Plastic Surgery Research Council

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High-Resolution Gene Expression Analysis of RNA Splicing Regulators Esrp1 and Esrp2 In Palate Development
Shannon H. Carroll, MS-PhD1,2, Claudio Macias-Trevino, MD-PhD Candidate1, Nora Alhazmi, BDS-MS1, Edward Li, MD-PhD Candidate1, Eric Liao, MD-PhD1,2.
1Massachusetts General Hospital, Boston, MA, USA, 2Shriners Hospital for Children, Boston, MA, USA.

PURPOSE: Alternative RNA splicing is a fundamental process that amplifies protein diversity and fine-tunes cell communications. Epithelial splicing regulatory proteins 1 and 2 (Esrp1 and Esrp2) are the only known tissue-specific regulators of RNA splicing. Disruption of Esrp1/2 results in cleft lip and palate in the mouse. Recently, human cohorts with ESRP2 gene variants were identified to be associated with cleft lip and palate. In order to gain mechanistic understanding of how epithelial cells regulate morphological changes during palate development, we applied a new gene expression detection method, RNAscope, to analyze the spatiotemporal expression of Esrp1 and Esrp2 during mouse and zebrafish palate development. METHODS: We applied a novel RNA in situ technology (RNAscope) to carry out detailed gene expression analysis of Esrp1 and Esrp2 in mouse and zebrafish craniofacial structures with unprecedented single cell resolution. RESULTS: Esrp1 and Esrp2 mRNA are found to be highly co-expressed in the periderm and basal epithelia of mouse as well as zebrafish epithelium during early embryogenesis. In the mouse, this included the epithelial layers surrounding the palatal shelves prior to and during fusion. With this enhanced RNA expression detection technique, we have identified new expression profiles for Esrp1/2 as well as other cleft lip/palate associated genes. Furthermore, we can differentiate between the expressions of protein isoforms (i.e. Esrp1 and 2), which were not previously possible with immunofluorescence. CONCLUSION: Detailed gene expression data of Esrp1 and Esrp2 resolved at the single-cell level supports a role for epithelial-specific alternative RNA splicing in the regulation of craniofacial and palate development. We are now applying RNAscope to delineate spatiotemporal changes in Esrp1 and Esrp2 gene expressions during impaired palatogenesis in various orofacial mutant mouse and zebrafish models. Investigating Esrp1/2 expression during craniofacial development will expand our understanding of cleft lip/palate etiology, specifically the role of the embryonic epithelium in cleft pathogenesis.


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