Frem1 activity regulated by Sonic Hedgehog signaling in the cranial neural crest mesenchyme guides midfacial morphogenesis

The Frem/Fras family of extracellular matrix proteins has been linked to human face shape variation and malformation, but little is known about their regulation and biological roles in facial development. During midfacial morphogenesis in mice, we observed Frem1 expression in the embryonic growth centers that form the median upper lip, nose, and palate. Expansive spatial gradients of Frem1 expression in the cranial neural crest cell (cNCC) mesenchyme of these tissues suggested transcriptional regulation by a secreted morphogen. Accordingly, Frem1 expression paralleled that of the conserved Sonic Hedgehog (Shh) target gene Gli1 in the cNCC mesenchyme. Suggesting direct transcriptional regulation by Shh signaling, we found that Frem1 expression is induced by SHH ligand stimulation or downstream pathway activation in cNCCs and observed GLI transcription factor binding at the Frem1 transcriptional start site during midfacial morphogenesis. Shh pathway antagonism reduced Frem1 expression during pathogenesis of midfacial hypoplasia, and FREM1 was sufficient to induce cNCC proliferation in a concentration-dependent manner. These findings provide novel insight into the mechanism by which the Shh pathway drives midfacial morphogenesis and reveal a functional role for Frem1 in cNCC biology that establishes the developmental basis for FREM1-associated face shape variation and malformation.