Dissecting the neural stem cell state during regeneration using zebrafish and planarians - Project Summary/Abstract The regenerative capacity of the central nervous system (CNS) is severely limited in humans and other mammals, such that neural injury is often irreparable. Thus, damage to the CNS is a catastrophic event and often results in permanent disability, as regenerative medicine lacks an effective strategy to overcome neural loss. To potentially overcome this damage, new neurons are ultimately made from stem cells, yet the genetic programs that stem cells use to regenerate lost neurons remains unknown. Understanding how damaged cells are replaced and how new cells functionally integrate into the existing tissue is of fundamental importance in understanding neural plasticity, and neural injury or degeneration. This proposal utilizes zebrafish and planarians, both powerful models of CNS regeneration. First, I will use larval zebrafish to identify the factors that define neural stem cells (NSCs) in the spinal cord that are activated in response to neural injury. I used a single-cell RNA-sequencing approach; over 40,000 cells were sequenced in total from spinal tissue spanning a week of recovery after injury. This dataset serves as an important guide for the experiments proposed here because neural cells can be isolated using known markers and I have identified injury-enriched neural cell clusters. In Aim 1, I seek to validate transcriptomic data with in vivo gene expression analysis to determine the molecular heterogeneity of NSCs and to perform clonal analysis, using a multicolor cell labeling technique, to understand if heterogeneous NSC populations give rise to diverse cell types. I will also use a conditional cell ablation strategy to determine how each stem cell population is involved in spinal cord regeneration. Experiments described in Aim 2 will investigate one candidate gene whose expression is upregulated in SCI: cytokine receptor-like factor 1a (crlf1a). I have found that crlf1a labels a unique population of NSCs that are potentially de-differentiated ERGs, activated in response to injury. I will determine what stem cell factors are co-expressed in crlf1a+ cells, and I will use a conditional mutagenesis strategy to remove crlf1a from NSCs to determine a functional role during spinal cord regeneration. Aim 3 will determine if cytokine signaling through the ancestral receptor unit, GP130, is required for whole-brain regeneration in planarians. The findings generated from this research will elucidate the complicated biology of stem cell lineage development in the CNS and, in turn, impact understanding of how the neural stem cell state can instruct proper neurogenesis following neural injury. Neural cell types are highly conserved, so we expect these findings to be broadly applicable to human neural injuries, diseases, and therapies. The experiments, lab environment, and additional training opportunities outlined in this proposal will present a phenomenal training experience that will enhance my technical skillset, further develop my knowledge of stem cell biology, and allow me to carve out a research area to lead as an independent investigator with my own laboratory.
