Search

Background: Frem1 has been linked to human face shape variation, dysmorphology, and malformation, but little is known about its regulation and biological role in facial development., Results: 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. Finally, we found that FREM1 is sufficient to induce cNCC proliferation in a concentration-dependent manner and that Shh pathway antagonism reduces Frem1 expression during pathogenesis of midfacial hypoplasia., Conclusions: By demonstrating that the Shh signaling pathway regulates Frem1 expression in cNCCs, these findings provide novel insight into the mechanisms underlying variation in midfacial morphogenesis., (© 2022 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: The evolutionarily conserved Sonic Hedgehog (Shh) signaling pathway is essential for embryogenesis and orofacial development. SHH ligand secreted from the surface ectoderm activates pathway activity in the underlying cranial neural crest cell (cNCC)-derived mesenchyme of the developing upper lip and palate. Disruption of Shh signaling causes orofacial clefts, but the biological action of Shh signaling and the full set of Shh target genes that mediate normal and abnormal orofacial morphogenesis have not been described., Results: Using comparative transcriptional profiling, we have defined the Shh-regulated genes of the cNCC-derived mesenchyme. Enrichment analysis demonstrated that in cultured cNCCs, Shh-regulated genes are involved in smooth muscle and chondrocyte differentiation, as well as regulation of the Forkhead family of transcription factors, G1/S cell cycle transition, and angiogenesis. Next, this gene set from Shh-activated cNCCs in vitro was compared to the set of genes dysregulated in the facial primordia in vivo during the initial pathogenesis of Shh pathway inhibitor-induced orofacial clefting. Functional gene annotation enrichment analysis of the 112 Shh-regulated genes with concordant expression changes linked Shh signaling to interdependent and unique biological processes including mesenchyme development, cell adhesion, cell proliferation, cell migration, angiogenesis, perivascular cell markers, and orofacial clefting., Conclusions: We defined the Shh-regulated transcriptome of the cNCC-derived mesenchyme by comparing the expression signatures of Shh-activated cNCCs in vitro to primordial midfacial tissues exposed to the Shh pathway inhibitor in vivo. In addition to improving our understanding of cNCC biology by determining the identity and possible roles of cNCC-specific Shh target genes, this study presents novel candidate genes whose examination in the context of human orofacial clefting etiology is warranted.

Sonic Hedgehog (Shh) signaling plays key regulatory roles in embryonic development and postnatal homeostasis and repair. Modulation of the Shh pathway is known to cause malformations and malignancies associated with dysregulated tissue growth. However, our understanding of the molecular mechanisms by which Shh regulates cellular proliferation is incomplete. Here, using mouse embryonic fibroblasts, we demonstrate that the Forkhead box gene Foxd1 is transcriptionally regulated by canonical Shh signaling and required for downstream proliferative activity. We show that Foxd1 deletion abrogates the proliferative response to SHH ligand while FOXD1 overexpression alone is sufficient to induce cellular proliferation. The proliferative response to both SHH ligand and FOXD1 overexpression was blocked by pharmacologic inhibition of cyclin-dependent kinase signaling. Time-course experiments revealed that Shh pathway activation of Foxd1 is followed by downregulation of Cdkn1c, which encodes a cyclin-dependent kinase inhibitor. Consistent with a direct transcriptional regulation mechanism, we found that FOXD1 reduces reporter activity of a Fox enhancer sequence in the second intron of Cdkn1c. Supporting the applicability of these findings to specific biological contexts, we show that Shh regulation of Foxd1 and Cdkn1c is recapitulated in cranial neural crest cells and provide evidence that this mechanism is operational during upper lip morphogenesis. These results reveal a novel Shh-Foxd1-Cdkn1c regulatory circuit that drives the mitogenic action of Shh signaling and may have broad implications in development and disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cleft lip is one of the most common human birth defects, yet our understanding of the mechanisms that regulate lip morphogenesis is limited. Here, we show in mice that sonic hedgehog (Shh)-induced proliferation of cranial neural crest cell (cNCC) mesenchyme is required for upper lip closure. Gene expression profiling revealed a subset of Forkhead box (Fox) genes that are regulated by Shh signaling during lip morphogenesis. During cleft pathogenesis, reduced proliferation in the medial nasal process mesenchyme paralleled the domain of reduced Foxf2 and Gli1 expression. SHH ligand induction of Foxf2 expression was dependent upon Shh pathway effectors in cNCCs, while a functional GLI-binding site was identified downstream of Foxf2 Consistent with the cellular mechanism demonstrated for cleft lip pathogenesis, we found that either SHH ligand addition or F OXF2 overexpression is sufficient to induce cNCC proliferation. Finally, analysis of a large multi-ethnic human population with cleft lip identified clusters of single-nucleotide polymorphisms in FOXF2 These data suggest that direct targeting of Foxf2 by Shh signaling drives cNCC mesenchyme proliferation during upper lip morphogenesis, and that disruption of this sequence results in cleft lip., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)