Molecular mechanisms underlying sensory neuron regeneration and function

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Project Summary/Abstract SoxB1 transcription factors, which play prominent roles in maintaining stem cell potency and organismal development, are expressed in adult tissues and have key roles in regenerative processes. Although SoxB1 genes have been implicated in diverse processes in adult animals, their mechanism of action in regulating stem cells and regeneration in vivo is poorly understood. A major obstacle in the field is that most model organisms have limited regenerative capacity or scarce stem cell populations. We propose to use the planarian Schmidtea mediterranea as a model to investigate the function of SoxB1 genes in tissue regeneration. Planarians are capable of regenerating complete worms from very small body fragments, an ability that is conferred by a population of adult pluripotent stem cells. My laboratory discovered that inhibiting the S. mediterranea SoxB1 gene, soxB1-2, causes animals to exhibit striking seizure-like movements. Molecular analysis revealed that soxB1-2 is expressed in planarian stem cells and is required for regeneration and maintenance of epidermal and sensory neuron populations in planarians. However, the mechanism underlying soxB1-2+ stem cell differentiation remains largely unknown. We hypothesize that soxB1-2 functions as a pioneer transcription factor that primes stem cells for acquiring ectodermal cell fates and its sustained activity is required for differentiation and function of sensory neuron subpopulations. Analysis of soxB1-2 function will provide insights into conserved gene targets required for stem cell regulation and mechanisms by which terminal differentiated cells maintain their fates throughout life. Aim 1 will determine which stem cell and differentiated cell types express soxB1-2 in S. mediterranea by mining >100,000 new single-cell gene transcriptomes. We will create predictions of soxB1-2+ cell developmental trajectories that can be experimentally assessed with high-throughput in situ hybridization combined with established cell-type specific markers. Aim 2 will identify genes regulated by and co-expressed with soxB1-2 in distinct sensory neuron populations by performing RNA-sequencing experiments after surgically isolating sensory organ regions from control and soxB1-2 RNAi-treated planarians. Differentially expressed genes will be compared to single cell transcriptomes to determine cell type-specificity, and validated by in situ hybridization. Additionally, we will establish an ATAC-seq or employ a ChIP-seq approach to identify direct genomic targets of SoxB1-2 in planarian stem cells. Aim 3 will use RNAi experiments to analyze soxB1-2-regulated genes that are required to confer specialized sensory cell fate and function. To define which genes are required for restoring specific senses, novel behavioral assays will be employed to establish the gene knockdowns that impair sensory modalities like chemo- and mechanosensation. Given the wide range of cell types that express SoxB1 genes in mammals, the proposed work will offer insights into how its sustained transcription co-regulates maintenance of cell type-specific gene modules indispensable for normal tissue homeostasis or repair.