Project Summary/Abstract The Nr2f nuclear receptors are essential for the formation of the facial primordia and for patterning the upper jaw, though their specific role(s) remain incompletely defined. Zebrafish suffer a broad spectrum of phenotypes with nr2f loss of function, ranging from a striking upper-jaw-to-lower-jaw transformation in nr2f2/5 double mutants, to a severe reduction of the pharyngeal arches and an almost total loss of facial skeleton in quadruple nr2f1a/1b/2/5 and nr2f2/5/6a/6b mutants. In mice, preliminary data show that early conditional ablation of Nr2f1/2 in the cranial NC (CNC) results in a similar hypoplasticity of the pharyngeal arches and a severe reduction in the dorsal facial skeleton. The overarching hypothesis of this proposal is that the Nr2fs function in at least two discrete steps of CNC development: First, they are predicted to confer ectomesenchyme fate to a subset of CNC via activation of Twist1. Loss of Nr2f function appears to cause this population of CNC to die instead. To test this model, the fellowship candidate will compare expression of Twist1 and its downstream targets in mouse mutants and controls and attempt rescue of the fish mutant phenotypes with twist1a misexpression in the neural crest. He will perform lineage-tracing experiments in both fish and mice to determine when loss of CNC occurs and use a combination of whole mount in situ and immunofluorescence experiments to examine possible causes. The results of these experiments will be bolstered with RNA-Seq of FACS-sorted mutant mouse CNC. Completing this portion of the proposed study will add the Cre-lox conditional mutation system to his ~3 years of experience in mouse genetics, train him in zebrafish genetics, and provide broad experience in modern imaging techniques. Second, the Nr2fs are proposed to pattern post-migratory CNC ectomesenchyme to make the skeletal structures of the upper jaw distinct from those of the lower jaw. There is evidence for this later patterning role in zebrafish, but early CNC loss in the conditional mouse mutants has confounded attempts to determine conservation of function. To separate this putative patterning role from the earlier NC role, the candidate proposes to use a later-acting Cre driver to ablate Nr2f1/2 and then to examine conditional mutants for homeotic jaw phenotypes. He will also perform RNA-Seq on post-migratory CNC in these mutants to determine whether a different set of targets is dysregulated at this later stage, consistent with these Nr2f roles being discrete. Together, the proposed studies will train the candidate in a wide range of laboratory techniques and analyses, preparing him for future research as an independent PI. Consistent with the stated mission of the NIDCR, this study will add to the body of knowledge on neural crest development and craniofacial anomalies, laying the groundwork for future development of non-surgical therapies to ameliorate patient conditions.