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During mammalian development, gonadal sex determination results from the commitment of bipotential supporting cells to Sertoli or granulosa cell fates. Typically, this decision is coordinated across the gonad to ensure commitment to a single organ fate. When unified commitment fails in an XY mouse, an ovotestis forms in which supporting cells in the center of the gonad typically develop as Sertoli cells, while supporting cells in the poles develop as granulosa cells. This central bias for Sertoli cell fate was thought to result from the initial expression of the drivers of Sertoli cell fate, SRY and/or SOX9, in the central domain, followed by paracrine expansion to the poles. However, we show here that the earliest cells expressing SRY and SOX9 are widely distributed across the gonad. In addition, Sertoli cell fate does not spread among supporting cells through paracrine relay. Instead, we uncover a center-biased pattern of supporting cell precursor ingression that occurs in both sexes and results in increased supporting cell density in the central domain. Our findings prompt a new model of gonad patterning in which a density-dependent organizing principle dominates Sertoli cell fate stabilization.

Mammalian sex is determined by opposing networks of ovarian and testicular genes that are well characterized. However, its epigenetic regulation is still largely unknown, thus limiting our understanding of a fundamental process for species propagation. Here we explore the 3D chromatin landscape of sex determinationin vivo, by profiling FACS-sorted embryonic mouse gonadal populations, prior and after sex determination, in both sexes. We integrate Hi-C with ChIP-seq experiments usingMETALoci, a novel genome spatial auto-correlation analysis that identifies 3D enhancer hubs across the genome. We uncover a prominent rewiring of chromatin interactions during sex determination, affecting the enhancer hubs of hundreds of genes that display temporal- and sex-specific expression. Moreover, the identification of the 3D enhancer hubs allows the reconstruction of regulatory networks, revealing key transcription factors involved in sex determination. By combining predictive approaches and validations in transgenic mice we identify a novelFgf9regulatory hub, deletion of which results in male-to-female sex reversal with the upregulation of ovarian-specific markers and the initiation of meiosis. Thus, spatial auto-correlation analysis is an effective strategy to identify regulatory networks associated to biological processes and to further characterize the functional role of the 3D genome.

Testis differentiation is initiated when Sry in pre-Sertoli cells directs the gonad toward a male-specific fate. Sertoli cells are essential for testis development, but cell types within the interstitial compartment, such as immune and endothelial cells, are also critical for organ formation. Our previous work implicated macrophages in fetal testis morphogenesis, but little is known about genes underlying immune cell development during organogenesis. Here we examine the role of the immune-associated genes Mafb and Maf in mouse fetal gonad development, and we demonstrate that deletion of these genes leads to aberrant hematopoiesis manifested by supernumerary gonadal monocytes. Mafb;Maf double knockout embryos underwent initial gonadal sex determination normally, but exhibited testicular hypervascularization, testis cord formation defects, Leydig cell deficit, and a reduced number of germ cells. In general, Mafb and Maf alone were dispensable for gonad development; however, when both genes were deleted, we observed significant defects in testicular morphogenesis, indicating that Mafb and Maf work redundantly during testis differentiation. These results demonstrate previously unappreciated roles for Mafb and Maf in immune and vascular development and highlight the importance of interstitial cells in gonadal differentiation.Summary statementDeletion of Mafb and Maf genes leads to supernumerary monocytes in fetal mouse gonads, resulting in vascular, morphogenetic, and differentiation defects during testicular organogenesis.

In vertebrates, the RNA-binding protein (RBP) dead end 1 (DND1) is essential for primordial germ cell (PGC) survival and maintenance of cell identity. In multiple species, Dnd1 loss or mutation leads to severe PGC loss soon after specification or, in some species, germ cell transformation to somatic lineages. Our investigations into the role of DND1 in PGC specification and differentiation have been limited by the absence of an available antibody. To address this problem, we used CRISPR/Cas9 gene editing to establish a transgenic mouse line carrying a DND1GFP fusion allele. We present imaging analysis of DND1GFP expression showing that DND1GFP expression is heterogeneous among male germ cells (MGCs) and female germ cells (FGCs). DND1GFP was detected in MGCs throughout fetal life but lost from FGCs at meiotic entry. In postnatal and adult testes, DND1GFP expression correlated with classic markers for the premeiotic spermatogonial population. Utilizing the GFP tag for RNA immunoprecipitation (RIP) analysis in MGCs validated this transgenic as a tool for identifying in vivo transcript targets of DND1. The DND1GFP mouse line is a novel tool for isolation and analysis of embryonic and fetal germ cells, and the spermatogonial population of the postnatal and adult testis.

At embryonic day (E) 10.5, prior to gonadal sex determination, XX and XY gonads are bipotential and able to differentiate into either a testis or an ovary. At this point, they are transcriptionally and morphologically indistinguishable. Sex determination begins around E11.5 in the mouse when the supporting cell lineage commits to either Sertoli or granulosa cell fate. Testis-specific factors such as SRY and SOX9 drive differentiation of bipotential-supporting cells into the Sertoli cell pathway, whereas ovary-specific factors like WNT4 and FOXL2 guide differentiation into granulosa cells. It is known that these 2 pathways are mutually antagonistic, and repression of the alternative fate is critical for maintenance of the testis or ovary programs. While we understand much about the transcription factor networks guiding the process of sex determination, it is only more recently that we have begun to understand how this process is epigenetically controlled. Studies in the past decade have demonstrated the importance of the chromatin state for gene expression and cell fate commitment, with histone modifications and DNA accessibility having a direct role in gene regulation. It is now clear that the chromatin state during sex determination is dynamic and likely critical for the establishment and/or maintenance of the transcriptional programs. Prior to sex determination, supporting cells have similar chromatin structure and histone modification profiles, reflecting the bipotential nature of these cells. After differentiation to Sertoli or granulosa cells, the chromatin state acquires sex-specific profiles. The proteins that regulate the deposition of histone modifications or the opening of compact chromatin likely play an important role in Sertoli and granulosa cell fate commitment and gonad development. Here, we describe studies profiling the chromatin state during gonadal sex determination and one example in which depletion of Cbx2, a member of the Polycomb Repressive Complex 1 (PRC1), causes male-to-female sex reversal due to a failure to repress the ovarian pathway.

How egg temperature sets sex In many reptiles, sex is determined by nest temperature during egg incubation. Temperature regulates the expression of an epigenetic modifier gene called Kdm6b , which is responsible for testis development. However, the molecular connection between temperature and sex-specific expression of this factor was previously unknown. Weber et al. have identified a link between temperature and the activation of a key regulator of Kdm6b called signal transducer and activator of transcription 3 (STAT3). After an influx of Ca 2+ at the warmer, female-producing temperature, STAT3 is phosphorylated and silences Kdm6b transcription to repress testis development. Science , this issue p. 303

At any given time, the ovary contains a number of follicles in distinct growth stages, each with a set of identifying characteristics. Although follicle counting and staging using histological stains on paraffin-embedded ovary sections has been the gold standard in assessing ovarian health in fertility studies, the final counts rely on extrapolation factors that diverge greatly among studies. These methods also limit our ability to investigate spatial aspects of ovary organization. Recent advances in optical tissue clearing and lightsheet microscopy have permitted comprehensive analysis of intact tissues. In this study, we set out to determine the best clearing and imaging methods to generate 3D images of the complete adult mouse ovary that could be used for accurate assessments of ovarian follicles. We found that a combination of iDISCO and CUBIC was the best method to clear the immunostained ovary. Using lightsheet microscopy, we generated 3D images of the intact ovary and performed qualitative assessments of follicles at all stages of development. This study is an important step toward developing quantitative computational models that allow rapid and accurate assessments of growing and quiescent primordial follicles, and to investigate the integrity of extrinsic ovarian components including vascular and neuronal networks.

Work that established the testis as the driver of male development, and the Y chromosome as the bearer of the male-determining gene, established a working model, and set the stage for the molecular age of mammalian sex determination. The discovery and characterization of Sry/SRY at the top of the hierarchy in mammals launched the field in two major directions. The first was to identify the downstream transcription factors and other molecular players that drive the bifurcation of Sertoli and granulosa cell differentiation. The second major direction was to understand organogenesis of the early bipotential gonad, and how divergence of its two distinct morphogenetic pathways (testis and ovary) is regulated at the cellular level. This review will summarize the early discoveries soon after Sry was identified and focus on my study of the gonad as a model of organogenesis.

Triggers and biological processes controlling male or female gonadal differentiation vary in vertebrates, with sex determination (SD) governed by environmental factors or simple to complex genetic mechanisms that evolved repeatedly and independently in various groups. Here, we review sex evolution across major clades of vertebrates with information on SD, sexual development and reproductive modes. We offer an up-to-date review of divergence times, species diversity, genomic resources, genome size, occurrence and nature of polyploids, SD systems, sex chromosomes, SD genes, dosage compensation and sex-biased gene expression. Advances in sequencing technologies now enable us to study the evolution of SD at broader evolutionary scales, and we now hope to pursue asexomicsintegrative research initiative across vertebrates. The vertebratesexomecomprises interdisciplinary and integrated information on sexual differentiation, development and reproduction at all biological levels, from genomes, transcriptomes and proteomes, to the organs involved in sexual and sex-specific processes, including gonads, secondary sex organs and those with transcriptional sex-bias. Thesexomealso includes ontogenetic and behavioural aspects of sexual differentiation, including malfunction and impairment of SD, sexual differentiation and fertility. Starting from data generated by high-throughput approaches, we encourage others to contribute expertise to building understanding of thesexomesof many key vertebrate species.This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)’.

With or without sex chromosomes, sex determination is a synthesis of many molecular events that drives a community of cells towards a coordinated tissue fate. In this review, we will consider how a sex determination pathway can be engaged and stabilized without an inherited genetic determinant. In many reptilian species, no sex chromosomes have been identified, yet a conserved network of gene expression is initiated. Recent studies propose that epigenetic regulation mediates the effects of temperature on these genes through dynamic post-transcriptional, post-translational and metabolic pathways. It is likely that there is no singular regulator of sex determination, but rather an accumulation of molecular events that shift the scales towards one fate over another until a threshold is reached sufficient to maintain and stabilize one pathway and repress the alternative pathway. Investigations into the mechanism underlying sex determination without sex chromosomes should focus on cellular processes that are frequently activated by multiple stimuli or can synthesize multiple inputs and drive a coordinated response. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)’.

Morphogenetic events during the development of the fetal ovary are crucial to the establishment of female fertility. However, the effects of structural rearrangements of the ovary and surrounding reproductive tissues on ovary morphogenesis remain largely uncharacterized. Using tissue clearing and lightsheet microscopy, we found that ovary folding correlated with regionalization into cortex and medulla. Relocation of the oviduct to the ventral aspect of the ovary led to ovary encapsulation, and mutual attachment of the ovary and oviduct to the cranial suspensory ligament likely triggered ovary folding. During this process, the rete ovarii (RO) elaborated into a convoluted tubular structure extending from the ovary into the ovarian capsule. Using genetic mouse models in which the oviduct and RO are perturbed, we found the oviduct is required for ovary encapsulation. This study reveals novel relationships among the ovary and surrounding tissues and paves the way for functional investigation of the relationship between architecture and differentiation of the mammalian ovary.In humans and other mammals, the female reproductive organs, or ovaries, develop early in life, while the young are still in their mother’s womb. Ovaries contain several different compartments, including the ovarian follicles. These are small groups of cells that produce reproductive hormones, and each follicle also has the potential to produce one egg for fertilisation. The ovaries are further surrounded by different tissues that develop alongside them. These include the oviducts, which carry fertilised eggs from the ovaries into the womb, and ligaments, which anchor the ovaries to the wall of the body cavity. During the development of ovaries, ovarian follicles are sorted into two distinct groups. The first, called medullary follicles, are lost before puberty. The second group, or cortical follicles, remain in a state of ‘suspended animation’ until puberty. After that, they act as a ‘reserve’ of eggs for the rest of the reproductive lifespan. Once each cortical follicle has produced an egg, it is not replenished. This means that proper follicle sorting is crucial for establishing female fertility, and therefore the ability to conceive. The mechanisms behind follicle sorting, however, are still poorly understood. McKey et al. set out to determine how the ovary’s structure changed during its development. In the experiments, high-resolution microscopy techniques were used to reconstruct ovaries of mice in 3D across different stages of development. This revealed that the ends of each ovary started folding towards each other just before birth, and that the folding also happened at the same time as follicle sorting. Simultaneous changes in the shape and orientation of the ligaments suggested that these tissues might direct the folding, for example by pushing or pulling on the rest of the ovary. These results suggest that the changes in ovary structure in early life are critically linked to the establishment of the ovary’s egg reserves. McKey et al. hope that this study will pave the way to a better understanding of infertility and, ultimately, better treatments.

During development of the mouse urogenital complex, the gonads undergo changes in three-dimensional structure, body position and spatial relationship with the mesonephric ducts, kidneys and adrenals. The complexity of genital ridge development obscures potential connections between morphogenesis and gonadal sex determination. To characterize the morphogenic processes implicated in regulating gonad shape and fate, we used whole-embryo tissue clearing and light sheet microscopy to assemble a time course of gonad development in native form and context. Analysis revealed that gonad morphology is determined through anterior-to-posterior patterns as well as increased rates of growth, rotation and separation in the central domain that may contribute to regionalization of the gonad. We report a close alignment of gonad and mesonephric duct movements as well as delayed duct development in a gonad dysgenesis mutant, which together support a mechanical dependency linking gonad and mesonephric duct morphogenesis.

Testes and ovaries undergo sex-specific morphogenetic changes and adopt strikingly different morphologies, despite the fact that both arise from a common precursor, the bipotential gonad. Previous studies showed that recruitment of vasculature is critical for testis patterning. However, vasculature is not recruited into the early ovary. Peripheral innervation is involved in patterning development of many organs but has been given little attention in gonad development. In this study, we show that while innervation in the male reproductive complex is restricted to the epididymis and vas deferens and never invades the interior of the testis, neural crest-derived innervation invades the interior of the ovary around E16.5. Individual neural crest cells colonize the ovary, differentiate into neurons and glia, and form a dense neural network within the ovarian medulla. Using a sex-reversing mutant mouse line, we show that innervation is specific to ovary development, is not dependent on the genetic sex of gonadal or neural crest cells, and may be blocked by repressive guidance signals elevated in the male pathway. This study reveals another aspect of sexually dimorphic gonad development, establishes a precise timeline and structure of ovarian innervation, and raises many questions for future research.

Sertoli cells proliferate and construct seminiferous tubules during fetal life, then undergo differentiation and maturation in the prepubertal testes. In the adult testes, mature Sertoli cells maintain spermatogonia and support spermatogenesis during the entire lifetime. Although Sertoli-like cells have been derived from iPS cells, they tend to remain immature. To investigate whether Sertoli cells can spontaneously acquire the ability to support spermatogenesis when transferred into the adult testis, we transplanted mouse fetal testicular cells into a Sertoli-depleted adult testis. We found that donor E12.5, E14.5 and E16.5 Sertoli cells colonized adult seminiferous tubules and supported host spermatogenesis 2 months after transplantation, demonstrating that immature fetal Sertoli cells can undergo sufficient maturation in the adult testis to become functional. This technique will be useful to analyze the developmental process of Sertoli cell maturation and to investigate the potential of iPS-derived Sertoli cells to colonize, undergo maturation, and support spermatogenesis within the testis environment.

Gonadal sex determination represents a unique model for studying cell fate decisions. However, a complete understanding of the different cell lineages forming the developing testis and ovary remains elusive. Here, we investigated the origin, specification and subsequent sex-specific differentiation of a previously uncharacterized population of supporting-like cells (SLC) in the developing mouse gonads. The SLC lineage is closely related to the coelomic epithelium and specified as early as E10.5, making it the first somatic lineage to be specified in the bipotential gonad. SLC progenitors are localized within the genital ridge at the interface with the mesonephros and initially co-express Wnt4 and Sox9. SLCs become sexually dimorphic around E12.5, progressively acquire a Sertoli- or granulosa-like identity and contribute to the formation of the rete testis and rete ovarii. Finally, we found that WNT4 is a crucial regulator of the SLC lineage and is required for the formation of the rete testis.TeaserDescription of an uncharacterized multipotent gonadal cell lineage involved in testis and ovary development

Spermatogonia, which produce sperm throughout the male lifetime, are regulated inside a niche composed of Sertoli cells, and other testis cell types. Defects in Sertoli cells often lead to infertility, but replacement of defective cells has been limited by the inability to deplete the existing population. Here, we use an FDA-approved non-toxic drug, benzalkonium chloride (BC), to deplete testis cell types in vivo. Four days after BC administration, Sertoli cells are preferentially depleted, and can be replaced to promote spermatogenesis from surviving (host) spermatogonia. Seven days after BC treatment, multiple cell types can be engrafted from fresh or cryopreserved testicular cells, leading to complete spermatogenesis from donor cells. These methods will be valuable for investigation of niche-supporting cell interactions, have the potential to lead to a therapy for idiopathic male infertility in the clinic, and could open the door to production of sperm from other species in the mouse., Sertoli cells and other somatic cells of the testis comprise the germ cell niche and are critical to regulate spermatogenesis. Here the authors present a method in which Sertoli cells are selectively targeted for ablation by the compound benzalkonium chloride (BC) in mice, and the spermatogenic niche is subsequently repopulated in regions that have been affected by BC treatment.

Sex determination of the gonads begins with fate specification of gonadal supporting cells into either ovarian pre-granulosa cells or testicular Sertoli cells. This fate specification hinges on a balance of transcriptional control. Here we report that expression of the transcription factor RUNX1 is enriched in the fetal ovary in rainbow trout, turtle, mouse, goat, and human. In the mouse, RUNX1 marks the supporting cell lineage and becomes pre-granulosa cell-specific as the gonads differentiate. RUNX1 plays complementary/redundant roles with FOXL2 to maintain fetal granulosa cell identity and combined loss of RUNX1 and FOXL2 results in masculinization of fetal ovaries. At the chromatin level, RUNX1 occupancy overlaps partially with FOXL2 occupancy in the fetal ovary, suggesting that RUNX1 and FOXL2 target common sets of genes. These findings identify RUNX1, with an ovary-biased expression pattern conserved across species, as a regulator in securing the identity of ovarian-supporting cells and the ovary., Proper ovarian differentiation requires RUNX1. Here, the authors show that double knockout of Runx1/Foxl2 results in masculinization of fetal ovaries, and that RUNX1 and FOXL2 jointly occupy common chromatin regions to maintain pre-granulosa cell identity in the fetal ovary.

Making males and back again Temperature-dependent sex determination occurs in many reptilian species. An epigenetic mechanism is presumed to be at work, but thus far it has not been identified. Ge et al. show that in the red-eared slider turtle, an epigenetic modifier, the histone demethylase KDM6B, binds to the promoter of the dominant male gene to activate male development (see the Perspective by Georges and Holleley). Knock down the expression of KDM6B, and embryos destined to be male turn into females. Science , this issue p. 645 ; see also p. 601

Summary: Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size but also selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy. : Using a mouse testicular germ cell tumor model, Pierpont et al. establish that male germ cells are susceptible to malignant transformation during a restricted window of embryonic development. The cancer stem cells of the resulting testicular cancers demonstrate genotoxin hypersensitivity, rendering these malignancies highly responsive to conventional chemotherapy. Keywords: testicular germ cell tumor, TGCT, cancer stem cells, CSCs, chemotherapy, embryonal carcinoma, EC, DNA damage response, DDR

Weber and Capel discuss various aspects of sex reversal, including how it happens, why it happens in some species but not others, and whether it is adaptive.

Temperature dependent sex determination (TSD) is the process by which the environmental temperature experienced during embryogenesis influences the sex of an organism, as in the red-eared slider turtle Trachemys scripta elegans. In accord with current paradigms of vertebrate sex determination, temperature is believed to exert its effects on sexual development in T. scripta entirely within the middle third of development, when the gonad is forming. However, whether temperature regulates the transcriptome in T. scripta early embryos in a manner that could influence secondary sex characteristics or establish a pro-male or pro-female environment has not been investigated. In addition, apart from a handful of candidate genes, very little is known about potential similarities between the expression cascade during TSD and the genetic cascade that drives mammalian sex determination. Here, we conducted an unbiased transcriptome-wide analysis of the effects of male- and female-promoting temperatures on the turtle embryo prior to gonad formation, and on the gonad during the temperature sensitive period. We found sexually dimorphic expression reflecting differences in steroidogenic enzymes and brain development prior to gonad formation. Within the gonad, we mapped a cascade of differential expression similar to the genetic cascade established in mammals. Using a Hidden Markov Model based clustering approach, we identified groups of genes that show heterochronic shifts between M. musculus and T. scripta. We propose a model in which multiple factors influenced by temperature accumulate during early gonadogenesis, and converge on the antagonistic regulation of aromatase to canalize sex determination near the end of the temperature sensitive window of development.

A window to the embryo Mammalian embryonic development is a complex process, continuously changing in space and time. Q. Huang et al. designed an abdominal window to image mouse embryos in utero from embryonic day 9.5 to birth. Using this technique, they visualized dynamic activities during embryonic organ formation, including neurotransmission and cell division in the brain, autophagy in the retina, viral gene delivery, and placental drug transfer. They also tracked diverging fates of human and mouse neural crest cells in interspecies chimeras. Science , this issue p. 181

In many reptiles, including the red-eared slider turtle

XX and XY fetal gonads are initially bipotential, poised between the ovary and testis fate. Multiple lines of evidence suggest that commitment to testis fate requires the repression of genes associated with ovary fate. It was previously shown that loss of CBX2, the subunit of the Polycomb Repressive Complex 1 (PRC1) that binds H3K27me3 and mediates silencing, leads to ovary development in XY mice and humans. While it had been proposed that CBX2 is an activator of the testis-determining gene Sry, we investigated the alternative possibility that CBX2 has a direct role as a repressor of the antagonistic ovary-promoting pathway. To investigate this possibility, we developed a quantitative genome-wide profile of the repressive histone mark H3K27me3 and its active counterpart H3K4me3 in isolated XY and XX gonadal supporting cells before and after sex determination. We show that testis and ovary sex-determining (SD) genes are bivalent before sex determination, providing insight into how the bipotential state of the gonad is established at the epigenetic level. After sex determination, many SD genes of the alternate pathway remain bivalent, possibly contributing to the ability of these cells to transdifferentiate even in adults. The finding that many genes in the Wnt signaling pathway were targeted for H3K27me3-mediated repression in Sertoli cells led us to test whether deletion of Wnt4 could rescue testis development in Cbx2 mutants. We show that Sry expression and testis development were rescued in XY Cbx2-/-;Wnt4-/- mice. Furthermore, we show that CBX2 directly binds the downstream Wnt signaler Lef1, an ovary-promoting gene that remains bivalent in Sertoli cells. Our results suggest that stabilization of the testis fate requires CBX2-mediated repression of bivalent ovary-determining genes, which would otherwise block testis development., Author summary During development, the bipotential fetal gonad can commit to the testis fate or to the ovary fate. Mutation of the epigenetic regulator CBX2 leads to ovary development in XY embryos, suggesting a critical role for chromatin remodeling during sex determination. However, the epigenetic mechanisms that regulate the testis vs. ovary cell-fate decision in the mammalian bipotential gonad are poorly understood. In this study, we developed a genome-wide profile of two histone modifications that play critical roles during development: H3K27me3 (repressive) and H3K4me3 (active). We find that sex-determining genes that are initially co-expressed in XX and XY bipotential gonads are bivalent (marked by both H3K4me3 and H3K27me3) prior to sex determination, poised to engage either the testis or ovary fate. Remarkably, after sex determination, repressed genes that promote the alternate fate remain bivalent. We show that stabilization of the testis fate requires CBX2-mediated repression of bivalent ovary-determining genes, which would otherwise block testis development. Our study provides insight into how the bipotential state of the gonad is established at the epigenetic level, and how the testis fate is stabilized by repression of the ovary fate during sex determination.

INTRODUCTION AND OBJECTIVES:Sertoli cells proliferate and construct seminiferous tubules during fetal life, then undergo differentiation in prepubertal testes. In adult testes, differentiated matur...

The adult spermatogonial stem cell population arises from pluripotent primordial germ cells (PGCs) that enter the fetal testis around embryonic day 10.5 (E10.5). These cells undergo rapid mitotic proliferation, then enter a prolonged period of cell cycle arrest (G1/G0) during which they transition to pro-spermatogonia. In mice homozygous for theTermutation in the RNA-binding proteinDND1(DND1Ter/Ter), many germ cells fail to enter G1/G0, and give rise to teratomas, tumors in which many embryonic cell types are represented. To investigate the origin of these tumors, we sequenced the transcriptome of male germ cells inDND1Ter/Termutants at E12.5, E13.5, and E14.5, just prior to the formation of teratomas, and correlated this information with direct targets of DND1identified by DO-RIP-Seq. Consistent with previous results, we found that DND1controls the down regulation of many genes associated with pluripotency and active cell cycle, including elements of the mTor, Hippo and Bmp/Nodal signaling pathways. However, DND1targets also include genes associated with male differentiation including a large group of chromatin regulators activated in wild type but not mutant germ cells during the transition between E13.5and E14.5. These results suggest multiple functions of DND1, and link DND1to the initiation of epigenetic modifications in male germ cells.

Sex determination of the gonads begins with fate specification of gonadal supporting cells into either ovarian granulosa cells or testicular Sertoli cells. This process of fate specification hinges on a balance of transcriptional control. We discovered that expression of the transcription factor RUNX1 is enriched in the fetal ovary in rainbow trout, turtle, mouse, goat and human. In the mouse, RUNX1 marks the supporting cell lineage and becomes granulosa cell-specific as the gonads differentiate. RUNX1 plays complementary/redundant roles with FOXL2 to maintain fetal granulosa cell identity, and combined loss of RUNX1 and FOXL2 results in masculinization of the fetal ovaries. At the chromatin level, RUNX1 occupancy overlaps partially with FOXL2 occupancy in the fetal ovary, suggesting that RUNX1 and FOXL2 target a common set of genes. These findings identify RUNX1, with an ovary-biased pattern conserved across species, as a novel regulator in securing the identity of ovarian supporting cells and the ovary.

The epigenetic mechanisms that regulate the male vs. female cell-fate decision in the mammalian bipotential gonad are poorly understood. In this study, we developed a quantitative genome-wide profile of H3K4me3 and H3K27me3 in isolated XY and XX gonadal supporting cells before and after sex determination. We show that male and female sex-determining (SD) genes are bivalent before sex determination, providing insight into how the bipotential state of the gonad is established at the epigenetic level. After sex determination, many SD genes of the alternate pathway remain bivalent, possibly contributing to the ability of these cells to transdifferentiate even in adults. It was previously shown that loss of CBX2, the Polycomb group subunit that binds H3K27me3 and mediates silencing, leads to loss of Sry and male-to-female sex reversal. The finding that many genes in the Wnt signaling pathway were targeted for H3K27me3-mediated repression in Sertoli cells led us to test whether deletion of Wnt4 could rescue male development in Cbx2 mutants. We show that Sry expression and testis development were rescued in XY Cbx2-/-;Wnt4-/- mice. Furthermore, we show that CBX2 directly binds the downstream Wnt signaler Lef1, a female-promoting gene that remains bivalent in Sertoli cells. Our results suggest that stabilization of the male fate requires CBX2-mediated repression of bivalent female-determining genes, which would otherwise block Sry expression and male development.