DEV012849 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Table S2 -

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• Supplemental Table S3 -

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• Supplemental Table S4 -

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• Supplemental Figure S1 -

  Fig. S1. GFP-expressing myotubes in primary culture derived from embryos carrying Hand-Gal4/UAS-2EGFP transgenes. Primary cells were stained for Dmef2 (blue in merge), Mhc (red in merge) and GFP (green in merge). Note that GFP-positive cells are also stained for Mhc, which shows periodic sarcomeric organization (red arrow), but myofibrils in these cells are thinner and less bundled (red arrow) compared with those in non-GFP primary muscles (white arrow). The number of nuclei per cell in the cells positive for both GFP (derived from Hand-Gal4, UAS-2EGFP) and Dmef2 was ∼1.2±0.4, many fewer than in the GFP-negative Dmef2-positive primary myotubes presumably derived from somatic muscles (∼3.7±0.2).

• Supplemental Figure S2 -

  Fig. S2. Myogenesis in culture with a serum starvation step. (A) Freshly isolated cells were stained for Actin with phalloidin. (B) Primary cells were stained for Actin with phalloidin and with DAPI (blue) immediately before the addition of serum. Note that multinucleated myotubes began to form, but that the periodic pattern of Actin filaments was not obvious (arrowhead). Phalloidin stainings of primary myotubes at 2 days (C) and 10 days (D) after serum addition at 18°C in culture. Note that the periodic pattern of Actin filaments is obvious in C and D, whereas myofibrils are not bundled in C (short arrows), but are in D (long arrows). Scale bar: 40 µm.

• Supplemental Figure S3 -

  Fig. S3. RNAi phenotypes of Fit1 and Fit2 in primary culture. Primary cells treated with dsRNAs targeting (A) lacZ, (B) Fit1 and (C) Fit2 stained with phalloidin. (D-F) Primary cells were isolated from embryos carrying Dmef2-Gal4, D42-Gal4, UAS-mito-GFP and treated with dsRNAs targeting both Fit1 and Fit2. Primary muscles in culture were detected by phalloidin staining for Actin (D and red in F). mito-GFP (E and green in F) revealed both neuronal axons (white arrows in E,F) and rounded-up muscles (yellow in F).

• Movie 1 -

  Movie 1. Time-lapse movie of a contracting primary myotube in culture. The myotube expresses GFP as a result of the fusion between Dmef2-Gal4 and UAS-2EGFP cells.

• Movie 2 -

  Movie 2. Time-lapse movie of contracting primary myotubes shown at lower magnification.