These signals include Sonic hedgehog and BMP4 (Munsterberg et al., 1995; Pourqui et al., 1996). of the somite into specific cell types is usually under the influence of inductive signals from surrounding tissues, such as notochord, neural tube, and the surface ectoderm (for review, observe Hauschka, 1994; Christ and Ordahl, 1995). A variety of extracellular signaling molecules, including users of (Fan and Tessier-Lavigne, 1994; Johnson et al., 1994), (Munsterberg et al., 1995), and (Pourqui et al., 1996) gene families, have been implicated in patterning the somite. Ventral midline tissues express Sonic hedgehog, which plays a critical role in sclerotome and myotome induction (Fan and Tessier-Lavigne, 1994; Johnson et al., 1994). Wnts, Tonabersat (SB-220453) which are expressed in the neural tube, act in combination with Hedgehog to induce myogenesis in vitro (Munsterberg et al., 1995). Lateral plate mesoderm in chick embryos expresses BMP4, a gene family member that is a candidate for inducing the differentiation of the lateral myogenic precursors in the somite, which give rise to the muscles of the limbs and body wall (Pourqui et al., 1996). This effect of BMP4 is usually opposed by an unknown diffusible factor expressed in the neural tube (Pourqui et al., 1996). Vertebrate skeletal muscle mass contains muscle mass fibers of several types, which can be broadly classified as slow or fast fibers on the basis of differences in contraction speeds, metabolic activities, and motoneuron innervation. The earliest developing embryonic muscle mass fibers have intrinsic fiber type properties (Butler et al., 1982; Thornell et al., 1984; Crow and Stockdale, 1986; Harris et al., 1989; Fredette and Landmesser, 1991and gene families in the development of different muscle mass fiber types in zebrafish. We provide evidence that slow muscle mass cells are induced by Hedgehogs, and that this induction is likely due to respecification of fast muscle mass precursor cells into slow muscle mass cells. We also show that ectopic expression of Hedgehogs induces supernumerary muscle mass pioneer cells. This induction of muscle mass pioneers is usually repressed by Tonabersat (SB-220453) ectopic expression in the notochord of Dorsalin-1, a BMP4-related protein. Our data suggest that members of the and gene families play opposing functions in patterning the developing somite. Tonabersat (SB-220453) MATERIALS AND METHODS Animals Embryos were staged by hours (h) after fertilization at 28.5C (Kimmel et al., 1995; available at World Wide Web address Chorions were removed with watchmaker’s forceps, and embryos were managed in Ringer’s answer (Westerfield, 1995). Older embryos were anesthetized in a 0.6 mM solution of tricaine ((gene (Ekker et al., 1995promoter, was partially digested with BstXI and EcoRI to release the DNA place. The EcoRI site at the 5 end of the place was then blunted by Klenow DNA polymerase and subcloned into the pBluescript-SK BstXI site that had been Tonabersat (SB-220453) partially blunted by T4 DNA polymerase. The producing plasmid, pBluescript-SK-promoter sequence was purified and cloned into plasmid pCS-(a gift from D. Turner, R. Rupp, J. Lee, and H. Weintraub, Fred Hutchinson Malignancy Research Center, Seattle, WA) at the SalI and HindIII sites, the Tonabersat (SB-220453) latter site having been blunted by Klenow DNA polymerase. The producing plasmid, pCS-promoter and the nuclear reporter gene. pCS-twhh–gal-vec. To make the promoter into a convenient expression vector for expressing heterologous cDNAs, the BamHI site upstream of the promoter in the plasmid pCS-was deleted. The producing plasmid, which retains the BamHI site between the promoter and the sequence. pCS-twhh-bGFP. The reporter gene, bright green fluorescent protein (bGFP) with a serine 65 to threonine mutation (Heim et al., 1995), was cloned into vector pCS-cDNA was amplified from 9-d chick embryos by reverse transcriptase PCR using primers based on the published chick DNA sequence (Basler et al., 1993). The sequences for the 5 and 3 PCR primers were 5 CTCTGTCTGTAAAGATTCAAC 3 and 5 GTACAGTTTCACAGACAGCAG 3, respectively. The PCR product was subcloned into the pCR-II vector (Invitrogen, San Diego, CA). The c-mycCtagged derivative (promoter, the DNA place of promoter, the sequence. The final construct, pCS-promoter and and pT7TScontain the zebrafish and cDNAs, respectively (Ekker et al., 1995contains the with a single base pair insertion, resulting in a frame shift in amino acid position No. 39 (Ekker et al., 1995contains the dominant negative form of PKA regulatory subunit (Ungar and Moon, 1996). IL-15 Plasmid pSP64T-and RNAs were transcribed from DNA plasmid T7TSor T7TSas explained (Ekker et al., 1995and genes at high levels (Hatta et al., 1991; Ekker et al., 1992). Fast muscle mass precursors, in contrast, develop from lateral presomitic cells and remain deep within.