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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.02.010 Byline: Lindsey A. Barske, Blanche Capel Keywords: Sex determination; Turtle; SOX9; Estrogen; Gonad Abstract: Production of male offspring in viviparous eutherian mammals requires a sex-determining mechanism resistant to maternal hormones. This constraint is relaxed in egg-laying species, which are sensitive to hormones during sex determination and often use an increase in aromatase, the estrogen-synthesizing enzyme, as a key feminizing signal. In the turtle Trachemys scripta, sex is normally determined by temperature, but estrogen treatment overrides this cue and leads exclusively to female development. We assessed whether the expression of SOX9, a central male sex-determining gene in mammals, or three other conserved transcription factors (WT1, GATA4, and LHX9) was regulated by estrogen signaling in the turtle. As in mice, all somatic cell types in the immature turtle gonad initially expressed WT1 and GATA4, whereas SOX9 was restricted to the Sertoli precursors and LHX9 to the coelomic epithelium and interstitium. After the bipotential period, SOX9 was abruptly down-regulated at the female temperature. Strikingly, embryos treated with [beta]-estradiol at the male temperature lost SOX9 expression more than two stages earlier than controls, though WT1, GATA4, and LHX9 were unaffected. Conversely, inhibition of estrogen synthesis and signaling prevented or delayed SOX9 down-regulation at the female temperature. These results suggest that endogenous estrogen feminizes the medulla of the bipotential turtle gonad by inhibiting SOX9 expression. This mechanism may be involved in the male-to-female sex reversal in wild populations exposed to environmental estrogens, and is consistent with results showing that the estrogen receptor represses Sox9 to block transdifferentiation of granulosa cells into Sertoli-like cells in the adult mouse ovary. Author Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, NC, USA Article History: Received 11 December 2009; Revised 5 February 2010; Accepted 5 February 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.01.041 Byline: Matthew S. Cook (a), Douglas Coveney (a), Iordan Batchvarov (a), Joseph H. Nadeau (b), Blanche Capel (a) Keywords: Dnd1; Ter; Testicular teratoma; Teratocarcinoma; Testicular germ cell tumor Abstract: A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1.sup.Ter/Ter) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1.sup.Ter/Ter embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1.sup.Ter/Ter XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background. Author Affiliation: (a) Department of Cell Biology, Duke University, Durham, NC, USA (b) Department of Genetics, Case Western Reserve University, Cleveland, OH, USA Article History: Received 25 November 2008; Revised 28 January 2009; Accepted 28 January 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.03.001 Byline: Hao Tang, Andrea Ross, Blanche Capel Keywords: Testis; Spermatogonial stem cells; Bam; GM114 Abstract: In the testis, the continuous production of sperm is maintained by a small population of stem cells called germ line stem cells (GSCs) in Drosophila, or spermatogonial stem cells (SSCs) in mammals. This stem cell population can self-renew or produce daughter cells that differentiate into mature sperm. In Drosophila, BMP signals inhibit GSC differentiation by blocking transcription of the gene bag of marbles (bam). Once bam is activated, germ cells initiate differentiation. We identified a novel gene in mouse, Gm114, that shows homology to Drosophila bam. In male germ cells, expression of Gm114 begins at 12.5-13.5 days post coitum (dpc), the stage in mice when germ cells cease proliferation and begin differentiation into prospermatogonia. In the adult testis, Gm114 is highly expressed in differentiated spermatocytes and spermatids but not in undifferentiated spermatogonia, strongly suggesting that, similar to Bam, GM114 plays an important role in mammalian germ line stem cell self-renewal and differentiation. Interestingly, deletion of the majority of the GM114 protein does not affect mouse viability or fertility. This suggests that either there is a function for the remaining N-terminal of GM114, or that there are alternative mechanisms in the mammalian system that control germ cell differentiation. Author Affiliation: The Department of Cell Biology, Duke University Medical Center, Box 3471, Genome Science Research Building II, Room 4026, Research Drive, Durham, NC 27710, USA Article History: Received 8 January 2008; Revised 29 February 2008; Accepted 3 March 2008

Cell proliferation has been shown to have multiple functions in development and pattern formation, including roles in growth, morphogenesis, and gene expression. Previously, we determined that the earliest known morphological event downstream of the male sex determining gene, Sry, is the induction of proliferation. In this study, we used proliferation inhibitors to block cell division during early gonad development, at stages before the XY gonad has committed to the testis pathway. Using the expression of sex-specific genes and the formation of testis morphology as markers of testis determination, we found that proliferation within a specific 8-h window was critical for the establishment of the male pathway and the formation of the testis. Inhibition of proliferation before or after this critical period led to smaller gonads, but did not block testis formation. The critical period of proliferation coincides with the initiation of Sry expression and is essential for the differentiation of Sertoli cells, suggesting that proliferation is a vital component of the initiation of the male pathway by Sry. We believe these studies suggest that proliferation is involved not only in the elaboration of organ pattern, but also in the choice between patterns (male and female) in the bipotential gonad. Keywords: Sry; Sex determination; Cell proliferation; Organogenesis; Testis; Gonad; Sertoli cell

Most studies to date indicate that the formation of testis cords is critical for proper Sertoli cell differentiation, inhibition of germ cell meiosis, and regulation of Leydig cell differentiation. However, the connections between these events are poorly understood. The objective of this study was to dissect the molecular and cellular relationships between these events in testis formation. We took advantage of the different effects of two hedgehog signaling inhibitors, cyclopamine and forskolin, on gonad explant cultures. Both hedgehog inhibitors phenocopied the disruptive effect of [Dhh.sup.-/-] on formation of testis cords without influencing Sertoli cell differentiation. However, they exhibited different effects on other cellular events during testis development. Treatment with cyclopamine did not affect inhibition of germ cell meiosis and mesonephric cell migration but caused defects in Leydig cell differentiation. In contrast, forskolin treatment induced germ cell meiosis, inhibited mesonephric cell migration, and had no effect on Leydig cell differentiation. By carefully contrasting the different effects of these two hedgehog inhibitors, we demonstrate that, although formation of testis cords and development of other cell types normally take place in a tightly regulated sequence, each of these events can occur independent of the others. Key Words: desert hedgehog; Patched1; Sertoli; Leydig; germ cells; meiosis; PKA.

Although the primitive vasculature is identical in XX and XY genital ridges until 11.5 days postcoitum (dpc), by 12.5 dpc the XY gonad develops a distinct vasculature. This male-specific vasculature, which includes the development of a large coelomic vessel, develops coincident with expression of Sry and formation of testis cords. We show that similar levels of proliferation and vasculogenesis expand the primary vasculature in XX and XY gonads. However, soon after Sry expression begins, the XY gonad recruits a large number of endothelial cells from the adjacent mesonephros, a mechanism totally absent in XX gonads. These migrating cells do not contribute to venous or lymphatic development. Instead, these cells contribute to the arterial system, as indicated by expression of ephrinB2 and by elements of the Notch signaling pathway. This newly formed arterial system establishes a new pattern of blood flow in the XY gonad, which we speculate may have an important role in export of testosterone to masculinize the XY embryo. Key Words: Sry, vasculature; gonadogenesis; organogenesis; arterial system.

Sex reversal from male to female in mice having no Fibroblast growth factor 9 (Fgf9) is discussed. The phenomenon shows that FGF signaling in testicular embryogenesis has a novel role. Embryogenesis of several organs is directed by Fgfs, and they may act in reproductive system development and sex determination in many species.

The Sry gene is found at higher levels in the adult mouses testis than in the genital ridge which is at a critical site of action. RNAase protection assays and reverse transcription polymerase chain reactions reiterate that these unusual RNA molecules are the most abundant transcript in testis. Oligonucleotide hybridization and RNAase H digestion confirm that these Sry RNA molecules are circular. The Sry RNA molecules are circular due to the unique genomic structure encircling the Sry locus in the mouse.

Mammalian sex determination begins with a primordial gonad that can develop into a testis or an ovary depending on the expression of Sry. Sry induces a testis pathway leading to cell migration from mesonephros, formation of testis cords, and inhibition of germ cell entering meiosis. Without Sry, primordial gonads develop into ovaries with germ cells entering meiosis at 13.5 dpc. The sex dimorphic development of germ cells in meiosis suggests potential roles of meiotic germ cells in sex determination. To understand how meiotic germ cells influence sex determination, we examined effects of meiotic germ cells on testis characteristics (cell migration from mesonephros, cord formation, and Sox9 expression) by recombinant organ and reconstituted gonad culture. We were able to induce testis characteristics in 11.5 dpc XX gonads before germ cells enter meiosis but not in 13.5 dpc XX gonads containing meiotic germ cells. Germ cells but not somatic cells appear to be responsible for the incapability to induce testis characteristics in 13.5 dpc XX gonad because testis characteristics were induced in 13.5 dpc germ-cell-depleted XX gonad. When we combined somatic cells from XY gonads and germ cells from 11.5 dpc XX gonads to generate reconstituted gonads, reconstituted gonads differentiated into testicular tissues with cord formation. In contrast, cord formation in reconstituted gonads was inhibited when we combined somatic cells from XY gonads and meiotic germ cells from 13.5 dpc XX gonads. Our results indicate that meiotic germ cells in XX gonads produce signals to maintain ovary development and to antagonize the Sry-induced testis development.

In mammals the primary step in male sex determination is the initiation of testis development in the bipotential gonad primordium. This step depends on the Y-linked male sex-determining gene, Sry, a DNA binding protein. We investigated Sry-dependent changes in gonad morphology that mark the divergence of development of the bipotential gonad toward testis organogenesis. These experiments revealed that Sry induces the male-specific proliferation of Sertoli cell precursors. Recent analysis of Fgf9-/- gonads suggest that Fgf9 is involved in this step. Sry also induces the migration of cells from the mesonephros that are critical for the differentiation of Sertoli ceils and the organization of germ cells within testis cords. Ovarian fate is initiated in the absence of specification of the male pathway. Although ovarian fate determinates have not been defined, ovarian follicle formation is known to depend on the presence of germ cells in the gonad. We present evidence that the entry of germ cells into meiosis blocks the male pathway. Sry opposes the ovarian pathway by blocking the entry of germ cells into meiosis in the XY gonad through the formation of testis cords. Much evidence suggests that it is the relative timing of Sry-mediated pathways and germ cell entry into meiosis that controls the fate of the bipotential gonad.

During the critical period of mouse sex determination, mesenchymal cells migrate from the mesonephros into the adjacent developing testis. This process is thought to initiate cord development and is dependent on Sry. The presence of Sry, however, does not always guarantee normal testis development. For example, transfer of certain Mus domesticus-derived Y chromosomes, i.e., M. domesticus Sry alleles, onto the C57BL/6J (B6) inbred mouse strain results in abnormal testis development. We tested the hypothesis that mesonephric cell migration was impaired in three cases representing a range of aberrant testis development: B6 [XY.sup.AKR], B6 [XY.sup.POS], and (BXD-21 x [B6-Y.sup.POS])F1 [XY.sup.POS]. In each case, mesonephric cell migration was abnormal. Furthermore, the timing, extent, and position of migrating cells in vitro and cord development in vivo were coincident, supporting the hypothesis that mesonephric cells are critical for cord development. Additional experiments indicated that aberrant testis development results from the inability of [Sry.sup. M. domesticus] to initiate normal cell migration, but that downstream signal transduction mechanisms are intact. These experiments provide new insight into the mechanism of C57BL/6J-[Y.sup.M domesticus] sex reversal. We present a model incorporating these findings as they relate to mammalian sex determination. [Copyright] 2000 Academic Press Key Words: mesonephros; cell migration; sex determination; sex reversal.

In mouse the initiation of testis development in the bipotential gonad depends on the Y-linked male sex-determining gene, Sry. Sry specifies male differentiation and instructs gonadal cells to take on the architecture of testis cords; however, no targets of this transcription factor have been identified. We reasoned that a reverse approach would be informative: The investigation of early Sry dependent changes in gonad morphology would allow the deduction of molecular and cellular pathways initiated by Sry. Using this approach, we identified two critical male-specific pathways, proliferation and cell migration. We showed that Sry induces proliferation of Sertoli cell precursors in the coelomic epithelium. Single coelomic epithelial cells give rise to Sertoli cells as well as other lineages in the gonad. The mechanism of this asymmetric fate decision is under investigation in the lab. A second cellular pathway induced by Sry is the migration of somatic cells from the adjacent mesonephros into the gonad. Migration of mesonephric cells is required for testis cord formation in XY gonads. Moreover, induction of cell migration into an XX gonad results in organization of XX cells into cord structures and up-regulation of the male specific marker Sox9. Experiments are underway to identify the signals downstream of Sry that mediate the proliferation and cell migration pathways. This work is supported by grants from the National Institutes of Health and the National Science Foundation.

Vascular development in XX and XY gonads diverges early in gonadogenesis. The gonad originates as a bipotential tissue where the vasculature is initially identical between males and females. Sry expression in the XY gonad initiates signaling events and morphological changes that promote the formation of a testis. One mechanism activated by Sry is the sexually dimorphic recruitment of endothelial and smooth muscle cells from the attached mesonephros into the XY and not the XX gonad. It is evident that the function of the vasculature is tightly coordinated with the structure of organs, yet very little is known about the relationship between vascular development and organogenesis. We are using expression analysis and molecular markers as well as functional assays to investigate the role migrating endothelial cells play in the development of the testis. The bipotential nature of gonadal development, combined with the culture system utilized in our lab provides a unique approach to comparatively analyze the structural differences in the vasculature of XX and XY gonads and examine how their different vascular phenotypes are signaled and integrated with function.

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. [ABSTRACT FROM AUTHOR]

Summary The testis produces sperm throughout the male reproductive lifespan by balancing self-renewal and differentiation of spermatogonial stem cells (SSCs). Part of the SSC niche is thought to lie outside the seminiferous tubules of the testis; however, specific interstitial components of the niche that regulate spermatogonial divisions and differentiation remain undefined. We identified distinct populations of testicular macrophages, one of which lies on the surface of seminiferous tubules, in close apposition to areas of tubules enriched for undifferentiated spermatogonia. These macrophages express spermatogonial proliferation- and differentiation-inducing factors, such as colony-stimulating factor 1 (CSF1) and enzymes involved in retinoic acid (RA) biosynthesis. We show that transient depletion of macrophages leads to a disruption in spermatogonial differentiation. These findings reveal an unexpected role for macrophages in the spermatogonial niche in the testis and raise the possibility that macrophages play previously unappreciated roles in stem/progenitor cell regulation in other tissues. [ABSTRACT FROM AUTHOR]

The gonads form bilaterally as bipotential organs that can develop as testes or ovaries. All secondary sex characteristics that we associate with ‘maleness’ or ‘femaleness’ depend on whether testes or ovaries form. The fate of the gonads depends on a cell fate decision that occurs in a somatic cell referred to as the ‘supporting cell lineage’. Once supporting cell progenitors commit to Sertoli (male) or granulosa (female) fate, they propagate this decision to the other cells within the organ. In this review, we will describe what is known about the bipotential state of somatic and germ cell lineages in the gonad and the transcriptional and antagonistic signaling networks that lead to commitment, propagation, and maintenance of testis or ovary fate. [ABSTRACT FROM AUTHOR]

During mouse development, the gonad begins to form shortly before 10.5 days postcoitum (dpc) on the ventromedial side of the mesonephros. The XY gonad consists of germ cells and somatic cells. The origin of the germ cells is clearly established; however, the origin of the somatic cells, especially the epithelial supporting cell lineages, called Sertoli cells, is still unclear. Sertoli cells are the first somatic cell type to differentiate in the testis and are thought to express Sry, the male sex-determining gene, and to play a crucial role in directing testis development. Previous data have suggested that the somatic cells of the gonad may arise from the mesonephric tubules, the mesonephric mesenchyme, or the coelomic epithelium. Immunohistochemical staining of the gonad at 11.5 dpc showed that the basement membrane barrier under the coelomic epithelium is discontinuous, suggesting that cells in the coelomic epithelium at this stage might move inward. To test this possibility directly, cells of the coelomic epithelium were labeled using the fluorescent lipophilic dye, DiI. We show that when labeled at tail somite 15-17 stages, corresponding to 11.2-11.4 dpc, the coelomic epithelial cells of both sexes migrated into the gonad. In XY gonads, the migrating coelomic epithelial cells became Sertoli cells, as well as interstitial cells. This ability of the coelomic epithelium to give rise to Sertoli cells was developmentally regulated. When labeled at tail somite 18-20 stages, corresponding to 11.5-11.7 dpc, the coelomic epithelial cells no longer became Sertoli cells. Instead, cells that migrated into the gonad stayed outside testis cords, in the interstitium. Migration gradually decreased and ceased by tail somite 30 stage, corresponding to 12.5 dpc, after testis cords had formed and the basement membrane layer underlying the coelomic epithelium had thickened to form the tunica albuginea. In XX gonads, coelomic epithelial cells also migrated into the gonad, but there was no obvious fate restriction during the same developmental period. Taken together, our data show that the coelomic epithelium is a source of Sertoli cells as well as other somatic cells of the gonad in the developing mouse testis.

Sex in vertebrates is determined by genetically or environmentally based signals. These signals initiate molecular cascades and cell–cell interactions within the gonad that lead to the adoption of the male or female fate. Previously, genetically and environmentally based mechanisms were thought to be distinct, but this idea is fading as a result of the unexpected discovery of coincident genetic and thermal influences within single species. Together with accumulating phylogenetic evidence of frequent transitions between sex-determining mechanisms, these findings suggest that genetic and environmental sex determination actually represent points on a continuum rather than discrete categories, and that populations may shift in one direction or the other in response to mutations or changing ecological conditions. Elucidation of the underlying molecular basis of sex determination in mice has yielded a bistable model of mutually antagonistic signaling pathways and feedback regulatory loops. This system would be highly responsive to changes in the upstream primary signal and may provide a basis for the rapid evolution of and transitions between different methods of sex determination. [Copyright &y& Elsevier]

Abstract: In mammals, germ cells are induced from a population of cells at the base of the allantois. This regulative mechanism of germ line induction depends on Bmp signals and a combination of epigenetic changes that silence somatic differentiation genes and activate pluripotency genes. RNA binding proteins are a conserved feature of germ cell development in mammals, and play critical roles in the establishment and maintenance of pluripotency. After their specification, germ cells move through the gut to the gonads under the influence of migratory and attractive cues. In the gonad, germ cells initiate sex-specific differentiation. Germ cells that arrive in the ovary enter meiosis, whereas germ cells that arrive in the testis undergo mitotic arrest. Entry into meiosis is controlled by retinoic acid signals that are blocked in the testis. The signals regulating mitotic arrest in the testis are still not completely understood, but likely involve RNA-binding proteins. Epigenetic reprograming occurs during specification, migratory stages, and sex-specific stages, when maternal and paternal imprints are established. The facility of transitions between germ cells and stem cells suggests a close relationship among their genomic programs. [Copyright &y& Elsevier]

Abstract: Sex determination in mammals results in two discrete sexes, male and female. The sexes are genetically distinct at fertilization (XY = male and XX = female). However, there is little evidence for differences in their development until mid-gestation when the gonadal primordium forms. Recent research suggests that signals within this tissue maintain the gonad in a bipotential state, balanced between two alternative fates, testis or ovary. At mid-gestation, expression of the Y-linked gene Sry in gonadal cells triggers the divergence of gonad development toward the testis pathway. The critical role of Sry may be simply to promote the stable expression of SOX9 in the key cell population in the gonad. Establishment of SOX9 in this lineage is opposed by female promoting factors that push the gonad toward an ovarian fate. Both the male and female sides of these antagonistic pathways are augmented by feedback loops and reinforcing signals that canalize development, once the initial choice is made. Hormones and growth factors produced by the developing testis regulate the male differentiation of the sex ducts and the external genitalia during fetal life. In contrast, the ovary is not required for the fetal development of female genital ducts or female external genitalia, as these organs develop in the absence of a gonad. At puberty, hormones produced by the testis or the ovary control the sex-specific differentiation of the musculature, mammary tissue, and body hair. Male or female development of the brain was previously thought to depend on hormones produced by the testis or ovary. However, recent evidence reveals expression differences between XX and XY brains prior to the time that hormones are circulating, suggesting that some influences on brain development may be autonomous to the cells of the brain. [Copyright &y& Elsevier]

In mammals, the gonads arise as bipotential primordia that are capable of developing as either testes or ovaries. Expression of the Y-linked gene Sry in the XY gonad initiates testis differentiation; in the absence of Sry, ovarian pathways prevail. Although the molecular targets of SRY are not known, many of the early cellular and morphological events that occur downstream of Sry have been characterized. These include increased cell proliferation, Sertoli cell differentiation, testis cord formation, development of a testis-specific vasculature and differentiation of interstitial lineages. Recently, several of the molecular signals that direct these processes have been identified. In addition, the pathways that suppress features of testis development in the XX gonad are being elucidated. [Copyright &y& Elsevier]

A fundamental goal in biology is to understand how distinct cell types containing the same genetic information arise from a single stem cell throughout development. Sex determination is a key developmental process that requires a unidirectional commitment of an initially bipotential gonad towards either the male or female fate. This makes sex determination a unique model to study cell fate commitment and differentiation in vivo. We have focused this review on the accumulating evidence that epigenetic mechanisms contribute to the bipotential state of the fetal gonad and to the regulation of chromatin accessibility during and immediately downstream of the primary sex-determining switch that establishes the male fate. [ABSTRACT FROM AUTHOR]

In all vertebrates sex determination is the step at which development of a testis or ovary is initiated in the bipotential gonad. Although Mus musculus and the red-eared slider turtle, Trachemys scripta, use different mechanisms to initiate organogenesis of the testis (the Y-linked gene, Sry, in the mouse vs. the incubation temperature of the egg in the turtle), the structure of the adult testis is strikingly similar. We have identified several cellular mechanisms involved in testis organogenesis in mouse. Here we investigated whether these cellular mechanisms are conserved in T. scripta downstream of the temperature-dependent switch. Cell tracing experiments indicated that the coelomic epithelium in T. scripta contributes precursors for Sertoli cells and interstitial cells as in mouse. However, we detect no male-specific mesonephric cell migration, a process required for the de novo testis cord-forming process in mouse. In contrast to mouse gonads, where no cord structure is discernible until after the divergence of testis development, we find that primitive sex cords continuous with the coelomic epithelium exist in all T. scripta gonads from the earliest bipotential stages examined. We conclude that typical testis architecture results from the maintenance and elaboration of primitive sex cords in T. scripta rather than the assembly of de novo structures as in mouse. [Copyright &y& Elsevier]

Abstract: After their arrival in the fetal gonad, mammalian germ cells express E-cadherin and are found in large clusters, similar to germ cell cysts in Drosophila. In Drosophila, germ cells in cysts are connected by ring canals. Several molecular components of intercellular bridges in mammalian cells have been identified, including TEX14, a protein required for the stabilization of intercellular bridges, and several associated proteins that are components of the cytokinesis complex. This has led to the hypothesis that germ cell clusters in the mammalian gonad arise through incomplete cell divisions. We tested this hypothesis by generating chimeras between GFP-positive and GFP-negative mice. We show that germ cell clusters in the fetal gonad arise through aggregation as well as cell division. Intercellular bridges, however, are likely restricted to cells of the same genotype. [Copyright &y& Elsevier]

Abstract: One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4−/− and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses. [Copyright &y& Elsevier]

Abstract: Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17–27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided. [Copyright &y& Elsevier]

Abstract: One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4+/+ and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses. [Copyright &y& Elsevier]

Migration of mesonephric cells into XY gonads is a critical early event in testis cord formation. Based on the fact that anti-Mu¨llerian hormone (AMH) can induce testis cord formation in XX gonads, we investigated whether AMH plays a role in the induction of cell migration. Addition of recombinant AMH induced mesonephric migration into XX gonads in culture. AMH-treated XX gonads displayed increased vascular development and altered morphology of the coelomic epithelium, both features of normal testis differentiation. AMH did not induce markers of Sertoli or Leydig cell differentiation. We examined early testis development in Amh-deficient mice, but found no abnormalities, suggesting that any function AMH may have in vivo is redundant. Other transforming growth factor (TGF-β) family proteins, bone morphogenetic proteins (BMP2 and BMP4) show similar inductive effects on XX gonads in culture. Although neither BMP2 nor BMP4 is expressed in embryonic XY gonads, our findings suggest that a TGF-β signalling pathway endogenous to the XY gonad may be involved in regulation of mesonephric cell migration. The factors involved in this process remain to be identified. [Copyright &y& Elsevier]