UC Merced

Regional signals along the anteroposterior (AP) body axis influence the growth of normal and neoplastic cells. The regenerative response, cellular turnover and the aggressiveness of proliferative cells are superior in the top-half of the body than in the posterior region. However, the mechanisms underlying regional differences in the adult body have yet to be elucidated. Studies in several model systems have suggested that SUMO modification system may be responsible for regulating the regional survival of cells. Small ubiquitin-like modifier (SUMO) proteins can alter function of other proteins through reversible post-translational modification. SUMOylation has been implicated in a range of processes associated with the regulation of cellular fate decisions during cell cycle progression and apoptosis. The planarian flatworm Schmidtea mediterranea is an emerging model organism that allows analysis of stem cell-mediated tissue turnover and regeneration in the context of the whole body. We identify that post-translational modifications through SUMOylation are evolutionarily conserved in the Lophotrochozoan Schmidtea mediterranea. Disruption of SUMOylation with RNA-interference of the E2 conjugating enzyme UBC9 lead to regional defects characterized by loss of the posterior half of the body and lethality. In addition, UBC9 RNAi uniformly reduced the number of adult stem cells in planaria, which was linked to an inability for cells to undergo proper cell cycle transition. In addition, the loss of tail phenotype was determined to be caused by a marked increase in cell death specific to the posterior. Further evidence indicates that UBC9-induced regional cell death is due to inhibition of Hedgehog signaling through an early increase in expression of its repressor Patched, a regional-specific cue. Furthermore, UBC9 is essential for planarians to mount a robust regenerative response to injury, suggesting a novel function for the SUMOylation pathway. Our results evaluating UBC9 function in the adult organism uncover that in addition to organ specific and systemic events, SUMOylation also regulates regional cell fate during tissue renewal and regeneration.

Intriguingly, UBC9 downregulation leads to loss of genomic integrity characterized by the appearance of double stranded breaks and abnormal chromosomes throughout the planarian body axis. It was found that UBC9 knockdown restricts nuclear translocation of RAD51, a key regulator of homologous recombination repair. However, some cells in near the head continued to proliferate with DNA damage, leading to a cancer-like state. To dissect the molecular events driving anterior tissues survival in the presence of DNA damage, we performed RNA sequencing on anterior and posterior fragments from control and UBC9 RNAi animals. Our analysis revealed 37 differentially regulated genes that are specific to the anterior region of UBC9 animals. One of these genes, MADD (MAP kinase activating death domain), is known to act as a negative regulator of apoptosis and is overexpressed in neoplastic cells. In planarians, MADD is predominantly expressed in the nervous system and confined to post-mitotic cells. To better characterize MADD function, we downregulated its expression via RNAi. Knockdown of MADD led planarians to oversecrete mucous, fission excessively and exhibit poor motor function. MADD RNAi animals displayed a regional difference in mitotic activity across the AP axis, where stem cells were lost in the anterior but not in the posterior. In addition, MADD knockdown animals were unable to fully regenerate their cephalic ganglia and displayed reduced mitotic activity after amputation. Additionally, double knockdown of MADD and UBC9 lead to a rescue of the morphological defects associated with the UBC9 phenotype. Superficially, MADD knockdown attenuates the dramatic increase in posterior cell death in UBC9 RNAi animals that leads to posterior tissue loss. Taken together, these findings suggest MADD is a regional regulator of cell death and long-range neural signals play an important role in determining cell fate across the planarian body axis. Furthermore, this finding is critical as it provides validation of our transcriptomic analysis and approach to identify novel regulators of regional tissue maintenance.