Regulation of stem cell numbers
Controlling the number of stem cells is a primary mechanism by which homeostasis is maintained and oncogenesis is prevented. Stem cells divide to produce one daughter cell that self-renews and one that differentiates. A critical regulator of stem cell numbers in mammals is the JAK/STAT pathway. However, the mechanisms utilized by this pathway to regulate stem cell numbers have not yet been elucidated.
Drosophila provides an ideal system to address this important issue, as this function is conserved in several tissues. Unlike the redundancy of the mammalian system, Drosophila has only one jak and one stat gene (Stat92E), which allows facile in vivo analysis. Despite these advantages, nothing is known mechanistically about how this pathway controls stem cell populations in Drosophila. Previous work has shown that over-expression of the cytokine Unpaired (Upd), which activates JAK/STAT signaling, leads to an expansion of stem/progenitor cells in the eye and testis. Our current hypothesis is that activated Stat92E must regulate three distinct processes in stem cells in order to regulate their numbers: (1) increase cellular mass; (2) accelerate cell cycle progression; and (3) promote self-renewal. Since Stat92E is a transcription factor, discrete Stat92E target genes should mediate its effects on these processes. We have identified several genes with human homologs that may lie directly downstream of Stat92E and may regulate cellular growth, cell cycle and self-renewal in stem/progenitor cells. The following are current research projects in the lab:
Characterize the role of the Stat92E in the cellular growth of stem/progenitor cells. Stat92E is required for clone survival and cells lacking it are eliminated by cell competition, a process by which cells with lower metabolic activity are killed and replaced by their healthier neighbors. A central regulator of cellular growth and cell competition is dMyc, the sole Drosophila c-Myc homolog. Cells with higher levels of dMyc are super-competitors and grow at the expense of their (lower dMyc-expressing) neighbors. In contrast, cells with reduced dMyc (low-dMyc cells) are eliminated by surrounding wild type cells. We found that activating the JAK/STAT pathway rescued low-dMyc cells from competition. In addition, we determined that cells with activated Stat92E are super-competitors. We found that activated Stat92E and dMyc act in parallel to regulate cellular growth/competition. In contrast to dMyc, activated Stat92E does not increase ribosomal biogenesis. These results demonstrate for the first time that (1) activated Stat92E is a super-competitor and (2) Stat92E is the first super-competitor that acts independently of dMyc. We are currently focused on identifying the molecular mechanisms by which activated Stat92E allows cells to out-compete their neighbors.
Determine the targets of Stat92E in stem cell proliferation. Activation of JAK/STAT signaling leads to increased proliferation. Our data suggest that Stat92E regulates both G1/S and G2 cell cycle checkpoints. We are currently using genetic approaches to determine if Stat92E regulates cell cycle regulators, like Cyclin E and Cdc25, which control cell cycle progression. We are also assessing the roles of potential Stat92E target genes identified by our micro-array in regulating proliferation of stem cells.
Identify stem cell self-renewal target genes of Stat92E. Whole genome micro-array analysis identified chinmo as novel Stat92E target gene (Flaherty, 2009, 2010). Loss and gain of function in Stat92E or chinmo produce similar phenotypes in multiple tissues. To further characterize Chinmo’s actions downstream of Stat92E, we turned to the Drosophila testis, an ideal tissue in which to characterize self-renewal factors. Niche cells in the testis produce Upd, which activates Stat92E and induces the self-renewal of germline stem cells (GSCs) and Cyst stem cells (CySCs). Factors that act downstream of Stat92E to promote self-renewal in these cells are largely unknown. We find that Chinmo is expressed in GSCs and CySCs but is only required in the CySC lineage for self-renewal. Mis-expression of Chinmo in CySCs, like hyperactivation of Stat92E in these cells, results in expansion of GCSs and CySCs outside the niche. Chinmo has one BTB domain and two Zinc finger domains. The presence of these domains suggests that Chinmo may function as a transcriptional repressor or as an adaptor for Cullin 3 E3 ligases. We are now using micro-array, biochemical and genetic approaches to determine the cellular function of Chinmo and to identify its targets.