DEV02765 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 -

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• Supplemental Table 2 -

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• Supplemental Figure 1 -

  Fig. S1. Comparative sequence analysis of the zic2a-zic5 genomic locus in zebrafish (Danio rerio) and Fugu rubripes. (A) Graphic representation of the region, with the conserved regions identified using PipMaker. Detailed views of the conserved regions IG and D5 are shown as thick gray lines. Asterisks mark sequences matching the consensus Tcf/Lef-binding site SWWSAAAS (Dorsky et al., 2000, Ryu et al., 2001). Blue asterisks mark sites that are found only in the zebrafish genome; red asterisks mark sites that are conserved in Fugu. (B) Sequence alignment of the IG conserved region. IG contains an 850 bp region that is 49% conserved with Fugu. Blue highlighting indicates the Tcf/Lef consensus sites found only in the zebrafish genome; red marks conserved sites. (C) Sequence alignment of the D5 region. Two conserved regions were found in D5, one of 450 bp and 73% conserved (top), and one of 250 bp and 66% conserved (bottom). The Tcf/Lef consensus sites contained in these regions are shown in red to indicate conservation in Fugu.

• Supplemental Figure 2 -

  Fig. S2. Comparison of TOP:GFP and zic2aD5:gfp expression. Embryos were processed by ISH to detect gfp RNA. (A) Transgenics carrying the Wnt-responsive transgene TOP:GFP (Dorsky et al., 2002) show strong midbrain and weak hindbrain expression at 10 somites. (B) zic2aD5:gfp is weakly expressed in the midbrain and strongly expressed in the hindbrain at the same stage. (C,D) At a later stage (20 somites) TOP:GFP expression overlaps with zic2aD5:gfp, primarily in the midbrain. All views are lateral, anterior to the left. Arrowheads mark the tectal borders.

• Supplemental Figure 3 -

  Fig. S3. Cycloheximide inhibits translation. Transgenic flh:gfp (Gamse et al., 2003) embryos were treated with vehicle (DMSO) (A,C) or CHX (B,D) and scored for expression of Gfp by fluorescence. (A,B) Embryos treated at sphere stage and scored at 80% epiboly. Vehicle-treated control embryos (A) show normal levels of Gfp, while expression is barely detectable in embryos treated with CHX (B). (C,D) Embryos treated at 50% epiboly and scored at 100% epiboly. Gfp expression was much reduced in CHX-treated embryos compared to vehicle-treated controls. Note that transgenic Gfp expression had already begun by the start of the earliest CHX treatment at sphere stage. Gfp protein is very stable, making it likely that some of the remaining Gfp fluorescence is due to pretreatment synthesis.

• Supplemental Figure 4 -

  Fig. S4. Tcf3 reduction affects midbrain size. (A-C) Uninjected control embryos showing normal zic2aD5:gfp (A), zic2a (B) and wnt3a (C) expression at 18-somites. (D-O) Embryos injected with tcf3 MOs and assayed for zic2aD5:gfp (G,J,M), zic2a (H,K,N), or wnt3a (I,L,O) expression at 18-somites. Knockdown of Tcf3 resulted in expanded expression of all markers in the midbrain at low MO concentrations. This is most likely due to the previously documented overall midbrain expansion (D-I). At higher MO concentrations, zic2aD5:gfp and zic2a were reduced in midbrain (J,M,K,N), whereas wnt3a expression remained strong (L,O). Embryos are shown in lateral view, anterior to the left. Arrowheads mark anterior and posterior limits of the midbrain.

• Supplemental Figure 5 -

  Fig. S5. Tectal wnt gene expression domain is shorter in zic morphants. Embryos injected with the morpholino listed on the y-axis were measured along the AP axis using Zeiss Axiovision software. Tectal wnt gene expression domains were statistically shorter in zic2a and zic2a+zic5 morphants than in controls (*P&λτ;0.05). Error bars represent the s.e.m.

• Supplemental Figure 6 -

  Fig. S6. TOP:GFP expression in zic morphants. As zic1 was recently shown to regulate wnt genes (Merzdorf and Sive, 2006), we asked if Wnt signaling was affected in zic2a-zic5 morphants by assaying expression of a Wnt reporter transgene. This analysis, although not quantitative, suggested that Wnt signaling was not altered in zic morphants. (A) TOP:GFP transgene contains a wnt/β-catenin responsive synthetic promoter (TOP) composed of four consensus Lef-binding sites (blue stars) and a minimal promoter sequence, driving a destablized gfp reporter (Dorsky et al., 2002b). TOP:GFP transgenic embryos were injected with 2 MOC+5MOC (C,E), and gfp expression was compared with uninjected embryos (B,D) at 20-somites or at 25 hpf. (B,C) At 20-somites, TOP:GFP expression in morphants (n=24) was comparable to that in uninjected embryos (n=18). (D,E) At 25 hpf, the TOP:GFP expression domain was misshapen in morphants (n=20/82), probably as a result of morphological defects, as compared with controls (n=18). However, there was no significant expansion or reduction in TOP:GFP expression. All embryos are positioned in lateral view, with anterior to the left.