Frs2-deficiency in cardiac progenitors disrupts a subset of FGF signals required for outflow tract morphogenesis

DEV025361 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Diminished Frs2α expression occurs earlier in Frs2α^cn/Nkx than in Frs2α^cn/Mef mutants. (A,B) Paraffin sections were stained with anti-FRS2α antibody (green) and To-Pro3 (red). High-magnification views of the boxed areas are shown in the lower panels. Note that Frs2α expression is reduced in the OFT, PE and SM of Frs2α^cn/Nkx at the 8-9 ss, and in the OFT and SM of Frs2α^cn/Mef at the 11-12 ss. OFT-M, outflow tract myocardium; PE, pharyngeal endoderm; SM, splanchnic mesoderm; ss, somite stage.

• Supplemental Figure S2 -

  Fig. S2. Ablation of Frs2α with Tie2^Cre does not cause OFT defect. (A) Sections from E10.5 embryos were stained with anti-FRS2α antibody and To-Pro3. The boxed areas are shown at high-magnification beneath. Note that Frs2α expression is diminished in the OFT endocardium of Frs2α^cn/Tie2 mutants. (B,C) Frs2α^cn/Tie2 mutants have a normal OFT. Sections of E14.5 embryos were stained with H&E. Note that no obvious defect was observed in the Frs2α mutant OFT. Data from ten individuals are listed in C. Ao, aorta; PT, pulmonary trunk; En, endocardium.

• Supplemental Figure S3 -

  Fig. S3. Ablation of Frs2α reduces SHF lineage cells in E9.5 OFT. (A) Sections of E9.5 embryos were stained with anti-ISL1 antibodies and To-Pro3, demonstrating that ISL1⁺ cells are reduced in both the OFT myocardium and endocardium of Frs2α^cn/Nkx mutants, but only in the OFT myocardium of Frs2α^cn/Mef mutants. (B) Statistical analyses of ISL1⁺ cells in the OFT myocardium and endocardium of five individuals. Data are presented as mean ±s.d. OFT, outflow tract; F/F, Frs2α^f/f; Mef, Frs2α^cn/Mef mutant; Nkx, Frs2α^cn/Nkx mutant.

• Supplemental Figure S4 -

  Fig. S4. Ablation of Frs2α does not increase apoptosis in the SHF. (A-G) Embryo sections at the indicated stages were stained with TUNEL reagents (green) and To-Pro3 (red). Inserts are high-magnification views of the boxed areas in the same sections. No obvious increase in apoptosis is observed in mutant versus wild-type OFT, SM and NCCs located in pharyngeal arches 3, 4 and 6, although increased apoptosis is found in the PE of Frs2α^cn/Nkx mice. PE, pharyngeal arch; SM, splanchnic mesoderm; OFT, outflow tract; TP3, To-Pro3.

• Supplemental Figure S5 -

  Fig. S5. Pitx2 expression is not changed in the OFT of the Frs2α mutant. Whole-mount in situ hybridization of E9.5 embryos. Note that Pitx2 expression in the OFT is unchanged in the Frs2α mutant (indicated by arrows). OFT, outflow tract.

• Supplemental Figure S6 -

  Fig. S6. Frs2α mutant has normal AV cushions. (A-C) Expression of Frs2α in E9.5 embryos was assessed by immunostaining with anti-FRS2α antibody on paraffin sections (green), and nuclei were stained with To-Pro3 (red). Note that expression of Frs2α in the atrioventricular myocardium is diminished in Frs2α^cn/Nkx embryos and partially reduced in Frs2α^cn/Mef embryos at E9.5. (D-F) H&E staining demonstrates no obvious defect in the AV cushion of Frs2α^cn mutants at E10.5. Cu, cushion; My, myocardium.

• Supplemental Figure S7 -

  Fig. S7. Malformed OFTs in mutants with defective FGF signaling. (A,B) Whole-mount images of embryonic hearts dissected from E17.5 embryos with the indicated genotypes. RCC and LCC, right and left common carotid artery; RSA and LSA, right and left subclavian artery; Ao, aorta; Mis, misaligned OFT; PT, pulmonary truck; TA, truncus arteriosus; PTA, persistent truncus arteriosus.