DEV007088 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Morpholino knockdown of Mef2s. Immunodetection with anti-Mef2 antibody visualized with fluorescent (A,E,F) or DAB staining (B,C) or in situ mRNA hybridisation (D) viewed in dorsal flatmount (A-C,D right; anterior to top), lateral wholemount (D left; anterior to top, dorsal to left) or lateral flatmount (E,F, anterior to left, dorsal to top). (A) Adaxial cells in presomitic mesoderm show nuclear staining that is abolished by injection of mef2d MO, but not mef2c MO. (B) At 15 somites, mef2d MO ablates adaxial expression (arrowhead) and reduces expression in differentiated slow fibres (arrows), whereas mef2c MO has little effect. Note that mef2d-specific MO had no effect on anti-Mef2c signal (compare to Fig. S2B). (C) Heart anlagen in midline beneath neural tube shows nuclear reaction (arrows) in control that is abolished by mef2c MO, but not by mef2d MO. (D) At 14-15 somites, neither mef2d nor mef2c MOs affect levels of the cognate mRNA. (E) At 24 hpf, Mef2 immunoreactivity present in fibre nuclei is abolished by mef2d/c MO. (F) Confocal stack of Mef2 immunoreactivity (green) and slow MyHC (red) in embryos from a hoover heterozygote cross. mef2d MO-injected embryos show three phenotypes: severely affected with no detectable Mef2 (bottom), mildly affected with weak Mef2 (middle), or indistinguishable from uninjected control embryos, all of which contain strong Mef2 and well-organised slow fibres (top). Scale bars: 20 μm.

• Supplemental Figure S2 -

  Fig. S2. Morpholino knockdown of mef2c. Immunofluorescent detection of anti-Mef2c antibody (A, red; B-D, green) or slow MyHC (C, red) in skeletal muscle viewed in dorsal (A, presomitic mesoderm, anterior to top) or lateral (B,C, anterior to left, dorsal to top) flatmount. (A) Confocal stack showing unstained adaxial cells in presomitic mesoderm at tailbud stage. (B) At 24 hpf, confocal stacks reveal that Mef2c immunoreactivity is predominantly nuclear in both fast and slow muscle fibres. mef2c MO ablates nuclear signal, whereas mef2d MO has little effect. mef2c/d, mef2d/c and mef2c + mef2d MOs also ablate Mef2c immunoreactivity. (C) Single confocal slices of 24-hpf embryos from a cross of hoover heterozygotes. Mef2c immunoreactivity is ablated in superficial slow and medial fast fibres of 13/48 (27%) hoo^tn213 mutant embryos, but slow muscle is unaffected. (D) mef2c mRNA is reduced in putative hoo^tn213 mutants (21/67, 31%) and partially reduced in heterozygotes (27/67, 40%). (E) At 24 hpf, Mef2c immunoreactivity is predominantly nuclear in cardiomyocytes. mef2c MO ablates nuclear signal, whereas mef2d MO has no effect. Note that mef2c-specific MO greatly reduced anti-Mef2c stain in heart and muscle and anti-Mef2 in heart without diminishing muscle anti-Mef2 signal or cognate mRNA (compare to Fig. S1A-C). Scale bars: 20 μm.

• Supplemental Figure S3 -

  Fig. S3. Several morpholino combinations produce the same skeletal muscle phenotype. Confocal stacks of fluorescent immunodetection of slow MyHC (F59, red) and anti-Mef2 (green) antibodies of 24-hpf somites of control and embryos injected with different MO combinations, viewed in lateral flatmount. Phenotype severity in slow muscle fibres correlates with levels of Mef2 in fibre nuclei. Single morpholinos show no detectable defects in slow fibres and wild-type levels of Mef2 in nuclei. Double knockdown with either mef2d/c MO, mef2c+mef2d MOs or mef2c/d MO, shows a similar fibre phenotype. Note the correlation of residual Mef2 (arrows) with better MyHC accumulation. Scale bars: 20 μm.

• Supplemental Figure S4 -

  Fig. S4. Mef2 knockdown reduces myhz1 but not tnnc mRNA in fast muscle. In situ mRNA hybridisation for myhz1 (A,B) and tnnc (C) (lateral view, anterior to left, dorsal to top). (A) Expression of myhz1 at 24 hpf in wild-type embryos injected with the indicated MOs. (B) myhz1 mRNA in wild type (top row) or embryos from a hoo heterozygote cross (bottom row) injected with mef2d MO. In wild type, mef2d MO had no effect, whereas ∼25% of the hoo lay appeared wild type, ∼50% mildly affected and ∼25% severely affected. (C) No change in level of tnnc in 24-hpf embryos injected with the indicated MO combinations.