Figure 2. Complementary in vivo and in vitro functional assays to test human ESRP1 and ESRP2 gene variants.

(A) Microdissected ANC of Alcian-blue stained embryos at 4 dpf for wild-type, esrp1^−/−; esrp2^+/+, esrp1^−/−; esrp2^−/−, and esrp1^−/−; esrp2 MO embryos. (B) Schematic for the esrp morphant variant assay in zebrafish. Variants that robustly rescued the cleft ANC phenotype were scored as benign, while variants that failed to rescue the cleft ANC phenotype were scored as pathogenic. (C) RT-PCR was performed using primers spanning exons 36–38 of Arhgef11 on cDNA isolated from wild-type mouse Py2T cells, Esrp1/2 double-knockout Py2T cells, or Esrp1/2 double-knockout PyY2T cells electroporated with plasmids encoding for either Esrp1 or Esrp2 genes. Arrow markers point to the epithelial (short) isoform and mesenchymal (long) isoform retaining exon 37.