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. Author manuscript; available in PMC: 2018 Jul 16.
Published in final edited form as: Dev Biol. 2017 Mar 2;424(2):221–235. doi: 10.1016/j.ydbio.2017.02.006

Figure 4. SHH-E176A is highly active when ectopically expressed in the developing mouse forebrain.

Figure 4

A. Diagram of retroviral backbone (pCLE) used to express the human SHH protein. Full length human cDNA sequence was inserted into cloning site (CS), producing N-and C-lipid modified SHH proteins. Placental alkaline phosphatase (PLAP) is bicistronic with the Shh cDNA, allowing detection of virally infected cells (Gaiano et al., 1999; Kohtz et al., 2001). B. Whole cell extracts of C17 cells infected with wtSHH and SHH-E176A expressing viruses are Western-blotted and probed with H160 antibody, showing that both viruses express monomeric, cell associated SHH proteins. C–K. Analysis of embryos injected with retroviruses at E9.5 and analyzed at E12.5 (C–E) uninfected littermates, (F–H) wtSHH, (I–K) Shh-E176A. Whole embryos (C, F, I), and sections (D, E, G, H, J, K) of E12.5 mouse brains 3 days after infection with SHH expressing. Infected viral clusters are visualized by alkaline phosphatase staining in the dorsal midline of the telencephalon (G, J). Uninfected littermate control does not contain alkaline phosphatase-expressing clusters of cells (D). In situ hybridization of adjacent sections probed for ectopic expression of Dlx2 (E, H, K). wtSHH, n=8/8, SHH-E176A, n=4/4 display gross morphological defects. Scale bar in D, (same for E, G, H, J, K) is 300 μm. Arrows indicate ventralization of dorsal telencephalon by SHH overexpression and ectopic expression of Dlx2.