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. PDGFRalpha and PDGFRBeta have recently been shown to genetically and physically interact in the craniofacial mesenchyme to form functional heterodimers with 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.
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. Second, the effect of SHP-2 binding to PDGFRalpha on downstream signaling will be determined through genetic epistasis experiments and, in parallel, BiFC and affinity purification will be employed to selectively purify PDGFRalpha/Beta 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. Additional experiments, technologies and training opportunities are introduced in this Independent Scientist Award application that significantly extend and enhance the original research plan and allow for career development. These include cell biology and superresolution microscopy training to characterize the subcellular localization and internalization dynamics of the various PDGFR dimers; training in the application of bioinformatics approaches for RNA-sequencing analysis; and participation in leadership and scientific communication training programs. This training plan will take place within the School of Dental Medicine at the University of Colorado Anschutz Medical Campus, which has a strong, well-established research program in craniofacial biology and provides ample opportunities for training and collaboration. University support includes guaranteed salary support beyond the award period; provision of funds for post-doctoral fellow recruitment, shared equipment usage, course tuition and travel to scientific meetings; permission for the candidate to spend essentially full-time conducting research; and participation in joint lab meetings and working groups designed to facilitate the development of the candidate's research program. Combined, these activities will contribute towards the goal of establishing a recognized research laboratory committed to understanding the signaling hierarchies that govern mammalian craniofacial development and how disruption of these signaling events leads to 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.
