MicroRNA miR-219 is required for neural border and neural crest development in Xenopus neurulas

At head levels, neural crest (NC) multipotent stem cells are specified in the ectoderm territory located between the anterior neural plate ectoderm and the future pre-placodal and lateral non-neural ectoderm. While the fine-tuning of NC specification is increasingly being elucidated, many questions remain, including how microRNAs may govern expression of gene programs during these processes. We have identified miR-219 as a candidate regulator of craniofacial development in the vertebrate Xenopus laevis. Here, we characterize this phenotype and explore miR-219-dependent molecular pathways by morpholino knock-down. The development of the NC and adjacent ectoderm was evaluated using whole mount in situ hybridization (WISH) of key markers (pax3, zic1, xhe2, sox10, snai2, sox2), alcian blue cartilage staining, phenotype analysis, RNA sequencing of microdissected dorsal ectoderm and microRNA rescue experiments. These complementary approaches show that while neural induction is mainly unaffected, miR-219 depletion alters gene expression programs associated with neural border development, explaining the phenotype. WISH and RNA sequencing of the miR-219 morphant tissue highlights that NC progenitor programs (pax3, zic1, pou5f3, sall3) are maintained or increased at the expense of the progression of NC development (twist1, rpe65), resulting in defective NC specification. While increased pax3 expression seems an important part of this program, reducing pax3 in miR-219 morphant embryos is not sufficient to rescue the phenotype, highlighting that other key partners remain to be identified. This study supports an important and complex role of miR-219 in the early steps of neural crest formation.