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First published online 21 December 2006
doi: 10.1242/dev.02742

Development 134, 491-501 (2007)
Published by The Company of Biologists 2007
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Antagonistic roles of full-length N-cadherin and its soluble BMP cleavage product in neural crest delamination

Irit Shoval1, Andreas Ludwig2 and Chaya Kalcheim1,*

1 Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, Jerusalem 91120 - PO Box 12272, Israel.
2 Institute for Molecular Cardiovascular Research, University Hospital, RWTH Aachen, 52074, Germany.


Figure 1
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  		Fig. 1. Axial-level-dependent downregulation of N-cadherin protein, but not mRNA, in the dorsal NT is correlated with loss of noggin transcripts. (A-C) N-cadherin immunoreactivity. Arrowheads point to the dorsal NT of a 25-somite-stage embryo that expresses N-cadherin immunoreactivity opposite the segmental plate (C), but lacks the protein in the region facing epithelial somites (B) and dissociated somites (A). Arrows in B depict the onset of NC emigration from a N-cadherin-deficient dorsal NT. (D-F) N-cadherin mRNA showing no change in dorsal NT along similar axial levels. (G) Axial-dependent downregulation of noggin mRNA coincides with a similar pattern of N-cadherin disappearance. Abbreviations: DS, dissociated somites; ES, epithelial somites; M, myotome; N, notochord; NT, neural tube; Scl, sclerotome; SP, segmental plate. Scale bar: 80 µm.

 

Figure 2
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  		Fig. 2. Overexpression of N-cadherin inhibits NC delamination. (A,C,E) Electroporation of control-GFP. (A) Segmentally migrating labeled NC (arrows) in whole-mounts. (C) Early delaminating NC (arrows) opposite a dissociating somite (S) in transverse section. (E) GFP-labeled mesenchymal NC cells emigrate from NT explants along with unlabeled progenitors evident with phase contrast optics. (B,D,F) Electroporation of full-length N-cadherin. No NC delamination occurs in whole-mounts (B), in sections opposite an already dissociated somite (D), or from NT explants (F). Note that unlabeled cells emigrate normally, yet transfected cells round up as in vivo, with no evidence of cell death (F and data not shown). (G,H) Control and N-cadherin-treated embryos, respectively, fixed 35 hours after transfection when peripheral ganglia are already coalesced. (G) Note the presence of GFP+ cells (arrows, green) that colonize the HNK-1+ DRG and SG (red). (H) By contrast, N-cadherin+ cells remain in the dorsal NT where they express ectopic HNK-1 (arrow). n=6 for each treatment. Abbreviations: DM, dermomyotome; DRG, dorsal root ganglion; NT, neural tube; S, somite; scl, sclerotome; SG, sympathetic ganglion. Scale bar: 28 µm for C,D; 55 µm for E,F; 25 µm for G,H.

 

Figure 3
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  		Fig. 3. N-cadherin mutants do not affect NC delamination. (A) Schematic of N-cadherin mutants used. (B-G) Transverse sections through embryos electroporated with: (B,D) control-GFP; (C,E) cN390{Delta}-GFP; (F) CBR-GFP; (G) JMD-GFP. (H) NT explanted on fibronectin following transfection with cN390{Delta}-GFP. (I) Quantification of NC delamination. Efficiency of electroporation was greater than 70%. Results represent the mean±s.d. of five embryos per treatment normalized to control values. The extent of NC emigration was not affected by the mutant DNAs (arrows in D-G, and I), but labeled emigrating NC cells appeared more dispersed than controls (arrows in B,C). Transfected NT cells lost epithelial morphology in cN390{Delta}-GFP and CBR-GFP-treated cases, and NC cells emigrated as round cells from NT explants (H). Scale bar: 38 µm for D-G; 45 µm for H.

 

Figure 4
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  		Fig. 4. N-cadherin-mediated inhibition of NC delamination is associated with a downregulation of cyclin D1 transcription and G1-S transition. (A-C) Brdu incorporation (red nuclei) following electroporation (green) with control-GFP (A), wild-type N-cadherin (B) and cN390{Delta}-GFP (C). (D) Quantification of the percentage of Brdu+/GFP+ nuclei in the various treatments (data are the mean±s.d. of at least five embryos per treatment). Cells that received wild-type N-cadherin failed to incorporate Brdu+ and no GFP+ NC cells exited the treated side of the tube. By contrast, Brdu+ NC cells emigrate normally from control-GFP and cN390{Delta}-GFP-treated hemi-tubes (arrows in A,C). Note that Brdu+ nuclei are spread across the apico-basal thickness of the NT (C) contrary to their normal localization to the basal half of the epithelium (A). (E-H) cyclin D1 mRNA (blue) is reduced in cells that received wild-type N-cadherin-GFP (G,H, green) but not in control-GFP-treated hemi-tubes (E,F) where delaminating NC cells express cyclin D1 (arrows). Scale bar: 25 µm for A-C,E,F; 30 µm for G,H.

 

Figure 5
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  		Fig. 5. The normal downregulation of N-cadherin protein along the axis is regulated by BMP4. (A-D) Treatment with BMP4-coated beads (* in B,C; n=7) or electroporation with BMP4-DNA/GFP (arrowhead in D; insert depicts localization of transfected BMP/GFP; n=8) for 8 hours downregulates N-cadherin immunoreactivity at levels of rostral and caudal segmental plates. At these levels, N-cadherin is still expressed in the dorsal NT under normal conditions (A) and in matching controls (not shown). (E,F) Sections through a dissociating somite level 15 hours after electroporation of control-GFP (E; n=8) and noggin-GFP (F; n=9). (E) Note the absence of N-cadherin in the dorsal NT from which N-cadherin-negative NC cells emigrate (arrows depict phase-bright NC in main image and GFP+ NC in insert). (F) By contrast, noggin overexpression (green cells in insert and surrounding area) maintains N-cadherin and no NC emigration is apparent. Similar results were obtained by staining with anti-N-cadherin antibodies directed to either the extracellular or intracellular domains of the protein; the data presented stem from the latter. Abbreviations: CSP, caudal segmental plate; RSP, rostral segmental plate; scl, sclerotome. Scale bar: 25 µm.

 

Figure 6
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  		Fig. 6. BMP4 stimulates N-cadherin cleavage and NC delamination via ADAM10. Explants of neural primordia that received control medium, BMP4, GI254023X or BMP4+GI254023X. (A,D,G,J) Phase contrast. (B,E,H,K) GC4 anti-N-cadherin antibody that recognizes the extracellular domain. (C,F,I,L) N-cadherin antibody against intracellular region. (M) Quantification of the number of mesenchymal NC cells that exited from explanted NTs (mean±s.d. of 4-5 similar cultures per treatment). Note that whereas BMP4 dramatically enhanced NC EMT, GI254023X inhibited both basal and BMP-induced NC delamination and maintained full-length membrane-bound N-cadherin under both conditions (n≥12 explants per treatment stained with each antibody). The broken white line (B,C) delineates the border between the explant and emigrated cells. E and F exhibit only emigrated cells, whereas H,I,K and L exhibit only densely packed but non-mesenchymal cells. Scale bar: 100 µm in A,D,G,J; 10 µm in B,C,E,F,H,I,K,L.

 

Figure 7
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  		Fig. 7. N-cadherin cytoplasmic tail (CTF2) stimulates cyclin D1 transcription and NC emigration. (A) Delamination of NC cells that received CTF2-GFP (arrows); CTF2-GFP+ delaminating cells co-express HNK-1 (arrows, left insert) and Brdu (arrows, right insert). (B) Quantification of NC delamination. Results represent the mean±s.d. of 5 embryos per treatment normalized to control values. (C,D) The transfected hemi-tubes and delaminating NC (arrows) reveal enhanced ß-catenin and cyclin D1 transcription (n=4 and 7, respectively); note the round appearance of the overexpressing cells. (E,F) Anti-GFP and phase-contrast images, respectively, showing that CTF2-GFP is enriched in nuclei of emigrated NC cells (arrows; n=8). (G-J) Electroporation of neural tubes with CTF2 followed by explantation in the presence of GI254023X. All explants were stained with antibodies directed to the intracellular domain of N-cadherin that also react with transfected CTF2 evident in cell nuclei. (G,H) Explants that received control-GFP and GI254023X (n=13). Note that cells retain membrane-bound N-cadherin immunoreactivity and are adhered to each other. (I,J) Explants treated with CTF2 and GI254023X (n=14). Note that cells expressing CTF2 in their nucleus (arrows in I and higher magnification in J) have emigrated from the tube, lack membrane immunostaining and are detached from each other. By contrast, untransfected cells in the same field still express membrane N-cadherin and are epithelial (I, compare left and right sides). Similar results were obtained upon transfection of CTF2 alone (data not shown; n=12). Scale bar: 35 µm in A (25 µm in left insert; 33 µm in right insert); 25 µm in C,D; 15 µm in E,F; 40 µm in G,I; 17 µm in H,J.

 

Figure 8
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  		Fig. 8. An updated model for NC delamination. Opposite the segmental plate mesoderm, high levels of noggin result in low BMP activity, no Wnt1 transcription, low cyclin D1 transcription in dorsal NT and no NC cells emigrating from the caudal NT. N-cadherin at this stage is expressed in the dorsal NT where it contributes to maintaining low cyclin D1 transcription and lack of NC emigration. With ongoing development, opposite mature epithelial and dissociating somites, a factor emitted by the dorsomedial portion of the paraxial mesoderm inhibits noggin transcription in the dorsal NT, thereby releaving BMP activity. BMP4 in turn positively regulates Wnt1 transcription. Wnt signaling, via the canonical pathway, positively modulates transcription of cyclin D1, G1-S transition and NC cell delamination. In parallel, BMP4, via ADAM10, promotes N-cadherin protein cleavage into CTF1. CTF1 is in turn cleaved by {gamma}-secretase to generate soluble CTF2. CTF2 may act in at least two ways: by upregulating levels of ß-catenin transcription and by binding ß-catenin protein. We propose that the CTF2-ß-catenin complex translocates into the cell nucleus where transcription of target genes such as cyclin D1, followed by G1-S transition and EMT of NC, are stimulated. Hence, BMP activity transforms N-cadherin from an inhibitory cue into a stimulatory signal. Altogether, these data suggest that NC emigration from the NT is the result of at least two separate yet converging pathways, both stimulated by BMP signaling in coordination with somite development.

 

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© The Company of Biologists Ltd 2007