Fig. 6. DLHP formation requires the presence of surface ectoderm on the neural fold, whereas paraxial mesoderm is not required. (A,B) Diagrams of the surgical method used to remove surface ectoderm unilaterally from the E9.5 caudal region. The boxed region in A is shown enlarged in B,G. (C,D) Transverse sections (H and E) through the rostral end of the posterior neuropore of embryos fixed immediately (C; n=5) and 5 hours (D; n=7) after surface ectoderm removal. Immediately after surgery, surface ectoderm is absent from the operated side to the tip of the neural fold (arrowheads in C), but is present contralaterally (asterisk in C). Note the neuroepithelium of the neural fold is intact following surgery, with DLHPs on both sides of the neural plate (arrows in C). After 5 hours culture, the neuroepithelium on the operated side still lacks contact with surface ectoderm (between arrowheads in D) and does not form a DLHP. There is no evidence of cell death within the neural plate on the operated side, suggesting that absence of DLHP represents suppression of bending by surface ectoderm removal. A prominent DLHP is present contralaterally (arrow in D) where surface ectoderm contact is maintained (asterisk). (E,F) In situ hybridisation for Bmp2, a marker of dorsal surface ectoderm (arrows in E; n>10). Unilateral removal of the surface ectoderm abolishes Bmp2 expression ipsilaterally (arrowhead in F; n=6), whereas expression is maintained contralaterally (arrow in F), where the surface ectoderm is intact (asterisk). (G-I) Surgical method for removing paraxial mesoderm from the caudal region of the E9.5 embryo, using cuts shown by dotted lines in G and I. Immediately after the operation, the open posterior neuropore is maintained dorsally (arrowhead in H) despite surgical removal of most of the ventrolateral tissue of the caudal region (arrows in H). (J,K) Transverse sections (H and E) through the rostral end of the posterior neuropore (plane of section shown by dotted line in H) of embryos following bilateral removal of the paraxial mesoderm. DLHPs are present in the neural plate immediately following removal of ventrolateral tissue (J; n=5). Note the presence of surface ectoderm on the outer aspects of the neural folds (arrows in J). After 3 hours culture, bending at the DLHPs has progressed with incipient closure of the isolated neural plate (arrows in K; n=5). The neural plate appears thickened after removal of paraxial mesoderm, perhaps indicating a slowing of axial elongation, with more cells appearing in transverse section. Scale bars: C-F and I-K, 50 µm; H, 0.2 mm.