Fig. 6. Hedgehog-independent secondary superficial slow fibres require Prdm1, whereas smyhc2 expression does not. (A) Lateral views of smyhc1-CDS in situ hybridisation on 24 hpf, 36 hpf and 48 hpf smo mutants, ubo (prdm1)^tp39 mutants, nrd (prdm1)^m805 and wild-type (wt) siblings; and transverse sections of 24 hpf ubo (prdm1)^tp39 mutants and wild-type siblings. The near absence of Smyhc-expressing primary slow fibres in smo mutants at 24 hpf allows secondary slow fibres to be identified unambiguously at later stages. At 24 hpf, ubo (prdm1)^tp39 or nrd (prdm1)^m805 mutants have abundant, misplaced, weakly Smyhc-expressing fibres. Thus, it is unclear whether Smyhc-expressing fibres at later stages in prdm1 mutants are simply the remnants of those present at 24 hpf. (B) When primary slow fibre specification is prevented by cyclopamine treatment, 48 hpf genotyped ubo (prdm1)^tp39 mutants lack almost all smyhc1-CDS in situ hybridisation and slow troponin C (stnnC) in situ hybridisation in secondary superficial slow fibres, whereas genotyped wild-type siblings have robust expression. (C) smyhc2 (1 to 155 bp) in situ hybridisation showing 96 hpf nrd (prdm1)^m805 mutants have near wild-type levels of expression in the sca, iob, scp, icp and craniofacial muscles, but lack smyhc2-expressing els fibres. High magnification dorsal views show sca and lateral views show iob and scp/icp. Scale bars: 25 µm. Abbreviations: els, embryonic lateralis superficialis; icp, infracarinalis posterior; iob, inferior obliquus; sca, supracarinalis anterior; scp, supracarinalis posterior.