Craniofacial development is a critical morphological event during embryogenesis, defects in which result in highly prevalent human birth defects such as cleft lip and palate (CL/P). In both humans and mice this process relies on signaling through the platelet-derived growth factor receptor alpha (PDGFRa). Mutations in human PDGFRA are associated with CL/P and mouse models with mutations in this gene similarly display facial clefting phenotypes. Identification of phosphorylation targets of PI3K/Akt downstream of PDGFRa signaling in E13.5 primary mouse embryonic palatal mesenchyme cells revealed an enrichment for proteins that regulate RNA splicing, including the RNA-binding protein (RBP) Srsf3. While dysregulation of alternative RNA splicing has been shown to cause numerous diseases in humans, the role of RBPs in regulating alternative splicing in neural crest cells (NCCs) and their derivatives in the facial mesenchyme has been understudied.
The aim of this proposal is to examine the tissue-specific alternative RNA splicing mediated by Srsf3 in the facial mesenchyme and the effect of PI3K/Akt-mediated PDGFRa signaling on Srsf3 activity during midface development. First, the role of Srsf3 during craniofacial development in vivo will be characterized using a conditional Srsf3fl allele in combination with the NCC-specific Wnt1-CreTg driver. The timing and extent of NCC migration will be analyzed in this setting as well as the expression of markers specific to individual facial processes. Second, an RNA- sequencing experiment will be performed to identify transcripts that are differentially alternatively spliced between E11.5 Srsf3fl/fl;Wnt1-Cre+/+ and Srsf3fl/fl;Wnt1-Cre+/Tg maxillary processes. Validation of the differential alternative splicing of select transcripts will be performed using semi-quantitative PCR. Finally, the effect of Akt phosphorylation on Srsf3 subcellular localization, RNA binding and sequence specificity will be assessed through the transfection of Srsf3 wild-type and phosphomutant GFP fusion constructs and enhanced crosslinking and immunoprecipitation followed by sequencing, respectively, in the presence or absence of PDGF-AA ligand. Further, genetic epistasis experiments will be performed between PdgfraPI3K and Srsf3fl mice and the resulting craniofacial phenotypes analyzed to determine if the PdgfraPI3K/PI3K palatal clefting defect is rescued or exacerbated upon loss of Srsf3 in the NCC lineage. This project will investigate a potentially novel role for Srsf3 as a splicing regulator in NCCs and their derivatives and explore how post-translational modification of this protein downstream of PDGFRa signaling contributes to midface development. These studies will provide significant insight into the mechanisms underlying mammalian craniofacial morphogenesis and new therapeutic directions for the treatment of human craniofacial birth defects such as CL/P.
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. Dysregulation of alternative RNA splicing has been shown to cause numerous diseases in humans, ranging from muscular dystrophies to neurological disorders, dilated cardiomyopathy, retinitis pigmentosa and cancer. The studies proposed here will be the first to address a potentially novel role for the splicing factor Srsf3 in regulating alternative RNA splicing in neural crest cells and their derivatives in the facial mesenchyme, as well as the first to examine the role of post-translational modifications of an RNA-binding protein in this setting and, ultimately, provide new therapeutic directions for the treatment of human craniofacial birth defects.
