Mechanisms of neural specification and patterning during planarian brain regeneration

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Decades of elegant work have described neural development in the embryos of many model organisms, which has shown that neural development is the precise coordination of neural stem cells in space and time in order to make a functioning nervous system with appropriate cell-type ratios. However, it is not known how these developmental principles apply to adult organisms as neurons are turned over (homeostasis) or are replaced following injury (regeneration). In our first NSERC funding term, we started to investigate adult neural homeostasis and regeneration using the freshwater planarian (flatworm; Schmidtea mediterranea) due to its high level of neural homeostasis and the ability to regenerate a new brain from scratch within 7 days. We hypothesized that we could take a candidate approach to finding a transcription factors responsible for neural fates in planarians. Using molecular tools and RNA-interference to test gene function, we discovered that planarians specify and regenerate serotonergic neurons using the homeobox transcription factors lhx1/5 and pitx. Furthermore, in the central brain, bHLH transcription factors specify neural cell types and are expressed in some of the stem cells adjacent to the brain. It is this last observation that drives the current proposal: How are brain-adjacent stem cells coordinated by signaling pathways to make the proper number and type of neurons, and do the stem cells differ in potential between each other (i.e. heterogeneity)? Here we will test this biological question using both biased and unbiased approaches. Biased on our previous work, we know that a conserved signaling pathway, Hedgehog, is transcribed by a specific population of neurons in the brain. We propose to test whether Hedgehog is being used as a signal from neurons, to adjacent stem cells, in order to control rates of neuronal homeostasis. Furthermore, we hypothesize that not all brain-adjacent stem cells respond to Hedgehog, which implies that they are heterogeneous in their competence to respond. We propose that this can be tested by taking an unbiased approach using single-cell RNA-deep sequencing of brain-adjacent stem cells to determine whether these stem cells are 1) heterogeneous, and 2) whether we can find a novel populations of neural stem cells in planarians. From this, we will also find novel stem cell regulators involved in brain homeostasis and regeneration in planarians. Impact of NSERC: We are the only lab in Canada to use planarians to study brain regeneration, and all of our neural regeneration research is exclusively funded by NSERC.