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    Matthew Francois Pech.
    Telomerase is required for maintaining the male germline in worms, fish and mice. Underlying the continuous production of sperm in these species are poorly characterized germline stem and progenitors. Telomere attrition is strictly avoided during sperm production, thus avoiding the inter-generational transmission of ageing phenotypes. The work described in this dissertation aims to answer fundamental questions regarding the behavior of germline stem cells and the control of telomerase within those cells. Firstly, we describe the generation of telomerase reverse transcriptase reporter mice, allowing the prospective isolation of germ cells with discrete telomerase levels, and uncovering the basis principles of telomerase regulation in vivo. We discover that high telomerase levels are a hallmark of the male germline stem cell lineage, regardless of proliferation status, and that telomere attrition depletes the stem cell pool. Secondly, we exploit the pattern of telomerase expression in the adult testis to understand germline stem cell heterogeneity. We fractionate purified spermatogonia into GFRa1+ and GFRa1- populations, enabling functional studies by transplantation and molecular characterization by RNA-Seq. Although GFRa1+ cells were thought to represent the predominant self-renewing population by lineage tracing, GFRa1+ and GFRa1- cells exhibit comparably high stem cell activity by transplantation. The transition from GFRa1+ to GFRa1- is accompanied by a reduction in GDNF and FGF signaling and a marked decrease in proliferation. Upon transplantation, GFRa1- cells replenish the GFRa1+ population, revealing that GFRa1- cells are in a poised state, retaining stem cell potential and the ability to be reprogrammed by a vacant niche. In the final part of this dissertation, we again exploit TERT-reporter mice to determine the transcriptomes of defined spermatogonial subtypes. These datasets established a pipeline for the rapid discovery and validation of new markers for undifferentiated spermatogonia (MSI2 and SDC4), differentiated spermatogonia (ALCAM), GFRa1- cells (OCT4-delta-PE-GFP), and finally GFRa1+ cells (MCAM).
    Digital Access   2015