Biology subjects

Fantauzzo, K. A.

Publications and source records attributed to Fantauzzo, K. A..

7 recordsLinked to original sources

bioRxiv2025

PDGFRα is required for postnatal cerebral perivascular fibroblast development

Perivascular fibroblasts (PVFs) are a cell type associated with large diameter blood vessels in the brain and spinal cord parenchyma and leptomeninges. PVFs have previously defined roles in injury and neuroinflammatory diseases and predicted roles in supporting neurovascular function. The temporal dynamics of PVF development in the pre- and postnatal cerebral cortex have recently been described, however the molecular mechanisms that underly PVF development have not been identified. PVFs express both platelet derived growth factor receptors (PDGFRs), PDGFR and PDGFR{beta}. Here we investigate the role of PDGF signaling in PVF development. We use immunohistochemistry and RNA transcript detection methods to show developmental expression of PDGFRs by PVFs and examine distribution of PDGF ligand expression in the brain. We show that postnatal deletion of PDGFR in fibroblasts using the Col1a2-CreERT mouse line significantly impairs PVF coverage of cerebral vessels at postnatal day 10. Perivascular macrophages, a cell type previously shown to co-develop with PVFs, have impaired cerebral vessel coverage in conditional mutants that is similar to PVF coverage defects. This work establishes a requirement for PDGFR signaling in PVF development and may shed light upon the potential pathways that are over-activated in PVFs in injury and disease contexts.

developmental biology↗

bioRxiv2025

Additive effects on craniofacial development upon conditional ablation of PDGFRα and SHP2 in the mouse neural crest lineage

BackgroundActivity of the receptor tyrosine kinase PDGFR and the tyrosine phosphatase SHP2 are critical for vertebrate craniofacial development. We sought to determine the effect of SHP2 binding to PDGFR via phenotypic and biochemical analyses of an allelic series of mouse embryos with combined loss of both proteins in the neural crest lineage. ResultsWe demonstrated that SHP2 preferentially binds PDGFR/ homodimers among the three PDGFR dimers. Analysis of allelic series mutant embryos revealed increased cell death in the lateral nasal and maxillary processes at E10.5, variably penetrant facial blebbing, facial hemorrhaging, midline clefting and loss of the mandibular region at E13.5, and widespread craniofacial bone and cartilage defects at birth. Further, we showed that loss of SHP2 leads to increased phosphorylation of PDGFR and the downstream effector Erk1/2 in E10.5 allelic series mutant embryo lysates. ConclusionsTogether, our findings demonstrate additive effects on craniofacial development upon conditional ablation of PDGFR and SHP2 in the mouse neural crest lineage and indicate that SHP2 may negatively and positively regulate PDGFR signaling through distinct mechanisms.

developmental biology↗

bioRxiv2024

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking

Signaling through the platelet-derived growth factor receptor alpha (PDGFR) plays a critical role in craniofacial development, as mutations in PDGFRA are associated with cleft lip/palate in humans and Pdgfra mutant mouse models display varying degrees of facial clefting. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFR signaling during skeletal development in the mouse. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFR signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting, due to defects in proliferation and survival of cranial neural crest cells, and widespread alternative RNA splicing (AS) changes. Here, we sought to determine the molecular mechanisms by which Srsf3 activity is regulated downstream of PDGFR signaling to control AS of transcripts necessary for craniofacial development. We demonstrated via enhanced UV-crosslinking and immunoprecipitation (eCLIP) of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. Moreover, we found that the subset of transcripts that are bound by Srsf3 and undergo AS upon PDGFR signaling commonly encode regulators of PI3K signaling and early endosomal trafficking. Functional validation studies further confirmed that Srsf3 activity downstream of PDGFR signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.

molecular biology↗

bioRxiv2023

PDGFRα/β heterodimer activation negatively affects downstream ERK1/2 signaling and cellular proliferation

The platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases allows cells to communicate with one another by binding to growth factors at the plasma membrane and activating intracellular signaling pathways to elicit responses such as migration, proliferation, survival and differentiation. The PDGFR family consists of two receptors, PDGFR and PDGFR{beta}, that dimerize to form PDGFR homodimers, PDGFR/{beta} heterodimers and PDGFR{beta} homodimers. Here, we overcame prior technical limitations in visualizing and purifying PDGFR/{beta} heterodimers by generating a cell line stably expressing C-terminal fusions of PDGFR and PDGFR{beta} with bimolecular fluorescence complementation fragments corresponding to the N-terminal and C-terminal regions of the Venus fluorescent protein, respectively. We found that these receptors heterodimerize relatively quickly in response to PDGF-BB ligand treatment, with a peak of receptor autophosphorylation following 5 minutes of ligand stimulation. Moreover, we demonstrated that PDGFR/{beta} heterodimers are rapidly internalized into early endosomes, particularly signaling endosomes, where they dwell for extended lengths of time. We showed that PDGFR/{beta} heterodimer activation does not induce downstream phosphorylation of ERK1/2 and significantly inhibits cell proliferation. Further, we characterized the PDGFR dimer-specific interactome and identified MYO1D as a novel protein that preferentially binds PDGFR/{beta} heterodimers. We demonstrated that knockdown of MYO1D leads to retention of PDGFR/{beta} heterodimers at the plasma membrane, resulting in increased phosphorylation of ERK1/2 and increased cell proliferation. Collectively, our findings impart valuable insight into the molecular mechanisms by which specificity is introduced downstream of PDGFR activation to differentially propagate signaling and generate distinct cellular responses. One-sentence summaryPDGFR/{beta} heterodimer binding to MYO1D contributes to rapid internalization of the dimerized receptors, thereby negatively affecting downstream ERK1/2 signaling and cellular proliferation.

cell biology↗

bioRxiv2021

PDGFR dimer-specific activation, trafficking and downstream signaling dynamics

Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in multiple cellular processes during development. The two PDGFRs, PDGFR and PDGFR{beta}, dimerize to form homodimers and/or heterodimers. Here, we overcome previous limitations in studying PDGFR dimer-specific dynamics by generating cell lines stably expressing C-terminal fusions of each PDGFR with bimolecular fluorescence complementation (BiFC) fragments corresponding to the N-terminal or C-terminal regions of the Venus fluorescent protein. We find that PDGFR{beta} receptors homodimerize more quickly than PDGFR receptors in response to PDGF ligand, with increased levels of autophosphorylation. Further, we demonstrate that PDGFR homodimers are trafficked and degraded more quickly, while PDGFR{beta} homodimers are more likely to be recycled back to the cell membrane. We show that PDGFR{beta} homodimer activation results in a greater amplitude of phospho-ERK1/2 and phospho-AKT signaling, as well as increased proliferation and migration. Collectively, our findings provide significant insight into how biological specificity is introduced to generate unique responses downstream of PDGFR engagement. SummaryThe authors utilize a novel bimolecular fluorescence complementation approach to investigate PDGFR homodimer-specific dynamics. They uncover differences in the timing and extent of receptor dimerization, activation and trafficking, which lead to changes in downstream signaling and cellular activity.

cell biology↗

bioRxiv2020

Srsf3 mediates alternative RNA splicing downstream of PDGFRα signaling

Signaling through the platelet-derived growth factor receptor alpha (PDGFR) is critical for mammalian craniofacial development, though the mechanisms by which the activity of downstream intracellular effectors is regulated to mediate gene expression changes have not been defined. We find that the RNA-binding protein Srsf3 is phosphorylated at Akt consensus sites downstream of PI3K-mediated PDGFR signaling in palatal mesenchyme cells, leading to its nuclear translocation. We further demonstrate that ablation of Srsf3 in the neural crest lineage leads to facial clefting due to defective cranial neural crest cell specification and survival. Finally, we show that Srsf3 regulates the alternative RNA splicing of transcripts encoding protein kinases in the facial process mesenchyme to negatively regulate PDGFR signaling. Collectively, our findings reveal that PI3K/Akt-mediated PDGFR signaling primarily modulates gene expression through alternative RNA splicing in the facial mesenchyme and identify Srsf3 as a critical regulator of craniofacial development.

developmental biology↗

bioRxiv2020

Pdgfra and Pdgfrb genetically interact in the murine neural crest cell lineage to regulate migration and proliferation

Cranial neural crest cells (cNCCs) are migratory, multipotent cells that originate from the forebrain to the hindbrain and eventually give rise to the bone and cartilage of the frontonasal skeleton, among other derivatives. Signaling through the two members of the platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases, alpha and beta, plays critical roles in the cNCC lineage to regulate craniofacial development during murine embryogenesis. Further, the PDGFRs have been shown to genetically interact during murine craniofacial development at mid-to-late gestation. Here, we examined the effect of ablating both Pdgfra and Pdgfrb in the murine NCC lineage on earlier craniofacial development and determined the cellular mechanisms by which the observed phenotypes arose. Our results confirm a genetic interaction between the two receptors in this lineage, as phenotypes observed in an allelic series of mutant embryos often worsened with the addition of conditional alleles. The defects observed here were shown to stem from reduced cNCC stream size and aberrant cNCC directional migration, as well as decreased proliferation of the facial mesenchyme upon combined decreases in PDGFR and PDGFR{beta} signaling. Importantly, we found that PDGFR plays a predominant role in cNCC migration whereas PDGFR{beta} primarily contributes to proliferation of the facial mesenchyme. Our findings provide insight into the distinct mechanisms by which PDGFR and PDGFR{beta} signaling regulate cNCC activity and subsequent craniofacial development in the mouse embryo.

developmental biology↗