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Abstract
Adult stem cells undergo asymmetric cell division to self-renew and to produce differentiated cells throughout the life of an organism. This increases the risk of replicative senescence or neoplastic transformation due to mutations that accumulate over many rounds of DNA replication. The immortal strand hypothesis proposes that stem cells reduce the accumulation of replication-induced mutations by retaining all the older template DNA strands. In addition, other models have also been proposed in which stem cells non-randomly segregate only a subset of chromosomes for different reasons, such as retention of epigenetic memories. However, the mechanism and the biological relevance of these chromosome asymmetries remain elusive. This is primarily due to the lack of model systems in which chromosome asymmetries can be assessed in the context of other asymmetries, such as cell fate.
The Drosophila melanogaster testis is one of the few well-characterized model systems that enable a detailed study of the regulation of stem cells. To elaborate, unlike many other model systems Drosophila male germline stem cells (GSCs) can be unambiguously identified at single-cell resolution. Further, GSCs divide asymmetrically giving rise to a stem cell and a differentiating cell, which can be readily identified in vivo enabling unambiguous identification of both asymmetric stem cell division and any other potential asymmetries such as nonrandom sister chromatid segregation.
In this thesis, I describe work where I first showed that the bulk of chromosomes are not segregated asymmetrically in dividing Drosophila GSCs, suggesting that GSCs do not retain all the older template DNA strands to maintain their genomic integrity. However, these initial results did not exclude the possibility that GSCs might be non-randomly segregating individual chromosomes. In order to unambiguously study the segregation patterns of individual chromosomes, I adapted the CO-FISH (chromosome orientation fluorescence in situ hybridization) protocol, which allows strand-specific identification of sister chromatids. Using this method, I found that sister chromatids of X and Y chromosomes, but not autosomes, are segregated non-randomly during asymmetric divisions of GSCs. These results provide the first direct evidence that sister chromatids of certain chromosomes can be distinguished and segregated non-randomly during asymmetric stem cell divisions. Further, in this work I also showed that centrosomal proteins, nuclear envelope proteins, and methyltransferase are all required for non-random sister chromatid segregation of X and Y chromosomes. This study establishes the first genetically tractable experimental model system to study chromosome strand segregation pattern with unprecedented resolution during cell division. Finally, this work suggests that non-random sister chromatid segregation in asymmetrically dividing stem cells is potentially an evolutionarily conserved mechanism that is critical for diversification of cell fates—thus establishing a new paradigm for understanding stem cell regulation.
