Fig 2: Wnt11 acts through the Wnt/Ca2+ signaling pathway during dorsal somite extension.(A) Schematic representation of KN-93 treatment, embryos in the control group were treated with DMSO. (B) Embryos treated with KN-93 (n = 65.5 somites from 6 embryos) have significantly more pH3-positive cell in the dorsal DM compared to embryos of the control group (n = 52.5 somites from 5 embryos) (median, first and third quartiles, p = 0.045). (C-C’’) Quantification of protrusions formed by the 8th-12th somites of Tg(zic1:GFP) embryos treated with DMSO (n = 10 embryos) or KN-93 (n = 13 embryos) (mean ± SD, p = 0.65 for small protrusions, p = 0.014 for large protrusions, p = 0.0017 for total protrusions). *p < 0.05, **p < 0.01, ns, not significant.

Fig 3: Zic1 regulates dorsal-specific expression of genes in the somites.(A) Schematic representation of preparation of dorsal and ventral somite cells for RNA-seq and ATAC-seq. (B) Schematic representation of generating the transgenic line Tg(zic1:zic1-Myc);Da. Somites expressing zic1-Myc are subjected to ChIPmentation against Myc. (C) Analysis of RNA-seq revealed that 694 genes are expressed specifically in the dorsal somites (among these genes are zic1, zic4 and wnt11) and 724 genes are expressed specifically in ventral somites. Magenta indicates differentially expressed genes (adjusted p-value < 0.01). (D) The ChIPmentation against Zic1 revealed that 324 of dorsal-high genes, 192 dorsal-low genes and 2716 non differential expressed genes in the somites are potential Zic1 target genes.

Fig 4: Knock-down of wnt11 in Wt embryos recapitulates the Da dorsal somite phenotype.(A) Schematic outline of PhotoMO mediated knock-down of wnt11. (B, C) Dorsal view of maximum projection of whole-mount Phalloidin staining of 9 dpf embryos injected with PhotoMO but not photocleaved (B) and photo-cleaved (C). The epaxial myotomes of the wnt11 morphant are affected. (D) Quantification of pH3-positive cells in the dorsal DM of 22 ss embryos. Embryos with reduced levels of wnt11 in their dorsal somites (n = 40.5 somites from 5 Da embryos (adopted from Figure 2E), n = 61.5 somites of 6 wnt11 PhotoMO-Morphant embryos and n = 52 somites from 5 wnt11 morphant embryos) have significantly more pH3-positive cells in their dorsal DM compared to the respective controls (n = 66 somites from 6 Wt embryos (adopted from Figure 2E), n = 51 somites of 5 uninduced PhotoMO embryos and n = 65 somites from 6 Control MO embryos; p = 0.0035, 0.0091, 0.021, respectively). Median, first and third quartiles are shown. (E) Dorsal view of 24 ss Tg(zic1:GFP) embryo injected with wnt11 PhotoMO and photo-cleaved at 4 ss. Z-stacks were recorded every 10 min, time is displayed in min, and 15th somite is positioned in the center. (F, G) Lateral view of dorsal somites (colored magenta) of Tg(zic1:GFP) injected with Control MO (F) and wnt11 MO (G). (H-H’’) Quantification of protrusions formed by the 8th-12th somite of Tg(zic1:GFP) injected with Control MO (n = 10 embryos) or wnt11 MO (n = 13 embryos) (mean ± SD, p = 0.069 for small protrusions, p = 7.7e-08 for large protrusions, p = 0.00064 for total protrusions). Anterior to the left. NT, neural tube. Scale bar = 50 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Fig 5: wnt11 is a direct downstream target of Zic1 and down-regulated in the dorsal somites of the Da mutant.(A) GO analysis of dorsal-high (left) and dorsal-low (right) Zic1 target genes. (B) Pathway enrichment analysis indicated that genes associated with Wnt signaling pathway are specifically enriched in dorsal-high Zic1 target genes. (C) Analysis of the wnt11 locus. Peaks form the ChIPmentation against Zic1 (magenta track) overlap with open chromatin regions in the dorsal somites (black track), while this genomic region is less open in ventral somites (grey track). RNA-seq data revealed that wnt11 is highly expressed in dorsal somites (dark green track) and minimally in ventral somites (light green track). (D, E) Dorsal view of tails of whole-mount in situ hybridization against wnt11 performed in Wt (D) and Da (E) 12 ss embryos. The expression of wnt11 is reduced in the Da mutant. (F) RT-PCR performed on pooled tails of Wt and Da 12 ss embryos indicates that zic1 expression is reduced by 8.8-fold and wnt11 expression by 1.4-fold in the Da mutant. Anterior to the left. Scale bar = 100 µm.