DEV003905 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Table S2 -

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• Supplemental Figure 1 -

  Fig. S1. Pbx expression during early myogenesis stages is lost in pbx2-MO; pbx4-MO embryos. (A-D) Anti-zebrafish pan-Pbx staining in (A-C) whole wild-type control and (D) pbx2-MO; pbx4-MO embryos at (A) bud stage/10 hpf, (B) 10 somite stage (s)/14 hpf, or (C) 24 hpf. 24 hpf embryos were also stained with Sytox Green to identify nuclei. (A,B) Show dorsal views, anterior towards the left, of (A) early paraxial mesoderm and (B) somites 3-5. (A) Arrowheads label the rows of adaxial cells next to the notochord. (C,D) Show lateral views, anterior towards the left, centered on somites 8-11. (E) Western blot using zebrafish pan-Pbx antibody and Histone H4 antibody. Western analysis with a Pbx4-specific antibody identified the two Pbx4 bands (data not shown).

• Supplemental Figure S2 -

  Fig. S2. pbx2-MO; pbx4-MO embryos are rescued by exd. (A-D) RNA in situ expression of myog and krox-20 (blue) and myod (red). A and B are wild-type control siblings of C and D, which were injected with pbx2-MO; pbx4-MO. A and C were injected with gfp mRNA, and B and D were injected with exd mRNA. In this experiment, embryos were fixed at the same time, about 10s-stage for A,B,D and about 8s-stage for C. exd mRNA rescues krox-20 and myog expression in pbx2-MO; pbx4-MO embryos. exd mRNA also rescues the developmental delay (scored by myod expression) in pbx2-MO; pbx4-MO embryos. Embryos are shown in dorsal view, anterior towards the left. (E) Quantification of the rescue experiment by counting the number of somites expressing myog or myod. Error bars represent standard deviation.

• Supplemental Figure S3 -

  Fig. S3. myod splice-blocking and translation-blocking morpholinos knock down normal myod mRNA or protein levels. (A) Diagrams of functional domains of Myod, including the acidic activation domain, and the histidine- and cysteine-rich domain (H/C). The amino acid sequence of helix III is 100% identical between zebrafish, mouse and human. Exon 1-exon 2 (E1/E2) and exon 2-exon 3 (E2/E3) junctions are shown. (B) RT-PCR analysis of myod mRNA products (using primers in exon 1 and exon 3) from wild-type control, myod-splMO E1I1 and myod-splMO E2I2 embryos. E1I1 targets the myod exon 1-intron 1 boundary and E2I2 targets the exon 2-intron 2 boundary. cDNA sequencing confirms that the E1I1 splice variant retains intron 1 and the E2I2 splice variants retain all (variant a) or most (variant b) of intron 2. qRT-PCR shows that less than about 10-15% of normally spliced myod transcripts remain in myod-splMO embryos (Figs 2P and S3 and data not shown), whereas the total level of myod mRNA remains similar or increased compared to that in control embryos (Fig. S3 and data not shown). (C) Predicted amino acid sequences based on cDNA sequencing of splice variants. Helix III domain is underlined; amino acid sequence from translated intron sequences are shown in italics. When intron 1 or intron 2 splicing is inhibited, in-frame stop codons in intron 1 or intron 2 predict the generation of truncated Myod proteins lacking helix III. E2I2 variants a and b share the same intron sequence up to the stop codon. (D-G) Mammalian anti-Myf5 antibody (which detects zebrafish Myod protein; Hammond et al., 2007) staining in (D) control, (E) myod-MO, (F) myod-splMO E1I1, and (G) myod-splMO E2I2 embryos. The anti-Myf5 antibody was generated against the C-terminus of the protein. Therefore, although myod-splMO embryos appear to have little or no wild-type Myod protein, we cannot determine whether the predicted truncated product is present.

• Supplemental Figure S4 -

  Fig. S4. Fast-muscle differentiation is delayed and reduced in pbx2-MO; pbx4-MO and in myod-MO embryos. (A-C) Antibody staining of fast-muscle myosins with F310 (green in A-C) or pan-muscle myosins with MF20 (red in A-C) in (A) wild-type control, (B) pbx2-MO; pbx4-MO, or (C) myod-MO embryos, all at 16s stage. Somites 4-11 of each embryo are shown in dorsal view, anterior towards the left. MF20 is expressed most strongly in adaxial cells at this stage and appears normal in pbx2-MO; pbx4-MO and in myod-MO embryos. F310 staining is reduced in more posterior somites of pbx2-MO; pbx4-MO and myod-MO embryos.

• Supplemental Figure S5 -

  Fig. S5. cxcr4a expression is maintained in anterior somites in pbx2-MO; pbx4-MO and in myod-MO embryos. (A-C) RNA in situ hybridization of cxcr4a at 18s in (A) wild-type control, (B) pbx2-MO; pbx4-MO, or (C) myod-MO embryos. cxcr4a is normally expressed in the anterior-lateral cells of only tail somites at this stage (Hollway et al., 2007). Embryos are shown in dorsal view, anterior towards the left.

• Supplemental Figure S6 -

  Fig. S6. Pbx-Meis heterodimers bind to the zebrafish mylz2 promoter. EMSA using in vitro translated zebrafish Pbx4 and Meis3 proteins and a probe from sequences −392/−366 of the zebrafish mylz2 promoter (Ju et al., 2003). An excess of unlabeled wild-type probe effectively competes the shifted species, whereas a competitor with a mutant Pbx site does not.