DEV025437 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Table S2 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Quantitation of cushion thickness and cellularity in Fgf8;Isl1Cre mutants versus controls. The thickness of the cardiac jelly and cell numbers in the OFT cushions were measured from three different sections of each of four individual embryos. Data below the graph indicate mean ± s.d. (A) The thickness of the proximal cardiac jelly at E10.5 is significantly reduced in Fgf8;Isl1Cre mutants. (B) Cell number in E10.5 proximal OFT cushions was reduced in Fgf8;Isl1Cre mutants as compared with controls. Cell numbers were counted in 100 µm² of the proximal OFT cushions. (C,D) Significantly reduced thickness of the proximal and distal OFT cushions at E11.5.

• Supplemental Figure S2 -

  Fig. S2. Ubiquitous expression of Fgfr1 and Fgfr2 in the embryonic pharynx. Co-immunostaining of transverse sectioned 10- to 12-somite stage embryos bearing either Fgfr1;IRESCFP (A-D) or Fgfr2;IRESYFP alleles (G-J). nt, neural tube; oft, outflow tract; fg, foregut; sm, splanchnic mesoderm. (A,G) Nuclear staining with Hoechst. (B,H) Anti-AP2α protein in ectoderm and neural crest. The outline observed in the foregut lumen is an artifact resulting from non-specific secondary antibody binding to proteinaceous substance. (C,I) Texas Red fluorescence obtained with anti-FP antibody detects either CFP (C, FGFR1) or YFP (I, FGFR2). Note the ubiquitous signal of varying intensity in pharyngeal tissues and OFT. (D,J) Merged view; yellow-white signal is apparent in neural crest/ectoderm; purple signal in all other cell types. (E,F) Section (E) or right-sided view of whole-mount embryo (F) after in situ hybridization with Fgfr1 antisense probe and prolonged exposure. White line in F shows approximate plane of section in E. (K,L) Section (K) or right-sided view of whole-mount embryo (L) after in situ hybridization with Fgfr2 antisense probe and prolonged exposure. White line in L shows approximate plane of section in K.

• Supplemental Figure S3 -

  Fig. S3. Disrupted FGF signaling to neural crest disrupts craniofacial, but not OFT, development. R1, Fgfr1; R2, Fgfr2. (A,B) Thorax dissection of E18.5 Fgfr1^c/c;Fgfr2^c/c;Wnt1Cre mutant heart (B) is indistinguishable from littermate control (A). (C,D) Alkaline phosphatase staining of control and Spry2-GOF;Pax3Cre hearts at E12.5. CNC cells of the Pax3Cre lineage are labeled and contribute to the smooth muscle investing the great arteries (arrowheads in D). Note normal OFT in Spry2-GOF;Pax3Cre mutant. (E,E′) Normal craniofacial structures in a control embryo. (F,F′) Fgfr1^c/c;Fgfr2^c/c;Wnt1Cre mutant with severe facial clefting. (G-H′) Craniofacial structures of control and Spry2-GOF/Pax3Cre embryo at E16.5. Note the shortened snout (white line) and mandible of mutant.

• Supplemental Figure S4 -

  Fig. S4. Quantitation of EMT failure and rescue in isolated and co-cultured OFT explants. (A) Decreased number of cells migrating from explants in Fgf8;Isl1Cre mutants. The number of migrating cells was normalized for total explant area (n=11 explants). (B) The maximum distance of cell migration is decreased in Fgf8;Isl1Cre explants (n=11). (C) Rescue of number of migrating cells in Fgf8;Isl1Cre OFT explants by co-culture with control OFT explants. Total cell number was counted from control OFT explant co-cultured with control (n=6), Fgf8;Isl1Cre (mutant) explant co-cultured with control (n=3), and mutant explants co-cultured with mutant (n=4). Numbers were normalized for explant area. (D) Rescued maximum distance of migration in Fgf8;Isl1Cre explants by co-culture with control explant. (E) Control OFT explant co-cultured with control. (F) Fgf8;Isl1Cre OFT explants co-cultured with Fgf8;Isl1Cre OFT explant fails to rescue EMT.

• Supplemental Figure S5 -

  Fig. S5. Fgfr1/2 conditional mutants phenocopy the EMT and CNC invasion defects seen in Fgf8;Isl1Cre mutants. R1, Fgfr1; R2, Fgfr2. Sagittal sections through the OFTs of an E10.5 Fgfr;Mesp1Cre mutant allelic series. Embryos that will have OFT alignment or septation defects (C,D) have short, aberrantly angulated OFTs, hypoplastic OFT cushions (arrowheads) relative to control (A) and unaffected Fgfr1^c/+;R2^c/c;MesP1Cre (B) mutants.