Analysis of transcriptional priming of progenitors in the vertebrate neural plate border

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Project Summary/Abstract The neural crest and ectodermal placodes, unique and defining features of the vertebrate embryo, arise from the region of the ectoderm termed the neural plate border, which is juxtaposed between the neural plate and the non-neural ectoderm of the gastrula. They constitute the origins of the craniofacial structures as well as the cranial ganglia, in addition to other diverse cell and tissue types. Malformation of these cells or their derivatives can have devastating effects on embryonic development, resulting in pathologies including craniofacial syndromes, congenital heart defects, and cancers. Nonetheless, exceedingly little is known of the genetic and epigenetic mechaniasms by which neural crest and ectodermal placode cells become specified. The goal of this proposal is to investigate the epigenetic regulation of micro RNAs (miRNAs) during specification of neural plate border cells in order to understand how this diverse pool of progenitors becomes differentially primed for the acquisition of distinct fates. Importantly, we will address this question in the context of developmentally critical vitamins, folate and Vitamin C, which are essential cofactors for DNA methylation and demethylation upstream of miRNA expression. We will combine advanced sequencing technologies with classical techniques in embryology to test the hypothesis that Vitamin C and folate are required for establishing proper DNA methylation patterns on miRNA regulatory elements to prime neural plate border cells for fate specification. Specifically, we will (1) perform transcriptome-wide profiling of miRNAs in progenitors of the neural plate and neural plate border at different developmental timepoints to uncover spatiotemporal miRNA signatures underlying differential specification, (2) perform scATAC-seq to identify differential chromatin accessibilty (i.e. putative regulatory elements), and perform oxBS-seq to reveal differential mehtyation states of these elements, and (3) determine the respective roles of the essential DNA (de)methylation cofactors folate and Vitamin C in regulating methylation states on miRNA regulatory elements during fate specifcation. This proposal seeks to provide the first transcriptome-wide analysis of miRNAs and the corresponding mRNA targets that they regulate, and the first comprehensive assessment of chromatin landscape (i.e. chromatin accessibility revealed by ATAC-seq and 5hmC/5mC revealed by oxBS-seq) in neural plate and neural plate border tissues. Moreover, results from the proposed experiments will contribute fundamental knowledge of the complex mechanisms regulating fate specification at the neural plate border. Our findings will also have important implications for the understanding of birth defects arising from malformation of the neural tube, neural crest, and ectodermal placodes. The proposed project will be carried out under the guidance of Dr. Marianne Bronner who is a leader in the field of developmental biology and an expert in neural crest development. She is the director of Caltech’s Beckman Institute where her lab has access to outstanding core facilities (e.g. advanced microscopy and sequencing centers) that will be instrumental to the successful completion of the proposed study.