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Spermatogonial Stem Cell Establishment

精原干细胞的建立

基本信息

批准号：
9251326
负责人：
MILES Frome WILKINSON
金额：
$ 40.46万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2020-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9251326
关键词：
Address Adult Anatomy Automobile Driving Biological Biological Assay Cell Maintenance Cell Transplantation Cells ChIP-seq Coupled Data Defect Development Drosophila melanogaster Embryo Future Gametogenesis Gene Cluster Gene Targeting Genes Germ Cells Homeobox Genes Human Hypermethylation Impairment Infertility Knock-out Knockout Mice Laboratories Life Link Mammals Methods Mitotic Activity Modeling Molecular Morphology Mus Mutant Strains Mice Nature Pathway interactions Patients Perinatal Phenotype Population Positioning Attribute Reporter Reproduction Role Severities Source Spermatogenesis Spermatogonia Stem cells Testing Testis Transgenic Mice Undifferentiated Wild Type Mouse base cell type clinically relevant critical period experimental study in vivo insight knowledge base member mouse model postnatal precursor cell programs public health relevance reproductive tract sperm cell success transcription factor transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): Spermatogonial stem cells (SSCs) are critical for maintaining spermatogenesis throughout adult life. While considerable progress has been made in our understanding of SSCs, little is known about the regulatory mechanisms that initially establish these cells in mammals. In this application, we propose to fill this gap through study of a homeobox gene-Rhox10-that we find is essential for the normal initial establishment of SSCs in the mouse testis. Rhox10 is a member of a large X-linked homeobox gene cluster that my laboratory discovered some years ago. The genes in this Rhox gene cluster are preferentially expressed in the reproductive tract, raising the possibility that they encode transcription factors critical for reproduction, but there has been little direct evidence for this As one means to test their role, we recently generated whole Rhox cluster knockout (KO) mice that lack all 33 homeobox genes in the Rhox cluster. These mice have a "progressive spermatogenic decline" phenotype that strongly resembles well-established mouse models harboring SSC defects. Conditional KO of the single Rhox gene that we regarded as most likely to be responsible - Rhox10 - yielded essentially the same phenotype as KO of the entire Rhox cluster. In-depth analysis of Rhox10-KO mice revealed they have a strong defect in the ability to generate SSCs during the postnatal period when SSCs are normally first established. This deficit likely results from a differentiation defect in the precursor cells that give rise to SCCs-Pro- spermatogonia (ProSG)-based on several lines of evidence, including single-cell (SC)-RNAseq analysis. Aim 1 of this proposal is to elucidate the mechanisms by which RHOX10 promotes ProSG differentiation and SSC establishment. We will first precisely define the cell subsets and developmental pathways operating during the SSC establishment period using SC-RNAseq analysis and other methods. We will then perform the same analyses on Rhox10-KO mice to define the specific roles of the RHOX10 transcription factor in ProSG differentiation and SSC establishment. As part of this analysis, we will verify candidate ProSG marker genes we have recently identified, and screen for new ones. This is critical for the field, as, to our knowledge, no well- established ProSG marker genes have previously been identified. Aim 2 of this proposal is to delineate the molecular mode of action of RHOX10 in germ cells. Our preliminary SC-RNAseq experiments have already identified candidate RHOX10-regulated genes in germ cell subsets. We will verify these candidate targets and expand our analysis to the specific germ cell subsets that we show RHOX10 acts in in vivo. RHOX10 direct targets will be identified by a battery of approaches, including ChIP-seq analysis. Functional studies will be performed to screen for target genes important for RHOX10's function in ProSG and SSC establishment. Given that little is known about this critical period of gametogenesis, these experiments will fill a large gap in the field and will provide an unprecedented view of the role o a transcription factor in vivo.