Craniofacial development is a complex morphogenic process, disruptions in which result in highly prevalent birth defects in humans. Signaling through platelet-derived growth factor receptor alpha (PDGFR?) plays a critical role in craniofacial development; mutant mouse models of the gene display phenotypes such as facial clefting, subepidermal blebbing and facial hemorrhaging. While the other receptor tyrosine kinase in the family, PDGFR?, does not have an established role in craniofacial development, limited evidence from the literature indicates that the two receptors may be able to form functional heterodimers with distinct properties from homodimeric receptor complexes. Our preliminary in vitro studies reveal that PDGFR? and PDGFR? are expressed in primary mouse embryonic palatal mesenchyme (MEPM) cells and are capable of forming functional heterodimers with the capacity to activate downstream signaling pathways. Further preliminary in vivo analyses demonstrate that the two receptors interact in the neural crest cell lineage during craniofacial development, as double homozygous mutant embryos exhibit an overt facial clefting phenotype with subepidermal blebbing that is more severe than that observed in either single mutant embryo. The aim of this application is to explore the role of PDGFR? in craniofacial development, with a long-term goal of identifying novel effector molecules operating downstream of this receptor during midline development and characterizing their role in the etiology of facial clefting and subepidermal blebbing. First, a homologous recombination knock- in approach will be used to generate a PdgfrbmCherry null reporter allele. Reporter expression will be analyzed in craniofacial structures in whole mount using a fluorescent stereomicroscope and in coronal frozen sections by immunofluorescence analysis. Second, the activation of signaling pathways, proliferation, survival and migration will be assessed downstream of PDGFR? homodimer versus PDGFR?/? heterodimer activation by Western blotting, BrdU incorporation assays, cell growth curves and Transwell assays, respectively. Finally, the interaction of PDGFR? and PDGFR? will be characterized during craniofacial development in vivo, by employing conditional alleles for both genes together with a Wnt1-Cre driver to target ablation in the neural crest. The phenotypes of compound mutant mice will be compared to those of single homozygous mutant embryos by morphological, histological and marker expression analyses. The research strategy outlined in this application will take advantage of the vast array of reagents available for the PDGF signaling pathway, as well as generate vital new tools, to explore novel aspects of craniofacial biology. The innovative studies proposed here will explore the expression of PDGFR? in the facial mesenchyme and identify the signaling pathways and cellular processes induced downstream of PDGFR? homodimer and PDGFR?/? heterodimer activation during craniofacial development, ultimately providing new therapeutic directions aimed at the prevention of craniofacial birth defects.

R03DE025263

2015-07-01

2018-05-31