Fig. 4

Direct labeling of the deep layer confirms that cumulus cells internalize first in L. mactans. a External view just after CM-DiI injection through blastopore into early deep layer, arrowhead indicates label (red) in cumulus. b Confocal section of similar embryo fixed 1 day after injection; red dye is limited to cumulus mesenchyme (arrowhead). A few cells appear to have been pushed deeper into the embryo during injection. Tubulin is green, nuclei are blue, arrowheads mark labeled cells in b–f. c, d Injection into early deep layer. c Confocal stack of embryo fixed 1 week after injection; label is in posterior germ band consistent with normal migration path of cumulus. d Confocal section of a similar embryo fixed 1 week after injection; labeled cells are in deep layer at posterior. Asterisk marks fluorescent debris attached to exterior of embryo. e, f. Injection into later deep layer below blastopore, after cumulus has migrated away from blastopore (~1 day later than early injection). e Confocal stack of embryo fixed 2 weeks after injection; labeled cells are in right germ band and extraembryonic area. f Confocal section of similar embryo fixed 2 weeks after injection; labeled cells are in deep layer of right germ band. Position of label is consistent with non-cumulus fate. Scale bars 200 µm