Fig. 7. Vg1 RBP is asymmetrically localized in explanted neural crest cells and required for their migration. Stage 20-22 embryos were cut horizontally in the middle of the embryo, and a caudal piece of trunk neural tube (indicated schematically by red lines in A) containing premigratory neural crest cells was placed in a petri dish coated with fibronectin. Twenty-four hours of incubation at 25°C led to an emigration of up to several hundred cells, with several different morphologies. Generally, the tubes rolled onto their sides, leading to an asymmetric outgrowth of the neural crest on one side of the explanted tube. (B) Phase-contrast image; (C) the fluorescence micrograph of an explanted neural tube stained with the anti-HNK1 antibody. (D) Emigrating neural crest cells and neural fibers stain positive for HNK1. (E,F) Explanted neural tube and outgrowing cells were stained with anti-Vg1 RBP antibody and viewed using phase-contrast (E) or fluorescence (F) microscopy. For both the anti-HNK1 and anti-Vg1 RBP antibodies, note the fluorescence in the neural tube and in the majority of the cells that have migrated out of the tube. (Melanophores, because of their pigment, do not show noticeable fluorescence.) (G-I) Representative patterns of asymmetric distribution of Vg1 RBP in migratory neural crest cells. Vg1 RBP is observed at different sites in neural crest cells, including along the membrane (G), and in processes that generally point away from the explanted tissue (H,I). (J-L) Injection of AMO reduces the migration of neural crest cells in explants. Neural tubes from stage 20-22 embryos, injected with either CMO (J) or AMO (K), were cultured as described in A. The number of cells that migrate out of the explant from AMO-injected embryos (K) was reduced approximately threefold when compared with that from CMO-injected embryos (J). Immunostaining of the AMO-injected explants shows that these migrating cells are positive for Vg1 RBP (L). Note the presence of differentiated neural crest derivatives in the explant cultures, suggesting that both the cells and the tube remained viable during the course of the assay.