Investigation of the doublesex and mab-3 related transcription factors (Dmrts) in mammalian germ cell development

My thesis work is focused on mammalian germ cell development, specifically focusing on the Dmrt (Doublesex and mab-3 related transcription factor) genes, which play key roles in gonad and germ cell development in a broad range of species. Male mammals produce prodigious quantities of sperm continuously over a long reproductive life. What makes this possible is a population of spermatogonial stem cells (SSCs), which support a complex differentiation program that includes a series of proliferative spermatogonial divisions followed by meiosis and spermiation. My work has focused on how spermatogonial development is coordinated and controlled to sustain fertility.;For my thesis research, I have aimed to understand how two related transcription factors, DMRT1 and DMRT6, coordinate mammalian spermatogenesis, from spermatogonial stem cell (SSC) development to meiotic entry.;DMRT6 is expressed only in differentiating spermatogonia and is a direct target of DMRT1. Therefore, I hypothesized that DMRT6 functions downstream of DMRT1 in spermatogenesis to regulate the final steps leading to meiotic entry. I generated a conditional Dmrt6 allele and found that mutants on the C57BL/6J strain have severe defects in spermatogonial development leading to infertility. This phenotype was caused by inappropriate expression of spermatogonial differentiation factors and resulted in most late spermatogonia undergoing apoptosis. On the 129Sv background, mutant germ cells could complete spermatogonial differentiation and enter meiosis, but they showed defects in meiotic chromosome pairing, establishment of the XY body, and processing of recombination foci, and they mainly arrested in mid-pachynema. I also performed mRNA profiling of Dmrt6 mutant testes together with DMRT6 ChIP-seq to show that DMRT6 represses genes involved in spermatogonial differentiation and activates genes required for meiotic prophase. My results indicated that DMRT6 promotes meiotic entry by shutting down the spermatogonial differentiation program, and at the same time activates key meiotic genes to coordinate the transition from mitosis to meiosis. This finding was significant because previous studies only identified genes that function in either mitotic or meiotic development but I identified a gene that bridges this gap to coordinate the transition between these two developmental programs.;DMRT1 is expressed in spermatogonia, and its expression is silenced when cells enter meiosis. Previous work from our lab showed that DMRT1 controls the mitosis/meiosis switch. I focused on its roles early in spermatogonial development. Because DMRT1 is expressed both in SSCs and committed progenitor cells (spermatogonia), I hypothesized that DMRT1 is also required for SSC development. My work with Dmrt1 in mice has revealed that Dmrt1 is required to maintain SSCs during steady state spermatogenesis, where it appears to transcriptionally activate critical regulators of SSC maintenance including Plzf. I also found that Dmrt1 is required for regeneration of SSCs after cytotoxic stress. Committed progenitors (Ngn3-positive cells) normally do not contribute to SSCs, but can do so when spermatogonia are chemically depleted. I found that when Dmrt1 is lost in committed progenitor cells, there is no regeneration of SSCs and no recovery from germ cell depletion. Thus my data reveal that Dmrt1 plays two distinct roles supporting SSC homeostasis, allowing SSCs to remain in the stem cell pool under normal conditions, and allowing committed progenitors to return to stemness when germ cells are depleted. My data also show that Ngn3-positive germ cells can re-express Id4 under conditions of cytotoxic stress. This finding helps reconcile competing SSC models that are likely to be discussed by other groups at the 2016 meeting.;Together these studies of Dmrt1 and Dmrt6 will increase our understanding how germ cells are shepherded through the important process of spermatogenesis and how the stem cell population supporting these processes is maintained. My research will be significant to the field of reproductive biology because it furthers our mechanistic understanding of two key points in germ cell development: regulation of the stem cell pool and control of the transition from mitotic to meiotic development. My work ultimately may help provide novel treatments for infertility and new strategies for contraception.