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Sonic hedgehog and the molecular regulation of mouse neural tube closure

Neural Development Unit, Institute of Child Health, University College London, London WC1N 1EH, UK

View larger version (84K): [in a new window]   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.

View larger version (114K): [in a new window]   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.

View larger version (87K): [in a new window]   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.

View larger version (100K): [in a new window]   Fig. 4. Homozygosity for a null allele of Shh results in closure of the neural tube by bending at DLHPs, at E8.5 (9-10 somites) when wild-type and heterozygous embryos of this strain exhibit bending only at the MHP (Mode 1 neurulation). Transverse sections (H and E) through the rostral end of the posterior neuropore of embryos from Shh+/– x Shh+/– litters. (A-D) MHP (arrows in A,B) but no DLHPs are present in wild-type (A; n=10) and Shh+/– (B; n>20) embryos. In contrast, Shh–/– embryos (C,D; n=7) exhibit DLHPs (black arrowheads). MHP is present in some Shh–/– embryos (arrow in C) but not in others (white arrowhead in D). (E-G) E8.5 embryos from Shh+/– x Shh+/– litters, cultured for 6 hours in the presence of 0.05 µg/ml cytochalasin D, to reveal bending points in the neural plate, which are resistant to cytochalasin (Ybot-Gonzalez and Copp, 1999). Wild-type (E) and Shh+/– (F) embryos exhibit a clear MHP (arrows in E,F) but rarely show DLHPs (1/6 and 1/13 respectively). Cytochalasin treatment causes the normally straight neural folds of Mode 1 to flare outwards. In contrast, cytochalasin D-treated Shh–/– embryos exhibit DLHPs in 6/13 cases (arrowheads in G), and all have an MHP (arrow in G). Scale bar: 50 µm.

View larger version (95K): [in a new window]   Fig. 5. Local release of Shh from implanted beads is sufficient to inhibit DLHP formation but does not induce an MHP. (A) E9.5 embryos after 3 hours culture following implantation with a single AffiGel blue bead (arrows), adjacent to the neural fold on the right side of the posterior neuropore. The middle embryo has a caudally located bead whereas other embryos have more rostral beads. (B) Sub-division of posterior neuropore into rostrocaudal regions for analysis. (C) Mean ‘asymmetry scores’ of sections containing PBS- or Shh-soaked beads located at each neuropore level. A mean score of 1 (dotted line) indicates symmetrical DHLPs. Scores of 2 and 3 indicate partial and complete suppression of the ipsilateral DLHP respectively. Mean angles of DLHP bending in neural plate with a score of 1, 2 or 3 are shown in Table 1. Mean scores for Shh beads significantly exceed values for PBS beads at rostral and intermediate levels (P<0.001) but not at the caudal position (P>0.05). (D-K) Transverse sections through the posterior neuropore of eight different cultured embryos containing beads (asterisk in D) soaked in PBS (D,F,H,J) or Shh (E,G,I,K). (D,F,H) PBS beads are associated with symmetrical DLHPs in all neuropore regions (27/29 beads in 23/25 embryos) whereas Shh beads regularly suppress formation/maintenance of the ipsilateral DLHP (19/38 beads in 16/31 embryos) in rostral and intermediate neuropore regions (E,G). (I) Complete suppression of DLHP by a caudally located Shh bead. (J,K) Caudal sections of embryos in which a bead has been implanted in the midventral region, beneath the Mode 3 neural plate. There is no sign of MHP formation in embryos containing either PBS (n=2) or Shh beads (n=3). Scale bars: A, 0.5 mm; D-K, 100 µm.

View larger version (105K): [in a new window]   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.

View larger version (23K): [in a new window]   Fig. 7. Proposed interactions regulating neural tube closure at three progressive stages of mouse spinal neurulation. Solid arrows indicate active signaling influences, whereas dotted arrows show inactive/ineffective signals, as identified in the present study. See text for further explanation.