Figure 9. Ectopic Shh signaling disrupts lateral cell remodeling and causes exencephaly.

(A,B) Expression of the activated Shh receptor Smoothened (SmoM2) using the midbrain-specific Wnt1-Cre2 driver causes exencephaly (12/12 Wnt1-Cre2; SmoM2 embryos vs. 0/13 littermate controls). Control embryos were Wnt1-Cre2 or SmoM2 alone. (C,D) Wnt1-Cre2 drives SmoM2-YFP expression in the midbrain and induces ectopic Nkx6.1 expression throughout the mediolateral axis. Boxes, regions shown in (E,F). (E,F) Cells expressing SmoM2-YFP have larger apical areas compared with cells outside of the Wnt1-Cre2 expression domain (cells below the dashed line) and cells from equivalent regions in controls (E). SmoM2-YFP signal at the lateral edge of the N-cadherin region is shown. (G) Lateral and midline cells labeled with N-cadherin are color coded by area in control and SmoM2-expressing embryos. (H–K) Average apical cell area (H,J) and apical area distributions (I,K) in lateral and midline cells in control and SmoM2-expressing embryos. (L) Schematics of the pattern and intensity of the Shh response in WT, Gli2 mutant, IFT-A mutant, and SmoM2-expressing embryos. (M) Model. The different shapes of lateral and midline cells correlate with different levels of Shh signaling. A high Shh response inhibits apical remodeling and apicobasal elongation in midline cells, whereas a low Shh response allows apical constriction in lateral cells. A single value was obtained for each embryo and the mean ± SD between embryos is shown, n = 3 embryos/genotype, **p<0.01 (Welch’s t-test). See Supplementary file 1 for n and p values. Embryos are E10.5 in (A,B), 6–7 somites in (E–K). Anterior up. Bars, 100 μm in (C,D), and 20 μm in (E–G).

Figure 9—figure supplement 1. Cell proliferation is not affected by neuroepithelial expression of SmoM2.

(A) Lateral cell proliferation in embryos labeled with pHH3 to mark mitotic cells and phalloidin (F-actin) to mark cell outlines in embryos expressing activated Smoothened using the midbrain-specific Wnt1-Cre driver. Control embryos were Wnt1-Cre2 or SmoM2 alone. (B) The percentage of proliferative (pHH3+) cells in a 100 μm x 100 μm lateral region. No significant difference was observed between SmoM2-expressing embryos and controls. A single value was obtained for each embryo and the mean ± SD between embryos is shown, n = 4 embryos/genotype, Welch’s t-test. Embryos are 7–9 somites. See Supplementary file 1 for n and p values. Anterior up. Bars, 20 μm.

Figure 9—figure supplement 2. Analysis of mediolateral cell orientation in SmoM2-expressing embryos.

(A,C) Percentage of lateral cells (A) and midline cells (C) with a mediolateral (ML) orientation (0–45° relative to the ML axis) or an anterior-posterior (AP) orientation (45–90° relative to the ML axis) in embryos expressing activated Smoothened using the midbrain-specific Wnt1-Cre2 driver. Control embryos were Wnt1-Cre2 or SmoM2 alone. (B,D) Average ratio of cell length along the ML and AP axes. n = 3 embryos per genotype, two-way ANOVA test (A,C) or Welch’s t-test (B,D). See Supplementary file 1 for n and p values.