listed.

P4

Gene Name Fold Change Gene Symbol RARE 1428460_at 2.731 Syn2 DR-4 1418990_at 2.471 Ms4a4d DR-4 1427306_at 2.403 Ryr1 DR-0 1419324_at 2.307 Lhx9 DR-2 1419184_a_at 2.232 Fhl2 DR-7 1416454_s_at 2.202 Acta2 DR-4 1454969_at 2.123 Lypd6 DR-8 1453226_at 2.113 Kif18b DR-2 1417184_s_at 2.074 Hbb-b1 DR-8 1447833_x_at 2.013 Mfap2 DR-7 1416846_a_at 1.982 Pdzrn3 DR-7 1449254_at -2.027 Spp1 DR-3 1416178_a_at -2.196 Plekhb1 DR-5 1426873_s_at -2.446 Jup DR-7 1425177_at -2.481 Shmt1 DR-0

P8

Gene Name Fold Change Gene Symbol RARE 1449254_at 3.191 Spp1 DR-3, DR-9 1426632_at/ 1426633_s_at 2.064 Kctd14 DR-4 1452954_at 2.018 Ube2c DR-2 1438273_at -1.961 Gm13718 DR-0 1443483_at, 1422933_at -1.964 Xlr5a/Xlr5b/Xlr5c DR-2 1428460_at -1.977 Syn2 DR-4 1422592_at -1.978 Ctnnd2 DR-2 1453310_at -1.985 Ppil6 DR-2 1429566_a_at -2.070 Hipk2 DR-3 1424530_at -2.101 Sec14l2 DR-2, DR-8 1435132_at -2.104 Disp1 DR-2 1435793_at/ 1429466_s_at -2.113 Aph1b DR-3 1419394_s_at -2.208 S100a8 DR-4 1450542_s_at -2.571 Magea1/Magea3 DR-4 1418617_x_at/ 1430240_a_at -2.603 Clgn DR-6 1416178_a_at -2.667 Plekhb1 DR-5 1421424_a_at -2.730 Anpep DR-0 1421566_at -3.452 Pet2 DR-0 1427603_at/ 1430364_at/1442796_at -3.882 Atf7ip2 DR-2 1419147_at -3.931 Rec8 DR-1 1440692_at -3.957 Gm364 DR-0 1444122_at -3.975 Sycp2 DR-10 1449103_at -4.713 Tex101 DR-5 1431648_at -4.747 4930528F23Rik DR-0

170 Table 3-5. Top ten Pathway Studio subnetworks for the P4 gene set.

Group Term Gene #s p value Genes 1 Cell 114 4.13E-15 ↑Cd24a,↑Igf2,↑Entpd2,↑Mrvi1,↑Cav1,↑Cdh1,↑Sct,↑Myl9, Differentiation ↑Bmp7,↑Thbs2,↑Fhl2,↑Tnnt2,↑Ednrb,↑Fn1,↑Rapgef3,↑M yh11,↓Lepr,↑Tfap2A,↓Spp1,↑Prkg1,↑Cyp1b1,↑Pou5f1,↑C artpt,↑Actn3,↑Il18,↑Foxc2,↑Itga1,↑Igfbp6,↓Acan,↑Actg2, ↑Cyp11a1,↑Slc6a4,↑Igfbp2,↑Ptger4,↑Tcf23,↑Hbb-b1/Hbb -b2/LOC100503605,↑Tagln,↑Cyp17a1,↑Cxcl16,↑Bgn,↓Ju p,↑Mbl2,↑Gjc1,↑Pdgfra,↑Ccnd3,↑Tgfbr3,↑Thbd,↑H2-Q7, ↑Sorl1,↑Plcg2,↑Igfbp7,↓Shmt1,↑Icosl,↓Ccnd2,↑Apoc2,↑E tv4,↑Tfap2C,↑Ascl2,↑Hoxd10,↑Lama2,↑Ogn,↑Parp14,↓N fia,↑Crxos1,↑Il2rg,↑Itgae,↑Gfra1,↓Frzb,↑Lsp1,↑Hba-a1/H ba-a2,↑Sfrp1,↑Fap,↑Tbx2,↑Tcf21,↑Col1a2,↑Gfap,↑Twist2 ,↑Casq2,↑Meis2,↓Smarce1,↑S100a6,↓Ndel1,↑Lhx1,↑Itga8 ,↑Lin28a,↑Ifitm3,↓Trim25,↑Cnn2,↑Cd248,↓Rdh10,↑Piwil 4,↑Tnp1,↑Arx,↑Sall1,↑Hoxd8,↑Plb1,↑Matn2,↑Pdzrn3,↑Fb lim1,↑Syt13,↑Sepw1,↑Lhx9,↑Cbln1,↑Dppa2,↑Krt12,↑Hox d9,↓Msi2,↑Pkdcc,↑Slxl1,↓Cyb5d2,↑Tmem119,↑Cbln2,↑D pep1,↓Zfhx4 2 Cell Adhesion 50 1.46E-09 ↑Cd24a,↑Igf2,↑Entpd2,↑Cav1,↑Cdh1,↑Bmp7,↑Thbs2,↑Fh l2,↑Myh6,↑Ednrb,↑Fn1,↑Rapgef3,↓Spp1,↑Prkg1,↑Il18,↑F oxc2,↑Itga1,↑Igfbp6,↓Acan,↑Igfbp2,↑Ptger4,↑Cyp17a1,↑ Cxcl16,↑Bgn,↓Jup,↑Mbl2,↑Pdgfra,↑Thbd,↑Sorl1,↑Plcg2,↑ Igfbp7,↓Ccnd2,↑Col4a3,↑Ascl2,↑Lama2,↑Mfap2,↑Gfra1, ↑Sytl2,↑Lsp1,↑Sfrp1,↑Fap,↑Tbx2,↑Col1a2,↑S100a6,↑Col 4a4,↑Cd248,↑Matn2,↑Fblim1,↑Lgals2,↑Hoxd9 3 Cell Migration 60 4.40E-08 ↑Cd24a,↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Thbs2,↑Fhl2,↑Fn1,↑ Rapgef3,↑Tfap2A,↓Spp1,↑Prkg1,↑Cyp1b1,↑Pou5f1,↑Pnli p,↑Il18,↑Foxc2,↑Itga1,↑Igfbp6,↓Acan,↑Actg2,↑Igfbp2,↑Pt ger4,↑Tagln,↑Abcc9,↑Cxcl16,↑Bgn,↓Jup,↑Pdgfra,↑Tgfbr3 ,↑Thbd,↑Sorl1,↑Hif3a,↑Plcg2,↑Igfbp7,↑Col4a3,↑Etv4,↑Tf ap2C,↑Hoxd10,↑Lama2,↑Ecscr,↑Plvap,↑Itgae,↑Gfra1,↓Fr zb,↑Tm4sf5,↑Sfrp1,↑Fap,↑Coro1a,↑S100a6,↓Ndel1,↑Itga 8,↑Cnn2,↑Cd248,↑Arx,↑Adamts20,↑Matn2,↑Mcf2l,↑Fbli m1,↑Arhgap9 4 Pregnancy 37 8.21E-08 ↑Igf2,↑Entpd2,↑Cav1,↑Cdh1,↑Sct,↑Bmp7,↑Thbs2,↑Ednrb ,↑Fn1,↓Lepr,↓Spp1,↑Pou5f1,↑Il18,↑Igfbp6,↑Actg2,↑Cyp1 1a1,↑Slc6a4,↑Igfbp2,↑Serping1,↑Elovl6,↑Ptgis,↑Ptger4,↑ Cyp17a1,↑Mbl2,↑Gjc1,↑Thbd,↓Shmt1,↓Ccnd2,↑Etv4,↑Ld hc,↑Tfap2C,↑Ascl2,↓Nfia,↑Hba-a1/Hba-a2,↑S100a6,↑Kc ne4,↑Htra3 5 Contraction 37 1.67E-06 ↑Ryr1,↑Igf2,↑Cav1,↑Sct,↑Myl9,↑Thbs2,↑Tacr3,↑Fhl2,↑M yh6,↑Tnnt2,↑Ednrb,↑Fn1,↑Rapgef3,↑Myh11,↓Spp1,↑Prkg 1,↑Cartpt,↑Actn3,↑Il18,↑Itga1,↑Igfbp6,↑Actg2,↑Slc6a4,↑I gfbp2,↑Serping1,↑Ptgis,↑Ptger4,↑Hbb-b1/Hbb-b2/LOC10 0503605,↑Tagln,↑Abcc9,↑Gjc1,↑Pdgfra,↑Tpm2,↑Thbd,↓ Ccnd2,↑Lama2,↑Casq2 6 Cell Proliferation 101 2.42E-06 ↑1700026L06Rik,↑Igf2,↑Entpd2,↑Cav1,↑Cdh1,↑Sct,↑Bm p7,↑Thbs2,↑Fhl2,↑Ednrb,↑Fn1,↑Rapgef3,↑Myh11,↓Lepr, ↑Tfap2A,↓Spp1,↑Prkg1,↑Cyp1b1,↑Pou5f1,↑Cartpt,↑Pnlip ,↑Il18,↑Stc2,↑Foxc2,↑Itga1,↑Igfbp6,↓Acan,↑Actg2,↑Cyp1 1a1,↑Slc6a4,↑Igfbp2,↑Serping1,↑Ptgis,↑Ptger4,↑Tagln,↑C yp17a1,↑Cxcl16,↑Bgn,↓Jup,↑Pdgfra,↑Ccnd3,↑Tgfbr3,↑Th bd,↑H2-Q7,↑Sorl1,↑Plcg2,↑Igfbp7,↓Shmt1,↑Icosl,↓Ccnd2 ,↑Col4a3,↑Etv4,↑Tfap2C,↑Ascl2,↑Figla,↑Hoxd10,↑Lama 2,↑Ogn,↓Kcnj3,↑Il2rg,↑Itgae,↑Gfra1,↓Frzb,↑Tm4sf5,↑Gb p3,↑Sfrp1,↑Fap,↑Tbx2,↑Coro1a,↑Tcf21,↑Col1a2,↑Apcdd 1,↑Gfap,↑Twist2,↑Meis2,↑Kcnc4,↓Smarce1,↑S100a6,↓Nd el1,↑Itga8,↑Lin28a,↑Ifitm3,↑Col4a4,↓Trim25,↑Cnn2,↑Cd

171 248,↑Arx,↑Bcat1,↑Tbata,↑Unc45b,↓Plxna4,↑Mcf2l,↑Prr5, ↑Lhx9,↑Lgals2,↑Dppa2,↑Hoxd9,↑Htra3,↓Msi2,↑Osr2,↓Cy b5d2 7 Osteoblast 19 2.71E-06 ↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Thbs2,↑Fhl2,↑Fn1,↓Spp1,↑F Differentiation oxc2,↑Igfbp6,↑Igfbp2,↑Ptger4,↑Bgn,↓Frzb,↑Sfrp1,↑Prrx1, ↑Twist2,↑S100a6,↑Tmem119 8 Extracellular 12 6.99E-06 ↑Igf2,↑Cav1,↑Bmp7,↑Thbs2,↑Fhl2,↑Ednrb,↑Fn1,↓Spp1,↑ Matrix Pdgfra,↑Fap,↑Col1a2,↑Col5a2 Polymerization 9 Ovulation 15 7.75E-06 ↑Igf2,↑Cav1,↑Bmp7,↑Tnnt2,↑Ednrb,↑Fn1,↓Lepr,↓Acan,↑ Cyp11a1,↑Igfbp2,↑Ptger4,↑Igfbp7,↓Ccnd2,↑Etv4,↑Gfap 10 Apoptosis 95 8.74E-06 ↑Ryr1,↑Igf2,↑Entpd2,↑Cav1,↑Cdh1,↑Sct,↑Myl9,↑Bmp7,↑ Thbs2,↑Fhl2,↑Tnnt2,↑Ednrb,↑Fn1,↑Rapgef3,↑Myh11,↓Le pr,↑Tfap2A,↓Spp1,↑Prkg1,↑Cyp1b1,↑Pou5f1,↑Cartpt,↑Pn lip,↑Il18,↑Stc2,↑Itga1,↑Igfbp6,↓Acan,↑Actg2,↑Cyp11a1,↑ Slc6a4,↑Igfbp2,↑Serping1,↑Elovl6,↑Ptgis,↑Ptger4,↑Hbb- b1/Hbb-b2/LOC100503605,↑Tagln,↑Cyp17a1,↑Cxcl16,↑ Bgn,↓Jup,↑Mbl2,↑Gjc1,↑Adcy5,↑Pdgfra,↑Ccnd3,↑Tgfbr3, ↑Thbd,↑H2-Q7,↑Hif3a,↑Plcg2,↑Igfbp7,↑Icosl,↓Ccnd2,↑C ol4a3,↑Etv4,↑Tfap2C,↑Hoxd10,↑Lama2,↑Parp14,↑Plvap, ↑Il2rg,↑Itgae,↓Frzb,↑Lsp1,↑Hba-a1/Hba-a2,↑Sfrp1,↑Fap, ↑Tbx2,↑Coro1a,↑Tcf21,↑Gfap,↑Twist2,↑Dleu7,↑Kcnc4,↓ Smarce1,↑S100a6,↑Itga8,↑Lin28a,↑Col4a4,↑Cnn2,↑Parm 1,↑Tnp1,↑Sall1,↑Bcat1,↑Adamts20,↑Tshz3,↑Crygs,↑Fbli m1,↑Lgals2,↓Msi2,↑Fabp9,↑Gml,↑C1qb

172 Table 3-6. Top ten Pathway Studio subnetworks for the P8 gene set.

Group Term Gene #s p value Genes 1 Synapsis 17 1.36E-14 ↓Stra8,↓Dmc1,↓Spo11,↓Rec8,↓Msh5,↓Stag3,↓Sycp1,↓Sycp 2,↓Smc1b,↓Tex11,↓Mei1,↓Sycp3,↓Tex15,↓Tex12,↓Mov10l 1,↓Hormad1,↓Syce1 2 Male Meiosis 10 1.43E-12 ↓Boll,↓Stag3,↓Sycp2,↓Tex11,↓Mei1,↓Sycp3,↓Tex15,↓Slc25 a31,↓Mov10l1,↓Dmrtc2 3 Spermatogenesis 32 3.05E-09 ↓Timp2,↑Spp1,↓Lmna,↓Krt18,↓Stra8,↓Clgn,↓Tex101,↓Mtl5, ↓Dmc1,↓Spo11,↓Boll,↓Taf4b,↑Adamts2,↑Gfra1,↓Msh5,↓Prs s50,↓Sycp2,↓Gfer,↓Mei1,↓Sycp3,↓Prdm9,↑Hagh,↓Tex15,↓X lr,↓Slc25a31,↓Mov10l1,↓Hormad1,↓Dmrtc2,↓Syce1,↓Mael, ↓Hsf2bp,↓Zfp541 4 Synaptonemal 7 6.99E-09 ↓Dmc1,↓Spo11,↓Rec8,↓Msh5,↓Smc1b,↓Sycp3,↓Hormad1 Complex Formation 5 Crossover 6 9.24E-07 ↓Dmc1,↓Spo11,↓Msh5,↓Sycp1,↓Smc1b,↓Tex11 Formation 6 Pachytene 8 6.47E-06 ↓Dmc1,↓Spo11,↓Rec8,↓Tex11,↓Mei1,↓Gm98,↓Hormad1,↓R nf17 7 Synaptonemal 3 7.98E-06 ↓Stag3,↓Sycp2,↓Tex15 Complex Assembly 8 Meiosis 25 1.39E-05 ↓Pak1,↓Stra8,↓Dmc1,↓Spo11,↓Boll,↓Rec8,↓Msh5,↓Spdya,↓ Stag3,↓Sycp1,↓Sycp2,↓Brsk2,↓Smc1b,↓Tex11,↓Mei1,↓Gm9 8,↓Sycp3,↓Prdm9,↓Tex15,↓Slc25a31,↓Tex12,↓Hormad1,↓D mrtc2,↓Mael,↓Rnf17 9 Neurite 33 3.61E-05 ↑P2rx7,↓Trpc1,↓Timp2,↓Pak1,↑Spp1,↓Crk,↓Tfrc,↓Klf9,↓So Outgrowth cs3,↓H2-Q10,↑Itga9,↓Atf3,↑Wnt5a,↓Itgb3,↓Nes,↓Braf,↑Ptpr z1,↓Scn8a,↓Hck,↓Alcam,↑Gfra1,↓Kidins220,↑Gfap,↓Pou4f1 ,↓Ndrg4,↓Nck2,↓Rab6b,↑Stx3,↑Negr1,↑Rgmb,↓Nptx2,↓Mic all2,↓Gpc2 10 Cell Growth 74 9.68E-05 ↑P2rx7,↓Entpd2,↓Trpc1,↓Timp2,↓Chga,↓Rel,↓Nr4a3,↑Igfbp 5,↓Pak1,↑Spp1,↓Crk,↓Tfrc,↓Prkd3,↓Klf9,↑Fgfr2,↓Pth1r,↓So cs3,↓H2-Q10,↑Itga9,↓Igfbp2,↓Spint1,↓Vldlr,↓Ptger1,↓Nmb, ↓Atf3,↑Wnt5a,↓S100a8,↓Itgb3,↑Cth,↓Nes,↓Braf,↓Lmna,↓Kr t18,↓Sgpl1,↑Sox18,↓Anpep,↑Ptprz1,↓Dmrtb1,↑Fbln2,↓Rgs1 6,↓Egr3,↓Hck,↑Ube2c,↓Tnfrsf13c,↓Alcam,↓Epcam,↑Gfra1,↓ Runx3,↓Spdya,↓Dkk3,↑,↑Synpo2,↑Lgals7,↓Hipk2,↓Rif 1,↓Sec14l2,↓Crabp1,↑Gfap,↓Tshb,↓Plekhf1,↓Gfer,↓Kif1b,↑ Hagh,↓Daam1,↓Ptpn4,↓Slc25a31,↓Ebf3,↑Negr1,↑Ctr9,↓Rps 6ka6,↓Tktl1,↑1500015O10Rik,↓Arrdc4,↑Adamts16

173 Table 3-7. Top ten Pathway Studio Gene Ontology (GO) biological processes for the P4 gene set.

Group Term Gene #s p value Genes 1 Multicellular 35 4.19E-13 ↑Cd24a,↓Zfp39,↑Igf2,↑Bmp7,↓Lepr,↑Pou5f1,↑Foxc2,↑Igfbp Organismal 2,↑Tcf23,↑Sorl1,↑Ascl2,↑Figla,↑Hoxd10,↑Ecscr,↑Aff3,↓Frzb Development ,↑Sfrp1,↑Tbx2,↑Prrx1,↑Twist2,↓Ndel1,↑Lhx1,↑Itga8,↑Piwil4 ,↑Tnp1,↑Arx,↓Plekhb1,↑Tshz3,↑Tbata,↑Unc45b,↑Hoxd8,↓Pl xna4,↑Hoxd9,↑Pkdcc,↑Olfml3 2 Muscle 11 1.92E-09 ↑Actg2,↑Ryr1,↑Myl9,↑Myh6,↑Tnnt2,↑Myh11,↑Actn3,↑Itga1 Contraction ,↑Actg2,↑Gjc1,↑Tpm2 3 Cell 23 3.78E-09 ↓Herc4,↑Cd24a,↓Zfp39,↑Bmp7,↓Spp1,↑Tcf23,↑Tfap2c,↑Asc Differentiation l2,↑Figla,↑Ecscr,↓Frzb,↑Sfrp1,↑Twist2,↓Ndel1,↑Lhx1,↑Itga8 ,↑Piwil4,↑Tnp1,↑Arx,↑Tbata,↑Unc45b,↑Pkdcc,↑Gli2 4 Ureteric Bud 7 3.66E-08 ↑Bmp7,↑Foxc2,↑Sfrp1,↑Tcf21,↑Lhx1,↑Sall1,↑Tshz3 Development 5 Skeletal System 10 2.54E-07 ↑Igf2,↑Bmp7,↑Foxc2,↓Acan,↑Hoxd10,↓Frzb,↑Col1a2,↑Dnm Development 3os,↑Col5a2,↑Gli2 6 Osteoblast 7 2.73E-07 ↑Igf2,↑Fhl2,↓Spp1,↑Il18,↑Sfrp1,↑Twist2,↑Gli2 Differentiation 7 Metanephros 6 7.02E-07 ↑Bmp7,↑Foxc2,↑Itga8,↓Rdh10,↑Tshz3,↑Osr2 Development 8 Cell Adhesion 18 2.52E-06 ↑Islr,↑Col22a1,↑Cd24a,↑Cdh1,↑Thbs2,↑Fn1,↓Spp1,↑Itga1,↓ Acan,↓Jup,↑Igfbp7,↑Col4a3,↑Lama2,↑Itgae,↑Itga8,↑Mfap4,↑ Fblim1,↑Lamc3 9 Response To 9 2.86E-06 ↑Cdh1,↓Spp1,↑Cyp1b1,↑Cyp11a1,↑Slc6a4,↑Cyp17a1,↑Pdgfr Organic a,↑Ccnd3,↑C1qb Substance 10 Axon Guidance 12 4.82E-06 ↑Bmp7,↑Myh11,↑Itga1,↑Col4a3,↑Gfra1,↓Spna1,↑Col1a2,↑C ol4a4,↑Arx,↓Plxna4,↑Col5a2,↑Gli2

174 Table 3-8. Top ten Pathway Studio Gene Ontology (GO) biological processes for the P8 gene set.

Group Term Gene #s p value Genes 1 Meiosis 84 1.48E-23 ↓Stra8,↓Clgn,↓Dmc1,↓Spo11,↓Boll,↓Rec8,↓Msh5,↓Stag3,↓S ycp1,↓Sycp2,↓Smc1b,↓Mei1,↓Sycp3,↓Prdm9,↓Zfp318,↓Hor mad1,↓4930528F23Rik,↓Syce1,↓Mael,↓Dpep3 2 Spermatogenesis 423 5.82E-15 ↓Taf7l,↓Timp2,↓Rbp4,↓Lmna,↓Sgpl1,↓Stra8,↓Clgn,↓Mtl5,↓ Dmc1,↓Spo11,↓Boll,↓Rec8,↑Adamts2,↓Sycp1,↓Gfer,↓Sycp3 ,↑Hagh,↓Tex15,↓Mov10l1,↓Dazl,↓D6mm5e,↓Mael,↓Adad1, ↓Rnf17,↓Hsf2bp,↓Zfp541 3 Multicellular 1146 4.64E-12 ↓4930447C04Rik,↓Taf7l,↑Foxc2,↓Igfbp2,↑Wnt5a,↓Itgb3,↓N Organismal es,↓Clgn,↓Anpep,↓Mtl5,↓Nobox,↓Boll,↓Spdya,↓Dkk3,↓Krt8 Development ,↓Crabp1,↓Hopx,↓Cdk13,↓Pou4f1,↓Ndrg4,↓Plekhb1,↓Sema4 f,↓Ebf3,↓Npnt,↓Radil,↓Ctnnd2,↓Mov10l1,↑Mkx,↓Dazl,↓Mb nl3,↓Mael,↓Adad1,↓Rnf17,↓Disp1,↓Zfp541,↓Olfml3,↓Ebf4 4 Synaptonemal 13 5.19E-12 ↓Stag3,↓Sycp1,↓Sycp2,↓Tex11,↓Sycp3,↓Tex15,↓Syce1 Complex Assembly 5 Synapsis 9 3.10E-11 ↓Stra8,↓Rec8,↓Msh5,↓Sycp3,↓Tex15,↓Mael 6 Male Meiosis I 16 1.81E-07 ↓Dmc1,↓Spo11,↓Rec8,↓Mei1,↓Sycp3 7 Reciprocal 35 4.96E-07 ↓Stra8,↓Dmc1,↓Spo11,↓Rec8,↓Msh5,↓Sycp1 Meiotic Recombination 8 Fertilization 36 5.91E-07 ↓Stra8,↓Sycp2,↓Tex11,↓Tex15,↓Mael,↓Ooep 9 Axon Guidance 328 1.10E-06 ↓Trpc1,↓Nr4a3,↓Pak1,↑Itga9,↓Itgb3,↓Cacna1h,↓Scn8a,↓Alc am,↑Gfra1,↓Runx3,↓Nck2,↓Sema4f,↑Rgmb,↓Rps6ka6 10 Meiotic 11 1.67E-06 ↓Sycp2,↓Prdm9,↓Syce1,↓Mael Prophase I

175 Table 3-9. Genes whose gene products bind to DMRT1 in the P4 gene set.

Gene Name Fold Change Gene Symbol Gene Title 1450871_a_at 3.696 Bcat1 Branched Chain Aminotransferase 1, Cytosolic 1425090_s_at 3.193 Kcnc4 Potassium Voltage Gated Channel, Shaw-Related Subfamily, Member 4 1425528_at 2.302 Prrx1 Pair Related Homeobox 1 1456901_at 2.300 Adamts20 A Disintegrin-Like And Metallopeptidase (Reprolysin Type) with Thrombospondin Type 1 Motif, 20 1416454_s_at 2.202 Acta2 Actin, Alpha 2, Smooth Muscle, Aorta 1444646_at 2.127 Bnc2 Basonuclin 2 1445328_at 2.092 Col4a4 Collagen, Type IV, Alpha 4 1433939_at 2.086 Aff3 AF4/FMR2 Family, Member 3 1417933_at 2.029 Igfbp6 Insulin-Like Growth Factor Binding Protein 6 1438779_at 1.989 Col4a3 Collagen, Type IV, Alpha 3 1419212_at 1.980 Icosl Icos Ligand 1440250_at 1.974 Col4a4 Collagen, Type IV, Alpha 4 1437399_at -1.978 Cldnd1 Claudin Domain Containing 1 1449254_at -2.027 Spp1 Secreted Phosphoprotein 1 1431293_a_at -2.055 Cldnd1 Claudin Domain Containing 1 1434990_at -2.145 Ppm1e Protein Phosphatase 1E (PP2C Domain Containing) 1446742_at -2.262 Nfia Nuclear Factor I/A

176 Table 3-10. Pathway Analysis of transcripts that are differentially regulated at P8 and related to DNA repair.

GO-biological process/subnetworks # of Genes Pvalue Genes DNA repair 25 2.05E-05 ↑210018m11rik, ↓Mms22L, ↑Giyd2, ↓Eme2, ↓Atm, ↓Rad51, ↓Bard1, ↓Dmc1, ↓Brca2, ↓Fancd2, ↑Sfpq, ↑Fancb, ↑Lig4, ↑Polh, ↓Recql4, ↑Rad23A, ↓Rad9B, ↑Pola1, ↓Zbtb32, ↓Poln, ↓Pole2, ↓Pole, ↓Obfc2a, ↓Pold3 DNA synthesis involved in DNA repair 4 1.29E-04 ↑Polh, ↑Pola1, ↓Pole, ↓Pold3 error-prone post-replication DNA repair 3 3.45E-02 ↓Trp53, ↓Rad51, ↑Polh DNA repair 48 3.51E-02 ↑Kdr, ↓Trp53, ↑Cdh1, ↓Prkcd, ↓Ccnd1, ↑Cdk1, ↑Cdk2, ↑Plk1, ↓Atm, ↑Sp1, ↑Igf1R, ↑Pten, ↓Cebpd, ↓Gadd45B, ↓Atf3, ↓Braf, ↑Nr4a2, ↓Lmna, ↓Sgpl1, ↓Adarb1, ↓Rad51, ↓Bard1, ↓Dmc1, ↓Spo11, ↑Dnmt3a, ↓Rec8, ↓Rrm2, ↓Brca2, ↓Fancd2, ↑Sfpq, ↑2810417H13Rik, ↓Msh5, ↑Fancb, ↓Htatip2, ↑Lig4, ↓Hipk2, ↑Polh, ↓Recql, ↓Recql4, ↑Rad23A, ↑Cebpg, ↑Pola1, ↓Tex11, ↓Mtss1, ↓Sycp3, ↓Tex15, ↓Syce2, ↓Pole DNA ligation involved in DNA repair 1 1.86E-01 ↑Lig4 regulation of DNA repair 1 2.55E-01 ↑Polh

177 CHAPTER FOUR

CONCLUSIONS AND FUTURE EXPERIMENTS

178 CONCLUSION CHAPTER

Action of retinoic acid (RA) and retinoic acid receptors on spermatogenesis

Spermatogenesis is a complex and unique process with various phases of proliferation, differentiation, meiosis, and chromatin compaction that continuously produce mature sperm over a lifetime in healthy mammals. Many factors through autocrine or paracrine pathways are known to direct this process. Retinoic acid (RA), a biological active form of dietary vitamin A regulates spermatogenesis in a paracrine manner.

Through studying the vitamin A deficient (VAD) animal model system, RA was found to be the key factor in spermatogonial differentiation. The spermatogenic arrest of VAD mice and rats is primarily at the undifferentiated to differentiating spermatogonial transition (Griswold et al., 1989; Huang and Hembree, 1979; Morales et al., 1987; Thomson et al., 1964; van Pelt and de

Rooij, 1990; van Pelt and de Rooij, 1991). RA is also involved in the meiotic entry regulation as shown by the ability of embryonic testis to enter meiosis after exogenous RA stimulation

(Bowles et al., 2006; Koubova et al., 2006; MacLean et al., 2007; Trautmann et al., 2008).

Retinoic acid acts through retinoic acid receptors to regulate spermatogenesis. Rara-null mice analysis has revealed the central role of this receptor in mediating the function of RA

(Lufkin et al., 1991; Chung et al., 2004; Chung et al., 2005; Doyle et al., 2007). Dramatic lost of early meiotic spermatocytes by apoptosis (Doyle et al., 2007) in Rara-null mice corresponds to the same cell types lost in VAD rats (van Pelt and de Rooij, 1990). These results suggest that

RARA acts to support early meiotic germ cell survival. Additionally, the germ cell lost is spermatogenic stage-dependent (Doyle et al., 2007). While normal testicular morphology was observed for tubules at stages I-V, testicular degeneration was moderate for tubules at stages

VII-IX and severe for stages X-XII. The highest expression of Rara mRNA in stages VII and

179 VIII of the spermatogenic cycle in rats further supports RARA function at these middle stages

(Akmal et al., 1997; Kim and Griswold 1990), and explains the sequential arrest of step 8 and 9 spermatids at stages VIII and IX (Chung et al., 2005). Consistent with the lost of early meiotic germ cells and elongating spermatids, the epididymal sperm number of Rara-null mice is only

1.7% of the wild-type counterpart (Doyle et al., 2007). Furthermore, the remaining Rara-null sperms had fairly low motility (Doyle et al., 2007). These results collectively explain why Rara- null males are infertile (Lufkin et al., 1991)

RARA function in germ cells or in Sertoli cells was specifically investigated using spermatogonial stem cell transplantation assay. When Rara-null germ cells were transplanted into wild type recipients without endogenous germ cells, there was no regeneration of spermatogenesis. It was concluded that RARA in germ cells is important for the efficacy of stem cell proliferation and differentiation (Doyle et al., 2007). This conclusion was supported by a study showing a decrease of 87% in the number of stem cells from VAD mice compared to adult control mice (Mclean et al., 2002).

When wild type germ cells were transplanted into the testis of Rara-null recipient, so that all recipient somatic cells including Sertoli cells were deficient of RARA, there were only 52% of the seminiferous tubules with donor-derived spermatogenesis (Doyle et al., 2007). This indicates that the stem cell function was decreased to about 50%. Examining the tubules, the tubules contained early meiotic spermatocytes, round and elongated spermatids, although at lower numbers. Hence, RARA in Sertoli cells is supporting stem cell function, the survival of early meiotic spermatocytes, and progression of spermiogenesis. The supportive role of Sertoli cell was also suggested by Sertoli cell-specific Rara cKO testicular phenotypes. Sertoli cell- specific cKO has fewer tubules with early meiotic spermatocytes and round spermatids

180 compared to the wild types (Vernet et al., 2006). In addition, there was fewer tubule with spermatogonia expressing STRA8 at P5. The decrease implies that RARA in Sertoli cells may potentiate the differentiation of spermatogonia. STRA8 is expressed highly in type A and B differentiating spermatogonia at P5. These results are in contrast to the results we obtained when

Rara-null germ cells were transplanted into the wild type niche. There was no donor-derived spermatogenesis, suggesting that the stem cells function is 100% impaired when RARA is missing from germ cells.

Conclusions from the characterization of the germ cell-specific knockout mice and a working model

Morphological and immunological studies with germ cell-specific Rara conditional knockout (Rara cKO) mice have shed light on RARA function in germ cells, before the time of meiosis and during meiosis. Rara cKO mice showed an increase in abnormalities of germ cell layers that included severely degenerated testes with lack of advanced germ cells, sloughing of germ cells, and vacuole formation in the seminiferous epithelium. Further investigation of early postnatal Rara cKO mice revealed reduction in the mitosis of undifferentiated (including stem cells) and differentiating spermatogonia by immunohistochemistry using a protein marker phospho-histone H3 (pHH3) and a nucleotide analog bromodeoxyuridine (BrdU), and in the number of reproductive homeobox gene RHOX13-expressing differentiating spermatogonia.

During meiosis, our investigation demonstrated that there were reductions in the quantity and quality of meiotic cells by immunohistochemistry using protein markers phosphorylated histone

2 variant (γH2AX) and synaptonemal complex protein 3 (SYCP3). Consequently, there was

181 decreased post-meiotic spermatid formation. Thus, absence of RARA specifically in germ cells led to decreased spermatogenic output (Fig. 4-1).

Spermatogonial stem cell transplantation assays were conducted by transplanting Rara cKO germ cells from various ages of donor mice into busulfan-treated wild type testes. When wild type germ cells from postnatal day 8 (P8) and P16 mice were transplanted, there was no difference in the spermatogenic regeneration rate. However, when Rara cKO germ cells from P8,

P11, and P25 were transplanted, there was a decrease in the spermatogenic regeneration rate. The rate was similar to the wild types’ at P8, but declined to virtually no regeneration in the recipient with P25 germ cells. It seems that some events occurred during germ cell development between

P8 and P11 in cKO that has affected either the quantity or quality of stem cells. pHH3 results suggested stem cell quantity is compromised. Further investigation is required to increase the power of this analysis. Nonetheless, this interesting result opens up new doors to understanding how RARA affects stem cell function in an age-dependent manner.

In conclusion, as shown in the working model (Fig. 4-3A), in the normal testis, RARA mediates RA action to maintain the integrity of germ cell layers in the seminiferous epithelium and ensure quantitatively normal spermatogonial proliferation and differentiation. These processes require the cross talk between Sertoli cells and germ cells, which are in close physical contacts with each other.

We found that RARA in germ cells promote mitosis of undifferentiated and differentiating spermatogonia, and drive undifferentiated spermatogonia into the differentiated state as shown by the decrease in the number of RHOX13-expressing differentiating spermatogonia when RARA is deleted in germ cells (Fig. 4-3 B). There is also a reduction in the efficacy of stem cell colonization and proliferation in spermatogonial stem cell transplantation

182 experiments. During meiosis, RARA in germ cells appears to be involved in the resolution of

DSBs. Germ cells, especially the ones in the different compartments of seminiferous tubules, may talk to each other through Sertoli cells. Adluminal germ cells could signals to Sertoli cells to indicate their healthiness and feedback to undifferentiated germ cells to advance into differentiation and meiosis.

When RARA is absent from Sertoli cells (Fig. 4-3C) (Vernet et al., 2006), mutant Sertoli cells failed to support the full development of spermatogenesis, especially there was a decrease in the number of tubules with early meiotic spermatocytes. This is also shown by transplantation of wild type germ cells into Rara-null testes (Doyle et al., 2007). Normally, Sertoli cells are thought to count the number of differentiating and premeiotic germ cells and maintain a finite number of germ cells they can support. If there were too many germ cells, Sertoli cells are shown to eliminate them by apoptosis. Similarly, RARA in Sertoli cell may potentiate germ cells to differentiate, if more germ cells are required. Without RARA in Sertoli cells, Sertoli cells may not be healthy and not work efficiently as the cell counter of sperm production.

Therefore, both cell-autonomous and cooperative efforts in germ cells are needed to mediate RA action through RARA. In cell autonomous effort in germ cells maintains the quantity of stem cells, undifferentiated and differentiating spermatogonia, and police genomic stability during meiosis. In cooperative manner, germ cells talk to Sertoli cells to potentiate spermatogonial differentiation and subsequent entry into meiosis. The cross talks between Sertoli cells and germ cells are both important to maintain full spermatogenesis.

183 Conclusions from microarray experiments

RARA is a transcription factor that regulates target gene transcription by binding to

retinoic acid response element (RARE) in front of primary target genes (Fig. 1-6 and 4-2A). To

activate gene transcription in this canonical pathway, newly synthesized RARA is transported

into the nucleus as either an inactive form (represented as a rectangle in Fig. 4-2 A) or active

form (represented as an oval in Fig. 4-2 A).

In the presence of RA, newly synthesized cytoplasmic inactive RARA is activated and

sumoylated by a small ubiquitin-like modifier (SUMO2) to facilitate the transport into the

nucleus (Fig. 4-2A) (Zhu et al., 2009). The nuclear localization of RARA is inhibited by protein

kinase A (PKA) (Santos et al., 2010). Once inside the nucleus, active RARA heterodimerizes to

one retinoid X receptor (RXR), recruits coactivator complexes (CoA), and the complex then

binds to a RARE in front of target genes and turns on the transcription (Bastien and Rochette-

Egly, 2004). When activated RARA is no longer needed, a chaperone protein disulfide isomerase

glucose-regulated protein 58 (GRp58) brings RARA to the endoplasmic reticulum (ER) for the

ER-associated degradation (ERAD) (Zhu et al., 2010).

From the transcriptome profiling studies, we have identified three genes from the P4 gene set and ten genes from the P8 gene set that contain canonical RAREs of DR-1, DR-2 and DR-5

(Table 3-4). From the P4 gene set, LIM homeobox gene 9 (Lhx9) is up-regulated 2.3 fold in the cKO. It is a transcription factor that is involved in gonadal development. Kinesin family member

18B (Kif18b) is up-regulated 2.1 fold in the cKO and participates in microtubule depolymerization and cell division process. Pleckstrin homology domain containing, family B

(evectins) member 1 (Plekhb1) is down-regulated 2.2 fold in the cKO and plays a role in cell differentiation. From the P8 gene set, the top three genes with RAREs are: testis-expressed

184 sequence 101 protein (Tex101) that is down-regulated 4.7 fold in the cKO and plays a role to promote protein tyrosine phosphorylation; meiotic recombination protein REC8 homolog (Rec8) is down-regulated 3.9 fold in the cKO and is required for separating homologous chromosome and sister chromatids during meiosis; activating transcription factor 7-interacting protein 2

(Atf7ip2) is down-regulated 3.9 fold in the cKO and acts as a recruiter to modulate transcription regulation and chromatin formation. These genes are the primary targets for RARA, and may affect the transcription of their target genes.

Additionally, it was remarkable that we found 15 genes in the P4 gene set (Table 3-9), which interact with a double sex and mab-3 related transcription factor 1 (DMRT1), a transcription factor that is known to enhance spermatogonial proliferation and differentiation and inhibit meiosis (Matson et al., 2010). Previously, our laboratory has identified using a yeast-two- hybrid system DMRT1 as a binding partner of RARA in the absence of RA (Zhu et al., 2010).

RARA may act as a co-regulator of DMRT1, similar to RARA acting as a co-regulator of activation protein 1 (AP1), another transcription factor composed of fos- and jun-related proteins, that mediates the trans-repressive activity of retinoic acid (Benkoussa et al., 2002) (Fig.

4-2B). This type of transcriptional regulation is possible because protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) can promote the nuclear localization of inactive

RARA in the absence of RA ligand (Braun et al., 2002).

The top three differentially regulated genes among these 15 common genes are: branched chain aminotransferase 1, cytosolic (Bcat1), up-regulated 3.7 fold in the cKO; potassium voltage gated channel, shaw-related subfamily, member 4 (Kcnc4), up-regulated in the cKO for 3.2 fold; and pair related homeobox 1 (Prrx1), up-regulated 2.3 fold in the cKO. BCAT1 catalyzes the

185 first reaction in the catabolism of leucine, isoleucine and valine. KCNC4 forms a potassium- selective channel and PRRX1 is a transcriptional regulator of muscle creatine kinase.

Furthermore, there were 58 genes that are involved in the DNA repair pathway from the

P8 gene set (Table 3-10), potentially regulated by RARA indirectly (without any canonical

RAREs). The top three differentially regulated genes are Eme2, Dmc1 and Spo11. Essential meiotic endonuclease 1 homolog 2 (Eme2) is down-regulated 26.2 fold in the cKO. Forming a complex with MUS81, the complex cleaves specific DNA structures. Meiotic recombination protein DMC1/LIM15 homolog (Dmc1) is down-regulated 3.9 fold in the cKO. It is required for the resolution of meiotic double-strand breaks (DSBs). Meiotic recombination protein SPO11 is down-regulated 3.6 fold in the cKO and mediates DNA cleavage that forms DSBs during meiosis.

In conclusion, consistent with the finding that RARA plays a distinct role in the mitosis and differentiation of spermatogonia, and resolution of DSBs in meiosis, transcriptome analysis of RNA from enriched germ cells of wild type and cKO testes from animals at P4 show that

RARA directs the transcription of primary, secondary and tertiary target genes enriched in cell differentiation, cell adhesion and cell migration functional clusters, whereas Rara regulates the gene transcription centered around synapsis, male meiosis and spermatogenesis at P8 (Table 3-

5~3-8). Through transcriptome profiling and mining of the direct and indirect targets of RARA, we can elucidate the circuitry of RARA regulation network in spermatogenesis.

186 FUTURE EXPERIMENTS

Stem cell function regulated by RARA

Our findings from the spermatogonial stem cell transplantation assay indicate that RARA affects stem cell function in an age-dependent manner, possibly by feedback signaling mechanisms from differentiated and meiotic germ cells around P8 and P11 to the stem cells.

Further investigation with more animals and using immunohistochemistry with protein markers for different populations of neonatal germ cells will be needed to solidify this finding.

Previously, RARA was shown to have a function in potentiating spermatogonial stem cells (SSCs) for colonization and proliferation (Doyle et al., 2007; Chapter Two). Taking advantage of early deletion of Rara by STRA8-iCRE in the gonocytes before the resumption of mitosis and stem cell formation, germ cell-specific RARA function in stem cells can be further investigated. Preliminary data suggest that stem cell function maybe normal in the first wave of spermatogenesis at P8, but defective in the subsequent waves at P11 and P25. As the spermatocytes encounter roadblocks during differentiation and meiosis in the cKO testes around

P11, we hypothesize that normal Sertoli cells sense the defects and feedback to undifferentiated spermatogonia and stem cells sending them into quiescence. To determine the percent undifferentiated spermatogonia that were able to colonize but not differentiate, we can immunostain undifferentiated spermatogonia with DMRT1, a protein marker that expresses highly in undifferentiated spermatogonia, in comparison to immunostaining with GCNA, a germ cell nuclear antigen expressed in all spermatogonia. In addition, increasing the power of analysis with more animal replicates (more spermatogonial transplantation assays) would help solidify the role of RARA in the regulation of stem cell function.

187 Follow-up of transcriptome differentially regulated in the cKO vs. wild type germ cells

Spermatogonial proliferation and differentiation

Biological functions of cell proliferation and differentiation are the top functional clusters enriched for the target genes regulated by RARA in germ cells at P4, highlighting the action of

RARA as a transcription factor regulating the transcription of RARE-containing primary target genes, and the transcription of subsequent secondary and tertiary target genes; and as a co- regulator regulating genes that are involved in the proliferation and differentiation of spermatogonia. Further studies on RARE-dependent and independent RARA regulation will contribute to the understanding of RA action through this receptor in controlling the commitment from undifferentiated to differentiating spermatogonia.

For the P4 set of genes, we will focus on DMRT1, a double sex and mab-3 related transcription factor 1. Among the 283 unique gene differentially regulated by RARA, 15 of them were found to be directly regulated by DMRT1 (Fig 4-4 A) to activate the transcription. One of these 15 genes is Bcat1. It is a germ cell-specific gene that catalyzes the first reaction in the catabolism of the essential branched chain amino acids leucine, isoleucine, and valine, and takes part in the pantothenate and CoA biosynthesis pathway. This is a target gene of c-myc (Eden et al., 1996) and may be involved in the G1/S transition of the cell cycle (Schuldiner et al., 1996). It is regulated by DMRT1, a transcription factor that was shown to play a role in germ cell proliferation, differentiation, migration, and in the pluripotency of germ cells (Kim et al., 2007;

Krentz et al., 2009). Furthermore, when DMRT1 is deleted in germ cells, Bcat1 is up-regulated

(Murphy et al., 2010). Bcat1 transcript is up-regulated upon Rara deletion, implying RARA is a co-activator of DMRT1. Using immunohistochemistry, staining for BCAT1 and DMRT1 at the neonatal ages, we may be able to investigate the expression pattern of this novel regulator of

188 spermatogenesis and determine if it is a negative regulator of spermatogonial proliferation and differentiation. Using chromatin immunoprecipitation and genomic wide-DNA sequencing, promoters of these 15 genes bound by DMRT1 (Murphy et al., 2010) were identified. These

DMRT1-regulated genes present a noncanonical regulation mechanism of RARA in spermatogonial differentiation.

Meiosis and the resolution of DSBs

Biological function activities in meiosis are the top functional clusters enriched for the target genes regulated by RARA in germ cells at P8, underscoring the action of RARA in resolving DSBs during meiosis. More analysis on RARE-dependent and independent RARA regulation will shed light on the understanding of RA action through this receptor in sustaining efficient meiosis.

There were 58 unique genes that are involved in the DNA repair functional clusters.

During meiosis, DNA repair plays a big role and ensures the progression of DNA recombination.

One of the enzymes, essential in meiosis endonuclease 1 homolog 2 (Eme2) is down-regulated

26.2 fold in the cKO germ cells, at the initiation of meiosis when transcripts are synthesized in preparation for meiosis. In a complex with MUS81 (xeroderma pigmentosa complementation group F, also known as ERCC4, XPF-type), EME2 is a DNA structure-specific endonuclease.

Phenotypic analysis of pachytene spermatocyte chromosome spreads exhibited three fold higher unrepaired double strand breaks in the cKO, potentially a consequence of the lack of this EME2-

MUS81 endonuclease (Ciccia et al., 2008). Immunostaining of MUS81 and EME2 on pachytene spermatocyte chromosome spreads will provide us with information of the expression pattern/level of this enzyme. RA action has not been linked to DSB pathways. This added

189 information of RARA regulating meiotic endonuclease would provide exciting mechanism of

RA action in meiosis.

190 REFERENCE

Akmal, K.M., Dufour, J.M., Kim, K.H., 1997. Retinoic acid receptor alpha gene expression in

the rat testis: potential role during the prophase of meiosis and in the transition from

round to elongating spermatids. Biology of reproduction 56, 549-556.

Bastien, J., Rochette-Egly, C., 2004. Nuclear retinoid receptors and the transcription of retinoid-

target genes. Gene 328, 1-16.

Benkoussa, M., Brand, C., Delmotte, M., Formstecher, P., Lefebvre, P., 2002. Extracellular

signal-regulated kinase and CBP recruitment to an AP1-responsive promoter. Mol and

Cel Bio 22, 13, 4522-4534

Bowles, J., Knight, D., Smith, C., Wilhelm, D., Richman, J., Mamiya, S., Yashiro, K.,

Chawengsaksophak, K., Wilson, M.J., Rossant, J., Hamada, H., Koopman, P., 2006.

Retinoid signaling determines germ cell fate in mice. Science 312, 596-600.

Braun, K.W., Tribley, W.A., Griswold, M.D., Kim, K.H., 2000. Follicle-stimulating hormone

inhibits all-trans-retinoic acid-induced retinoic acid receptor alpha nuclear localization

and transcriptional activation in mouse Sertoli cell lines. The Journal of biological

chemistry 275, 4145-4151.

Braun, K.W., My-Nuong, V., Kim, K.H., 2002. Positive regulation of retinoic acid receptor

alpha by protein kinase C and mitogen-activated protein kinase in Sertoli cells. Biol

Repro 67, 29-37.

Chung, S.S., Sung, W., Wang, X., Wolgemuth, D.J., 2004. Retinoic acid receptor alpha is

required for synchronization of spermatogenic cycles and its absence results in

progressive breakdown of the spermatogenic process. Dev Dyn 230, 754-766.

191 Chung, S.S., Wang, X., Wolgemuth, D.J., 2005. Male sterility in mice lacking retinoic acid

receptor alpha involves specific abnormalities in spermiogenesis. Differentiation 73, 188-

198.

Chung, S.S., Wang, X., Wolgemuth, D.J., 2009. Expression of retinoic acid receptor alpha in the

germline is essential for proper cellular association and spermiogenesis during

spermatogenesis. Dev 136, 2091-2100.

Ciccia, A., McDonald, N., West, S.C., 2008. Structural and functional relationships of the

XPF/MUS81 family of proteins. Annual review of biochemistry 77, 259-287.

Doyle, T.J., Braun, K.W., McLean, D.J., Wright, R.W., Griswold, M.D., Kim, K.H., 2007.

Potential Functions of Retinoic Acid Receptor A in Sertoli Cells and Germ Cells During

Spermatogenesis. Annals of the New York Academy of Sciences 1120, 114-130.

Eden, A., Simchen, G., Benvenisty, N., 1996. Two yeast homologs of ECA39, a target for c-Myc

regulation, code for cytosolic and mitochondrial branched-chain amino acid

aminotransferases. The Journal of biological chemistry 271, 20242-20245.

Griswold, M.D., Bishop, P.D., Kim, K.H., Ping, R., Siiteri, J.E., Morales, C., 1989. Function of

vitamin A in normal and synchronized seminiferous tubules. Annals of the New York

Academy of Sciences 564, 154-172.

Howell, J.M., Thompson, J.N., Pitt, G.A., 1963. Histology of the lesions produced in the

reproductive tract of animals fed a diet deficient in vitamin A alcohol but containing

vitamin A acid. I. The male rat. Journal of reproduction and fertility 5, 159-167.

Huang, H.F., Dyrenfurth, I., Hembree, W.C., 1983. Endocrine changes associated with germ cell

loss during vitamin A-induced recovery of spermatogenesis. Endocrinology 112, 1163-

1171.

192 Huang, H.F., Hembree, W.C., 1979. Spermatogenic response to vitamin A in vitamin A deficient

rats. Biology of reproduction 21, 891-904.

Kim, S., Bardwell, V.J., Zarkower, D., 2007. Cell type-autonomous and non-autonomous

requirements for Dmrt1 in postnatal testis differentiation. Developmental biology 307,

314-327.

Kim, K.H., Griswold, M.D., 1990. The regulation of retinoic acid receptor mRNA levels during

spermatogenesis. Mol Endocrinol 4, 1679-1688.

Krentz, A.D., Murphy, M.W., Kim, S., Cook, M.S., Capel, B., Zhu, R., Matin, A., Sarver, A.L.,

Parker, K.L., Griswold, M.D., Looijenga, L.H., Bardwell, V.J., Zarkower, D., 2009. The

DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation

and pluripotency. Proceedings of the National Academy of Sciences of the United States

of America 106, 22323-22328.

Koubova, J., Menke, D.B., Zhou, Q., Capel, B., Griswold, M.D., Page, D.C., 2006. Retinoic acid

regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci U S A 103,

2474-2479.

Lufkin, T., Lohnes, D., Mark, M., Dierich, A., Gorry, P., Gaub, M.P., LeMeur, M., Chambon, P.,

1993. High postnatal lethality and testis degeneration in retinoic acid receptor alpha

mutant mice. Proc Natl Acad Sci USA 90, 7225-7229.

MacLean, G., Li, H., Metzger, D., Chambon, P., Petkovich, M., 2007. Apoptotic extinction of

germ cells in testes of Cyp26b1 knockout mice. Endocrinology 148, 4560-4567.

McLean, D.J., Russell, L.D., Griswold, M.D., 2002. Biological activity and enrichment of

spermatogonial stem cell in vitamin A-deficient and hyperthermia exposed testes from

193 mice based on colonization following germ cell transplantation. Biol Reprod 66, 1374-

1379.

Morales, C., Griswold, M.D., 1987. Retinol-induced stage synchronization in seminiferous

tubules of the rat. Endocrinology 121, 432-434.

Matson, C.K., Murphy, M.W., Griswold, M.D., Yoshida, S., Bardwell, V.J., Zarkower, D., 2010.

The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls

the mitosis versus meiosis decision in male germ cells. Developmental cell 19, 612-624.

Murphy, M.W., Sarver, A.L., Rice, D., Hatzi, K., Ye, K., Melnick, A., Heckert, L.L., Zarkower,

D., Bardwell, V.J., 2010. Genome-wide analysis of DNA binding and transcriptional

regulation by the mammalian Doublesex homolog DMRT1 in the juvenile testis. Proc

Natl Acad Sci U S A 107, 13360-13365.

Ohinata, Y., Payer, B., O'Carroll, D., Ancelin, K., Ono, Y., Sano, M., Barton, S.C., Obukhanych,

T., Nussenzweig, M., Tarakhovsky, A., Saitou, M., Surani, M.A., 2005. Blimp1 is a

critical determinant of the germ cell lineage in mice. Nature 436, 207-213.

Ross-Innes, C.S., Stark, R., Holmes K.A., Schmidt, D., Spyrou, C., Russell, R., Massie, C.E.,

Vowler, S.L., Eldridge, M., Carroll J.S., 2010. Cooperative interaction between retinoic

acid receptor-α and estrogen receptor. Genes & Development 24, 171-182.

Sadate-Ngatchou, P.I., Payne, C.J., Dearth, A.T., Braun, R.E., 2008. Cre recombinase activity

specific to postnatal, premeiotic male germ cells in transgenic mice. Genesis 46, 738-742.

Santos, N.C., Kim, K.H., 2010. Activity of retinoic acid receptor-alpha is directly regulated at its

protein kinase A sites in response to follicle-stimulating hormone signaling.

Endocrinology 151, 2361-2372.

194 Schuldiner, O., Eden, A., Ben-Yosef, T., Yanuka, O., Simchen, G., Benvenisty, N., 1996.

ECA39, a conserved gene regulated by c-Myc in mice, is involved in G1/S cell cycle

regulation in yeast. Proc Natl Acad Sci U S A 93, 7143-7148.

Trautmann, E., Guerquin, M.J., Duquenne, C., Lahaye, J.B., Habert, R., Livera, G., 2008.

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes. Cell cycle 7, 656-

664.

Thomson, J.N., Howell, J.M., Pitt, G.A.J., 1964. Vitamin A and reproduction in rats. Proc R Soc

Lond (Biol) 159, 510-535.

van Pelt, A.M., de Rooij, D.G., 1990. Synchronization of the seminiferous epithelium after

vitamin A replacement in vitamin A-deficient mice. Biology of reproduction 43, 363-367. van Pelt, A.M., de Rooij, D.G., 1991. Retinoic acid is able to reinitiate spermatogenesis in

vitamin A-deficient rats and high replicate doses support the full development of

spermatogenic cells. Endocrinology 128, 697-704.

Vernet, N., Dennefeld, C., Guillou, F., Chambon, P., Ghyselinck, N.B., Mark, M., 2006.

Prepubertal testis development relies on retinoic acid but not rexinoid receptors in Sertoli

cells. Embo J 25, 5816-5825.

Vincent, S.D., Dunn, N.R., Sciammas, R., Shapiro-Shalef, M., Davis, M.M., Calame, K., Bikoff,

E.K., Robertson, E.J., 2005. The zinc finger transcriptional repressor Blimp1/Prdm1 is

dispensable for early axis formation but is required for specification of primordial germ

cells in the mouse. Development 132, 1315-1325.

Zhu, L., Santos, N.C., Kim, K.H., 2009. Small ubiquitin-like modifier-2 modification of retinoic

acid receptor-alpha regulates its subcellular localization and transcriptional activity.

Endocrinology 150, 5586-5595.

195 Zhu, L., Santos, N.C., Kim, K.H., 2010. Disulfide isomerase glucose-regulated protein 58 is

required for the nuclear localization and degradation of retinoic acid receptor alpha.

Reproduction 139, 717-731.

196 Figure 4-1. The three commitment steps of spermatogenesis tightly regulated by RA and

RARA.

197 Figure 4-1. The three commitment steps of spermatogenesis tightly regulated by RA and

RARA.

Spermatogenesis consists of three continuous phases: the mitosis and differentiation of stem cells, undifferentiated and differentiated spermatogonia, meiosis of spermatocytes, and spermiogenesis of spermatids. This process takes 35 days to complete in the mouse. Every 8.6 days, a new life cycle or spermatogenic wave begins, providing continuous supply of sperm.

Retinoic acid (RA), a biological active form of vitamin A is shown to be critical at each commitment step transitioning from spermatogonia to spermatocytes, and from spermatocytes to spermatids mediated by RARA (red arrows). Spermatogenesis encounters roadblocks at mitosis, differentiation and meiosis steps in the absence of RARA in germ cells, consequently leading to the reduction in the output of spermatogenesis in the first, second, third and fourth waves in cKO testes, compared to the wild type (WT), represented by the blue and grey triangles.

198 Figure 4-2. Regulation of RARA in the canonical transcription pathway, and RARA acting in non-canonical transcription and regulation pathways.

A

B

199 C

200 Figure 4-2. Regulation of RARA in the canonical transcription pathway, and RARA acting in non-canonical transcription and regulation pathways.

(A) Retinoic acid (RA) action is mediated by retinoid receptors, through regulating transcription of target genes (represented by gene X) by binding to retinoic acid response element (RARE) in the promoter region of target genes (Bastien and Rochette-Egly, 2004). There are two families of retinoid receptors, RARs (retinoic acid receptors) and RXRs (retinoid X receptors). The transcriptional activity of one of the retinoid receptors, retinoic acid receptor alpha (RARA) is illustrated in (A). Newly synthesized RARA could be transported into the nucleus in its inactive form (rectangle) or active form (oval). RARA is regulated positively by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the absence of RA (Braun et al., 2002) and negatively by protein kinase A (PKA) in the presence of RA (Santos et al., 2010). RA can activate the inactive RARA inside the nucleus. Small ubiquitin-like modifier (SUMO2) is known to sumoylate RARA and promotes nuclear localization of RARA (Zhu et al., 2009), and then activates the transcriptional activity of RARA. Once RARA is activated and used in the transcription of target genes, a chaperon protein, GRp58 (disulfide isomerase glucose-regulated protein 58) will shepherd RARA to the endoplasmic reticulum (ER) for degradation (Zhu et al.,

2009). The inactive form of nuclear RARA could also be degraded, possibly through a proteasomal pathway involving SUG1 (proteasomal ATPase), which is known to bind to RARA in the absence of RA (Zhu et al., 2010). (B) Gene X represents primary target genes of RARA, and gene Y and Z represent secondary and tertiary target genes, regulated by genes which in turn are regulated by RARA. If the primary target gene X was a transcription factor, it can turn on gene Y transcription, and gene Y, possibly a transcription factor, then turns on the transcription

201 of gene Z. (C) Apart from the RARE-dependent transcriptional activity, RARA can act as a co- transcription factor to estrogen receptor (ER) and enhance the transcription of estrogen (E) responsive genes. This action is exerted through the binding of estrogen response element (ERE)

(Ross-Innes et al., 2010). Additionally, RARA can bind to activation protein 1 (AP1), another transcription factor composed of fos- and jun-related proteins, and act as a co-regulator and mediate the trans-repressive activity of retinoic acid (Benkoussa et al., 2002).

202 Figure 4-3. Proposed model of RARA regulation of spermatogonial mitosis, at the transition of undifferentiated spermatogonia to differentiating spermatogonia and meiosis.

A B C

203 Figure 4-3. Proposed model of RARA regulation of spermatogonial mitosis, at the

transition of undifferentiated spermatogonia to differentiating spermatogonia and meiosis.

The expression of RARA is indicated with the red color fillings for Sertoli and germ cells. In the

normal situation (A), retinoic acid (RA) signals to maintain the mitosis of stem cells and

undifferentiated spermatogonia and direct meiosis of spermatocytes. Communications between

germ cells and Sertoli cells at different developmental stages regulate the full development of

stem cell function and meiotic progression, represented by thick green arrows. When RARA is

deleted in germ cells (B), RA is not able to maintain the mitosis of spermatogonia and

subsequent differentiation of spermatogonia. RARA in germ cells is also thought to preserve the

quality of pachytene spermatocytes, as shown by increased meiotic defects in germ cell-specific

Rara cKO pachytene spermatocytes. Cross talk from germ cells to wild type Sertoli cells is

broken, but cross talk from Sertoli cells to germ cells were normal (green arrows). So that Sertoli

cells are able to detect these abnormalities and cross talk back to the undifferentiated

spermatogonia and reduce the output of stem cells, potentially causing the decrease in

colonization rate of stem cells from germ cell-specific cKO. When RARA is deleted in Sertoli

cells (C), Sertoli cells are not able to cross talk with germ cells, but germ cells can cross talk to

Sertoli cells. However, the cross talk between germ cells in the adluminal compartment and germ

cells in the basal compartment through Sertoli cells is missing (black arrows). These cross talks

between the two cell types are important in the regulation of quantitatively normal sperm production. RaraSC cKO: Sertoli cell-specific Rara cKO. RaraGC cKO: germ cell-specific Rara

cKO. Aundif: undifferentiated A spermatogonia. Adif: differentiating A spermatogonia. pl:

204 preleptotene spermatocytes. p: pachytene spermatocytes. r: round spermatids. e: elongated spermatids.

205 Figure 4-4. Co-regulator activity of RARA in the transcriptional activity of DMRT1.

A

B

206 C

D

207 Figure 4-4. Co-regulator activity of RARA in the transcriptional activity of DMRT1.

(A) When Rara is deleted in the enriched germ cells from the cKO mice, 283 unique genes were

observed to change ± 2.0 fold or higher at P4. Among these 283 genes, 15 of them are regulated

by DMRT1 (double sex and mab-3 related transcription factor 1) (Murphy et al., 2010). Using

chromatin immunoprecipitation coupled with genome wide expression studies, promoters of

1439 unique genes were identified that are bound by DMRT1. (B) A yeast-two-hybrid study

found DMRT1 to be interacting with RARA in the absence of retinoic acid (RA) ligand (Zhu et

al., 2010). Without RA, RARA is not active, represented by the rectangular shape. The lack of

RARE in the promoter region of the 15 genes indicates RARA is not directly controlling these

genes, but as a co-regulator of DMRT1. (C) This model illustrates DMRT1, acting as a

transcription factor, regulates the transcription of another transcription factor, Sohlh1

(spermatogenesis and oogenesis-specific basic helix loop helix protein 1) and controls

spermatogonial proliferation and differentiation. DMRT1 also suppresses meiosis by inhibiting

the transcription of stimulated by retinoic acid gene 8 (Stra8). From the study by Murphy and

colleagues, DMRT1 may regulate spermatogonial proliferation and differentiation through the

transcriptional control of the 15 genes. (D) When Rara is deleted, DMRT1 transcriptional activity is also compromised.

208 APPENDIX ONE

SUPPLEMENTAL MICROARRAY DATA

209 Table A1-1. Total Pathway Studio subnetworks for P4 germ cell differentially regulated at ±

2.0 fold genes.

Group Term # of Genes Pvalue Genes 1 Cell 114 4.13E-15 ↑Cd24a,↑Igf2,↑Entpd2,↑Mrvi1,↑Cav1,↑Cdh1,↑Sct,↑Myl9,↑Bmp7, Differentiation ↑Thbs2,↑Fhl2,↑Tnnt2,↑Ednrb,↑Fn1,↑Rapgef3,↑Myh11,↓Lepr,↑Tfa p2a,↓Spp1,↑Prkg1,↑Cyp1b1,↑Pou5f1,↑Cartpt,↑Actn3,↑Il18,↑Foxc 2,↑Itga1,↑Igfbp6,↓Acan,↑Actg2,↑Cyp11a1,↑Slc6a4,↑Igfbp2,↑Ptge r4,↑Tcf23,↑Hbb-b1/Hbb-b2/LOC100503605,↑Tagln,↑Cyp17a1,↑ Cxcl16,↑Bgn,↓Jup,↑Mbl2,↑Gjc1,↑Pdgfra,↑Ccnd3,↑Tgfbr3,↑Thbd, ↑H2-Q7,↑Sorl1,↑Plcg2,↑Igfbp7,↓Shmt1,↑Icosl,↓Ccnd2,↑Apoc2,↑ Etv4,↑Tfap2c,↑Ascl2,↑Hoxd10,↑Lama2,↑Ogn,↑Parp14,↓Nfia,↑Cr xos1,↑Il2rg,↑Itgae,↑Gfra1,↓Frzb,↑Lsp1,↑Hba-a1/Hba-a2,↑Sfrp1,↑ Fap,↑Tbx2,↑Tcf21,↑Col1a2,↑Gfap,↑Twist2,↑Casq2,↑Meis2,↓Smar ce1,↑S100a6,↓Ndel1,↑Lhx1,↑Itga8,↑Lin28a,↑Ifitm3,↓Trim25,↑Cn n2,↑Cd248,↓Rdh10,↑Piwil4,↑Tnp1,↑Arx,↑Sall1,↑Hoxd8,↑Plb1,↑ Matn2,↑Pdzrn3,↑Fblim1,↑Syt13,↑Sepw1,↑Lhx9,↑Cbln1,↑Dppa2, ↑Krt12,↑Hoxd9,↓Msi2,↑Pkdcc,↑Slxl1,↓Cyb5d2,↑Tmem119,↑Cbln 2,↑Dpep1,↓Zfhx4 2 Cell Adhesion 50 1.46E-09 ↑Cd24a,↑Igf2,↑Entpd2,↑Cav1,↑Cdh1,↑Bmp7,↑Thbs2,↑Fhl2,↑Myh 6,↑Ednrb,↑Fn1,↑Rapgef3,↓Spp1,↑Prkg1,↑Il18,↑Foxc2,↑Itga1,↑Igf bp6,↓Acan,↑Igfbp2,↑Ptger4,↑Cyp17a1,↑Cxcl16,↑Bgn,↓Jup,↑Mbl2 ,↑Pdgfra,↑Thbd,↑Sorl1,↑Plcg2,↑Igfbp7,↓Ccnd2,↑Col4a3,↑Ascl2,↑ Lama2,↑Mfap2,↑Gfra1,↑Sytl2,↑Lsp1,↑Sfrp1,↑Fap,↑Tbx2,↑Col1a2 ,↑S100a6,↑Col4a4,↑Cd248,↑Matn2,↑Fblim1,↑Lgals2,↑Hoxd9 3 Cell Migration 60 4.40E-08 ↑Cd24a,↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Thbs2,↑Fhl2,↑Fn1,↑Rapgef3 ,↑Tfap2a,↓Spp1,↑Prkg1,↑Cyp1b1,↑Pou5f1,↑Pnlip,↑Il18,↑Foxc2,↑I tga1,↑Igfbp6,↓Acan,↑Actg2,↑Igfbp2,↑Ptger4,↑Tagln,↑Abcc9,↑Cxc l16,↑Bgn,↓Jup,↑Pdgfra,↑Tgfbr3,↑Thbd,↑Sorl1,↑Hif3a,↑Plcg2,↑Igf bp7,↑Col4a3,↑Etv4,↑Tfap2c,↑Hoxd10,↑Lama2,↑Ecscr,↑Plvap,↑Itg ae,↑Gfra1,↓Frzb,↑Tm4sf5,↑Sfrp1,↑Fap,↑Coro1a,↑S100a6,↓Ndel1, ↑Itga8,↑Cnn2,↑Cd248,↑Arx,↑Adamts20,↑Matn2,↑Mcf2l,↑Fblim1, ↑Arhgap9 4 Pregnancy 37 8.21E-08 ↑Igf2,↑Entpd2,↑Cav1,↑Cdh1,↑Sct,↑Bmp7,↑Thbs2,↑Ednrb,↑Fn1,↓ Lepr,↓Spp1,↑Pou5f1,↑Il18,↑Igfbp6,↑Actg2,↑Cyp11a1,↑Slc6a4,↑Ig fbp2,↑Serping1,↑Elovl6,↑Ptgis,↑Ptger4,↑Cyp17a1,↑Mbl2,↑Gjc1,↑ Thbd,↓Shmt1,↓Ccnd2,↑Etv4,↑Ldhc,↑Tfap2c,↑Ascl2,↓Nfia,↑Hba- a1/Hba-a2,↑S100a6,↑Kcne4,↑Htra3 5 Contraction 37 1.67E-06 ↑Ryr1,↑Igf2,↑Cav1,↑Sct,↑Myl9,↑Thbs2,↑Tacr3,↑Fhl2,↑Myh6,↑Tn nt2,↑Ednrb,↑Fn1,↑Rapgef3,↑Myh11,↓Spp1,↑Prkg1,↑Cartpt,↑Actn 3,↑Il18,↑Itga1,↑Igfbp6,↑Actg2,↑Slc6a4,↑Igfbp2,↑Serping1,↑Ptgis, ↑Ptger4,↑Hbb-b1/Hbb-b2/LOC100503605,↑Tagln,↑Abcc9,↑Gjc1, ↑Pdgfra,↑Tpm2,↑Thbd,↓Ccnd2,↑Lama2,↑Casq2 6 Cell Proliferation 101 2.42E-06 ↑1700026L06Rik,↑Igf2,↑Entpd2,↑Cav1,↑Cdh1,↑Sct,↑Bmp7,↑Thb s2,↑Fhl2,↑Ednrb,↑Fn1,↑Rapgef3,↑Myh11,↓Lepr,↑Tfap2a,↓Spp1,↑ Prkg1,↑Cyp1b1,↑Pou5f1,↑Cartpt,↑Pnlip,↑Il18,↑Stc2,↑Foxc2,↑Itga 1,↑Igfbp6,↓Acan,↑Actg2,↑Cyp11a1,↑Slc6a4,↑Igfbp2,↑Serping1,↑ Ptgis,↑Ptger4,↑Tagln,↑Cyp17a1,↑Cxcl16,↑Bgn,↓Jup,↑Pdgfra,↑Ccn d3,↑Tgfbr3,↑Thbd,↑H2-Q7,↑Sorl1,↑Plcg2,↑Igfbp7,↓Shmt1,↑Icosl, ↓Ccnd2,↑Col4a3,↑Etv4,↑Tfap2c,↑Ascl2,↑Figla,↑Hoxd10,↑Lama2, ↑Ogn,↓Kcnj3,↑Il2rg,↑Itgae,↑Gfra1,↓Frzb,↑Tm4sf5,↑Gbp3,↑Sfrp1, ↑Fap,↑Tbx2,↑Coro1a,↑Tcf21,↑Col1a2,↑Apcdd1,↑Gfap,↑Twist2,↑ Meis2,↑Kcnc4,↓Smarce1,↑S100a6,↓Ndel1,↑Itga8,↑Lin28a,↑Ifitm3 ,↑Col4a4,↓Trim25,↑Cnn2,↑Cd248,↑Arx,↑Bcat1,↑Tbata,↑Unc45b,↓ Plxna4,↑Mcf2l,↑Prr5,↑Lhx9,↑Lgals2,↑Dppa2,↑Hoxd9,↑Htra3,↓Ms i2,↑Osr2,↓Cyb5d2 7 Osteoblast 19 2.71E-06 ↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Thbs2,↑Fhl2,↑Fn1,↓Spp1,↑Foxc2,↑I Differentiation gfbp6,↑Igfbp2,↑Ptger4,↑Bgn,↓Frzb,↑Sfrp1,↑Prrx1,↑Twist2,↑S100 a6,↑Tmem119 8 Extracellular 12 6.99E-06 ↑Igf2,↑Cav1,↑Bmp7,↑Thbs2,↑Fhl2,↑Ednrb,↑Fn1,↓Spp1,↑Pdgfra,↑ Matrix Fap,↑Col1a2,↑Col5a2 Polymerization 9 Ovulation 15 7.75E-06 ↑Igf2,↑Cav1,↑Bmp7,↑Tnnt2,↑Ednrb,↑Fn1,↓Lepr,↓Acan,↑Cyp11a1 ,↑Igfbp2,↑Ptger4,↑Igfbp7,↓Ccnd2,↑Etv4,↑Gfap 10 Apoptosis 95 8.74E-06 ↑Ryr1,↑Igf2,↑Entpd2,↑Cav1,↑Cdh1,↑Sct,↑Myl9,↑Bmp7,↑Thbs2,↑

210 Fhl2,↑Tnnt2,↑Ednrb,↑Fn1,↑Rapgef3,↑Myh11,↓Lepr,↑Tfap2a,↓Spp 1,↑Prkg1,↑Cyp1b1,↑Pou5f1,↑Cartpt,↑Pnlip,↑Il18,↑Stc2,↑Itga1,↑Ig fbp6,↓Acan,↑Actg2,↑Cyp11a1,↑Slc6a4,↑Igfbp2,↑Serping1,↑Elovl 6,↑Ptgis,↑Ptger4,↑Hbb-b1/Hbb-b2/LOC100503605,↑Tagln,↑Cyp 17a1,↑Cxcl16,↑Bgn,↓Jup,↑Mbl2,↑Gjc1,↑Adcy5,↑Pdgfra,↑Ccnd3,↑ Tgfbr3,↑Thbd,↑H2-Q7,↑Hif3a,↑Plcg2,↑Igfbp7,↑Icosl,↓Ccnd2,↑Co l4a3,↑Etv4,↑Tfap2c,↑Hoxd10,↑Lama2,↑Parp14,↑Plvap,↑Il2rg,↑Itg ae,↓Frzb,↑Lsp1,↑Hba-a1/Hba-a2,↑Sfrp1,↑Fap,↑Tbx2,↑Coro1a,↑T cf21,↑Gfap,↑Twist2,↑Dleu7,↑Kcnc4,↓Smarce1,↑S100a6,↑Itga8,↑L in28a,↑Col4a4,↑Cnn2,↑Parm1,↑Tnp1,↑Sall1,↑Bcat1,↑Adamts20,↑ Tshz3,↑Crygs,↑Fblim1,↑Lgals2,↓Msi2,↑Fabp9,↑Gml,↑C1qb 11 Ossification 21 3.55E-05 ↑Igf2,↑Bmp7,↑Fhl2,↑Fn1,↑Rapgef3,↓Lepr,↓Spp1,↑Cartpt,↑Igfbp6, ↑Slc6a4,↑Igfbp2,↑Ptger4,↑Bgn,↑H2-Q7,↑Igfbp7,↑Ogn,↓Frzb,↑Sfr p1,↑Col1a2,↑Twist2,↑Osr2 12 Bone 13 3.71E-05 ↑Igf2,↑Bmp7,↑Thbs2,↓Spp1,↑Il18,↑Stc2,↓Acan,↑Igfbp2,↑Bgn,↓Fr Development zb,↑Sfrp1,↑Tbx2,↑Pkdcc 13 Contractile 17 3.85E-05 ↑Igf2,↑Cav1,↑Myl9,↑Myh6,↑Tnnt2,↑Ednrb,↑Fn1,↑Myh11,↑Prkg1, Activity ↑Il18,↑Actg2,↑Serping1,↑Ptger4,↑Bgn,↑Tpm2,↑Thbd,↑Unc45b 14 Heart Function 21 4.20E-05 ↑Igf2,↑Cav1,↑Thbs2,↑Fhl2,↑Myh6,↑Tnnt2,↑Ednrb,↑Fn1,↑Rapgef3 ,↓Lepr,↓Spp1,↑Il18,↑Actg2,↑Serping1,↑Ptger4,↑Gjc1,↑Adcy5,↑Sfr p1,↑Casq2,↑Unc45b,↑Pik3r6 15 Vascularization 43 5.81E-05 ↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Thbs2,↑Fhl2,↑Tnnt2,↑Ednrb,↑Fn1,↑ Rapgef3,↓Lepr,↑Tfap2a,↓Spp1,↑Prkg1,↑Cyp1b1,↑Pou5f1,↑Il18,↑F oxc2,↑Itga1,↑Igfbp6,↑Igfbp2,↑Ptgis,↑Ptger4,↑Cxcl16,↑Bgn,↓Jup,↑ Mbl2,↑Pdgfra,↑Tgfbr3,↑Thbd,↑Hif3a,↑Igfbp7,↑Col4a3,↑Etv4,↑As cl2,↑Hoxd10,↑Plvap,↑Tm4sf5,↑Sfrp1,↑Fap,↑Tcf21,↑Cd248,↑Sall1 16 Decidualization 11 6.16E-05 ↑Igf2,↑Myl9,↑Bmp7,↑Fn1,↓Spp1,↑Il18,↑Stc2,↑Ptger4,↑Ccnd3,↑Ig fbp7,↑Htra3 17 Muscle Function 13 7.40E-05 ↑Ryr1,↑Tacr3,↑Myh6,↑Tnnt2,↑Ednrb,↑Fn1,↑Prkg1,↑Actn3,↑Igfbp 2,↑Bgn,↑Tpm2,↑Lama2,↑Unc45b 18 Diastolic Function 7 8.08E-05 ↑Thbs2,↑Myh6,↑Tnnt2,↑Ednrb,↑Fn1,↓Spp1,↑Tpm2 19 Bone Remodeling 13 8.65E-05 ↑Igf2,↑Sct,↑Bmp7,↑Thbs2,↑Fn1,↓Lepr,↓Spp1,↑Cartpt,↑Il18,↑Igfb p2,↑Ptger4,↑Bgn,↑Mfap2 20 Relaxation 22 0.000101 ↑Ryr1,↑Igf2,↑Mrvi1,↑Cav1,↑Sct,↑Myl9,↑Tacr3,↑Myh6,↑Tnnt2,↑E dnrb,↑Rapgef3,↓Spp1,↑Prkg1,↑Cyp1b1,↑Actg2,↑Ptgis,↑Ptger4,↑A dcy5,↑Tpm2,↑Thbd,↑Lama2,↓Kcnj3 21 Lumen Formation 21 0.000102 ↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Thbs2,↑Ednrb,↑Fn1,↓Spp1,↑Il18,↑Pt gis,↑Ptger4,↑Cxcl16,↓Jup,↑Hif3a,↑Igfbp7,↑Col4a3,↑Hoxd10,↑Ecs cr,↑Plvap,↑Sfrp1,↑Cd248 22 Lymphocyte 8 0.000119 ↑Cav1,↑Cdh1,↑Fn1,↓Spp1,↑Itga1,↑Serping1,↑Cxcl16,↑Itgae Adhesion 23 Endothelial Cell 13 0.000128 ↑Entpd2,↑Thbs2,↑Fn1,↓Spp1,↑Il18,↑Foxc2,↑Itga1,↓Acan,↑Serpin Adhesion g1,↑Cxcl16,↓Jup,↑Thbd,↑Cd248 24 Trophoblast 8 0.000171 ↑Igf2,↑Cdh1,↑Fn1,↑Tfap2a,↑Pou5f1,↑Cyp11a1,↑Tfap2c,↑Ascl2 Differentiation 25 Lymphangiogenes 9 0.000175 ↑Igf2,↑Thbs2,↑Ednrb,↑Fn1,↑Foxc2,↑Itga1,↑Ptger4,↑Hoxd10,↑Hox is d8 26 Epithelial Cell 14 0.000179 ↑Igf2,↑Cdh1,↑Bmp7,↑Ednrb,↑Fn1,↑Igfbp6,↑Igfbp2,↑Ptger4,↑Tagl Proliferation n,↓Jup,↑Ccnd3,↓Ccnd2,↑Tbata,↑Crygs 27 Placenta 12 0.000201 ↑Igf2,↑Cdh1,↑Fn1,↑Tfap2a,↓Spp1,↑Cyp17a1,↑Cxcl16,↑Mbl2,↑Th Development bd,↑Tfap2c,↑Ascl2,↑Htra3 28 Epithelial To 21 0.00021 ↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Fhl2,↑Ednrb,↑Fn1,↓Spp1,↑Pou5f1,↑I Mesenchymal l18,↑Stc2,↑Foxc2,↑Actg2,↑Tagln,↑Cxcl16,↑Tgfbr3,↑Thbd,↑Etv4,↑ Transition Tm4sf5,↑Tbx2,↑Twist2 29 Dentinogenesis 4 0.000259 ↑Bmp7,↓Spp1,↑Bgn,↑Lama2 30 Heart 14 0.000267 ↑Igf2,↑Bmp7,↑Myh6,↑Tnnt2,↑Fn1,↑Tfap2a,↑Pou5f1,↑Slc6a4,↓Jup Development ,↑Gjc1,↑Pdgfra,↓Kcnj3,↑Tbx2,↑Casq2 31 Neural Crest Cell 6 0.000348 ↑Bmp7,↑Ednrb,↑Tfap2a,↑Pdgfra,↑Etv4,↓Rdh10 Development 32 Trophoblast 7 0.00038 ↑Igf2,↑Bmp7,↓Spp1,↑Cxcl16,↑Igfbp7,↑Tfap2c,↑Ascl2 Growth 33 Vasculogenesis 11 0.000388 ↑Igf2,↑Fhl2,↑Fn1,↓Spp1,↑Prkg1,↑Pou5f1,↑Itga1,↑Pdgfra,↑Thbd,↑ Mfap2,↑Sfrp1 34 Chondrogenesis 13 0.000407 ↑Igf2,↑Entpd2,↑Bmp7,↑Fn1,↑Tfap2a,↑Pou5f1,↑Itga1,↓Acan,↑Bgn ,↑Igfbp7,↓Frzb,↑Sfrp1,↑Twist2 35 Organogenesis 13 0.000449 ↑Igf2,↑Cdh1,↑Bmp7,↑Fn1,↓Spp1,↑Pdgfra,↑Tgfbr3,↑Plcg2,↑Etv4,↑ Ogn,↑Tbx2,↑Tcf21,↑Lhx9 36 Morphogenesis 32 0.000509 ↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Ednrb,↑Fn1,↑Tfap2a,↓Spp1,↑Cyp1b 1,↑Pou5f1,↑Foxc2,↓Acan,↑Actg2,↑Bgn,↓Jup,↑Pdgfra,↑Thbd,↑Col 4a3,↑Etv4,↑Tfap2c,↑Hoxd10,↑Gfra1,↓Frzb,↑Tbx2,↑Tcf21,↑Twist2

211 ,↑Casq2,↓Ndel1,↑Col4a4,↓Rdh10,↑Adamts20,↑Osr2 37 Kidney Function 14 0.000561 ↑Sct,↑Bmp7,↑Thbs2,↑Tnnt2,↑Ednrb,↑Fn1,↑Actg2,↑Serping1,↑Ptg er4,↑Mbl2,↑Tgfbr3,↑Thbd,↑Col4a3,↑Col4a4 38 Tear Flow 5 0.000714 ↑Bmp7,↑Fn1,↑Cyp1b1,↑Ptger4,↑Sfrp1 39 Muscle Strength 6 0.000717 ↑Ryr1,↑Igf2,↑Myl9,↑Tnnt2,↑Igfbp2,↑Lama2 40 Dedifferentiation 12 0.000719 ↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Fhl2,↑Fn1,↓Spp1,↑Pou5f1,↑Itga1,↑T fap2c,↑Sfrp1,↑Krt12 41 Regeneration 26 0.000795 ↑Igf2,↑Cdh1,↑Bmp7,↑Thbs2,↑Ednrb,↑Fn1,↑Rapgef3,↓Spp1,↑Prkg 1,↑Pou5f1,↑Il18,↑Igfbp6,↓Acan,↑Igfbp2,↑Ptgis,↑Ptger4,↑Pdgfra,↑ Ccnd3,↓Ccnd2,↑Lama2,↑Ogn,↑Gfap,↑Twist2,↑S100a6,↓Ndel1,↑M atn2 42 Skeletal System 3 0.000852 ↑Bmp7,↑Col1a2,↑Col5a2 Development 43 Epithelial Cell 4 0.000944 ↑Igf2,↑Fn1,↓Spp1,↑Cxcl16 Interaction 44 SMC Proliferation 17 0.001143 ↑Cav1,↑Bmp7,↑Fhl2,↑Ednrb,↑Fn1,↓Lepr,↓Spp1,↑Prkg1,↑Il18,↑Sl c6a4,↑Ptgis,↑Tagln,↑Cxcl16,↑Bgn,↑Pdgfra,↑Thbd,↑Icosl 45 Metanephros 2 0.001235 ↓Rdh10,↑Sall1 Development 46 Muscle 10 0.001282 ↑Ryr1,↑Myl9,↑Myh6,↑Tnnt2,↑Ednrb,↑Rapgef3,↑Prkg1,↑Actn3,↑P Contraction tger4,↑Tpm2 47 Myogenesis 16 0.001473 ↑Ryr1,↑Igf2,↑Cdh1,↑Bmp7,↑Fn1,↑Igfbp2,↑Ccnd3,↑Igfbp7,↑Lama 2,↓Frzb,↑Tcf21,↑Twist2,↑Lin28a,↑Arx,↑Dnm3os,↑Olfml3 48 Germ-Cell 7 0.001631 ↑Cdh1,↑Pou5f1,↓Ccnd2,↑Lin28a,↑Ifitm3,↑Piwil4,↑Dppa2 Development 49 Histogenesis 10 0.001796 ↑Igf2,↑Cdh1,↑Bmp7,↑Fn1,↓Lepr,↑Actg2,↑Bgn,↑Pdgfra,↑Figla,↓N del1 50 Embryonal 38 0.002031 ↑Igf2,↑Cav1,↑Cdh1,↑Myl9,↑Bmp7,↑Tnnt2,↑Ednrb,↑Fn1,↑Myh11, Development ↓Lepr,↑Tfap2a,↓Spp1,↑Prkg1,↑Cyp1b1,↑Pou5f1,↑Cyp11a1,↑Slc6a 4,↑Igfbp2,↑Hbb-b1/Hbb-b2/LOC100503605,↑Tagln,↑Cyp17a1, ↑Bgn,↓Jup,↑Gjc1,↑Pdgfra,↑Thbd,↓Shmt1,↑Etv4,↑Tfap2c,↓Kcnj3,↓ Nfia,↑Sfrp1,↑Tbx2,↑Gfap,↓Rdh10,↑Unc45b,↑Dppa2,↑Dnm3os 51 Luteinization 8 0.002033 ↑Igf2,↑Bmp7,↑Ednrb,↓Lepr,↓Spp1,↑Stc2,↑Cyp11a1,↑Igfbp7 52 Germ-Cell 4 0.002227 ↑Igf2,↑Cdh1,↑Pou5f1,↑Ifitm3 Migration 53 Immunoreactivity 16 0.002248 ↑Cdh1,↑Sct,↑Bmp7,↑Tacr3,↑Fn1,↑Prkg1,↑Cyp1b1,↑Cartpt,↑Pnlip, ↑Igfbp2,↓Jup,↑Mbl2,↑Pdgfra,↓Ccnd2,↑Gfra1,↑Gfap 54 Wound Healing 19 0.00232 ↑Igf2,↑Cav1,↑Cdh1,↑Thbs2,↑Fhl2,↑Ednrb,↑Fn1,↓Spp1,↑Foxc2,↑A ctg2,↑Ptgis,↓Jup,↑Tgfbr3,↑Thbd,↑Sfrp1,↑S100a6,↑Tnp1,↑Matn2,↑ Lgals2 55 Response To 5 0.00246 ↑Sct,↑Rapgef3,↓Spp1,↑Foxc2,↓Ccnd2 Camp 56 Oncogenesis 17 0.002496 ↑Igf2,↑Cav1,↑Cdh1,↑Fn1,↑Tfap2a,↑Cyp1b1,↑Pou5f1,↑Ptger4,↓Ju p,↑Ccnd3,↓Ccnd2,↑Etv4,↑Tfap2c,↑Parp14,↓Frzb,↑Sfrp1,↑Tbx2 57 Leydig Cell 3 0.002697 ↑Igf2,↑Pdgfra,↑Tcf21 Differentiation 58 Growth Rate 20 0.002751 ↑Igf2,↑Cav1,↑Cdh1,↑Ednrb,↑Fn1,↓Lepr,↓Spp1,↑Igfbp2,↑Ptger4,↑ Bgn,↓Jup,↑Ccnd3,↑Tgfbr3,↑Tpm2,↑Igfbp7,↓Shmt1,↓Ccnd2,↑Sfrp 1,↑Fap,↑Rasgrp2 59 Smooth Muscle 7 0.00288 ↑Fhl2,↑Fn1,↑Actg2,↑Tagln,↑Lama2,↑Prrx1,↑Tshz3 Cell Differentiation 60 Actomyosin 4 0.003183 ↑Myl9,↑Myh6,↑Tnnt2,↑Myh11 Based Movement 61 Sebaceous Gland 2 0.003381 ↑Tfap2a,↑Tfap2c Development 62 Osteocyte 7 0.003391 ↑Igf2,↑Fhl2,↑Fn1,↓Spp1,↑Slc6a4,↑Sfrp1,↑Osr2 Function 63 Vasoconstriction 13 0.003526 ↑Cav1,↑Tacr3,↑Ednrb,↑Fn1,↑Cartpt,↑Slc6a4,↑Ptgis,↑Ptger4,↑Gjc1 ,↑Tpm2,↑Thbd,↑Kcnc4,↑Tmem204 64 Cell Invasion 24 0.004032 ↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Thbs2,↑Ednrb,↑Fn1,↓Spp1,↑Pnlip,↑I gfbp2,↑Ptger4,↑Tagln,↑Cxcl16,↓Jup,↑Ccnd3,↑Tgfbr3,↑Thbd,↑H2- Q7,↑Etv4,↑Hoxd10,↑Fap,↑Tbx2,↑S100a6,↑Fblim1 65 Muscle 7 0.004093 ↑Sct,↑Myl9,↑Tacr3,↑Ednrb,↑Prkg1,↑Ptger4,↑Tpm2 Relaxation 66 Collagen Fibril 2 0.004315 ↑Col1a2,↑Col5a2 Organization 67 T-Cell Adhesion 7 0.00435 ↑Cdh1,↑Ednrb,↑Fn1,↑Il18,↑Itga1,↑Cxcl16,↑Itgae 68 Cell Motility 27 0.00439 ↑Igf2,↑Cav1,↑Cdh1,↑Myl9,↑Bmp7,↑Fhl2,↑Fn1,↑Rapgef3,↓Spp1,↑ Stc2,↓Acan,↑Actg2,↑Igfbp2,↑Ptger4,↓Jup,↑Pdgfra,↑Tgfbr3,↑Thbd,

212 ↑Plcg2,↑Etv4,↑Lsp1,↑Coro1a,↑S100a6,↑Cnn2,↑Adamts20,↑Mcf2l ,↑Fblim1 69 Heart Contraction 10 0.004594 ↑Cav1,↑Myh6,↑Tnnt2,↑Rapgef3,↑Prkg1,↑Il18,↑Actg2,↑Serping1, ↑Abcc9,↑Gjc1 70 Trophoblast 5 0.004607 ↑Igf2,↑Cdh1,↑Fn1,↑Tfap2a,↑Cxcl16 Migration 71 Alveolus 5 0.004607 ↑Igf2,↑Cav1,↑Cdh1,↓Ccnd2,↑Sfrp1 Formation 72 Blood Vessel 11 0.004628 ↑Cav1,↑Fn1,↑Cyp1b1,↑Il18,↑Foxc2,↑Igfbp2,↑Gjc1,↑Pdgfra,↑Mfa Development p2,↑Col1a2,↑Tmem204 73 Innervation 8 0.00552 ↑Cav1,↑Bmp7,↑Ednrb,↑Fn1,↓Acan,↑Etv4,↑Hoxd10,↑Cbln1 74 Spermatogenesis 17 0.006206 ↑Igf2,↑Bmp7,↑Fn1,↓Spp1,↑Il18,↑Cyp17a1,↑Ccnd3,↓Ccnd2,↑Col4 a3,↑Ldhc,↑Gfra1,↑Col1a2,↑Lhx1,↑Mei1,↑Piwil4,↓Pdha2,↑Fabp9 75 Peptide Hormone 2 0.006495 ↑Bmp7,↑Rapgef3 Secretion 76 Placenta Function 4 0.006534 ↑Igf2,↓Lepr,↓Spp1,↑Tfap2c 77 Membrane 19 0.006691 ↑Ryr1,↑Igf2,↑Cav1,↑Cdh1,↑Sct,↑Tacr3,↑Myh6,↑Ednrb,↑Rapgef3, Depolarization ↓Spp1,↑Cartpt,↑Il18,↑Slc6a4,↑Ptger4,↑Abcc9,↑Hif3a,↓Shmt1,↑Ca sq2,↑Kcnc4 78 Kidney 7 0.006868 ↑Igf2,↑Bmp7,↓Spp1,↑Etv4,↑Lhx1,↑Itga8,↑Sall1 Development 79 Muscle Cell 16 0.007016 ↑Ryr1,↑Igf2,↑Bmp7,↑Fhl2,↑Tnnt2,↑Fn1,↑Pou5f1,↑Ccnd3,↑Tgfbr3 Differentiation ,↑Igfbp7,↑Lama2,↑S100a6,↑Lin28a,↑Tshz3,↑Unc45b,↑Pdzrn3 80 Mesenchyma 5 0.007258 ↑Cdh1,↑Bmp7,↑Fn1,↑Prrx1,↑Tcf21 Interaction 81 Cell Growth 59 0.007471 ↑Igf2,↑Entpd2,↑Mrvi1,↑Cav1,↑Cdh1,↑Sct,↑Bmp7,↑Thbs2,↑Fhl2,↑ Ednrb,↑Fn1,↑Rapgef3,↓Lepr,↑Tfap2a,↓Spp1,↑Prkg1,↑Pou5f1,↑Pnl ip,↑Il18,↑Stc2,↑Itga1,↑Igfbp6,↑Slc6a4,↑Igfbp2,↑Serping1,↑Ptgis,↑ Ptger4,↑Tagln,↑Cxcl16,↑Bgn,↓Jup,↑Pdgfra,↑Ccnd3,↑Tpm2,↑Thbd ,↑Plcg2,↑Igfbp7,↓Shmt1,↑Icosl,↓Ccnd2,↑Col4a3,↑Etv4,↑Tfap2c,↑ Ogn,↑Gfra1,↓Frzb,↑Sfrp1,↑Gfap,↑Twist2,↓Smarce1,↑S100a6,↑Itg a8,↑Lin28a,↓Trim25,↑Cnn2,↓Exoc6,↑Tbata,↑Rasgrp2,↑Ctr9 82 Endometrium 4 0.007636 ↑Igf2,↑Cdh1,↑Il18,↑Igfbp7 Function 83 Fibroblast 4 0.007636 ↑Thbs2,↑Fn1,↓Spp1,↑Actg2 Adhesion 84 Artery 5 0.008135 ↑Fn1,↓Spp1,↑Actg2,↑Ptgis,↑Thbd Remodeling 85 Osteoclast 10 0.00841 ↑Igf2,↑Bmp7,↑Fhl2,↓Spp1,↑Il18,↑Slc6a4,↑Igfbp2,↑Ptger4,↑Bgn,↑ Differentiation Plcg2 86 Leiomyocyte 3 0.00889 ↑Fn1,↓Spp1,↑Itga1 Adhesion 87 Chondrocyte 2 0.009076 ↑Fn1,↓Acan Adhesion 88 Ureteric Bud 2 0.009076 ↑Bmp7,↑Lhx1 Development 89 Cementogenesis 2 0.009076 ↑Bmp7,↓Spp1 90 Cell Development 18 0.009273 ↑Igf2,↑Bmp7,↓Lepr,↓Spp1,↑Il18,↑Igfbp6,↑Ptger4,↑Mbl2,↑Pdgfra, ↑Ccnd3,↑Tgfbr3,↑Plcg2,↓Ccnd2,↑Etv4,↑Il2rg,↑Hba-a1/Hba-a2,↑ Arx,↑Sall1 91 Lung Function 7 0.009369 ↑Cav1,↑Fn1,↑Slc6a4,↑Serping1,↑Mbl2,↑Thbd,↑Col4a3 92 Homotypic Cell- 5 0.009414 ↑Cdh1,↑Tfap2a,↓Spp1,↑Itgae,↑Ifitm3 Cell Adhesion 93 Calcium Ion 24 0.009426 ↑Ryr1,↑Cadps,↑Igf2,↑Mrvi1,↑Cav1,↑Cdh1,↑Sct,↑Tnnt2,↑Ednrb,↑ Homeostasis Fn1,↑Rapgef3,↓Spp1,↑Prkg1,↑Pou5f1,↑Cartpt,↑Il18,↑Stc2,↑Cxcl1 6,↑Bgn,↑Sorl1,↑Plcg2,↓Frzb,↑Casq2,↑Lypd6 94 Stress Fiber 11 0.009699 ↑Cav1,↑Cdh1,↑Myl9,↑Fhl2,↑Fn1,↓Spp1,↑Itga1,↑Actg2,↑Tpm2,↑ Assembly Mcf2l,↑Fblim1 95 Endothelial Cell 12 0.009762 ↑Cav1,↑Ednrb,↑Fn1,↓Lepr,↑Prkg1,↑Cyp1b1,↑Il18,↑Ptger4,↑Pdgfr Function a,↑Thbd,↑Sfrp1,↑Sepw1 96 Opsinization 4 0.010193 ↑Fn1,↓Spp1,↑Il18,↑Mbl2 97 Gland 3 0.010242 ↑Igf2,↑Cdh1,↓Jup Development 98 Branching 9 0.010348 ↑Igf2,↑Cdh1,↑Bmp7,↑Fn1,↑Tgfbr3,↑Etv4,↑Tfap2c,↑Sfrp1,↑Itga8 Morphogenesis 99 Inflammatory 27 0.010554 ↑Igf2,↑Cav1,↑Bmp7,↑Thbs2,↑Tacr3,↑Fn1,↑Rapgef3,↓Lepr,↓Spp1, Response ↑Il18,↑Slc6a4,↑Serping1,↑Ptgis,↑Ptger4,↑Cxcl16,↑Bgn,↑Mbl2,↑T hbd,↑H2-Q7,↑Plcg2,↑Igfbp7,↓Ccnd2,↑Col4a3,↑Apoc2,↑Il2rg,↑Gb p3,↑Twist2 100 Hatching 6 0.010763 ↑Igf2,↑Fn1,↓Lepr,↑Igfbp2,↑Ldhc,↑Mei1

213 101 Ovarian Follicle 5 0.01197 ↑Igf2,↑Cdh1,↑Fn1,↓Lepr,↑Cyp11a1 Development 102 Response To 2 0.01204 ↑Bmp7,↓Ccnd2 Estradiol Stimulus 103 Fibroblast 9 0.01247 ↑Cdh1,↑Fn1,↑Rapgef3,↓Spp1,↑Igfbp2,↑Pdgfra,↑Igfbp7,↓Frzb,↑S1 Proliferation 00a6 104 Blood Circulation 13 0.01303 ↑Igf2,↑Cav1,↑Sct,↑Ednrb,↑Fn1,↓Spp1,↑Il18,↑Actg2,↑Slc6a4,↑Ptgi s,↑Ptger4,↑Mbl2,↑Sfrp1 105 Cell Fate 17 0.01397 ↑Cav1,↑Cdh1,↑Bmp7,↑Fn1,↓Spp1,↑Pou5f1,↑Il18,↑Foxc2,↑Igfbp6, ↓Jup,↑Pdgfra,↑Tfap2c,↑Ascl2,↑Tbx2,↑Lin28a,↑Arx,↑Sall1 106 T-Cell 5 0.01407 ↓Spp1,↑Il18,↑Serping1,↑Cxcl16,↑Itgae Recruitment 107 Neutrophil 4 0.01497 ↑Fn1,↑Prkg1,↑Il18,↑Plcg2 Degranulation 108 Bone 4 0.01497 ↑Fn1,↓Spp1,↑Ptger4,↑Bgn Calcification 109 Blastocyst 3 0.01498 ↑Fn1,↓Spp1,↑Ptgis Development 110 Mesenteric 2 0.01536 ↑Ednrb,↑Serping1 Circulation 111 Quiescence 9 0.01651 ↑Cav1,↑Cdh1,↑Fn1,↓Spp1,↑Prkg1,↑Ccnd3,↓Ccnd2,↑Hoxd10,↑Osr 2 112 Cardiovascular 5 0.0174 ↑Fhl2,↑Fn1,↑Foxc2,↑Igfbp2,↑Prrx1 Development 113 Multicellular 4 0.01748 ↑Cd24a,↑Bmp7,↑Pkdcc,↑Olfml3 Organismal Development 114 Cartilage 3 0.01774 ↑Bmp7,↓Acan,↓Frzb Homeostasis 115 Mesoderm 5 0.01791 ↑Igf2,↑Bmp7,↑Pou5f1,↑Tbx2,↑Lhx1 Formation 116 Immune System 7 0.01808 ↑Il18,↑Slc6a4,↑Cxcl16,↑Mbl2,↓Slc15a2,↑H2-Q7,↑Gbp3 Activation 117 Glycoprotein 2 0.01904 ↑Sct,↑Ptger4 Secretion 118 Midbrain 2 0.01904 ↑Bmp7,↑Sfrp1 Development 119 Capillary 6 0.0194 ↑Cav1,↑Sct,↑Tacr3,↑Ednrb,↑Serping1,↑Lsp1 Permeability 120 Mesenchymal 3 0.02077 ↑Bmp7,↑Fhl2,↑Pdgfra Cell Differentiation 121 Glial Cell 2 0.021 ↑Pdgfra,↑Gfap Development 122 Tubulogenesis 7 0.02167 ↑Cav1,↑Cdh1,↑Bmp7,↑Fn1,↓Spp1,↑Itga1,↑Ptger4 123 Lens 4 0.02247 ↑Bmp7,↑Tfap2a,↑Gjc1,↑Pdgfra Development 124 Leukocyte Cell 8 0.02252 ↑Cav1,↑Fn1,↑Rapgef3,↓Spp1,↑Serping1,↑Mbl2,↑Thbd,↑Plcg2 Adhesion 125 Osteoclast 7 0.02253 ↑Igf2,↑Bmp7,↑Fn1,↓Spp1,↑Il18,↑Ptger4,↑Pdgfra Function 126 Cell Survival 38 0.02275 ↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Ednrb,↑Fn1,↑Rapgef3,↑Tfap2a,↓Spp 1,↑Cyp1b1,↑Pou5f1,↑Cartpt,↑Il18,↑Stc2,↑Foxc2,↑Itga1,↑Igfbp6,↑I gfbp2,↑Ptgis,↑Ptger4,↑Hbb-b1/Hbb-b2/LOC100503605,↑Cxcl 16,↓Jup,↑Pdgfra,↑Plcg2,↓Ccnd2,↑Etv4,↑Hoxd10,↑Itgae,↑Gfra1,↑S frp1,↑Fap,↑Coro1a,↑Twist2,↑S100a6,↑Hoxd9,↑Htra3,↓Cyb5d2 127 Nerve 6 0.02277 ↑Igf2,↑Cdh1,↑Fn1,↑Gfra1,↑Gfap,↑Matn2 Regeneration 128 Biomineral 10 0.02384 ↑Igf2,↑Bmp7,↑Thbs2,↑Fhl2,↑Fn1,↓Spp1,↓Acan,↑Bgn,↑Gjc1,↑Col Formation 1a2 129 Cell-Cell 14 0.02406 ↑Cd24a,↑Cav1,↑Cdh1,↑Bmp7,↑Fn1,↓Spp1,↑Cxcl16,↓Jup,↑Thbd,↑ Adhesion Sorl1,↑Icosl,↑Ecscr,↑Tm4sf5,↑Cd248 130 Endochondral 5 0.02424 ↑Igf2,↑Bmp7,↑Fn1,↓Spp1,↑Prkg1 Ossification 131 Hemolysis 6 0.02432 ↑Cav1,↑Fn1,↑Serping1,↑Hbb-b1/Hbb-b2/LOC100503605,↑Mbl2 ,↑Hba-a1/Hba-a2 132 Skeletal 6 0.02432 ↑Entpd2,↑Bmp7,↑Pdgfra,↑Hoxd10,↑Dnm3os,↑Pkdcc Development 133 T-Cell Function 12 0.0248 ↑Fn1,↓Lepr,↓Spp1,↑Il18,↑Mbl2,↑Ccnd3,↑Plcg2,↑Icosl,↑Il2rg,↑Itg ae,↑Coro1a,↑Lgals2

214 134 Endothelial Cell 6 0.02485 ↑Cav1,↑Fn1,↑Il18,↑Ptgis,↑Bgn,↑Thbd Activation 135 Anoikis 9 0.02503 ↑Igf2,↑Cav1,↑Cdh1,↑Fn1,↑Tpm2,↑Lama2,↑Gfra1,↑Fblim1,↑Fabp 9 136 Trophoblast 2 0.02515 ↑Fn1,↓Spp1 Adhesion 137 Menarche 2 0.02515 ↑Cyp1b1,↑Cyp17a1 138 Osteocyte 2 0.02515 ↑Fn1,↓Spp1 Adhesion 139 Monocyte 8 0.02535 ↑Bmp7,↑Ednrb,↑Fn1,↓Spp1,↑Il18,↑Serping1,↑Mbl2,↑Sorl1 Adhesion 140 Endothelialization 4 0.02568 ↑Fn1,↓Spp1,↑Il18,↑Ptgis 141 Eating Behavior 11 0.02585 ↑Igf2,↑Cav1,↑Sct,↑Bmp7,↓Lepr,↓Spp1,↑Cartpt,↑Il18,↑Slc6a4,↑Pt ger4,↑Cbln1 142 Stem Cell 9 0.02657 ↑Cdh1,↑Bmp7,↑Fhl2,↑Myh6,↑Myh11,↑Pou5f1,↓Jup,↑Pdgfra,↑Lin Differentiation 28a 143 Granulocyte 3 0.02762 ↑Fn1,↑Serping1,↑Thbd Production 144 Keratinocyte 3 0.02887 ↑Cdh1,↑Fn1,↓Jup Adhesion 145 Myoblast 5 0.02893 ↑Igf2,↑Fn1,↑Tfap2a,↑Igfbp6,↑Igfbp2 Proliferation 146 Calcium- 2 0.02962 ↑Cdh1,↑Thbd Dependent Cell- Cell Adhesion 147 Response To Food 2 0.02962 ↑Prkg1,↑Slc6a4 148 Endometrium 2 0.02962 ↑Igf2,↑Igfbp6 Proliferation 149 Organ 2 0.02962 ↑Bmp7,↓Rdh10 Morphogenesis 150 Response To 2 0.02962 ↑Bmp7,↓Ccnd2 Peptide Hormone Stimulus 151 Smooth Muscle 8 0.0298 ↑Mrvi1,↑Cav1,↑Myl9,↑Ednrb,↑Myh11,↑Prkg1,↑Ptger4,↑Tagln Contraction 152 18 0.03065 ↑Cadps,↑Cav1,↑Cdh1,↑Thbs2,↑Fhl2,↑Fn1,↑Rapgef3,↓Spp1,↑Prkg Organization And 1,↑Actg2,↑Tagln,↑Sorl1,↑Plcg2,↑Ecscr,↑Lsp1,↑Coro1a,↑S100a6,↑ Biogenesis Cnn2 153 Osteoclast 8 0.03075 ↑Igf2,↑Fhl2,↑Fn1,↓Spp1,↑Il18,↑Igfbp2,↑Ptger4,↑Sfrp1 Formation 154 Stem Cell 5 0.03111 ↑Pou5f1,↑Pdgfra,↑Lhx1,↑Lin28a,↑Sall1 Maintenance 155 Adipocyte 11 0.03191 ↑Igf2,↑Bmp7,↑Fn1,↑Rapgef3,↑Tfap2a,↑Prkg1,↑Foxc2,↑Igfbp2,↑Pt Differentiation ger4,↑Ccnd3,↑Sfrp1 156 Mesenchymal 3 0.03278 ↑Igf2,↑Cdh1,↑Bmp7 Transition 157 B-Cell Receptor 2 0.03437 ↑Ptger4,↑Plcg2 Signaling Pathway 158 Ear Development 2 0.03437 ↑Etv4,↓Rdh10 159 Ca++ ER Import 3 0.03553 ↑Ryr1,↑Rapgef3,↑Casq2 160 Muscle 3 0.03553 ↑Igf2,↑Ednrb,↑Matn2 Innervation 161 Chemotaxis 20 0.03585 ↑Igf2,↑Cav1,↑Sct,↑Bmp7,↑Thbs2,↑Fn1,↑Rapgef3,↓Lepr,↓Spp1,↑P rkg1,↑Il18,↑Igfbp6,↑Cxcl16,↑Pdgfra,↑Plcg2,↑Ecscr,↑Lsp1,↑Sfrp1, ↑Coro1a,↑Cnn2 162 Complement 6 0.03651 ↑Fn1,↑Serping1,↑Bgn,↑Mbl2,↑Thbd,↑Col4a3 Activation 163 Invasive Growth 5 0.03658 ↑Cdh1,↑Fhl2,↑Fn1,↓Spp1,↑Igfbp2 164 Tissue 8 0.03691 ↑Cdh1,↑Fhl2,↑Fn1,↓Spp1,↑Il18,↑Ptger4,↑Pdgfra,↑Fap Remodeling 165 Cell 3 0.03695 ↑Cdh1,↑Fhl2,↑Fn1 Dedifferentiation 166 Myelination 9 0.03727 ↑Igf2,↑Cav1,↓Spp1,↑Il18,↓Acan,↑Igfbp2,↑Tfap2c,↑Lama2,↓Nfia 167 ECM Degradation 6 0.03791 ↑Cav1,↑Bmp7,↑Fn1,↑Il18,↑Fap,↑Adamts20 168 Cardiovascular 5 0.03824 ↑Ednrb,↑Slc6a4,↑Abcc9,↑Gjc1,↑Pnmt Function 169 Thymus Function 2 0.0394 ↑Igf2,↑Tbata 170 Cell Death 50 0.04009 ↑Ryr1,↑Cadps,↑Igf2,↑Entpd2,↑Cav1,↑Cdh1,↑Myl9,↑Bmp7,↑Tnnt 2,↑Ednrb,↑Fn1,↑Rapgef3,↓Lepr,↑Tfap2a,↓Spp1,↑Cyp1b1,↑Pou5f1

215 ,↑Cartpt,↑Il18,↑Stc2,↑Foxc2,↑Cyp11a1,↑Ptgis,↑Ptger4,↑Cxcl16,↑ Bgn,↓Jup,↑Mbl2,↑Pdgfra,↑Ccnd3,↑Thbd,↓Shmt1,↓Ccnd2,↑Tfap2c ,↑Lama2,↓Kcnj3,↓Nfia,↑Gfra1,↑Sfrp1,↑Fap,↑Tbx2,↑Gfap,↑Twist2 ,↓Smarce1,↑S100a6,↓Ndel1,↑Tshz3,↑Lgals2,↑Bnc2,↑Htra3 171 Oocyte 8 0.04086 ↑Igf2,↑Cav1,↓Lepr,↑Pou5f1,↑Ptger4,↑Cyp17a1,↑Ccnd3,↑Tgfbr3 Maturation 172 G1 Phase 14 0.0411 ↑Igf2,↑Cav1,↑Cdh1,↑Bmp7,↑Fn1,↑Tfap2a,↓Spp1,↑Bgn,↑Ccnd3,↑I gfbp7,↓Ccnd2,↑Col4a3,↑Sfrp1,↑Sepw1 173 Gap Junction 3 0.04138 ↑Cdh1,↑Rapgef3,↑Gjc1 Assembly 174 Capillary 3 0.04138 ↑Thbs2,↑Fn1,↑Cyp1b1 Morphogenesis 175 Sertoli Cell 2 0.04201 ↑Pdgfra,↑Tcf21 Differentiation 176 Homophilic Cell 2 0.04201 ↑Cdh1,↑Gfra1 Adhesion 177 Gland 2 0.04201 ↑Igf2,↑Cdh1 Morphogenesis 178 Thymus 2 0.04201 ↑Il18,↑Il2rg Maturation 179 Leukocyte 4 0.04337 ↓Spp1,↑Thbd,↑Icosl,↑Cd248 Accumulation 180 Vasodilation 12 0.04378 ↑Cav1,↑Sct,↑Ednrb,↑Fn1,↓Lepr,↑Prkg1,↑Itga1,↑Ptgis,↑Ptger4,↑Ab cc9,↑Igfbp7,↑Aldoc 181 G1/S Transition 15 0.04381 ↑Igf2,↑Cdh1,↑Fn1,↑Myh11,↑Tfap2a,↓Spp1,↑Cyp1b1,↑Ptger4,↑Cc nd3,↓Ccnd2,↑Tfap2c,↑Tm4sf5,↓Smarce1,↑Bcat1,↑Gli2 182 Gastrulation 8 0.04384 ↑Cdh1,↑Bmp7,↑Fn1,↑Tfap2a,↑Pou5f1,↑Ptger4,↓Ccnd2,↓Frzb 183 Mucosal Barrier 3 0.04447 ↑Il18,↑Ptger4,↑Lgals2 184 Vasculature 2 0.04468 ↑Bmp7,↑Thbs2 Development 185 Osteoclast 2 0.04468 ↓Spp1,↑Plcg2 Adhesion 186 Actin 19 0.0467 ↑Cdh1,↑Myl9,↑Bmp7,↑Ednrb,↑Fn1,↑Rapgef3,↓Spp1,↑Actg2,↑Tag Organization ln,↓Jup,↑Plcg2,↑Tm4sf5,↑Lsp1,↑Coro1a,↓Ndel1,↑Syn2,↑Fblim1,↑ Prr5,↑Rcsd1 187 Hemostasis 6 0.04704 ↑Thbs2,↑Fn1,↑Slc6a4,↑Mbl2,↑Thbd, ↑Mfap2 188 Neurosteroidogen 2 0.04742 ↑Cyp11a1,↑Cyp17a1 esis 189 Blood Clotting 14 0.04762 ↑Igf2,↑Entpd2,↑Mrvi1,↑Ednrb,↑Fn1,↓Lepr,↑Prkg1,↑Slc6a4,↑Serpi ng1,↑Ptgis,↑Ptger4,↑Mbl2,↑Thbd,↑Plcg2 190 Mesenchymal To 3 0.04768 ↑Cdh1,↑Bmp7,↑Twist2 Epithelial Transition 191 Agglutination 4 0.04803 ↑Fn1,↑Mbl2,↑Thbd,↑Plcg2 192 Cell Fusion 7 0.04851 ↑Igf2,↑Cav1,↑Cdh1,↑Fn1,↑Prkg1,↑Il18,↑Ascl2 193 Platelet Response 4 0.04924 ↑Fn1,↑Prkg1,↑Thbd,↑Plcg2 194 Regulation Of 4 0.04924 ↑Igf2,↑Prkg1,↑Igfbp2,↑Mfap2 Body Size 195 Locomotion 13 0.04982 ↑Cadps,↑Cav1,↑Cdh1,↑Tacr3,↑Ednrb,↑Fn1,↓Lepr,↓Spp1,↑Cartpt,↑ Il18,↑Slc6a4,↑Bgn,↓Ndel1

216 Table A1-2. Total Pathway Studio subnetworks for P8 germ cell differentially regulated at ±

2.0 fold genes.

Group Term # of Genes Pvalue Genes 1 Synapsis 17 1.36E-14 ↓Stra8,↓Dmc1,↓Spo11,↓Rec8,↓Msh5,↓Stag3,↓Sycp1,↓Sycp2,↓S mc1b,↓Tex11,↓Mei1,↓Sycp3,↓Tex15,↓Tex12,↓Mov10l1,↓Horma d1,↓Syce1 2 Male Meiosis 10 1.43E-12 ↓Boll,↓Stag3,↓Sycp2,↓Tex11,↓Mei1,↓Sycp3,↓Tex15,↓Slc25a31,↓ Mov10l1,↓Dmrtc2 3 Spermatogenesis 32 3.05E-09 ↓Timp2,↑Spp1,↓Lmna,↓Krt18,↓Stra8,↓Clgn,↓Tex101,↓Mtl5,↓Dm c1,↓Spo11,↓Boll,↓Taf4b,↑Adamts2,↑Gfra1,↓Msh5,↓Prss50,↓Syc p2,↓Gfer,↓Mei1,↓Sycp3,↓Prdm9,↑Hagh,↓Tex15,↓Xlr,↓Slc25a31, ↓Mov10l1,↓Hormad1,↓Dmrtc2,↓Syce1,↓Mael,↓Hsf2bp,↓Zfp541 4 Synaptonemal 7 6.99E-09 ↓Dmc1,↓Spo11,↓Rec8,↓Msh5,↓Smc1b,↓Sycp3,↓Hormad1 Complex Formation 5 Crossover 6 9.24E-07 ↓Dmc1,↓Spo11,↓Msh5,↓Sycp1,↓Smc1b,↓Tex11 Formation 6 Pachytene 8 6.47E-06 ↓Dmc1,↓Spo11,↓Rec8,↓Tex11,↓Mei1,↓Gm98,↓Hormad1,↓Rnf17 7 Synaptonemal 3 7.98E-06 ↓Stag3,↓Sycp2,↓Tex15 Complex Assembly 8 Meiosis 25 1.39E-05 ↓Pak1,↓Stra8,↓Dmc1,↓Spo11,↓Boll,↓Rec8,↓Msh5,↓Spdya,↓Stag3 ,↓Sycp1,↓Sycp2,↓Brsk2,↓Smc1b,↓Tex11,↓Mei1,↓Gm98,↓Sycp3, ↓Prdm9,↓Tex15,↓Slc25a31,↓Tex12,↓Hormad1,↓Dmrtc2,↓Mael,↓ Rnf17 9 Neurite Outgrowth 33 3.61E-05 ↑P2rx7,↓Trpc1,↓Timp2,↓Pak1,↑Spp1,↓Crk,↓Tfrc,↓Klf9,↓Socs3,↓ H2-Q10,↑Itga9,↓Atf3,↑Wnt5a,↓Itgb3,↓Nes,↓Braf,↑Ptprz1,↓Scn8a ,↓Hck,↓Alcam,↑Gfra1,↓Kidins220,↑Gfap,↓Pou4f1,↓Ndrg4,↓Nck2 ,↓Rab6b,↑Stx3,↑Negr1,↑Rgmb,↓Nptx2,↓Micall2,↓Gpc2 10 Cell Growth 74 9.68E-05 ↑P2rx7,↓Entpd2,↓Trpc1,↓Timp2,↓Chga,↓Rel,↓Nr4a3,↑Igfbp5,↓P ak1,↑Spp1,↓Crk,↓Tfrc,↓Prkd3,↓Klf9,↑Fgfr2,↓Pth1r,↓Socs3,↓H2- Q10,↑Itga9,↓Igfbp2,↓Spint1,↓Vldlr,↓Ptger1,↓Nmb,↓Atf3,↑Wnt5a ,↓S100a8,↓Itgb3,↑Cth,↓Nes,↓Braf,↓Lmna,↓Krt18,↓Sgpl1,↑Sox18, ↓Anpep,↑Ptprz1,↓Dmrtb1,↑Fbln2,↓Rgs16,↓Egr3,↓Hck,↑Ube2c,↓ Tnfrsf13c,↓Alcam,↓Epcam,↑Gfra1,↓Runx3,↓Spdya,↓Dkk3,↑E4f1 ,↑Synpo2,↑Lgals7,↓Hipk2,↓Rif1,↓Sec14l2,↓Crabp1,↑Gfap,↓Tshb ,↓Plekhf1,↓Gfer,↓Kif1b,↑Hagh,↓Daam1,↓Ptpn4,↓Slc25a31,↓Ebf3 ,↑Negr1,↑Ctr9,↓Rps6ka6,↓Tktl1,↑1500015O10Rik,↓Arrdc4,↑Ada mts16 11 Cell Differentiation 94 0.000199 ↑P2rx7,↓Entpd2,↓Trpc1,↓Timp2,↓Rel,↓Nr4a3,↓Enpp1,↑Igfbp5,↓ Pak1,↓Dsp,↑Spp1,↓Crk,↓Tfrc,↓Actn3,↓Klf9,↓Acsl1,↑Foxc2,↑Fgf r2,↓Pth1r,↓Socs3,↓H2-Q10,↑Itga9,↓Igfbp2,↓Spint1,↓Vldlr,↓Ptger 1,↓Nmb,↓Rbp1,↓Atf3,↑Wnt5a,↓S100a8,↓Itgb3,↓Nes,↓Braf,↓Abc c5,↓Lmna,↓Krt18,↓Sgpl1,↓Stra8,↑Sox18,↓Anpep,↑Fabp4,↑Ptprz1 ,↓Boll,↓Cd3e,↑Fbln2,↓Rgs16,↓Egr3,↓Hck,↓Tnfrsf13c,↓Taf4b,↓Al cam,↓Epcam,↑Gfra1,↓Cd83,↓Runx3,↓Dkk3,↓Krt8,↑Synpo2,↑Lga ls7,↓Hipk2,↓Crabp1,↓Hopx,↓Kidins220,↑Gfap,↓Pbx3,↓Pou4f1,↓ Ndrg4,↓Elavl2,↓Nck2,↓Apba1,↓Sycp3,↓Daam1,↓Ptpn4,↓Sema4f, ↑Tesc,↓Ebf3,↓Npnt,↑Tpbg,↑Rgmb,↓Zfp318,↓Ctnnd2,↑Mkx,↑Mp zl2,↓Dnase1l2,↓Mbnl3,↓Nut,↓Mael,↓Necab3,↑Tchh,↓Rnf17,↓Oo ep,↓Disp1 12 Organ 4 0.0002195 ↑Fgfr2,↑Wnt5a,↓Braf,↓Sycp2 Morphogenesis 13 Epithelial To 22 0.0004172 ↓Timp2,↓Rel,↑Igfbp5,↓Pak1,↓Dsp,↑Spp1,↓Crk,↑Foxc2,↑Fgfr2,↓S Mesenchymal pint1,↓Vldlr,↓Atf3,↑Wnt5a,↓Itgb3,↑Cth,↓Krt18,↑Fbln2,↓Epcam,↓ Transition Runx3,↓Dkk3,↑Tpbg,↓Galnt6 14 Cell Migration 53 0.0004392 ↑P2rx7,↓Trpc1,↓Timp2,↑Igfbp5,↓Pak1,↑Spp1,↓Crk,↓Klf9,↑Foxc 2,↑Fgfr2,↓Socs3,↓H2-Q10,↑Itga9,↓Igfbp2,↓Spint1,↓Vldlr,↓Ptger1 ,↓Atf3,↑Wnt5a,↓S100a8,↓Itgb3,↓Nes,↓Braf,↓Abcc5,↓Lmna,↓Krt 18,↓Sgpl1,↑Sox18,↓Anpep,↑Ptprz1,↑Fbln2,↓Rgs16,↓Egr3,↓Hck,↓ Taf4b,↓Alcam,↓Epcam,↑Gfra1,↓Cd83,↓Dkk3,↓Krt8,↑Synpo2,↑L gals7,↓Kidins220,↓Ly6k,↓Mt1,↓Nck2,↓Gfer,↓Daam1,↓Npnt,↓Ctn nd2,↑1500015O10Rik,↓Gpc2 15 Positive Regulation 2 0.0004767 ↑P2rx7,↓H2-Q10

217 Of Natural Killer Cell Mediated Cytotoxicity 16 Snaptonemal 2 0.0004767 ↓Stag3,↓Sycp2 Complex Assembly 17 Meiotic Prophase I 3 0.0005246 ↓Mtl5,↓Msh5,↓Sycp3 18 Premeiotic DNA 4 0.0006064 ↓Stra8,↓Spo11,↓Rec8,↓Msh5 Synthesis 19 Cell Motility 32 0.000879 ↓Trpc1,↓Timp2,↓Rel,↓Enpp1,↑Igfbp5,↓Pak1,↑Spp1,↓Crk,↓Socs3, ↓Igfbp2,↓Vldlr,↓Atf3,↑Wnt5a,↓S100a8,↓Nes,↓Braf,↓Lmna,↓Sgpl 1,↓Anpep,↑Ptprz1,↑Fbln2,↓Alcam,↓Epcam,↓Dkk3,↓Krt8,↑Synpo 2,↓Hipk2,↓Kidins220,↓Nck2,↑Tpbg,↓Rps6ka6,↓Ctnnd2 20 Cell Proliferation 101 0.0008861 ↑P2rx7,↓Entpd2,↓Trpc1,↓Timp2,↓Chga,↓Rel,↓Nr4a3,↑Igfbp5,↓P ak1,↓Dsp,↑Spp1,↓Crk,↓Tfrc,↓Klf9,↓Acsl1,↑Foxc2,↑Fgfr2,↓Pth1r, ↓Socs3,↓H2-Q10,↑Itga9,↓Igfbp2,↓Spint1,↓Vldlr,↓Ptger1,↓Nmb, ↓Rbp1,↓Atf3,↑Wnt5a,↓S100a8,↓Itgb3,↑Cth,↑Rbm3,↓Nes,↓Braf,↓ Lmna,↓Krt18,↓Sgpl1,↓Ramp1,↓Anpep,↑Fabp4,↓Dmc1,↑Ptprz1,↓ Cd3e,↓Rab3b,↑Fbln2,↑Tle4,↓Rgs16,↓Egr3,↓Hck,↑Ube2c,↓Tnfrsf 13c,↓Taf4b,↑Adamts2,↓Alcam,↓Epcam,↑Gfra1,↓Cd83,↓Runx3,↓ Prss50,↓Spdya,↓Dkk3,↑E4f1,↑Synpo2,↑Lgals7,↓Hipk2,↓Rif1,↑Po lh,↓Sec14l2,↓Crabp1,↓Hopx,↑Gfap,↓Aak1,↑Kcnc4,↓Ndrg4,↓Plek hf1,↓Tex11,↓Gfer,↑Bcat1,↓Daam1,↓Ptpn4,↓Sema4f,↑Tesc,↓Npnt ,↓Aifm3,↑Tpbg,↑Rgmb,↓Tuba4a,↓Rps6ka6,↓Ctnnd2,↓Nptx2,↓M ov10l1,↓Tktl1,↑1500015O10Rik,↓Eras,↑Adamts16,↓Slc41a2,↓In ca1,↓Galnt6, ↓Car11 21 DNA 24 0.0011358 ↓Entpd2,↓Rel,↑Fgfr2,↓Pth1r,↓Nes,↓Braf,↓Dmc1,↓Spo11,↓Cd3e,↓ Recombination Rec8,↓Tnfrsf13c,↓Msh5,↓Rif1,↑Polh,↑Gfap,↓Sycp1,↓Smc1b,↓Te x11,↓Mei1,↓Gm98,↓Sycp3,↓Prdm9,↓Tex15,↓Hormad1 22 Meiotic Entry 5 0.0013044 ↓Stra8,↓Boll,↓Spdya,↓Mei1,↓Gm98 23 Cell Adhesion 38 0.0013739 ↓Entpd2,↓Timp2,↓Chga,↓Rel,↑Igfbp5,↓Pak1,↓Dsp,↑Spp1,↓Crk,↓ Tfrc,↓Klf9,↑Foxc2,↓H2-Q10,↑Itga9,↓Igfbp2,↓Vldlr,↓Atf3,↑Wnt5 a,↓Itgb3,↓Nes,↓Lmna,↓Krt18,↓Anpep,↑Ptprz1,↓Hck,↓Alcam,↓Ep cam,↑Gfra1,↓Cd83,↓Krt8,↓Npnt,↑Rgmb,↓Radil,↑Cytip,↓Ctnnd2, ↑Mpzl2,↓Gpc2,↓Galnt6 24 Pachytene 3 0.0014282 ↓Spo11,↓Rec8,↓Gm98 Checkpoint 25 Meiotic 2 0.0015626 ↓Stra8,↓Spo11 Chromosome Condensation 26 Embryonal 41 0.0031616 ↑P2rx7,↓Timp2,↓Rel,↑Igfbp5,↓Dsp,↑Spp1,↓Crk,↓Rbp4,↑Fgfr2,↓ Development Pth1r,↑Itga9,↓Igfbp2,↓Spint1,↑Wnt5a,↓S100a8,↓Nes,↓Braf,↓Abc c5,↓Lmna,↓Sgpl1,↑Sox18,↓Ramp1,↓Anpep,↑Ptprz1,↓Nobox,↑Fbl n2,↓Alcam,↓Runx3,↑E4f1,↓Rif1,↓Hopx,↑Gfap,↓Pbx3,↑Ppm1k,↓ Rad9b,↓Npnt,↑Tpbg,↓Tuba4a,↓Rps6ka6,↑Mkx,↓Ooep 27 Morphogenesis 32 0.0036957 ↓Timp2,↓Chga,↓Rel,↑Igfbp5,↓Pak1,↓Dsp,↑Spp1,↓Crk,↓Klf9,↑Fo xc2,↑Fgfr2,↓Socs3,↑Wnt5a,↓Itgb3,↓Krt18,↓Sgpl1,↓Mtl5,↑Ptprz1, ↓Egr3,↓Alcam,↓Epcam,↑Gfra1,↓Dkk3,↓Kidins220,↓Nck2,↓Gm9 8,↓Npnt,↓Ctnnd2,↓Whrn,↑Mkx,↓Adad1,↓Galnt6 28 Wound Healing 20 0.0039522 ↓Timp2,↓Rel,↓Pak1,↑Spp1,↑Foxc2,↑Fgfr2,↓Socs3,↑Itga9,↓Atf3,↑ Wnt5a,↓S100a8,↓Itgb3,↑Cth,↓Sgpl1,↑Sox18,↓Anpep,↑Fbln2,↓Dk k3,↑Lgals7,↓Nck2 29 Female Meiosis 3 0.0042234 ↓Spo11,↓Sycp2,↓Sycp3 30 Axon Guidance 10 0.0047565 ↓Trpc1,↓Pak1,↑Wnt5a,↓Alcam,↑Gfra1,↓Runx3,↓Kidins220,↓Pou 4f1,↓Sema4f,↑Rgmb 31 Transmission Of 20 0.0052164 ↑P2rx7,↓Entpd2,↓Chga,↓Pak1,↑Spp1,↓H2-Q10,↓Ptger1,↑Wnt5a, Nerve Impulse ↓Slc22a3,↓Lmna,↓Cacna1h,↓Scn8a,↓Grin2d,↓Kidins220,↓Brsk2, ↓Apba1,↓Pcsk1n,↓Kif1b,↑Stx3,↓Nptx2 32 Osteoclast 3 0.0063543 ↑Spp1,↓Itgb3,↓Plekhm1 Adhesion 33 Fatty Acid 8 0.0071595 ↓Chga,↓Nr4a3,↓Dsp,↓Rbp4,↓Acsl1,↓Igfbp2,↓Vldlr,↑Fabp4 Metabolism 34 Cellular Response 2 0.0081731 ↓Pak1,↓Vldlr To Insulin Stimulus 35 G1/S Transition 19 0.0082669 ↓Rel,↓Pak1,↑Spp1,↓Tfrc,↓Socs3,↓Atf3,↓S100a8,↓Nes,↓Braf,↓Krt 18,↓Spo11,↓Rec8,↓Spdya,↓Dkk3,↓Krt8,↑E4f1,↓Rad9b,↑Bcat1,↑1 500015O10Rik 36 Retinoic Acid 3 0.0083049 ↓Rbp1,↓Stra8,↓Crabp1 Metabolism

218 37 Bone Remodeling 10 0.0089393 ↑P2rx7,↓Timp2,↑Igfbp5,↑Spp1,↓Pth1r,↓Socs3,↓Igfbp2,↓Ptger1,↓ S100a8,↓Lmna 38 Chromosome 5 0.009302 ↓Tfrc,↓Dmc1,↓Spo11,↓Rec8,↓Eif2c3 Breakage 39 Endochondral 6 0.0105394 ↓Enpp1,↑Igfbp5,↑Spp1,↑Fgfr2,↓Pth1r,↓Runx3 Ossification 40 B-Cell 4 0.011464 ↓Rel,↓Sgpl1,↓Tnfrsf13c,↓Cd83 Homeostases 41 Leiomyocyte 3 0.0122607 ↓Chga,↑Spp1,↓Itgb3 Adhesion 42 Skeletal 7 0.0130263 ↑P2rx7,↓Entpd2,↑Fgfr2,↓Pth1r,↑Wnt5a,↑Sox18,↓Runx3 Development 43 Notochord 2 0.0131889 ↓Rel,↓Daam1 Formation 44 Axonogenesis 9 0.0149372 ↑P2rx7,↑Itga9,↑Wnt5a,↓Cacna1h,↓Alcam,↓Kidins220,↓Kif1b,↓S ema4f,↑Rgmb 45 Triacylglycerols 4 0.0152027 ↑Spp1,↓Acsl1,↑Agpat9,↓Lpgat1 Biosynthesis 46 Apoptosis 89 0.018339 ↑P2rx7,↓Entpd2,↓Trpc1,↓Timp2,↓Chga,↓Rel,↓Nr4a3,↓Enpp1,↑Ig fbp5,↓Pak1,↓Dsp,↑Spp1,↓Crk,↓Tfrc,↓Fam3b,↓Prkd3,↓Rbp4,↓Klf 9,↓Acsl1,↑Fgfr2,↓Pth1r,↓Socs3,↓H2-Q10,↓Igfbp2,↓Ptger1,↓Rbp1 ,↓Atf3,↑Wnt5a,↓S100a8,↓Itgb3,↑Cth,↑Rbm3,↓Nes,↓Braf,↓Lmna, ↓Krt18,↓Sgpl1,↓Anpep,↑Fabp4,↓Spo11,↑Ptprz1,↓Cd3e,↑Tle4,↓E gr3,↓Rec8,↓Hck,↑Ube2c,↓Tnfrsf13c,↓Taf4b,↓Epcam,↓Cd83,↓Ru nx3,↓Msh5,↓Prss50,↓Spdya,↓Dkk3,↓Krt8,↑E4f1,↑Lgals7,↓Hipk2, ↓Rif1,↓Gm3776/Gsta1/Gsta2,↑Polh,↓Sec14l2,↓Crabp1,↓Hopx,↓K idins220,↑Gfap,↑Kcnc4,↓Sycp1,↓Mt1,↓Pou4f1,↓Ndrg4,↑Ppm1k, ↓Brsk2,↓Plekhf1,↓Gfer,↓Kif1b,↑Hagh,↑Bcat1,↓Ptpn4,↓Ebf3,↓170 0007E06Rik,↓Aifm3,↓Nptx2,↑1500015O10Rik,↓Atp11c,↓Gml,↓ Rassf10 47 Synaptogenesis 9 0.0184214 ↓H2-Q10,↓Nes,↓Cacna1h,↑Ptprz1,↓Kidins220,↑Gfap,↑Gda,↓Syn 2,↓Nptx2 48 Inner Ear 2 0.0192258 ↓Nr4a3,↑Fgfr2 Morphogenesis 49 Telomere 2 0.0192258 ↓Rec8,↓Brsk2 Clustering 50 Strand Invasion 3 0.0193355 ↓Dmc1,↓Spo11,↑Polh 51 Fatty Acids Import 5 0.0197022 ↓Rbp4,↓Acsl1,↓Vldlr,↑Fabp4,↓Slc27a2 52 Response To 2 0.0214505 ↓Enpp1,↓Krt8 Cadmium Ion 53 Neuron 5 0.0222999 ↑Fgfr2,↑Wnt5a,↑Gfra1,↓Runx3,↓Hipk2 Development 54 Neuromuscular 2 0.0237764 ↓Timp2,↓Kidins220 Junction Development 55 Ureteric Bud 4 0.0238716 ↓Timp2,↑Fgfr2,↑Wnt5a,↑Gfra1 Branching 56 Sister Chromatid 4 0.0238716 ↓Dmc1,↓Rec8,↓Smc1b,↓Sycp3 Cohesion 57 Vasodilation 14 0.0240001 ↑P2rx7,↓Trpc1,↓Timp2,↓Crk,↓Rbp4,↓Pth1r,↓H2-Q10,↓Ptger1,↓It gb3,↑Cth,↓Abcc5,↓Cacna1h,↓Ramp1,↑Fabp4 58 S Phase 22 0.0244481 ↓Entpd2,↓Timp2,↓Rel,↑Igfbp5,↑Spp1,↓Tfrc,↓Acsl1,↓Pth1r,↓Socs 3,↓Atf3,↓Lmna,↓Dmc1,↓Spo11,↓Rec8,↑Ube2c,↓Epcam,↓Spdya,↑ E4f1,↓Rif1,↑Polh,↑1500015O10Rik, ↓Inca1 59 Multicellular 4 0.0257112 ↑Wnt5a,↓Dkk3,↓Zfp541,↓Olfml3 Organismal Development 60 Genital 2 0.0262007 ↑Fgfr2,↑Gfra1 Development 61 Long-Chain Fatty 2 0.0287204 ↓Acsl1,↓Slc27a2 Acid Transport 62 Meiotic 2 0.0287204 ↓Rec8,↓Sycp3 Chromosome Segregation 63 Cell Invasion 23 0.0291844 ↑P2rx7,↓Timp2,↓Pak1,↑Spp1,↓Crk,↑Fgfr2,↓Igfbp2,↓Ptger1,↓Atf3 ,↑Wnt5a,↓S100a8,↓Itgb3,↓Braf,↓Tex101,↓Anpep,↓Egr3,↑Ube2c, ↓Alcam,↓Epcam,↓Runx3,↓Dkk3,↓Hipk2,↑Tpbg 64 Mitochondrion 3 0.0296636 ↑Foxc2,↓Gfer,↓Kif1b Fusion 65 Ossification 15 0.0302455 ↑P2rx7,↓Timp2,↑Igfbp5,↑Spp1,↑Fgfr2,↓Pth1r,↓Igfbp2,↓Ptger1,↓

219 Rbp1,↑Wnt5a,↓Itgb3,↓Lmna,↓Taf4b,↓Runx3,↓Dkk3 66 Osteocyte 2 0.0313328 ↑Spp1,↑Fgfr2 Adhesion 67 Myelination 10 0.0315042 ↑P2rx7,↑Spp1,↓Tfrc,↓Klf9,↓H2-Q10,↓Igfbp2,↓Gm98,↓Kif1b, ↓C1orf130,↓Ugt8 68 G0/G1 Transition 12 0.0339405 ↓Trpc1,↓Timp2,↓Rel,↑Igfbp5,↓Acsl1,↓Socs3,↑Ube2c,↓Dkk3,↓Cr abp1,↑Gfap,↓Pou4f1,↓Nptx2 69 Karyogamy 2 0.0340351 ↓Rec8,↓Mei1 70 3 0.0341524 ↓Nr4a3,↑Igfbp5,↓Socs3 Growth 71 Cell Survival 41 0.0352046 ↑P2rx7,↓Trpc1,↓Timp2,↓Rel,↓Nr4a3,↑Igfbp5,↓Pak1,↑Spp1,↓Crk, ↑Foxc2,↑Fgfr2,↓Socs3,↓H2-Q10,↓Igfbp2,↓Spint1,↓Rbp1,↓Atf3, ↑Wnt5a,↓S100a8,↓Itgb3,↑Cth,↓Nes,↓Braf,↓Krt18,↓Sgpl1,↓Dmc1, ↑Tle4,↓Tnfrsf13c,↑Gfra1,↓Cd83,↓Runx3,↓Spdya,↓Dkk3,↓Hipk2, ↓Sec14l2,↓Kidins220,↓Pou4f1,↑Ppm1k,↓Nck2,↓Gfer,↓Ebf3 72 Spermatid 2 0.0368247 ↓Boll,↓Dazl Differentiation 73 Wnt Receptor 2 0.0368247 ↑Wnt5a,↓Dkk3 Signaling Pathway 74 Glucose 10 0.0368659 ↓Chga,↑Igfbp5,↑Spp1,↓Fam3b,↓Rbp4,↓Socs3,↓Igfbp2,↓Atf3,↑Fa Homeostasis bp4,↓Hipk2 75 Osteoclast 3 0.0373292 ↑Igfbp5,↑Spp1,↓Pth1r Activation 76 Spermatid 5 0.0395387 ↓Pak1,↓Mtl5,↓Spo11,↓Boll,↓Rnf17 Development 77 Protein-Protein 4 0.0397153 ↑Fgfr2,↓Pth1r,↓S100a8,↑Cth Cross-Linking Via L-Cystine 78 G2/M Transition 17 0.0404128 ↓Rel,↑Igfbp5,↑Spp1,↓Tfrc,↑Wnt5a,↓Nes,↓Braf,↓Lmna,↓Boll,↑Ub e2c,↓Spdya,↑E4f1,↑Lgals7,↓Rif1,↑Kcnc4,↓Brsk2,↓Gm98 79 Beta Selection 3 0.040652 ↓Socs3,↓Cd3e,↓Egr3 80 Macrophage 10 0.0422638 ↑P2rx7,↓Nr4a3,↑Spp1,↓Socs3,↓Atf3,↑Wnt5a,↓S100a8,↓Hck,↓Ste Activation ap1,↑1500015O10Rik 81 Placenta 8 0.0432361 ↑Spp1,↓Socs3,↓Spint1,↑Wnt5a,↓Braf,↓Abcc5,↓Krt18,↓H2-Q10 Development 82 Regeneration 22 0.0454602 ↑P2rx7,↓Timp2,↓Rel,↑Igfbp5,↑Spp1,↓Klf9,↑Fgfr2,↓Socs3,↓H2- Q10,↑Itga9,↓Igfbp2,↓Spint1,↓Rbp1,↓Atf3,↓Nes,↑Ptprz1,↓Scn8a,↓ Prss50,↑Gfap,↓Gfer,↑Hagh,↓Npnt 83 Cytoskeleton 19 0.047183 ↑P2rx7,↓Chga,↑Igfbp5,↓Pak1,↑Spp1,↓Crk,↑Wnt5a,↓S100a8,↓Itg Organization And b3,↓Nes,↓Braf,↓Krt18,↑Sox18,↓H2-Q10,↓Epcam,↓Krt8,↓Nck2,↑ Biogenesis Tpbg,↑Cytip 84 Mesenchyma 4 0.0473897 ↑Fgfr2,↓Pth1r,↓Atf3,↑Wnt5a Interaction

220 Table A1-3. Total Pathway Studio Gene Ontology (GO) for P4 germ cell differentially regulated at ± 2.0 fold genes.

Group Term # of Genes Pvalue Genes 1 Multicellular 35 4.19E-13 ↑Cd24a,↓Zfp39,↑Igf2,↑Bmp7,↓Lepr,↑Pou5f1,↑Foxc2,↑Igfbp2,↑ Organismal Tcf23,↑Sorl1,↑Ascl2,↑Figla,↑Hoxd10,↑Ecscr,↑Aff3,↓Frzb,↑Sfr Development p1,↑Tbx2,↑Prrx1,↑Twist2,↓Ndel1,↑Lhx1,↑Itga8,↑Piwil4,↑Tnp1, ↑Arx,↓Plekhb1,↑Tshz3,↑Tbata,↑Unc45b,↑Hoxd8,↓Plxna4,↑Hox d9,↑Pkdcc,↑Olfml3 2 Muscle 11 1.92E-09 ↑Actg2,↑Ryr1,↑Myl9,↑Myh6,↑Tnnt2,↑Myh11,↑Actn3,↑Itga1,↑ Contraction Actg2,↑Gjc1,↑Tpm2 3 Cell 23 3.78E-09 ↓Herc4,↑Cd24a,↓Zfp39,↑Bmp7,↓Spp1,↑Tcf23,↑Tfap2c,↑Ascl2, Differentiation ↑Figla,↑Ecscr,↓Frzb,↑Sfrp1,↑Twist2,↓Ndel1,↑Lhx1,↑Itga8,↑Pi wil4,↑Tnp1,↑Arx,↑Tbata,↑Unc45b,↑Pkdcc,↑Gli2 4 Ureteric Bud 7 3.66E-08 ↑Bmp7,↑Foxc2,↑Sfrp1,↑Tcf21,↑Lhx1,↑Sall1,↑Tshz3 Development 5 Skeletal System 10 2.54E-07 ↑Igf2,↑Bmp7,↑Foxc2,↓Acan,↑Hoxd10,↓Frzb,↑Col1a2,↑Dnm3o Development s,↑Col5a2,↑Gli2 6 Osteoblast 7 2.73E-07 ↑Igf2,↑Fhl2,↓Spp1,↑Il18,↑Sfrp1,↑Twist2,↑Gli2 Differentiation 7 Metanephros 6 7.02E-07 ↑Bmp7,↑Foxc2,↑Itga8,↓Rdh10,↑Tshz3,↑Osr2 Development 8 Cell Adhesion 18 2.52E-06 ↑Islr,↑Col22a1,↑Cd24a,↑Cdh1,↑Thbs2,↑Fn1,↓Spp1,↑Itga1,↓Ac an,↓Jup,↑Igfbp7,↑Col4a3,↑Lama2,↑Itgae,↑Itga8,↑Mfap4,↑Fbli m1,↑Lamc3 9 Response To 9 2.86E-06 ↑Cdh1,↓Spp1,↑Cyp1b1,↑Cyp11a1,↑Slc6a4,↑Cyp17a1,↑Pdgfra, Organic Substance ↑Ccnd3,↑C1qb 10 Axon Guidance 12 4.82E-06 ↑Bmp7,↑Myh11,↑Itga1,↑Col4a3,↑Gfra1,↓Spna1,↑Col1a2,↑Col4 a4,↑Arx,↓Plxna4,↑Col5a2,↑Gli2 11 Response To 8 7.19E-06 ↑Bmp7,↑Pnlip,↑Il18,↑Stc2,↑Cyp11a1,↑Ccnd3,↓Ccnd2,↑Pnliprp Peptide Hormone 2 Stimulus 12 Response To 10 7.89E-06 ↑Igf2,↑Ednrb,↑Cyp11a1,↑Cyp17a1,↑Igfbp7,↓Ccnd2,↓Nfia,↑Sfr Organic Cyclic p1,↑Aldoc,↑Ugt1a1/Ugt1a10/Ugt1a2/Ugt1a5/Ugt1a6a/Ugt1a6b Compound /Ugt1a7c/Ugt1a9 13 Response To 7 9.01E-06 ↑Cd24a,↑Cav1,↓Lepr,↑Cyp11a1,↑Igfbp2,↑Pdgfra,↓Trim25 Estrogen Stimulus 14 Response To 10 1.02E-05 ↑Cd24a,↑Ryr1,↑Cav1,↑Il18,↑Slc6a4,↑Ptgis,↑Tgfbr3,↑Hif3a,↑A Hypoxia scl2,↑Aldoc 15 In Utero 10 1.10E-05 ↑Meg3,↑Cdh1,↑Myh6,↑Pdgfra,↑Tgfbr3,↑Ascl2,↑Hba-a1/Hba- Embryonic a2,↓Rdh10,↑Tshz3,↑Gli2 Development 16 Ossification 7 1.23E-05 ↑Igf2,↑Bmp7,↑Fn1,↓Spp1,↑Foxc2,↑Pkdcc,↑Col5a2 17 Extracellular 7 1.39E-05 ↑Fn1,↑Pdgfra,↑Lama2,↑Gfap,↑Itga8,↑Adamts20,↑Egflam Matrix Organization 18 Response To 4 3.36E-05 ↑Bmp7,↓Spp1,↑Stc2,↓Trim25 Vitamin D 19 Embryonic Cranial 4 3.36E-05 ↑Tfap2a,↑Foxc2,↑Prrx1,↑Twist2 Skeleton Morphogenesis 20 Palate 5 3.53E-05 ↑Pdgfra,↑Tgfbr3,↑Prrx1,↑Osr2,↑Pkdcc Development 21 Platelet Activation 9 6.64E-05 ↑Entpd2,↑Mrvi1,↑Fn1,↑Prkg1,↑Serping1,↑Plcg2,↑Col1a2,↑Ras grp2,↑Pik3r6 22 Ventricular 4 6.87E-05 ↑Myh6,↑Tnnt2,↑Foxc2,↑Tgfbr3 Cardiac Muscle Tissue Morphogenesis 23 Embryonic 5 7.08E-05 ↑Foxc2,↑Hoxd10,↑Prrx1,↑Hoxd9,↑Osr2 Skeletal System Morphogenesis 24 Cell Migration 7 7.29E-05 ↑Cd24a,↑Fn1,↓Jup,↑Tgfbr3,↑Sorl1,↑Coro1a,↓Ndel1 25 Dibenzo-P-Dioxin 3 7.56E-05 ↑Cyp1b1,↑Cyp11a1,↑Cyp17a1 Metabolic Process

221 26 Response To 7 7.97E-05 ↑Igf2,↑Bmp7,↑Tacr3,↑Slc6a4,↑Igfbp2,↑Pdgfra,↓Ccnd2 Estradiol Stimulus 27 Negative 13 7.98E-05 ↑Cav1,↑Ednrb,↑Pou5f1,↑Foxc2,↑Ascl2,↑Tbx2,↑Prrx1,↑Tcf21,↑ Regulation Of Twist2,↑Meis2,↑Sall1,↑Hoxd8,↑Gli2 Transcription From RNA Polymerase II Promoter 28 Somatic Stem Cell 4 0.0001395 ↑Pou5f1,↑Tfap2c,↑Ascl2,↑Sfrp1 Maintenance 29 Positive 14 0.0001396 ↑Igf2,↑Bmp7,↑Tfap2a,↑Pou5f1,↑Foxc2,↓Jup,↑Hif3a,↑Tfap2c,↑ Regulation Of Hoxd10,↓Nfia,↑Meis2,↑Sall1,↑Hoxd9,↑Gli2 Transcription From RNA Polymerase II Promoter 30 Endothelial Cell- 2 0.0001519 ↑Cyp1b1,↓Jup Cell Adhesion 31 Peripheral Nervous 2 0.0001519 ↑Hoxd10,↑Hoxd9 System Neuron Development 32 Retina Vasculature 2 0.0001519 ↑Cyp1b1,↑Pdgfra Development In Camera-Type Eye 33 Leydig Cell 3 0.0001539 ↑Cyp11a1,↑Cyp17a1,↑Pdgfra Differentiation 34 Negative 11 0.0001541 ↑H19,↑Meg3,↑Cav1,↑Bmp7,↑Igfbp6,↑Igfbp7,↑Col4a3,↓Frzb,↑ Regulation Of Cell Sfrp1,↑Ifitm3,↑Gml Proliferation 35 Kidney 6 0.0001588 ↑Bmp7,↑Foxc2,↑Tcf21,↑Lhx1,↑Sall1,↑Gli2 Development 36 Nervous System 12 0.0001718 ↑Ednrb,↓Jup,↑Ascl2,↑Gfra1,↓Smarce1,↓Ndel1,↑Lhx1,↑Itga8,↑ Development Arx,↓Plxna4,↓Cbln1,↓Cyb5d2 37 Blood Coagulation 12 0.0001821 ↑Mrvi1,↑Cav1,↑Fn1,↑Rapgef3,↑Prkg1,↑Serping1,↑Hbb-b1/Hbb -b2/LOC100503605,↑Thbd,↑Plcg2,↑Col1a2,↑Rasgrp2,↑Pik3r6 38 Trophectodermal 3 0.0001885 ↑Cdh1,↑Pou5f1,↑Tfap2c Cell Differentiation 39 Forelimb 3 0.0001885 ↑Hoxd10,↑Sall1,↑Hoxd9 Morphogenesis 40 Regulation Of 5 0.0002051 ↑Myh6,↑Ednrb,↑Actg2,↑Hbb-b1/Hbb-b2/LOC100503605,↑Col Blood Pressure 1a2 41 Muscle Filament 4 0.0002092 ↑Myh6,↑Tnnt2,↑Actn3,↑Tpm2 Sliding 42 Hindlimb 3 0.0002276 ↑Hoxd10,↑Sall1,↑Hoxd9 Morphogenesis 43 Positive 3 0.0002717 ↑Cd24a,↑Il18,↑Icosl Regulation Of Activated T Cell Proliferation 44 Keratinocyte 2 0.0003024 ↑Tfap2a,↑Tfap2c Development 45 Embryonic 3 0.000321 ↑Sall1,↑Pkdcc,↑Gli2 Digestive Tract Development 46 Anterior-Posterior 6 0.000356 ↑Ifitm1,↑Hoxd10,↑Sfrp1,↑Lhx1,↑Hoxd8,↑Hoxd9 Pattern Formation 47 Negative 11 0.0003762 ↑Bmp7,↑Fhl2,↑Pou5f1,↑Sfrp1,↑Tbx2,↑Tcf21,↑Twist2,↓Smarce Regulation Of 1,↑Tnp1,↑Tshz3,↑Elk3 Transcription, DNA-Dependent 48 Transcription, 31 0.0003779 ↓Zfp39,↑Fhl2,↑Tfap2a,↑Pou5f1,↑Foxc2,↑Hif3a,↑Etv4,↑Tfap2c, DNA-Dependent ↑Ascl2,↑Figla,↑Hoxd10,↑Parp14,↓Nfia,↑Aff3,↑Tbx2,↑Tcf21,↑ Twist2,↑Meis2,↑Lhx1,↓Trim25,↑Arx,↑Sall1,↑Tshz3,↑Hoxd8,↑ Elk3,↑Bnc2,↑Hoxd9,↑Vgll3,↓Crtc3,↑Gli2,↓Zfhx4 49 Male Gonad 5 0.0004319 ↑Cyp11a1,↑Cyp17a1,↑Tfap2c,↑Sfrp1,↑Lhx9 Development 50 Vasoconstriction 3 0.0004359 ↑Cav1,↑Ednrb,↑Slc6a4 51 Germ-Line Stem 2 0.0005017 ↑Pou5f1,↑Tfap2c Cell Maintenance

222 52 Neural Crest Cell 2 0.0005017 ↑Foxc2,↑Sfrp1 Fate Commitment 53 Cellular Response 2 0.0005017 ↑Cdh1,↓Jup To Indole-3- Methanol 54 Lipid Digestion 2 0.0005017 ↑Pnlip,↑Pnliprp2 55 Positive 2 0.0005017 ↑Cd24a,↑Foxc2 Regulation Of Integrin Activation 56 Regulation Of Cell 5 0.0005298 ↑Igfbp6,↑Igfbp2,↑Igfbp7,↑Rasgrp2,↑Htra3 Growth 57 Response To 4 0.0005657 ↓Spp1,↑Cyp11a1,↑Igfbp2,↑Cyp17a1 Steroid Hormone Stimulus 58 Embryonic 3 0.0005744 ↓Rdh10,↑Hoxd9,↑Osr2 Forelimb Morphogenesis 59 Skin Development 4 0.0006076 ↑Tfap2a,↓Jup,↑Tfap2c,↑Col5a2 60 Heart 7 0.000656 ↑Pdlim3,↑Tnnt2,↑Foxc2,↑Gjc1,↑Casq2,↑Sall1,↑Gli2 Development 61 Cellular Response 3 0.0007381 ↑Il18,↑Cyp11a1,↑Sfrp1 To Tumor Necrosis Factor 62 Regulation Of 3 0.0007381 ↑Foxc2,↑Hbb-b1/Hbb-b2/LOC100503605,↑Tgfbr3 Blood Vessel Size 63 Regulation Of 2 0.0007489 ↑Myh6,↑Tpm2 Atpase Activity 64 Atrial Cardiac 2 0.0007489 ↑Myh6,↑Tnnt2 Muscle Tissue Morphogenesis 65 Positive 3 0.00083 ↑Bmp7,↑Tfap2a,↑Pkdcc Regulation Of Bone Mineralization 66 Female Gonad 3 0.00083 ↑Pdgfra,↑Sfrp1,↑Lhx9 Development 67 Positive 4 0.0009061 ↑Bmp7,↑Lin28a,↑Sall1,↑Gli2 Regulation Of Neuron Differentiation 68 Positive 3 0.0009289 ↑Bmp7,↑Hbb-b1/Hbb-b2/LOC100503605,↑Hba-a1/Hba-a2 Regulation Of Cell Death 69 Proximal-Distal 3 0.0009289 ↑Hoxd10,↑Hoxd9,↑Gli2 Pattern Formation 70 Adrenal Gland 3 0.0010349 ↑Cyp17a1,↑Pdgfra,↑Sall1 Development 71 Cardiac Muscle 2 0.0010436 ↑Myh6,↑Myh11 Fiber Development 72 Phthalate 2 0.0010436 ↑Cyp11a1,↑Cyp17a1 Metabolic Process 73 Cellular Response 2 0.0010436 ↑Cyp11a1,↑Sfrp1 To Fibroblast Growth Factor Stimulus 74 Glomerular 2 0.0010436 ↑Col4a3,↑Col4a4 Basement Membrane Development 75 Regulation Of 2 0.0010436 ↑Bmp7,↑Sfrp1 Branching Involved In Prostate Gland Morphogenesis 76 Cellular Response 4 0.0011546 ↑Ednrb,↑Il18,↑Cyp11a1,↑Cyp17a1 To Lipopolysaccharid e 77 Regulation Of 35 0.0012673 ↓Zfp39,↑Igf2,↑Fhl2,↑Tfap2a,↑Pou5f1,↑Foxc2,↑Tcf23,↑Hif3a,↑ Transcription, Etv4,↑Tfap2c,↑Ascl2,↑Figla,↑Hoxd10,↑Parp14,↓Nfia,↑Crxos1,

223 DNA-Dependent ↑Aff3,↑Tbx2,↑Prrx1,↑Tcf21,↑Twist2,↑Meis2,↑Lhx1,↑Lin28a,↑ Arx,↑Sall1,↑Tshz3,↑Hoxd8,↑Elk3,↑Lhx9,↑Bnc2,↑Hoxd9,↑Vgll 3,↓Crtc3,↓Zfhx4 78 Gonad 3 0.0012691 ↓Frzb,↓Rdh10,↑Sall1 Development 79 Positive 2 0.0013849 ↑Il2rg,↑Gli2 Regulation Of T Cell Differentiation In Thymus 80 Astrocyte 2 0.0013849 ↑Gfap,↑Lamc3 Development 81 Peptide Cross- 2 0.0013849 ↑Bgn,↑Egflam Linking Via Chondroitin 4- Sulfate Glycosaminoglyca n 82 Embryonic 2 0.0013849 ↑Foxc2,↓Rdh10 Viscerocranium Morphogenesis 83 Visual Perception 7 0.0013947 ↑Cyp1b1,↑Gjc1,↓Rdh10,↑Gnat2,↑Krt12,↑Pitpnm3,↑Lamc3 84 Cellular Response 3 0.0013977 ↑Cav1,↑Cartpt,↑Sfrp1 To Starvation 85 Pituitary Gland 3 0.0013977 ↑Cdh1,↑Sall1,↑Gli2 Development 86 Skeletal Muscle 4 0.0014474 ↑Pdlim3,↑Cav1,↑Hoxd10,↑Hoxd9 Tissue Development 87 Response To 5 0.001505 ↑Pou5f1,↑Cyp17a1,↑Cxcl16,↑Pdgfra,↑Coro1a Cytokine Stimulus 88 Lipid Catabolic 5 0.001505 ↑Pnlip,↑Plcg2,↑Apoc2,↑Pnliprp2,↑Plb1 Process 89 Regulation Of 3 0.0015343 ↑Ryr1,↑Tnnt2,↑Casq2 Muscle Contraction 90 Collagen Fibril 3 0.0016788 ↑Foxc2,↑Col1a2,↑Col5a2 Organization 91 Vasculature 3 0.0016788 ↓Frzb,↑Sfrp1,↑Tcf21 Development 92 Steroid Metabolic 5 0.0017476 ↑Pnlip,↑Cyp11a1,↑Cyp17a1,↑Sorl1,↑Plb1 Process 93 Negative 2 0.0017722 ↑Sfrp1,↑Tcf21 Regulation Of Androgen Receptor Signaling Pathway 94 Negative 2 0.0017722 ↑Tgfbr3,↑Sfrp1 Regulation Of Epithelial To Mesenchymal Transition 95 Hormone 4 0.0017883 ↑Cyp11a1,↑Ptgis,↑Cyp17a1,↑Pnmt Biosynthetic Process 96 Organ 6 0.0019318 ↑Igf2,↑Bmp7,↑Pdgfra,↑Tcf21,↑Lhx1,↓Rdh10 Morphogenesis 97 Response To 4 0.0019781 ↑Cyp11a1,↑Cyp17a1,↑Thbd,↓Ccnd2 Camp 98 Neural Tube 3 0.0019929 ↑Sfrp1,↑Sall1,↑Gli2 Development 99 Synaptic 9 0.002126 ↑Tacr3,↑Cartpt,↑Gjc1,↑Adcy5,↓Kcnj3,↑Kcnc4,↑Syn2,↓Cbln1,↑ Transmission Sv2c 100 Lung Alveolus 3 0.0021627 ↑Meg3,↑Tcf21,↑Pkdcc Development 101 Regulation Of Cell 4 0.0021813 ↑Fn1,↓Spna1,↑Coro1a,↑Fblim1 Shape 102 Biphenyl 2 0.0022048 ↑Cyp11a1,↑Cyp17a1 Metabolic Process 103 Nucleosome 2 0.0022048 ↓Smarce1,↑Tnp1

224 Disassembly 104 Mammary Gland 2 0.0022048 ↑Cav1,↓Frzb Involution 105 Metabolic Process 30 0.0022549 ↓Arsk,↓Abhd3,↑Cox8c,↑Entpd2,↑Myl9,↑Myh6,↑Myh11,↑Prkg 1,↑Ptgis,↑Cyp17a1,↑Abcc9,↑Plcg2,↑Fap,↓Ndel1,↓Trim25,↓Rdh 10,↑Bcat1,↑Adamts20,↑Syn2,↑Pdzrn3,↑Pi4k2b,↑Aldoc,↓Abcc1 0,↓Pdha2,↑Ugt1a1/Ugt1a10/Ugt1a2/Ugt1a5/Ugt1a6a/Ugt1a6b/ Ugt1a7c/Ugt1a9,↑Gfpt2,↑Oasl2,↑Dnahc8,↑Kif18b,↑Lyz1 106 Eye Development 3 0.0023412 ↑Bmp7,↑Sfrp1,↑Meis2 107 Positive 3 0.0025286 ↑Cav1,↓Jup,↑Sfrp1 Regulation Of Canonical Wnt Receptor Signaling Pathway 108 Cellular Response 3 0.0025286 ↑Bmp7,↑Stc2,↑Sfrp1 To Hypoxia 109 Mammary Gland 3 0.0025286 ↑Cav1,↑Hoxd9,↑Gli2 Development 110 Positive 2 0.0026821 ↑Cd24a,↑Foxc2 Regulation Of Cell Adhesion Mediated By Integrin 111 Phenol-Containing 2 0.0026821 ↑Cyp11a1,↑Cyp17a1 Compound Metabolic Process 112 Cellular Response 2 0.0026821 ↑Cyp11a1,↑Cyp17a1 To Gonadotropin Stimulus 113 Heart Trabecula 2 0.0026821 ↑Fhl2,↑Tgfbr3 Formation 114 Lymph Vessel 2 0.0026821 ↑Foxc2,↑Tmem204 Development 115 Response To Drug 10 0.0026873 ↑Igf2,↑Cav1,↑Cdh1,↑Cyp11a1,↑Slc6a4,↑Igfbp2,↑Cyp17a1,↑Ab cc9,↑Apoc2,↑Sfrp1 116 Transport 24 0.002737 ↑Kdelr3,↑Arf2,↑Dbil5,↑Ryr1,↑Cadps,↑Slc6a4,↑Hbb-b1/Hbb-b 2/LOC100503605,↑Abcc9,↑Gjc1,↓Slc15a2,↑Sorl1,↑Apoc2,↓Kc nj3,↑Hba-a1/Hba-a2,↑Kcnc4,↓Ndel1,↑Slc38a4,↓Exoc6,↑Kcne4 ,↓Slc25a17,↑Sv2c,↓Abcc10,↑Fabp9,↑Pkdcc 117 Response To 5 0.0029174 ↑Cav1,↑Stc2,↑Cyp11a1,↑Slc6a4,↑Igfbp2 Nutrient 118 Response To 4 0.0031383 ↑Pou5f1,↑Igfbp2,↑Cyp17a1,↑Igfbp7 Retinoic Acid 119 Response To 4 0.0031383 ↑Cav1,↑Igfbp2,↑Meis2,↑Gli2 Mechanical Stimulus 120 Positive 2 0.0032034 ↑Ccnd3,↓Ccnd2 Regulation Of Cyclin-Dependent Protein Kinase Activity 121 Cellular Response 2 0.0032034 ↑Cyp11a1,↑Cyp17a1 To Antibiotic 122 Mesonephros 2 0.0032034 ↑Bmp7,↑Osr2 Development 123 Angiogenesis 6 0.0032608 ↑Fn1,↓Lepr,↑Cyp1b1,↑Il18,↑Ecscr,↑Elk3 124 Neuron Projection 3 0.0033697 ↑Bmp7,↑Itga1,↓Plxna4 Morphogenesis 125 Embryo 3 0.0033697 ↓Spp1,↑Stc2,↑Igfbp7 Implantation 126 Neural Crest Cell 2 0.003768 ↑Foxc2,↓Rdh10 Development 127 Regulation Of 8 0.0039319 ↑Fhl2,↑Tfap2a,↑Tfap2c,↑Ascl2,↓Frzb,↑Sfrp1,↑Twist2,↓Smarce Transcription 1 From RNA Polymerase II Promoter 128 Cell-Cell Signaling 7 0.0042546 ↑Cd24a,↑Cartpt,↑Il18,↑Stc2,↑Gjc1,↑Tfap2c,↑Lhx1 129 Hemopoiesis 4 0.004348 ↑Hbb-b1/Hbb-b2/LOC100503605,↑Sfrp1,↓Spna1,↑Cnn2 130 Skeletal System 3 0.0043634 ↑Tfap2a,↑Pdgfra,↑Hoxd8

225 Morphogenesis 131 Protein 3 0.0043634 ↑Cav1,↑Slc6a4,↑Mbl2 Oligomerization 132 Organic Acid 2 0.0043754 ↑Cyp11a1,↑Cyp17a1 Metabolic Process 133 Cardiac Muscle 2 0.0043754 ↑Foxc2,↑Tgfbr3 Cell Proliferation 134 Response To 2 0.0043754 ↑Cyp11a1,↑Cyp17a1 Fungicide 135 Positive 2 0.0043754 ↑Cav1,↑Fn1 Regulation Of Peptidase Activity 136 Oxygen Transport 2 0.0043754 ↑Hbb-b1/Hbb-b2/LOC100503605,↑Hba-a1/Hba-a2 137 Positive 9 0.0044844 ↑Igf2,↑Ednrb,↑Pdgfra,↑Ccnd3,↓Ccnd2,↑Sfrp1,↑Tbx2,↑Osr2,↑G Regulation Of Cell li2 Proliferation 138 Innate Immune 7 0.0045918 ↑Nlrc5,↑Serping1,↑Mbl2,↑Coro1a,↑Ifitm3,↓Trim25,↑C1qb Response 139 Immune Response 10 0.0046288 ↑Cd24a,↑Il18,↑Ptger4,↑Tgfbr3,↑H2-Q7,↑Il2rg,↑Ifitm3,↑Oasl2, ↑Gpsm2,↑C1qb 140 Post-Embryonic 4 0.0046933 ↑Meg3,↑Pnlip,↑Lhx1,↑Pnliprp2 Development 141 Protein 4 0.0046933 ↑Tnnt2,↑Hbb-b1/Hbb-b2/LOC100503605,↓Jup,↑Hba-a1/Hba- Heterooligomeriza a2 tion 142 Xenobiotic 5 0.0048309 ↑Cyp1b1,↑Cyp11a1,↑Ptgis,↑Cyp17a1,↑Ugt1a1/Ugt1a10/Ugt1a Metabolic Process 2/Ugt1a5/Ugt1a6a/Ugt1a6b/Ugt1a7c/Ugt1a9 143 Heart 3 0.00492 ↑Foxc2,↑Tgfbr3,↑Tbx2 Morphogenesis 144 Regulation Of 8 0.0049582 ↑Efcab10,↑Rapgef3,↑Slc6a4,↑Apoc2,↑Sytl2,↑Rasgrp2,↑Mcf2l, Catalytic Activity ↑Exph5 145 Oocyte 2 0.0050248 ↓Jup,↑Figla Development 146 Pathway- 2 0.0050248 ↑Bmp7,↑Tgfbr3 Restricted SMAD Protein Phosphorylation 147 Vesicle 2 0.0050248 ↑Cadps,↑Cav1 Organization 148 Response To 2 0.0050248 ↑Cyp11a1,↑Cyp17a1 Insecticide 149 Cochlea 2 0.0050248 ↓Frzb,↑Gli2 Morphogenesis 150 Negative 3 0.0052136 ↓Frzb,↑Sfrp1,↑Apcdd1 Regulation Of Wnt Receptor Signaling Pathway 151 Lipid Metabolic 8 0.0055428 ↑Pnlip,↑Cyp11a1,↑Ptgis,↑Sorl1,↑Plcg2,↑Apoc2,↑Pnliprp2,↑Plb Process 1 152 Regulation Of 4 0.0056359 ↑Pou5f1,↑Plcg2,↑Il2rg,↑Lhx1 Gene Expression 153 Positive 2 0.0057157 ↑Sfrp1,↑Prrx1 Regulation Of Smoothened Signaling Pathway 154 Estrogen 2 0.0057157 ↑Cyp1b1,↑Pdgfra Metabolic Process 155 Defense Response 3 0.0061566 ↑Mbl2,↑Gbp3,↑Lyz1 To Gram-Positive Bacterium 156 Somitogenesis 3 0.0061566 ↑Ifitm1,↑Foxc2,↑Sfrp1 157 Wound Healing 4 0.006258 ↑Fn1,↑Pdgfra,↑Cnn2,↑Elk3 158 Stem Cell 2 0.0064475 ↑Etv4,↑Lin28a Differentiation 159 Cellular Response 2 0.0064475 ↑Il18,↑Cyp11a1 To Camp 160 Olfactory Bulb 2 0.0064475 ↑Arx,↑Sall1 Development 161 Cardiac Muscle 2 0.0064475 ↑Gjc1,↑Tbx2 Tissue

226 Development 162 Digestive Tract 2 0.0064475 ↑Il18,↑Sfrp1 Morphogenesis 163 Integrin-Mediated 4 0.0064751 ↑Itga1,↑Itgae,↑Itga8,↑Adamts20 Signaling Pathway 164 Positive 1 0.0071494 ↑Il18 Regulation Of Male Germ Cell Proliferation 165 Ectodermal Cell 1 0.0071494 ↑Pou5f1 Fate Commitment 166 Substrate- 1 0.0071494 ↑Abcc9 Dependent Cell Migration, Cell Contraction 167 Gonad 1 0.0071494 ↑Lhx9 Morphogenesis 168 Fertilization, 1 0.0071494 ↑Tnp1 Exchange Of Chromosomal Proteins 169 Stromal-Epithelial 1 0.0071494 ↑Sfrp1 Cell Signaling Involved In Prostate Gland Development 170 Inductive Cell-Cell 1 0.0071494 ↑Sall1 Signaling 171 Negative 1 0.0071494 ↑Sfrp1 Regulation Of Canonical Wnt Receptor Signaling Pathway Involved In Controlling Type B Pancreatic Cell Proliferation 172 Interleukin-4- 1 0.0071494 ↑Il2rg Mediated Signaling Pathway 173 Negative 1 0.0071494 ↓Spp1 Regulation Of Collateral Sprouting Of Intact Axon In Response To Injury 174 Positive 1 0.0071494 ↑Apoc2 Regulation Of Phospholipid Catabolic Process 175 Regulation Of 1 0.0071494 ↑Pou5f1 Methylation- Dependent Chromatin Silencing 176 Negative 1 0.0071494 ↓Frzb Regulation Of Hepatocyte Differentiation 177 Negative 1 0.0071494 ↑Cd24a Regulation Of T Cell Homeostatic Proliferation 178 Negative 1 0.0071494 ↑Cd24a Regulation Of Erythrocyte Aggregation 179 Negative 1 0.0071494 ↑Slc6a4 Regulation Of Cerebellar Granule

227 Cell Precursor Proliferation 180 Negative 1 0.0071494 ↑Coro1a Regulation Of Actin Nucleation 181 Negative 1 0.0071494 ↑Sfrp1 Regulation Of Fibroblast Apoptosis 182 Positive 1 0.0071494 ↑Gfap Regulation Of Schwann Cell Proliferation 183 Positive 1 0.0071494 ↑Cd24a Regulation Of T Cell Homeostatic Proliferation 184 Positive 1 0.0071494 ↑Cartpt Regulation Of Epinephrine Secretion 185 Positive 1 0.0071494 ↑Sfrp1 Regulation Of Fibroblast Apoptosis 186 Follicular B Cell 1 0.0071494 ↑Plcg2 Differentiation 187 RNA Folding 1 0.0071494 ↑Meg3 188 Chemorepulsion 1 0.0071494 ↓Plxna4 Of Branchiomotor Axon 189 Cellular Response 1 0.0071494 ↑Slc6a4 To Cgmp 190 Cellular Response 1 0.0071494 ↑Sfrp1 To BMP Stimulus 191 Desmosome 1 0.0071494 ↓Jup Assembly 192 Negative 1 0.0071494 ↑Cd24a Regulation Of Erythrocyte Clearance 193 Ureteric Peristalsis 1 0.0071494 ↑Tshz3 194 Regulation Of 1 0.0071494 ↑Cdh1 Water Loss Via Skin 195 Somite 1 0.0071494 ↓Frzb Development 196 Ventral Spinal 1 0.0071494 ↑Gli2 Cord Development 197 Visceral Muscle 1 0.0071494 ↑Myh6 Development 198 Convergent 1 0.0071494 ↑Sfrp1 Extension Involved In Somitogenesis 199 Vagus Nerve 1 0.0071494 ↓Plxna4 Morphogenesis 200 Facial Nerve 1 0.0071494 ↓Plxna4 Morphogenesis 201 Dipeptide 1 0.0071494 ↓Slc15a2 Transport 202 Negative 3 0.0071952 ↑Cd24a,↑Bmp7,↑Slc6a4 Regulation Of Neuron Differentiation 203 Response To 2 0.0072195 ↑Cyp11a1,↑Cyp17a1 Gonadotropin Stimulus 204 Liver 4 0.0078829 ↑Meg3,↑Tgfbr3,↓Ccnd2,↑Ugt1a1/Ugt1a10/Ugt1a2/Ugt1a5/Ugt Development 1a6a/Ugt1a6b/Ugt1a7c/Ugt1a9

228 205 Regulation Of 3 0.0079416 ↑Cd24a,↑Efcab10,↑Bmp7 Phosphorylation 206 Negative 2 0.008031 ↑Cd24a,↑Bmp7 Regulation Of Neurogenesis 207 Retinoid Metabolic 2 0.008031 ↑Pnlip,↑Plb1 Process 208 Regulation Of 2 0.008031 ↑Ptger4,↑Sfrp1 Ossification 209 Decidualization 2 0.008031 ↓Spp1,↑Stc2 210 Artery 2 0.008031 ↑Foxc2,↑Prrx1 Morphogenesis 211 Positive 4 0.0083933 ↑Cd24a,↑Ednrb,↑Ccnd3,↓Ccnd2 Regulation Of Protein Phosphorylation 212 Response To 4 0.0083933 ↑Cdh1,↑Cyp1b1,↑Slc6a4,↑Cyp17a1 Toxin 213 Signal 38 0.0085031 ↑Kdelr3,↑Ms4a4d,↑Efcab10,↑Ryr1,↑Tacr3,↑Ednrb,↑Rapgef3,↓ Transduction Lepr,↑Prkg1,↑Cartpt,↑Il18,↑Igfbp6,↑Igfbp2,↑Ptger4,↑Abcc9,↑C xcl16,↑Ccnd3,↑Tgfbr3,↑Thbd,↑Sorl1,↑Hif3a,↑Plcg2,↑Icosl,↑Il2 rg,↑Gfra1,↓Frzb,↑Lsp1,↑Sfrp1,↑S100a6,↑Mfap4,↓Plekhb1,↑Ras grp2,↓Plxna4,↑Elk3,↑Sv2c,↑Gnat2,↑Arhgap9,↑Gpsm2 214 Energy Reserve 4 0.0086565 ↑Rapgef3,↓Lepr,↑Adcy5,↑Gfpt2 Metabolic Process 215 Positive 4 0.0086565 ↑Slc6a4,↑Il2rg,↑Cnn2,↑Osr2 Regulation Of Gene Expression 216 Actin Filament 3 0.0087321 ↑Pdlim3,↓Spna1,↑Coro1a Organization 217 Complement 3 0.0087321 ↑Serping1,↑Mbl2,↑C1qb Activation, Classical Pathway 218 Odontogenesis Of 3 0.0087321 ↑Bmp7,↑Pdgfra,↑Gli2 Dentine- Containing Tooth 219 Positive 2 0.0088816 ↓Frzb,↑Sfrp1 Regulation Of Fat Cell Differentiation 220 Cytosolic Calcium 2 0.0088816 ↑Ryr1,↑Cav1 Ion Homeostasis 221 Cellular Response 2 0.0088816 ↑Cyp11a1,↑Sfrp1 To Interleukin-1 222 Ectoderm 2 0.0088816 ↑Tfap2a,↓Jup Development 223 Middle Ear 2 0.0088816 ↑Prrx1,↑Osr2 Morphogenesis 224 Aging 5 0.0092658 ↑Tacr3,↑Ednrb,↑Igfbp2,↑Aldoc,↑C1qb 225 Muscle Organ 4 0.0094782 ↑Tcf23,↑Tagln,↑Lama2,↑Unc45b Development 226 Cholesterol 3 0.0095671 ↑Cd24a,↑Cav1,↑Apoc2 Homeostasis 227 Protein 4 0.0097629 ↑Cav1,↑Cdh1,↑Slc6a4,↑C1qtnf2 Homooligomerizat ion 228 Positive 2 0.0097707 ↑Sfrp1,↑Sall1 Regulation Of Wnt Receptor Signaling Pathway 229 Positive 2 0.0097707 ↑Tgfbr3,↑Itga8 Regulation Of Transforming Growth Factor Beta Receptor Signaling Pathway 230 Cellular Response 2 0.0097707 ↑Il18,↑Cyp11a1 To Peptide Hormone Stimulus 231 Cellular Response 2 0.0097707 ↑Cyp11a1,↑Sfrp1

229 To Transforming Growth Factor Beta Stimulus 232 Anatomical 3 0.0100014 ↑Bmp7,↑Lhx1,↑Gli2 Structure Formation Involved In Morphogenesis 233 Positive 4 0.0100532 ↑Cxcl16,↑Pdgfra,↑Coro1a,↑Prr5 Regulation Of Cell Migration 234 Negative 3 0.010447 ↑Cav1,↓Frzb,↑Sfrp1 Regulation Of Canonical Wnt Receptor Signaling Pathway 235 Cell Activation 2 0.0106975 ↑Cd24a,↑Pdgfra 236 Negative 2 0.0106975 ↑Cartpt,↑Sfrp1 Regulation Of Osteoclast Differentiation 237 2 0.0106975 ↑Myh6,↑Tnnt2 Organization 238 Positive 3 0.010904 ↓Ccnd2,↑Sfrp1,↑Osr2 Regulation Of Epithelial Cell Proliferation 239 Cellular Response 3 0.010904 ↑Cyp11a1,↑Cyp17a1,↓Plxna4 To Protein Stimulus 240 Blood Vessel 3 0.010904 ↑Foxc2,↑Tgfbr3,↑Col1a2 Development 241 Cell Surface 6 0.0110974 ↑Cd24a,↑Ednrb,↓Lepr,↑Stc2,↑Col4a3,↑Gfra1 Receptor Linked Signaling Pathway 242 Leukocyte 4 0.01127 ↑Cav1,↑Fn1,↑Thbd,↑Col1a2 Migration 243 Lung Development 4 0.01127 ↑Il18,↑Pdgfra,↑Tshz3,↑Gli2 244 Vasculogenesis 3 0.0118523 ↑Cav1,↑Foxc2,↑Gjc1 245 Cell Proliferation 8 0.0124562 ↑Igf2,↑Rapgef3,↑Foxc2,↑Sorl1,↑Col4a3,↑Bcat1,↑Lhx9,↑Gli2 246 Substrate 2 0.0126624 ↑Fn1,↑Sfrp1 Adhesion- Dependent Cell Spreading 247 Endothelial Cell 2 0.0126624 ↑Cyp1b1,↑Fap Migration 248 Vesicle Docking 2 0.0126624 ↑Sytl2,↓Exoc6 Involved In Exocytosis 249 Striated Muscle 2 0.0126624 ↑Myh6,↑Casq2 Contraction 250 Glycolysis 3 0.0128466 ↑Ldhc,↑Aldoc,↓Pdha2 251 Response To 3 0.0128466 ↑Cyp11a1,↑Hbb-b1/Hbb-b2/LOC100503605,↑Hba-a1/Hba-a2 Hydrogen Peroxide 252 Inner Ear 3 0.0128466 ↓Frzb,↑Prrx1,↑Itga8 Morphogenesis 253 Embryonic Limb 3 0.0133611 ↑Bmp7,↑Hoxd10,↑Prrx1 Morphogenesis 254 Vascular 2 0.0136993 ↑Foxc2,↑Pdgfra Endothelial Growth Factor Receptor Signaling Pathway 255 Phospholipid 2 0.0136993 ↑Plcg2,↑Pnliprp2 Catabolic Process 256 Hydrogen 2 0.0136993 ↑Hbb-b1/Hbb-b2/LOC100503605,↑Hba-a1/Hba-a2 Peroxide Catabolic Process 257 Cellular Response 2 0.0136993 ↑Il18,↑Gbp3

230 To Interferon- Gamma 258 Negative 1 0.0142478 ↑Bmp7 Regulation Of Mesenchymal Stem Cell Apoptosis Involved In Nephron Morphogenesis 259 Muscle Cell Fate 1 0.0142478 ↑Tbx2 Determination 260 Endodermal Cell 1 0.0142478 ↑Pou5f1 Fate Commitment 261 Nuclear Envelope 1 0.0142478 ↓Ndel1 Disassembly 262 Spermatid Nucleus 1 0.0142478 ↑Tnp1 Elongation 263 Cerebellar 1 0.0142478 ↑Lhx1 Purkinje Cell- Granule Cell Precursor Cell Signaling Involved In Regulation Of Granule Cell Precursor Cell Proliferation 264 Regulation Of 1 0.0142478 ↑Pou5f1 Heart Induction By Regulation Of Canonical Wnt Receptor Signaling Pathway 265 Negative 1 0.0142478 ↑Nlrc5 Regulation Of Type I Interferon- Mediated Signaling Pathway 266 Negative 1 0.0142478 ↑Cartpt Regulation Of Glucagon Secretion 267 Positive 1 0.0142478 ↑Sfrp1 Regulation Of Non-Canonical Wnt Receptor Signaling Pathway 268 Semaphorin-Plexin 1 0.0142478 ↓Plxna4 Signaling Pathway 269 BMP Signaling 1 0.0142478 ↑Pou5f1 Pathway Involved In Heart Induction 270 Negative 1 0.0142478 ↑Meg3 Regulation Of DNA Biosynthetic Process 271 Negative 1 0.0142478 ↑Serping1 Regulation Of Complement Activation, Lectin Pathway 272 Negative 1 0.0142478 ↑Pkdcc Regulation Of Golgi To Plasma Membrane Protein Transport 273 Negative 1 0.0142478 ↑Ednrb Regulation Of Neuron Maturation 274 Negative 1 0.0142478 ↑Pou5f1

231 Regulation Of Gene Silencing By Mirna 275 Negative 1 0.0142478 ↑Bmp7 Regulation Of Striated Muscle Cell Apoptosis 276 Regulation Of 1 0.0142478 ↑Tnnt2 Muscle Filament Sliding Speed 277 Anterior 1 0.0142478 ↓Plxna4 Commissure Morphogenesis 278 Spinal Cord 1 0.0142478 ↑Gli2 Ventral Commissure Morphogenesis 279 Glomerular 1 0.0142478 ↑Foxc2 Mesangial Cell Development 280 Olfactory Bulb 1 0.0142478 ↑Sall1 Interneuron Differentiation 281 5-Phosphoribose 1 0.0142478 ↑Pygl 1-Diphosphate Biosynthetic Process 282 Retinoic Acid 1 0.0142478 ↓Rdh10 Biosynthetic Process 283 Glycine 1 0.0142478 ↓Shmt1 Biosynthetic Process From Serine 284 Galactolipid 1 0.0142478 ↑Pnliprp2 Catabolic Process 285 B Cell Receptor 1 0.0142478 ↑Cd24a Transport Into Membrane Raft 286 Chemokine 1 0.0142478 ↑Cd24a Receptor Transport Out Of Membrane Raft 287 Embryonic 1 0.0142478 ↑Arx Olfactory Bulb Interneuron Precursor Migration 288 Cellular Response 1 0.0142478 ↑Sfrp1 To Prostaglandin E Stimulus 289 Actin Crosslink 1 0.0142478 ↑Tnnt2 Formation 290 Uropod 1 0.0142478 ↑Coro1a Organization 291 Primary Lung Bud 1 0.0142478 ↓Rdh10 Formation 292 Negative 1 0.0142478 ↑Cd24a Regulation Of Transforming Growth Factor- Beta3 Production 293 Positive 1 0.0142478 ↑Apoc2 Regulation Of Very-Low-Density Lipoprotein Particle Remodeling 294 Negative 1 0.0142478 ↑Sfrp1 Regulation Of

232 Bone Remodeling 295 Positive 1 0.0142478 ↑Cd24a Regulation Of T Cell Mediated Immune Response To Tumor Cell 296 Metanephric 1 0.0142478 ↑Bmp7 Mesenchymal Cell Proliferation Involved In Metanephros Development 297 Response To 1 0.0142478 ↑Cyp17a1 Acetate 298 Response To 1 0.0142478 ↑Tgfbr3 Luteinizing Hormone Stimulus 299 Response To 1 0.0142478 ↑Cyp11a1 Genistein 300 Negative 1 0.0142478 ↑Sfrp1 Regulation Of Cysteine-Type Endopeptidase Activity 301 Ectoderm 1 0.0142478 ↑Lhx1 Formation 302 Metanephric 1 0.0142478 ↑Pdgfra Glomerular Capillary Formation 303 Ureteric Bud 1 0.0142478 ↑Sall1 Invasion 304 Globus Pallidus 1 0.0142478 ↑Arx Development 305 Posterior Midgut 1 0.0142478 ↑Ednrb Development 306 Olfactory Bulb 1 0.0142478 ↑Sall1 Mitral Cell Layer Development 307 Bronchiole 1 0.0142478 ↑Tcf21 Development 308 Postganglionic 1 0.0142478 ↓Plxna4 Parasympathetic Nervous System Development 309 Glomerular 1 0.0142478 ↑Foxc2 Endothelium Development 310 Olfactory Nerve 1 0.0142478 ↑Sall1 Development 311 Specification Of 1 0.0142478 ↑Gli2 Segmental Identity, Maxillary Segment 312 Anterior 1 0.0142478 ↑Tfap2a Neuropore Closure 313 Dichotomous 1 0.0142478 ↑Tfap2c Subdivision Of Terminal Units Involved In Mammary Gland Duct Morphogenesis 314 Nephrogenic 1 0.0142478 ↑Bmp7 Mesenchyme Morphogenesis 315 Trigeminal Nerve 1 0.0142478 ↓Plxna4 Morphogenesis 316 Notochord 1 0.0142478 ↑Gli2 Regression

233 317 Hyaluronan 1 0.0142478 ↑Ccnd3 Biosynthetic Process 318 Nitric Oxide 1 0.0142478 ↑Cav1 Homeostasis 319 Response To Lipid 2 0.0147718 ↑Pnlip,↑Pnliprp2 320 Blastocyst 2 0.0147718 ↑Pou5f1,↑Tgfbr3 Development 321 Elevation Of 4 0.0150922 ↑Cd24a,↑Ednrb,↑Plcg2,↑Gnat2 Cytosolic Calcium Ion Concentration 322 Response To 4 0.0158645 ↑Cav1,↑Igfbp2,↑Pnliprp2,↑C1qb Glucocorticoid Stimulus 323 Stem Cell 2 0.0158792 ↑Pou5f1,↑Lin28a Maintenance 324 Embryonic Pattern 2 0.0158792 ↑Bmp7,↑Lhx1 Specification 325 Cholesterol Efflux 2 0.0158792 ↑Cav1,↑Apoc2 326 Regulation Of 2 0.0170211 ↑Kcnc4,↑Syn2 Neurotransmitter Secretion 327 Negative 2 0.0181968 ↑Sfrp1,↑Twist2 Regulation Of Osteoblast Differentiation 328 Release Of 2 0.0181968 ↑Ryr1,↓Ibtk Sequestered Calcium Ion Into Cytosol 329 Cerebellum 2 0.0181968 ↑Cyp11a1,↑Lhx1 Development 330 Positive 2 0.0181968 ↑Tacr3,↑Cartpt Regulation Of Blood Pressure 331 Negative 2 0.0194059 ↑Cav1,↑Bmp7 Regulation Of MAP Kinase Activity 332 Cell-Cell Adhesion 4 0.0196455 ↑Cd24a,↑Cdh1,↓Jup,↑Itga8 333 Potassium Ion 4 0.0196455 ↑Abcc9,↓Kcnj3,↑Kcnc4,↑Kcne4 Transmembrane Transport 334 Cholesterol 3 0.0204482 ↓Lepr,↑Cyp11a1,↑Sorl1 Metabolic Process 335 Sex Differentiation 2 0.0206479 ↑Cyp17a1,↑Tcf21 336 Vasodilation 2 0.0206479 ↑Ednrb,↑Itga1 337 Response To 2 0.0206479 ↑Il18,↑Pdgfra Hyperoxia 338 Negative 1 0.0212958 ↑Sfrp1 Regulation Of Planar Cell Polarity Pathway Involved In Axis Elongation 339 Regulation Of 1 0.0212958 ↑Pou5f1 Asymmetric Cell Division 340 Spinal Cord Motor 1 0.0212958 ↑Hoxd10 Neuron Cell Fate Specification 341 Cardiac Cell Fate 1 0.0212958 ↑Pou5f1 Determination 342 Caveola Assembly 1 0.0212958 ↑Cav1 343 Development Of 1 0.0212958 ↑Sfrp1 Primary Male Sexual Characteristics 344 Negative 1 0.0212958 ↓Jup Regulation Of Wnt

234 Receptor Signaling Pathway Involved In Heart Development 345 Negative 1 0.0212958 ↑Sfrp1 Regulation Of Wnt Receptor Signaling Pathway Involved In Dorsal-Ventral Axis Specification 346 Negative 1 0.0212958 ↑Cav1 Regulation Of Tyrosine Phosphorylation Of Stat5 Protein 347 Positive 1 0.0212958 ↑Nlrc5 Regulation Of Interferon- Gamma-Mediated Signaling Pathway 348 Regulation Of 1 0.0212958 ↑Cnn2 Actin Filament- Based Process 349 Leptin-Mediated 1 0.0212958 ↓Lepr Signaling Pathway 350 Smoothened 1 0.0212958 ↑Gli2 Signaling Pathway Involved In Ventral Spinal Cord Interneuron Specification 351 Smoothened 1 0.0212958 ↑Gli2 Signaling Pathway Involved In Spinal Cord Motor Neuron Cell Fate Specification 352 Activation Of 1 0.0212958 ↓Ndel1 Cdc42 Gtpase Activity 353 Positive 1 0.0212958 ↑Mbl2 Regulation Of Complement Activation 354 Regulation Of Eif2 1 0.0212958 ↑Hbb-b1/Hbb-b2/LOC100503605 Alpha Phosphorylation By Heme 355 Regulation Of 1 0.0212958 ↑Tcf21 Histone Deacetylation 356 Negative 1 0.0212958 ↑Fap Regulation Of Extracellular Matrix Disassembly 357 Negative 1 0.0212958 ↑Sfrp1 Regulation Of Osteoblast Proliferation 358 Negative 1 0.0212958 ↑Bmp7 Regulation Of Glomerular Mesangial Cell Proliferation 359 Positive 1 0.0212958 ↑Il18 Regulation Of NK T Cell Proliferation 360 Positive 1 0.0212958 ↑Il2rg

235 Regulation Of CD4-Positive, CD25-Positive, Alpha-Beta Regulatory T Cell Differentiation 361 Epicardial Cell To 1 0.0212958 ↑Tgfbr3 Mesenchymal Cell Transition 362 Atrial Cardiac 1 0.0212958 ↑Fhl2 Muscle Cell Development 363 Granulosa Cell 1 0.0212958 ↑Cyp11a1 Differentiation 364 Glomerular 1 0.0212958 ↑Foxc2 Visceral Epithelial Cell Differentiation 365 Bergmann Glial 1 0.0212958 ↑Gfap Cell Differentiation 366 Branched Chain 1 0.0212958 ↑Bcat1 Family Amino Acid Biosynthetic Process 367 Epinephrine 1 0.0212958 ↑Pnmt Biosynthetic Process 368 L-Serine Catabolic 1 0.0212958 ↓Shmt1 Process 369 Mirna Catabolic 1 0.0212958 ↑Lin28a Process 370 Mirna Metabolic 1 0.0212958 ↑Lin28a Process 371 Phagolysosome 1 0.0212958 ↑Coro1a Assembly 372 Cerebral Cortex 1 0.0212958 ↑Arx Tangential Migration 373 Cellular Response 1 0.0212958 ↑Sfrp1 To Heparin 374 Cellular Response 1 0.0212958 ↑Il18 To Parathyroid Hormone Stimulus 375 Negative 1 0.0212958 ↑Bmp7 Regulation Of Prostatic Bud Formation 376 Positive 1 0.0212958 ↑Tfap2a Regulation Of Tooth Mineralization 377 Positive 1 0.0212958 ↑Osr2 Regulation Of Gastrulation 378 Positive 1 0.0212958 ↑Foxc2 Regulation Of Vascular Wound Healing 379 Regulation Of 1 0.0212958 ↑Il18 Blood Vessel Remodeling 380 Sensory Perception 1 0.0212958 ↑Tshz3 Of Touch 381 Regulation Of 1 0.0212958 ↓Plxna4 Negative Chemotaxis 382 Positive 1 0.0212958 ↑Cav1 Regulation Of Metalloenzyme

236 Activity 383 Floor Plate 1 0.0212958 ↑Gli2 Formation 384 Bud Elongation 1 0.0212958 ↓Rdh10 Involved In Lung Branching 385 Head Development 1 0.0212958 ↑Lhx1 386 Ventral Midline 1 0.0212958 ↑Gli2 Development 387 Metanephric 1 0.0212958 ↑Bmp7 Mesenchyme Morphogenesis 388 Atrioventricular 1 0.0212958 ↓Jup Valve Morphogenesis 389 Granulocyte 1 0.0212958 ↑Il18 Macrophage Colony- Stimulating Factor Biosynthetic Process 390 Interleukin-13 1 0.0212958 ↑Il18 Biosynthetic Process 391 Serotonin Uptake 1 0.0212958 ↑Slc6a4 392 Serotonin 1 0.0212958 ↑Slc6a4 Transport 393 Positive 8 0.0217745 ↑Fhl2,↑Pou5f1,↑Foxc2,↑Etv4,↑Sfrp1,↑Tcf21,↑Hoxd9,↑Gli2 Regulation Of Transcription, DNA-Dependent 394 Regulation Of 2 0.0219221 ↑Ednrb,↑Hba-a1/Hba-a2 Sensory Perception Of Pain 395 T Cell 3 0.0224856 ↑Cd24a,↑Cav1,↑Icosl Costimulation 396 Exocytosis 3 0.0231884 ↑Cadps,↑Sytl2,↓Exoc6 397 Response To 3 0.0239031 ↑Fn1,↑Pou5f1,↑Gfap Wounding 398 Negative 2 0.0245654 ↑Cav1,↑Sfrp1 Regulation Of BMP Signaling Pathway 399 Organ 3 0.0246296 ↑Tgfbr3,↑Aldoc,↑Ugt1a1/Ugt1a10/Ugt1a2/Ugt1a5/Ugt1a6a/Ug Regeneration t1a6b/Ugt1a7c/Ugt1a9 400 Positive 2 0.0259335 ↓Spp1,↑Egflam Regulation Of Cell-Substrate Adhesion 401 Adherens Junction 2 0.0259335 ↑Cdh1,↓Jup Organization 402 Response To 2 0.0259335 ↑Cav1,↑Cyp11a1 Gamma Radiation 403 Morphogenesis Of 2 0.0259335 ↑Crygs,↑Gli2 An Epithelium 404 Neuron Projection 3 0.0261181 ↑Cd24a,↑Cdh1,↓Ndel1 Development 405 Phototransduction 2 0.0273317 ↓Plekhb1,↑Gnat2 406 Gene Silencing By 2 0.0273317 ↑Lin28a,↑Piwil4 RNA 407 Positive 1 0.0282936 ↑Cd24a Regulation Of Cell-Cell Adhesion Mediated By Integrin 408 Cell Fate 1 0.0282936 ↑Pou5f1 Commitment Involved In Formation Of Primary Germ

237 Layers 409 Paraxial 1 0.0282936 ↑Foxc2 Mesodermal Cell Fate Commitment 410 Forebrain Neuron 1 0.0282936 ↑Tfap2c Fate Commitment 411 Killing By Host Of 1 0.0282936 ↑Mbl2 Symbiont Cells 412 Negative 1 0.0282936 ↑Slc6a4 Regulation Of Synaptic Transmission, Dopaminergic 413 Smoothened 1 0.0282936 ↑Gli2 Signaling Pathway Involved In Dorsal-Ventral Neural Tube Patterning 414 Smoothened 1 0.0282936 ↑Gli2 Signaling Pathway Involved In Regulation Of Cerebellar Granule Cell Precursor Cell Proliferation 415 Positive 1 0.0282936 ↑Icosl Regulation Of Interleukin-4 Biosynthetic Process 416 Positive 1 0.0282936 ↑Cyp17a1 Regulation Of Steroid Hormone Biosynthetic Process 417 Regulation Of 1 0.0282936 ↑Bmp7 Removal Of Superoxide Radicals 418 Negative 1 0.0282936 ↑Ascl2 Regulation Of Schwann Cell Proliferation 419 Positive 1 0.0282936 ↑Cd24a Regulation Of B Cell Apoptosis 420 Regulation Of 1 0.0282936 ↑Cdh1 Protein Localization At Cell Surface 421 Activation Of 1 0.0282936 ↑Plcg2 Store-Operated Calcium Channel Activity 422 Pre-B Cell 1 0.0282936 ↑Cd24a Differentiation 423 Platelet Formation 1 0.0282936 ↑Mfap2 424 Definitive 1 0.0282936 ↑Tgfbr3 Erythrocyte Differentiation 425 Neural Crest Cell 1 0.0282936 ↓Frzb Differentiation 426 Kidney Smooth 1 0.0282936 ↑Tshz3 Muscle Cell Differentiation 427 Spongiotrophoblas 1 0.0282936 ↑Ascl2 t Differentiation 428 Purine 1 0.0282936 ↑Entpd2 Ribonucleoside

238 Diphosphate Catabolic Process 429 Mrna 1 0.0282936 ↑Pou5f1 Transcription From RNA Polymerase II Promoter 430 Fatty Acid 1 0.0282936 ↑Elovl6 Elongation, Monounsaturated Fatty Acid 431 Adenine 1 0.0282936 ↓Slc25a17 Nucleotide Transport 432 Cellular Response 1 0.0282936 ↑Cyp11a1 To Cadmium Ion 433 Skeletal Muscle 1 0.0282936 ↑Myh11 Thick Filament Assembly 434 Positive 1 0.0282936 ↑Sytl2 Regulation Of Mucus Secretion 435 Vein Smooth 1 0.0282936 ↑Ednrb Muscle Contraction 436 Positive 1 0.0282936 ↑Parm1 Regulation Of Telomerase Activity 437 Regulation Of 1 0.0282936 ↑Cav1 Peptidase Activity 438 Mammary Placode 1 0.0282936 ↑Tbx2 Formation 439 Eyelid 1 0.0282936 ↑Osr2 Development In Camera-Type Eye 440 Pronephros 1 0.0282936 ↑Osr2 Development 441 Fractalkine 1 0.0282936 ↑Cyp11a1 Metabolic Process 442 Epithelial Fluid 1 0.0282936 ↑Ednrb Transport 443 Antibiotic 1 0.0282936 ↓Slc15a2 Transport 444 Nitric Oxide 1 0.0282936 ↑Hbb-b1/Hbb-b2/LOC100503605 Transport 445 Cell Junction 3 0.0284395 ↑Cdh1,↓Jup,↑Fblim1 Assembly 446 Epithelial To 2 0.0287598 ↑Bmp7,↑Tgfbr3 Mesenchymal Transition 447 Branching 2 0.030217 ↑Bmp7,↑Gli2 Morphogenesis Of A Tube 448 Response To 2 0.030217 ↑Ifitm1,↑Ifitm3 Biotic Stimulus 449 Neuron Migration 3 0.0308669 ↑Prkg1,↓Ndel1,↑Arx 450 Anatomical 4 0.0316685 ↑Pou5f1,↑Pdgfra,↑Gfra1,↑Lhx1 Structure Morphogenesis 451 Blood Vessel 2 0.0317031 ↑Foxc2,↑Bgn Remodeling 452 Spermatogenesis 7 0.0331417 ↓Herc4,↑Dbil5,↓Zfp39,↓Ccnd2,↑Piwil4,↑Tnp1,↑Tbata 453 Positive 2 0.0332174 ↑Il18,↑Itga1 Regulation Of Neuron Apoptosis 454 Odontogenesis 2 0.0332174 ↑Col1a2,↑Osr2 455 Response To 2 0.0347596 ↑Igf2,↓Lepr Nicotine

239 456 Cell-Matrix 3 0.0351466 ↑Fn1,↑Itga1,↑Itga8 Adhesion 457 Regulation Of 1 0.0352415 ↓Plxna4 Axon Extension Involved In Axon Guidance 458 Regulation Of 1 0.0352415 ↑Cd24a Cell-Cell Adhesion Mediated By Integrin 459 Cellular Response 1 0.0352415 ↑Sfrp1 To X-Ray 460 Negative 1 0.0352415 ↓Jup Regulation Of Heart Induction By Canonical Wnt Receptor Signaling Pathway 461 Regulation Of 1 0.0352415 ↑Gfap Neurotransmitter Uptake 462 Positive 1 0.0352415 ↑Nlrc5 Regulation Of Type I Interferon- Mediated Signaling Pathway 463 Regulation Of 1 0.0352415 ↑Igfbp2 Insulin-Like Growth Factor Receptor Signaling Pathway 464 Inhibition Of 1 0.0352415 ↑Adcy5 Adenylate Cyclase Activity By Dopamine Receptor Signaling Pathway 465 Negative 1 0.0352415 ↑Tnnt2 Regulation Of Atpase Activity 466 Positive 1 0.0352415 ↑Il18 Regulation Of Superoxide Anion Generation 467 Negative 1 0.0352415 ↓Frzb Regulation Of Cell Development 468 Negative 1 0.0352415 ↑Lin28a Regulation Of Glial Cell Differentiation 469 Negative 1 0.0352415 ↑Etv4 Regulation Of Mammary Gland Epithelial Cell Proliferation 470 Positive 1 0.0352415 ↑Adamts20 Regulation Of Melanocyte Differentiation 471 Protection From 1 0.0352415 ↑H2-Q7 Natural Killer Cell Mediated Cytotoxicity 472 Regulation Of 1 0.0352415 ↑Sall1 Neural Precursor Cell Proliferation 473 Stem Cell 1 0.0352415 ↓Msi2 Development 474 Ureter Smooth 1 0.0352415 ↑Tshz3

240 Muscle Cell Differentiation 475 Carnitine 1 0.0352415 ↓Shmt1 Biosynthetic Process 476 UDP-N- 1 0.0352415 ↑Gfpt2 Acetylglucosamine Biosynthetic Process 477 Long-Chain Fatty 1 0.0352415 ↑Elovl6 Acid Biosynthetic Process 478 Fatty Acid 1 0.0352415 ↑Elovl6 Elongation 479 Fatty Acid 1 0.0352415 ↑Elovl6 Elongation, Saturated Fatty Acid 480 Negative 1 0.0352415 ↑Apoc2 Regulation Of Very-Low-Density Lipoprotein Particle Clearance 481 Potassium Ion 1 0.0352415 ↑Abcc9 Import 482 Cerebral Cortex 1 0.0352415 ↑Arx Gabaergic Interneuron Migration 483 Transforming 1 0.0352415 ↑Tgfbr3 Growth Factor Beta Receptor Complex Assembly 484 Synaptic Vesicle 1 0.0352415 ↑Cadps Priming 485 Apoptotic Nuclear 1 0.0352415 ↑Cd24a Change 486 Regulation Of 1 0.0352415 ↑Ednrb Fever Generation 487 Regulation Of 1 0.0352415 ↑Etv4 Branching Involved In Mammary Gland Duct Morphogenesis 488 Regulation Of 1 0.0352415 ↑Cdh1 Branching Involved In Salivary Gland Morphogenesis 489 Positive 1 0.0352415 ↑Il18 Regulation Of Granulocyte Macrophage Colony- Stimulating Factor Production 490 Regulation Of 1 0.0352415 ↑Il18 Circadian Sleep- Wake Cycle, Non- REM Sleep 491 Vascular Smooth 1 0.0352415 ↑Actg2 Muscle Contraction 492 Positive 1 0.0352415 ↑Il18 Regulation Of Vascular Permeability 493 Positive 1 0.0352415 ↑Cd24a

241 Regulation Of Inflammatory Response To Antigenic Stimulus 494 Epithelial Cell 1 0.0352415 ↑Tfap2c Proliferation Involved In Mammary Gland Duct Elongation 495 Response To 1 0.0352415 ↑Igfbp7 Cortisol Stimulus 496 Response To Light 1 0.0352415 ↑Gnat2 Intensity 497 Paraxial 1 0.0352415 ↑Foxc2 Mesoderm Formation 498 Salivary Gland 1 0.0352415 ↑Cdh1 Cavitation 499 Spongiotrophoblas 1 0.0352415 ↑Ascl2 t Layer Development 500 Nose Development 1 0.0352415 ↓Rdh10 501 Sebaceous Gland 1 0.0352415 ↑Tfap2c Development 502 Kidney Epithelium 1 0.0352415 ↑Sall1 Development 503 Forebrain 1 0.0352415 ↑Lhx1 Regionalization 504 Hindgut 1 0.0352415 ↑Gli2 Morphogenesis 505 Glossopharyngeal 1 0.0352415 ↓Plxna4 Nerve Morphogenesis 506 Chemokine 1 0.0352415 ↑Il18 Biosynthetic Process 507 Inositol 1 0.0352415 ↑Plcg2 Trisphosphate Biosynthetic Process 508 Regulation Of Cell 2 0.0363291 ↑Il18,↑Lama2 Adhesion 509 Response To 2 0.0363291 ↑Igf2,↑Il18 Radiation 510 Positive 2 0.0379255 ↑Pdgfra,↑S100a6 Regulation Of Fibroblast Proliferation 511 Peripheral Nervous 2 0.0379255 ↑Ednrb,↑Ascl2 System Development 512 Mesoderm 2 0.0379255 ↑Foxc2,↑Tcf21 Development 513 Embryonic Digit 2 0.0379255 ↑Sall1,↑Gli2 Morphogenesis 514 Positive 2 0.0395483 ↑Pdgfra,↑Gli2 Regulation Of DNA Replication 515 Potassium Ion 4 0.0400156 ↑Abcc9,↓Kcnj3,↑Kcnc4,↑Kcne4 Transport 516 Acute-Phase 2 0.041197 ↑Fn1,↑Mbl2 Response 517 Negative 1 0.0421401 ↑Tgfbr3 Regulation Of Epithelial Cell Migration 518 Positive 1 0.0421401 ↓Ndel1 Regulation Of Axon Regeneration

242 519 Mesodermal Cell 1 0.0421401 ↑Pou5f1 Fate Commitment 520 Centrosome 1 0.0421401 ↓Ndel1 Localization 521 Regulation Of 1 0.0421401 ↑Sfrp1 Establishment Of Planar Polarity 522 Retrograde Axon 1 0.0421401 ↓Ndel1 Cargo Transport 523 Positive 1 0.0421401 ↑Lama2 Regulation Of Synaptic Transmission, Cholinergic 524 Positive 1 0.0421401 ↑Cartpt Regulation Of Transmission Of Nerve Impulse 525 Positive 1 0.0421401 ↑Igf2 Regulation Of Insulin Receptor Signaling Pathway 526 Wnt Receptor 1 0.0421401 ↑Sfrp1 Signaling Pathway Involved In Somitogenesis 527 Rhodopsin 1 0.0421401 ↑Gnat2 Mediated Phototransduction 528 Positive 1 0.0421401 ↑C1qtnf2 Regulation Of Fatty Acid Oxidation 529 Negative 1 0.0421401 ↓Ibtk Regulation Of Protein Tyrosine Kinase Activity 530 Negative 1 0.0421401 ↑Sfrp1 Regulation Of B Cell Differentiation 531 Positive 1 0.0421401 ↑Il18 Regulation Of Natural Killer Cell Proliferation 532 Positive 1 0.0421401 ↑Foxc2 Regulation Of Cell Migration Involved In Sprouting Angiogenesis 533 Positive 1 0.0421401 ↑Tshz3 Regulation Of Smooth Muscle Cell Differentiation 534 Retinal Cone Cell 1 0.0421401 ↑Gnat2 Development 535 Somatostatin 1 0.0421401 ↑Cartpt Secretion 536 Gonadotropin 1 0.0421401 ↑Meg3 Secretion 537 Progesterone 1 0.0421401 ↑Cyp11a1 Biosynthetic Process 538 Testosterone 1 0.0421401 ↑Cyp11a1 Biosynthetic Process 539 Pre-Microrna 1 0.0421401 ↑Lin28a Processing

243 540 Regulation Of 1 0.0421401 ↑Cartpt Bone Remodeling 541 Triglyceride-Rich 1 0.0421401 ↑Apoc2 Lipoprotein Particle Remodeling 542 Chylomicron 1 0.0421401 ↑Apoc2 Remodeling 543 Response To 1 0.0421401 ↑Tgfbr3 Follicle- Stimulating Hormone Stimulus 544 Detection Of 1 0.0421401 ↓Jup Mechanical Stimulus 545 Positive 1 0.0421401 ↑Apoc2 Regulation Of Phospholipase Activity 546 Menstrual Cycle 1 0.0421401 ↑Sfrp1 Phase 547 Prostatic Bud 1 0.0421401 ↑Gli2 Formation 548 Lung Vasculature 1 0.0421401 ↑Tcf21 Development 549 Spinal Cord 1 0.0421401 ↑Gli2 Dorsal-Ventral Patterning 550 Cranial Nerve 1 0.0421401 ↓Plxna4 Morphogenesis 551 Cerebellar Cortex 1 0.0421401 ↑Gli2 Morphogenesis 552 Outer Ear 1 0.0421401 ↑Sall1 Morphogenesis 553 Convergent 1 0.0421401 ↓Frzb Extension Involved In Organogenesis 554 Interferon-Gamma 1 0.0421401 ↑Il18 Biosynthetic Process 555 Interleukin-2 1 0.0421401 ↑Il18 Biosynthetic Process 556 Response To 3 0.0425953 ↑Fhl2,↑Foxc2,↑Pdgfra Hormone Stimulus 557 Negative 2 0.0428713 ↑Nlrc5,↑Bmp7 Regulation Of NF- Kappab Transcription Factor Activity 558 Triglyceride 2 0.0428713 ↑Cav1,↑Pnliprp2 Metabolic Process 559 Branching 2 0.0428713 ↑Tcf21,↑Sall1 Involved In Ureteric Bud Morphogenesis 560 Defense Response 2 0.0445706 ↑Nlrc5,↑Abcc9 To Virus 561 Negative 2 0.0445706 ↑Tgfbr3,↑Sfrp1 Regulation Of Epithelial Cell Proliferation 562 Hair Follicle 2 0.0445706 ↑Tfap2c,↑Apcdd1 Development 563 Actin Cytoskeleton 4 0.0449809 ↑Prkg1,↑Sfrp1,↑Coro1a,↑Prr5 Organization 564 G-Protein 2 0.0462945 ↑Ptger4,↑Gnat2 Signaling, Coupled To Camp

244 Nucleotide Second Messenger 565 Regulation Of 2 0.0462945 ↑Myh6,↑Tnnt2 Heart Contraction 566 Adult Locomotory 2 0.0462945 ↑Hoxd10,↑Hoxd9 Behavior 567 Positive 1 0.0489895 ↑Cdh1 Regulation Of Transcription Factor Import Into Nucleus 568 Gap Junction 1 0.0489895 ↑Gjc1 Assembly 569 Positive 1 0.0489895 ↑Ednrb Regulation Of Penile Erection 570 Spermatid 1 0.0489895 ↑Tbata Differentiation 571 Regulation Of 1 0.0489895 ↑Tmem204 Vascular Endothelial Growth Factor Receptor Signaling Pathway 572 Positive 1 0.0489895 ↑Apoc2 Regulation Of Triglyceride Catabolic Process 573 Regulation Of 1 0.0489895 ↑Nlrc5 Kinase Activity 574 Negative 1 0.0489895 ↑Cav1 Regulation Of Epithelial Cell Differentiation 575 Negative 1 0.0489895 ↑Thbd Regulation Of Platelet Activation 576 Negative 1 0.0489895 ↑Bmp7 Regulation Of NF- Kappab Import Into Nucleus 577 Negative 1 0.0489895 ↑Pou5f1 Regulation Of Calcium Ion- Dependent Exocytosis 578 Negative 1 0.0489895 ↑Apoc2 Regulation Of Receptor-Mediated Endocytosis 579 Positive 1 0.0489895 ↑Cadps Regulation Of Calcium Ion- Dependent Exocytosis 580 Regulation Of B 1 0.0489895 ↑Cd24a Cell Differentiation 581 Gliogenesis 1 0.0489895 ↑Pdgfra 582 Fatty Acid Alpha- 1 0.0489895 ↓Slc25a17 Oxidation 583 Branched Chain 1 0.0489895 ↑Bcat1 Family Amino Acid Metabolic Process 584 Transcytosis 1 0.0489895 ↑Cav1 585 Myelination In 1 0.0489895 ↑Lama2 Peripheral Nervous System 586 Myofibril 1 0.0489895 ↑Myh6

245 Assembly 587 Elastic Fiber 1 0.0489895 ↑Myh11 Assembly 588 Prostate Epithelial 1 0.0489895 ↑Sfrp1 Cord Arborization Involved In Prostate Glandular Acinus Morphogenesis 589 Positive 1 0.0489895 ↑Il18 Regulation Of Interleukin-1 Beta Production 590 Negative 1 0.0489895 ↑Thbd Regulation Of Coagulation 591 Sperm Ejaculation 1 0.0489895 ↑Slc6a4 592 Type 2 Immune 1 0.0489895 ↑Il18 Response 593 Respiratory 1 0.0489895 ↑Tcf21 System Development 594 Mesenchyme 1 0.0489895 ↑Bmp7 Development 595 Lymphangiogenesi 1 0.0489895 ↑Foxc2 s 596 Skin 1 0.0489895 ↑Col1a2 Morphogenesis 597 Eye 1 0.0489895 ↑Col5a2 Morphogenesis 598 Kidney 1 0.0489895 ↑Tshz3 Morphogenesis 599 Negative 1 0.0489895 ↑Twist2 Regulation Of Molecular Function 600 Pattern 3 0.0497077 ↑Bmp7,↑Lhx1,↑Gli2 Specification Process 601 Cellular Response 2 0.0498145 ↑Igfbp2,↑Igfbp7 To Hormone Stimulus 602 Hippocampus 2 0.0498145 ↑Cyp11a1,↑Cyp17a1 Development

246 Table A1-4. Total Pathway Studio Gene Ontology (GO) for P8 germ cell differentially regulated at ± 2.0 fold genes.

Group Term # of Genes Pvalue Genes 1 Meiosis 84 1.48E-23 ↓Stra8,↓Clgn,↓Dmc1,↓Spo11,↓Boll,↓Rec8,↓Msh5,↓Stag3,↓Sycp 1,↓Sycp2,↓Smc1b,↓Mei1,↓Sycp3,↓Prdm9,↓Zfp318,↓Hormad1,↓ 4930528F23Rik,↓Syce1,↓Mael,↓Dpep3 2 Spermatogenesis 423 5.82E-15 ↓Taf7l,↓Timp2,↓Rbp4,↓Lmna,↓Sgpl1,↓Stra8,↓Clgn,↓Mtl5,↓Dmc 1,↓Spo11,↓Boll,↓Rec8,↑Adamts2,↓Sycp1,↓Gfer,↓Sycp3,↑Hagh,↓ Tex15,↓Mov10l1,↓Dazl,↓D6mm5e,↓Mael,↓Adad1,↓Rnf17,↓Hsf 2bp,↓Zfp541 3 Multicellular 1146 4.64E-12 ↓4930447C04Rik,↓Taf7l,↑Foxc2,↓Igfbp2,↑Wnt5a,↓Itgb3,↓Nes,↓ Organismal Clgn,↓Anpep,↓Mtl5,↓Nobox,↓Boll,↓Spdya,↓Dkk3,↓Krt8,↓Crabp Development 1,↓Hopx,↓Cdk13,↓Pou4f1,↓Ndrg4,↓Plekhb1,↓Sema4f,↓Ebf3,↓N pnt,↓Radil,↓Ctnnd2,↓Mov10l1,↑Mkx,↓Dazl,↓Mbnl3,↓Mael,↓Ada d1,↓Rnf17,↓Disp1,↓Zfp541,↓Olfml3,↓Ebf4 4 Synaptonemal 13 5.19E-12 ↓Stag3,↓Sycp1,↓Sycp2,↓Tex11,↓Sycp3,↓Tex15,↓Syce1 Complex Assembly 5 Synapsis 9 3.10E-11 ↓Stra8,↓Rec8,↓Msh5,↓Sycp3,↓Tex15,↓Mael 6 Male Meiosis I 16 1.81E-07 ↓Dmc1,↓Spo11,↓Rec8,↓Mei1,↓Sycp3 7 Reciprocal 35 4.96E-07 ↓Stra8,↓Dmc1,↓Spo11,↓Rec8,↓Msh5,↓Sycp1 Meiotic Recombination 8 Fertilization 36 5.91E-07 ↓Stra8,↓Sycp2,↓Tex11,↓Tex15,↓Mael,↓Ooep 9 Axon Guidance 328 1.10E-06 ↓Trpc1,↓Nr4a3,↓Pak1,↑Itga9,↓Itgb3,↓Cacna1h,↓Scn8a,↓Alcam,↑ Gfra1,↓Runx3,↓Nck2,↓Sema4f,↑Rgmb,↓Rps6ka6 10 Meiotic Prophase I 11 1.67E-06 ↓Sycp2,↓Prdm9,↓Syce1,↓Mael 11 Spermatid 68 1.96E-06 ↓Taf7l,↓Dmc1,↓Spo11,↓Rec8,↓Mei1,↓Adad1,↓Rnf17 Development 12 Female Meiosis 4 2.48E-06 ↓Stra8,↓Stag3,↓Sycp3 Sister Chromatid Cohesion 13 Male Meiosis 14 4.95E-06 ↓Sycp2,↓Tex11,↓Tex15,↓Mael 14 Metabolic Process 2421 9.76E-06 ↓Zdhhc14,↓Zranb3,↓Otud7a,↓Ppil6,↓Nudt17,↓Entpd2,↓Enpp1,↓ Pak1,↓Acsl1,↓Prkag2,↓Spint1,↑Cth,↓Braf,↓Abcc5,↓Anpep,↑Ptpr z1,↓Nags,↑Adamts2,↓Prss50,↓Gm3776/Gsta1/Gsta2,↓Aak1,↑Gd a,↓Brsk2,↓Kif27,↓Kif1b,↑Hagh,↑Bcat1,↓Syn2,↓Slc27a2,↓Mov10 l1,↓Tktl1,↑Agpat9,↓Syce1,↓Ugt8,↓Dpep3,↓Gpat2,↓Rnf17,↓Atp1 3a3,↓1700106N22Rik,↓Galnt6,↓Lpgat1,↓Acpl2 15 Cell 735 2.26E-05 ↓Taf7l,↑Spp1,↑Wnt5a,↓Clgn,↓Anpep,↓Mtl5,↓Nobox,↓Boll,↑Lgal Differentiation s7,↓Ndrg4,↓Gm98,↓Sema4f,↓Npnt,↓Dmrtc2,↓Dazl,↓Mael,↓Adad 1,↓Rnf17,↓Zfp541 16 Female Gamete 20 2.30E-05 ↓Dmc1,↓Spo11,↓Msh5,↓D6mm5e Generation 17 Branching 8 3.39E-05 ↑Fgfr2,↓Socs3,↓Spint1 Involved In Embryonic Placenta Morphogenesis 18 Regulation Of 12 0.0001297 ↑Foxc2,↓Stra8,↓Mael Organ Growth 19 Ovarian Follicle 57 0.0001302 ↓Stra8,↓Dmc1,↓Spo11,↓Nobox,↓Tox2 Development 20 Positive 14 0.0002119 ↑Spp1,↓Tfrc,↓Itgb3 Regulation Of Bone Resorption 21 Regulation Of 2872 0.0002286 ↓Pcgf3,↓Sp5,↓Esx1,↑3110052M02Rik,↓Taf7l,↓Eaf2,↓A630033E Transcription, 08Rik,↓Rel,↓Nr4a3,↓Klf9,↑Foxc2,↓Atf3,↑Sox18,↓Dmrtb1,↓Nob DNA-Dependent ox,↑Tle4,↓Egr3,↓Taf4b,↓Runx3,↑E4f1,↓Hipk2,↓Sec14l2,↓Hopx, ↓Pbx3,↓Pou4f1,↓Elavl2,↓Gm98,↓Prdm9,↓Ebf3,↓Zfp318,↓Ctnnd 2,↓Dmrtc2,↑Mkx,↑Spic,↓Rhox13,↓Zbtb42,↓Zfp287,↓Zfp541,↓T ox2,↓Atf7ip2,↓Ccdc79,↓Scml2,↓Ebf4 22 Ion 540 0.0002641 ↑Kctd14,↓Clca1,↑P2rx7,↓Trpc1,↓Slc4a8,↓Slc22a3,↓Cacna1h,↓S Transmembrane cn8a,↓Grin2d,↑Kcnc4,↓Slc41a2,↓Atp11c,↓Atp13a3,↓Slc22a21

247 Transport 23 Positive 476 0.0002658 ↓Eaf2,↓Rel,↓Zp3,↑Foxc2,↑Wnt5a,↑Sox18,↓Hipk2,↓Sec14l2,↓G Regulation Of m98,↑Tesc,↓Ebf3,↑Rgmb,↓Zfp287 Transcription, DNA-Dependent 24 Regulation Of 112 0.0004218 ↑Rbm3,↓Boll,↓Eif2c3,↓Nck2,↓Dazl,↓Adad1 Translation 25 Urinary Bladder 4 0.000435 ↓Rbp4,↑Wnt5a Development 26 Muscle Organ 115 0.0004856 ↓Lmna,↓Cacna1h,↓Scn8a,↓Egr3,↓Mov10l1,↑Mkx Development 27 Transcription, 2265 0.0005297 ↓Pcgf3,↓Sp5,↓Taf7l,↓Eaf2,↓Rel,↓Nr4a3,↓Klf9,↑Foxc2,↓Atf3,↑S DNA-Dependent ox18,↓Dmrtb1,↓Nobox,↑Tle4,↓Egr3,↓Runx3,↑E4f1,↓Hipk2,↓Sec 14l2,↓Hopx,↓Pbx3,↓Pou4f1,↓Gm98,↓Prdm9,↓Ebf3,↓Zfp318,↓Ct nnd2,↓Dmrtc2,↑Spic,↓Zbtb42,↓Zfp287,↓Zfp541,↓Tox2,↓Atf7ip2 ,↓Scml2,↓Ebf4 28 Interspecies 325 0.0005427 ↓Tfrc,↓H2-Q10,↓Itgb3,↓Krt18,↓Anpep,↓Hck,↓Krt8,↑E4f1,↓Cdk Interaction 13 Between Organisms 29 Positive 46 0.000651 ↓Zp3,↓Cd3e,↓Tnfrsf13c,↓Nck2 Regulation Of T Cell Proliferation 30 Ureteric Bud 46 0.000651 ↑Foxc2,↑Fgfr2,↓Epcam,↓Npnt Development 31 Chiasma 5 0.0007209 ↓Msh5,↓Tex11 Assembly 32 Positive 5 0.0007209 ↓Zp3,↓Cacna1h Regulation Of Acrosome Reaction 33 Cell Projection 5 0.0007209 ↓Itgb3,↓Nes Morphogenesis 34 Protein 743 0.0007538 ↑Riok2,↓Stk32c,↑P2rx7,↓Pak1,↓Prkd3,↑Fgfr2,↑Wnt5a,↓Braf,↓H Phosphorylation ck,↓Runx3,↓Hipk2,↓Cdk13,↓Aak1,↓Brsk2,↓Rps6ka6,↓Ooep 35 Cell Cycle 604 0.0007958 ↓Krt18,↓Dmc1,↑Ube2c,↓Spdya,↑E4f1,↓Rif1,↓Stag3,↓Cdk13,↓Sy cp1,↓Sycp2,↓Smc1b,↓Sycp3,↓Rab11fip3,↓Syce1 36 Positive 22 0.0008521 ↑Foxc2,↑Wnt5a,↓Itgb3 Regulation Of Endothelial Cell Migration 37 Transport 1812 0.0009549 ↓Rims2,↑P2rx7,↓Trpc1,↓Slc4a8,↓Rbp4,↓Vldlr,↓Rbp1,↓Slc22a3, ↓Abcc5,↓Cacna1h,↓Ramp1,↑Fabp4,↓Scn8a,↓Grin2d,↓Rab3b,↓Se c14l2,↓Crabp1,↓Steap1,↑Kcnc4,↓Rab6b,↓Slc9a5,↓Rab11fip3,↓Sl c25a31,↑Stx3,↓Aifm3,↓Slc41a2,↓Syt17,↓Rims3,↓Slc22a21 38 Positive 6 0.0010752 ↑Wnt5a,↓Dazl Regulation Of Meiosis 39 Uterus 6 0.0010752 ↓Rbp4,↑Wnt5a Development 40 Placenta Blood 6 0.0010752 ↓Socs3,↓Spint1 Vessel Development 41 Embryonic Retina 6 0.0010752 ↓Rbp4,↓Hipk2 Morphogenesis In Camera-Type Eye 42 Negative 297 0.0011339 ↓Nr4a3,↑Wnt5a,↓Nes,↓Braf,↓Krt18,↓Pou4f1,↓Tex11,↓Sycp3,↓M Regulation Of ael Apoptosis 43 Neuron 136 0.0011684 ↑Wnt5a,↓Nes,↓Hipk2,↓Pou4f1,↓Brsk2,↓Gpc2 Differentiation 44 Apoptosis 778 0.0012166 ↓Clca2,↓Xaf1,↓Pak1,↓Dsp,↓Fam3b,↓Lmna,↓Krt18,↓Sgpl1,↓Krt8 ,↑Lgals7,↓Hipk2,↓Sycp2,↓Plekhf1,↓Kif1b,↓Aifm3,↓Gml 45 Protein 7 0.0014967 ↓Spo11,↓Tex15 Localization To Chromosome 46 Lymphangiogenes 7 0.0014967 ↑Foxc2,↑Sox18 is 47 Embryonic Pattern 27 0.001568 ↑Fgfr2,↓Ooep,↓Disp1 Specification

248 48 Signal Complex 8 0.0019843 ↓Cd3e,↓Nck2 Assembly 49 Positive 8 0.0019843 ↓Zp3,↓Cd3e Regulation Of Interleukin-4 Production 50 Hair Follicle 30 0.0021356 ↑Igfbp5,↑Fgfr2,↓Runx3 Morphogenesis 51 Transmembrane 831 0.0023618 ↑P2rx7,↓Trpc1,↓Slc4a8,↓Tfrc,↓Rbp4,↓Slc22a3,↓Abcc5,↓Cacna1 Transport h,↓Scn8a,↑Kcnc4,↓Slc9a5,↓Slc25a31,↓Slc41a2,↓Atp11c,↓Atp13 a3,↓Slc22a21 52 Phospholipid 68 0.0028044 ↑Agpat9,↓Gpat2,↓Lpgat1,↓Cds1 Biosynthetic Process 53 Response To 163 0.0029156 ↑P2rx7,↓Pak1,↑Spp1,↓Tfrc,↓Acsl1,↑Wnt5a Organic Substance 54 DNA Metabolic 70 0.0031156 ↓Eme2,↓Dmc1,↓Spo11,↓Dnase1l2 Process 55 Positive 10 0.0031532 ↓Prkag2,↑Wnt5a Regulation Of Peptidyl- Threonine Phosphorylation 56 Xenobiotic 10 0.0031532 ↓Acsl1,↓Gm3776/Gsta1/Gsta2 Catabolic Process 57 Vagina 10 0.0031532 ↓Rbp4,↑Wnt5a Development 58 Anatomical 166 0.0031901 ↓Krt18,↑Gfra1,↓Dkk3,↓Tshb,↑Mpzl2,↓Scml2 Structure Morphogenesis 59 Positive 35 0.0033359 ↑Spp1,↑Fbln2,↓Npnt Regulation Of Cell-Substrate Adhesion 60 Positive 35 0.0033359 ↓Zp3,↑Wnt5a,↓Cd3e Regulation Of Interferon-Gamma Production 61 Triglyceride 36 0.0036163 ↓Acsl1,↑Agpat9,↓Gpat2 Biosynthetic Process 62 Lung Alveolus 36 0.0036163 ↑Fgfr2,↑Wnt5a,↓Hopx Development 63 Positive 11 0.0038321 ↑Foxc2,↓Itgb3 Regulation Of Cell Adhesion Mediated By Integrin 64 Cellular Metal Ion 11 0.0038321 ↓Mtl5,↓Mt1 Homeostasis 65 Lymph Vessel 11 0.0038321 ↑Foxc2,↑Sox18 Development 66 Positive 38 0.0042193 ↓Pak1,↑Wnt5a,↓Braf Regulation Of Peptidyl-Serine Phosphorylation 67 Positive 38 0.0042193 ↓Zp3,↓Prkag2,↓Vldlr Regulation Of Protein Kinase Activity 68 Aging 178 0.0044841 ↓Timp2,↓Tfrc,↓Pth1r,↓Socs3,↓Igfbp2,↓Gm3776/Gsta1/Gsta2 69 Cellular Response 39 0.0045422 ↑Wnt5a,↓Stra8,↑Krt13 To Retinoic Acid 70 Cation Transport 125 0.0045525 ↑P2rx7,↓Grin2d,↓Slc9a5,↓Slc41a2,↓Atp13a3 71 Positive 12 0.0045726 ↓Boll,↓Dazl Regulation Of Translational Initiation 72 Positive 438 0.0048238 ↑Fgfr2,↓Pth1r,↓Nmb,↓Atf3,↑Wnt5a,↑Fabp4,↓Epcam,↓Spdya,↓Hi Regulation Of Cell pk2,↓Cdk13

249 Proliferation 73 Regulation Of 40 0.0048797 ↓Sycp1,↓Tex15,↓Rabgap1l Protein Localization 74 Embryonic Organ 13 0.0053735 ↓Rbp4,↑Fgfr2 Morphogenesis 75 Embryo 42 0.0055991 ↑Spp1,↓Klf9,↓Ooep Implantation 76 Single 42 0.0055991 ↓Zp3,↓Clgn,↓Dazl Fertilization 77 Regulation Of 84 0.0059737 ↑Igfbp5,↓Socs3,↓Igfbp2,↑E4f1 Growth 78 Glutathione 43 0.0059814 ↑Cth,↓Gm3776/Gsta1/Gsta2,↑Hagh Metabolic Process 79 Peripheral 43 0.0059814 ↓Scn8a,↓Egr3,↓Ugt8 Nervous System Development 80 Mesoderm 43 0.0059814 ↑Foxc2,↓Hck,↓Pou4f1 Development 81 Response To 134 0.006092 ↓Socs3,↓Igfbp2,↑Wnt5a,↑Fabp4,↓Pnliprp1 Glucocorticoid Stimulus 82 Organic Cation 14 0.0062338 ↑P2rx7,↓Slc22a3 Transport 83 Response To 135 0.006283 ↓Tfrc,↓Acsl1,↓Igfbp2,↓Vldlr,↓Cd3e Nutrient 84 Central Nervous 137 0.0066777 ↓Timp2,↓Nes,↑Ptprz1,↓Rps6ka6,↓Ugt8 System Development 85 Cell Adhesion 686 0.0066925 ↑Spp1,↑Itga9,↓Itgb3,↓Alcam,↓Apba1,↓Npnt,↑Negr1,↑Tpbg,↑Rg mb,↓Radil,↓Col27a1,↓Ctnnd2,↑Mpzl2 86 Smoothened 45 0.0067918 ↓Hipk2,↓Disp2,↓Disp1 Signaling Pathway 87 Male Gonad 88 0.0070305 ↓Rbp4,↑Wnt5a,↓Tex11,↑Tesc Development 88 Oocyte 15 0.0071523 ↓Zp3,↓Stra8 Development 89 Intermediate 15 0.0071523 ↑Gfap,↑Tchh Filament Organization 90 Negative 47 0.0076643 ↑Fgfr2,↑Wnt5a,↓Runx3 Regulation Of Epithelial Cell Proliferation 91 Positive 47 0.0076643 ↓Zp3,↑Wnt5a,↑Fabp4 Regulation Of Inflammatory Response 92 Activation Of 48 0.0081242 ↓Crk,↓Braf,↓Kidins220 MAPKK Activity 93 Adult Locomotory 48 0.0081242 ↓Grin2d,↓Hipk2,↓Pbx3 Behavior 94 Oocyte Maturation 16 0.0081281 ↓Dmc1,↓Rec8 95 Positive 16 0.0081281 ↑P2rx7,↓Acsl1 Regulation Of Protein Serine- Threonine Kinase Activity 96 Meiotic DNA 1 0.0085792 ↓Stra8 Double-Strand Break Formation 97 Meiotic Cell Cycle 1 0.0085792 ↓Stra8 DNA Replication Checkpoint 98 Zygotene 1 0.0085792 ↓Spo11 99 Meiotic Prophase 1 0.0085792 ↓Msh5 II 100 Achiasmate 1 0.0085792 ↓Sycp3 Meiosis I 101 Male Meiosis 1 0.0085792 ↓Stag3

250 Sister Chromatid Cohesion 102 Meiotic 1 0.0085792 ↓Stra8 Chromosome Condensation 103 Female Meiosis 1 0.0085792 ↓Sycp3 Chromosome Separation 104 Regulation Of 1 0.0085792 ↑E4f1 Mitotic Cell Cycle, Embryonic 105 Spermatogonial 1 0.0085792 ↓Rbp4 Cell Division 106 Positive 1 0.0085792 ↓Zp3 Regulation Of Acrosomal Vesicle Exocytosis 107 Positive 1 0.0085792 ↓Lmna Regulation Of Cell Aging 108 Regulation Of Rap 1 0.0085792 ↓Timp2 Protein Signal Transduction 109 Fibroblast Growth 1 0.0085792 ↑Fgfr2 Factor Receptor Signaling Pathway Involved In Positive Regulation Of Cell Proliferation In Bone Marrow 110 Fibroblast Growth 1 0.0085792 ↑Fgfr2 Factor Receptor Signaling Pathway Involved In Negative Regulation Of Apoptosis In Bone Marrow 111 Fibroblast Growth 1 0.0085792 ↑Fgfr2 Factor Receptor Signaling Pathway Involved In Hemopoiesis 112 Negative 1 0.0085792 ↑Spp1 Regulation Of Collateral Sprouting Of Intact Axon In Response To Injury 113 Negative 1 0.0085792 ↓Dkk3 Regulation Of Aldosterone Biosynthetic Process 114 Negative 1 0.0085792 ↓Dkk3 Regulation Of Cortisol Biosynthetic Process 115 Positive 1 0.0085792 ↓Zp3 Regulation Of Phosphatidylinosit ol Biosynthetic Process 116 Positive 1 0.0085792 ↑Gfap Regulation Of Schwann Cell Proliferation

251 117 Positive 1 0.0085792 ↓Nes Regulation Of Intermediate Filament Depolymerization 118 Positive 1 0.0085792 ↑Wnt5a Regulation Of Interleukin-8 Secretion 119 Positive 1 0.0085792 ↑Wnt5a Regulation Of Cytokine Secretion Involved In Immune Response 120 Peripheral 1 0.0085792 ↓Pou4f1 Nervous System Neuron Differentiation 121 CDP- 1 0.0085792 ↓Cds1 Diacylglycerol Biosynthetic Process 122 Protein-Pyridoxal- 1 0.0085792 ↑Cth 5-Phosphate Linkage Via Peptidyl-N6- Pyridoxal Phosphate-L- Lysine 123 Patched Ligand 1 0.0085792 ↓Disp1 Maturation 124 Positive 1 0.0085792 ↓H2-Q10 Regulation Of Tolerance Induction To Nonself Antigen 125 Positive 1 0.0085792 ↓Zp3 Regulation Of Ovarian Follicle Development 126 Positive 1 0.0085792 ↓Zp3 Regulation Of Antral Ovarian Follicle Growth 127 Proprioception 1 0.0085792 ↓Pou4f1 Involved In Equilibrioception 128 Positive 1 0.0085792 ↓Zp3 Regulation Of Type IV Hypersensitivity 129 Vestibular Reflex 1 0.0085792 ↓Nr4a3 130 Cervix 1 0.0085792 ↑Wnt5a Development 131 Anterior 1 0.0085792 ↓Pbx3 Compartment Pattern Formation 132 Posterior 1 0.0085792 ↓Pbx3 Compartment Specification 133 Histamine Uptake 1 0.0085792 ↓Slc22a3 134 Sterol Regulatory 1 0.0085792 ↓Lmna Element Binding Protein Import Into Nucleus 135 NAD Transport 1 0.0085792 ↑P2rx7 136 Retinol Transport 1 0.0085792 ↓Rbp4 137 Metestrus 1 0.0085792 ↑Wnt5a 138 Regulation Of G- 50 0.0090919 ↓Itgb3,↓Ramp1,↓Rgs16

252 Protein Coupled Receptor Protein Signaling Pathway 139 Cellular Amino 50 0.0090919 ↑Cth,↓Nags,↑Bcat1 Acid Biosynthetic Process 140 Neuromuscular 17 0.00916 ↓Egr3,↓Kif1b Synaptic Transmission 141 Positive 17 0.00916 ↑P2rx7,↑Wnt5a Regulation Of Interleukin-1 Beta Secretion 142 Defense Response 52 0.0101241 ↑P2rx7,↓Hck,↑Gbp1 To Gram-Positive Bacterium 143 Sister Chromatid 18 0.0102472 ↓Rec8,↓Smc1b Cohesion 144 Retinol Metabolic 18 0.0102472 ↓Rbp4,↓Rbp1 Process 145 Positive 18 0.0102472 ↑P2rx7,↑Wnt5a Regulation Of Ossification 146 Negative 19 0.0113884 ↓Enpp1,↓Socs3 Regulation Of Insulin Receptor Signaling Pathway 147 Wound Healing 102 0.0116794 ↓Dsp,↑Wnt5a,↓S100a8,↓Itgb3 148 Negative 506 0.0125708 ↓Rhox2a,↑Foxc2,↑Fgfr2,↑Wnt5a,↑Tle4,↓Hipk2,↓Hopx,↓Pou4f1, Regulation Of ↓Mael,↓Zbtb42 Transcription From RNA Polymerase II Promoter 149 Embryonic Heart 20 0.0125828 ↑Foxc2,↑Sox18 Tube Development 150 Positive 21 0.0138294 ↓Hipk2,↓Npnt Regulation Of Transforming Growth Factor Beta Receptor Signaling Pathway 151 Positive 21 0.0138294 ↑P2rx7,↓H2-Q10 Regulation Of T Cell Mediated Cytotoxicity 152 Regulation Of 21 0.0138294 ↑P2rx7,↓Scn8a Action Potential In Neuron 153 Regulation Of 365 0.0140035 ↓Cd55,↓Rims2,↓Timp2,↓Prkag2,↓Sec14l2,↓Kidins220,↓Rab11fi Catalytic Activity p3,↑Tesc 154 Positive 60 0.0149128 ↑Fgfr2,↓Itgb3,↓Braf Regulation Of ERK1 And ERK2 Cascade 155 Regulation Of 22 0.0151271 ↓Acsl1,↓Prkag2 Fatty Acid Oxidation 156 Positive 22 0.0151271 ↓Zp3,↓Itgb3 Regulation Of Leukocyte Migration 157 Establishment Of 22 0.0151271 ↓Lmna,↓Brsk2 Cell Polarity 158 Oogenesis 22 0.0151271 ↓Nobox,↓Dazl 159 Homeostasis Of 22 0.0151271 ↑P2rx7,↓Tex15 Number Of Cells Within A Tissue 160 Lipid Metabolic 372 0.0155253 ↓Gdpd1,↓Acsl1,↓Vldlr,↓Sgpl1,↑Fabp4,↓Slc27a2,↓Pnliprp1,↓Cds

253 Process 1 161 Neurotransmitter 61 0.0155867 ↓Rims2,↑Stx3,↓Rims3 Transport 162 Fatty Acid 23 0.016475 ↓Acsl1,↓Slc27a2 Transport 163 Positive 610 0.016932 ↓Nr4a3,↑Foxc2,↑Fgfr2,↑Wnt5a,↑Sox18,↓Nobox,↓Hipk2,↓Pou4f Regulation Of 1,↓Prdm9,↑Spic,↓Tox2 Transcription From RNA Polymerase II Promoter 164 Phosphorylation 531 0.0170308 ↓Pak1,↓Crk,↓Prkag2,↓Braf,↓Hipk2,↓Aak1,↓Brsk2,↓Nck2,↓Rps6 ka6,↓Pgm2l1 165 Regulation Of 2 0.0170851 ↑P2rx7 Killing Of Cells Of Other Organism 166 Female Meiosis 2 0.0170851 ↓Sycp3 Chromosome Segregation 167 Male Meiosis 2 0.0170851 ↓Tex11 Chromosome Segregation 168 Spermatogenesis, 2 0.0170851 ↓Sycp3 Exchange Of Chromosomal Proteins 169 Negative 2 0.0170851 ↓Zp3 Regulation Of Binding Of Sperm To Zona Pellucida 170 Squamous Basal 2 0.0170851 ↑Fgfr2 Epithelial Stem Cell Differentiation Involved In Prostate Gland Acinus Development 171 Fibroblast Growth 2 0.0170851 ↑Fgfr2 Factor Receptor Signaling Pathway Involved In Mammary Gland Specification 172 Fibroblast Growth 2 0.0170851 ↑Fgfr2 Factor Receptor Signaling Pathway Involved In Orbitofrontal Cortex Development 173 Phosphorylation 2 0.0170851 ↓Cdk13 Of RNA Polymerase II C- Terminal Domain 174 B Cell 2 0.0170851 ↓Tnfrsf13c Costimulation 175 Positive 2 0.0170851 ↑Wnt5a Regulation Of Cell-Cell Adhesion Mediated By Cadherin 176 Positive 2 0.0170851 ↑P2rx7 Regulation Of Cytolysis 177 Establishment Of 2 0.0170851 ↑Sox18 Endothelial Barrier

254 178 Glomerular 2 0.0170851 ↑Foxc2 Mesangial Cell Development 179 Lymphatic 2 0.0170851 ↑Sox18 Endothelial Cell Differentiation 180 Endocardial Cell 2 0.0170851 ↑Sox18 Differentiation 181 Mesenchymal Cell 2 0.0170851 ↑Fgfr2 Differentiation Involved In Lung Development 182 Transsulfuration 2 0.0170851 ↑Cth 183 Hydrogen Sulfide 2 0.0170851 ↑Cth Biosynthetic Process 184 Free Ubiquitin 2 0.0170851 ↑Ube2c Chain Polymerization 185 Multicellular 2 0.0170851 ↑P2rx7 Organismal Protein Catabolic Process 186 Antigen 2 0.0170851 ↓H2-Q10 Processing And Presentation Of Endogenous Peptide Antigen Via MHC Class I Via ER Pathway, TAP-Dependent 187 Viral Envelope 2 0.0170851 ↓Hsbp1l1 Fusion With Host Membrane 188 Mammary Gland 2 0.0170851 ↑Fgfr2 Bud Formation 189 Optic Cup 2 0.0170851 ↑Wnt5a Formation Involved In Camera-Type Eye Development 190 Branch Elongation 2 0.0170851 ↑Fgfr2 Involved In Salivary Gland Morphogenesis 191 Habenula 2 0.0170851 ↓Pou4f1 Development 192 Glomerular 2 0.0170851 ↑Foxc2 Endothelium Development 193 Hypophysis 2 0.0170851 ↑Wnt5a Morphogenesis 194 Pericardium 2 0.0170851 ↑Wnt5a Morphogenesis 195 Lateral Sprouting 2 0.0170851 ↑Wnt5a Involved In Mammary Gland Duct Morphogenesis 196 Cytoskeleton- 2 0.0170851 ↓Kif1b Dependent Intracellular Transport 197 Phospholipid 2 0.0170851 ↑P2rx7 Transfer To Membrane 198 Ion Transport 693 0.0172252 ↑P2rx7,↓Trpc1,↓Slc4a8,↓Slc22a3,↓Cacna1h,↓Scn8a,↓Grin2d,↓St eap1,↑Kcnc4,↓Slc9a5,↓Slc41a2,↓Slc22a21 199 Blood Vessel 64 0.0177103 ↓Esx1,↑Foxc2,↑Sox18 Development

255 200 Negative 24 0.0178722 ↓Enpp1,↑Wnt5a Regulation Of Fat Cell Differentiation 201 Vasculogenesis 66 0.019211 ↑Foxc2,↓Sgpl1,↑Sox18 202 Binding Of Sperm 25 0.0193178 ↓Zp3,↓Clgn To Zona Pellucida 203 Positive 25 0.0193178 ↓Pak1,↑Wnt5a Regulation Of JUN Kinase Activity 204 Synaptic Vesicle 25 0.0193178 ↑P2rx7,↓Apba1 Exocytosis 205 Calcium Ion- 25 0.0193178 ↓Rims2,↓Rims3 Dependent Exocytosis 206 Cellular Response 25 0.0193178 ↑Wnt5a,↑Gbp1 To Interferon- Gamma 207 Lung 121 0.0206052 ↓Rbp4,↑Fgfr2,↑Wnt5a,↑Adamts2 Development 208 Inner Ear 68 0.02078 ↓Nr4a3,↑Fgfr2,↑Wnt5a Morphogenesis 209 Peptidyl-Lysine 26 0.0208107 ↓Prdm9,↓1700106N22Rik Methylation 210 Antigen 26 0.0208107 ↓H2-Q10 Processing And Presentation Of Peptide Antigen Via MHC Class I 211 Peptide Hormone 26 0.0208107 ↓Tshb,↓Pcsk1n Processing 212 Positive 69 0.0215902 ↑P2rx7,↓Timp2,↓Itgb3 Regulation Of MAPKKK Cascade 213 Response To 27 0.0223501 ↓Rbp1,↓Tshb Vitamin A 214 Type I Interferon- 70 0.0224174 ↓Socs3,↓H2-Q10 Mediated Signaling Pathway 215 Intracellular 125 0.0228977 ↓Pak1,↓Prkag2,↓Socs3,↓Rps6ka6 Protein Kinase Cascade 216 Regulation Of 28 0.0239351 ↑Kcnc4,↓Syn2 Neurotransmitter Secretion 217 Tumor Necrosis 28 0.0239351 ↓Krt18,↓Krt8 Factor-Mediated Signaling Pathway 218 Establishment Or 3 0.0255183 ↓Lmna Maintenance Of Microtubule Cytoskeleton Polarity 219 Inhibition Of 3 0.0255183 ↓Itgb3 Adenylate Cyclase Activity By Opioid Receptor Signaling Pathway 220 Planar Cell 3 0.0255183 ↑Wnt5a Polarity Pathway Involved In Cardiac Right Atrium Morphogenesis 221 Planar Cell 3 0.0255183 ↑Wnt5a Polarity Pathway Involved In Cardiac Muscle

256 Tissue Morphogenesis 222 Planar Cell 3 0.0255183 ↑Wnt5a Polarity Pathway Involved In Pericardium Morphogenesis 223 Planar Cell 3 0.0255183 ↑Wnt5a Polarity Pathway Involved In Outflow Tract Morphogenesis 224 Planar Cell 3 0.0255183 ↑Wnt5a Polarity Pathway Involved In Ventricular Septum Morphogenesis 225 Positive 3 0.0255183 ↑Wnt5a Regulation Of Cgmp Metabolic Process 226 Regulation Of 3 0.0255183 ↓Necab3 Amyloid Precursor Protein Biosynthetic Process 227 Regulation Of 3 0.0255183 ↓Tex15 Double-Strand Break Repair Via Homologous Recombination 228 Negative 3 0.0255183 ↑Wnt5a Regulation Of Mesenchymal Cell Proliferation 229 Positive 3 0.0255183 ↑Tesc Regulation Of Megakaryocyte Differentiation 230 Positive 3 0.0255183 ↓Nr4a3 Regulation Of Leukocyte Apoptosis 231 Positive 3 0.0255183 ↑P2rx7 Regulation Of Interleukin-1 Alpha Secretion 232 Glomerular 3 0.0255183 ↑Foxc2 Visceral Epithelial Cell Differentiation 233 Bergmann Glial 3 0.0255183 ↑Gfap Cell Differentiation 234 Thiamine 3 0.0255183 ↓Tktl1 Metabolic Process 235 Antibiotic 3 0.0255183 ↓Necab3 Biosynthetic Process 236 Branched Chain 3 0.0255183 ↑Bcat1 Family Amino Acid Biosynthetic Process 237 Production Of 3 0.0255183 ↑Rbm3 Mirnas Involved In Gene Silencing By Mirna 238 Peptidyl-Cysteine 3 0.0255183 ↓Rab3b Methylation

257 239 Phagolysosome 3 0.0255183 ↑P2rx7 Assembly 240 Cell Migration In 3 0.0255183 ↓Pou4f1 Hindbrain 241 Ventricular Zone 3 0.0255183 ↑Fgfr2 Neuroblast Division 242 Golgi To Plasma 3 0.0255183 ↓Krt18 Membrane CFTR Protein Transport 243 Pore Complex 3 0.0255183 ↑P2rx7 Assembly 244 Negative 3 0.0255183 ↑Wnt5a Regulation Of Prostatic Bud Formation 245 Positive 3 0.0255183 ↓Hopx Regulation Of Skeletal Muscle Tissue Regeneration 246 Positive 3 0.0255183 ↑Foxc2 Regulation Of Vascular Wound Healing 247 Voluntary 3 0.0255183 ↓Hipk2 Musculoskeletal Movement 248 Mesenchymal Cell 3 0.0255183 ↑Fgfr2 Proliferation Involved In Lung Development 249 Detoxification Of 3 0.0255183 ↓Mt1 Copper Ion 250 Response To 3 0.0255183 ↓Krt8 Hydrostatic Pressure 251 Female Genitalia 3 0.0255183 ↓Rbp4 Morphogenesis 252 Development Of 3 0.0255183 ↑Wnt5a Primary Male Sexual Characteristics 253 Prostate Epithelial 3 0.0255183 ↑Fgfr2 Cord Elongation 254 Tube Closure 3 0.0255183 ↑Wnt5a 255 Bud Elongation 3 0.0255183 ↑Fgfr2 Involved In Lung Branching 256 Orbitofrontal 3 0.0255183 ↑Fgfr2 Cortex Development 257 Labyrinthine 3 0.0255183 ↓Esx1 Layer Morphogenesis 258 Inorganic 3 0.0255183 ↓Enpp1 Diphosphate Transport 259 Regulation Of 29 0.0255648 ↓H2-Q10,↓Hck Defense Response To Virus By Virus 260 Response To 193 0.0258508 ↑P2rx7,↓Socs3,↓Vldlr,↓S100a8,↓Abcc5 Lipopolysaccharid e 261 ATP Biosynthetic 74 0.0258971 ↓Prkag2,↓Atp11c,↓Atp13a3 Process 262 Cell Division 336 0.0265861 ↑Ube2c,↑E4f1,↓Sycp1,↓Sycp2,↓Sycp3,↓Rab11fip3,↓Syce1 263 Cellular Response 30 0.0272383 ↓Nr4a3,↓Pak1 To Stress 264 Neuromuscular 30 0.0272383 ↓Pak1,↑Fgfr2

258 Junction Development 265 Regulation Of 132 0.02727 ↓H2-Q10,↓Cd3e,↓Tnfrsf13c Immune Response 266 Interferon- 76 0.0277393 ↓Socs3,↓H2-Q10 Gamma-Mediated Signaling Pathway 267 Sex 31 0.0289548 ↓Dmrtb1,↓Dmrtc2 Differentiation 268 Ventricular 31 0.0289548 ↑Foxc2,↑Fgfr2 Cardiac Muscle Tissue Morphogenesis 269 Response To 78 0.0296496 ↑P2rx7,↓Trpc1,↓Tshb Calcium Ion 270 Response To 78 0.0296496 ↓Rbp4,↓Socs3,↓Abcc5 Insulin Stimulus 271 Heart 202 0.0306225 ↓Rbp4,↑Foxc2,↓Vldlr,↑Sox18,↓Hopx Development 272 Response To 138 0.0313883 ↓Nr4a3,↓Socs3,↓Braf,↓Pnliprp1 Peptide Hormone Stimulus 273 GTP Catabolic 204 0.031753 ↓Rab3b,↓Rab6b,↓Tuba4a,↓Eras,↑Gbp1 Process 274 Collagen Fibril 33 0.0325132 ↑Foxc2,↑Adamts2 Organization 275 Positive 33 0.0325132 ↓Aph1c,↑P2rx7 Regulation Of Catalytic Activity 276 Transcription 81 0.0326419 ↓Taf7l,↓4933416C03Rik,↓Taf4b Initiation From RNA Polymerase II Promoter 277 Homologous 4 0.0338794 ↓Msh5 Chromosome Segregation 278 Positive 4 0.0338794 ↑Wnt5a Regulation Of Protein Kinase C Signaling Cascade 279 Positive 4 0.0338794 ↓Gm98 Regulation Of Myelination 280 Negative 4 0.0338794 ↓Enpp1 Regulation Of Glycogen Biosynthetic Process 281 Negative 4 0.0338794 ↓Itgb3 Regulation Of Low-Density Lipoprotein Particle Receptor Biosynthetic Process 282 Negative 4 0.0338794 ↑Wnt5a Regulation Of Synaptogenesis 283 Positive 4 0.0338794 ↓Itgb3 Regulation Of Glomerular Mesangial Cell Proliferation 284 Spongiotrophoblas 4 0.0338794 ↓Socs3 t Differentiation 285 Paraxial 4 0.0338794 ↑Foxc2 Mesodermal Cell Fate Commitment 286 Cysteine 4 0.0338794 ↑Cth Biosynthetic

259 Process 287 Purine 4 0.0338794 ↓Entpd2 Ribonucleoside Diphosphate Catabolic Process 288 D-Gluconate 4 0.0338794 ↓Kif1b Metabolic Process 289 Very Long-Chain 4 0.0338794 ↓Slc27a2 Fatty Acid Catabolic Process 290 Phospholipid 4 0.0338794 ↑P2rx7 Translocation 291 Mitochondrial 4 0.0338794 ↓Aifm3 Depolarization 292 Chromatin 4 0.0338794 ↓D6mm5e Assembly 293 Positive 4 0.0338794 ↓Cd3e Regulation Of T Cell Anergy 294 Positive 4 0.0338794 ↑Wnt5a Regulation Of Response To Cytokine Stimulus 295 Endocardium 4 0.0338794 ↑Sox18 Formation 296 Lacrimal Gland 4 0.0338794 ↑Fgfr2 Development 297 Semicircular 4 0.0338794 ↓Nr4a3 Canal Morphogenesis 298 Lateral Sprouting 4 0.0338794 ↑Fgfr2 From An Epithelium 299 Outflow Tract 4 0.0338794 ↑Fgfr2 Septum Morphogenesis 300 Histamine 4 0.0338794 ↓Slc22a3 Transport 301 Regulation Of 4 0.0338794 ↓Slc22a3 Appetite 302 Negative 34 0.0343534 ↑Wnt5a,↓Hipk2 Regulation Of BMP Signaling Pathway 303 Adult Walking 34 0.0343534 ↓Scn8a,↓Hipk2 Behavior 304 Cellular Response 83 0.0347211 ↓Enpp1,↓Pak1,↓Vldlr To Insulin Stimulus 305 T Cell 84 0.0357858 ↓Pak1,↓Cd3e,↓Tnfrsf13c Costimulation 306 Response To 84 0.0357858 ↓Tfrc,↓Rbp4,↓Igfbp2 Retinoic Acid 307 Positive 35 0.0362333 ↓Nr4a3,↑Fgfr2 Regulation Of Cell Cycle 308 Positive 35 0.0362333 ↑P2rx7,↑Wnt5a Regulation Of Interleukin-6 Production 309 Bone 35 0.0362333 ↑Fgfr2,↓Pth1r Mineralization 310 Exocytosis 85 0.0368672 ↓Rims2,↑Stx3,↓Rims3 311 Gene Silencing By 36 0.0381519 ↓Eif2c3,↓Mael RNA 312 Response To Zinc 36 0.0381519 ↑P2rx7,↓S100a8 Ion 313 Biomineral Tissue 36 0.0381519 ↓Enpp1,↑Spp1 Development 314 Negative 443 0.0382748 ↓Zp3,↓Atf3,↑Wnt5a,↑Sox18,↑Fabp4,↑Tle4,↓Dkk3,↓Hipk2

260 Regulation Of Transcription, DNA-Dependent 315 Epithelial To 37 0.0401085 ↑Fgfr2,↑Wnt5a Mesenchymal Transition 316 Embryonic 37 0.0401085 ↓Rbp4,↑Wnt5a Skeletal System Development 317 ATP Catabolic 291 0.0402899 ↓Abcc5,↓Dmc1,↓Msh5,↓Kif1b,↓Atp11c,↓Atp13a3 Process 318 Neuron Projection 89 0.0413587 ↓Runx3,↑Stx3,↓Tbc1d24 Development 319 Insulin Receptor 151 0.0415021 ↓Crk,↑Foxc2,↑Fgfr2,↓Prkag2 Signaling Pathway 320 Cellular Response 38 0.0421024 ↓Vldlr,↓Lmna To Hypoxia 321 Protein Processing 38 0.0421024 ↓Aph1c,↑P2rx7 322 Positive 5 0.0421691 ↑Ube2c Regulation Of Exit From Mitosis 323 Resolution Of 5 0.0421691 ↓Tex11 Meiotic Recombination Intermediates 324 Regulation Of 5 0.0421691 ↑Gfap Neurotransmitter Uptake 325 Positive 5 0.0421691 ↑Wnt5a Regulation Of Type I Interferon- Mediated Signaling Pathway 326 Regulation Of 5 0.0421691 ↓Igfbp2 Insulin-Like Growth Factor Receptor Signaling Pathway 327 Regulation Of 5 0.0421691 ↑Agpat9 TOR Signaling Cascade 328 Adiponectin- 5 0.0421691 ↓Acsl1 Mediated Signaling Pathway 329 Negative 5 0.0421691 ↓Eif2c3 Regulation Of Translation Involved In Gene Silencing By Mirna 330 Negative 5 0.0421691 ↓Itgb3 Regulation Of Lipoprotein Metabolic Process 331 Activation Of 5 0.0421691 ↑Ube2c Anaphase- Promoting Complex Activity 332 Negative 5 0.0421691 ↓Rbp4 Regulation Of Cardiac Muscle Cell Proliferation 333 Positive 5 0.0421691 ↑Fgfr2 Regulation Of Epithelial Cell Proliferation Involved In Lung Morphogenesis 334 Positive 5 0.0421691 ↓Zp3 Regulation Of

261 Calcium Ion Import 335 Regulation Of 5 0.0421691 ↓Zp3 Calcium Ion Transport Via Store-Operated Calcium Channel Activity 336 Type B Pancreatic 5 0.0421691 ↑Wnt5a Cell Development 337 Mesodermal Cell 5 0.0421691 ↑Fgfr2 Differentiation 338 Neural Crest Cell 5 0.0421691 ↑Foxc2 Fate Commitment 339 Arginine 5 0.0421691 ↓Nags Biosynthetic Process 340 Glucose 1- 5 0.0421691 ↓Pgm2l1 Phosphate Metabolic Process 341 Pyrimidine Dimer 5 0.0421691 ↑Polh Repair 342 Cyclin Catabolic 5 0.0421691 ↑Ube2c Process 343 Membrane 5 0.0421691 ↑P2rx7 Budding 344 Very-Low-Density 5 0.0421691 ↓Vldlr Lipoprotein Particle Clearance 345 Bleb Assembly 5 0.0421691 ↑P2rx7 346 Regulation Of 5 0.0421691 ↑Wnt5a Branching Involved In Mammary Gland Duct Morphogenesis 347 Negative 5 0.0421691 ↓Cd83 Regulation Of Interleukin-4 Production 348 Positive 5 0.0421691 ↓Tnfrsf13c Regulation Of Germinal Center Formation 349 Epithelial Cell 5 0.0421691 ↑Wnt5a Proliferation Involved In Mammary Gland Duct Elongation 350 Response To Oleic 5 0.0421691 ↓Acsl1 Acid 351 Positive 5 0.0421691 ↑Foxc2 Regulation Of Integrin Activation 352 Prostate Gland 5 0.0421691 ↑Fgfr2 Morphogenesis 353 Male Genitalia 5 0.0421691 ↓Sycp2 Morphogenesis 354 Retina Layer 5 0.0421691 ↓Hipk2 Formation 355 Primitive Streak 5 0.0421691 ↑Wnt5a Formation 356 Paraxial 5 0.0421691 ↑Foxc2 Mesoderm Formation 357 Diaphragm 5 0.0421691 ↓Disp1 Development 358 Iris 5 0.0421691 ↓Hipk2 Morphogenesis 359 Hindgut 5 0.0421691 ↑Wnt5a

262 Morphogenesis 360 Membranous 5 0.0421691 ↑Fgfr2 Septum Morphogenesis 361 Positive 39 0.0441327 ↑Fgfr2,↑Wnt5a Regulation Of Mesenchymal Cell Proliferation 362 Response To Cold 39 0.0441327 ↑Rbm3,↓Pcsk1n 363 Regulation Of Cell 92 0.0448996 ↑Igfbp5,↓Socs3,↓Igfbp2 Growth 364 Positive 40 0.0461986 ↓Tbc1d24,↓Rabgap1l Regulation Of Rab Gtpase Activity 365 Regulation Of 40 0.0461986 ↑Fgfr2,↓Sgpl1 Multicellular Organism Growth 366 Negative 94 0.0473413 ↓Nr4a3,↓Braf,↓Hipk2 Regulation Of Neuron Apoptosis 367 Post-Embryonic 94 0.0473413 ↑Fgfr2,↓Sgpl1,↓Pnliprp1 Development 368 Positive 41 0.0482995 ↑Wnt5a,↓Hipk2 Regulation Of JNK Cascade 369 Embryo 159 0.0485404 ↓Rbp4,↑Wnt5a,↑E4f1,↓Kif1b Development 370 Meiotic Mismatch 6 0.0503879 ↓Msh5 Repair 371 Anterograde Axon 6 0.0503879 ↓Kif1b Cargo Transport 372 Mesenchymal- 6 0.0503879 ↑Wnt5a Epithelial Cell Signaling 373 Lens Induction In 6 0.0503879 ↓Hipk2 Camera-Type Eye 374 Negative 6 0.0503879 ↑Igfbp5 Regulation Of Insulin-Like Growth Factor Receptor Signaling Pathway 375 Positive 6 0.0503879 ↑P2rx7 Regulation Of Gamma- Aminobutyric Acid Secretion 376 ER-Nucleus 6 0.0503879 ↓Pak1 Signaling Pathway 377 Regulation Of 6 0.0503879 ↑Fgfr2 ERK1 And ERK2 Cascade 378 Regulation Of Rac 6 0.0503879 ↓Crk Protein Signal Transduction 379 Progesterone 6 0.0503879 ↓Klf9 Receptor Signaling Pathway 380 Positive 6 0.0503879 ↑P2rx7 Regulation Of Mitochondrial Depolarization 381 Positive 6 0.0503879 ↓Nes Regulation Of Neural Precursor Cell Proliferation 382 Positive 6 0.0503879 ↓Cd83 Regulation Of CD4-Positive, Alpha-Beta T Cell

263 Differentiation 383 Positive 6 0.0503879 ↑Foxc2 Regulation Of Cell Migration Involved In Sprouting Angiogenesis 384 Regulation Of Cell 6 0.0503879 ↑Fgfr2 Fate Commitment 385 Regulation Of 6 0.0503879 ↓Rbp1 Granulocyte Differentiation 386 Positive 6 0.0503879 ↓Zp3 Regulation Of Calcium Ion Transport Via Store-Operated Calcium Channel Activity 387 Pyramidal Neuron 6 0.0503879 ↑Fgfr2 Development 388 Dopaminergic 6 0.0503879 ↑Wnt5a Neuron Differentiation 389 Pentose-Phosphate 6 0.0503879 ↓Kif1b Shunt, Oxidative Branch 390 Sphingolipid 6 0.0503879 ↓Sgpl1 Catabolic Process 391 Cellular Phosphate 6 0.0503879 ↓Enpp1 Ion Homeostasis 392 Inner Ear Receptor 6 0.0503879 ↓Whrn Stereocilium Organization 393 Branching 6 0.0503879 ↑Fgfr2 Morphogenesis Of A Nerve 394 Mammary Gland 6 0.0503879 ↑Wnt5a Branching Involved In Thelarche 395 Positive 6 0.0503879 ↑Wnt5a Regulation Of Macrophage Cytokine Production 396 Positive 6 0.0503879 ↓Zp3 Regulation Of Humoral Immune Response 397 Innervation 6 0.0503879 ↓Pou4f1 398 Blastocyst 6 0.0503879 ↓Zp3 Formation 399 Axis Elongation 6 0.0503879 ↑Wnt5a 400 Face Development 6 0.0503879 ↑Wnt5a 401 Lung Lobe 6 0.0503879 ↑Fgfr2 Morphogenesis 402 Convergent 6 0.0503879 ↑Wnt5a Extension Involved In Organogenesis 403 Tongue 6 0.0503879 ↑Krt13 Morphogenesis 404 Carnitine 6 0.0503879 ↓Slc22a21 Transport 405 Sequestering Of 6 0.0503879 ↓Enpp1 Triglyceride 406 Response To 42 0.0504345 ↑P2rx7,↓Socs3 Bacterium 407 Neuron Projection 42 0.0504345 ↓Pak1,↓Brsk2

264 Morphogenesis 408 Response To DNA 309 0.0511881 ↓Eme2,↓Spdya,↓Rif1,↑Polh,↓Brsk2,↓Mael Damage Stimulus 409 Response To 234 0.0518672 ↑P2rx7,↓Acsl1,↓Socs3,↑Wnt5a,↓Cd83 Organic Cyclic Compound 410 Cell Maturation 43 0.0526029 ↓Pth1r,↓Runx3 411 Insulin Secretion 43 0.0526029 ↓Rims2,↓Fam3b 412 Heart Looping 43 0.0526029 ↑Wnt5a,↑Sox18 413 Synaptic 391 0.0527014 ↓Braf,↓Grin2d,↑Kcnc4,↓Apba1,↓Syn2,↓Rps6ka6,↓Nptx2 Transmission 414 Nervous System 474 0.0529927 ↓Vldlr,↓Nes,↓Scn8a,↑Gfra1,↑Gda,↓Pou4f1,↓Apba1,↓Sema4f Development 415 Activation Of JUN 44 0.0548041 ↓Crk,↑Wnt5a Kinase Activity 416 Metanephros 44 0.0548041 ↓Aph1c,↑Foxc2 Development 417 Induction Of 240 0.0566117 ↓Eaf2,↓Runx3,↑Lgals7,↓Plekhf1,↓Aifm3 Apoptosis 418 Germ Cell 45 0.0570372 ↓Mov10l1,↓Dazl Development 419 Negative 399 0.057495 ↓Timp2,↑Fgfr2,↓Pth1r,↑Cth,↓Nck2,↑Tesc,↓Gml Regulation Of Cell Proliferation 420 Response To Drug 484 0.0584215 ↑P2rx7,↓Timp2,↓Acsl1,↓Socs3,↓Igfbp2,↓Vldlr,↓Nes,↑Fabp4 421 Pachytene 7 0.0585364 ↓Dmc1 422 Regulation Of 7 0.0585364 ↑Fgfr2 Fibroblast Growth Factor Receptor Signaling Pathway 423 Negative 7 0.0585364 ↓H2-Q10 Regulation Of Natural Killer Cell Mediated Cytotoxicity 424 Positive 7 0.0585364 ↑Wnt5a Regulation Of Thymocyte Apoptosis 425 Positive 7 0.0585364 ↓Rbp4 Regulation Of Immunoglobulin Secretion 426 Glucose Catabolic 7 0.0585364 ↓Tktl1 Process 427 Fatty Acid Alpha- 7 0.0585364 ↓Slc27a2 Oxidation 428 Branched Chain 7 0.0585364 ↑Bcat1 Family Amino Acid Metabolic Process 429 Manga↓Nese Ion 7 0.0585364 ↓Zp3 Transmembrane Transport 430 Establishment Or 7 0.0585364 ↓Ooep Maintenance Of Apical-Basal Cell Polarity 431 Cellular Response 7 0.0585364 ↑Wnt5a To Molecule Of Bacterial Origin 432 Regulation Of 7 0.0585364 ↑Fgfr2 Branching Involved In Prostate Gland Morphogenesis 433 Prostate Epithelial 7 0.0585364 ↑Fgfr2 Cord Arborization Involved In Prostate Glandular

265 Acinus Morphogenesis 434 Positive 7 0.0585364 ↑P2rx7 Regulation Of Interleukin-1 Beta Production 435 Positive 7 0.0585364 ↑Wnt5a Regulation Of Cartilage Development 436 Saliva Secretion 7 0.0585364 ↓Trpc1 437 T Cell Tolerance 7 0.0585364 ↓H2-Q10 Induction 438 Antigen 7 0.0585364 ↓H2-Q10 Processing And Presentation Of Exogenous Peptide Antigen Via MHC Class I 439 Type B Pancreatic 7 0.0585364 ↑Igfbp5 Cell Proliferation 440 Epithelial Cell 7 0.0585364 ↑Fgfr2 Proliferation Involved In Salivary Gland Morphogenesis 441 Negative 7 0.0585364 ↓Itgb3 Regulation Of Lipid Transport 442 Positive 7 0.0585364 ↓Sec14l2 Regulation Of Cholesterol Biosynthetic Process 443 Genitalia 7 0.0585364 ↑Wnt5a Development 444 Limb Bud 7 0.0585364 ↑Fgfr2 Formation 445 Angiogenesis 7 0.0585364 ↓Itgb3 Involved In Wound Healing 446 Skeletal Muscle 7 0.0585364 ↑Igfbp5 Tissue Growth 447 Respiratory 7 0.0585364 ↑Wnt5a System Development 448 Integrin-Mediated 103 0.0591128 ↑Itga9,↓Itgb3,↑Adamts2 Signaling Pathway 449 Skeletal System 46 0.0593016 ↑P2rx7,↓Sgpl1 Morphogenesis 450 Gluconeogenesis 47 0.0615966 ↓Rbp4,↓Atf3 451 Regulation Of 48 0.0639215 ↓Cacna1h,↓Hopx Heart Contraction 452 Response To 249 0.0641817 ↓Prkag2,↓Igfbp2,↑Rbm3,↓Ndrg4,↓Hsf2bp Stress 453 Ossification 107 0.0647448 ↑Spp1,↑Foxc2,↓Pth1r 454 Response To 108 0.0661901 ↑Foxc2,↓Socs3,↓Vldlr Hormone Stimulus 455 Pathogenesis 8 0.0666153 ↑Tmem181 456 Adhesion To 8 0.0666153 ↑Gbp1 Symbiont 457 Exit From Mitosis 8 0.0666153 ↑Ube2c 458 Meiosis I 8 0.0666153 ↓Msh5 459 Negative 8 0.0666153 ↑Fgfr2 Regulation Of Mitosis 460 Female Meiosis 8 0.0666153 ↓Sycp2 461 Negative 8 0.0666153 ↓Eaf2 Regulation Of Epithelial Cell

266 Proliferation Involved In Prostate Gland Development 462 Positive 8 0.0666153 ↑P2rx7 Regulation Of Glutamate Secretion 463 Regulation Of 8 0.0666153 ↓Nck2 Epidermal Growth Factor Receptor Activity 464 Positive 8 0.0666153 ↓Rel Regulation Of Interleukin-12 Biosynthetic Process 465 Regulation Of 8 0.0666153 ↓Timp2 Camp Metabolic Process 466 Positive 8 0.0666153 ↓Itgb3 Regulation Of Fibroblast Migration 467 Positive 8 0.0666153 ↑Wnt5a Regulation Of T Cell Chemotaxis 468 Astrocyte 8 0.0666153 ↑Gfap Development 469 Megakaryocyte 8 0.0666153 ↑Tesc Differentiation 470 Gamma- 8 0.0666153 ↓Apba1 Aminobutyric Acid Secretion 471 Homocysteine 8 0.0666153 ↑Cth Metabolic Process 472 Glycolipid 8 0.0666153 ↓Ugt8 Biosynthetic Process 473 Carnitine Shuttle 8 0.0666153 ↓Prkag2 474 Response To 8 0.0666153 ↓Pcsk1n Dietary Excess 475 Regulation Of 8 0.0666153 ↓Itgb3 Bone Resorption 476 Maintenance Of 8 0.0666153 ↓Rbp4 Gastrointestinal Epithelium 477 Response To Fluid 8 0.0666153 ↑P2rx7 Shear Stress 478 Detection Of Light 8 0.0666153 ↓Rbp4 Stimulus Involved In Visual Perception 479 Regulation Of 8 0.0666153 ↓Prkag2 Glucose Import 480 Dorsal Spinal 8 0.0666153 ↓Pbx3 Cord Development 481 Embryonic 8 0.0666153 ↑Foxc2 Viscerocranium Morphogenesis 482 Convergent 8 0.0666153 ↑Wnt5a Extension 483 Dopamine 8 0.0666153 ↓Slc22a3 Transport 484 Regulation Of Ion 177 0.0666251 ↓Cacna1h,↓Scn8a,↓Grin2d,↑Kcnc4 Transmembrane Transport 485 Mitosis 252 0.0668255 ↓Hormad2,↑Ube2c,↑E4f1,↓Sycp3,↓Hormad1 486 Extracellular 109 0.0676502 ↓Spint1,↑Gfap,↓Npnt Matrix

267 Organization 487 Cytokine 50 0.0686583 ↓Rel,↑Fabp4 Production 488 DNA Replication 179 0.0688235 ↓Stra8,↑E4f1,↑Polh,↓Rad9b 489 Organ 180 0.0699366 ↑Fgfr2,↑Wnt5a,↓Braf,↓Sycp2 Morphogenesis 490 Positive 111 0.0706142 ↑P2rx7,↑Wnt5a,↓Itgb3 Regulation Of Protein Phosphorylation 491 Positive 51 0.0710688 ↓Zp3,↑Igfbp5 Regulation Of Protein Kinase B Signaling Cascade 492 Induction Of 51 0.0710688 ↓Eaf2,↓Hipk2 Apoptosis By Intracellular Signals 493 T Cell Activation 51 0.0710688 ↓Cd3e,↓Nck2 494 Cellular 51 0.0710688 ↓Dsp,↓Lmna Component Disassembly Involved In Apoptosis 495 Signal 3586 0.071519 ↓Trpc1,↓Zp3,↓Nr4a3,↓Enpp1,↑Igfbp5,↓Crk,↓Tfrc,↑Fgfr2,↓Pth1r, Transduction ↓Socs3,↓Igfbp2,↓Vldlr,↓Ptger1,↓Nmb,↑Wnt5a,↓Itgb3,↓Braf,↓Ra mp1,↑Ptprz1,↓Cd3e,↓Grin2d,↓Tnfrsf13c,↓Alcam,↑Gfra1,↓Cd83, ↓Crabp1,↓Nck2,↓Plekhb1,↓Sema4f,↑Rgmb,↓Radil,↓Rps6ka6,↓C tnnd2,↓Arrdc4,↓Eras,↓Caskin1,↓Disp1,↓Rassf10,↓Cds1 496 Axonogenesis 112 0.0721178 ↑Fgfr2,↑Ptprz1,↓Pou4f1 497 Cell Surface 259 0.0732273 ↑P2rx7,↓Pth1r,↓Vldlr,↓Cd3e,↑Gfra1 Receptor Linked Signaling Pathway 498 Positive 52 0.0735066 ↑Wnt5a,↓Itgb3 Regulation Of Endothelial Cell Proliferation 499 Somitogenesis 52 0.0735066 ↑Foxc2,↑Wnt5a 500 Palate 52 0.0735066 ↑Wnt5a,↓Sgpl1 Development 501 Mitochondrion 9 0.0746251 ↓Kif1b Transport Along Microtubule 502 Cellular Response 9 0.0746251 ↓Vldlr To Glucose Starvation 503 Regulation Of 9 0.0746251 ↓Crk Cellular Component Movement 504 Purinergic 9 0.0746251 ↑P2rx7 Nucleotide Receptor Signaling Pathway 505 Negative 9 0.0746251 ↑Wnt5a Regulation Of Axon Extension Involved In Axon Guidance 506 Negative 9 0.0746251 ↓Enpp1 Regulation Of Protein Autophosphorylati on 507 Regulation Of 9 0.0746251 ↓Prkag2 Fatty Acid Biosynthetic Process 508 Positive 9 0.0746251 ↓Timp2 Regulation Of

268 Adenylate Cyclase Activity 509 Positive 9 0.0746251 ↓Hopx Regulation Of Striated Muscle Cell Differentiation 510 Lens Fiber Cell 9 0.0746251 ↑Fgfr2 Development 511 Trophoblast Giant 9 0.0746251 ↓Socs3 Cell Differentiation 512 Cysteine 9 0.0746251 ↑Cth Metabolic Process 513 CDP-Choline 9 0.0746251 ↓Cds1 Pathway 514 Retinal Metabolic 9 0.0746251 ↓Rbp4 Process 515 Alternative 9 0.0746251 ↓Cdk13 Nuclear Mrna Splicing, Via Spliceosome 516 Postreplication 9 0.0746251 ↑Polh Repair 517 Nucleoside 9 0.0746251 ↓Enpp1 Triphosphate Catabolic Process 518 Plasma Membrane 9 0.0746251 ↑P2rx7 Organization 519 Nuclear Envelope 9 0.0746251 ↓Lmna Organization 520 Ameboidal Cell 9 0.0746251 ↑Wnt5a Migration 521 Cellular Sodium 9 0.0746251 ↑Tesc Ion Homeostasis 522 Cellular Zinc Ion 9 0.0746251 ↓Mt1 Homeostasis 523 Endocytic 9 0.0746251 ↓Rab11fip3 Recycling 524 Synaptic Vesicle 9 0.0746251 ↑Stx3 Docking Involved In Exocytosis 525 Branching 9 0.0746251 ↑Fgfr2 Involved In Prostate Gland Morphogenesis 526 Positive 9 0.0746251 ↓Cd83 Regulation Of Interleukin-2 Production 527 Detection Of 9 0.0746251 ↓Grin2d Mechanical Stimulus Involved In Sensory Perception Of Pain 528 Negative 9 0.0746251 ↓Enpp1 Regulation Of Glucose Import 529 Regulation Of 9 0.0746251 ↓Sec14l2 Cholesterol Biosynthetic Process 530 Prostate Gland 9 0.0746251 ↑Wnt5a Development 531 Lung-Associated 9 0.0746251 ↑Fgfr2 Mesenchyme Development 532 Gland 9 0.0746251 ↑Fgfr2 Morphogenesis 533 Epidermis 9 0.0746251 ↑Fgfr2

269 Morphogenesis 534 Quaternary 9 0.0746251 ↓Slc22a3 Ammonium Group Transport 535 Retina 9 0.0746251 ↓Whrn Homeostasis 536 Intracellular 261 0.075116 ↓Rims2,↓Zp3,↓Ramp1,↓Apba1,↑Stx3 Protein Transport 537 Negative 53 0.075971 ↓Runx3,↓Mov10l1 Regulation Of Cell Cycle 538 Myelination 53 0.075971 ↓Scn8a,↓Ugt8 539 Response To 53 0.075971 ↑Spp1,↓Igfbp2 Steroid Hormone Stimulus 540 Cell Proliferation 429 0.0778141 ↑Foxc2,↓Runx3,↑E4f1,↑Lgals7,↓Gfer,↑Bcat1,↓Mov10l1 541 Skin Development 54 0.0784613 ↓Dsp,↑Adamts2 542 Regulation Of Cell 55 0.080977 ↓Socs3,↓Plekhb1 Differentiation 543 Positive 55 0.080977 ↑Fgfr2,↓Itgb3 Regulation Of Smooth Muscle Cell Proliferation 544 Fatty Acid 118 0.0814362 ↓Acsl1,↑Fabp4,↓Slc27a2 Metabolic Process 545 Intermediate 10 0.0825664 ↑Gfap Filament-Based Process 546 Axon Cargo 10 0.0825664 ↓Apba1 Transport 547 Protein Kinase C 10 0.0825664 ↓Zp3 Signaling Cascade 548 Negative 10 0.0825664 ↓Cd55 Regulation Of Complement Activation 549 Positive 10 0.0825664 ↑Wnt5a Regulation Of Chemokine Biosynthetic Process 550 Regulation Of 10 0.0825664 ↑Polh DNA Repair 551 Negative 10 0.0825664 ↓Mbnl3 Regulation Of Myoblast Differentiation 552 Regulation Of Cell 10 0.0825664 ↑Tesc Adhesion Mediated By Integrin 553 Regulation Of 10 0.0825664 ↓Pou4f1 Neurogenesis 554 Ventricular 10 0.0825664 ↓Lmna Cardiac Muscle Cell Development 555 Cell 10 0.0825664 ↓Krt8 Differentiation Involved In Embryonic Placenta Development 556 DNA Synthesis 10 0.0825664 ↑Polh Involved In DNA Repair 557 Glycerol-3- 10 0.0825664 ↓Gpat2 Phosphate Metabolic Process 558 Pirna Metabolic 10 0.0825664 ↓Mael Process

270 559 DNA Catabolic 10 0.0825664 ↓Dnase1l2 Process 560 Histone 10 0.0825664 ↑Ctr9 Monoubiquitinatio n 561 Histone H2B 10 0.0825664 ↑Ctr9 Ubiquitination 562 Smooth Muscle 10 0.0825664 ↓Itgb3 Cell Migration 563 Platelet 10 0.0825664 ↓Itgb3 Aggregation 564 Central Nervous 10 0.0825664 ↓Gm98 System Myelination 565 Cell-Substrate 10 0.0825664 ↓Itgb3 Junction Assembly 566 Negative 10 0.0825664 ↑P2rx7 Regulation Of Bone Resorption 567 Mammary Gland 10 0.0825664 ↑Igfbp5 Involution 568 Adult Feeding 10 0.0825664 ↓Nmb Behavior 569 Hair Cycle 10 0.0825664 ↑Sox18 Process 570 Hemopoietic Stem 10 0.0825664 ↑Wnt5a Cell Proliferation 571 Response To UV- 10 0.0825664 ↑Polh C 572 Positive 10 0.0825664 ↑P2rx7 Regulation Of Prostaglandin Secretion 573 Otic Vesicle 10 0.0825664 ↑Fgfr2 Formation 574 Embryonic 10 0.0825664 ↓Nes Camera-Type Eye Development 575 Manganese Ion 10 0.0825664 ↓Zp3 Transport 576 Monoamine 10 0.0825664 ↓Slc22a3 Transport 577 Luteinization 10 0.0825664 ↓Sgpl1 578 Negative 56 0.0835175 ↓Prkag2,↑Fabp4 Regulation Of Protein Kinase Activity 579 Keratinocyte 56 0.0835175 ↓Dsp,↑Wnt5a Differentiation 580 Microtubule- 121 0.0862809 ↓Kif27,↓Kif1b,↓Tuba4a Based Movement 581 Negative 122 0.0879225 ↓Eaf2,↓Enpp1,↑Cth Regulation Of Cell Growth 582 Gamete 58 0.0886701 ↓Dmc1,↓Mei1 Generation 583 Mitotic Cell Cycle 11 0.0904398 ↑Bcat1 G1-S Transition DNA Damage Checkpoint 584 Negative 11 0.0904398 ↑Wnt5a Regulation Of Fibroblast Growth Factor Receptor Signaling Pathway 585 Negative 11 0.0904398 ↓Sema4f Regulation Of Axon Extension 586 Regulation Of 11 0.0904398 ↓Prkag2 Glycolysis

271 587 Regulation Of 11 0.0904398 ↑Igfbp5 Glucose Metabolic Process 588 Negative 11 0.0904398 ↑Igfbp5 Regulation Of Smooth Muscle Cell Migration 589 Regulation Of 11 0.0904398 ↑Fgfr2 Smooth Muscle Cell Differentiation 590 Neuron Projection 11 0.0904398 ↑Gfap Regeneration 591 Vasculogenesis 11 0.0904398 ↑Fgfr2 Involved In Coronary Vascular Morphogenesis 592 Urea Cycle 11 0.0904398 ↓Nags 593 Vitamin A 11 0.0904398 ↓Rbp1 Metabolic Process 594 Fibroblast 11 0.0904398 ↓Sgpl1 Migration 595 Neuron Cell-Cell 11 0.0904398 ↓Ctnnd2 Adhesion 596 Cellular Response 11 0.0904398 ↑Wnt5a To Calcium Ion 597 Regulation Of 11 0.0904398 ↑Fgfr2 Morphogenesis Of A Branching Structure 598 Collagen 11 0.0904398 ↑P2rx7 Metabolic Process 599 Heart Trabecula 11 0.0904398 ↓Rbp4 Formation 600 Anterior-Posterior 11 0.0904398 ↑Wnt5a Axis Specification, Embryo 601 Embryonic 11 0.0904398 ↓Hipk2 Camera-Type Eye Morphogenesis 602 Magnesium Ion 11 0.0904398 ↓Slc41a2 Transport 603 Response To 59 0.0912812 ↓Nes,↓Brsk2 Ionizing Radiation 604 Regulation Of Cell 60 0.0939146 ↓Itgb3,↓Lmna Migration 605 Muscle 126 0.0946192 ↑Actg2,↓Actn3,↓Cacna1h Contraction 606 Cell-Cell 281 0.0954323 ↑Fgfr2,↓Nmb,↑Wnt5a,↓Tshb,↓Sema4f Signaling 607 Osteoblast 61 0.0965697 ↑Igfbp5,↑Spp1 Differentiation 608 Positive 12 0.0982459 ↓Spdya Regulation Of Cyclin-Dependent Protein Kinase Activity 609 Neuron-Neuron 12 0.0982459 ↓Kif1b Synaptic Transmission 610 DNA Damage 12 0.0982459 ↓Hipk2 Response, Signal Transduction By P53 Class Mediator Resulting In Transcription Of P21 Class Mediator

272 611 Positive 12 0.0982459 ↑Sh3d19 Regulation Of Membrane Protein Ectodomain Proteolysis 612 Activation Of 12 0.0982459 ↑Wnt5a Protein Kinase B Activity 613 Positive 12 0.0982459 ↑Fgfr2 Regulation Of Cardiac Muscle Cell Proliferation 614 Positive 12 0.0982459 ↑P2rx7 Regulation Of Cytoskeleton Organization 615 Mesenchymal Cell 12 0.0982459 ↑Fgfr2 Differentiation 616 Lymphocyte 12 0.0982459 ↓Cd3e Activation 617 Synaptic Vesicle 12 0.0982459 ↑Fgfr2 Transport 618 Regulation Of 12 0.0982459 ↓Pbx3 Respiratory Gaseous Exchange By Neurological System Process 619 Negative Thymic 12 0.0982459 ↓Cd3e T Cell Selection 620 Response To 12 0.0982459 ↓Tfrc Manganese Ion 621 RNA Processing 129 0.099775 ↑Rbm3,↓D6mm5e,↓Adad1 622 Protein Complex 130 0.101518 ↓Clgn,↓Cd3e,↓Apba1 Assembly 623 Negative 63 0.101943 ↑Wnt5a,↓Dkk3 Regulation Of Canonical Wnt Receptor Signaling Pathway 624 Cell Growth 64 0.10466 ↑Fgfr2,↓Ndrg4 625 Positive 64 0.10466 ↑Fgfr2,↑Wnt5a Regulation Of Epithelial Cell Proliferation 626 Cell 64 0.10466 ↑P2rx7,↓Mael Morphogenesis 627 Cellular Response 64 0.10466 ↓Vldlr,↑Wnt5a To Lipopolysaccharid e 628 Elevation Of 132 0.105041 ↓Cd55,↓Pth1r,↓Nmb Cytosolic Calcium Ion Concentration 629 Suckling Behavior 13 0.105985 ↓Pou4f1 630 Positive 13 0.105985 ↑Igfbp5 Regulation Of Insulin-Like Growth Factor Receptor Signaling Pathway 631 Regulation Of 13 0.105985 ↓Prkag2 Fatty Acid Metabolic Process 632 Activation Of 13 0.105985 ↓Aifm3 Caspase Activity By Cytochrome C 633 Neural Crest Cell 13 0.105985 ↑Foxc2 Development 634 Morphogenesis Of 13 0.105985 ↓Ctnnd2 A Branching

273 Structure 635 Negative 13 0.105985 ↓Enpp1 Regulation Of Ossification 636 Startle Response 13 0.105985 ↓Grin2d 637 Tube 13 0.105985 ↓Itgb3 Development 638 Regulation Of Rab 66 0.110153 ↓Tbc1d24,↓Rabgap1l Gtpase Activity 639 Multicellular 66 0.110153 ↑Fgfr2,↓Apba1 Organism Growth 640 Negative 14 0.113659 ↓Itgb3 Regulation Of Macrophage Derived Foam Cell Differentiation 641 Positive 14 0.113659 ↓Cd3e Regulation Of Alpha-Beta T Cell Proliferation 642 Positive 14 0.113659 ↑Wnt5a Regulation Of Macrophage Activation 643 Regulation Of 14 0.113659 ↓Disp1 Protein Secretion 644 White Fat Cell 14 0.113659 ↑Fabp4 Differentiation 645 Glutathione 14 0.113659 ↑Hagh Biosynthetic Process 646 Glycosphingolipid 14 0.113659 ↓Ugt8 Biosynthetic Process 647 Blood Vessel 14 0.113659 ↑Sox18 Endothelial Cell Migration 648 Sensory 14 0.113659 ↓Whrn Perception Of Light Stimulus 649 Cardiac Muscle 14 0.113659 ↑Foxc2 Cell Proliferation 650 Response To 14 0.113659 ↓Gm3776/Gsta1/Gsta2 Stilbenoid 651 Regulation Of 14 0.113659 ↑P2rx7 Sodium Ion Transport 652 Midgut 14 0.113659 ↑Wnt5a Development 653 Peptide Transport 14 0.113659 ↓Disp1 654 Proteolysis 657 0.114364 ↓Nrip3,↑Fam111a,↓Tfrc,↓Anpep,↑Adamts2,↓Prss50,↑Adamts16, ↓Dpep3,↓Prss41 655 MAPKKK 68 0.11572 ↓Pak1,↓Braf Cascade 656 Regulation Of 68 0.11572 ↓4930447C04Rik,↑Foxc2 Sequence-Specific DNA Binding Transcription Factor Activity 657 Intracellular 388 0.118423 ↓Zp3,↑Igfbp5,↓Prkd3,↓Socs3,↓Braf,↓Plekhm1 Signal Transduction 658 Response To 15 0.121266 ↓Krt8 Other Organism 659 Leydig Cell 15 0.121266 ↓Sgpl1 Differentiation 660 Positive 15 0.121266 ↓Itgb3 Regulation Of Vascular

274 Endothelial Growth Factor Receptor Signaling Pathway 661 Positive 15 0.121266 ↓Cd3e Regulation Of Interleukin-2 Biosynthetic Process 662 Positive 15 0.121266 ↓Tnfrsf13c Regulation Of Interferon-Gamma Biosynthetic Process 663 3'- 15 0.121266 ↓Enpp1 Phosphoadenosine 5'-Phosphosulfate Metabolic Process 664 NADP Metabolic 15 0.121266 ↓Kif1b Process 665 Regulation Of 15 0.121266 ↓Plekhf1 Mitochondrial Membrane Permeability 666 Cellular Response 15 0.121266 ↑Gbp1 To Interferon-Beta 667 Humoral Immune 15 0.121266 ↓Zp3 Response Mediated By Circulating Immunoglobulin 668 Cochlea 15 0.121266 ↑Wnt5a Morphogenesis 669 Notch Signaling 70 0.121356 ↓Aph1c,↑Foxc2 Pathway 670 Positive 70 0.121356 ↓Itgb3,↓Cd3e Regulation Of Peptidyl-Tyrosine Phosphorylation 671 Cellular Lipid 141 0.121469 ↓Acsl1,↓Prkag2,↓Slc27a2 Metabolic Process 672 Response To 143 0.125242 ↓Socs3,↓Igfbp2,↑Wnt5a Estradiol Stimulus 673 Transcription 309 0.128041 ↑Tle4,↓Taf4b,↓Runx3,↓Pou4f1,↓Hsf2bp From RNA Polymerase II Promoter 674 Defense Response 16 0.128809 ↑Gbp1 To Protozoan 675 Mitotic 16 0.128809 ↓Rec8 Metaphase- Anaphase Transition 676 DNA Methylation 16 0.128809 ↓Mael Involved In Gamete Generation 677 Long-Term 16 0.128809 ↑Gfap Synaptic Potentiation 678 Wnt Receptor 16 0.128809 ↑Wnt5a Signaling Pathway, Planar Cell Polarity Pathway 679 Planar Cell 16 0.128809 ↑Wnt5a Polarity Pathway Involved In Neural Tube Closure 680 Positive 16 0.128809 ↑P2rx7

275 Regulation Of Calcium Ion Transport Into Cytosol 681 Positive 16 0.128809 ↓Trpc1 Regulation Of Release Of Sequestered Calcium Ion Into Cytosol 682 Trophectodermal 16 0.128809 ↓Hopx Cell Differentiation 683 UTP Biosynthetic 16 0.128809 ↓Rab11fip3 Process 684 Glutamate 16 0.128809 ↓Nags Metabolic Process 685 Estrogen 16 0.128809 ↓Sgpl1 Metabolic Process 686 Cellular Response 16 0.128809 ↑Fabp4 To Lithium Ion 687 Branching 16 0.128809 ↑Fgfr2 Involved In Salivary Gland Morphogenesis 688 Labyrinthine 16 0.128809 ↓Esx1 Layer Blood Vessel Development 689 Bone 16 0.128809 ↑Fgfr2 Morphogenesis 690 Arachidonic Acid 16 0.128809 ↓Nmb Secretion 691 Bicarbonate 16 0.128809 ↓Slc4a8 Transport 692 Glucose 73 0.129934 ↑Igfbp5,↓Rbp4 Homeostasis 693 Chromosome 75 0.135728 ↓Rec8,↓Stag3 Segregation 694 Myoblast Fusion 17 0.136287 ↓Cacna1h 695 Regulation Of 17 0.136287 ↑Fgfr2 Smoothened Signaling Pathway 696 Positive 17 0.136287 ↓Vldlr Regulation Of Protein Tyrosine Kinase Activity 697 GTP Biosynthetic 17 0.136287 ↓Rab11fip3 Process 698 CTP Biosynthetic 17 0.136287 ↓Rab11fip3 Process 699 Androgen 17 0.136287 ↓Sgpl1 Metabolic Process 700 Cell Volume 17 0.136287 ↑P2rx7 Homeostasis 701 B Cell 17 0.136287 ↓Tnfrsf13c Homeostasis 702 Chronic 17 0.136287 ↓S100a8 Inflammatory Response 703 Negative 17 0.136287 ↓Itgb3 Regulation Of Lipid Storage 704 Reproductive 17 0.136287 ↑Fgfr2 Structure Development 705 Cardiac Muscle 17 0.136287 ↓Rbp4 Tissue Development 706 Dorsal-Ventral 17 0.136287 ↑Wnt5a

276 Axis Specification 707 Digestive Tract 17 0.136287 ↑Wnt5a Morphogenesis 708 Establishment Of 17 0.136287 ↑Wnt5a Planar Polarity 709 Protein 17 0.136287 ↓Disp1 Homotrimerizatio n 710 Visual Perception 230 0.136594 ↓Rgs16,↓Whrn,↑Pitpnm3,↓Cds1 711 Calcium Ion 150 0.138768 ↑P2rx7,↓Trpc1,↓Cacna1h Transport 712 Phosphatidylinosit 77 0.141579 ↓Zp3,↑Ube2c ol-Mediated Signaling 713 Negative 18 0.143701 ↓Cd3e Regulation Of Smoothened Signaling Pathway 714 Negative 18 0.143701 ↓Nmb Regulation Of Hormone Secretion 715 SMAD Protein 18 0.143701 ↓Hipk2 Signal Transduction 716 Regulation Of 18 0.143701 ↓Socs3 Interferon- Gamma-Mediated Signaling Pathway 717 Regulation Of 18 0.143701 ↓Timp2 MAPKKK Cascade 718 Regulation Of 18 0.143701 ↓Mbnl3 RNA Splicing 719 Osteoblast 18 0.143701 ↓Pth1r Development 720 Central Nervous 18 0.143701 ↓Pou4f1 System Neuron Differentiation 721 Long-Chain Fatty- 18 0.143701 ↓Acsl1 Acyl-Coa Biosynthetic Process 722 Histone H3-K4 18 0.143701 ↓Prdm9 Methylation 723 Nucleus 18 0.143701 ↓Lmna Organization 724 Positive 18 0.143701 ↓Cd83 Regulation Of Interleukin-10 Production 725 Bone Remodeling 18 0.143701 ↓Enpp1 726 Negative 18 0.143701 ↑Wnt5a Chemotaxis 727 Outflow Tract 18 0.143701 ↑Sox18 Morphogenesis 728 Embryonic 18 0.143701 ↑Fgfr2 Digestive Tract Morphogenesis 729 Sodium Ion 154 0.14671 ↓Slc4a8,↓Scn8a,↓Slc9a5 Transport 730 Platelet 80 0.150454 ↓Itgb3,↓Tuba4a Degranulation 731 Virus-Host 19 0.151051 ↓Hipk2 Interaction 732 G2-M Transition 19 0.151051 ↓Brsk2 DNA Damage Checkpoint 733 Negative 19 0.151051 ↓Timp2 Regulation Of

277 Mitotic Cell Cycle 734 Negative 19 0.151051 ↑P2rx7 Regulation Of MAPKKK Cascade 735 Positive 19 0.151051 ↓Nmb Regulation Of Hormone Secretion 736 Negative 19 0.151051 ↓Prkag2 Regulation Of Protein Serine- Threonine Kinase Activity 737 Striated Muscle 19 0.151051 ↑Igfbp5 Cell Differentiation 738 Ceramide 19 0.151051 ↑P2rx7 Biosynthetic Process 739 Cellular Response 19 0.151051 ↑Bcat1 To UV 740 Response To ATP 19 0.151051 ↑P2rx7 741 Motor Axon 19 0.151051 ↓Alcam Guidance 742 Decidualization 19 0.151051 ↑Spp1 743 Tail 19 0.151051 ↑Wnt5a Morphogenesis 744 Artery 19 0.151051 ↑Foxc2 Morphogenesis 745 Response To 241 0.153832 ↓Pak1,↓Tfrc,↓Socs3,↓Vldlr Hypoxia 746 Anti-Apoptosis 243 0.157051 ↑Spp1,↑Foxc2,↓Socs3,↓Braf 747 In Utero 243 0.157051 ↑Fgfr2,↑Sox18,↓Apba1,↓Ooep Embryonic Development 748 Negative 20 0.158339 ↓Timp2 Regulation Of Ras Protein Signal Transduction 749 Nitric Oxide 20 0.158339 ↓Mt1 Mediated Signal Transduction 750 Positive 20 0.158339 ↑Wnt5a Regulation Of Cell-Cell Adhesion 751 Cell 20 0.158339 ↓Krt8 Morphogenesis Involved In Differentiation 752 Very Long-Chain 20 0.158339 ↓Slc27a2 Fatty Acid Metabolic Process 753 Receptor 20 0.158339 ↓Pak1 Clustering 754 Cytosolic Calcium 20 0.158339 ↓Trpc1 Ion Homeostasis 755 Cellular Response 20 0.158339 ↓Vldlr To Interleukin-1 756 Regulation Of 20 0.158339 ↓Enpp1 Bone Mineralization 757 Response To 20 0.158339 ↓Igfbp2 Lithium Ion 758 Retinal Ganglion 20 0.158339 ↓Sema4f Cell Axon Guidance 759 Bone 20 0.158339 ↑Fgfr2 Development

278 760 Face 20 0.158339 ↓Sgpl1 Morphogenesis 761 Endocytosis 160 0.158892 ↓Tfrc,↓Vldlr,↓Micall2 762 Response To 84 0.162451 ↑P2rx7,↓Igfbp2 Mechanical Stimulus 763 Positive 21 0.165565 ↑Fgfr2 Regulation Of Wnt Receptor Signaling Pathway 764 Positive 21 0.165565 ↑P2rx7 Regulation Of Protein Secretion 765 Positive 21 0.165565 ↓Itgb3 Regulation Of Osteoclast Differentiation 766 Regulation Of 21 0.165565 ↑Fgfr2 Epithelial Cell Proliferation 767 Purine Nucleotide 21 0.165565 ↑Gda Catabolic Process 768 Branched Chain 21 0.165565 ↑Bcat1 Family Amino Acid Catabolic Process 769 Long-Chain Fatty 21 0.165565 ↓Slc27a2 Acid Metabolic Process 770 Mrna Catabolic 21 0.165565 ↓Eif2c3 Process 771 Cellular Response 21 0.165565 ↑Wnt5a To Transforming Growth Factor Beta Stimulus 772 Epithelial Cell 21 0.165565 ↑Fgfr2 Proliferation 773 Male Genitalia 21 0.165565 ↓Tex15 Development 774 Morphogenesis Of 21 0.165565 ↑Fgfr2 Embryonic Epithelium 775 Membrane Protein 21 0.165565 ↑P2rx7 Ectodomain Proteolysis 776 Organ Growth 21 0.165565 ↑Fgfr2 777 Calcium Ion 86 0.168513 ↓Trpc1,↓Cacna1h Transmembrane Transport 778 Protein 166 0.171372 ↑Ptprz1,↑Ppm1k,↓Ptpn4 Dephosphorylatio n 779 Negative 166 0.171372 ↓Timp2,↓Spint1,↓Pcsk1n Regulation Of Endopeptidase Activity 780 Organ 87 0.171559 ↓Nr4a3,↓Socs3 Regeneration 781 Spindle 22 0.172728 ↑Ube2c Organization 782 Osteoclast 22 0.172728 ↓Tfrc Differentiation 783 Glutamate 22 0.172728 ↓Apba1 Secretion 784 Sarcomere 22 0.172728 ↓Krt8 Organization 785 Adult Behavior 22 0.172728 ↓Nr4a3 786 T Cell 22 0.172728 ↑P2rx7 Homeostasis 787 Response To Iron 22 0.172728 ↓Tfrc

279 Ion 788 Acute 22 0.172728 ↓S100a8 Inflammatory Response 789 Wnt Receptor 168 0.175593 ↑Wnt5a,↑Tle4,↓Dkk3 Signaling Pathway 790 Negative 89 0.177677 ↓Socs3,↓Rgs16 Regulation Of Signal Transduction 791 Cellular Response 23 0.179831 ↑P2rx7 To Extracellular Stimulus 792 Cellular Aromatic 23 0.179831 ↓Anpep Compound Metabolic Process 793 Glycerol 23 0.179831 ↓Gdpd1 Metabolic Process 794 Ceramide 23 0.179831 ↓Sgpl1 Metabolic Process 795 Embryonic Organ 23 0.179831 ↑Fgfr2 Development 796 Locomotory 91 0.183828 ↓Scn8a,↓Apba1 Behavior 797 Chloride Transport 91 0.183828 ↓Clca1,↓Clca2 798 Wnt Receptor 24 0.186873 ↑Wnt5a Signaling Pathway, Calcium Modulating Pathway 799 DNA Damage 24 0.186873 ↓Hipk2 Response, Signal Transduction By P53 Class Mediator Resulting In Induction Of Apoptosis 800 Camp-Mediated 24 0.186873 ↓Rims2 Signaling 801 Negative 24 0.186873 ↑Gfap Regulation Of Neuron Projection Development 802 Positive 24 0.186873 ↓Itgb3 Regulation Of Smooth Muscle Cell Migration 803 Triglyceride 24 0.186873 ↑Fabp4 Catabolic Process 804 Retrograde 24 0.186873 ↓Rab6b Vesicle-Mediated Transport, Golgi To ER 805 Focal Adhesion 24 0.186873 ↓Actn3 Assembly 806 Positive 24 0.186873 ↓Hipk2 Regulation Of DNA Binding 807 Hemopoiesis 92 0.186916 ↓Sgpl1,↓Cdk13 808 Regulation Of 263 0.19054 ↑P2rx7,↓Actn3,↓Lmna,↓Cd3e Apoptosis 809 T Cell Receptor 94 0.193112 ↓Pak1,↓Cd3e Signaling Pathway 810 Cellular Calcium 94 0.193112 ↓Pth1r,↓Itgb3 Ion Homeostasis 811 Defense Response 25 0.193854 ↓Cotl1 To Fungus 812 Positive 25 0.193854 ↓Cd3e Regulation Of

280 Calcium-Mediated Signaling 813 Vascular 25 0.193854 ↑Foxc2 Endothelial Growth Factor Receptor Signaling Pathway 814 Bile Acid 25 0.193854 ↓Slc27a2 Biosynthetic Process 815 Protein O-Linked 25 0.193854 ↓Galnt6 Glycosylation 816 Carboxylic Acid 25 0.193854 ↓Sgpl1 Metabolic Process 817 Retinoic Acid 25 0.193854 ↓Rbp1 Metabolic Process 818 Lamellipodium 25 0.193854 ↓Nck2 Assembly 819 Regulation Of 25 0.193854 ↑Foxc2 Blood Vessel Size 820 Response To Food 25 0.193854 ↓Socs3 821 Digestive Tract 25 0.193854 ↑Fgfr2 Development 822 Embryonic 25 0.193854 ↓Spint1 Placenta Development 823 Regulation Of Ph 25 0.193854 ↓Slc9a5 824 Regulation Of 26 0.200776 ↓Cdk13 Mitosis 825 Positive 26 0.200776 ↓Cd3e Regulation Of T Cell Activation 826 Positive 26 0.200776 ↑P2rx7 Regulation Of Cytokine Secretion 827 Oligodendrocyte 26 0.200776 ↓Gm98 Development 828 T Cell 26 0.200776 ↑P2rx7 Proliferation 829 Mismatch Repair 26 0.200776 ↓Msh5 830 Histone 26 0.200776 ↓Prdm9 Methylation 831 Positive 26 0.200776 ↑P2rx7 Regulation Of Bone Mineralization 832 Response To 26 0.200776 ↑Spp1 Vitamin D 833 Female Gonad 26 0.200776 ↓Sgpl1 Development 834 Blastocyst 26 0.200776 ↑Spic Development 835 Embryonic Cranial 26 0.200776 ↑Foxc2 Skeleton Morphogenesis 836 Bone Resorption 26 0.200776 ↓Pth1r 837 Iron Ion 26 0.200776 ↓Steap1 Homeostasis 838 Stem Cell 27 0.207638 ↓Rif1 Maintenance 839 Negative 27 0.207638 ↑Igfbp5 Regulation Of Smooth Muscle Cell Proliferation 840 Positive 27 0.207638 ↓Nck2 Regulation Of Actin Filament Polymerization 841 Regulation Of 27 0.207638 ↓Rab3b

281 Exocytosis 842 Chondrocyte 27 0.207638 ↓Pth1r Differentiation 843 Reactive Oxygen 27 0.207638 ↑P2rx7 Species Metabolic Process 844 Protein K11- 27 0.207638 ↑Ube2c Linked Ubiquitination 845 Cellular Response 27 0.207638 ↑Stx3 To Oxidative Stress 846 Collagen 27 0.207638 ↑Adamts2 Catabolic Process 847 Protein Metabolic 27 0.207638 ↓Necab3 Process 848 Activation Of 99 0.208711 ↑P2rx7,↑Wnt5a MAPK Activity 849 Cell-Matrix 100 0.211847 ↓Itgb3,↓Npnt Adhesion 850 Negative 28 0.214442 ↓Timp2 Regulation Of Proteolysis 851 Negative 28 0.214442 ↓Itgb3 Regulation Of Cell Death 852 Cell Fate 28 0.214442 ↓Atf3 Determination 853 Protein Secretion 28 0.214442 ↓Necab3 854 Membrane 28 0.214442 ↑P2rx7 Depolarization 855 Adrenal Gland 28 0.214442 ↓Dkk3 Development 856 Response To 102 0.218132 ↓Igfbp2,↓Tshb Estrogen Stimulus 857 Epidermis 102 0.218132 ↓Dsp,↑Krt13 Development 858 Positive 188 0.219227 ↓Rgs16,↓Tbc1d24,↓Rabgap1l Regulation Of Gtpase Activity 859 Small Gtpase 375 0.220978 ↓Pak1,↓Braf,↓Rab3b,↓Rab6b,↓Eras Mediated Signal Transduction 860 Negative 29 0.221188 ↑Igfbp5 Regulation Of Osteoblast Differentiation 861 Regulation Of 29 0.221188 ↓Pak1 Actin Cytoskeleton Organization 862 Release Of 29 0.221188 ↑P2rx7 Sequestered Calcium Ion Into Cytosol 863 Phosphate 29 0.221188 ↓Enpp1 Metabolic Process 864 Sodium Ion 29 0.221188 ↓Scn8a Transmembrane Transport 865 Heterophilic Cell- 29 0.221188 ↑Lgals7 Cell Adhesion 866 Protein Maturation 29 0.221188 ↑Adamts2 By Peptide Bond Cleavage 867 Regulation Of 30 0.227876 ↓Socs3 Type I Interferon- Mediated Signaling Pathway 868 Activation Of 30 0.227876 ↓Itgb3

282 Protein Kinase Activity 869 Peptide Cross- 30 0.227876 ↓Dsp Linking 870 Histone 30 0.227876 ↓Hopx Deacetylation 871 Response To 30 0.227876 ↓Tfrc Copper Ion 872 Mesoderm 30 0.227876 ↓Nr4a3 Formation 873 Sterol 30 0.227876 ↓Prkag2 Biosynthetic Process 874 Transferrin 30 0.227876 ↓Tfrc Transport 875 Regulation Of 31 0.234506 ↓Zfp287 Cytokine Biosynthetic Process 876 Phosphatidylinosit 31 0.234506 ↑Pitpnm3 ol Metabolic Process 877 Cytolysis 31 0.234506 ↑P2rx7 878 Response To 31 0.234506 ↑Wnt5a Hyperoxia 879 Response To 31 0.234506 ↑P2rx7 Electrical Stimulus 880 Positive 31 0.234506 ↑Wnt5a Regulation Of Protein Catabolic Process 881 Limb 31 0.234506 ↑Wnt5a Morphogenesis 882 Transmembrane 109 0.240243 ↓Cd3e,↑Gfra1 Receptor Protein Tyrosine Kinase Signaling Pathway 883 Synaptic 32 0.24108 ↓Grin2d Transmission, Glutamatergic 884 Platelet-Derived 32 0.24108 ↓Sgpl1 Growth Factor Receptor Signaling Pathway 885 Regulation Of 32 0.24108 ↓Grin2d Excitatory Postsynaptic Membrane Potential 886 Regulation Of 32 0.24108 ↓Grin2d Sensory Perception Of Pain 887 Cellular 32 0.24108 ↓Daam1 Component Organization 888 Angiogenesis 200 0.246384 ↑Fgfr2,↑Sox18,↓Anpep 889 Antigen 111 0.246582 ↓H2-Q10 Processing And Presentation 890 Regulation Of 33 0.247598 ↓Timp2 Neuron Differentiation 891 T Cell 33 0.247598 ↓Cd3e Differentiation In Thymus 892 Protein K48- 33 0.247598 ↑Ube2c Linked Ubiquitination 893 Organelle 33 0.247598 ↓Chga Organization

283 894 Vasculature 33 0.247598 ↑Sox18 Development 895 Kidney 112 0.249754 ↑Foxc2,↓Sgpl1 Development 896 Mitotic Cell Cycle 34 0.25406 ↑Ube2c Spindle Assembly Checkpoint 897 Regulation Of 34 0.25406 ↓Cacna1h Calcium Ion Transport Via Voltage-Gated Calcium Channel Activity 898 Cellular 34 0.25406 ↑Stx3 Membrane Fusion 899 Lens Development 34 0.25406 ↑Wnt5a In Camera-Type Eye 900 Midbrain 34 0.25406 ↑Fgfr2 Development 901 Phospholipid 34 0.25406 ↓Atp11c Transport 902 Negative 114 0.256102 ↓Expi,↓Spint1 Regulation Of Peptidase Activity 903 Negative 35 0.260467 ↓Prkag2 Regulation Of Phosphorylation 904 Brown Fat Cell 35 0.260467 ↑Fabp4 Differentiation 905 Histone Lysine 35 0.260467 ↓Prdm9 Methylation 906 Adherens Junction 35 0.260467 ↓Dsp Organization 907 Response To 35 0.260467 ↓Socs3 Gamma Radiation 908 Neural Tube 35 0.260467 ↑Wnt5a Development 909 Morphogenesis Of 35 0.260467 ↑Wnt5a An Epithelium 910 Response To 116 0.262452 ↓Timp2,↓Socs3 Cytokine Stimulus 911 Electron Transport 117 0.265628 ↓Steap1,↓Aifm3 Chain 912 Phototransduction 36 0.266818 ↓Plekhb1 913 Sperm Motility 36 0.266818 ↓Taf7l 914 Respiratory 36 0.266818 ↓Pbx3 Gaseous Exchange 915 Cellular Nitrogen 209 0.267081 ↓Nags,↓Tshb,↑Bcat1 Compound Metabolic Process 916 Cytoskeleton 118 0.268804 ↓Pak1,↓Krt8 Organization 917 Patterning Of 37 0.273116 ↑Foxc2 Blood Vessels 918 Neuromuscular 37 0.273116 ↓Nr4a3 Process Controlling Balance 919 Pancreas 37 0.273116 ↓Pnliprp1 Development 920 Eye Development 37 0.273116 ↓Rbp4 921 Nitrogen 37 0.273116 ↓Sgpl1 Compound Metabolic Process 922 Dephosphorylatio 212 0.274027 ↑Ptprz1,↓Ptpn4,↓Acpl2 n 923 Positive 38 0.27936 ↑Fgfr2 Regulation Of Canonical Wnt

284 Receptor Signaling Pathway 924 Positive 38 0.27936 ↓Tnfrsf13c Regulation Of B Cell Proliferation 925 Bile Acid 38 0.27936 ↓Slc27a2 Metabolic Process 926 De Novo' 39 0.28555 ↓Tuba4a Posttranslational Protein Folding 927 Blood Vessel 39 0.28555 ↑Foxc2 Remodeling 928 Positive 40 0.291687 ↓Rbp4 Regulation Of Insulin Secretion 929 Neuroprotection 40 0.291687 ↓Rel 930 Chromosome 40 0.291687 ↓Smc1b Organization 931 Odontogenesis 40 0.291687 ↑Fgfr2 932 Regulation Of 41 0.297772 ↓Ctnnd2 Synaptic Plasticity 933 JAK-STAT 41 0.297772 ↓Socs3 Cascade 934 Negative 41 0.297772 ↑Igfbp5 Regulation Of Translation 935 Regulation Of 41 0.297772 ↓Socs3 Protein Phosphorylation 936 Muscle Filament 41 0.297772 ↓Actn3 Sliding 937 Cellular Protein 41 0.297772 ↑Wnt5a Localization 938 Dendrite 41 0.297772 ↓Pak1 Development 939 Anion Transport 41 0.297772 ↓Slc4a8 940 Nerve Growth 224 0.301973 ↓Crk,↓Braf,↓Kidins220 Factor Receptor Signaling Pathway 941 Regulation Of Cell 42 0.303804 ↑Cytip Adhesion 942 Positive 42 0.303804 ↑Rbm3 Regulation Of Translation 943 Glycogen 42 0.303804 ↓Prkag2 Metabolic Process 944 Phagocytosis 42 0.303804 ↓Hck 945 Response To 42 0.303804 ↑Krt13 Radiation 946 Anterior-Posterior 130 0.306831 ↑Wnt5a,↓Hipk2 Pattern Formation 947 G-Protein 43 0.309785 ↓Pth1r Signaling, Coupled To Cyclic Nucleotide Second Messenger 948 Activation Of 43 0.309785 ↓Prkd3 Protein Kinase C Activity By G- Protein Coupled Receptor Protein Signaling Pathway 949 Positive 43 0.309785 ↑Wnt5a Regulation Of Fibroblast Proliferation 950 Purine Base 43 0.309785 ↑Gda Metabolic Process 951 Learning 43 0.309785 ↓Ctnnd2 952 Response To 43 0.309785 ↓Mtl5

285 Metal Ion 953 Embryonic Digit 43 0.309785 ↑Wnt5a Morphogenesis 954 Cytokine- 228 0.311326 ↓Socs3,↓H2-Q10 Mediated Signaling Pathway 955 Neuron 44 0.315715 ↓Pbx3 Development 956 Response To 44 0.315715 ↓Socs3 Progesterone Stimulus 957 Organic Anion 44 0.315715 ↓Abcc5 Transport 958 Neuropeptide 134 0.319437 ↓Nmb,↓Pcsk1n Signaling Pathway 959 Glucose Metabolic 134 0.319437 ↑Igfbp5,↓Pgm2l1 Process 960 Activation Of 45 0.321594 ↓Pth1r Phospholipase C Activity By G- Protein Coupled Receptor Protein Signaling Pathway Coupled To IP3 Second Messenger 961 Positive 45 0.321594 ↑Fgfr2 Regulation Of Cell Division 962 Post-Golgi 45 0.321594 ↑Sh3d19 Vesicle-Mediated Transport 963 Response To 45 0.321594 ↓Tfrc Inorganic Substance 964 Response To 45 0.321594 ↑Wnt5a Testosterone Stimulus 965 Acute-Phase 45 0.321594 ↓Tfrc Response 966 Response To 135 0.32258 ↓Rbp4,↓S100a8 Ethanol 967 Triglyceride 46 0.327423 ↓Acsl1 Metabolic Process 968 Synapse Assembly 46 0.327423 ↓Pou4f1 969 Branching 46 0.327423 ↓Npnt Involved In Ureteric Bud Morphogenesis 970 Cerebral Cortex 46 0.327423 ↓Vldlr Development 971 Protein 46 0.327423 ↑P2rx7 Oligomerization 972 DNA Repair 337 0.328154 ↓Eme2,↓Dmc1,↑Polh,↓Rad9b 973 Cellular Protein 47 0.333202 ↓Ooep Complex Assembly 974 Hair Follicle 47 0.333202 ↑Sox18 Development 975 Humoral Immune 47 0.333202 ↓Cd83 Response 976 Protein 48 0.338931 ↓Tuba4a Polymerization 977 Response To UV 48 0.338931 ↓Brsk2 978 Heart 48 0.338931 ↑Foxc2 Morphogenesis 979 Protein Folding 240 0.339412 ↓Ppil6,↓Clgn,↓Tuba4a 980 Cell Cycle 141 0.341357 ↑Ube2c,↓Rad9b Checkpoint 981 Cell Migration 141 0.341357 ↑Wnt5a,↓Nck2 982 Platelet Activation 242 0.34409 ↓Entpd2,↓Itgb3,↓Tuba4a

286 983 Negative 49 0.344611 ↓Dkk3 Regulation Of Wnt Receptor Signaling Pathway 984 I-Kappab Kinase- 49 0.344611 ↓Rel NF-Kappab Cascade 985 Cilium Assembly 49 0.344611 ↓Kif27 986 Vesicle-Mediated 244 0.348765 ↓Rab6b,↓Rab11fip3,↑Stx3 Transport 987 Cellular Response 50 0.350243 ↓Igfbp2 To Hormone Stimulus 988 Mitochondrion 50 0.350243 ↑P2rx7 Organization 989 Hippocampus 50 0.350243 ↓Nr4a3 Development 990 Intracellular 50 0.350243 ↑Stx3 Transport 991 Methylation 144 0.350686 ↓Prdm9,↓1700106N22Rik 992 Activation Of 51 0.355827 ↓Pth1r Adenylate Cyclase Activity By G- Protein Signaling Pathway 993 Memory 51 0.355827 ↓Vldlr 994 Protein 51 0.355827 ↑Cth Homotetramerizati on 995 Positive 52 0.361362 ↓Socs3 Regulation Of Cell Differentiation 996 Determination Of 53 0.366851 ↓Disp1 Left-Right Symmetry 997 Viral 362 0.376658 ↓H2-Q10,↓Hck,↓Taf4b,↓Zfp287 Reproduction 998 Steroid Hormone 55 0.377687 ↓Nr4a3 Mediated Signaling Pathway 999 Negative 55 0.377687 ↓Hopx Regulation Of Cell Differentiation 1000 Negative 55 0.377687 ↓Mael Regulation Of Gene Expression 1001 G1-S Transition 154 0.381434 ↓Spdya,↑Bcat1 Of Mitotic Cell Cycle 1002 Microtubule- 57 0.388338 ↓Tuba4a Based Process 1003 Regulation Of 57 0.388338 ↓Kidins220 Phosphorylation 1004 Sphingolipid 57 0.388338 ↓Sgpl1 Metabolic Process 1005 Sensory 58 0.393595 ↑P2rx7 Perception Of Pain 1006 Dorsal-Ventral 58 0.393595 ↓Disp1 Pattern Formation 1007 Water-Soluble 58 0.393595 ↓Enpp1 Vitamin Metabolic Process 1008 Actin Filament 59 0.398807 ↓Nck2 Organization 1009 Complement 59 0.398807 ↓Cd55 Activation, Classical Pathway 1010 Keratinization 59 0.398807 ↑Tchh 1011 Skeletal System 160 0.399587 ↑Foxc2,↓Pth1r Development

287 1012 60 0.403975 ↓Rab11fip3 1013 BMP Signaling 60 0.403975 ↑Rgmb Pathway 1014 Positive 60 0.403975 ↓Timp2 Regulation Of Neuron Differentiation 1015 Embryonic 60 0.403975 ↑Foxc2 Skeletal System Morphogenesis 1016 Regulation Of 61 0.409099 ↓Nr4a3 Transcription From RNA Polymerase II Promoter By Nuclear Hormone Receptor 1017 Cholesterol 61 0.409099 ↑Fabp4 Homeostasis 1018 Protein Transport 602 0.413303 ↓Ramp1,↓Rab3b,↓Rab6b,↓Apba1,↓Rab11fip3,↑Zg16 1019 DNA Damage 62 0.414178 ↓Gml Response, Signal Transduction By P53 Class Mediator Resulting In Cell Cycle Arrest 1020 JNK Cascade 62 0.414178 ↑Wnt5a 1021 Peptidyl-Serine 62 0.414178 ↓Brsk2 Phosphorylation 1022 Neural Tube 62 0.414178 ↑Wnt5a Closure 1023 Vitamin Metabolic 63 0.419214 ↓Enpp1 Process 1024 Cellular Response 64 0.424207 ↑Fgfr2 To Protein Stimulus 1025 Fibroblast Growth 65 0.429157 ↑Fgfr2 Factor Receptor Signaling Pathway 1026 Epithelial Cell 65 0.429157 ↑Fgfr2 Differentiation 1027 Camera-Type Eye 65 0.429157 ↑Foxc2 Development 1028 Retina 67 0.438931 ↓Rbp4 Development In Camera-Type Eye 1029 Generation Of 68 0.443755 ↓Enpp1 Precursor Metabolites And Energy 1030 Amino Acid 69 0.448537 ↓Slc22a21 Transmembrane Transport 1031 Digestion 69 0.448537 ↓Acsl1 1032 Positive 69 0.448537 ↑Wnt5a Regulation Of Angiogenesis 1033 Embryonic Limb 69 0.448537 ↑Wnt5a Morphogenesis 1034 Iron Ion Transport 69 0.448537 ↓Steap1 1035 Cell Fate 70 0.453279 ↑Fgfr2 Commitment 1036 Transcription 70 0.453279 ↓Taf4b Elongation From RNA Polymerase II Promoter 1037 Potassium Ion 179 0.455319 ↑Kctd14,↑Kcnc4 Transport 1038 Negative 71 0.45798 ↑Ube2c

288 Regulation Of Ubiquitin-Protein Ligase Activity Involved In Mitotic Cell Cycle 1039 Positive 72 0.462641 ↓Crk Regulation Of Signal Transduction 1040 Response To 72 0.462641 ↓Nes Nutrient Levels 1041 Hormone 72 0.462641 ↓Tshb Biosynthetic Process 1042 Circadian Rhythm 72 0.462641 ↓Egr3 1043 Negative 73 0.467262 ↑Igfbp5 Regulation Of Cell Migration 1044 Neurotransmitter 73 0.467262 ↓Syn2 Secretion 1045 Response To 74 0.471843 ↓Braf Camp 1046 Actin 186 0.4751 ↓Crk,↓Daam1 Cytoskeleton Organization 1047 Translational 75 0.476386 ↓Eif2c3 Initiation 1048 Regulation Of 75 0.476386 ↓Chga Blood Pressure 1049 Response To Heat 76 0.480889 ↓Socs3 1050 Regulation Of 77 0.485353 ↑Ube2c Ubiquitin-Protein Ligase Activity Involved In Mitotic Cell Cycle 1051 Cellular Amino 77 0.485353 ↑Cth Acid Metabolic Process 1052 Positive 78 0.48978 ↑Ube2c Regulation Of Ubiquitin-Protein Ligase Activity Involved In Mitotic Cell Cycle 1053 Response To 78 0.48978 ↑P2rx7 Protein Stimulus 1054 Cell Redox 80 0.498519 ↓Aifm3 Homeostasis 1055 Cholesterol 81 0.502832 ↓Vldlr Metabolic Process 1056 Epidermal Growth 82 0.507109 ↓Nck2 Factor Receptor Signaling Pathway 1057 Positive 82 0.507109 ↑Wnt5a Regulation Of NF- Kappab Transcription Factor Activity 1058 Mitotic Cell Cycle 316 0.510713 ↓Rec8,↑Ube2c,↓Tuba4a 1059 Inflammatory 316 0.510713 ↑P2rx7,↑Spp1,↓S100a8 Response 1060 Nucleobase, 83 0.511349 ↑Gda Nucleoside And Nucleotide Metabolic Process 1061 Cartilage 83 0.511349 ↑Wnt5a Development 1062 Canonical Wnt 84 0.515553 ↑Wnt5a Receptor Signaling Pathway

289 1063 Response To 86 0.523852 ↑Gfap Wounding 1064 Transforming 88 0.53201 ↓Hipk2 Growth Factor Beta Receptor Signaling Pathway 1065 Nucleobase, 88 0.53201 ↑Gda Nucleoside, Nucleotide And Nucleic Acid Metabolic Process 1066 DNA 88 0.53201 ↓Eme2 Recombination 1067 Fatty Acid 89 0.536036 ↓Prkag2 Biosynthetic Process 1068 Mitotic 90 0.540028 ↓Rec8 Prometaphase 1069 Peptidyl-Tyrosine 90 0.540028 ↓Ptpn4 Dephosphorylatio n 1070 Anaphase- 90 0.540028 ↑Ube2c Promoting Complex- Dependent Proteasomal Ubiquitin- Dependent Protein Catabolic Process 1071 Protein 94 0.555657 ↓Braf Heterooligomeriza tion 1072 Ubiquitin- 217 0.557218 ↓Usp43,↑Ube2c Dependent Protein Catabolic Process 1073 M Phase Of 96 0.563272 ↓Rec8 Mitotic Cell Cycle 1074 Regulation Of 99 0.574451 ↓Apba1 Gene Expression 1075 Regulation Of 351 0.581601 ↓Crk,↓Klf9,↓Pou4f1 Transcription From RNA Polymerase II Promoter 1076 Cellular Iron Ion 101 0.581745 ↓Tfrc Homeostasis 1077 Positive 232 0.593559 ↑P2rx7,↓Pou4f1 Regulation Of Apoptosis 1078 Proton Transport 110 0.613056 ↓Slc9a5 1079 Lipid Transport 110 0.613056 ↓Vldlr 1080 Carbohydrate 369 0.615443 ↓Kif1b,↑Hagh,↓Pgm2l1 Metabolic Process 1081 Response To 111 0.616388 ↓Scn8a Toxin 1082 Energy Reserve 112 0.619691 ↓Prkag2 Metabolic Process 1083 Positive 112 0.619691 ↑Tesc Regulation Of Gene Expression 1084 Brain 245 0.623223 ↓Nes,↓Pcsk1n Development 1085 Pattern 115 0.629431 ↓Disp1 Specification Process 1086 G2-M Transition 116 0.632622 ↓Tuba4a Of Mitotic Cell Cycle 1087 Cellular 118 0.638922 ↑Sh3d19 Membrane

290 Organization 1088 Induction Of 118 0.638922 ↓Cd3e Apoptosis By Extracellular Signals 1089 Protein 118 0.638922 ↓Pak1 Autophosphorylati on 1090 Steroid Metabolic 120 0.645115 ↓Vldlr Process 1091 Leukocyte 121 0.648172 ↓Itgb3 Migration 1092 Female Pregnancy 123 0.654206 ↓Igfbp2 1093 Sensory 126 0.663066 ↓Whrn Perception Of Sound 1094 Lipid Biosynthetic 128 0.668846 ↓Prkag2 Process 1095 Rrna Processing 131 0.677331 ↓Utp18 1096 Gene Expression 415 0.693291 ↑P2rx7,↓Nr4a3,↓Taf4b 1097 Potassium Ion 143 0.709161 ↑Kcnc4 Transmembrane Transport 1098 Cell-Cell 143 0.709161 ↓Dsp Adhesion 1099 Cell Cycle Arrest 148 0.721482 ↓Prkag2 1100 Positive 149 0.723883 ↓Rel Regulation Of I- Kappab Kinase- NF-Kappab Cascade 1101 Xenobiotic 152 0.730963 ↓Gm3776/Gsta1/Gsta2 Metabolic Process 1102 Chemotaxis 153 0.733283 ↓S100a8 1103 Cell Death 161 0.751136 ↑P2rx7 1104 Homophilic Cell 168 0.765778 ↑Mpzl2 Adhesion 1105 RNA Splicing 323 0.76689 ↓Rbmy1a1,↓Mbnl3 1106 Mrna Processing 331 0.778558 ↓Rbmy1a1,↓Mbnl3 1107 Blood Coagulation 477 0.778647 ↓Crk,↓Itgb3,↓Tuba4a 1108 Regulation Of Cell 180 0.78891 ↑Fgfr2 Proliferation 1109 Post-Translational 187 0.801337 ↑Ube2c Protein Modification 1110 Immune Response 523 0.828561 ↓Enpp1,↓H2-Q10 1111 Defense Response 204 0.828567 ↓Cd83 1112 Defense Response 205 0.830047 ↓H2-Q10 To Bacterium 1113 Protein 220 0.850787 ↑Ube2c Ubiquitination 1114 Chromatin 262 0.89639 ↓Prdm9 Modification 1115 Innate Immune 285 0.915168 ↓Cd55 Response 1116 Cellular Protein 302 0.92683 ↓Tuba4a Metabolic Process 1117 Response To 748 0.956684 ↓Rbp4,↓Whrn,↑Pitpnm3 Stimulus 1118 Oxidation- 840 0.976427 ↓Steap1,↓Gfer,↓Aifm3 Reduction Process 1119 Translation 814 0.99326 ↑Rbm3,↓Rab11fip3 1120 G-Protein Coupled 2183 0.999568 ↓Entpd2,↓Pth1r,↓Ptger1,↓Ramp1,↓Cd3e,↓Rgs16,↓Tshb Receptor Protein Signaling Pathway

291

Table A1-5. Total gene list for ± 2.0 or higher fold change of RARA regulated genes in P4 germ cells.

Gene Name Fold Gene Symbol Gene Title Change 1415846_a_at 2.280 Ldhc Lactate Dehydrogenase C 1415924_at 2.110 Tnp1 Transition Protein 1 1416034_at 3.070 Cd24a CD24a Antigen 1416123_at -1.976 Ccnd2 Cyclin D2 1416178_a_at -2.196 Plekhb1 Pleckstrin Homology Domain Containing, Family B (Evectins) Member 1 1416246_a_at 2.879 Coro1a Coronin, Actin Binding Protein 1A 1416295_a_at 2.026 Il2rg Interleukin 2 Receptor, Gamma Chain 1416405_at 2.033 Bgn Biglycan 1416431_at 2.152 Tubb6 , Beta 6 1416454_s_at 2.202 Acta2 Actin, Alpha 2, Smooth Muscle, Aorta 1416473_a_at 1.980 Igdcc4 Immunoglobulin Superfamily, DCC Subclass, Member 4 1416521_at 1.970 Sepw1 Selenoprotein W, Muscle 1 1416594_at 1.990 Sfrp1 Secreted Frizzled-Related Protein 1 1416612_at 2.586 Cyp1b1 Cytochrome P450, Family 1, Subfamily B, Polypeptide 1 1416625_at 2.338 Serping1 Serine (Or Cysteine) Peptidase Inhibitor, Clade G, Member 1 1416658_at -3.262 Frzb Frizzled-Related Protein 1416693_at 2.334 Foxc2 Forkhead Box C2

292 1416846_a_at 1.982 Pdzrn3 PDZ Domain Containing RING Finger 3 1417017_at 2.389 Cyp17a1 Cytochrome P450, Family 17, Subfamily A, Polypeptide 1 1417076_at 2.012 Fabp9 Fatty Acid Binding Protein 9, Testis 1417078_at 2.415 Lgals2 Lectin, Galactose-Binding, Soluble 2 1417079_s_at 3.258 Lgals2 Lectin, Galactose-Binding, Soluble 2 1417150_at 3.546 Slc6a4 Solute Carrier Family 6 (Neurotransmitter Transporter, Serotonin), Member 4 1417184_s_at 2.074 Hbb-b1 /// Hbb-b2 /// LOC100503605 Hemoglobin, Beta Adult Major Chain /// Hemoglobin, Beta Adult Minor Chain /// Hemoglobin Subunit Beta- 1-Like 1417439_at 1.964 Cd248 CD248 Antigen, Endosialin 1417447_at 2.190 Tcf21 Transcription Factor 21 1417552_at 2.183 Fap Fibroblast Activation Protein 1417600_at -2.258 Slc15a2 Solute Carrier Family 15 (H+/Peptide Transporter), Member 2 1417741_at 2.086 Pygl Liver Glycogen Phosphorylase 1417756_a_at 2.590 Lsp1 Lymphocyte Specific 1 1417932_at 2.497 Il18 Interleukin 18 1417933_at 2.029 Igfbp6 Insulin-Like Growth Factor Binding Protein 6 1417945_at 2.340 Oct3/4 POU Domain, Class 5, Transcription Factor 1 1417946_at -1.970 Abhd3 Abhydrolase Domain Containing 3 1418069_at 3.174 Apoc2 Apolipoprotein C-II 1418090_at 2.485 Plvap Plasmalemma Vesicle Associated Protein 1418147_at 2.138 Tfap2c Transcription Factor AP-2, Gamma 1418156_at 2.074 Kcne4 Potassium Voltage-Gated Channel, Isk-Related Subfamily, Gene 4 1418259_a_at 2.156 Entpd2 Ectonucleoside Triphosphate Diphosphohydrolase 2 1418392_a_at 3.255 Gbp3 Guanylate Binding Protein 3 1418396_at 2.069 Gpsm3 G-Protein Signalling Modulator 3 (AGS3-Like, C. Elegans) 1418450_at 2.440 Islr Immunoglobulin Superfamily Containing Leucine-Rich Repeat 1418536_at 2.037 H2-Q7 Histocompatibility 2, Q Region Locus 7

1418538_at 2.004 Kdelr3 KDEL (Lys-Asp-Glu-Leu) Endoplasmic Reticulum Protein Retention Receptor 3 1418606_at 2.561 Hoxd10 Homeobox D10 1418677_at 2.495 Actn3 Alpha 3 1418726_a_at 3.506 Tnnt2 Troponin T2, Cardiac 1418753_at 2.220 Gfpt2 Glutamine Fructose-6-Phosphate Transaminase 2 1418787_at 2.002 Mbl2 Mannose-Binding Lectin (Protein C) 2 1418805_at 2.160 Sct Secretin 1418910_at 2.688 Bmp7 Bone Morphogenetic Protein 7 1418990_at 2.471 Ms4a4d Membrane-Spanning 4-Domains, Subfamily A, Member 4D 1419126_at 2.286 Hoxd9 Homeobox D9 1419184_a_at 2.232 Fhl2 Four And A Half LIM Domains 2 1419212_at 1.980 Icosl Icos Ligand 1419215_at 2.262 Aox4 Aldehyde Oxidase 4 1419231_s_at 2.213 Krt12 12 1419292_at 2.147 Htra3 Htra Serine Peptidase 3 1419324_at 2.307 Lhx9 LIM Homeobox Protein 9 1419663_at 3.202 Ogn Osteoglycin 1419738_a_at 2.343 Tpm2 2, Beta 1419810_x_at 2.509 Arhgap9 Rho Gtpase Activating Protein 9 1420408_a_at 2.781 Abcc9 ATP-Binding Cassette, Sub-Family C (CFTR/MRP), Member 9 1420411_a_at 10.200 Pi4k2b Phosphatidylinositol 4-Kinase Type 2 Beta 1420453_at 2.104 Crygs Crystallin, Gamma S 1421078_at 2.158 Tcf23 Transcription Factor 23 1421183_at 2.066 Tex12 Testis Expressed Gene 12 293 1421277_at -2.568 Spna1 Alpha 1 1421375_a_at 2.517 S100a6 S100 Calcium Binding Protein A6 (Calcyclin) 1421433_at -1.966 Zfhx4 Zinc Finger Homeodomain 4 1421594_a_at 2.307 Sytl2 Synaptotagmin-Like 2 1421865_at 2.715 Dbil5 Diazepam Binding Inhibitor-Like 5 1421951_at 2.359 Lhx1 LIM Homeobox Protein 1 1422340_a_at 2.877 Actg2 Actin, Gamma 2, Smooth Muscle, Enteric 1422400_a_at 2.110 Gml /// Hemt1 GPI Anchored Molecule Like Protein /// Hematopoietic Cell Transcript 1 1422437_at 2.129 Col5a2 Collagen, Type V, Alpha 2 1422529_s_at 2.054 Casq2 Calsequestrin 2 1422545_at 2.095 Tbx2 T-Box 2 1422606_at 3.053 C1qtnf3 C1q And Tumor Necrosis Factor Related Protein 3 1422617_at 2.379 Gm10058 /// Gm10230 /// Gm10486 /// Gm10488 /// Predicted Gene 10058 /// Predicted Gene 10230 /// Predicted Gene 10486 /// Predicted Gene 10488 /// Gm14525 /// Gm14632 /// Gm14819 /// Gm2012 /// Predicted Gene 14525 /// Predicted Gene 14632 /// Predicted Gene 14819 /// Predicted Gene 2012 /// Predicted Gm2030 /// Gm4297 /// Gm5169 /// Gm5934 /// Gene 2030 /// Predicted Gene 4297 /// Predicted Gene 5169 /// Predicted Gene 5934 /// Predicted Gene 6121 Gm6121 1422618_x_at 2.279 Gm10058 /// Gm10230 /// Gm10486 /// Gm10488 /// Predicted Gene 10058 /// Predicted Gene 10230 /// Predicted Gene 10486 /// Predicted Gene 10488 /// Gm14525 /// Gm14632 /// Gm14819 /// Gm2012 /// Predicted Gene 14525 /// Predicted Gene 14632 /// Predicted Gene 14819 /// Predicted Gene 2012 /// Predicted Gm2030 /// Gm4297 /// Gm5169 /// Gm5934 /// Gene 2030 /// Predicted Gene 4297 /// Predicted Gene 5169 /// Predicted Gene 5934 /// Predicted Gene 6121 Gm6121 1422675_at -2.393 Smarce1 SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily E, Member 1 1422679_s_at 6.434 Ctr9 Ctr9, Paf1/RNA Polymerase II Complex Component, Homolog (S. Cerevisiae) 1422825_at 3.126 Cartpt CART Prepropeptide 1422907_at 2.107 Gnat2 Guanine Nucleotide Binding Protein, Alpha Transducing 2 1423232_at 2.384 Etv4 Ets Variant Gene 4 (E1A Enhancer Binding Protein, E1AF) 1423287_at -2.424 Cbln1 Cerebellin 1 Precursor Protein

1423288_s_at -2.554 Cbln1 Cerebellin 1 Precursor Protein 1423505_at 2.327 Tagln Transgelin 1423585_at 2.159 Igfbp7 Insulin-Like Growth Factor Binding Protein 7 1423754_at 2.067 Ifitm3 Interferon Induced Transmembrane Protein 3 1424010_at 2.150 Mfap4 Microfibrillar-Associated Protein 4 1424194_at 2.219 Rcsd1 RCSD Domain Containing 1 1424208_at 2.402 Ptger4 Prostaglandin E Receptor 4 (Subtype EP4) 1424214_at 2.115 Parm1 Prostate Androgen-Regulated Mucin-Like Protein 1 1424254_at 2.009 Ifitm1 Interferon Induced Transmembrane Protein 1 1424445_at 5.098 Tm4sf5 Transmembrane 4 Superfamily Member 5 1424451_at 2.410 Acaa1b Acetyl-Coenzyme A Acyltransferase 1B 1424470_a_at 2.091 Rapgef3 Rap Guanine Nucleotide Exchange Factor (GEF) 3 1424713_at 2.181 Calml4 Calmodulin-Like 4 1424783_a_at 2.320 Ugt1a1 /// Ugt1a10 /// Ugt1a2 /// Ugt1a5 /// Ugt1a6a UDP Glucuronosyltransferase 1 Family, Polypeptide A1 /// UDP Glycosyltransferase 1 Family, Polypeptide /// Ugt1a6b /// Ugt1a7c /// Ugt1a9 A10 /// UDP Glucuronosyltransferase 1 Family, Polypeptide A2 /// UDP Glucuronosyltransferase 1 Family, Polypeptide A5 /// UDP Glucuronosyltransferase 1 Family, Polypeptide A6A /// UDP Glucuronosyltransferase 1 Family, Polypeptide A6B /// UDP Glucuronosyltransferase 1 Family, Polypeptide A7C /// UDP Glucuronosyltransferase 1 Family, Polypeptide A9 1424893_at -7.524 Ndel1 Nuclear Distribution Gene E-Like Homolog 1 (A. Nidulans) 1424912_at -5.061 Slc25a17 Solute Carrier Family 25 (Mitochondrial Carrier, Peroxisomal Membrane Protein), Member 17 1424936_a_at 2.696 Dnahc8 , Axonemal, Heavy Chain 8 1424967_x_at 3.369 Tnnt2 Troponin T2, Cardiac 1425028_a_at 2.295 Tpm2 Tropomyosin 2, Beta 1425090_s_at 3.193 Kcnc4 Potassium Voltage Gated Channel, Shaw-Related Subfamily, Member 4 294 1425108_a_at 2.284 Smagp Small Cell Adhesion Glycoprotein 1425177_at -2.481 Shmt1 Serine Hydroxymethyltransferase 1 (Soluble) 1425428_at 2.204 Hif3a Hypoxia Inducible Factor 3, Alpha Subunit 1425528_at 2.302 Prrx1 Paired Related Homeobox 1 1425594_at 2.173 Lamc3 Laminin Gamma 3 1425957_x_at 2.430 Tbata Thymus, Brain And Testes Associated 1425974_a_at -2.401 Trim25 Tripartite Motif-Containing 25 1426048_s_at 2.435 Tfap2a Transcription Factor AP-2, Alpha 1426155_a_at 2.152 Osr2 Odd-Skipped Related 2 (Drosophila) 1426258_at 2.128 Sorl1 Sortilin-Related Receptor, LDLR Class A Repeats-Containing 1426285_at 2.200 Lama2 Laminin, Alpha 2 1426509_s_at 5.798 Gfap Glial Fibrillary Acidic Protein 1426642_at 1.993 Fn1 Fibronectin 1 1426873_s_at -2.446 Jup Junction 1426926_at 2.459 Plcg2 Phospholipase C, Gamma 2 1427076_at -2.087 Mpeg1 Macrophage Expressed Gene 1 1427298_at 2.065 Dnm3os 3, Opposite Strand 1427306_at 2.403 Ryr1 Ryanodine Receptor 1, Skeletal Muscle 1427865_at 2.203 LOC100503322 /// LOC100503686 Hypothetical LOC100503322 /// Hypothetical LOC100503686 1428361_x_at 2.355 Hba-a1 /// Hba-a2 Hemoglobin Alpha, Adult Chain 1 /// Hemoglobin Alpha, Adult Chain 2 1428459_at 2.282 Pramef12 PRAME Family Member 12 1428460_at 2.731 Syn2 Synapsin II 1428891_at 2.326 Parm1 Prostate Androgen-Regulated Mucin-Like Protein 1 1429074_at -4.477 1700026D08Rik RIKEN Cdna 1700026D08 Gene 1429280_at 1.973 Col22a1 Collagen, Type XXII, Alpha 1 1429803_at 2.422 1700021F07Rik RIKEN Cdna 1700021F07 Gene

1429891_at 2.044 Capsl Calcyphosine-Like 1429929_at 2.527 Mei1 Meiosis Defective 1 1430401_at 2.064 3110045C21Rik RIKEN Cdna 3110045C21 Gene 1430582_at -2.043 Shprh SNF2 Histone Linker PHD RING Helicase 1430596_s_at 2.387 Vgll3 Vestigial Like 3 (Drosophila) 1430774_at -2.545 A430106A12Rik RIKEN Cdna A430106A12 Gene 1430867_at 2.491 4933431M02Rik RIKEN Cdna 4933431M02 Gene 1430946_at 2.006 2600014E21Rik RIKEN Cdna 2600014E21 Gene 1431079_at 2.217 C1qtnf2 C1q And Tumor Necrosis Factor Related Protein 2 1431099_at 2.334 Hoxd8 Homeobox D8 1431293_a_at -2.055 Cldnd1 Claudin Domain Containing 1 1431418_at -2.164 C030026M15Rik RIKEN Cdna C030026M15 Gene 1432018_at 2.024 Ascl2 Achaete-Scute Complex Homolog 2 (Drosophila) 1433077_at -2.015 2700078F05Rik RIKEN Cdna 2700078F05 Gene 1433431_at 2.403 Pnlip Pancreatic Lipase 1433638_s_at 2.166 Hoxd8 Homeobox D8 1433795_at 2.093 Tgfbr3 Transforming Growth Factor, Beta Receptor III 1433939_at 2.086 Aff3 AF4/FMR2 Family, Member 3 1433945_at 2.004 Fam189a1 Family With Sequence Similarity 189, Member A1 1434140_at 1.978 Mcf2l Mcf.2 Transforming Sequence-Like 1434282_at -2.046 Ibtk Inhibitor Of Bruton Agammaglobulinemia Tyrosine Kinase 1434470_at 2.174 Syt13 Synaptotagmin XIII 1434647_at 2.204 Egflam EGF-Like, Fibronectin Type III And Laminin G Domains 1434779_at 2.794 Cbln2 Cerebellin 2 Precursor Protein 295 1434990_at -2.145 Ppm1e Protein Phosphatase 1E (PP2C Domain Containing) 1435318_at 2.760 E130218I03Rik RIKEN Cdna E130218I03 Gene 1435337_at 2.144 Tshz3 Teashirt Zinc Finger Family Member 3 1435521_at -2.133 Msi2 Musashi Homolog 2 (Drosophila) 1435665_at -2.157 Trim30d Tripartite Motif-Containing 30D 1435943_at 2.920 Dpep1 Dipeptidase 1 (Renal) 1436392_s_at 2.575 Tfap2c Transcription Factor AP-2, Gamma 1436419_a_at 2.775 1700097N02Rik RIKEN Cdna 1700097N02 Gene 1436453_at 1.991 BB144871 Expressed Sequence BB144871 1436939_at 2.422 Unc45b Unc-45 Homolog B (C. Elegans) 1436943_at -2.180 Cyb5d2 Cytochrome B5 Domain Containing 2 1436948_a_at -2.432 Fam70a Family With Sequence Similarity 70, Member A 1436996_x_at 3.014 Lyz1 Lysozyme 1 1437029_at 2.333 Tacr3 Tachykinin Receptor 3 1437176_at 3.708 Nlrc5 NLR Family, CARD Domain Containing 5 1437347_at 2.406 Ednrb Endothelin Receptor Type B 1437399_at -1.978 Cldnd1 Claudin Domain Containing 1 1437438_x_at 1.959 Pnliprp2 Pancreatic Lipase-Related Protein 2 1437451_at 2.167 Ecscr Endothelial Cell-Specific Chemotaxis Regulator 1437502_x_at 2.424 Cd24a CD24a Antigen 1437726_x_at 2.295 C1qb Complement Component 1, Q Subcomponent, Beta Polypeptide 1437752_at 2.568 Lin28a Lin-28 Homolog A (C. Elegans) 1437798_at -2.588 6720422M22Rik RIKEN Cdna 6720422M22 Gene 1437893_at 2.253 Plb1 Phospholipase B1 1437983_at 2.224 Sall1 Sal-Like 1 (Drosophila) 1437988_x_at 2.328 1700003E24Rik RIKEN Cdna 1700003E24 Gene

1438661_a_at 2.365 Arf2 ADP-Ribosylation Factor 2 1438779_at 1.989 Col4a3 Collagen, Type IV, Alpha 3 1438805_at 2.752 Ccnd3 Cyclin D3 1438814_at -2.225 Herc4 Hect Domain And RLD 4 1438933_x_at 2.228 Rasgrp2 RAS, Guanyl Releasing Protein 2 1438946_at 2.486 Pdgfra Platelet Derived Growth Factor Receptor, Alpha Polypeptide 1439015_at 2.057 Gfra1 Glial Cell Line Derived Neurotrophic Factor Family Receptor Alpha 1 1439033_at -2.069 Zcchc7 Zinc Finger, CCHC Domain Containing 7 1439426_x_at 3.166 Lyz1 Lysozyme 1 1439480_at 2.841 Gm10857 Predicted Gene 10857 1439543_at 2.070 1110064A23Rik RIKEN Cdna 1110064A23 Gene 1439575_at 2.756 Tmem232 Transmembrane Protein 232 1440250_at 1.974 Col4a4 Collagen, Type IV, Alpha 4 1440342_at -2.612 G530011O06Rik RIKEN Cdna G530011O06 Gene 1440752_at 2.226 ------1440803_x_at 2.698 Tacr3 Tachykinin Receptor 3 1440804_at -2.544 Nktr Natural Killer Tumor Recognition Sequence 1441198_at -2.383 Zfp39 Zinc Finger Protein 39 1441214_at 1.989 Exph5 Exophilin 5 1441498_at -2.254 ------1441917_s_at 1.986 Tmem40 Transmembrane Protein 40 1441972_at 2.303 6230424C14Rik RIKEN Cdna 6230424C14 Gene 1442180_at 2.693 Dleu7 Deleted In Lymphocytic Leukemia, 7 1442344_at -2.052 ------296 1442447_at -2.490 ------1442656_at 1.955 Elovl6 ELOVL Family Member 6, Elongation Of Long Chain Fatty Acids (Yeast) 1443271_at -1.968 ------1443302_at -2.097 6720403M19Rik RIKEN Cdna 6720403M19 Gene 1443458_at 2.939 D630033O11Rik RIKEN Cdna D630033O11 Gene 1443534_at -2.256 ------1443621_at 8.008 Xaf1 XIAP Associated Factor 1 1443675_at -2.101 ------1443816_s_at 2.268 Pik3r6 Phosphoinositide-3-Kinase, Regulatory Subunit 6 1444177_at 2.025 E330020D12Rik Predicted Gene 6648 1444232_at 2.120 Prkg1 Protein Kinase, Cgmp-Dependent, Type I 1444482_at -2.220 A130078K24Rik RIKEN Cdna A130078K24 Gene 1444646_at 2.127 Bnc2 Basonuclin 2 1444677_at 3.034 C77673 Expressed Sequence C77673 1445186_at 2.080 Stc2 Stanniocalcin 2 1445281_a_at 1.963 B230311B06Rik RIKEN Cdna B230311B06 Gene 1445328_at 2.092 Col4a4 Collagen, Type IV, Alpha 4 1445363_at -2.085 2810055G20Rik RIKEN Cdna 2810055G20 Gene 1445749_at -2.217 ------1446375_at -2.456 Exoc6 Exocyst Complex Component 6 1446742_at -2.262 Nfia Nuclear Factor I/A 1447476_at -2.245 Abcc10 ATP-Binding Cassette, Sub-Family C (CFTR/MRP), Member 10 1447541_s_at 2.565 Itgae Integrin Alpha E, Epithelial-Associated 1447564_x_at 2.138 Piwil4 Piwi-Like Homolog 4 (Drosophila) 1447833_x_at 2.013 Mfap2 Microfibrillar-Associated Protein 2 1447862_x_at 2.089 Thbs2 Thrombospondin 2

1447915_x_at 1.980 Tmem204 Transmembrane Protein 204 1448152_at 2.352 Igf2 Insulin-Like Growth Factor 2 1448182_a_at 2.765 Cd24a CD24a Antigen 1448194_a_at 2.387 H19 H19 Fetal Liver Mrna 1448261_at 2.154 Cdh1 Cadherin 1 1448323_a_at 2.066 Bgn Biglycan 1448475_at 2.283 Olfml3 Olfactomedin-Like 3 1448529_at 2.097 Thbd Thrombomodulin 1448797_at 2.020 Elk3 ELK3, Member Of ETS Oncogene Family 1448804_at 2.314 Cyp11a1 Cytochrome P450, Family 11, Subfamily A, Polypeptide 1 1448816_at 2.009 Ptgis Prostaglandin I2 (Prostacyclin) Synthase 1448826_at 2.212 Myh6 Myosin, Heavy Polypeptide 6, Cardiac Muscle, Alpha 1448889_at 2.518 Slc38a4 Solute Carrier Family 38, Member 4 1448925_at 2.334 Twist2 Twist Homolog 2 (Drosophila) 1448955_s_at 2.248 Cadps Ca2+-Dependent Secretion Activator 1448962_at 2.756 Myh11 Myosin, Heavy Polypeptide 11, Smooth Muscle 1449094_at 2.027 Gjc1 Gap Junction Protein, Gamma 1 1449141_at 2.184 Fblim1 Binding LIM Protein 1 1449145_a_at 2.176 Cav1 Caveolin 1, Caveolae Protein 1449178_at 2.283 Pdlim3 PDZ And LIM Domain 3 1449195_s_at 2.529 Cxcl16 Chemokine (C-X-C Motif) Ligand 16 1449254_at -2.027 Spp1 Secreted Phosphoprotein 1 1449553_at 2.016 Nkain1 Na+/K+ Transporting Atpase Interacting 1 1449577_x_at 2.252 Tpm2 Tropomyosin 2, Beta 297 1449619_s_at 2.178 Arhgap9 Gtpase Activator For The Rho-Type Gtpases By Converting Them To An Inactive GDP-Bound State. Has A Substantial GAP Activity Toward CDC42 And RAC1 And Less Toward RHOA. Has A Role In Regulating Adhesion Of Hematopoietic Cells To The Extracellular Matrix. 1449804_at 2.950 Pnmt Phenylethanolamine-N-Methyltransferase 1449827_at -3.432 Acan Aggrecan 1450042_at 2.247 Arx Aristaless Related Homeobox 1450281_a_at 2.319 Tbata Thymus, Brain And Testes Associated 1450369_at 1.956 Figla Folliculogenesis Specific Basic Helix-Loop-Helix 1450606_at 3.740 Pnmt Phenylethanolamine-N-Methyltransferase 1450857_a_at 1.951 Col1a2 Collagen, Type I, Alpha 2 1450871_a_at 3.696 Bcat1 Branched Chain Aminotransferase 1, Cytosolic 1450962_at -2.359 Pdha2 Pyruvate Dehydrogenase E1 Alpha 2 1450981_at 2.262 Cnn2 Calponin 2 1451344_at 2.561 Tmem119 Transmembrane Protein 119 1451461_a_at 2.299 Aldoc Aldolase C, Fructose-Bisphosphate 1451463_at 2.003 Prr5 Proline Rich 5 (Renal) 1451564_at 2.093 Parp14 Poly (ADP-Ribose) Polymerase Family, Member 14 1451758_at 2.452 Lamc3 Laminin Gamma 3 1451778_at -2.006 Crtc3 CREB Regulated Transcription Coactivator 3 1452183_a_at 2.145 Meg3 Maternally Expressed 3 1452324_at 2.577 Pvt1 Plasmacytoma Variant Translocation 1 1452330_a_at 2.071 Mxra8 Matrix-Remodelling Associated 8 1452670_at 2.220 Myl9 Myosin, Light Polypeptide 9, Regulatory 1452731_x_at 2.185 ENSMUSG00000068790 /// Gm10128 /// Gm2897 /// Predicted Gene, ENSMUSG00000068790 /// Alpha-Takusan Pseudogene /// Predicted Gene 2897 /// Alpha- Gm3002 /// Gm3373 /// Gm3558 /// Gm3696 /// Takusan Pseudogene /// Predicted Gene 3373 /// Predicted Gene 3558 /// Predicted Gene 3696 /// Predicted Gm8348 Pseudogene 8348

1452757_s_at 2.522 Hba-a1 /// Hba-a2 Hemoglobin Alpha, Adult Chain 1 /// Hemoglobin Alpha, Adult Chain 2 1452904_at 2.408 1700026L06Rik RIKEN Cdna 1700026L06 Gene 1453003_at 2.488 Sorl1 Sortilin-Related Receptor, LDLR Class A Repeats-Containing 1453084_s_at 2.369 Col22a1 Collagen, Type XXII, Alpha 1 1453108_at -1.994 Arsk Arylsulfatase K 1453196_a_at 1.981 Oasl2 2'-5' Oligoadenylate Synthetase-Like 2 1453217_at 3.435 Slxl1 Slx-Like 1 1453223_s_at 2.016 Dppa2 Developmental Pluripotency Associated 2 1453226_at 2.113 Kif18b Kinesin Family Member 18B 1453540_at -3.296 5430404G13Rik RIKEN Cdna 5430404G13 Gene 1453558_at 2.268 Efcab10 EF-Hand Calcium Binding Domain 10 1453715_at 2.316 Sv2c Synaptic Vesicle Glycoprotein 2c 1453749_at -6.357 2610507I01Rik RIKEN Cdna 2610507I01 Gene 1454159_a_at 2.044 Igfbp2 Insulin-Like Growth Factor Binding Protein 2 1454822_x_at 2.006 Apcdd1 Adenomatosis Polyposis Coli Down-Regulated 1 1454838_s_at 1.975 Pkdcc Protein Kinase Domain Containing, Cytoplasmic 1454966_at 2.483 Itga8 Integrin Alpha 8 1454969_at 2.123 Lypd6 LY6/PLAUR Domain Containing 6 1455140_at 2.448 Pitpnm3 PITPNM Family Member 3 1455167_at 1.955 Cox8c Cytochrome C Oxidase, Subunit Viiic 1455251_at 2.219 Itga1 Integrin Alpha 1 1455269_a_at 2.800 Coro1a Coronin, Actin Binding Protein 1A 1455296_at 2.080 Adcy5 Adenylate Cyclase 5 1455374_at -3.024 Kcnj3 Potassium Inwardly-Rectifying Channel, Subfamily J, Member 3 298 1455692_x_at 2.152 1700097N02Rik RIKEN Cdna 1700097N02 Gene 1455978_a_at 2.127 Matn2 Matrilin 2 1456156_at -4.630 Lepr Leptin Receptor 1456857_at 2.060 1500011B03Rik RIKEN Cdna 1500011B03 Gene 1456901_at 2.300 Adamts20 A Disintegrin-Like And Metallopeptidase (Reprolysin Type) With Thrombospondin Type 1 Motif, 20 1456973_at -2.477 ------1457632_s_at 2.423 Meis2 Meis Homeobox 2 1457717_at -2.028 D14Ertd449e DNA Segment, Chr 14, ERATO Doi 449, Expressed 1457840_at -1.953 Plxna4 Plexin A4 1458083_at -1.965 ------1458496_at -1.993 ------1458773_at -2.144 ------1459211_at 2.216 Gli2 GLI-Kruppel Family Member GLI2 1459665_s_at 2.023 Mrvi1 MRV Integration Site 1 1459989_at 2.428 ------1460259_s_at 1.976 Clca1 /// Clca2 Chloride Channel Calcium Activated 1 /// Chloride Channel Calcium Activated 2 1460269_at 2.890 Pnmt Phenylethanolamine-N-Methyltransferase 1460605_at 2.359 Crxos1 Crx Opposite Strand Transcript 1 1460632_at -1.963 Rdh10 Retinol Dehydrogenase 10 (All-Trans)

Table A1-6. Total gene list for ± 2.0 or higher fold change of RARA regulated genes in P8 germ cells.

Fold Gene Name Gene Symbol Gene Title Change 1426509_s_at 10.324 Gfap Glial Fibrillary Acidic Protein 1443621_at 9.042 Xaf1 XIAP Associated Factor 1 1425090_s_at 6.710 Kcnc4 Potassium Voltage Gated Channel, Shaw-Related Subfamily, Member 4 1460285_at 6.539 Itga9 Integrin Alpha 9 1425530_a_at 5.082 Stx3 Syntaxin 3 1420549_at 4.181 Gbp1 Guanylate Binding Protein 1 1448265_x_at 3.796 Mpzl2 Myelin Protein Zero-Like 2 1444677_at 3.460 C77673 Expressed Sequence C77673 1424045_at 3.261 5730437N04Rik RIKEN Cdna 5730437N04 Gene 1449254_at 3.191 Spp1 Secreted Phosphoprotein 1 1429169_at 3.161 Rbm3 RNA Binding Motif Protein 3 1422679_s_at 3.090 Ctr9 Ctr9, Paf1/RNA Polymerase II Complex Component, Homolog (S. Cerevisiae) 1440006_at 3.060 BC026600 Cdna Sequence BC026600 1428359_s_at 2.762 Zg16 Zymogen Granule Protein 16 1423261_at 2.756 1500015O10Rik RIKEN Cdna 1500015O10 Gene 1434137_x_at 2.755 Zg16 Zymogen Granule Protein 16 1455140_at 2.602 Pitpnm3 PITPNM Family Member 3 1440381_at 2.585 Riok2 RIO Kinase 2 (Yeast) 1460049_s_at 2.579 1500015O10Rik RIKEN Cdna 1500015O10 Gene 299 1442384_at 2.543 ------1435748_at 2.533 Gda Guanine Deaminase 1449134_s_at 2.533 Spic Spi-C Transcription Factor (Spi-1/PU.1 Related) 1451364_at 2.496 Polr3gl Polymerase (RNA) III (DNA Directed) Polypeptide G Like 1436789_at 2.467 Ccnjl Cyclin J-Like 1423310_at 2.451 Tpbg Trophoblast Glycoprotein 1448002_x_at 2.435 2610001J05Rik RIKEN Cdna 2610001J05 Gene 1437418_at 2.428 Gm3515 Predicted Gene 3515 1450871_a_at 2.415 Bcat1 Branched Chain Aminotransferase 1, Cytosolic 1440780_x_at 2.407 1500015O10Rik RIKEN Cdna 1500015O10 Gene 1434425_at 2.404 Tchh Trichohyalin 1430384_at 2.389 Tle4 Transducin-Like Enhancer Of Split 4, Homolog Of Drosophila E(Spl) 1444743_at 2.369 ------1452284_at 2.330 Ptprz1 Protein Tyrosine Phosphatase, Receptor Type Z, Polypeptide 1 1428358_at 2.308 Zg16 Zymogen Granule Protein 16 1452114_s_at 2.302 Igfbp5 Insulin-Like Growth Factor Binding Protein 5 1435665_at 2.280 Trim30d Tripartite Motif-Containing 30D 1451263_a_at 2.210 Fabp4 Fatty Acid Binding Protein 4, Adipocyte 1444169_at 2.207 3110052M02Rik RIKEN Cdna 3110052M02 Gene 1418743_a_at 2.201 Tesc Tescalcin 1454799_at 2.197 Agpat9 1-Acylglycerol-3-Phosphate O-Acyltransferase 9 1455748_at 2.197 Tmem181a Transmembrane Protein 181A 1441863_x_at 2.185 Krt13 1455336_at 2.167 Thap2 THAP Domain Containing, Apoptosis Associated Protein 2 1422628_at 2.163 Fam111a Family With Sequence Similarity 111, Member A 1442335_at 2.159 ------

1440384_at 2.149 Tmcc1 Transmembrane And Coiled Coil Domains 1 1418460_at 2.146 Sh3d19 SH3 Domain Protein D19 1450464_at 2.134 E4f1 E4F Transcription Factor 1 1420988_at 2.121 Polh Polymerase (DNA Directed), Eta (RAD 30 Related) 1443928_at 2.092 Cytip Cytohesin 1 Interacting Protein 1440992_at 2.086 3110052M02Rik RIKEN Cdna 3110052M02 Gene 1423407_a_at 2.079 Fbln2 Fibulin 2 1447443_at 2.067 ------1426632_at 2.064 Kctd14 Potassium Channel Tetramerisation Domain Containing 14 1439604_at 2.060 Adamts16 A Disintegrin-Like And Metallopeptidase (Reprolysin Type) With Thrombospondin Type 1 Motif, 16 1433489_s_at 2.056 Fgfr2 Fibroblast Growth Factor Receptor 2 1455240_x_at 2.055 Gm7969 Predicted Gene 7969 1446104_at 2.054 ------1426243_at 2.042 Cth Cystathionase (Cystathionine Gamma-Lyase) 1435399_at 2.041 Synpo2 Synaptopodin 2 1449135_at 2.033 Sox18 SRY-Box Containing Gene 18 1456392_at 2.031 Negr1 Neuronal Growth Regulator 1 1427410_at 2.030 Dleu2 Deleted In Lymphocytic Leukemia, 2 1452954_at 2.018 Ube2c Ubiquitin-Conjugating Enzyme E2C 1416693_at 2.017 Foxc2 Forkhead Box C2 1459978_x_at 2.016 Ccnjl Cyclin J-Like 1424171_a_at 2.016 Hagh Hydroxyacyl Glutathione Hydrolase 1437492_at 2.014 Mkx Mohawk Homeobox 1459366_at 2.013 Rgmb RGM Domain Family, Member B 300 1422308_a_at 2.005 Lgals7 Lectin, Galactose Binding, Soluble 7 1419812_s_at 2.005 Ccdc56 Coiled-Coil Domain Containing 56 1441988_at 2.003 Ppm1k Protein Phosphatase 1K (PP2C Domain Containing) 1437673_at 1.988 Wnt5a Wingless-Related MMTV Integration Site 5A 1439015_at 1.984 Gfra1 Glial Cell Line Derived Neurotrophic Factor Family Receptor Alpha 1 1439787_at 1.975 P2rx7 Purinergic Receptor P2X, Ligand-Gated Ion Channel, 7 1426633_s_at 1.970 Kctd14 Potassium Channel Tetramerisation Domain Containing 14 1455720_at 1.969 Adamts2 A Disintegrin-Like And Metallopeptidase (Reprolysin Type) With Thrombospondin Type 1 Motif, 2 1452953_at 1.961 Fam18b Family With Sequence Similarity 18, Member B 1440319_at -1.953 Rbm44 RNA Binding Motif Protein 44 1423530_at -1.955 Stk32c Serine/Threonine Kinase 32C 1417481_at -1.955 Ramp1 Receptor (Calcitonin) Activity Modifying Protein 1 1438295_at -1.958 ------1452445_at -1.958 Slc41a2 Solute Carrier Family 41, Member 2 1455265_a_at -1.959 Rgs16 Regulator Of G-Protein Signaling 16 1435302_at -1.960 Taf4b TAF4B RNA Polymerase II, TATA Box Binding Protein (TBP)-Associated Factor 1438273_at -1.961 Gm13718 Predicted Gene 13718 1456212_x_at -1.962 Socs3 Suppressor Of Cytokine Signaling 3 1442273_at -1.963 Ccdc158 Coiled-Coil Domain Containing 158 1439506_at -1.964 Gm98 Predicted Gene 98 1422933_at -1.964 Xlr5a /// Xlr5b /// Xlr5c X-Linked Lymphocyte-Regulated 5A /// X-Linked Lymphocyte-Regulated 5B /// X-Linked Lymphocyte- Regulated 5C 1419851_at -1.966 Slc4a8 Solute Carrier Family 4 (Anion Exchanger), Member 8 1427352_at -1.967 Krt79 Keratin 79 1453375_at -1.969 4930422N03Rik RIKEN Cdna 4930422N03 Gene 1454159_a_at -1.969 Igfbp2 Insulin-Like Growth Factor Binding Protein 2

1440156_s_at -1.973 Tox2 TOX High Mobility Group Box Family Member 2 1431946_a_at -1.974 Necab3 N-Terminal EF-Hand Calcium Binding Protein 3 1436838_x_at -1.977 Cotl1 Coactosin-Like 1 (Dictyostelium) 1428460_at -1.977 Syn2 Synapsin II 1422592_at -1.978 Ctnnd2 Catenin (Cadherin Associated Protein), Delta 2 1453310_at -1.985 Ppil6 Peptidylprolyl Isomerase (Cyclophilin)-Like 6 1452107_s_at -1.986 Npnt Nephronectin 1451140_s_at -1.988 Prkag2 Protein Kinase, AMP-Activated, Gamma 2 Non-Catalytic Subunit 1426615_s_at -1.988 Ndrg4 N-Myc Downstream Regulated Gene 4 1441089_at -1.988 Eif2c3 Eukaryotic Translation Initiation Factor 2C, 3 1449537_at -1.991 Msh5 Muts Homolog 5 (E. Coli) 1449215_at -1.994 Slc22a21 Solute Carrier Family 22 (Organic Cation Transporter), Member 21 1421330_at -1.994 Ptpn4 Protein Tyrosine Phosphatase, Non-Receptor Type 4 1429013_at -1.996 Mtap7d2 MAP7 Domain Containing 2 1442328_at -1.997 Grin2d Glutamate Receptor, Ionotropic, NMDA2D (Epsilon 4) 1421040_a_at -1.998 Gsta2 Glutathione S-Transferase, Alpha 2 (Yc2) 1456596_at -1.999 Fam70a Family With Sequence Similarity 70, Member A 1457195_at -2.004 Plekhm1 Pleckstrin Homology Domain Containing, Family M (With RUN Domain) Member 1 1427607_at -2.004 Cacna1h Calcium Channel, Voltage-Dependent, T Type, Alpha 1H Subunit 1424838_at -2.006 A330049M08Rik RIKEN Cdna A330049M08 Gene 1425800_at -2.011 Rad9b RAD9 Homolog B (S. Cerevisiae) 1434742_s_at -2.011 Aifm3 Apoptosis-Inducing Factor, Mitochondrion-Associated 3 1460471_at -2.015 Ooep Oocyte Expressed Protein Homolog (Dog) 1419340_at -2.019 Mov10l1 Moloney Leukemia Virus 10-Like 1 301 1427746_x_at -2.022 H2-K1 Histocompatibility 2, K1, K Region 1433909_at -2.027 Syt17 Synaptotagmin XVII 1417373_a_at -2.029 Tuba4a Tubulin, Alpha 4A 1449455_at -2.031 Hck Hemopoietic Cell Kinase 1460666_a_at -2.035 Ebf3 Early B-Cell Factor 3 1455899_x_at -2.036 Socs3 Suppressor Of Cytokine Signaling 3 1440203_at -2.037 Pcgf3 Polycomb Group Ring Finger 3 1438796_at -2.037 Nr4a3 Subfamily 4, Group A, Member 3 1460287_at -2.041 Timp2 Tissue Inhibitor Of Metalloproteinase 2 1436837_at -2.042 Mael Maelstrom Homolog (Drosophila) 1455760_at -2.046 Slc9a5 Solute Carrier Family 9 (Sodium/Hydrogen Exchanger), Member 5 1425137_a_at -2.048 H2-Q10 Histocompatibility 2, Q Region Locus 10 1416111_at -2.048 Cd83 CD83 Antigen 1428662_a_at -2.050 Hopx HOP Homeobox 1458440_at -2.054 Specc1 Sperm Antigen With Calponin Homology And Coiled-Coil Domains 1 1425855_a_at -2.055 Crk V-Crk Sarcoma Virus CT10 Oncogene Homolog (Avian) 1460259_s_at -2.057 Clca1 /// Clca2 Chloride Channel Calcium Activated 1 /// Chloride Channel Calcium Activated 2 1450371_at -2.057 Tshb Thyroid Stimulating Hormone, Beta Subunit 1453191_at -2.058 Col27a1 Collagen, Type XXVII, Alpha 1 1418617_x_at -2.059 Clgn Calmegin 1456857_at -2.060 1500011B03Rik RIKEN Cdna 1500011B03 Gene 1442529_at -2.061 ------1431393_at -2.061 4930447C04Rik RIKEN Cdna 4930447C04 Gene 1438286_at -2.065 Otud7a OTU Domain Containing 7A 1444790_at -2.066 Hsbp1l1 Binding Protein 1-Like 1 1421654_a_at -2.067 Lmna A

1422011_s_at -2.068 3830403N18Rik /// Xlr RIKEN Cdna 3830403N18 Gene /// X-Linked Lymphocyte-Regulated Complex 1429566_a_at -2.070 Hipk2 Homeodomain Interacting Protein Kinase 2 1429745_at -2.071 DXBay18 /// Gm14685 /// Gm5640 /// Gm5936 DNA Segment, Chr X, Baylor 18 /// Predicted Gene 14685 /// Predicted Gene 5640 /// Predicted Gene 5936 1453184_at -2.076 Fam83g Family With Sequence Similarity 83, Member G 1424671_at -2.079 Plekhf1 Pleckstrin Homology Domain Containing, Family F (With FYVE Domain) Member 1 1424866_at -2.082 Usp43 Ubiquitin Specific Peptidase 43 1418043_at -2.083 Abcc5 ATP-Binding Cassette, Sub-Family C (CFTR/MRP), Member 5 1450761_s_at -2.085 Rims2 Regulating Synaptic Membrane Exocytosis 2 1424759_at -2.086 Arrdc4 Arrestin Domain Containing 4 1424077_at -2.087 Gdpd1 Glycerophosphodiester Phosphodiesterase Domain Containing 1 1439842_at -2.090 Zfp287 Zinc Finger Protein 287 1453872_at -2.091 Dmrtc2 Doublesex And Mab-3 Related Transcription Factor Like Family C2 1457715_at -2.092 1010001B22Rik RIKEN Cdna 1010001B22 Gene 1455369_at -2.094 Apba1 Amyloid Beta (A4) Precursor Protein Binding, Family A, Member 1 1419405_at -2.096 Nmb Neuromedin B 1430433_at -2.097 4933406J08Rik RIKEN Cdna 4933406J08 Gene 1424530_at -2.101 Sec14l2 SEC14-Like 2 (S. Cerevisiae) 1435132_at -2.104 Disp1 Dispatched Homolog 1 (Drosophila) 1435494_s_at -2.105 Dsp 1424379_at -2.106 Car11 Carbonic Anhydrase 11 1424349_a_at -2.107 Lpgat1 Lysophosphatidylglycerol Acyltransferase 1 1434399_at -2.109 Galnt6 UDP-N-Acetyl-Alpha-D-Galactosamine:Polypeptide N-Acetylgalactosaminyltransferase 6 1440833_at -2.112 Cdk13 Cyclin-Dependent Kinase 13 1443941_at -2.113 Gm447 Predicted Gene 447 302 1429466_s_at -2.113 Aph1b /// Aph1c Anterior Pharynx Defective 1b Homolog (C. Elegans) /// Anterior Pharynx Defective 1c Homolog (C. Elegans) 1446119_at -2.114 LOC100503783 Hypothetical LOC100503783 1435404_at -2.115 Disp2 Dispatched Homolog 2 (Drosophila) 1425759_at -2.117 Nobox NOBOX Oogenesis Homeobox 1452496_at -2.117 Atp11c Atpase, Class VI, Type 11C 1422914_at -2.119 Sp5 Trans-Acting Transcription Factor 5 1438516_at -2.120 Rif1 Rap1 Interacting Factor 1 Homolog (Yeast) 1422340_a_at -2.121 Actg2 Actin, Gamma 2, Smooth Muscle, Enteric 1448326_a_at -2.126 Crabp1 Cellular Retinoic Acid Binding Protein I 1456891_at -2.126 Dennd2c DENN/MADD Domain Containing 2C 1459626_at -2.127 ------1438275_at -2.128 C130046K22Rik RIKEN Cdna C130046K22 Gene 1447907_x_at -2.130 Necab3 N-Terminal EF-Hand Calcium Binding Protein 3 1459546_s_at -2.131 Enpp1 Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 1425157_x_at -2.136 Tspan33 Tetraspanin 33 1445404_at -2.137 Kif27 Kinesin Family Member 27 1456846_at -2.138 Zbtb42 Zinc Finger And BTB Domain Containing 42 1420710_at -2.139 Rel Reticuloendotheliosis Oncogene 1448956_at -2.140 Stard10 START Domain Containing 10 1426300_at -2.143 Alcam Activated Leukocyte Cell Adhesion Molecule 1419307_at -2.145 Tnfrsf13c Tumor Necrosis Factor Receptor Superfamily, Member 13c 1450070_s_at -2.157 Pak1 P21 Protein (Cdc42/Rac)-Activated Kinase 1 1419542_at -2.158 Dazl Deleted In Azoospermia-Like 1456984_at -2.160 Scml2 Sex Comb On Midleg-Like 2 (Drosophila) 1448028_at -2.164 Tbc1d24 TBC1 Domain Family, Member 24 1441336_at -2.168 Nut Nuclear Protein In Testis

1441957_x_at -2.170 2410076I21Rik RIKEN Cdna 2410076I21 Gene 1456750_at -2.172 B230303O12Rik RIKEN Cdna B230303O12 Gene 1421514_a_at -2.173 Scml2 Sex Comb On Midleg-Like 2 (Drosophila) 1435044_at -2.180 Ebf4 Early B-Cell Factor 4 1436485_s_at -2.181 Whrn Whirlin 1451287_s_at -2.181 Aif1l Allograft Inflammatory Factor 1-Like 1454052_at -2.182 Hormad2 HORMA Domain Containing 2 1442537_at -2.184 ------1456242_at -2.189 Gm7325 Predicted Gene 7325 1435617_at -2.191 1700106N22Rik RIKEN Cdna 1700106N22 Gene 1416316_at -2.193 Slc27a2 Solute Carrier Family 27 (Fatty Acid Transporter), Member 2 1422583_at -2.194 Rab3b RAB3B, Member RAS Oncogene Family 1418289_at -2.204 Nes 1419394_s_at -2.208 S100a8 S100 Calcium Binding Protein A8 (Calgranulin A) 1449363_at -2.209 Atf3 Activating Transcription Factor 3 1431826_a_at -2.212 Brsk2 BR Serine/Threonine Kinase 2 1453617_at -2.212 Zranb3 Zinc Finger, RAN-Binding Domain Containing 3 1437578_at -2.217 Clca2 Chloride Channel Calcium Activated 2 1451762_a_at -2.223 Kif1b Kinesin Family Member 1B 1435365_at -2.224 4732415M23Rik RIKEN Cdna 4732415M23 Gene 1421183_at -2.227 Tex12 Testis Expressed Gene 12 1458662_at -2.230 Daam1 Dishevelled Associated Activator Of Morphogenesis 1 1418149_at -2.233 Chga Chromogranin A 1440275_at -2.234 Runx3 Runt Related Transcription Factor 3 303 1453133_at -2.237 Slc25a31 Solute Carrier Family 25 (Mitochondrial Carrier; Adenine Nucleotide Translocator), Member 31 1438820_at -2.237 Rnf17 Ring Finger Protein 17 1441782_at -2.249 Aak1 AP2 Associated Kinase 1 1441806_at -2.261 ------1436433_at -2.265 BC049762 Cdna Sequence BC049762 1424394_at -2.266 Selm Selenoprotein M 1433523_at -2.266 Radil Ras Association And DIL Domains 1447266_at -2.266 Utp18 UTP18, Small Subunit (SSU) Processome Component, Homolog (Yeast) 1449502_at -2.269 Dazl Deleted In Azoospermia-Like 1447939_a_at -2.271 Gm3893 /// Gm5859 /// Gm7819 Predicted Gene 3893 /// Predicted Pseudogene 5859 /// Predicted Gene 7819 1423668_at -2.277 Zdhhc14 Zinc Finger, DHHC Domain Containing 14 1425122_at -2.282 Fam3b Family With Sequence Similarity 3, Member B 1421882_a_at -2.287 Elavl2 ELAV (Embryonic Lethal, Abnormal Vision, Drosophila)-Like 2 (Hu Antigen B) 1431896_at -2.291 4930447C04Rik RIKEN Cdna 4930447C04 Gene 1434465_x_at -2.292 Vldlr Very Low Density Lipoprotein Receptor 1460040_at -2.294 4933407H18Rik RIKEN Cdna 4933407H18 Gene 1442145_at -2.299 Atp13a3 Atpase Type 13A3 1429196_at -2.300 Rabgap1l RAB Gtpase Activating Protein 1-Like 1420980_at -2.300 Pak1 P21 Protein (Cdc42/Rac)-Activated Kinase 1 1418259_a_at -2.302 Entpd2 Ectonucleoside Triphosphate Diphosphohydrolase 2 1453201_at -2.302 Rassf10 Ras Association (Ralgds/AF-6) Domain Family (N-Terminal) Member 10 1440339_at -2.306 Enpp1 Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 1425283_a_at -2.313 Mtl5 Metallothionein-Like 5, Testis-Specific (Tesmin) 1437596_at -2.316 Lypd4 Ly6/Plaur Domain Containing 4 1443698_at -2.324 Xaf1 XIAP Associated Factor 1 1457426_at -2.327 1700048O20Rik RIKEN Cdna 1700048O20 Gene

1426107_at -2.333 Prdm9 PR Domain Containing 9 1448475_at -2.338 Olfml3 Olfactomedin-Like 3 1420979_at -2.341 Pak1 P21 Protein (Cdc42/Rac)-Activated Kinase 1 1416627_at -2.344 Spint1 Serine Protease Inhibitor, Kunitz Type 1 1456521_at -2.357 ------1425693_at -2.363 Braf Braf Transforming Gene 1432159_a_at -2.365 Tex13 Testis Expressed Gene 13 1415777_at -2.366 Pnliprp1 Pancreatic Lipase Related Protein 1 1418682_at -2.367 Adad1 Adenosine Deaminase Domain Containing 1 (Testis Specific) 1437659_at -2.373 Als2cr11 Amyotrophic Lateral Sclerosis 2 (Juvenile) Chromosome Region, Candidate 11 (Human) 1421478_a_at -2.373 Zfp318 Zinc Finger Protein 318 1425230_at -2.378 Nags N-Acetylglutamate Synthase 1423691_x_at -2.395 Krt8 1429293_at -2.398 Gpc2 Glypican 2 (Cerebroglycan) 1420444_at -2.401 Slc22a3 Solute Carrier Family 22 (Organic Cation Transporter), Member 3 1453591_at -2.410 5730437N04Rik RIKEN Cdna 5730437N04 Gene 1455762_at -2.415 Kidins220 Kinase D-Interacting Substrate 220 1435989_x_at -2.444 Krt8 Keratin 8 1422526_at -2.449 Acsl1 Acyl-Coa Synthetase Long-Chain Family Member 1 1437264_at -2.450 BC051142 Cdna Sequence BC051142 1448669_at -2.464 Dkk3 Dickkopf Homolog 3 (Xenopus Laevis) 1447339_at -2.466 ------1451612_at -2.467 Mt1 Metallothionein 1 1434914_at -2.469 Rab6b RAB6B, Member RAS Oncogene Family 304 1440672_at -2.476 Zfp541 Zinc Finger Protein 541 1438151_x_at -2.476 Zdhhc14 Zinc Finger, DHHC Domain Containing 14 1452272_a_at -2.482 Gfer Growth Factor, Erv1 (S. Cerevisiae)-Like (Augmenter Of Liver Regeneration) 1439006_x_at -2.483 Fam70a Family With Sequence Similarity 70, Member A 1421041_s_at -2.485 Gm3776 /// Gsta1 /// Gsta2 Predicted Gene 3776 /// Glutathione S-Transferase, Alpha 1 (Ya) /// Glutathione S-Transferase, Alpha 2 (Yc2) 1429759_at -2.490 Rps6ka6 Ribosomal Protein S6 Kinase Polypeptide 6 1432312_a_at -2.497 Prss41 Protease, Serine, 41 1456514_at -2.501 ------1459875_x_at -2.507 5730494M16Rik RIKEN Cdna 5730494M16 Gene 1420786_a_at -2.510 Rbmy1a1 RNA Binding Motif Protein, Y Chromosome, Family 1, Member A1 1441985_at -2.519 4933416C03Rik RIKEN Cdna 4933416C03 Gene 1445445_s_at -2.526 Ptger1 Prostaglandin E Receptor 1 (Subtype EP1) 1449534_at -2.537 Sycp3 Synaptonemal Complex Protein 3 1423515_at -2.537 Scn8a Sodium Channel, Voltage-Gated, Type VIII, Alpha 1425336_x_at -2.540 H2-K1 Histocompatibility 2, K1, K Region 1417092_at -2.544 Pth1r Parathyroid Hormone 1 Receptor 1441655_at -2.561 Gm884 Predicted Gene 884 1429197_s_at -2.563 Rabgap1l RAB Gtpase Activating Protein 1-Like 1450542_s_at -2.571 Magea1 /// Magea2 /// Magea3 /// Magea5 /// Magea6 Melanoma Antigen, Family A, 1 /// Melanoma Antigen, Family A, 2 /// Melanoma Antigen, Family A, 3 /// /// Magea8 Melanoma Antigen, Family A, 5 /// Melanoma Antigen, Family A, 6 /// Melanoma Antigen, Family A, 8 1438774_s_at -2.589 Pgm2l1 Phosphoglucomutase 2-Like 1 1457140_s_at -2.592 Rassf10 Ras Association (Ralgds/AF-6) Domain Family (N-Terminal) Member 10 1440192_at -2.597 Ttc39b Tetratricopeptide Repeat Domain 39B 1436329_at -2.597 Egr3 Early Growth Response 3 1430240_a_at -2.603 Clgn Calmegin 1421096_at -2.612 Trpc1 Transient Receptor Potential Cation Channel, Subfamily C, Member 1

1431905_s_at -2.617 4933427G17Rik RIKEN Cdna 4933427G17 Gene 1418875_at -2.620 Syngr4 Synaptogyrin 4 1444797_at -2.658 8030474K03Rik RIKEN Cdna 8030474K03 Gene 1429668_at -2.661 Pou4f1 POU Domain, Class 4, Transcription Factor 1 1416178_a_at -2.667 Plekhb1 Pleckstrin Homology Domain Containing, Family B (Evectins) Member 1 1460226_at -2.670 Trap1a Tumor Rejection Antigen P1A 1456114_at -2.678 Cds1 CDP-Diacylglycerol Synthase 1 1451608_a_at -2.680 Tspan33 Tetraspanin 33 1453365_at -2.686 Rabgap1l RAB Gtpase Activating Protein 1-Like 1456735_x_at -2.694 Acpl2 Acid Phosphatase-Like 2 1457864_at -2.704 Rab11fip3 RAB11 Family Interacting Protein 3 (Class II) 1417160_s_at -2.715 Expi Extracellular Proteinase Inhibitor 1447100_s_at -2.717 5730508B09Rik RIKEN Cdna 5730508B09 Gene 1421424_a_at -2.730 Anpep Alanyl (Membrane) Aminopeptidase 1456208_at -2.733 Gpat2 Glycerol-3-Phosphate Acyltransferase 2, Mitochondrial 1438619_x_at -2.756 Zdhhc14 Zinc Finger, DHHC Domain Containing 14 1420720_at -2.761 Nptx2 Neuronal Pentraxin 2 1434730_at -2.776 AI854517 Expressed Sequence AI854517 1416965_at -2.777 Pcsk1n Proprotein Convertase Subtilisin/Kexin Type 1 Inhibitor 1419007_at -2.777 Zp3 Zona Pellucida Glycoprotein 3 1420065_at -2.778 Tktl1 Transketolase-Like 1 1447640_s_at -2.778 Pbx3 Pre B-Cell Leukemia Transcription Factor 3 1420647_a_at -2.779 Krt8 Keratin 8 1449567_at -2.787 Tktl1 Transketolase-Like 1 305 1445576_at -2.793 Rsph10b2 Radial Spoke Head 10 Homolog B (Chlamydomonas) 1422836_at -2.806 Mbnl3 Muscleblind-Like 3 (Drosophila) 1440342_at -2.807 G530011O06Rik RIKEN Cdna G530011O06 Gene 1429667_at -2.816 Pou4f1 POU Domain, Class 4, Transcription Factor 1 1455618_x_at -2.821 Tspan33 Tetraspanin 33 1453749_at -2.829 2610507I01Rik RIKEN Cdna 2610507I01 Gene 1439768_x_at -2.841 Sema4f Sema Domain, Immunoglobulin Domain (Ig), TM Domain, And Short Cytoplasmic Domain 1451532_s_at -2.845 steap1 Six Transmembrane Epithelial Antigen Of The Prostate 1 1420433_at -2.847 Taf7l TAF7-Like RNA Polymerase II, TATA Box Binding Protein (TBP)-Associated Factor 1440531_at -2.847 Rbm11 RNA Binding Motif Protein 11 1429795_at -2.851 1700001L05Rik RIKEN Cdna 1700001L05 Gene 1420064_s_at -2.856 Tktl1 Transketolase-Like 1 1449347_a_at -2.879 LOC100505359 /// Xlr4a /// Xlr4b /// Xlr4c X-Linked Lymphocyte-Regulated Protein 3A-Like /// X-Linked Lymphocyte-Regulated 4A /// X-Linked Lymphocyte-Regulated 4B /// X-Linked Lymphocyte-Regulated 4C 1449358_at -2.896 D6Mm5e DNA Segment, Chr 6, Miriam Meisler 5, Expressed 1450936_a_at -2.899 Dnase1l2 Deoxyribonuclease 1-Like 2 1415893_at -2.903 Sgpl1 Sphingosine Phosphate Lyase 1 1448169_at -2.911 Krt18 1421479_at -2.916 Zfp318 Zinc Finger Protein 318 1422400_a_at -2.924 Gml /// Hemt1 GPI Anchored Molecule Like Protein /// Hematopoietic Cell Transcript 1 1443831_s_at -2.928 Mtl5 Metallothionein-Like 5, Testis-Specific (Tesmin) 1432458_at -2.943 1700011F14Rik RIKEN Cdna 1700011F14 Gene 1435971_at -2.949 Rims3 Regulating Synaptic Membrane Exocytosis 3 1418677_at -2.973 Actn3 1422325_at -3.003 Magea5 Melanoma Antigen, Family A, 5 1448954_at -3.023 Nrip3 Nuclear Receptor Interacting Protein 3

1431338_at -3.027 Caskin1 CASK Interacting Protein 1 1421883_at -3.036 Elavl2 ELAV (Embryonic Lethal, Abnormal Vision, Drosophila)-Like 2 (Hu Antigen B) 1456842_at -3.042 Boll Bol, Boule-Like (Drosophila) 1428685_at -3.046 Syce1 Synaptonemal Complex Central Element Protein 1 1431809_at -3.046 4932442L08Rik RIKEN Cdna 4932442L08 Gene 1420719_at -3.058 Tex15 Testis Expressed Gene 15 1444691_at -3.068 Prkd3 Protein Kinase D3 1428640_at -3.080 Hsf2bp Heat Shock Transcription Factor 2 Binding Protein 1429074_at -3.090 1700026D08Rik RIKEN Cdna 1700026D08 Gene 1422120_at -3.111 Eaf2 ELL Associated Factor 2 1450555_at -3.112 Tex13 Testis Expressed Gene 13 1420448_at -3.153 Rhox2a Reproductive Homeobox 2A 1455257_at -3.159 Itgb3 Integrin Beta 3 1422264_s_at -3.171 Klf9 Kruppel-Like Factor 9 1420568_at -3.184 Stra8 Stimulated By Retinoic Acid Gene 8 1435412_at -3.192 1700007E06Rik RIKEN Cdna 1700007E06 Gene 1448034_at -3.223 Inca1 Inhibitor Of CDK, Cyclin A1 Interacting Protein 1 1460229_at -3.228 Stag3 Stromal Antigen 3 1426225_at -3.253 Rbp4 Retinol Binding Protein 4, Plasma 1431904_at -3.269 4933427G17Rik RIKEN Cdna 4933427G17 Gene 1441045_at -3.270 Ddx43 DEAD (Asp-Glu-Ala-Asp) Box Polypeptide 43 1416579_a_at -3.278 Epcam Epithelial Cell Adhesion Molecule 1419729_at -3.287 Tex11 Testis Expressed Gene 11 1460242_at -3.314 Cd55 CD55 Antigen 30 6 1458496_at -3.337 ------1456511_x_at -3.390 Eras ES Cell-Expressed Ras 1427023_at -3.416 Phyhipl Phytanoyl-Coa Hydroxylase Interacting Protein-Like 1421566_at -3.452 Pet2 Plasmacytoma Expressed Transcript 2 1446051_at -3.531 Gm1140 Predicted Gene 1140 1452995_at -3.531 Nudt17 Nudix (Nucleoside Diphosphate Linked Moiety X)-Type Motif 17 AFFX- -3.567 Tfrc Transferrin Receptor TransRecMur/ X57349_3_at 1448754_at -3.677 Rbp1 Retinol Binding Protein 1, Cellular 1429481_at -3.689 LOC100503894 /// Nck2 Hypothetical Protein LOC100503894 /// Non-Catalytic Region Of Tyrosine Kinase Adaptor Protein 2 1460667_at -3.694 U90926 Cdna Sequence U90926 1430364_at -3.695 Atf7ip2 Activating Transcription Factor 7 Interacting Protein 2 1417021_a_at -3.713 Spo11 Sporulation Protein, Meiosis-Specific, SPO11 Homolog (S. Cerevisiae) 1422966_a_at -3.793 Tfrc Transferrin Receptor 1431403_a_at -3.854 Mtap7d2 MAP7 Domain Containing 2 1442796_at -3.882 Atf7ip2 Activating Transcription Factor 7 Interacting Protein 2 1420335_at -3.908 Dmc1 DMC1 Dosage Suppressor Of Mck1 Homolog, Meiosis-Specific Homologous Recombination (Yeast) 1419147_at -3.931 Rec8 REC8 Homolog (Yeast) 1459420_at -3.944 ------1427252_at -3.945 Dmrtb1 DMRT-Like Family B With Proline-Rich C-Terminal, 1 1429470_at -3.951 Spdya Speedy Homolog A (Xenopus Laevis) 1440692_at -3.957 Gm364 Predicted Gene 364 1449819_at -3.963 Dmc1 DMC1 Dosage Suppressor Of Mck1 Homolog, Meiosis-Specific Homologous Recombination (Yeast) 1444122_at -3.975 Sycp2 Synaptonemal Complex Protein 2 1449253_at -4.106 Smc1b Structural Maintenance Of Chromosomes 1B

1436761_s_at -4.129 Fam13c Family With Sequence Similarity 13, Member C 1427291_at -4.291 Sycp1 Synaptonemal Complex Protein 1 1429929_at -4.353 Mei1 Meiosis Defective 1 1457614_at -4.414 Micall2 MICAL-Like 2 1437062_s_at -4.563 Phyhipl Phytanoyl-Coa Hydroxylase Interacting Protein-Like 1449103_at -4.713 Tex101 Testis Expressed Gene 101 1431648_at -4.747 4930528F23Rik RIKEN Cdna 4930528F23 Gene 1437128_a_at -4.770 A630033E08Rik RIKEN Cdna A630033E08 Gene 1441390_at -4.794 Spdya Speedy Homolog A (Xenopus Laevis) 1434739_at -4.846 Fmr1nb Fragile X Mental Retardation 1 Neighbor 1419063_at -4.872 Ugt8a UDP Galactosyltransferase 8A 1420602_a_at -4.903 Esx1 Extraembryonic, Spermatogenesis, Homeobox 1 1453942_at -4.973 1700123I01Rik RIKEN Cdna 1700123I01 Gene 1443933_at -4.980 Tc2n Tandem C2 Domains, Nuclear 1437678_at -5.215 Gm1564 Predicted Gene 1564 1422105_at -5.274 Cd3e CD3 Antigen, Epsilon Polypeptide 1452855_at -5.283 Ly6k Lymphocyte Antigen 6 Complex, Locus K 1451838_a_at -5.316 Tc2n Tandem C2 Domains, Nuclear 1443844_at -5.321 Rhox13 Reproductive Homeobox 13 1450292_a_at -5.378 Hormad1 HORMA Domain Containing 1 1439045_x_at -5.534 Tc2n Tandem C2 Domains, Nuclear 1442013_at -5.580 Ccdc79 Coiled-Coil Domain Containing 79 1453544_at -5.842 Dmrtc1c /// Dmrtc1c2 DMRT-Like Family C1c /// DMRT-Like Family C1c2 1457100_at -5.861 AW552889 Expressed Sequence AW552889 307 1429035_at -6.393 Dpep3 Dipeptidase 3 1453331_at -6.417 1700013H16Rik RIKEN Cdna 1700013H16 Gene 1455739_at -6.776 Gm4980 Predicted Gene 4980 1441886_at -6.816 Ccdc79 Coiled-Coil Domain Containing 79 1438975_x_at -7.067 Zdhhc14 Zinc Finger, DHHC Domain Containing 14 1439739_at -7.326 Prss50 Protease, Serine, 50 1437614_x_at -8.765 Zdhhc14 Zinc Finger, DHHC Domain Containing 14 1460628_at -26.598 Eme2 Essential Meiotic Endonuclease 1 Homolog 2 (S. Pombe)