1,721,017 research outputs found
Hijacking Germ Cells for Cancer: Examining a 'Dead End' in Male Germ Cell Development
No full textGerm cells represent the immortal line: they are guardians of a totipotent genome and are essential for the genetic survival of an individual organism and ultimately a species. An error at any stage in development (specification, migration, colonization, differentiation, adult maintenance) can lead to one of two disastrous outcomes: (1) germ cell death or (2) unchecked growth and proliferation leading to tumorigenesis. The work in this dissertation utilizes a classic mouse model (Ter) resulting in both of these phenotypes to further explore the molecular mechanisms important for development of germ cells.
A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1Ter/Ter) results in a significant (but not complete) 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 129/SvJ 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, develop into teratomas, or what factors directly control the strain-specific phenotype variation.
Work here demonstrates that Dnd1 expression is restricted to germ cells and that the Ter mutant defect is cell autonomous. The early loss of germ cells is due in part to BAX–mediated apoptosis which also affects the incidence of tumorigenesis on a mixed genetic background. Moreover, tumor formation is-specific to the male developmental pathway and not dependent on sex chromosome composition of the germ cell (XX vs. XY). Despite normal initiation of the male somatic pathway, mutant germ cells fail to differentiate as pro–spermatogonia and instead prematurely enter meiosis.
Results here also reveal that, on a 129/SvJ background, many mutant germ cells fail to commit to the male differentiation pathway, instead maintain expression of the pluripotency markers, NANOG, SOX2, and OCT4, and initiate teratoma formation at the stage when male germ cells normally enter mitotic arrest. RNA immunoprecipitation experiments reveal that mouse DND1 directly binds a group of transcripts that encode negative regulators of the cell cycle, including p27Kip1, which is not translated in Dnd1Ter/Ter germ cells. Additionally, overexpression of DND1 in a teratocarcinoma cell line leads to significant alteration of pathways controlling the G1/S checkpoint and the RB tumor suppressor protein. This strongly suggests that DND1 regulates mitotic arrest in male germ cells through regulation of cell cycle genes, serving as a gatekeeper to prevent the activation of a pluripotent program leading to teratoma formation. Furthermore, strain–specific morphological and expression level differences possibly account for sensitivity to tumor development.</p
A Systems Level Analysis of Temperature-Dependent Sex Determination in the Red-Eared Slider Turtle Trachemys Scripta Elegans.
No full textSex determination is a critical biological process for all sexually reproducing animals. Despite its significance, evolution has provided a vast array of mechanisms by which sexual phenotype is determined and elaborated even within amniote vertebrates. The most prevalent systems of sex determination in this clade are genetic and temperature dependent sex determination. These two systems are sometimes consistent within large groups of species, such as the mammals who nearly ubiquitously utilize XY genetic sex determination, or they can be much more mixed as in reptiles that use genetic or temperature dependent systems and even both simultaneously. The turtles are a particularly diverse group in the way they determine sex with multiple different genetic and temperature based systems having been described. We investigated the nature of the temperature based sex determination system in Trachemys scripta elegans to ascertain whether it behaved as a purely temperature based system or if some other global source of sex determining information might be apparent within thermal regions insufficient to fully induce male or female development. These experiments found that sex determination in this species is much more complex and early acting than previously thought and that each gonad within an individual has the same sexual fate established enough that it can persist even without further communication between. We established a best practice for the assembly and annotation of de novo whole transcriptomes from T. scripta RNA-seq and utilized the technique to quantify the gene regulatory events that occur across the thermal sensitive period.Evidence is entirely lacking on the resolution of TSD when eggs are incubated at the pivitol temperature in which equal numbers or males and females are produces. We have produced a timecourse data set that allowed for the elucidation of the gene expression events that occur at both the MPT and FPT over the course of the thermal sensitive period. Our data suggests that early establishment of a male or female fate is possible when temperature is sufficiently strong enough as at MPT and FPT. We see a strong pattern of mutually antagonistic gene expression patterns emerging early and expanding over time through the end of the period of gonad plasticity. In addition, we have identified a strong pattern of differential expression in the early embryo at stages prior to the formation of the gonad. Even without the known systemic signaling attributed to sex hormones emanating from the gonad, the early embryo has a clear male and female gene expression pattern. We discuss how this early potential masculinization or feminization of the embryo may indicate that the influence of temperature may extend beyond the determination of gonadal sex or even metabolic adjustments and how this challenges the well-defined paradigm in which gonadal sex determines peripheral sexual characteristics.</p
A systems-level view of mammalian sex determination.
No full textPathologies of sexual development are common in humans and reflect the precarious processes of sex determination and sexual differentiation. The gonad forms as a bipotential organ, and recent results from the Capel lab revealed that it is initially balanced between testis and ovarian fates by opposing and antagonistic signaling networks. In XY embryos, this balance is disrupted by the transient expression of the Y-linked gene, Sry, which activates genes that promote the testis pathway and oppose the ovarian pathway. While the roles of a few genes have been defined by mutation, current evidence suggests that the interactions of many genes and signaling pathways are involved in the establishment of sexual fate. For example, most cases of disorders of sexual development (DSDs) are unexplained by mutations in known sex determination genes. In addition, recent microarray studies in the mouse revealed that nearly half the transcriptome is expressed in the gonad at the time of sex determination (Embryonic day 11.5, or E11.5), and as many as 1,500 genes are expressed in a sexually dimorphic pattern at this early stage. Thus the sexual fate decision in the developing gonad likely depends on a complex network of interacting factors that converge on a critical threshold. To begin to elucidate the transcription network topology underlying sex determination, we exploited two inbred mouse strains with well-characterized differences in sex reversal. The common inbred strain C57BL/6J (B6) is uniquely sensitive to XY male-to-female sex reversal in response to a number of genetic perturbations, while other strains, including 129S1/SvImJ (129S1) and DBA/2J (D2) are resistant to sex reversal. We hypothesized that these strain differences in gonad phenotype likely result from underlying expression differences in the gonad at the critical timepoint of E11.5. Using microarrays, we identified significant, reproducible differences in the transcriptome of the E11.5 XY gonad between B6 and 129S1 indicating that the reported sensitivity of B6 to sex reversal is consistent with a higher expression of a female-like transcriptome in B6 XY gonads. Surprisingly, a well-characterized master regulator of the testis pathway, Sox9, was found to be upregulated in the sensitive B6 background, which may serve as a compensatory mechanism to counteract the female-leaning transcriptome and activate the testis pathway in wild type B6 XY gonads.We extended our expression analysis to a large set of F2 XY gonads from B6 and 129S1 intercrosses. From each pair of gonads, we analyzed the expression of 56 sex-associated genes by nanoliter-scale quantitative RT-PCR (qRT-PCR). The expression levels of most genes were highly variable across the F2 population, yet strong correlations among genes emerged. We employed a First-Order Conditional Independence (FOCI) algorithm to estimate the F2 coexpression network. From this unbiased analysis of XY expression data, we uncovered two subnetworks consisting of primarily male and female genes. Furthermore, we predicted roles for genes of unknown function based on their connectivity and position within the network. To identify the genes responsible for these strain expression differences, we genotyped each F2 embryo at 128 single nucleotide polymorphisms (SNPs) located evenly throughout the 19 autosomes and X chromosome. We then employed linkage analysis to detect autosomal regions that control the expression of one or more of the 56 genes in the F2 population. These regions are termed expression quantitative trait loci, or eQTLs. We identified eQTLs for many sex-related genes, including Sry and Sox9, the key regulators of male sex determination. In addition, we identified multiple prominent trans-band eQTLs that controlled the expression of many genes. My work represents the first eQTL analysis of a developing vertebrate organ, the mouse gonad. This systems-level approach revealed the complex transcription architecture underlying sex determination, and provides a mechanistic explanation for sensitivity to sex reversal seen in some individuals.</p
Vascular Influence During Patterning and Differentiation of the Gonad
No full textThe 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.</p
Morphogenesis and Female Fate Determination in Vertebrates
No full textA unique feature of the fetal gonad is its ability to form two distinct organs, the testis and the ovary, from a single bipotential primordium. The outcome of this decision, which is made by a population of somatic cells known as the bipotential supporting cell precursors, determines whether an embryo will develop as a phenotypic male or female. Though several molecular pathways have been shown to be required for female fate determination in vertebrates, the intricacies of ovarian morphogenesis are not well understood. A key event in ovarian development occurs around birth, when meiotic germ cells and somatic granulosa cells organize into primordial follicles, the structures that generate mature oocytes for ovulation in adult females. We investigated the embryonic origins and proliferative properties of granulosa cells in the fetal mouse ovary and found that the precursors emerge from the ovarian surface epithelium and then enter mitotic arrest in a specification process that extends from the bipotential stage to the end of the postnatal follicle assembly period. Maintenance of cell cycle arrest in granulosa cell precursors appears to be regulated by Wnt signaling. The first granulosa cells to be specified were exclusively incorporated into the subset of follicles that begin to grow immediately upon assembly. We show that this first group of granulosa progenitors derives from the supporting cell precursors present in the bipotential gonad. Interestingly, both XX and XY supporting cell precursors were mitotically arrested towards the end of the bipotential period, indicating that adoption of supporting cell fate might be regulated by the cell cycle. We also show that antagonism of Notch signaling may be required for these precursor cells to exit the cell cycle and differentiate.In Witschi's classic model of vertebrate gonad development, the cortex and medulla of the undifferentiated gonad expand and differentiate in a mutually exclusive manner to yield the mature ovary and testis (Witschi 1951). Estrogen acts on both the cortex and medulla to promote female fate determination and ovary development in non-mammalian vertebrates. However, the downstream receptors and targets through which estrogen exerts its effects on the gonad have not yet been elucidated. We selected the red-eared slider turtle Trachemys scripta as a model with which to address this question. We first characterized the cellular composition of the turtle gonad before and after sex determination, identifying four populations of somatic cells distinguishable by their location within the gonad as well as the complement of transcription factors expressed. This information was then applied to an investigation of estrogen signaling pathways in the turtle ovary. We show that i) estrogen likely acts through its canonical receptors rather than a non-canonical pathway involving ERK signaling; ii) early exposure to estrogen resulted in the premature downregulation of a testis-specific gene, SOX9, in the medulla; iii) less estrogen is needed to promote ovarian differentiation in the cortex of the gonad than to repress testicular differentiation of the medulla, consistent with the localized production of estrogen in the medulla; and iv) estrogen's repressive effect on SOX9 expression may be mediated by Wnt signaling. Our findings add complexity to the standard model of how the male and female supporting cell lineages are established in mice, reveal evolutionary conservation between mice and turtles in the timing of granulosa cell specification relative to sex determination., and refine our understanding of how estrogen acts to promote ovarian development in non-mammalian species.</p
Understanding Cell Fate Decisions in the Embryonic Gonad
No full textThe 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.</p
Initiation and Maintenance of Temperature-Dependent Sex Determination in the Red-Eared Slider Turtle
No full textThe 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.</p
Reinterpreting the organizing principles of sex determination and gonadogenesis in the mouse
No full textThe 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.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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