PROJECT SUMMARY/ABSTRACT Intracellular pH (pHi) dynamics are a critical regulator of cell fate changes. Our lab and others demonstrated that an increase in pHi is necessary for the differentiation and lineage specification of multiple cell types, including mouse embryonic stem cells, Drosophila ovarian follicle stem cells, mouse intestinal stem cells, and chick paraxial mesoderm. Our lab has also shown how pHi dynamics regulate cell adhesion and migration behaviors by modifying the protonation state of pH-sensing proteins, including -catenin, cofilin, talin, and focal adhesion kinase. However, the mechanisms underlying pHi regulation of cell fate changes remain unresolved. Furthermore, our understanding of pHi dynamics during embryonic development in vivo is limited, largely due to lack of appropriate models. To address this, I generated a novel system for interrogating the functional significance of pHi dynamics during embryonic development in vivo using zebrafish, a genetically tractable organism suited for in vivo live cell imaging of embryos. Using this new model, I will determine the role of pHi dynamics in the development of cranial neural crest (NC), a highly conserved vertebrate embryonic cell population that gives rise to diverse cell types, including chondrocytes, osteocytes, and odontoblasts. Many cell behaviors involved in cranial NC development are regulated by pHi dynamics in other cell types, including lineage specification, cell migration, and epithelial to mesenchymal transition. Thus, cranial NC represents an ideal model for addressing the gaps in our understanding of pHi dynamics during embryonic development in vivo. By following early NC cells through the differentiation of cranial lineages, I will test the central hypothesis that pHi dynamics regulate cranial NC development, at the stage of delamination, migration, or lineage specification. In Aim 1, I will resolve spatial and temporal pHi dynamics during zebrafish cranial NC development by in vivo live cell imaging. My preliminary data indicate a higher pHi in migratory compared with premigratory cranial NC cells. In Aim 2, I will experimentally perturb pHi in zebrafish NC cells through pharmacologic and genetic modulation of plasma membrane ion transporters and determine the effect on cranial NC cell behaviors and transcription, thus establishing the functional significance of pHi dynamics during zebrafish cranial NC development. My findings have promise to reveal new insight on the cellular and molecular factors controlling craniofacial development, with important implications for human congenital diseases and tissue repair.