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Ephrin-B ligands play a dual role in the control of neural crest cell migration

Section of Molecular and Cellular Biology, University of California-Davis, Davis, California 95616, USA

View larger version (106K): [in a new window]   Fig. 1. Expression of ephrin-B proteins in chick embryos during early (A-C) and late (D-F) stages of neural crest cell migration. Whole-mount staining of stage-14 (A) and stage-23 (D) embryos with EphB2-Fc, visualized with anti-human Fc antibody coupled to alkaline phosphatase. Labelling revealed a similar pattern of expression in both developmental stages. Lateral views of the trunk region (B,E) show strong expression of ephrin-B ligands in the posterior half of the somites. Transverse sections of stage-14 and stage-23 embryos at midtrunk level show distribution of ephrin-B protein in the posterior sclerotome (C) (sc) and in the dorsolateral pathway (C,F) (arrows).

View larger version (54K): [in a new window]   Fig. 2. PCR shows EphB1, EphB2 and EphB3 receptors are expressed in early outgrowths and melanoblasts. EphB3 is highly expressed in both subpopulations of neural crest cells. GAPDH was used as an internal standard for PCR amplification.

View larger version (119K): [in a new window]   Fig. 3. Expression of EphB1, EphB2, EphB3, EphB5 and EphA4 in the developing chicken embryo. On the left are whole-mount in situ hybridisation on stage 14- and stage-23 embryos. (A-E) Transverse sections through the trunk region of a stage-14 embryo showing EphB3 expression in the sclerotome (A,B), and EphA4 expression in the unsegmented mesoderm and ventral edge of the dermomyotome (B). The transverse sections in B and D were also labelled with HNK-1 antibody showing neural crest cells in the ventral pathway (C,E, arrows). (F-Y) Transverse sections through the trunk region of stage-23 embryos showing EphB1 (F,H), EphB2 (J,L), EphB3 (N,P), and EphB5 (R,T) expression in the dorsal neural tube and dermomyotome (arrows), and EpA4 (V,X) in the dorsal neural tube and dorsal edge of the dermomyotome (arrow). Transverse sections in F,J,N,R and V labelled were also labelled with HNK-1 antibody showing neural crest cells in the ventral pathway (G,K,O,S,W). Transverse sections in H,L,P,T and X were also stained to show melanoblasts migrating along the dorsolateral pathway (I,M,Q,U,Y, arrows).

View larger version (165K): [in a new window]   Fig. 4. Detection of EphB receptors in explanted trunk neural crest cells. Early outgrowths (A,B) and melanoblasts (C,D) were incubated with ephrin-B1-Fc (A,C) or Fc (B,D) proteins and visualized with anti-human Fc antibody coupled to alkaline phosphatase. Control cultures display no staining (B,D). Ephrin-B1-Fc bound to both early outgrowths (A) and melanoblasts (C), indicating that the receptor remains expressed in vitro.

View larger version (68K): [in a new window]   Fig. 5. Effect of ephrin-B1-Fc on the migration of neural crest cells into the dorsolateral pathway in whole-trunk explants. Ephrin-B1-Fc (A,C), or Fc alone (B,D) were added to stage-12 (A,B) and stage-18 (C,D) explants and the pattern of neural crest migration was visualized by anti-HNK-1 antibody staining. (A) Addition of soluble ephrin-B1-Fc to stage-12 trunk explants results in the inappropriate migration of neural crest cells (arrows) into the dorsolateral pathway. Control explants (B) show the typical pattern of neural crest cell migration through the ventral pathway (arrow). (C) Addition of soluble ephrin-B1-Fc at the onset of melanoblast migration (stage 18) results in the lack of cells in the dorsolateral pathway. Migration of melanoblasts (arrows) along the dorsolateral pathway is normal in control explants (D).

View larger version (20K): [in a new window]   Fig. 6. Effects of soluble ephrin-B1-Fc on neural crest cell migration. Pre-clustered Fc or ephrin-B1-Fc (10 µg/ml) was added to the bottom well of a chemotaxis chamber and neural crest cells were added to the top well. In some control experiments preclustered ephrin-B1-Fc was also added to the upper well. Cells that migrated to the bottom side of the porous filter were stained, solubilised and quantified by OD measurements. Ephrin-B1-Fc induces a significant decrease of early migratory neural crest cell migration (white bars). In contrast, clustered ephrin-B1-Fc causes a significant increase in the transfilter migration of melanoblast cells (grey bars). This is a representative experiment and each data point is the mean ± s.e.m. of triplicate wells (P<0.05).

View larger version (22K): [in a new window]   Fig. 7. Effects of ephrin-B1-Fc on neural crest cell adhesion to fibronectin. (A) Cells were plated on surfaces coated with fibronectin plus Fc (10 µg/ml) or fibronectin plus ephrin-B1-Fc (10 µg/ml), as described in Materials and Methods. Ephrin-B1-Fc significantly increases adhesion of melanoblasts to fibronectin (A,C), but decreases the attachment of early outgrowths to fibronectin (A,B). Asterisks represent no significant difference between values (P<0.05). Early outgrowths (B). Melanoblasts (C).

View larger version (66K): [in a new window]   Fig. 8. Effects of ephrin-B1-Fc on neural crest cell morphology. Cells were plated on coverslips coated with ephrin-B1-Fc (10 µg/ml) or fibronectin (10 µg/ml). After 4 hours, cells were fixed and immunolabelled with rhodamine-conjugated phalloidin and anti-vinculin antibody. The figure shows cells representative of each treatment.