Esrp1 Loss Disrupts MicroRNAs Which Target Wnt Signaling Required For Palatogenesis
Nathan Wilson, PhD1, SungKyoung Lee, PhD2, Russ Carstens, MD2, Eric Liao, MD, PhD1.
1Massachusetts General Hospital, Boston, MA, USA, 2University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA.
Orofacial clefting is one of the most prevalent congenital defects, affecting 1/800 live births. Surgical repair of palate deformities constitutes a costly challenge to pediatric care which the reconstructive surgical community encounters commonly. In previous studies, reduced expression of an epithelium-specific alternative splicing regulator, Esrp1, caused cleft palate in a mouse model. Upon noting that Esrp1 physically interacts with a battery of microRNAs, small non-coding transcripts which negatively regulate mRNA expression, we investigated a possible role for Esrp1 in microRNA expression to explore the central hypothesis that Esrp1 loss results in disruption of microRNAs required for palate formation.
Due to the epithelium-specific expression and function of Esrp1, we employed an in vitro model of mouse epithelium, the Py2T mammary epithelial cell line. Our collaborator had previously employed the CRISPR-Cas9 gene editing system to disrupt Esrp1 expression in this cell line. We employed a microarray-based approach for all known mouse microRNAs, between unmodified Py2T cells and Esrp1: CRISPR Py2T cells.
Here we report that upon CRISPR-induced disruption of Esrp1 in a mouse epithelial cell line there is a distinct upregulation of three micro-RNAs, miR-342-5p, miR-374c-5p and miR-181c-5p, each of which have been shown to target the Wnt pathway, whose necessity in palatogenesis has already been extensively documented. Specifically, miR-342-5p downregulates the expression of Wnt3 and Wnt7b, as well as a variety of downstream effectors of Wnt signaling which drive the progression of the cell cycle required for the outgrowth of the palatal shelves.
This work establishes that Esrp1 negatively regulates the expression of key microRNAs which must be suppressed in order for orofacial primordia to undergo successful proliferation and migration to form the mature secondary palate. Ongoing work is focused upon identifying the mechanism by which Esrp1 loss disrupts specific microRNA expression levels and how such disruptions interfere with the process of palatogenesis.
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