Fast-muscle-fibre-specific genes are ectopically expressed in the slow domain of ubo^tp39 mutant embryos. In situ hybridization showing fast-muscle-specific expression of the fast myosin heavy chain gene (fMyHCx) at the 18-somite stage in (A) lateral and (B) transverse views. (C) An fMyHCx:GFP construct expresses GFP specifically in the fast-muscle fibres in the trunk. Transient expression of fMyHCx:GFP in the fast fibres in a 30 hpf (hours post fertilization) embryo, as shown by the colocalization with the fast-fibre marker F310 antigen. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) to highlight the polynuclear arrangement (arrowheads) of the fast fibres expressing fMyHCx:GFP. (D) The construct fMyHCx:GFP is detected in the adaxial cells of ubo^tp39 mutants in transient assays. The fMyHCx:GFP construct is not expressed at the 12-somite stage in wild-type embryos, but is ectopically expressed in the adaxial cells of a 12-somite-stage ubo^tp39 embryo. Lyn tdTomato (red) was used to highlight cellular membranes and the outline of the notochord. (E,F) Ectopic expression of fMyHCx messenger RNA (red) and F310 (green) is detected in the adaxial cells of a 12-somite-stage ubo^tp39 embryo (100% of embryos assayed; n>30). In wild-type siblings (wt sib), adaxial cells are only labelled with the slow MyHC S58 (blue) antibody. Genes that normally are expressed exclusively in the fast-muscle domain are ectopically expressed in the adaxial cells of the ubo^tp39 mutant: (G) mylz2, (H) tnni2 and (I) tnnt3a. Scale bars, 25 μm. GFP, green fluorescent protein; mylz2, myosin light chain 2; tnni2, troponin I2; tnnt3a, troponin T3a; ubo, u-boot; wt, wild type.

Slow-fibre differentiation requires the repressive function of Prdm1. (A) Prox1 expression in slow-muscle fibres of wild-type (wt) embryos at 24 h. (B) ubo^tp39 mutant embryos lack Prox1-expressing slow fibres at 24 h. (C) Eng–Prdm1 expression, marked by tdTomato (tdTOM), rescues mononucleate fibre differentiation and Prox1 expression in ubo^tp39 homozygotes (49 out of 94 Eng–Prdm1-expressing fibres were Prox1 positive; n=8). (D) VP16–Prdm1 expression, marked by tdTOM, was found exclusively in multinucleate fibres lacking Prox1 staining in ubo^tp39 homozygotes (none of the 134 Vp16–Prdm1-expressing fibres was Prox1 positive; n=11). (E) Precocious labelling with the fast-specific F310 antibody in VP16–Prdm1-expressing muscle precursors at the 12-somite stage (12s; 12 out of 30 F310-positive cells; n=4). Scale bars, 25 μm. Eng, Engrailed; Prox1, prospero-related homeobox gene 1; sib, sibling; ubo, u-boot.

Slow-specific gene expression is upregulated in sox6 morphant embryos. (A) sox6 is expressed in the fast-muscle precursors (red arrowheads) in wild-type (wt) embryos and ectopically expressed in the adaxial cells (green arrowheads) in ubo^tp39 embryos at the 12-somite (12s) and 15-somite (15s) stage. (B) Heat-shock-induced expression of Sox6 (marked with tdTomato (tdTOM)) inhibits Prox1 expression at 24 hpf (hours post fertilization) in adaxially derived cells marked by prdm1:GFP (arrowhead; 100% of the adaxially derived cells lacked Prox1 when ectopically expressing Sox6 (from the analysis of 14 fibres in 4 embryos)). (C) Lateral views of the tail at 24 hpf showing partial rescue of Prox1 expression in ubo^tp39 embryos injected with sox6MO (ubo^tp39: 0 rescued fibres in 20 embryos; ubo^tp39; sox6MO: mean of 58 rescued fibres per embryo, n=5, s.d.=29). (D) At 12s, ubo^tp39 mutants have reduced expression of smyhc1 and ectopic expression of fast-MyLC (recognized by the F310 antibody) in the adaxial cells marked with prdm1. sox6MO causes rescue of the smyhc1 expression in ubo^tp39 mutants but does not prevent ectopic fast-MyLC expression. (E) Transverse views of 24 hpf wt and smoothened (smo) embryos carrying the prdm1:GFP reporter gene (upper panel) and siblings of the same genotypes that have been injected with the sox6 morpholino (sox6MO; lower panels), showing that loss of sox6 causes Hedgehog-independent ectopic expression of stnnC (red) in fast muscle (identified by F310 antigen (blue)). (F) Lateral views of stnnC expression (red) in wt, smo and smo;sox6MO embryos at 24 hpf (9 out of 9 smo;sox6MO embryos, and 15 out of 15 wt embryos showed strong ectopic stnnC expression). Scale bars, 0.25 μm. GFP, green fluorescent protein; Prox1, prospero-related homeobox gene 1; smyhc1, slow myosin heavy chain 1; sox6MO, sox6 morpholino; stnnC, slow-specific Troponin C; ubo, u-boot.

Prdm1 acts as a direct repressor of fast-fibre-specific gene expression in adaxial cells. (A) prdm1:GFP expression is specific to adaxial cells in the somite area at the eight- to ten-somite stage. A Prdm1-specific polyclonal antibody detects Prdm1 protein in adaxial cells, consistent with the prdm1:GFP expression pattern. Scale bar, 0.25 μm. (B) Prdm1 is specifically immunoprecipitated using the Prdm1 antibody. (C) Amplification of the fast-fibre-specific genes fMyHCx, tnnt3a, tnni2 and mylz2 using Taqman real-time PCR showed significant enrichment compared with the pre-immune controls. Three slow-muscle-specific genes, prox1, sMyHC1 and stnnC, were tested as negative controls and were not enriched in the chromatin immunoprecipitated (ChIP) chromatin. (D) The mylz2:GFP promoter fusion is not expressed in wild-type embryos at the 12-somite stage, but is expressed in the adaxial cells in prdm1 morphants at the 12-somite stage. Mutation of the Prdm1-binding sites (5M) resulted in ectopic adaxial GFP expression in both control and prdm1 morphant embryos. Scale bar, 0.25 μm. fMyHC, fast myosin heavy chain; GFP, green fluorescent protein; MO, morpholino; mylz2, myosin light chain 2; prox1, prospero-related homeobox gene 1; sMyHC1, slow myosin heavy chain 1; stnnC, slow-specific Troponin C; tnni2, troponin I2; tnnt3a, troponin T3a.