Du SJ, Devoto SH, Westerfield M, Moon RT.

P01 HD022486 NICHD NIH HHS , HD22486 NICHD NIH HHS , HD29360 NICHD NIH HHS

	Figure 2. Induction of slow muscle cells by zebrafish Sonic hedgehog (Shh) and Tiggy-winkle hedgehog (Twhh). (A, B, and C) Sections (dorsal to the top) showing fluorescence localization of slow muscle cells labeled with F59, an anti–myosin heavy chain antibody, in embryos injected with frame shifted sonic hedgehog (Shhfs) (A), Shh (B), or Twhh (C). (D, E, and F) Sections (dorsal to the top) showing fluorescence localization of muscle pioneer cells labeled with 4D9, an anti-engrailed antibody, in embryos injected with Shhfs (D), Shh (E), or Twhh (F). (G, H, and I) Whole-mount Nomarski images showing muscle pioneer cells labeled with the 4D9 antibody in embryos injected with Shhfs (G), Shh (H), or Twhh (I). Embryos in G, H, and I are oriented in side views, with anterior to the left and dorsal to the top. Bars, 50 μm.
	Figure 3. Inhibition of slow muscle cells by a constitutively active isoform of PKA. (A) Whole-mount Nomarski images showing slow muscle cells labeled with the F59 antibody in a representative control embryo. (B) Whole-mount Nomarski images showing slow muscle cells labeled with the F59 antibody in a representative embryo injected with a constitutively active form of PKA. (C) Section (dorsal to the top) showing localization of slow muscle cells labeled with F59 in a control embryo. (D) Sections (dorsal to the top) showing local loss of slow muscle cells in an embryo injected with a constitutively active form of PKA.
	Figure 4. Dorsalin-1 blocks the development of muscle pioneer cells. (A) β-gal expression in embryos injected with twhh-β-gal was monitored by enzyme activity and Nomarski microscopy at early pharyngula stage (∼24 h). At this stage, the expression of β-galactosidase was notochord specific in >90% (n = 120) of the injected embryos that expressed the construct. We first detected β-galactosidase expression in the early segmentation stage (∼12 h), specifically in notochord cells of injected embryos (84%, n = 57, data not shown). (B) Immunolocalization with anti– c-myc antibody in embryos injected with the DNA construct twhh-dsl-1myc. The twhh promoter drives expression of dsl-1myc in notochord cells (arrowheads). (C and D) Double labeling with anti–c-myc antibody and anti-engrailed antibody, 4D9, in embryos injected with either the DNA construct twhh-bGFP (C) or twhh-dsl-1myc (D). The bracket in D marks the region affected by Dorsalin-1. The dsl-1myc expressing notochord cells in D are indicated by the arrowheads. Muscle pioneer cells in C and D are indicated by arrows. Cells in only some regions of the embryos expressed the transgenes (B and D, arrowheads), consistent with the mosaic expression of other injected DNAs (Westerfield et al., 1992). In 93% (n = 54) of the embryos lacking muscle pioneer cells in some of their somites, nearby notochord cells expressed Dorsalin-1. Embryos are oriented in side views, with anterior to the left and dorsal to the top. Bar, 50 μm.
	Figure 5. Dorsalin-1 specifically affects the development of muscle pioneer cells but not the non–muscle pioneer slow muscle cells. (A) Whole-mount staining of embryo injected with DNA construct twhh-dsl-1myc using F59 to monitor slow muscle cells. The bracket marks the region affected by Dorsalin-1. (B) Section through an unaffected region with both muscle pioneer cells (arrows) and non–muscle pioneer slow muscle cells (arrowhead) labeled with F59. (C) Section through an affected region with normal non–muscle pioneer slow muscle cells labeled with F59 antibody (arrowhead) but no muscle pioneer cells. The orientation is anterior to the left (A) and dorsal to the top (A–C). Bars: (A) 100 μm; (B and C) 50 μm.
	Figure 6. Ectopic Dorsalin-1 represses muscle pioneer cell induction by Hedgehogs. Embryos were double labeled with anti-myc antibody to monitor expression of dsl-1myc and with the 4D9 antibody to monitor muscle pioneer cells. (A) Embryos injected with Shhfs RNA as control. (B) Embryos coinjected with twhh-bGFP DNA and zebrafish Shh RNA show induction of extra muscle pioneer cells. (C) Embryos coinjected with twhh-dsl-1myc DNA and zebrafish Shh RNA also show induction of extra muscle pioneer cells, but not in the region (bracket) near the notochord cell expressing Dorsalin-1 (arrowhead). Embryos are oriented in side views, with anterior to the left and dorsal to the top. Bar, 50 μm.
	Figure 7. dnPKA induces slow muscle cells. (A–C) Sections showing slow muscle cells labeled with F59 in embryos injected with Shhfs (A), dnPKA (B), or dnPKA/twhh-dsl-1myc (C). Extra slow muscle cells are induced in both B and C. (D–F) Sections showing muscle pioneer cells labeled with 4D9 in embryos injected with Shhfs (D), dnPKA (E), or dnPKA/twhh-dsl-1myc (F). Muscle pioneer cells are induced by dnPKA (E) but are repressed by dsl-1 (F). (G–I) Whole-mount labeling with anti–c-myc and 4D9 antibodies in embryos injected with Shhfs (G), dnPKA (H), or dnPKA/twhh-dsl-1myc (I). The bracket in I marks the region affected by Dorsalin-1. The dsl-1myc expressing cell in I is indicated by the arrowhead. The orientation is dorsal to the top (A–I) and anterior to the left (G–I). Bar, 50 μm.
	Figure 8. Opposing actions of Hedgehog and BMP4-like proteins regulate slow muscle cell identity. (A) At the tail bud stage, paraxial mesoderm cells are induced by Hedgehog secreted from notochord precursor cells to become adaxial cells, the slow muscle precursors. A subset of the adaxial cells located adjacent to the notochord are induced to become muscle pioneer cells by continued Hedgehog signal from the notochord cells and floor plate cells. In contrast, other slow muscle precursors migrate to the lateral surface of the myotome and differentiate into non–muscle pioneer slow muscle cells. BMP4-like inhibitory signal antagonizes the hedgehog signals in dorsal and ventral regions of the somite and blocks the induction of muscle pioneer cells in these regions. (B) Schematic illustration of this model in cross section at the tailbud stage (upper embryo, arrow denotes Hedgehog signal), and at the somitogenesis stage (lower embryo, arrow denotes Hedgehog signal and stippling denotes the BMP4-like signal). The distribution of muscle pioneers is determined by the distribution of these competing signals.

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