The Silent Treatment: miR199 Family Silences Shh during Craniofacial Development

Abstract

Early craniofacial development requires precise timing and spatial regulation of key signaling pathways in order for proper facial structures to form. Perturbations, particularly in the sonic hedgehog (Shh) signaling pathway, can result in craniofacial birth defects that cause significant harm and financial costs. microRNAs (miRNA) play many roles in developing systems by repressing gene expression, but despite the enormous number of miRNAs that have been identified, few have been related to biological processes through direct targets. Here we identified the miR199 family as Shh‐regulated miRNAs during development of the upper jaw in a chick model.We performed a miRNA microarray analysis on chick embryonic ectoderm before (stage HH18) and after (stage HH22) formation of the Frontonasal Ectodermal Zone (FEZ) and after activating or blocking SHH signaling in the brain. We identified a family of miRNAs (miR199b, mir199* and miR199) that appeared to be regulated by SHH signaling in the brain. These are increased between stages HH18 and HH22, and increase upon activation of Shh signaling and decrease upon blockade of Shh signaling, and these data were confirmed by qPCR. To assess the function of this family of miRNAs during facial development, we performed miR199 knock‐down and overexpression experiments and examined the phenotypic and molecular changes. Up‐regulation of each miRNA led to decreased Shh expression in the FEZ and facial malformations that are consistent with decreased Shh signaling in the face. Using antagomirs to knockdown each miRNA, we observed that Shh expression was expanded and the faces were wider, reminiscent of SHH gain‐of‐function experiments. Hence, it appears that expression of these miRNAs in the FEZ are regulated by SHH signaling from the brain, and these miRNAs appear to regulate SHH signaling from the FEZ. We then identified Smurf2, Map3k4, Hif1alpha, and Dyrk1a as targets of the miR‐199 family of miRNAs via mir‐base. We confirmed that each of these genes behaved like targets in miR‐gain and loss‐of‐function experiments described above. Using pmirGLO luciferase, we identified MAP3K4 and HIF as direct targets of miR199. Finally, we generated a miRNA knockdown cassette to reduce expression of Hif1α and Map3k4. Blocking HIF1α resulted in a large reduction of Shh expression in the FEZ, and resulted in severe craniofacial malformations, while blocking Map3k4 altered facial morphology, resulting in altered mediolateral beak development at later stages. Overall, our results demonstrate that Shh signaling within the brain contributes to facial development by regulating a family of miRNAs that then regulate the expression of Shh in the FEZ via targeting the transcription factor Hif1α.Support or Funding InformationThis work was supported by the National Institutes of Health (R01 DE018234‐A1)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.