Understanding stem cell programs of regenerative neurogenesis using planarians and zebrafish - Project Abstract Many adult organisms, including humans, turnover a significant number of neurons during life, and many non- human organisms can regenerate substantial neural tissue following injury. New neurons in both cases are ultimately made from adult stem cells (ASCs), yet it is largely unknown how ASCs make the correct diversity of neural cell types at any given time while maintaining proper patterning and function in an otherwise healthy tissue. Understanding, at the single-cell level, how ASCs can access neural fates, make appropriate numbers and types of new cells, and then restore brain function is of fundamental importance in devising regenerative therapies for human neural injuries. However, in order to understand the mechanisms of successful neural regeneration, model organisms capable of both the biology and gene-function analyses must be used. To understand complex biology of adult neural regeneration we use complementary model organisms that can regenerate substantial parts of the nervous system following injury: the freshwater planarian and zebrafish. Planarians have the key advantages of being able to regenerate their entire brain following decapitation, as well as test gene function rapidly by RNAi during the regeneration process in vivo. Zebrafish are the best genetic vertebrate model of neural regeneration and have the advantages of live-imaging and the ability to recover from complete spinal cord transections. Only now do we have the tools in both systems to determine the conserved mechanisms of neural regeneration at the single-stem cell level. Despite much work from our lab and others, we understand little about stem cell heterogeneity or conserved stem cell programs of regenerative neurogenesis in either system. This proposal addresses these unknowns. In Aim 1, we will test four conserved transcription factors that we hypothesize drive the neural stem cell state in planarian brain regeneration. In Aim 2, we will test 57 novel factors that we have discovered in planarians that turn on specifically in neural-fated stem cells during brain regeneration, including two homologs of the conserved musashi family of RNA-binding genes. In Aim 3, we will determine the first roles of the musashi-1b gene in stem cells during spinal cord regeneration in zebrafish using conditional genetic strategies and live-clonal analyses. Similar to planarians, musashi-1b also turns on in zebrafish neural stem cells in response to injury and we will determine the RNA targets of musashi-1b in spinal cord regeneration. In total, this proposal will deliver conserved mechanisms of adult neural regeneration from stem cells that will be of broad relevance to designing regenerative therapies for human neural injuries.
