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Cranial neural crest (CNC) cells migrate extensively, typically in a pattern of cell streams. In Xenopus, these cells express the adhesion molecule Xcadherin-11 (Xcad-11) as they begin to emigrate from the neural fold. In order to study the function of this molecule, we have overexpressed wild-type Xcad-11 as well as Xcad-11 mutants with cytoplasmic (deltacXcad-11) or extracellular (deltaeXcad-11) deletions. Green fluorescent protein (GFP) was used to mark injected cells. We then transplanted parts of the fluorescent CNC at the premigratory stage into non-injected host embryos. This altered not only migration, but also the expression of neural crest markers. Migration of transplanted cranial neural crest cells was blocked when full-length Xcad-11 or its mutant lacking the beta-catenin-binding site (deltacXcad-11) was overexpressed. In addition, the expression of neural crest markers (AP-2, Snail and twist) diminished within the first four hours after grafting, and disappeared completely after 18 hours. Instead, these grafts expressed neural markers (2G9, nrp-I and N-Tubulin). Beta-catenin co-expression, heterotopic transplantation of CNC cells into the pharyngeal pouch area or both in combination failed to prevent neural differentiation of the grafts. By contrast, deltaeXcad-11 overexpression resulted in premature emigration of cells from the transplants. The AP-2 and Snail patterns remained unaffected in these migrating grafts, while twist expression was strongly reduced. Co-expression of deltaeXcad-11 and beta-catenin was able to rescue the loss of twist expression, indicating that Wnt/beta-catenin signalling is required to maintain twist expression during migration. These results show that migration is a prerequisite for neural crest differentiation. Endogenous Xcad-11 delays CNC migration. Xcad-11 expression must, however, be balanced, as overexpression prevents migration and leads to neural marker expression. Although Wnt/beta-catenin signalling is required to sustain twist expression during migration, it is not sufficient to block neural differentiation in non-migrating grafts.
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 (δe) or cytoplasmic (δc) 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.
Fig. 4. Xcadherin-11 affects the AP-2 pattern through its adhesion effect, while twist expression is influenced by its interference with Wnt/β-catenin signalling. (A) AP-2 and twist in situ hybridisation of embryos injected with 1 ng Xcad-11, 1 ng δeXcad-11 or 2.5-ng δ cXcad-11 RNA in one blastomere at the two-cell stage. (B) twist in situ hybridisation of an embryo injected with 1 ng δ eXcad-11 and 80 pg β-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.
Fig. 1. The extracellular Xcadherin-11 domain regulates adhesion of CNC cells in the transplantation assay, independently of β-catenin-binding. (A) Wild-type and Xcad-11 deletion constructs (black, transmembrane segment; dots, β -catenin-binding site). (B) GST-β-catenin pull-down assay. Western blot showing TNT lysates of full-length (X11pcDNA3.1/Myc-His-A) and cytoplasmic-deleted Xcad-11 (δcX11pcDNA3.1/Myc-His-A), all stained with 9E10 Myc antibody (left). Only the full-length Xcad-11 binds the GST-β-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, δeXcad-11 (δe), started migration earlier than GFP controls. The graph illustrates the comparison of 49 migrating GFP with 46 migrating δeXcad-11 (δe) grafts 0, 7 and 18 hours after transplantation. (G) Lateral views of a transplant expressing δeXcad-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).
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 (δe) or cytoplasmic (δc) 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.
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 δeXcad-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.
Fig. 4. Xcadherin-11 affects the AP-2 pattern through its adhesion effect, while twist expression is influenced by its interference with Wnt/β-catenin signalling. (A) AP-2 and twist in situ hybridisation of embryos injected with 1 ng Xcad-11, 1 ng δeXcad-11 or 2.5-ng δ cXcad-11 RNA in one blastomere at the two-cell stage. (B) twist in situ hybridisation of an embryo injected with 1 ng δ eXcad-11 and 80 pg β-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.
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).
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). δcXcad-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.
Fig. 7. Homotopic transplants show that non-migrating CNC cells adopt a neural fate. (A) Non-migrating transplant overexpressing δcXcad-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 δcXcad-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 δcXcad-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 δcXcad-11 and β-catenin RNA. (H) Section shown in G immunostained with 2G9 (red). Donor embryos were injected either with 1 ng Xcad-11, 2.5 ng δcXcad-11 or 2.5 ng δ cXcad-11 plus 80 pg β-catenin RNA. Asterisks mark the graft centres. Scale bars: 50 μm.
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 δcXcad-11 and β-catenin RNA was twist negative in whole-mount in situ hybridisation but (D) positive for 2G9. (E) Transplant from a donor co-injected with δcXcad-11 and β -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 δcXcad-11 RNA. (I) GFP-Myc immunostaining of the section shown in H. (J) NeuroD-positive transplant from a donor injected with δcXcad-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 δcXcad-11 or 2.5 ng δcXcad-11 plus 80 pg β-catenin RNA. Scale bars: 50 μm.
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 δcXcad-11 RNA. (D,E) Stage 20 embryos injected with δ 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.
