Fig. 1. Transition from midline to dorsolateral bending correlates with reduced strength of notochordal Shh signalling beneath the posterior neuropore. Modes 1-3: the three morphological patterns of neural plate bending. Mode 1 is characterised by an MHP only (E8.5; 7-15 somites; A,D,G,J,M); Mode 2 has both MHP and DLHPs (early E9.5; 16-24 somites; B,E,H,K,N); Mode 3 has only DLHPs (E10; 25-30 somites; C,F,I,L,O). (A-F) Whole-mount in situ hybridisation for Shh at Modes 1-3 (A-C: whole embryos; D-F: isolated caudal regions, rostral to left). Neural tube fusion begins at the hindbrain/cervical boundary (arrowhead in A) at the 6- to 7-somite stage, and then spreads in rostral and caudal directions (arrows in A). The notochord expresses Shh along its length (A-C), but the caudal termination point of the Shh domain varies with increasing developmental stage (D-F). Caudal regions in D-F are aligned against a vertical marker (asterisk): the posterior neuropore extends to the right of the marker (curved arrows). Shh-positive notochord (white arrows) extends far into the posterior neuropore in Mode 1 (D) whereas it barely reaches the rostral end of the relatively shorter neuropore in Mode 3 (F). Mode 2 shows an intermediate condition (E). (G-O) Transverse sections through rostral end of the posterior neuropore, at levels of equivalent neural fold elevation (dotted lines in D-F), in embryos of Modes 1-3. (G-I) H and E stained sections show the location of MHP (arrows in G,H) and DLHPs (arrowheads in H,I). Method of measuring DLHP angle (Table 1) is shown by red lines in H. (J-L) Sections of embryos hybridised as whole mounts for Shh expression: transcripts are present in the notochord (arrows) and ventral hindgut (arrowhead in J), but not in the presumptive floor plate, with a declining intensity of expression from Mode 1 to 3. (M-O) In situ hybridisation reveals Ptc1 expression in the ventral midline neural plate (arrows), with expression declining in intensity from Mode 1 to Mode 3. Embryos for gene expression comparison hybridised under identical conditions, with at least 10 embryos studied at each stage, by each method. Scale bars: A-C, 0.5 mm; D-F, 0.25 mm; G-O, 50 µm.