Figure 3.

Specific Dvl^1–3 knockdown induces developmental delays post-implantation, and anterior midline and neural fold defects. (A) Average Dvl^1–3 mRNA levels (Dvl^1–3/GAP3DH ratio) of 10 embryos electroporated with dsDvl^1–3 shown as a percentage of those of 10 dsGFP-treated controls, following 24 h in culture. Standard deviation bars are indicated. (B) Morphological phenotypes of dsDvl^1–3 (c-g) and control dsGFP -electroporated embryos (a, b) recovered at E7.5 and E8.0 (brightfield micrographs). Anterior faces left when it can be identified, except in b-e, where an anterior view is presented. E7.5 and E8.0 represent the embryonic time at collection; actual embryonic stages may vary due to Dvl RNAi. dsDvl^1–3-treated embryos are smaller than normal and retarded in development (c-f) (compare with f in Fig 2C, note scale bars). Note the severe delay in f and the early developmental arrest in g. Notably, in some E8.0 embryos, the anterior midline was grossly distorted and, in some cases, was connected anteriorly to a single neural fold of either side (d, e), giving to the anterior view the appearance of a slightly-lateralized view. Bar, 200 μm. (C) Distribution of morphological phenotypes observed past gastrulation. Emb. day, embryonic day; Nor, normal; Def, defective; Dl'd, delayed, Art'd, arrested. (D) Whole-mount in situ hybridization with anti-sense probes of indicated genes on dsDvl^1–3-electroporated embryos. Anterior faces left. Expression of Brachyury (n = 10), Eomes (n = 8), Lhx1 (n = 8), Cer-l (n = 11) and Oct4 (n = 6) is seemingly unaffected in these embryos (compare to Fig 2e, top row). Note the severe developmental delay, particularly evident in b, f and h, and the abnormal extraembryonic region in h. Bar, 200 μm.