Craniofacial development is a complex morphogenetic process, disruptions in which result in highly prevalent human birth defects. Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in this process in humans and mice. Pdgfra mutant mouse models display a range of craniofacial phenotypes such as midline clefting, subepidermal blebbing and hemorrhaging. PDGFRa signaling promotes migration of cranial neural crest cells (NCCs), proliferation of the NCC-derived craniofacial mesenchyme and osteoblast differentiation. Recently, a role for PDGFRb has been uncovered in murine craniofacial development, as ablation of Pdgfrb in the NCC lineage results in increased nasal septum width, delayed palatal shelf development and subepidermal blebbing. Further, PDGFRa and PDGFRb have recently been shown to genetically and physically interact in the craniofacial mesenchyme to form functional heterodimers. These PDGFRa/b heterodimers have unique signal molecule binding properties and the ability to generate more robust intracellular signaling and mitogenic responses in vitro than those generated by homodimeric receptor complexes. Combined, these findings have shifted the paradigm on how receptor tyrosine kinases act to regulate craniofacial morphogenesis and warrant a full reconsideration of PDGF signaling in midface development.
The aim of this proposal is to examine the in vivo dynamics of PDGFR dimer-specific formation, as well as the resulting effects on gene expression and cell activity in the craniofacial mesenchyme. First, PDGFR-bimolecular fluorescence complementation (BiFC) fragment alleles will be generated containing the N- or C-terminal regions of the Venus fluorescent protein. Venus expression will be analyzed in craniofacial structures by fluorescence microscopy to examine the spatiotemporal dynamics of PDGFR homodimer versus heterodimer formation. These alleles will be combined with ectoderm-specific ablation of PDGF-BB ligand to examine the effect on heterodimer formation. Second, the effect of SHP-2 binding to PDGFRa on downstream signaling will be determined through genetic epistasis experiments and, in parallel, BiFC and affinity purification will be employed to selectively purify PDGFRa/b heterodimers and identify PDGFR dimer-specific interacting proteins by mass spectrometry. Finally, RNA-sequencing will be performed to define the transcriptional program induced downstream of PDGFR dimer-specific activation in the maxillary processes. Transcriptional responses involved in proliferation and osteoblast differentiation will be validated through in vivo marker expression analysis to dissect the etiology of the midline defects observed upon ablation of one or both PDGFRs in the NCC lineage. This project will employ innovative techniques to pinpoint the timing and localization of PDGFR dimer-specific activation and analyze the resulting effects on the proteome and transcriptome. These studies will provide significant insight into the mechanisms underlying midface development and new therapeutic directions for the treatment of human craniofacial birth defects.
Platelet-derived growth factor (PDGF) signaling has been shown to regulate numerous processes throughout the body, both during development and adult life, and aberrant PDGF signaling has been causally associated with defects in craniofacial development, cancer, vascular disorders and fibrotic diseases. Defects in craniofacial development comprise one of the most prevalent birth defects in humans, with an estimated incidence in the United States of 1/940 live births for cleft lip and 1/1,574 live births for cleft palate. The studies proposed here will impart valuable insight into the mechanisms underlying craniofacial development by examining the spatiotemporal dynamics of PDGFR dimer-specific formation and the resulting effects on cell activity in the craniofacial mesenchyme, and, ultimately, provide new therapeutic directions for the treatment of human craniofacial birth defects.
