Figure 7. Wntless (Wls) in the floor plate is essential for A–P guidance of commissural axons.

(A) Schematic of Wls function in Wnt secretion. Wls is required for Wnts secretion. (B) Schematic of experimental design. Tamoxifen (0.1 mg/g of mother) was injected intraperitoneally at E8.5. Embryos were harvested and dissected at E11.5 for immunohistochemistry (IHC), in situ hybridization (ISH) and DiI tracing experiments. (C) Cre recombination (tdTomato signal) was detected specifically in the notochord and floor plate. After intraperitoneal injection of tamoxifen (0.1 mg/g mother) at E8.5, embryos were collected at E11.5 and transverse sections were immunostained with the indicated antibodies. FoxA2 is a floor plate marker and Nkx2.2 marks the cells immediately outside the floor plate. (D) Loss of Wls mRNA in the floor plate of Wls cKO (Wls ^fl/fl; Shh-CreER^T2). FP, floor plate. Scale bars, 50 μm. (E) Commissural axons, labeled by lipophilic DiI injection into the dorsal spinal cord, showed A–P guidance defects in Wls^fl/fl; Shh-CreER^T2. FP, floor plate. Scale bars, 50 μm. (F) Quantification of A–P guidance defects in (E). The graph represents the percentage of injection sites that showed normal anterior turning (correct turning). Gray bars indicate means of all data points, black bars indicate standard deviations, and diamond dots indicate individual data points (control = 13, cKO = 11). (G) A model for the midline switch of Wnt responsiveness. Before reaching the floor plate, commissural neurons express higher levels of Shisa2. Fzd3 is not glycosylated and not translocated to the cell surface. Therefore, growth cones are not able to sense the Wnt gradient. After commissural axons reach the floor plate, Shh–Smo signaling is activated in the cell bodies of commissural neurons and Shisa2 expression is decreased, allowing Fzd3 to be glycosylated and translocated to the surface of the commissural axon growth cones. After crossing the floor plate, a sufficient amount Fzd3 is on the cell surface and the growth cones can now detect the Wnt gradient and turn anteriorly.

Figure 7—figure supplement 1. Wntless is not required for cell-fate specification in the ventral spinal cord at E11.5.

(A) Schematics of an E11.5 mouse spinal cord. The pink line indicates the axons of the TAG-1 positive commissural neurons approaching the ventral midline. Ventral progenitor domains and postmitotic neuronal domain (ventral horn) are highlighted in the green box. (B, C) Ventral progenitors and post-mitotic neurons are not affected in Wls cKO. Dashed white lines represent postmitotic ventral horn where MMC, HMC and PGC neurons are located. Scale bars, 50 μm. (D) Quantification of cell numbers in (B) and (C). Gray lines indicate means of all data points, black bars indicate standard deviations, and diamond dots indicate individual data points (Wls^fl/fl; four embryos, Wls^fl/fl; Shh-CreER^T2; five embryos).