Protein/nucleic Acid Interactions In Vertebrate Embryogenesis

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In vertebrates, the ectoderm is subdivided during gastrulation into four primary fates: epidermis and central nervous system (CNS) on the ventral and dorsal surfaces, respectively, and neural crest and sensory placodes located between these two domains. The goal of this project is to understand the mechanisms that determine how ectodermal cells choose among these pathways, and how the ensuing tissue patterning and differentiation are regulated, with a primary focus on the neural crest. Neural crest induction in Xenopus requires two signals, a partially attenuated BMP signal and a Wntbeta catenin signal. We have found that the transcription factor TFAP2alpha is responsible for conveying the BMP signal to the genome, resulting in activation of a broad spectrum of neural crestspecific genes. We used a hormoneinducible version of AP2alpha as a tool in conjunction with microarray analysis to identifiy target genes in both epidermis and neural crest. We are now focusing our research on determining the function of three of these genes. One is a gene we named Inca (for Induced in Neural Crest by AP2). Inca is conserved in sequence and expression pattern in frogs, mice, and zebrafish. In the frog and zebrafish, we have used antisense oligonucleotides to show that Inca is required for proper development of the craniofacial cartilage and bones and other tissues where neural crest is involved. We have also found that in frog development Inca function is required for normal gastrulation movements. Yeast twohybrid analysis has identified a p21activated kinase, PAK4, as an interaction partner with Inca. We find that overexpression of Inca alters multiple signal transduction pathways, including Rhoclass GTPase and mitogenactivated protein kinase (MAPK) cascades. These findings support a model in which Inca functions to regulate cytoskeletal dynamics during both mesoderm induction and morphogenetic remodeling and neural crest cell migration and differentiation. The second gene encodes a novel protocadherin, PCNS, which is a potential adhesion molecule that is transiently expressed in mesoderm, neural crest and developing somites. Loss of PCNS results in failure of convergent extension in dorsal mesoderm, disrupted neural crest cell migration and also in abnormal somite and axial muscle development. The third gene is a nonmuscle myosin, MyosinX, that is expressed in neural crest and sensory placodes, and is also required for proper spreading of neural crest cells on fibronectin in vitro and for normal craniofacial development in vivo. Since these genes are conserved throughout vertebrate phylogeny, we expect our findings to broad relevance to biomedical research and human health and development.