Investigating the role of microRNAs in the development of Xenopus neural crest

The neural crest is a multipotent stem-cell population that is specified during early neurulation and that undergoes epithelial-mesenchymal-transition and proceeds to migrate to various points in the developing embryo where they give rise to several tissues including parts of the peripheral nervous system and craniofacial skeleton. The molecular background and detailed fine-tuning of neural crest specification is increasingly being elucidated but many questions remain. Dysregulation of neural crest results in several different diseases grouped under the term neurocristopathies. These include in-born defects like Waardenburg syndrome, presenting with Pax3 mutations, pigment defects and mild craniofacial dysmorphogenesis through to cancers such as neuroblastoma. MicroRNAs are short non-coding RNAs approximately 20 nucleotides long which affect gene expression through post-transcriptional repression and have known roles in development and disease. As part of an ongoing project microRNAs miR-196a and miR-219 were identified to be expressed in Xenopus neural crest. I have investigated the molecular pathways affected by these microRNAs by using morpholinos and developed a novel CRISPR-Cas9 knockout approach. Development of neural crest and other tissues has been evaluated using whole mount in situ hybridization of key neural crest, neural plate border and hatching gland markers including Sox10, Snail2, Pax3 and Xhe2; Alcian blue testing, q-RT-PCR, phenotype, and genotype analysis. Craniofacial and pigment phenotypes were observed following miRNA-knockouts, and miRNAknockdowns showed neural crest, neural plate, neural and placodal phenotypes