Project Summary/Abstract Cranial neural crest cells (CNCCs) form most of the craniofacial complex. Despite being derived from an ectodermal population, CNCCs are able to differentiate into both non-ectomesenchymal (e.g., neurons/glia) and ectomesenchymal derivatives (e.g., bone, cartilage, and smooth muscle). This unique ability to form a broad range of cell types from more than one germ layer has led to CNCCs being called “pleistopotent.” However, the signaling mechanisms that convey this pleistopotency and drive differentiation of CNCCs are not well understood. The Sonic hedgehog (Shh) signaling pathway has long been studied in the context of craniofacial development. The Shh signaling pathway is mediated by bimodal Gli transcription factors (Gli TFs), which activate or repress target genes associated with proper craniofacial development. Studies done on CNCC in vitro demonstrated that exposure to recombinant Shh protein increased CNCC potency by increasing the proportion of CNCCs that differentiated into both non-ectomesenchymal and ectomesenchymal derivatives and decreased the proportion of CNCCs that only differentiated into non-ectomesenchymal derivatives. To test the role of Shh signaling in CNCC potency, we generated a conditional mouse line lacking Gli activity in CNCCs (Gli2f/f;Gli3f/f;Wnt1-Cre). Single cell RNA-sequencing analysis showed that E13.5 Gli2f/f;Gli3f/f;Wnt1-Cre CNCCs were not separated into distinct clusters as compared to the wild-type CNCCs. Interestingly, while ectomesenchymal clusters were poorly defined molecularly, non-ectomesenchymal clusters were relatively unaffected. Thus, these data suggested that loss of Gli TFs led correlated with a loss of pleistopotency. Based on these preliminary data I hypothesize that Gli-mediated Shh signaling conveys CNCC potency via activation of pluripotent programs and that loss of Gli2/3 signaling limits potency, thus preventing differentiation into the full range of CNCC derivatives. In Aim 1, I will perform CUT&RUN for Gli2 and Gli3 on embryos during CNCC induction to look for binding of Gli TFs at traditional pluripotency markers. I will perform single cell RNA- sequencing on both wild-type and Gli2f/f;Gli3f/f;Wnt1-Cre embryos before, during, and after CNCC induction and specification and compare gene expression profiles to determine if loss of Gli TFs affects expression of potency factors in CNCC, which I will validate with RNAscope. Finally, I will perform Western blots for Gli2 and Gli3 to determine if Gli TFs are predominantly functioning as activators or repressors during CNCC induction. In Aim 2, I will determine if loss of Gli TFs restricts fate commitment of CNCCs. I will generate Gli2f/f;Gli3f/f;R26R-Confetti+/+; Wnt1-Cre embryos, where Wnt1-Cre drives Cre-recombination before establishment of CNCC pleistopotency, and Gli2f/f;Gli3f/f;R26R-Confetti+/+;Sox10-Cre embryos, where Sox10-Cre drives Cre-recombination after establishment of CNCC pleistopotency. This study is impor...