Fig. 2. Suppression of notochord development, by ablation of the node at E7.5, inhibits MHP development and induces premature DLHPs, permitting neural tube closure. (A,B) E7.5 (allantoic bud stage) conceptuses before (arrow in A) and immediately after (arrowhead in B) ablation of the node using a hot tungsten needle. (C,D) Transverse sections through E7.5 embryos homozygous for the cordon-bleu gene-trap insertion, in which the node and its derivatives express lacZ constitutively. The node is clearly indicated by lacZ expression in the ventral midline cells of an intact embryo (arrow in C) whereas these cells have been removed (arrowhead in D) in an embryo fixed immediately after ablation. (E,F) Transverse sections (H and E) of embryos cultured for 42 hours following node ablation. A notochord developed (white arrow in E), despite the ablation procedure in 6/11 cases, with induction of a floor plate in the neural tube (black arrow in E). In the remaining 5/11 ablated embryos, notochord development was suppressed (white arrowhead in F) with absence of a floor plate structure (black arrowhead in F). The neural tube always closed in these notochordless embryos. (G,H) Transverse sections (H and E) through the rostral end of the posterior neuropore of embryos cultured for 24 hours following node ablation. Non-operated control embryo exhibits Mode I neurulation, with MHP (arrow in G) but no DLHPs, whereas an ablated embryo lacking a notochord has a thickened ventral midline neural plate (black arrowhead in H), and DLHPs (white arrowheads in H; 5/7 cases studied), similar to Mode 3. (I-K) Transverse sections through the rostral end of the neuropore of embryos cultured for 24 hours following node ablation, followed by in situ hybridisation for Shh. Where a Shh-positive notochord develops despite the ablation procedure (black arrow in I), the neural folds are straight with a clear MHP (white arrow; Mode I neurulation; n=14). In contrast, complete suppression of the notochord results in a neural plate with Mode 3 morphology (8/17 cases studied), exhibiting DLHPs (white arrowhead in J) but no MHP (black arrowhead in J). Asterisk in J indicates artefactual absence of paraxial mesoderm. (K) Node-ablated embryo with a midline defect, in which the two halves of the neural plate are separated. The left hemi-plate has a Shh-positive notochordal fragment attached (black arrow), and exhibits Mode I neurulation (no DLHP), whereas the right hemi-plate has no notochord attached and has a DLHP (white arrowhead). al, allantois; am, amnion; ch, chorion; ep, ectoplacental cone. Scale bars: A,B, 0.2 mm; C,D 50 µm; E-K, 50 µm.