Fig. 4. After r3 removal, r3* mesenchyme gradually loses its NCC repulsive character. Experiments were performed to determine whether maturational changes intrinsic to NCCs (A,C-E), or surgically induced changes within the mesenchyme (B,F-K) were responsible for the observed delay between r3 removal and the onset of the aberrant NCC migration phenotype. Broken lines mark the neuroepithelial outlines. (A) r3 was removed at 10ss, clusters of donor DiI-labelled 16ss r4 cells were grafted homotopically into r4 and the embryos incubated for a further 5 hours. (C-E) Phase, combined phase/DiI, and DiI images, respectively, reveal that grafted cells seldom migrate into r3* mesenchyme, indicating that the r3 mesenchymal repulsive activity persists for up to 5 hours after r3 removal and affects late-migrating NCCs (cluster of cells within r2 in D,E is a cell injection artefact and does not represent migration from r4). (B) r3 was removed at 10ss and embryos were incubated for 5 hours before either directly labelling host r4 cells with DiI (I; F-H) or homotopically grafting age-matched DiI labelled r4 cell clusters (II; I-K). Embryos were then incubated for a further 3 hours. (F-K) Phase (F,I), combined phase/DiI (G,J) and DiI (H,K) images reveal that several r4 cells rapidly migrate (within 3 hours) into rostral r3* mesenchyme (arrows) following this additional post-operative period. This indicates that the mesenchymal repulsive activity is absent within 8 hours of r3 removal.
