Research
Receptor tyrosine kinases (RTKs) bind to growth factors at the cell surface and activate various intracellular signaling pathways to elicit responses with broad roles in developmental cellular processes. A subset of RTK families has been shown to regulate the activity of neural crest cells (NCCs) and the development of their derivatives in mammalian systems. NCCs are migratory, multipotent cells that play a critical role in vertebrate development. Cranial NCCs originate from the forebrain to the hindbrain and populate the frontonasal prominence and pharyngeal arches 1-4. These cells give rise to the bone and cartilage of the frontonasal skeleton, among other derivatives.
Our lab is focused on investigating the mechanism and function of signaling through a particular RTK family, the platelet-derived growth factor (PDGF) receptor family, in development of the cranial NCC-derived craniofacial skeleton. Craniofacial development is a complex morphogenetic process, disruptions in which result in highly prevalent human birth defects. Signaling through the PDGFRs plays a critical role in this process in both humans and mice. Pdgfra mutant mouse models display a range of craniofacial phenotypes such as midline clefting, subepidermal blebbing and hemorrhaging. Functional analysis of PDGFRalpha signaling during mouse development by our lab and others has revealed roles in promoting migration of cranial NCCs, proliferation of the NCC-derived craniofacial mesenchyme and osteoblast differentiation. We showed that alternative RNA splicing is an important mechanism of gene expression regulation downstream of PDGFRalpha signaling in the facial mesenchyme and identified the RNA-binding protein Srsf3 as a critical regulator of craniofacial development downstream of this pathway. We have demonstrated a role for the other RTK in this family, PDGFRbeta, in murine craniofacial development, demonstrating that ablation of Pdgfrb in the NCC lineage results in increased nasal septum width, delayed palatal shelf development and subepidermal blebbing. These defects stem, at least in part, from decreased proliferation of the facial mesenchyme past mid-gestation. Further, we showed that PDGFRalpha and PDGFRbeta genetically and physically interact in the craniofacial mesenchyme to form functional heterodimers with unique signaling properties, thus uncovering a novel mode of signaling for the PDGF family during vertebrate development.
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Our goal is to characterize novel intracellular pathways and cellular processes engaged downstream of PDGFR induction. We have several ongoing projects in the lab, each of which will provide significant insight into the mechanisms underlying mammalian craniofacial development and new therapeutic directions for the treatment of human craniofacial birth defects.
1. Examining the spatiotemporal dynamics of PDGFR dimer-specific activation and internalization through bimolecular fluorescence complementation (BiFC) in cultured cells and in the craniofacial mesenchyme of developing mouse embryos.
2. Characterizing PDGFR dimer-specific interactomes through two approaches: a) genetic epistasis experiments between Pdgfra mutant mice and both null or constitutively active alleles of genes encoding signaling molecules that are known to bind the receptors; and b) coupling BiFC with affinity purification and mass spectrometry analysis to identify PDGFR dimer-specific interacting proteins.
3. Analyzing the effect of PDGFR dimer-specific activation on gene expression in the mouse embryonic craniofacial mesenchyme through RNA-sequencing and subsequent in vivo validation analyses.
4. Determining how the activity of RNA-binding proteins (RBPs) is regulated by PDGFRalpha signaling to generate protein isoforms necessary for midface development through two approaches: a) employing biochemical techniques to determine the effects of PI3K/Akt-mediated phosphorylation of these RBPs downstream of PDGFRalpha signaling on their subcellular localization, protein-protein interactions, RNA-binding and sequence specificity; and b) characterizing the role of these RBPs during craniofacial development in vivo through phenotypic analyses of conditional knock-out and phosphomutant genetic mouse models.
Our goal is to characterize novel intracellular pathways and cellular processes engaged downstream of PDGFR induction. We have several ongoing projects in the lab, each of which will provide significant insight into the mechanisms underlying mammalian craniofacial development and new therapeutic directions for the treatment of human craniofacial birth defects.
1. Examining the spatiotemporal dynamics of PDGFR dimer-specific activation and internalization through bimolecular fluorescence complementation (BiFC) in cultured cells and in the craniofacial mesenchyme of developing mouse embryos.
2. Characterizing PDGFR dimer-specific interactomes through two approaches: a) genetic epistasis experiments between Pdgfra mutant mice and both null or constitutively active alleles of genes encoding signaling molecules that are known to bind the receptors; and b) coupling BiFC with affinity purification and mass spectrometry analysis to identify PDGFR dimer-specific interacting proteins.
3. Analyzing the effect of PDGFR dimer-specific activation on gene expression in the mouse embryonic craniofacial mesenchyme through RNA-sequencing and subsequent in vivo validation analyses.
4. Determining how the activity of RNA-binding proteins (RBPs) is regulated by PDGFRalpha signaling to generate protein isoforms necessary for midface development through two approaches: a) employing biochemical techniques to determine the effects of PI3K/Akt-mediated phosphorylation of these RBPs downstream of PDGFRalpha signaling on their subcellular localization, protein-protein interactions, RNA-binding and sequence specificity; and b) characterizing the role of these RBPs during craniofacial development in vivo through phenotypic analyses of conditional knock-out and phosphomutant genetic mouse models.
