Regulation of proliferation and differentiation in the male germ line adult stem cell lineage

Stanford University, Stanford CA

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Abstract

Project Summary / Abstract Our research is aimed at understanding the molecular mechanisms that regulate proliferation and differentiation in adult stem cell lineages. These events are of crucial importance for both short and long term health, as adult stem cell lineages underlie continued replacement of high turn over cell types in blood, skin, and the epithelial lining of the gut, as well as maintenance and repair of many other tissues in the body. Our primary focus is on elucidating the molecular circuitry that regulates the switch from precursor cell proliferation of onset of lineage appropriate differentiation. Failure to cleanly switch from proliferation to differentiation may underlie genesis of cancer in stem cell lineages, while failure to establish cell type specific gene expression for the proper differentiation program may lead to dysgenesis, scarring, fibrosis, or tissue degeneration. We utilize spermatogenesis in Drosophila as a model adult stem cell lineage in which the switch from proliferation to differentiation is especially amenable to study in vivo using an integrated combination of powerful genetic, genomic, cytological and biochemical tools. During spermatogenesis, mitotically proliferating spermatogonia switch to onset of meiotic prophase, during which spermatocytes express hundreds of novel transcripts required to regulate and mediate the dramatic changes in cell behavior and morphogenesis that eventually produce mature sperm. Our goal under the MIRA is an integrated, systems level understanding of how the different molecular mechanisms we have uncovered act in a pathway to trigger a clean switch from precursor cell proliferation, establish a stable program of cell type-specific gene expression, and regulate its execution to time and drive key steps in differentiation. Our approach will build upon recent technical advances and new discoveries made in the current funding period. Springing from our discovery that the key role of the RNA binding protein Bam required to trigger the switch from mitosis to meiosis is to repress expression of the nuclear RNA binding protein How, we will identify RNAs bound by How by immunoprecipitation, score for alternative splicing events that change immediately after How protein disappears from spermatogonia, and assess function of candidate How targets in vivo, for example, whether they modulate expression of cell cycle regulators or bind to promoters of differentiation genes as potential transcriptional regulators. Based on our finding that cell type specific isoforms of RNA binding proteins expressed in the first wave of spermatocyte transcription modulate alternative splicing, stability or translation of selected mRNAs expressed in the second wave, we will investigate how early onset RNA binding proteins regulate timing and execution of the meiotic cell cycle and cell type specific post meiotic cellular differentiation. Exploiting our genome wide, stage specific transcriptomic analysis, we will work upstream from the earliest promoters that turn on in young spermatocytes to explore potential transcription factors, chromatin regulators and nuclear architecture that may link onset of the gene expression program for terminal differentiation with action of How to stop mitotic proliferation and trigger onset of the meiotic program.