Browsing by Subject "Gonad"

The mouse embryonic gonad provides an outstanding model to study the complex mechanisms involved in cell fate specification and maintenance. At the bipotential stage, both XX and XY gonads are capable of becoming testes or ovaries upon specific molecular cues. The specification of the supporting cell lineage (as either Sertoli cells in the male or granulosa cells in the female) initiates the testis or ovary program, leading to male or female fate. However, there are significant gaps in our understanding of how the somatic cells in the gonad arise, are competent to differentiate, and determine and maintain their fates. In this dissertation, we addressed these questions.

We found that NUMB (an antagonist of Notch signaling) serves as competence factor for somatic cell differentiation during early gonadogenesis. The asymmetric allocation of NUMB to the basolateral domain of actively dividing coelomic epithelial (CE) cells is indispensable to (1) maintain the totipotent stem cell-like reservoir at the CE domain, and (2) give rise to progenitor cells that can further differentiate into supporting and interstitial cell lineages. Deletion of Numb; Numbl resulted in disruption of cell polarity in the CE domain as well as a reduction of multiple differentiated cell lineages within XX and XY gonads, including supporting cells and male steroidogenic cells, which were most severely affected. We detected elevated Notch downstream signaling in the Numb; Numbl mutant gonads. Moreover, treatment of DAPT (which blocks Notch signaling) rescued the Numb; Numbl mutant phenotypes, strongly suggesting that upregulation of Notch is responsible.

Previous experiments indicate that when supporting cells commit to the male (Sertoli) fate, they must repress the alternative female (granulosa) cell fate. In another line of experiments, we investigated the hypothesis that the Polycomb repressive complex (PRC1) plays a critical role in repressing the female pathway during male gonad patterning. We found that loss of Ring1B (a component of PRC1) led to the disruption of XY gonad development specific to the posterior region of male gonads. Sry, the upstream driver of the male pathway, was not appropriately expressed in the posterior domain, which contained cells expressing female markers and, in some cases, small aggregates of undifferentiated cells. Using ChIP-Seq, we identified potential targets of PRC during male gonad development. Moreover, a key gene in the male pathway, SOX9, interacts with Ring1B, based on immunoprecipitation results, leading to the hypothesis that it may be involved in the recruitment of PRC to its target sites to execute the repression of female genes in male gonads.

Our findings provide insight into how somatic cell fate is determined and maintained during mammalian sex determination. Our results may be valuable for patients with disorders of sexual development with unidentified genetic contributions.

The vertebrate gonad is an excellent model to study organogenesis due to its unique ability to form two distinct organs from a common bipotential primordium. No single factor is responsible for activation of ovary or testis development in all vertebrate species, but these developmental pathways tend to converge on the same cohort of genetic regulators. The structures of testes and ovaries are extremely similar across vertebrates, and this high level of conservation is also observed in the gene regulatory processes underlying their differentiation. In heterogametic species such as mice and chickens, genes on the sex chromosomes activate the genes that drive differentiation of the testis of ovary. However, not all vertebrate species have sex chromosomes, and it’s unknown how the many genetic and cellular processes that direct gonad development are activated in the absence of a clear genetic signal. Temperature-dependent sex determination (TSD) is one of the primary sex determination strategies found in reptiles and has repeatedly evolved in multiple reptilian lineages. During TSD, the fate of the gonad is driven by nest temperatures experienced during embryonic development. In the decades since TSD was first described, the molecular processes underlying this phenomenon have remained a mystery.

The Red-Eared Slider turtle, Trachemys scripta elegans (T. scripta), is a widely-studied model for temperature-dependent sex determination. When eggs are incubated at a constant 26˚C, 100% of embryos will develop testes. Incubating eggs at a constant 31˚C produces only embryos with ovaries. Prior work has focused the regulation of aromatase, which is crucial to estrogen synthesis, but it is expressed relatively late in the sex determination window. A transcriptome analysis of T. scripta gonads through sex determination revealed a group of early, male-biased genes, including the H3K27 demethylase Kdm6b. In many vertebrates, the epigenetic state of key sex determining genes appears to be critical in the activation of testis or ovary specific-signaling. We investigated whether KDM6B mediates the effect of temperature on gene expression in T. scripta and we found that it activates a conserved regulator of male sex development, DMRT1.

One of the few identified transcriptional regulators of Kdm6b, the transcription factor STAT3, is only phosphorylated at the warmer, female-producing temperature (FPT). We show that pSTAT3 binds the Kdm6b locus to repress transcription and inhibition of pSTAT3 is sufficient to induce female-to-male sex reversal. Using primary cells derived from T. scripta gonads, we found that a heat-mediated influx of calcium at FPT promotes phosphorylation of STAT3. From these data we propose the model that heat-mediated influx of calcium at FPT promotes activation of STAT3, a transcriptional repressor of the male pathway. Our model is the first proposed mechanism of temperature-dependent sex determination supported by direct experimental evidence.

It is unknown how the gonad interprets environmental signals and coordinates cell fates across the tissue. The embryonic gonad coelomic epithelium is a common feature of many vertebrate gonads, and its development is critical to placement of the germ cells in the appropriate stem cell niche, which is required for germ cell survival and maturation. Previous studies of testis morphogenesis in T. scripta show that invaginations of the coelomic epithelium move germ cells into the gonad medulla to form the seminiferous tubules. We show that these invaginations only occur below germ cells, express the conserved Steroli cell marker SOX9, and are sensitive to the hormone environment of the gonad. These data suggest that signals between the germ cell, somatic cells in the coelomic epithelium, and somatic cells of the primordial cords collectively participate in the morphogenetic changes underlying testis development in T. scripta.

Our findings provide a framework for future investigations into the mechanism underlying temperature-dependent sex determination by identifying the initial signaling events that regulate the epigenetic state of sex-specific genes and describing how cellular fates are maintained during the sex determination window. STAT3 signaling can be activated by many inputs and have numerous downstream impacts, only some of which have been experimentally tested, providing direction and future lines of investigation for the field. The data presented here has laid the groundwork for identifying how temperature-sex determination operates in the turtle and how pieces of this process may be conserved among many animal phyla.

The mouse gonad begins its development as a bipotential primordia, capable of developing into a testis or ovary depending on the presence of the sex-determining gene, Sry. In the XY gonad, opposing pro-testis and pro-ovary pathways compete in gonadal supporting cells. While the individual cellular decision process is well understood, the higher-level process of coordination of cell fates across the gonad remains to be explained. The testis and ovary exhibit distinct patterns of differentiation, suggesting that either development of each organ requires a particular organizing principle or bipotentiality requires regional separation for fate specification or stabilization. The overall goal of this work is to improve characterizations of the spatiotemporal features of sex determination and gonadogenesis, including cell fate organization, morphogenic processes, and system context.Though several hypotheses have connected gonad morphogenesis to sex determination, the morphogenic processes that occur in the gonad have not been sufficiently characterized for formulating testable hypotheses. To capture and analyze the complexity of genital ridge morphogenesis, we generated a 3-dimensional time course of gonad development in native form and context using whole embryo tissue clearing and light sheet microscopy. Analysis revealed that the early gonad exhibits anterior-to-posterior patterns as well as increased rates of growth, rotation, and separation in the central domain. In extending characterization to the neighboring nephric ducts, we found a close alignment of gonad and mesonephric duct movements as well as delayed duct development in Cbx2 mutants, which undergo XY sex reversal and gonad dysgenesis. These data support mechanical integration of gonad and mesonephric duct morphogenesis. In investigating the mechanisms underlying the center-to-pole pattern of testis differentiation, we performed anteroposterior axis analyses and ex vivo gonad reconstruction cultures. These experiments allowed us to rule out two commonly accepted theories in the field: paracrine relay and center-first Sry expression. After searching for patterns in other cellular processes during gonadogenesis, including cell cycle arrest and coelomic epithelium proliferation, we uncovered a center-biased pattern of supporting cell precursor ingression. The updated model indicates that differences between the patterns of differentiation in the testis and ovary are due to features of their respective regulatory networks connecting their fate dynamics to different general gonadal organizing principles acting upstream of supporting cell differentiation. Following recent work on the rete testis and rete ovarii suggesting these structures contribute to gonadal supporting cell populations, we characterized early development of the rete and adjacent tissues in both sexes. Comparison of the GATA4+/PAX8+ presumptive rete with mesonephric and gonadal cells led to the identification of undescribed patterns in mesonephros development which may play a role in sexual dimorphism of the rete. Cells of the rete may derive from mesonephric condensates in a process similar to kidney nephron development. Cell cycle analysis revealed the mesonephric tubules and early rete to be a largely non-proliferating population of cells, suggesting expansion through recruitment of new cells. These results were used to establish preliminary theories for lineage relationships in early urogenital development. Initial attempts at lineage tracing to test the theory were unsuccessful. The findings presented here contribute to a more comprehensive and systems level understanding of sex determination and gonad development. In particular, the incorporation of high-resolution spatial information into theories of sex determination serves to connect individual cell fate decisions to organ level patterns of differentiation in space and time. These results will be useful for novel hypothesis generation as well as for designing more detailed models and simulations of sex determination and gonadogenesis.

The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. The early gonad is composed of four lineages: supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. Each lineage in the early gonad consists of bipotential progenitors capable of adopting either a male or female fate, which they do in a coordinated manner to form a functional testis or ovary. The supporting cell lineage is of particular interest because the decision of these cells to adopt the male or female fate dictates the fate of the gonad as a whole.

To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells of the four lineages from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. Our analysis identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We also determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. Multipotent progenitors that show lineage priming express markers of the various fates into which they can differentiate and subsequently silence genes associated with the fate not adopted as they differentiate. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cell progenitors were primed with a female bias. This yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates.

We also used a genetic approach to investigate how individual factors contribute to the adoption of the male supporting cell fate. We previously demonstrated that Fgf9 and Wnt4 act as mutually antagonistic factors to promote male or female development of the bipotential mammalian gonad. Fgf9 is necessary to maintain Sox9 expression, which drives male development. However, whether FGF9 acted directly on Sox9 or indirectly through repression of Wnt4, was unknown. Wnt4 is a female-primed gene, and is therefore repressed during male development. To determine how Fgf9 functioned, we generated double Fgf9/Wnt4 and Fgfr2/Wnt4 mutants. While single XY Fgf9 and Fgfr2 mutants showed partial or complete male-to-female sex reversal, loss of Wnt4 in an Fgf9 or Fgfr2 mutant background rescued normal testis development. We also found that Wnt4 and another female-associated gene (Rspo1) were derepressed in Fgf9 mutants prior to the down-regulation of Sox9. Thus, the primary function of Fgf9 is the repression of female genes, including Wnt4. We also tested the reciprocal possibility: that de-repression of Fgf9 was responsible for the aspects of male development observed in XX Wnt4 mutants. However, we show that loss of Fgf9 in XX Wnt4-/- gonads does not rescue the partial female-to-male sex reversal.

Based on the Fgf9/Wnt4 double mutant studies, we propose a two part model of male sex determination in which both the activation of male genes and repression of female genes is required. Also, this work demonstrates that the repression of the female-primed gene Wnt4 is required for male development, and Fgf9 is one factor that leads to the repression of female-primed genes.

The gonad is a unique primordial organ that retains the ability to adopt one of two morphological fates through much of mammalian embryonic development. Previous work in our lab found that dimorphic vascular remodeling was one of the earliest steps during sex-specific morphogenesis. In particular, vessels in XY gonads display highly ordered behavior that coincides with testis cord formation. It was unknown how the vasculature may influence testis cord morphogenesis and, if so, how this was mechanistically related to sex determination. The work in this thesis addresses a single over-arching hypothesis: Male-specific vascular remodeling is required for testis morphogenesis and orchestrates differentiation of the XY gonad.

To address this question we have modified and developed techniques that allow us to isolate aspects of vascular behavior, gene expression, and endothelial influence on surrounding cells. In particular, the application of live imaging was instrumental to understanding the behavior of various gonadal cell-types in relation to remodeling vessels. It is difficult to grasp the complexity of an organ without understanding the dynamics of its constituents. A critical aim of my work was to identify specific inhibitors of the vasculature that do not affect the early stages of sex determination. Combining inhibitors, live imaging, cell sorting, qRT-PCR, mouse models, and whole organ culture has led to a far richer understanding of how the vasculature behaves and the cell-types that mediate its influence on organ morphogenesis. The beauty of our system is that we do not have to settle for a snapshot of the fate of cells in vivo, but can document their journeys and their acquaintances along the way.

Vascular migration is required for testis cord morphogenesis. Specific inhibitors revealed that in the absence of vessels, testis cords do not form. The work below shows that vessels establish a feedback loop with mesenchymal cells that results in both endothelial migration and subsequent mesenchymal proliferation. Interstitial control of testis morphogenesis is a new model within the field. The mechanisms regulating this process include Vegf mediated vascular remodeling, Pdgf induced proliferation, and Wnt repression of coordinated endothelial-mesenchymal dynamics. Our work also suggests that vascular patterning underlies testis patterning and, again, is mediated by signals within the interstitial space not within testis cords themselves.

A final aspect of my work has been focused on how vessels continue to influence morphology of the testis and the fate of surrounding cells. Jennifer Brennan, a graduate student in our lab, previously showed that loss of Pdgfrα antagonizes cord formation and development of male-specific lineages. The mechanisms and cell-types related to this defect were not clear. I began to reanalyze Pdgfrα mutants after finding remarkable similarity to gonads after vascular inhibition. This work is providing data suggesting that vessels are not simply responsible for testis morphology but also for the fate of specialized cells within the testis. On the whole, this thesis describes specific roles for endothelial cells during gonad development and mechanisms by which they are regulated.