Fig. 7

Six1 is required for pSmad1/5/8 expression in Pax7⁺ cells in the peripheral dermomyotome and reduced levels of pSmad1/5/8 decrease the number of Pax7⁺ cells. (A) Expression of Pax7 (red) and pSmad1/5/8 (green) in 24-hpf embryos uninjected and injected with six1a and six1b morpholinos alone or in combination. (B) Average number of Pax7⁺/pSmad1/5/8^- and Pax7⁺/pSmad1/5/8⁺ cells per somite in control embryos (n = 6) and in six1a (n = 14), six1b (n = 11) and six1a and six1b double morphants (n = 13). Somite number 10-12 was counted. (C) Cross-sections of 24-hpf control embryos and six1a, six1b and six1a/six1b morphant embryos stained with Pax7 (red), pSmad1/5/8 (green) and DAPI (blue). Enlargements of Pax7⁺/pSmad1/5/8⁺ border are presented, with arrowheads indicating coexpression. (D) Expression of six1b (blue) and smyhc1 (red) in embryos treated with DMSO or 15µM dorsomorphin (DM) from shield stage to 24hpf, showing a maintained six1b expression; dashed lines indicate the level of cross-section. (E) Pax7 expression in embryos treated with DMSO or 5µM dorsomorphin from 2–3hpf to 24hpf. (F) Pax7 expression in embryos treated with 100 or 200µM dorsomorphin from 10ss to 24hpf. (G) Embryos treated with 5µM dorsomorphin from 2–3hpf to 24hpf have an average number of 32.8 Pax7⁺ cells/somite (n = 24) and DMSO-treated controls have 46.7 Pax7⁺ cells/somite (n = 18). Embryos treated with 100µM dorsomorphin from 10ss to 24hpf have an average number of 33.1 Pax7⁺ cells/somite (n = 19) and DMSO-treated controls have 41.4 Pax7⁺ cells/somite (n = 17). Embryos treated with 200µM dorsomorphin from 10ss to 24hpf have an average number of 34.8 Pax7⁺ cells/somite (n = 19) and DMSO-treated control have 41.3 Pax7⁺ cells/somite (n = 16). Somite number 10–12 was counted and cross-sections were made at somite number 10. *P<0.05, **P<0.01, ***P<0.001. Scale bars: 50μm and 10μm for enlargements.