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Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification

¹ Department of Genetics, Stanford University, Stanford, CA 94305-5120, USA
² Department of Biochemistry, University of Ulm, Germany
^* Author for correspondence at present address: Institute of Zoology II, University Karlsruhe (e-mail: doris.wedlich{at}zi2.uni-karlsruhe.de

View larger version (53K): [in a new window]   Fig. 1. The extracellular Xcadherin-11 domain regulates adhesion of CNC cells in the transplantation assay, independently of ß-catenin-binding. (A) Wild-type and Xcad-11 deletion constructs (black, transmembrane segment; dots, ß-catenin-binding site). (B) GST-ß-catenin pull-down assay. Western blot showing TNT lysates of full-length (X11pcDNA3.1/Myc-His-A) and cytoplasmic-deleted Xcad-11 (cX11pcDNA3.1/Myc-His-A), all stained with 9E10 Myc antibody (left). Only the full-length Xcad-11 binds the GST-ß-catenin fusion protein (right). (C) Transplantation assay. (D) Comparison of cranial neural fold transplants overexpressing different Xcad-11 constructs. Migratory phenotype analysed by GFP fluorescence 18 hours after transplantation. (E) Confocal analysis of transverse transplant sections stained with 9E10 Myc antibody. (F) Transplants expressing the extracellular deletion mutant, eXcad-11 (e), started migration earlier than GFP controls. The graph illustrates the comparison of 49 migrating GFP with 46 migrating eXcad-11 (e) grafts 0, 7 and 18 hours after transplantation. (G) Lateral views of a transplant expressing eXcad-11 7 hours after transplantation, showing farther migration compared with the GFP control (left). Dorsal views of the same grafts show no differences after 48 hours (right).

View larger version (69K): [in a new window]   Fig. 2. Migration behaviour controlled by Xcadherin-11 affects specific neural crest markers differently. (A) Xcad-11, (B) AP-2, (C) Snail, and (D) twist whole-mount in situ hybridisation of transplants injected with GFP alone, Xcad-11, extracellular (e) or cytoplasmic (c) deletion mutants. The transplant-containing side (+) is compared with the control side (-) of the same embryo. Arrowheads indicate areas of different marker expression, arrows mark the non-migrating graft.

View larger version (83K): [in a new window]   Fig. 3. Localisation of host and donor CNC cells at stage 28. (A) Transverse section showing a Xcad-11-expressing non-migrating transplant (pink), and migrating host CNC cells (blue). (B) Mixture of host (blue) and donor (pink) CNC cells in GFP-RNA injected control embryos. (C) Transverse section showing most of the eXcad-11-expressing, migrating donor CNC cells (pink) separated from the host CNC cells (blue). Pink, immunostaining of Myc-tagged GFP; blue, twist in situ hybridisation. ea, ear vesicle; ey, eye anlage; g, gut. Scale bar: 50 µm.

View larger version (55K): [in a new window]   Fig. 4. Xcadherin-11 affects the AP-2 pattern through its adhesion effect, while twist expression is influenced by its interference with Wnt/ß-catenin signalling. (A) AP-2 and twist in situ hybridisation of embryos injected with 1 ng Xcad-11, 1 ng eXcad-11 or 2.5-ng cXcad-11 RNA in one blastomere at the two-cell stage. (B) twist in situ hybridisation of an embryo injected with 1 ng eXcad-11 and 80 pg ß-catenin. The injected side (right) is no different from the control side. (C) RT-PCR of half heads of embryos injected as in A. K, control.

View larger version (61K): [in a new window]   Fig. 5. Double in situ hybridisation. (A) Xcad-11 (blue) and twist (red) at stage 26. (B-E) Double whole-mount in situ hybridisation of stage 20 (B,D), and stage 26 (C,E) embryos for AP-2 (B,C, red) and Snail (D,E, blue).

View larger version (100K): [in a new window]   Fig. 6. Inhibition of migration alters neural crest marker expression. (A-C) twist in situ hybridisation of grafts expressing Xcad-11 0.5 hours (A), 4 hours (B) and 18 hours (C) after transplantation. GFP-Myc immunostaining of the corresponding sections shown in (D-F). cXcad-11 overexpressing graft 4 hours (G) and 18 hours (H) after transplantation, all stained with twist in situ hybridisation probe. (I) Xcad-11-expressing graft 18 hours after transplantation stained with Snail in situ hybridisation probe. (J-L) GFP-Myc immunostaining of the corresponding sections. Asterisk marks the graft centre. Scale bar: 50 µm.

View larger version (91K): [in a new window]   Fig. 7. Homotopic transplants show that non-migrating CNC cells adopt a neural fate. (A) Non-migrating transplant overexpressing cXcad-11 stained with 2G9 antibody, a neural marker. (B) Migrating GFP-Myc-expressing donor CNC cells (green) are negative for 2G9 (red). (C) Whole-mount in situ hybridisation of a cXcad-11-expressing graft shows that sox3 is not expressed 1 hour post grafting. (D) GFP-Myc immunostaining of the section shown in C. (E) In situ hybridisation with nrp-1 probe 18 hours after grafting. Donor embryo was injected with cXcad-11 RNA. (F) GFP-Myc immunostaining of the section shown in E. Upper half of the transplant is nrp-1 positive. (G) In situ hybridisation with twist probe 18 hours after grafting. Donor embryo was co-injected with cXcad-11 and ß-catenin RNA. (H) Section shown in G immunostained with 2G9 (red). Donor embryos were injected either with 1 ng Xcad-11, 2.5 ng cXcad-11 or 2.5 ng cXcad-11 plus 80 pg ß-catenin RNA. Asterisks mark the graft centres. Scale bars: 50 µm.

View larger version (100K): [in a new window]   Fig. 8. Non-migrating CNC cells also adopt neural fate after heterotopic transplantation into the pharyngeal pouch area. (A) Xcad-11-expressing transplant was found to be twist negative in whole-mount in situ hybridisation but (B) positive for 2G9 neural marker expression in immunostaining. (C) Transplant from a donor co-injected with cXcad-11 and ß-catenin RNA was twist negative in whole-mount in situ hybridisation but (D) positive for 2G9. (E) Transplant from a donor co-injected with cXcad-11 and ß-catenin RNA shows nrp-1 expression in whole-mount in situ hybridisation. (F) Higher magnification of the transplant seen in E. (G) GFP-Myc immunostaining of the section shown in F. (H) N-Tubulin-positive transplant from a donor injected with cXcad-11 RNA. (I) GFP-Myc immunostaining of the section shown in H. (J) NeuroD-positive transplant from a donor injected with cXcad-11 RNA. (K) GFP-Myc immunostaining of the section shown in J. Donor embryos were injected either with 1 ng Xcad-11, 2.5 ng cXcad-11 or 2.5 ng cXcad-11 plus 80 pg ß-catenin RNA. Scale bars: 50 µm.

View larger version (63K): [in a new window]   Fig. 9. Expression of Xcadherin-11 constructs neither inhibits neural crest induction nor alters CNS and PNS pattern. (A-C) Stage 15 embryos single-side injected with cXcad-11 RNA. (D,E) Stage 20 embryos injected with eXcad-11 RNA. (F,G) Stage 28 embryo injected with Xcad-11 RNA. (H,I) Transverse sections of stage 28 embryo injected with Xcad-11: (H) in situ hybridisation with sox2 probe; (I) GFP-Myc immunostaining of the same section shown in H. Marker detection as indicated. b, brain; n, notochord; s, somites; asterisk, injected side. Scale bar: 50 µm.