The majority of the bone and cartilage in the head and the face originate from neural crest cells. These cells originate from the dorsal margins of the closing neural folds, delaminate through an epithelial-to-mesenchymal transition (EMT) and migrate extensively to multiple locations in the embryo where they give rise to a wide variety of cell types in different tissues. These cells are thought to have progenitor and stem cell properties because they are so multipotent. Craniofacial development involves not only complex morphogenetic movements of neural crest cells, but also specific interactions of these cells with the cranial mesoderm, vessels and nerves. As a result of this complexity, alterations in craniofacial development are among the most frequent birth defects. We are studying the development of the midface and processes governed by growth factors in neural crest cells that are involved in facial or palatal fusion.
Previous loss of function studies on the receptors for platelet derived growth factors (PDGFs) indicate a role in cranial and cardiac neural crest cells, in somite patterning, in the kidney and in vascular smooth muscle cells (Soriano, 1994; Soriano, 1997). Loss of PDGFRα signaling affects two distinct craniofacial processes, the development of the frontonasal masses and fusion at the midline, that originate from a cell autonomous defect in neural crest cells (Tallquist and Soriano, 2003). By generating allelic series of knock-in mutations preventing the docking of various intracellular effectors to the receptor, we have shown that PI3K is the main signaling pathway utilized by PDGFRα in craniofacial development (Klinghoffer et al., 2002). We have carried out a detailed analysis of the neural crest phenotype in Pdgfra conditional and PI3K mutants, using in vivo Cre lineage tracing and in vitro approaches (He and Soriano, 2013; He and Soriano, 2015). We are also studying how deregulation of PDGFRα signaling regulates development of the cranial bones. In another set of studies, we have used a proteomic approach and genetic epistasis to identify critical phosphorylation targets of PDGFRα signaling in the midface, and have thus identified an important role for p53 in mediating PDGFRα-dependent cell survival and proliferation (Fantauzzo and Soriano, 2014). Last, we have identified a role for PDGFRβ in craniofacial development, and demonstrated that it exerts its function in part through heterodimers with PDGFRα (Fantauzzo and Soriano, 2016).
More recently we have focused on signaling pathways engaged by Fibroblast Growth Factor (FGF) Receptors 1 and 2. In the mouse, 18 FGF ligands signal through 4 FGF receptors, of which only FGFR1 and FGFR2 play essential roles in embryonic development. Although FGF signaling has been extensively studied from a biochemical standpoint in many laboratories, remarkably little is known about how it regulates morphogenetic processes in craniofacial development. In contrast to PDGF receptors, which signal mainly by recruiting effectors with intrinsic enzymatic activity, FGFR1 and FGFR2 are thought to signal primarily through the multidocking protein FRS2 (and the more spatially restricted FRS3) which engage the ERK1/2 signaling pathway through a relay mechanism (Brewer et al., 2016). Although Fgfr1 null mutants have been shown to exhibit defects in gastrulation and somitogenesis, embryos in which the FRS2 and FRS3 binding sites on FGFR1 is deleted die during late embryogenesis and exhibit defects in neural tube closure and the development of the tail bud and pharyngeal arches (Hoch and Soriano, 2006). Fgfr1 and Fgfr2 interact genetically in craniofacial development, and double mutants show extensive defects in the formation of the frontonasal complexes and mandible (Ray et al., 2020). To further study FGFR1/2 signaling mechanisms, we have created extensive allelic series of mutations at both the Fgfr1 and Fgfr2 loci that prevent the binding of FRS2, CRK, PLCγ or GRB14, alone or in combination. For Fgfr1, disruption of these pathways in combination prevents ERK1/2 activation, affecting axis elongation and craniofacial and limb development, but does not recapitulate the null mutant phenotype (Brewer et al., 2015). A similar combination of signaling mutations in Fgfr2 leads to viable mice, in contrast to the null mutants which fail at midgestation. Further analysis shows that these signaling mutations abrogate FGFR1 and FGFR2 canonical signaling, preventing ERK1/2, PI3K/AKT, PLCγ and STAT3 activation. The failure to recapitulate the null mutant phenotypes for both receptors can be attributed, in part, to the fact that these receptors have evolved functions beyond canonical signaling, in particular in mediating cellular adhesion (Ray et al., 2020). We are also further interested in determining how FGFs promote differential signaling to regulate distinct branching morphogenesis patterns in submandibular salivary glands. Last, we are initiating studies to investigate the roles of core PI3K and ERK1/2 signaling components, as well the signaling dynamics of these effectors, in craniofacial development.
It has been known for many years that activation of growth factor signaling pathways leads to the expression of multiple Immediate Early Genes (IEGs), yet their role in specifying particular growth factor responses remained controversial as similar IEGs are often engaged following induction of other signaling pathways. We have used gene trap-coupled microarray analysis to identify targets of PDGF signaling, and their function in PDGF regulated processes (Chen et al., 2004). Mutations in these genes lead to a high frequency of craniofacial and other phenotypes that affect the same cell types and processes as those controlled by PDGF signaling (Schmahl et al., 2007; Schmahl et al., 2008). We have conducted expression profiling of primary mouse embryonic palatal mesenchyme (MEPM) cells, and identified distinct responses to PDGFs and FGFs, that have provided insights into the mechanisms encoding receptor tyrosine kinases specificity (Vasudevan et al., 2015). We have characterized several transcription factors that are regulated by growth factor signaling and that play important roles in midline fusion. One of these is SRF, a critical player in mediating IEG responses. Srf interacts genetically with Pdgfra in craniofacial development, and PDGF signaling promotes the interaction of SRF with its co-factor MRTF to activate a network of cytoskeletal genes (Vasudevan and Soriano, 2014). We are further investigating the how SRF operates through MRTF to regulate craniofacial development.