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First published online August 10, 2007
doi: 10.1242/10.1242/dev.008177

Development 134, 3203-3211 (2007)
Published by The Company of Biologists 2007
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Neural plate morphogenesis during mouse neurulation is regulated by antagonism of Bmp signalling

Patricia Ybot-Gonzalez1,*, Carles Gaston-Massuet1, Gemma Girdler1,{dagger}, John Klingensmith2, Ruth Arkell3, Nicholas D. E. Greene1 and Andrew J. Copp1,*

1 Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.
2 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
3 Molecular Genetics and Evolution Group, Research School of Biological Sciences, Australian National University, Canberra ACT 0200, Australia.


Figure 1
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  		Fig. 1. Bmp signalling during mouse spinal neurulation. (A) Diagrammatic transverse sections through the PNP of embryos at modes 1 (E8.5-E9), 2 (E9-E9.75) and 3 (E9.75-E10.5) of spinal neurulation. Yellow triangles, median hinge point (MHP); red triangles, dorsolateral hinge points (DLHPs). (B-I) In situ hybridisation for Bmp2 (B,C), Bmp7 (D,E), cadherin 6 (F,G) and Msx1 (H,I) during mode 1 (E8.5; B,D,F,H) and mode 3 (E9.5; C,E,G,I) neurulation as seen in transverse sections through the posterior neuropore (PNP) of whole-mount embryos. Arrows show intense in situ hybridisation signal. (J-O) Immunohistochemistry for phospho-Smad1, -Smad5 and -Smad 8 combined (phospho-Smad1,5,8; J,K); activated caspase 3 (L,M); and phospho-histone H3 (N,O) during mode 1 (J,L,N) and mode 3 (K,M,O) neurulation. (J) Arrows indicate high levels of phospho-Smad1,5,8 in dorsal neural folds. (L,M) Caspase 3-positive cells were accumulated over ten consecutive sections of the same embryo and projected onto a single section. (L) Arrow indicates intensive apoptosis in the dorsal neural fold at mode 1. (N,O; insets) Controls with the omission of the primary antibody. (P,Q) Diagrammatic right-sided view (P) of the PNP at mode 3. Transverse section (Q) at the level of the dotted line in P summarises the embryonic tissues that are visible in C,E,G,I,K,M,O: hg, hindgut; mes, mesoderm; np, neural plate; no, notochord; se, surface ectoderm; ver, ventral ectoderm ridge. Scale bars: 0.1 mm in B (also C-I), in K (also J), in M (also L), in O (also N) and in inset O (also inset N).

 

Figure 2
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  		Fig. 2. Bmp2 is necessary and sufficient to inhibit DLHPs. (A-F) Transverse H&E-stained sections through the posterior neuropore (PNP) region of E8.5 (A-D) and E9 (E,F) embryos. At 7 somites (7 s), when closure is first initiated, dorsolateral hinge points (DLHPs) are absent from wild-type (WT, A) and Bmp2-/- (B) embryos. Whereas wild-type embryos at 9 somites continue in mode 1 neurulation (C), Bmp2-/- embryos show marked DLHPs, characteristic of modes 2/3 (arrows in D). At 15 somites, wild-type embryos have entered mode 2 neurulation with clear DLHPs (E), whereas 15-somite Bmp2-/- embryos have undergone premature closure of the neural tube (F). (G-N) Transverse sections through the PNP of mode 1 (G,I,K,M) and mode 2/3 (H,J,L,N) embryos stained with H&E (G-J) or after immunohistochemistry for phospho-Smad1, -Smad5 and -Smad8 combined (K-N). A bead soaked in Bmp2 (G,H,K,L) or PBS (I,J,M,N) has been implanted next to one neural fold. Exposure to Bmp2 inhibits DLHP formation in mode 2/3 (arrows in H,L), whereas DLHPs remain in PBS-treated control embryos (arrowheads in J,N). Note that the period of phospho-Smad staining was longer in PBS control embryos (M,N) than in those implanted with a Bmp2 bead (K,L) in order to reveal endogenous expression, as a positive control. Scale bars: 0.1 mm in F (also A-E); 0.1 mm in J (also G-I) and N (also K-M).

 

Figure 3
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  		Fig. 3. Antagonism of Bmps is necessary and sufficient to induce DLHPs. (A-F) In situ hybridisation for neuralin (A,B), chordin (C,D) and noggin (E,F) gene expression in mode 1 (A,C,E), mode 2 (insets in A-F) and mode 3 (B,D,F), as seen in transverse sections through the posterior neuropore (PNP) of whole-mount embryos. Arrows indicate detection of a strong signal in the dorsal neural plate or notochord (B-F); arrowheads indicate detection of a diminished signal in the dorsal neural plate or notochord (E,F). (G,H) Transverse sections, stained with H&E, through the PNP of E9.5 Nog+/+ (G) and Nog-/- (H) embryos. Wild-type embryos show exaggerated dorsolateral hinge points (DLHPs; G, black arrows) whereas DLHPs in mutant embryos are absent (H, left white arrow) or markedly reduced (H, right white arrow). (I-P) Transverse sections through the PNP of mode 1 (I,K,M,N) and mode 2/3 (J,L,O,P) embryos after the implantation of beads soaked in noggin peptide. Sections were stained with H&E (I,J), or by immunohistochemistry for noggin (K,L) or phospho-Smad1,5,8 (M-P). Arrows, induction of DLHPs ipsilaterally (I,K,M) after exposure to exogenous noggin. Notice the reduced phospho-Smad staining around the noggin beads (arrowheads in M,O) compared with slightly more-rostral sections of the same embryos (N,P), which were not exposed to exogenous noggin and show normal endogenous levels of phospho-Smad1,5,8. (Q) Summary of experiments showing the induction of DLHPs by noggin (mode 1, second column, asterisk) and inhibition of DLHPs by Bmp2 (mode 2/3, second column, asterisk). PBS: implantation of saline control beads. Percentages of embryos with particular morphology are shown, together with total number (n) receiving bead implants. Out of 25 (43.1%) mode 1 embryos with noggin-induced DLHPs, 12 had an ipsilateral DLHP, nine had a contralateral DLHP and four had bilateral DLHPs. The DLHP was contralateral in both of the affected PBS-treated mode 1 embryos. The frequency of DLHP induction in mode 1 is significantly greater in noggin-treated embryos than in PBS controls (Fisher exact test; P=0.02). The frequency of DLHP inhibition in mode 2/3 is significantly greater in Bmp2-treated embryos than in PBS controls (P<0.001). Scale bars: 0.1 mm in B (also A,C-F and insets); 0.05 mm in H (also G); 0.1 mm in I (also J) in K, in L and in P (also M-O).

 

Figure 4
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  		Fig. 4. Noggin expression is induced by Bmp2 and inhibited by Shh. (A-C) In situ hybridisation (lateral views) for noggin expression in wild-type (WT, A) and Bmp2-/- (B,C) embryos. Insets (A-C) correspond to transverse sections of the embryos at the level of the dashed red lines. Noggin is expressed in the notochord of all genotypes (arrows in A-C and in insets) but in the dorsal neural tube of only the wild-type embryo (black arrowheads in A and inset) and not in Bmp2-/- embryos (white arrowheads in B,C and insets). (D,E) In situ hybridisation for noggin in the posterior neuropore (PNP) region of an E9.5 wild-type embryo after the implantation of a bead soaked in Bmp2. Whole mount (right lateral view) shows intense expression of noggin around the bead (arrowhead in D), and transverse section (E) shows induction of noggin expression throughout the ipsilateral neural fold. Note the sharp boundary of expression at the midline (arrow in E) and the absence of dorsolateral hinge point (DLHP) ipsilaterally after exposure to Bmp2 (arrowhead in E). (F,G) In situ hybridisation for noggin in E9.5 embryos after the implantation of a bead soaked in PBS (F) or Shh (G). Noggin is expressed at the dorsolateral tips of both neural folds in the embryo with a PBS bead (arrows in F), but is downregulated on the neural fold ipsilateral to the Shh bead, with suppression of the DLHP (asterisk in G). (H-K) Transverse sections through wild-type (H,I) and Shh-/- (J,K) embryos, showing the ventralised expression domain of noggin in Shh-/- embryos compared with wild type (arrows), in both open PNP (H,J) and in closed neural tube (I,K). Scale bars: 0.4 mm in A-C; 0.1 mm in B inset (also A inset); 50 µm in C inset; 30 µm in D; 50 µm in E; 0.15 mm in G (also F); 50 µm in K (also H-J).

 

Figure 5
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  		Fig. 5. DLHPs are absent from Zic2Ku/Ku embryos during the development of spina bifida. (A-E) Scanning electron micrographs of the E9.5 caudal region of wild-type (A,C) and Zic2Ku/Ku (B,D,E) embryos. (A) The wild-type embryo has a closed brain and an open posterior neuropore (PNP), as expected at this stage of development. (B) The Zic2Ku/Ku embryo has exencephaly (arrowheads) and a greatly enlarged PNP (arrow), indicative of incipient spina bifida. Higher-magnification views (C-E; see bracketed areas in A,B) show indentations, representing dorsolateral hinge points (DLHPs), in the dorso-medial region of wild-type neural folds (arrow in C), whereas these are absent from mutant neural folds (asterisks), both at rostral (D) and more caudal (E) levels of the PNP. (F,G) H&E-stained sections through the PNP show early DLHPs in a wild-type embryo (arrows in F) but entirely straight neural folds, with no DLHPs (asterisks in G), in a Zic2Ku/Ku embryo. The level of sections in F and G are indicated by dotted red lines in C and D, respectively. Scale bars: 0.2 mm (A,B); 50 µm (C,E); 25 µm (D); 0.2 mm (F,G).

 

Figure 6
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  		Fig. 6. Zic2Ku/Ku embryos show reduced expression of Bmp antagonists and over-stimulation of Bmp signalling. (A,B) In situ hybridisation for Zic2 expression in transverse sections through the posterior neuropore (PNP) of mode 1 (E8.5) and mode 3 (E9.5) wild-type embryos. Note the more intense expression of Zic2 in mode 3. (C,D) Immunohistochemistry for phospho-Smad1,5,8 in transverse sections of E9.5 wild-type (WT) and Zic2Ku/Ku embryos. The mutant neural plate shows more-intense phospho-Smad expression than wild type (arrows in D). (E-G) In situ hybridisation for Bmp2, noggin and neuralin in whole mounts of E9.5 wild-type and Zic2Ku/Ku embryos. Red arrowheads, Bmp2 expression in the dorsal surface ectoderm of both genotypes; dorsal view of PNP in mutant shows Bmp2 domains on both sides. Black arrowheads, noggin and neuralin expression in the dorsal neural plate of wild-type but not mutant (asterisks) embryos. (H-J) In situ hybridisation for Bmp2, noggin and neuralin in sections through the open neural tube of Zic2Ku/Ku embryos. Whereas Bmp2 is expressed (arrowheads), expression of noggin and neuralin is absent (asterisks). (K) Summary of dorsolateral hinge point (DLHP) regulation by Bmp2, noggin and Shh in mouse spinal neurulation. Yellow triangles, median hinge point (MHP); red triangles, DLHPs; green arrows, stimulatory interactions; red lines, inhibitory interactions; dashed lines, inactive influences. Scale bars: 0.1 mm in B (also A); 0.15 mm in D (also C); 0.5 mm in F (also E,G); 0.05 mm in J (also H,I).

 

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© The Company of Biologists Ltd 2007