Fig 2: Deregulation of Rbfox2-TGFβ-Tak1 signaling axis in Rbfox2 mutant embryos.Representative western blot and quantification from microdissected palatal shelves from control and Rbfox2Pax3-CKO embryos (represented as cKO) at E14.5 and analyzed for Tak1, phospho-Tak1, Smad2, phospho-Smad2-C, p38 Mapk and phospho- p38 Mapk (A). Western blot and quantification for fibronectin on control and Rbfox2 mutant embryos at E14.5 (B). Primary palatal mesenchymal cell cultures were established from E14.5 Pax3Cre/+; Rbfox2flox/+; R26mTmG/+ embryos. Representative bright field and fluorescent images were taken. The majority of the cultured cells are GFP positive demonstrating their neural crest origin (C). Representative western blot and quantification from control and Rbfox2 mutant primary palatal mesenchymal cells stimulated with recombinant TGFβ and analyzed for Tak1, phospho-Tak1, Smad2, phospho-Smad2-C, p38 Mapk and phospho-p38 Mapk (D). Quantification of western blot (E). Overexpression of Tak1 in Rbfox2 mutant palatal mesenchymal cells. Ki67 immunostaining to determine palatal mesenchymal cell proliferation (F). Representative western blot for Tak1 and pTak1 (G). Quantification of the percentage of Ki67 positive cells (H). β-Actin was used as loading control. PS, palatal shelves; NS, not significant.

Fig 3: Rbfox2 is expressed in the neural crest cells during mouse embryonic development.Immunostaining for Rbfox2 was performed on E9.5, E10.5, E11.5 and E12.5 transverse sections at different rostrocaudal axis (A–R). Magnified view of the neural tube shows Rbfox2 expression in the pre-migratory (white arrows) and migratory (red arrows) neural crest cells (K–M). Rbfox2 expression in neural crest-derived tissues such as OFT and palate shelves (D-E, I-J, and N-O). Non-specific autofluorescence due to blood cells is observed in DA, MDA, OFT, RA and LA. Immunostaining for Rbfox2 and Pax3 was performed on adjacent sections from E9.5, E10.5, and E11.5 mouse embryos (P–U). Rbfox2 is expressed in the premigratory neural crest cells (white arrows) of the dorsal neural tube as well as in the migratory neural crest cells (red arrows). Nuclei were visualized by DAPI staining (blue). CAC, common atrial chamber and CV, cardinal vein; DGR, dorsal root ganglion; DM, dorsal mesocardium; DA, dorsal aorta; FV, fourth ventricle; LA, left atrium; MDA, midline dorsal aorta; NT, neural tube; OFT, outflow tract; PS, palatal shelves; RA, right atrium; S, somite; T, Tongue; TG, trigeminal ganglion; UV, umbilical vein; V, ventricle. Scale bars are 100 μm respectively.

Fig 4: Rbfox2-dependent splicing and transcriptional changes in neural crest cells.Representative fluorescent images from E12.5 control (n = 3) and Rbfox2Pax3-CKO mutant (represented as cKO, n = 3) embryos showing the area (white, dotted line) of craniofacial tissue microdissected for RNA-Seq analysis (A). Heat map of 50 differentially expressed transcripts (out of a total of 81 transcripts) identified by MISO analysis of RNA-Seq data (B). Heat map of 33 alternatively spliced transcripts identified by Cuffdiff analysis of RNA-Seq data (C). Pathway enrichment analysis of Rbfox2 target genes (D). Motif (UGCAUG) enrichment analysis. Significant enrichment was observed only in the introns of the target genes (E). Location of the intronic UGCAUG sequences in the Rbfox2 target genes (F). Venn diagram showing the overlap of transcripts identified by MISO and Cuffdiff analysis of RNA-Seq data (G). Heat map representation of 11 transcripts identified by both MISO and Cuffdiff analysis (H). RT-PCR analysis of alternative splicing of Rbfox2 targets in control and Rbfox2 mutant cranial mesenchyme from two independent replicates for each genotype (I). The gene structure, illustrating the alternative exons is presented on left. RNA-IP on palatal mesenchymal cells (J). Differentially expressed genes control and Rbfox2 mutants (K). qRT-PCR validation of differentially spliced and differentially expressed genes (L).10.7554/eLife.45418.017Figure 5—source data 1.A complete list of transcripts identified by MISO analysis.10.7554/eLife.45418.018Figure 5—source data 2.A complete list of transcripts identified by Cuffdiff analysis.

Fig 5: Cleft palate defects in Rbfox2 mutant embryos result from impaired cell proliferation in the neural crest-derived palatal shelves.Immunohistochemistry for Ki67 was performed on transverse sections through the middle palatal regions of E12.5 (n = 4 controls, n = 4 Rbfox2Pax3-CKO) and E15.5 (n = 5 controls, n = 5 Rbfox2Pax3-CKO) control (A, D) and Rbfox2Pax3-CKO (B, E) embryos. Immunohistochemistry for Ki67 was performed on E15.5 control (G) and Rbfox2Wnt1-CKO (H) middle palatal shelves sections (n = 5 controls, n = 5 Rbfox2Wnt1-CKO). Quantification of cell proliferation was calculated as the ratio of Ki67-positive cells to the total number of cells as determined by DAPI counterstain in the defined area of palatal shelves (C, F and I). TUNEL assay was performed on E15.5 control (J) and Rbfox2Pax3-CKO (K) sections (n = 4 controls, n = 4 Rbfox2Pax3-CKO). E-cadherin immunostaining on E15.5 control (L) and Rbfox2Pax3-CKO (M) sections (n = 4 controls, n = 4 Rbfox2Pax3-CKO). GFP immunostaining on E15.5 Wnt1Cre/+;Rbfox2flox/+;R26mTmG/+ (N) and Wnt1Cre/+;Rbfox2flox/flox;R26mTmG/+ (O) sections showing neural crest derivatives cells in the palatal shelves (n = 4 each genotype). NS, Nasal septum; PS, palatal shelves; T, Tongue. Scale bars are 100 μm respectively.