The mechanisms of morphogenesis are not well understood, yet shaping structures during development is essential for establishing correct organismal form and function. Here, we examine mechanisms that help to shape the developing face during the crucial period of facial primordia fusion. This period of development is a time when the faces of amniote embryos exhibit the greatest degree of similarity, and it probably results from the necessity for fusion to occur to establish the primary palate. Our results show that hierarchical induction mechanisms, consisting of iterative signaling by Sonic hedgehog (SHH) followed by Bone morphogenetic proteins (BMPs), regulate a dynamic expression pattern of Shh in the ectoderm covering the frontonasal (FNP) and maxillary (MxP) processes. Furthermore, this Shh expression domain contributes to the morphogenetic processes that drive the directional growth of the globular process of the FNP toward the lateral nasal process and MxP, in part by regulating cell proliferation in the facial mesenchyme. The nature of the induction mechanism that we discovered suggests that the process of fusion of the facial primordia is intrinsically buffered against producing maladaptive morphologies, such as clefts of the primary palate, because there appears to be little opportunity for variation to occur during expansion of the Shh expression domain in the ectoderm of the facial primordia. Ultimately, these results might explain why this period of development constitutes a phylotypic stage of facial development among amniotes.

Fibroblast growth factor (FGF) signaling mutations are a frequent contributor to craniofacial malformations including midfacial anomalies and craniosynostosis. FGF signaling has been shown to control cellular mechanisms that contribute to facial morphogenesis and growth such as proliferation, survival, migration and differentiation. We hypothesized that FGF signaling not only controls the magnitude of growth during facial morphogenesis but also regulates the direction of growth via cell polarity. To test this idea, we infected migrating neural crest cells of chicken embryos with  replication-competent avian sarcoma virus expressing either FgfR2(C278F), a receptor mutation found in Crouzon syndrome or the ligand Fgf8. Treated embryos exhibited craniofacial malformations resembling facial dysmorphologies in craniosynostosis syndrome. Consistent with our hypothesis, ectopic activation of FGF signaling resulted in decreased cell proliferation, increased expression of the Sprouty class of FGF signaling inhibitors, and repressed phosphorylation of ERK/MAPK. Furthermore, quantification of cell polarity in facial mesenchymal cells showed that while orientation of the Golgi body matches the direction of facial prominence outgrowth in normal cells, in FGF-treated embryos this direction is randomized, consistent with aberrant growth that we observed. Together, these data demonstrate that FGF signaling regulates cell proliferation and cell polarity and that these cell processes contribute to facial morphogenesis.

Craniosynostosis causes malformations of the skull that are characterized by premature suture closure, as well as malformations of the brain and face. Recent work in mice, and our data, suggest that the malformations in the brain and face are a primary consequence of mutations that produce craniosynostosis. Mutations in FgfR1–3 account for ~25% of craniosynostosis syndromes. To investigate mechanisms underlying these facial malformations, we infected chick embryos with a retrovirus encoding FgfR2C278F. Compared to controls, treated embryos have widened‐faces and midfacial dysplasia resembling defects seen in craniosynostosis. Further, microarray analysis detected changes in gene expression. In particular, Crispld2 (cysteine rich secretory LCCL domain protein 2), a nonsyndromic cleft lip/palate related gene, is downregulated. The function of Crispld2, and how it may contribute to cleft lip, is unknown. We detected Crispld2 expression in facial mesenchyme in chicken embryos at HH 22. To assess function of this gene, we created an RCAS vector encoding Crispld2. Infected chicken fibroblasts appeared to migrate faster than controls cells, but embryos infected with the virus appeared normal. Therefore, we are currently investigating the effect of knocking‐down Crispld2. In conclusion, we have identified a novel gene Crispld2 and are determining the role this gene plays in craniofacial development. Grant Funding Source : NIH

Craniosynostosis causes malformations of the skull that are characterized by premature suture closure, as well as brain and facial malformations. Recent work in mice, and our preliminary data, suggest that the malformations in the brain and face are a primary consequence of mutations in craniosynostosis patients. Gain‐of‐function mutations in Fibroblast growth factor receptors (FgfR1‐3) account for ~25% of all craniosynostosis syndromes. To investigate mechanisms underlying facial malformations due to mutations in FgfR2, we infected chick embryos with a retrovirus encoding FgfR2C278F that causes craniosynostosis in humans. Compared to normal embryos, infected embryos have widened‐faces and midfacial dysplasia that resemble the defects seen in craniosynostosis. These alterations were associated with a significant reduction in cell proliferation. Further, changes in gene expression detected by microarray analysis may contribute to the phenotypic and cellular outcomes. In particular, Lin28, an inhibitor of Let7 microRNA maturation, was upregulated and sustained for longer periods. Crispld2, a nonsyndromic cleft lip palate related gene, is downregulated. Dlg1, a gene regulating cell polarity, is upregulated and its interacting protein Scribble shows increase membrane‐localization in neural crest cell in FgfR2C278F embryos compared to normal embryos.

In addition to premature suture closure, patients with craniosynostosis have a variety of structural deformities in the brain and face. Whether brain and face malformations result from premature suture closure, or are caused directly by disease‐causing mutations are unknown. Gain‐of‐function mutations in Fibroblast Growth Factor Receptors (FGFRs) account for ~20% of craniosynostosis syndromes, and mice harboring a ubiquitously expressed, disease‐causing allele of FGFR2 recapitulate craniosynostosis phenotypes. To test the hypothesis that facial malformations result directly from activation of FGFR2 in the face, we introduced the human Fgfr2C278F mutant allele into chick embryos using the RCAS virus. RCAS::Fgfr2C278F particles were injected into mesenchyme adjacent to the forebrain after neural crest emigration form the neural tube was complete (stage 10). After 72 hrs embryos were harvested for analysis. Infected embryos were microcephalic, appeared to have widened faces, and had more prominent and hypertrophic nasal pits compared to controls. Consistent with FGF activation, we observed increased pErk staining. Infected embryos also had fewer proliferating cells. In conclusion, our results demonstrate that activation of the FGF pathway produces dysmorphology independent of premature suture fusion that resembles the facial phenotype of people with craniosynostosis.Grant Funding Source: NIH