Sonic Hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis

Abstract

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 FOXF2 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.

Coordinated d-cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway

“…6A). We next examined a mouse cranial neural crest cell line (O9–1) that was recently shown to be transcriptionally and proliferatively responsive to SHH ligand [26]. In this developmentally-defined cell line, SHH-induced changes in Gli1 , Foxd1 , Ccnd1 , and Cdkn1c closely paralleled those observed in both MEFs and iMEFs (Fig.…”

“…We next examined the effect of SHH stimulation on the expression of Forkhead box family genes, which have been implicated as pathway target genes in multiple developmental contexts including gut, central nervous system and orofacial development [8–11]. Treatment with SHH ligand resulted in a significant induction of the conserved pathway target Gli1 , as well as Foxd1 in WT iMEFs (Fig.…”

“…Two independent studies demonstrated multiple Fox genes downstream of Shh signaling in the cranial neural crest-derived mesenchyme in vivo [7,11]. While Foxd1 was identified in both these studies, neither tested whether it is a direct target of Shh signaling.…”

“…Our cell-based evidence suggests that Foxd1 is directly targeted by canonical Shh signaling, as its response to SHH ligand requires both SMO and GLI protein function. To test whether this circuit is operational in a specific developmental context, we chose to examine upper lip morphogenesis because we recently showed that formation of the upper lip is regulated by Shh-Fox signaling [11]. Specifically, SHH in the surface ectoderm activates pathway activity and expression of multiple Fox genes, including Foxd1 , in the adjacent cranial neural crest-derived mesenchyme.…”

“…Embryos then underwent graded dehydration (1:3, 1:1, 3:1 v/v) into 100% methanol and stored at −20 °C indefinitely for subsequent ISH analysis. In situ hybridization was performed as previously described [11,17]. Embryo sections were imaged using a Nikon Eclipse E600 microscope.…”