DEV014563 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Comparison of Pitx2 and Fgf10 (Mlc1v-nlacZ-24) expression in the SHF. Pitx2 transcripts shown by whole-mount in situ hybridization (purple) after X-Gal staining (blue) on Mlc1v-nlacZ-24 embryos at the 6-somite stage. In addition to expression in head mesenchyme, asymmetric expression is seen in the left SHF (arrow). R, right; L, left.

• Supplemental Figure S2 -

  Fig. S2. Pitx2c expression in left and right explants. Semi-quantitative RT-PCR analysis on right (R) and left (L) explants of the posterior SHF from Mlc3f-nlacZ-2E embryos at the 4-somite stage (s) after 72 hours (h) of culture; at the 5-somite stage after 48 and 72 hours of culture; at the 5-somite stage after 72 hours of infection with an adenovirus expressing Pitx2c (5 s Pitx2c inf); and at the 6-somite stage after 12 hours of culture. β-actin transcripts serve as control for the amount of RNA.

• Supplemental Figure S3 -

  Fig. S3. Upregulation of right atrial transgene expression in left explants from Pitx2c^−/− mutant embryos. (A-D) Explants of left (A,C) and right (B,D) posterior SHF from Mlc3f-nlacZ-2E^+/−;Pitx2c^−/− embryos after 72 hours of culture. (A,B) Merge of Hoechst (blue) staining and myosin heavy chain (MHC) immunofluorescence (red). (C,D) β-galactosidase (β-gal) immunofluorescence of the explants shown, respectively, in A and B. (E) Quantitative analysis of β-gal and MHC double-positive cells as a percentage of the total number of cells in ten explants from each side of Mlc3f-nlacZ-2E^+/−;Pitx2c^−/− embryos. White, percentage in left Mlc3f-nlacZ-2E^+/−;Pitx2c^−/− explants; black, percentage in right Mlc3f-nlacZ-2E^+/−;Pitx2c^−/− explants.

• Supplemental Figure S4 -

  Fig. S4. Asymmetric expression of the right atrial transgene in explants of the sinus venosus myocardium. X-Gal staining on right (A) and left (B) explants of the sinus venosus region below the lines on the heart tube (grey) in the diagram shown to the left from 7-somite-stage Mlc3f-nlacZ-2E embryos, after 48 hours of culture. Asymmetric expression of the transgene is detectable in the forming sinus venosus at the 7-somite-stage, after explant culture.