1. Identify the sources of somatic cell lineages in the fetal gonads and investigate how they acquire their organ-specific identities This project identifies the transcription factor RUNX1 s a novel regulator of granulose cell fate The identity of the gonads hinges upon the balance between pro-testis and pro-ovary forces. It is clear that the fate determination process of the testis is linear and sequential: Removal of one of the top regulators (i.e. SRY or SOX9) has a domino effect that lead to complete testis to ovary sex-reversal. However, this is not the case in the mouse ovary, where no single-gene loss/mutation results in a complete ovary-to-testis sex-reversal. This sex difference implies a multi-component system in action in the ovary. In this study, we discovered unexpectedly the transcription factor RUNX1 as a new player in the fate specification process of the ovary. RUNX1 is known for its critical role in the differentiation of hematopoietic cells and its link to acute myeloid leukemia in humans. In the ovary, however, RUNX1 acts to maintain the identity of the ovary through an interplay with another ovarian transcription factor FOXL2. Using a combination of mouse genetic models and transcriptomic and genome-wide chromatin approaches, we uncovered RUNX1 functions as anti-testis factor that antagonizes the appearance of the testis program in the fetal mouse ovary. We also found that RUNX1 is enriched in the fetal ovary of various vertebrate species including humans, goats, rainbow trout, and turtles, suggesting its conserved role in ovarian differentiation. Defects in ovarian differentiation have dire consequences on reproductive outcomes of women, from sex-reversal to infertility, and the molecular mechanisms behind these defects are often not identified. Our findings provide new insights into the genomic control of ovarian differentiation, and pave the way for the identification of novel transcription factors and cis-signatures contributing to the normal functions and pathology of the ovary. The topic of granulosa cell development is fundamentally important, given that disorders in ovarian cell differentiation are implicated in ovarian diseases such as polycystic ovary syndrome (PCOS), premature ovarian failure, and ovarian cancers. 2. Define the cellular and molecular processes that lead to sexually dimorphic establishment of the reproductive tracts Before sexual differentiation occurs, embryos are anatomically bisexual as they possess both male and female reproductive tracts. These two tracts derive from two separate progenitor systems in the fetal mesonephros: Wolffian duct for the male tract and Mllerian duct for the female tract. In the male embryos, Wolffian ducts are maintained by testis-derived androgens while Mllerian duct undergo regression induced by anti-Mllerian hormone, also a product of testes. Female embryos, which do not produce androgens or anti-Mllerian hormone (AMH), experience the opposite where Mllerian ducts are maintained and Wolffian ducts undergo regression. In search of novel regulators in this process, we discovered the presence of orphan nuclear receptor COUPTFII in the mesenchyme of the mesonephros. Inactivation of COUPTFII specifically in the mesonephric mesenchyme leads to maintenance of both Wolffian and Mllerian ducts in the male and female mouse embryos, a typical case of pseudohermaphroditism. The affected embryos still have sex-specific production of hormones (androgen and AMH), indicating that hormonal abnormality is not responsible for the pseudohermaphroditic phenotypes of the reproductive tracts. Instead, COUPTFII in the mesenchyme of the mesonephros appears to be a molecular switch that controls the decision-making process of the identity of reproductive tract progenitors. This novel finding provides new mechanistic insights into the dimorphic establishment of reproductive tract. Ongoing experiments are to identify the upstream and downstream regulators of COUPTFII in this process. 3. Investigate the effects of in utero exposure to endocrine disruptors on the development of fetal reproductive organs and its lingering impacts on fertility in adulthood Formation of fetal reproductive organs relies on an intricate interaction between steroid hormones and signaling molecules, therefore making this process a prime target of endocrine disruptors. Chemicals or compounds that mimic or interfere with the action of steroid hormone and signaling molecules are known to have detrimental impacts on fetal reproductive organ formation and long-term impacts on fertility whenthe affected animals reach adulthood. Arsenic, a human carcinogen found in underground water and food products, has been shown to affect the Hedgehog pathway, one of the signaling pathways important for the formation of reproductive organs. Exposure of moues embryos to arsenic leads to cancer development in the ovary and reproductive tracts, organs where the Hedgehog pathway is functional. To investigate whether arsenic exposure targets the Hedgehog pathway in the fetal gonads and reproductive tract, we expose pregnant mice with various doses of inorganic arsenic in the drinking water. We found a dose-dependent and organ-specific effect of arsenic exposure on the Hh pathway: arsenic activates the Hedgehog pathway in the ovary whereas decrease its activity in the fetal testis. When some of the exposed female fetuses are allowed to develop to adulthood, they exhibit precocious onset of puberty (vaginal opening) compared to the control littermate. The Hedgehog pathway is known to control steroidogenesis in the ovary and testis. We therefore hypothesize that altered Hedgehog activity by in utero arsenic exposure could contribute to premature steroid (i.e. estrogen) production and consequent early onset of puberty in the affected female. Our findings are the first to demonstrate a potential impact of arsenic exposure on female reproductive systems. We are collaborating with Jean Harry and Mike Walkkes at NTP to study the neurological impacts of arsenic on these animals.
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