| Summary: | The neural crest (NC) is a multipotent, migratory cell population that is unique to vertebrate embryos and gives rise to many derivatives, such as the craniofacial skeleton, sensory neurons and pigment cells. A complex gene regulatory network underlies the process of NC formation, which involves the early induction of the neural plate border (NPB), specification of the NC, migration of the NC away from the neural tube along distinct pathways and differentiation into diverse cell types. microRNAs (miRNAs) are a class of non-coding regulatory genes, which act posttranscriptionally to regulate gene expression. They are of widespread significance and have been implicated in many biological processes. Many miRNAs have now been identified, however, as of yet, they have not been shown to have any direct roles in early NC development. Using various molecular techniques this study has placed specific miRNAs within the complex NC gene network. These miRNAs are miR-196a and miR-219. SRNA sequencing of induced Xenopus NC tissue generated a miRNA expression profile which in combination with whole mount in situ hybridisation (WISH) revealed multiple candidate miRNAs expressed in NC. Using knockdown (KD) experiments, the depletion of miR-196a and miR-219 resulted in aberrant NC development including abnormal craniofacial cartilage development. Using luciferase assays, this study shows for the first time that miR-219 directly targets the transcription factor Eya1 in vitro. This gene lies directly upstream of the NPB marker Pax3. When miR-219 is knocked down, Pax3 expression is expanded across the surface ectoderm of the embryo suggesting the miR-219 serves to inhibit the Pax3 domain. To begin to understand the molecular mechanisms behind both this phenotype and why the NC is lost, RNA sequencing on dissected NC tissue was employed. Results from this sequencing data demonstrated that following miR-219 KD the NPB and the placodes form whilst the NC is lost. This indicates that miR-219 is playing a role in ensuring the correct specification of NC. In comparison, following miR-196a KD the NPB development is impaired and derivatives are lost (placode and NC). This implies miR-196a has an earlier role in ensuring the correct induction of the NPB possibly through fine-tuning early inducing signals such as BMP and Notch. Using the data presented in this study, the first models of how specific miRNAs could function in NC development have been formulated.
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