Cleft lip with or without cleft palate (CL/P) is among the most highly prevalent birth defects in human patients. In contrast to isolated CP, CL/P is more common in human patients and yet there are few mouse models for CL/P to define mechanisms that give rise to CL/P. It is well established that crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, yet the basic molecular events underlying this crosstalk are poorly understood. While a number of key transcription factors and signaling pathways involved in craniofacial development have been identified, the role of alternative splicing is largely unexplored. My lab studied alternative splicing of fibroblast growt factor receptor 2 (Fgfr2), a gene associated with craniofacial abnormalities. We discovered two paralogous epithelial-cell-type-specific splicing factors, Esrp1 and Esrp2, which are required for expression of the epithelial Fgfr2-IIIb isoform and a broader epithelial program of alternative splicing. We generated mice with knockout (KO) of the Esrp genes and the primary defect in Esrp1 KO mice is 100% penetrant CL/P. These Esrp KO mice provide a fabulous new tool to probe the mechanisms behind CL/P and to identify novel genes and pathways required for normal facial development. We hypothesize that Esrp1 KO causes cell autonomous defects in oral epithelium as well as cell- non-autonomous defects in underlying mesenchyme that lead to CL/P. We further hypothesize that Esrp target transcripts encode epithelial-specific protein isoforms that are essential for maintenance of epithelial- mesenchymal interactions and signaling events that are required for lip and palate formation. We will comprehensively define Esrp-regulated targets in ectoderm and the mechanisms leading to CL/P in Esrp1 KO mice through the following aims: 1) Characterize the defects in lip and palate formation that occur with ablation of Esrp1 and Esrp1/Esrp2 in the developing face and palate. We will conditionally ablate the Esrps at two distinct time points in development to define the key steps and mechanisms by which Esrp1 KO leads to CL/P. 2) Identify global programs of Esrp regulated alternative splicing and polyadenylation in developing ectoderm and palatal epithelial cells. We will use high throughput sequencing (RNA-Seq) and splicing sensitive microarrays in Esrp1flox/flox/Esrp2-/- CKO embryos to define genome-wide changes in splicing, polyadenylation, and gene expression that occur with loss of Esrp expression in ectoderm and derivative. 3) Define genome-wide Esrp binding sites using crosslinking immunoprecipitation coupled with high throughput sequencing (CLIP-Seq). We will identify genome-wide binding sites for Esrp1 to define in vivo direct targets of Esrp regulation. 4) Identify key epithelial-speific splice forms whose loss leads to CL/P. We will use intra-amniotic injections of viral cDNAs of Esrp regulated epithelial isoforms to test for rescue of CL/P phenotypes during embryonic development. These innovative studies have the potential to unveil new candidate genes for CL/P as well as to define molecular mechanisms that go awry in these disorders.

Public Health Relevance

Cleft lip with or without cleft palate (CL/P) and isolated cleft palate (CP) are among the most common birth defects and yet the genetic factors associated with these defects remain poorly understood. We recently observed CL/P as the primary abnormality in mice in which we deleted the epithelial splicing factor Esrp1, suggesting that changes in epithelial patterns of alternative splicing can result in human clefting disorders. The proposed studies will further define genes regulated by Esrp1 in the developing face and thereby identify new candidate genes that are involved in the pathogenesis of CL/P and CP.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
High Priority, Short Term Project Award (R56)
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Skeletal Biology Development and Disease Study Section (SBDD)
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Scholnick, Steven
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University of Pennsylvania
Internal Medicine/Medicine
Schools of Medicine
United States
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