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Fig. 4 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.