Molecular and cellular pathways driving competency for human vagal neural crest specification

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Abstract/Summary In vitro neural crest-like cells (NCCs) can be generated from human pluripotent stem cells (hPSCs). In response to retinoic acid (RA), NCCs can be patterned towards vagal neural crest identity based on gene expression and based on lineage differentiation capacity. Exciting preliminary data from the lab, acquired through the use of CellTag lineage tracing technology, indicate that the process of vagal identity acquisition actually begins at a very early differentiation stage, prior to RA exposure. Our data suggest the spontaneous appearance of vagal- competent versus non-competent populations during those early differentiation time points. Furthermore, we observed that the competent population exhibits a gene expression pattern that matches cells of the primordial ectoderm in the region that becomes the hindbrain. Understanding the process by which these early vagal- competent precursors are established, as well as the mechanism that maintains and executes vagal competence has broad implications. For NCC specification, an improved understanding of spatial patterning in the cranial region may greatly enhance our ability to generate vagal NCCs for the treatment of diseases such as Hirschsprung’s Disease. Beyond NCC patterning, our study may reveal general mechanisms of axial patterning that impact many other developing embryonic tissues including the CNS. In Aim 1 we propose to use scRNAseq to fully characterize the population that exhibits vagal competence in this system before identifying signals that selectively enrich for these cells using a double reporter hPSC line. In Aim 2 we propose to dissect the mechanism of competence with the ambitious combination of (1) parallel scATAC and scRNA sequencing in a multiomics approach to uncover regions of differential accessibility that account for distinct RA response mechanisms, and (2) application of CRISPR-a and CRISPR-i to test the ability of candidate genes to respectively program or disrupt competence in populations with distinct spatial identities.