DEV02291 Supplementary Material

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• Supplemental Table 1 (Adobe PDF)
• Supplemental Figure 1 -

  Fig. S1. Early disruption of the laminin-containing basament membrane in DM cells treated with cN390D-GFP. (A,B) Electroporation of control GFP (n=5). (C,D) Electroporation of cN390D-GFP (n=5). Embryo sections were immunolabeled with GFP (green) and laminin (red) antibodies. B and C are overlays of GFP and laminin fluorescence. The basement membrane overlying the DM in A and B is continuous as assessed by strong laminin expression, whereas it is patchy and reveals lower laminin protein in the basal aspect of cells that received cN390D-GFP (arrowheads in A and C). This is associated with the onset of disruption of epithelial conformation of cN390D-GFP-treated DM cells (arrowheads in D). Laminin also strongly labels the basement membrane underneath the ectoderm (EC, arrows in A,C), and that surrounding the neural tube (NT, curved arrows in A,C). Scale bar: 27 mm.

• Supplemental Figure 2 -

  Fig. S2. cN390D-GFP prevents the entry of Pax7-positive cells into the myotome. Transfection of control GFP (A-C, sagittal section) or cN390D-GFP (D-F, semi-frontal section) to the rostral lip of dissociating somites. Approximately 15 hours later, control GFP-electroporated cells entered the myotome either as myofibers that already downregulated Pax7 protein (arrowheads in A) or as Pax7-positive myoblasts (arrows in A,B). The epithelial conformation of the lip is maintained at this stage with strong Pax7 expression (asterisk in B). By contrast, cN390D-GFP-expressing cells failed to colonize the myotome either as GFP-positive fibers or as Pax7-positive progenitors. The lip structure is distorted and labeled cells remain at the intersomite and disperse subectodermally, as observed for the DM sheet. Notably, mRNA expression is already fully absent in the transfected cells by this stage (see, for example, Fig. 5 for Pax3 mRNA; Pax7 mRNA, data not shown), whereas Pax7 immunoreactivity is still retained, albeit weaker, in these cells when compared with both control GFP and untransfected cells in adjacent regions (asterisks). Hoechst nuclear staining in F illustrates that cN390D-GFP-positive cells that prematurely dispersed under the ectoderm are vital, with frequent mitoses (arrows, F). DM, dermomyotome; EC, ectoderm; Myo, myotome; Scl, sclerotome. Scale bar: 20 mm.