Project Summary Worldwide, more than a third of congenital birth defects are classified as craniofacial disorders. These disorders present diversely, from abnormal formation of facial features such as the jaw or palate, to impaired sensory function. Despite advances in surgical reconstruction, there is still a poor understanding of the molecular pathophysiology that underlies distinct phenotypes. Four multipotent cell lineages are the precursors to all craniofacial cell types, forming in an anterior region of the embryo known as the neural plate border. The central-most lineages— pre-placodal and neural crest— are especially key to craniofacial development, with pre-placodal cells giving rise to supporting and sensorineural cell types, while neural crest cells become the neurons and glia of the peripheral nervous system and the bones and cartilage of all facial structures. Yet identifying the factors responsible for the initial segregation of pre-placodal and neural crest lineages has been difficult, given that the two populations intermingle closely. This study thus aims to use a genetic lineage- tracing approach to isolate the pre-placodal and crest lineages and investigate the roles of two candidate molecules in pre-placodal specification. Our lab previously discovered that the Foxi3 transcription factor is transiently expressed in border cells and that genetic deletion of Foxi3 primarily affects placode-derived structures, including loss of the inner ear. Preliminary lineage tracing with a Foxi3CreER conditional reporter mouse line generated in the lab revealed that normal Foxi3-expressing border cells mostly become placode derivatives, but some mutant cells take on alternative border lineage fates. From this data, we hypothesize that Foxi3 directly specifies the pre-placodal lineage. We will test this hypothesis by further analyzing the fate of Foxi3 mutant border cells, using lineage tracing with our Foxi3CreER mice. We will also use a single-cell multiomic approach to assess concurrent transcriptional and epigenetic changes in Foxi3 functionally null border cells. Extracellular signaling also influences border lineages: notably, BMP signals are known to induce neural crest cells. Given the similar BMP levels to which crest and pre-placodal cells are exposed, we hypothesize that BMP also affects placode cell specification. We will test this hypothesis by an in vivo, genetic manipulation of BMP targeted to pre-placodal (Foxi3CreER) versus crest (Zic5CreER) border cells. Our work introduces the first method to isolate and study the mammalian pre-placodal lineage and will shed light on critical factors that direct neural plate border cell fate. We will also gain insight to molecular changes that facilitate fate transitions at the border, with the potential to identify genes whose mutation could result in specific kinds of disruption to craniofacial development.