PDGFR dimer-specific dynamics in the craniofacial mesenchyme

Craniofacial development is a complex morphogenetic process, disruptions in which result in prevalent human birth differences. Signaling through the platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases (RTKs) plays critical roles in this process in humans and mice. Pdgfra mutant mouse models display midline clefting, subepidermal blebbing and hemorrhaging, due to aberrant cranial neural crest cell (NCC) migration and defective proliferation of the NCC-derived craniofacial mesenchyme at mid-gestation. Alternatively, conditional ablation of Pdgfrb in the NCC lineage results in increased nasal septum width, delayed palatal shelf development and subepidermal blebbing, primarily due to decreased proliferation of the craniofacial mesenchyme past mid-gestation. Use of a novel bimolecular fluorescence complementation approach enabled visualization and purification of individual PDGFR dimers both in vitro and in vivo, revealing differences in the timing and extent of dimer activation, signal molecule binding, internalization, trafficking and downstream signaling. These differences in PDGFR dimer-specific dynamics translated into changes in migration and cell proliferation. Further preliminary results suggest that these receptors signal at sites other than the plasma membrane and require internalization to maximally activate downstream signaling. Combined, these findings have shifted the paradigm on how biological specificity is achieved to generate unique responses downstream of PDGFR engagement. The goal of this proposal is to test the hypothesis that the differential internalization and trafficking dynamics of the various PDGFR dimers underlie differences in downstream intracellular signaling and cellular behavior during craniofacial development. First, the internalization dynamics of ligand-receptor complexes for the various PDGFR dimers will be quantified via flow cytometry to determine how ligand concentration affects receptor internalization. Separately, CITE-seq will be combined with spatial transcriptomics to quantify individual PDGFR dimer formation in the murine craniofacial mesenchyme and correlate this metric with gene expression changes and distance from the ectodermal PDGF ligand source. Second, Myo1d, a novel protein involved in trafficking shown to differentially interact with the various PDGFR dimers, will be knocked out and the effects on PDGFR localization, downstream signaling, proliferation and craniofacial development examined. Finally, the requirement for formation of various endosomal compartments on PDGFR activation and downstream signaling will be analyzed to identify the subcellular compartment(s) that serves as the major signaling platform for the various PDGFR dimers, and the effects of craniofacial disease-associated PDGFRA and PDGFRB variants on receptor trafficking will be characterized. These studies will employ innovative techniques to provide significant insight into what are likely broadly applicable mechanisms underlying the temporal and spatial regulation of RTK signaling during mammalian craniofacial development. Project Number: 1R01DE035831-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Katherine Fantauzzo | Institution: UNIVERSITY OF COLORADO DENVER, Aurora, CO | Award Amount: $513,174 | Activity Code: R01 | Study Section: Skeletal Biology Development and Disease Study Section[SBDD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11336514