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microRNAs Regulate Neural Crest Development and Epithelial-Mesenchymal Signaling in the First Pharyngeal Arch

  • Author(s): Sheehy, Neil Timothy
  • Advisor(s): Srivastava, Deepak
  • et al.
Abstract

Neural crest cells (NCCs) are a subset of multipotent, migratory stem cells that populate a large number of tissues during development and are important for craniofacial and cardiac morphogenesis. Although microRNAs (miRNAs) have emerged as important regulators of development and disease, little is known about their role in NCC development.

Here I show that a single miRNA, miR-145, when introduced in to multipotent, progenitor NCCs in vitro, induces the differentiation of these NCCs in to vascular smooth muscle cells (VSMCs). The fact that a single miRNA is capable of directing NCC fate down a specific differentiation path indicates a likely important role for miRNAs in directing the development of the neural crest. To expand on this idea, I go on to show that loss of miRNA biogenesis by NCC-specific disruption of Dicer results in embryos lacking craniofacial cartilaginous structures, cardiac outflow tract septation, and thymic and dorsal root ganglia development. Dicer mutant embryos had reduced expression of Dlx2, a transcriptional regulator of pharyngeal arch development, in the first pharyngeal arch (PA1). miR-452 was enriched in NCCs, was sufficient to rescue Dlx2 expression in Dicer mutant pharyngeal arches, and regulated non-cell-autonomous signaling involving Wnt5a, Shh, and Fgf8 that converged on Dlx2 regulation in PA1. Correspondingly, knockdown of miR-452 in vivo decreased Dlx2 expression in the mandibular component of PA1, leading to minor craniofacial defects. These results suggest that post-transcriptional regulation by miRNAs is required for differentiation of NCC-derived tissues and that miR-452 is involved in epithelial-mesenchymal signaling in the pharyngeal arch.

To further understand the mechanism by which Wnt5a inhibits Shh signaling, I present evidence that the inhibitory signal requires the activity of G-proteins. Downstream of G-protein activity, there is a Wnt5a-dependent increase in cyclic-AMP levels that induces an increase in PKA activity. Inhibiting PKA activity, even in the presence of Wnt5a, abolishes the downregulation of Shh-responsive genes suggesting that PKA activity is required for Wnt5a-mediated inhibition of Shh signaling. This work establishes a novel pathway connecting these two developmentally important signaling pathways and lays the groundwork for future studies that may shed light on the importance of this signaling interaction.

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