Temporal Transcriptional Profiling of Somatic and Germ Cells Reveals Biased Lineage Priming of Sexual Fate in the Fetal Mouse Gonad

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. 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 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. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates

Vascular Influence During Patterning and Differentiation of the Gonad

<p>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. </p><p>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. </p><p>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. </p><p>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 Pdgfr&#945; 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 Pdgfr&#945; 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>Dissertatio

Vascular-mesenchymal cross-talk through Vegf and Pdgf drives organ patterning

The initiation of de novo testis cord organization in the fetal gonad is poorly understood. Endothelial cell migration into XY gonads initiates testis morphogenesis. However, neither the signals that regulate vascularization of the gonad nor the mechanisms through which vessels affect tissue morphogenesis are known. Here, we show that Vegf signaling is required for gonad vascularization and cord morphogenesis. We establish that interstitial cells express Vegfa and respond, by proliferation, to endothelial migration. In the absence of vasculature, four-dimensional imaging of whole organs revealed that interstitial proliferation is reduced and prevents formation of wedge-like structures that partition the gonad into cord-forming domains. Antagonizing vessel maturation also reduced proliferation. However, proliferation of mesenchymal cells was rescued by the addition of PDGF-BB. These results suggest a pathway that integrates initiation of vascular development and testis cord morphogenesis, and lead to a model in which undifferentiated mesenchyme recruits blood vessels, proliferates in response, and performs a primary function in the morphogenesis and patterning of the developing organ

Nucleoporin-mediated regulation of cell identity genes

The organization of the genome in the three-dimensional space of the nucleus is coupled with cell type-specific gene expression. However, how nuclear architecture influences transcription that governs cell identity remains unknown. Here, we show that nuclear pore complex (NPC) components Nup93 and Nup153 bind superenhancers (SE), regulatory structures that drive the expression of key genes that specify cell identity. We found that nucleoporin-associated SEs localize preferentially to the nuclear periphery, and absence of Nup153 and Nup93 results in dramatic transcriptional changes of SE-associated genes. Our results reveal a crucial role of NPC components in the regulation of cell type-specifying genes and highlight nuclear architecture as a regulatory layer of genome functions in cell fate

Nucleoporin-mediated regulation of cell identity genes

The organization of the genome in the three-dimensional space of the nucleus is coupled with cell type-specific gene expression. However, how nuclear architecture influences transcription that governs cell identity remains unknown. Here, we show that nuclear pore complex (NPC) components Nup93 and Nup153 bind superenhancers (SE), regulatory structures that drive the expression of key genes that specify cell identity. We found that nucleoporin-associated SEs localize preferentially to the nuclear periphery, and absence of Nup153 and Nup93 results in dramatic transcriptional changes of SE-associated genes. Our results reveal a crucial role of NPC components in the regulation of cell type-specifying genes and highlight nuclear architecture as a regulatory layer of genome functions in cell fate

Research software engineering accelerates the translation of biomedical research for health

Research software engineering is central to data-driven biomedical research, but its role is often undervalued and poorly understood

Data from sorted <i>Sf1-EGFP</i> cells also supported female-biased priming for supporting cells.

<p>(A–B) Graphical illustrations of the genes included in our analysis of priming in the <i>Sf1-EGFP</i> data. Because the <i>Sf1</i>-positive population is a mixture of lineages, we used two methods to identify the primed genes associated with supporting cells. XY cells are illustrated in this example, but the same operations were also performed for XX cells. (A) “<i>Sf1</i> primed and supporting cell enriched” genes were both male-primed in the <i>Sf1-EGFP</i> data (comparing E11.0 and E12.5) and lineage-specifically enriched in our XY <i>Sry-EGFP/Sox9-ECFP</i> purified supporting cells at E12.5. Red indicates genes being removed from the analysis, and green indicates genes being retained. (B) For the “<i>Sf1</i> primed, removing interstitial/stromal genes”, we removed genes associated with the interstitial/stromal cells at E12.5 (i.e., sexually dimorphic in the interstitium/stroma) from the <i>Sf1-EGFP</i> primed genes. Genes that were expressed sexually dimorphically in both the interstitial/stromal cells and the supporting cells were removed only if expression was higher in the interstitial/stromal cells than in the <i>Sry-EGFP/Sox9-ECFP</i> supporting cells. The <i>Sf1-EGFP</i> primed genes that were enriched in the <i>Sry-EGFP/Sox9-ECFP</i> supporting cells (C, D, and G) and those that were identified by removing interstitial/stromal genes (E, F, and H) were analyzed separately. (C and E) The percentages of primed genes that were male-primed and female-primed. Both methods showed a female bias. The boxes contain the p-values from the binomial test with the expected percentages of the extreme models, and all extreme models could be rejected as having a p-value<0.05. (D and F) The percentage of male or female genes that were primed showed a significant (*) bias toward the female pathway, as determined by the hypergeometric test (p-value<0.05). (G and H) However, primed genes in both sexes were predominantly expressed at similar levels in progenitors and E12.5 supporting cells of one sex. While supporting cell progenitors have a female bias, they also express some markers of the male pathway at levels similar to male supporting cells at E12.5. Gene lists and permutation tests are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002575#pgen.1002575.s005" target="_blank">Dataset S5</a>.</p

Supporting cells showed lineage priming with a female bias.

<p>(A and H) Graphs of the log-transformed, normalized intensity values of genes. The error bars are standard error. Only the values for supporting cells are shown, except in the depleted and primed example where all cell types are shown. (A) <i>Mdk</i> and <i>Rasgrp1</i> are examples of male- and female-primed genes and <i>Cenpa</i> is an example of a female-primed depleted gene. As in the germ cell analysis, we examined all primed genes (B, C, and I), primed and lineage-specifically enriched genes (D, E, and J), and primed and lineage-specifically depleted genes (F, G, and K). (B, D, and F) The percentages of primed genes that were male-primed and female-primed. The boxes contain the p-values from the binomial test with the expected percentages of the extreme models. (B) Using the first method, all of the extreme models could be excluded because they had a p-value<0.05. (D and F) However, using the second and third methods, the balanced and female models could not be excluded, respectively. (C, E, and G) Nevertheless, examining the percentage of male or female genes that were primed, all methods showed a significant (*) bias toward the female pathway, as determined by the hypergeometric test (p-value<0.05). Taken together, the data supported female-biased priming. (H) Graphs illustrating two primed genes, whose expression in the progenitor is “similar” to the differentiated cell of one sex, or “intermediate” between the two sexes. (I–K) The female-primed genes were predominantly similarly expressed, but the male-primed genes showed more variability. Gene lists and permutation tests are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002575#pgen.1002575.s004" target="_blank">Dataset S4</a>.</p

Models of differentiation for the different gonadal lineages.

<p>The interstitial/stromal cells differentiate asymmetrically over the time period examined, as we detected few genes specific to the XX stroma by E13.5, whereas, the XY interstitial population acquired a larger set of lineage-specific genes. Supporting cells are primed with a female bias. The natural progression of the primed state may be to adopt the female differentiated state, but in the presence of <i>Sry</i> the cells repress the female program and adopt the male fate. Conversely, germ cells are primed with a male bias. An extrinsic signal may be required from the mesonephros to induce the adoption of the female fate; otherwise, germ cells adopt the male fate.</p