The epithelial splicing regulatory proteins, ESRP1 and ESRP2 are essential for mammalian development through regulation of a global program of alternative splicing of genes involved in maintenance of epithelial cell function. To further inform our understanding of the molecular functions of ESRP1 we performed enhanced crosslinking immunoprecipitation coupled with high throughput sequencing (eCLIP) in epithelial cells of mouse epidermis. The genome-wide binding sites of ESRP1 were integrated with RNA-Seq analysis of alterations in splicing and total gene expression that result from epidermal ablation of Esrp1 and Esrp2. These studies demonstrated that ESRP1 functions in splicing regulation occur primarily through direct binding in a position-dependent manner to either promote exon inclusion or skipping. In addition, we also identified widespread binding of ESRP1 in 3′ and 5′ untranslated regions (UTRs) of genes involved in epithelial cell function suggesting that its post-transcriptional functions extend beyond splicing regulation.

Running Title: Altered Wnt signaling and CL/P in Esrp1 KO mice (32 characters) SUMMARY STATEMENT 3Ablation of the epithelial-specific splicing factor Esrp1 leads to cleft lip and cleft palate (CL/P) and this 4 study identifies alterations in Wnt signaling during face formation that may partly underlie this defect. 5 6 7 ABSTRACT 8 9Cleft lip is one of the most highly prevalent birth defects in human patients. However, there remain a 10 limited number of mouse models of cleft lip and thus much work is needed to further characterize genes 11 and mechanisms that lead to this disorder. It is well established that crosstalk between epithelial and 12 mesenchymal cells underlies formation of the face and palate, yet the basic molecular events mediating 13 this crosstalk are still poorly understood. We previously demonstrated that mice with ablation of the 14 epithelial-specific splicing factor Esrp1 have fully penetrant bilateral CL/P. In this study we further 15 investigated the mechanisms by which ablation of Esrp1 leads to cleft lip as well as cleft palate. These 16 studies included a detailed analysis of the changes in splicing and total gene expression in embryonic 17 ectoderm during formation of the face as well as gene expression changes in adjacent mesenchyme. We 18 identified altered expression in components of pathways previously implicated in cleft lip and/or palate, 19 including numerous components of the Wnt signaling pathway. These findings illustrate that maintenance 20 of an Esrp1 regulated epithelial splicing program is essential for face development through regulation of 21 key signaling pathways. 22 23 24 25 26 27 28 29Cleft lip and/or palate (CL/P) are among the most common congenital birth defects, affecting 30 approximately 1 in 700 live births and affected children face a variety of health and psychosocial 31 problems as well as a need for extensive surgical and dental treatments (Dixon et al., 2011). The causes of 32 CL/P are heterogeneous and include a variety of environmental and genetic factors. Although CL/P can 33 be a component of disease syndromes, most cases of CL/P are non-syndromic. Cleft lip with or without 34 cleft palate (CL/P) is more common in human patients than isolated cleft palate (CP, or CPO) and these 35 disorders are largely genetically and etiologically distinct (Fraser, 1970; Gritli-Linde, 2008).The proper 36 development of the lip and palate is similar between humans and mice and thus mouse models have 37 served an important role in studies to identify genes and characterize pathways that, when disrupted, lead 38 to CL/P or CPO (Gritli-Linde, 2012; Juriloff and Harris, 2008). In mice, formation of the face commences 39 around E9.5 and involves the five facial prominences consisting of mostly neural crest-derived 40 mesenchyme and overlying epithelium; the frontonasal prominence (FNP) and the paired maxillary and 41 mandibular prominences (MXP and MdP) (Jiang et al., 2006). The FNP gives rise to the lateral and 42 medial nasal prominences (LNP and MNP) and these prominence...