Fig. 3. Inhibition of Shh signaling by dbcAMP induces ectopic DLHPs. (A-D) Transverse sections through the rostral end of the posterior neuropore (A,C) or through recently closed neural tube (B,D) of embryos cultured for 18-20 hours in the presence of either PBS (A,B) or 1 mM dbcAMP (C,D). PBS-treated embryos following culture (7-9 somites) exhibit Mode I neurulation, with an MHP but no DLHPs (A). The recently closed neural tube has a slit-shaped lumen (arrow in B). In contrast, dbcAMP-treated embryos with the same somite number exhibit DLHPs as well as an MHP (i.e. Mode 2 morphology). The recently closed neural tube in these embryos has a diamond-shaped lumen (arrows in D). (E-H) Sections of embryos exposed in culture to PBS (E,G) or dbcAMP (F,H), and then processed for whole mount in situ hybridisation for the Shh-regulated genes Hnf3ß (E,F; n=20) and Ptc1 (G,H; n=10). Both genes are expressed with reduced intensity in dbcAMP-treated embryos (arrowheads in F,H) compared with PBS-treated embryos (arrows in E,G). (I) Bar chart showing the premature appearance of DLHPs (premature transition to Mode 2 neurulation) in embryos treated with dbcAMP, compared with PBS-treated controls. Scale bars: A-D, 50 µm; E-H, 50 µm.