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. 2022 May 6;11:e71845. doi: 10.7554/eLife.71845

Figure 7. 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.

Figure 7.

Figure 7—figure supplement 1. wnt11 expression starts before gastrulation and increases with proceeding development.

Figure 7—figure supplement 1.

(A) Reads per kilo base per million mapped reads (RPKM) of wnt11 at 5 hpf (hours post fertilization, stage 9), 6 hpf (stage 10), 8 hpf (stage 11), 10 hpf (stage 12, pre-early gastrula stage), 12 hpf (stage 13), 14 hpf (stage 14), 17 hpf (stage 15, mid-gastrula stage), 24 hpf (stage 17), 30 hpf (6 ss, stage 21), and 54 hpf (24 ss, stage 27).
Figure 7—figure supplement 2. Injection of wnt11 sgRNAs results in delayed epiboly movement, and impaired body axis and trunk development.

Figure 7—figure supplement 2.

(A) Schematic representation of the promoter region and exons of wnt11 including the position of sgRNAs. (B-B’’) Quantification of phenotypes at 17 hpf (50% epiboly) (B), 26 hpf (stage 18, 100% epiboly) (B’) and 2.4 dpf (24 ss) (B’’) (n = 20 control embryos, n = 30 wnt11 crispant embryos). (C, D) Lateral view of 17 hpf control embryo injected with Cas9-protein only (C) and wnt11 crispant embryo (D). The progression of epiboly is delayed in the wnt11 crispant (D). Arrowheads indicate progression of epiboly. (E, F) Dorsal and lateral view of control embryos (E) and wnt11 crispant embryos (D) at 24 ss. wnt11 crispants show a variety of morphological defects including shortened body axis, impaired trunk (arrowhead in F’) and head (arrowhead in E’) development. Scale bar = 400 μm.