Year 4

The past decade has witnessed major breakthroughs in our ability to generate and manipulate embryonic stem (ES) cells or ES-like cells in vitro, and these cells are being used to model animal development, drug screening, and cell transplantation studies. Despite these advances, major gaps exist in our understanding of how adult stem cells are regulated in most organs and to what extent these cells could be directed to replace lost cells in vivo after injury or during disease. In the human central nervous system (CNS), neural stem cells, which reside in specific areas of the adult brain, are not capable of compensating for cells lost after injury or disease. Studies of organisms that can regenerate the CNS can improve our understanding how adult stem cells can be harnessed for therapeutic purposes could benefit from. The aim of our work is to gain insights into how pluripotent stem cells are regulated in vivo using freshwater planarians (non-parasitic flatworms) as model organisms. Adult planarians possess a large population of somatic stem cells, which serve to replace cells lost after wounding or during normal physiological turnover. One of the truly remarkable properties of planarians is their ability for repair and regeneration the CNS. Thus, planarians are excellent models in which to investigate fundamental mechanisms regulating stem cells. We are capitalizing on the molecular tools available to study planarian regeneration to identify and functionally characterize genes with critical roles in CNS repair.

To advance our knowledge of how regeneration and patterning is achieved in the CNS, we are analyzing when and where neurons are replaced during regeneration. During the reporting period, we have identified neural-specific transcription factor genes that are co-expressed with stem cell or progeny markers and are gamma irradiation sensitive, which strongly suggests these transcription factors label cells with characteristics of neural progenitors. We also performed experiments to label cells replicating their DNA (cycling cells) followed by in situ hybridization and found we can readily detect cycling cells expressing neural-specific genes. This raises the possibility that, similar to vertebrates, planarians may maintain a population of neural progenitor cells that act as a source for new neurons during normal physiological cell turnover. Our new results represent a step forward because we can now examine how external stimulation or genetic perturbations might influence neurogenesis in planarians and gain insights into the gene networks involved in adult neurogenesis, both of which could have important implications for understanding basic mechanisms regulating human neural stem cells in vivo. To identify genes that are differentially expressed during regeneration of the CNS, we completed high-throughput gene expression studies (microarray and RNA-seq) using samples obtained from tissue replaced in the early phases of regeneration of the planarian head after amputation. To date, we have performed gene inhibition studies for 168 genes chosen from the microarray list based on expression pattern or homology. Knockdown of 25 of these genes produced phenotypes, including loss of the stem cells, reduced or delayed regeneration, and patterning defects. We are currently performing extended knockdown experiments for 42 of the genes. Results from these on-going experiments have already revealed interesting conserved genes with potential roles in stem cell regulation. This information has the potential to contribute to our ability to manipulate pluripotent stem cells to divide and acquire neuronal fates in vivo.