Whole Mount In Situ Hybridization Of An E10.5 ESRP1-/- Model

Objectives: The purpose of this study is to observe if an ESRP1-/- novel mouse model results in decreased genetic expression of Lef1, Axin2, and Wnt9b, and to examine its role in the pathogenesis of cleft lip and/or palate (CL/P).
Methods: An ESRP1+/- mouse line was already in the possession of the Nah laboratory. ESRP1+/- breeder mice were time mated to produce E10.5 embryos. Tails were saved for genotyping of the embryos. This study has been approved by the IACUC at CHOP. Whole mount in situ hybridization was carried out via a published protocol and immunological-staining was analyzed for gene expression patterns for each probe. Images were captured for further observation.
Results: In mice, palatal and lip fusion begins at E10.5. While wildtype ESRP1+/+ (and ESRP1+/-) presents with normal development, ESRP-/- exhibits the CL/P phenotype. Following analysis of the immunological-staining patterns of the various whole mount in situ hybridizations, the genetic expression of Lef1, Axin2, and Wnt9b decreased characteristically in E10.5 ESRP1-/- mice. This occurred in areas critical for proper craniofacial development, such as the nasal and maxillary processes.
Conclusions: Upon creation of an ESRP1 knockout (KO) mouse model, it was observed that ESRP1-/- mice developed CL/P at a rate of 100%. ESRP1-/- mice results in decreased genetic expression of important regulators of the Wnt signaling pathway, such as Lef1, Axin2, and Wnt9b. Normal expression of these genes is required during the critical fusion time frame of E10.5 in order to facilitate proper craniofacial formation. ESRP1 may be regulating the activity of these genes, thereby preventing the fusion of craniofacial processes during development. Therefore, identifying the downstream targets of ESRP1 proteins and how they interact with Lef1, Axin2, and Wnt9b may unveil the most important splicing events involved in craniofacial development. This may ultimately uncover pathways that have potential therapeutic targets worldwide.