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Fig. S7 Myotome patterning as a proxy of notochord maturity. (A-C) myod1 expression in 9-somite stage embryos is increased in adaxial and medial fast muscle cells of Fstl1/2 morphants (B), and is reduced in the medial fast muscle domain of tBR embryos heat-shocked at 80% epiboly (C). (D-F) Immunostaining against Myod1 and F-actin reveals an increase in Myod1-positive cells of the medial fast muscle domain of Fstl1/2 morphants (E), and a decrease in Myod1-positive cells in tBR embryos (F). Images are of the anterior-most three somites. The mean number of Myod1-stained cells from five experimental embryos was subjected to one-way ANOVA, followed by a two-tailed, equal variance t-test. Asterisks indicate significance (P<0.001). (G-I) Immunostaining with the F59 antibody, which recognizes mainly slow muscle, reveals that the horizontal myoseptum is increased in 24 hpf Fstl1/2 morphant embryos (H). Muscle fibers are disorganized in tBR embryos (I). (J-L) Immunostaining with antibodies against 4D9, which labels Engrailed-positive cells (red), and Prox1, which labels slow muscle cells (green), reveal an increase in medial fast muscle (red) and slow muscle pioneers (yellow) in 24 hpf Fstl1/2 morphants (K), and a reduction of Engrailed positive cells in tBR embryos (L). (M-O) Sections through the trunk level of 24 hpf embryos confirm the results in J-L. Antibodies against PSMAD1/5/8 (red) and Engrailed (green) reveal an increase in PSMAD1/5/8-positive chondrocytes as well as medial fast muscle fibers and slow muscle pioneers (green) in Fstl1/2 morphants (N). Chondrocytes do not form in tBR embryos, and the domain of medial fast muscle fibers and slow muscle precursors is reduced (O).