The major aims of this proposal are to identify signaling mechanisms underlying midface development that are controlled by PDGF and FGF. Loss of the Pdgfra or Fgfr1 receptors leads to facial clefting and improper development of the frontonasal process, whereas hypomorphic mutations in these pathways result in cleft palate. Pdgfra and Fgfr1 regulate craniofacial development mainly in cranial neural crest cells (cNCCs) through PI3K and Erk, respectively. PDGF induces a short duration or Erk signaling cell differentiation, whereas FGF promotes cell proliferation and perdurance of the Erk signal. Last, PDGF and FGF regulate craniofacial development by engaging immediate early genes (IEGs) through serum response factor (Srf), a critical transcription factor activated by these growth factors itself required for midface closure. This application proposes: 1. To establish the roles and locations of PI3K and Erk signaling in craniofacial development. We will analyze midface development in conditional PI3K and Erk core component genes in NCCs. To identify sites of PDGF and FGF driven signaling activity in vivo, we will breed PI3K and Erk biosensors into wild type, Pdgfr and Fgfr mutant backgrounds. 2. To determine how PDGF and FGF signaling differences differentially regulate craniofacial development. We will alter the duration of Erk signaling in primary MEPMs using Mek/Erk and PKC inhibitors, and investigate how this affects cell differentiation and proliferation. To establish the relative importance of combinatorial vs. dynamic signaling and downstream responses in craniofacial development, we will analyze expression of known PDGF and FGF transcriptional targets that are PI3K and Erk dependent and linked to differentiation or proliferative responses, in Pdgfr/PI3K or Fgfr/Erk neural crest specific mutants. 3. To determine the signaling mechanisms through which Srf, a shared PDGF and FGF transcriptional target, regulates differential transcriptional outputs. Srf interacts with two classes of co-factors, Myocardin Related Transcription Factors (MRTFs) or Ternary Complex Factors (TCFs). PDGF promotes the association of Srf with MRTFs through PI3K to regulate the expression of cytoskeletal target genes critical for craniofacial development, whereas both PDGF and FGF allow Srf to interact with TCFs through PI3K and Erk signaling to facilitate the expression of core IEGs. To establish the molecular pathways and targets by which growth factors regulate midface development through Srf, we will generate mice carrying mutations in Srf that abrogate its ability to associate with MRTFs, while maintaining its interactions with TCFs. These proposed studies explore novel territories in the area of growth factor signaling in craniofacial biology and open new directions for the prevention of craniofacial birth defects.
Craniofacial developmental diseases including cleft lip or palate are the most prevalent birth defects in the human population worldwide. The major goal of this proposal is to characterize signaling mechanisms controlled by PDGFs and FGFs that underlie normal development of the midface, by investigating the functions of PI3K and Erk, their activation domains, their kinetics of activation, and how they regulate transcription through Serum Response Factor. These studies will identify mechanisms underlying formation of the midface and establish paradigms for the prevention of birth defects.
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