Fig. 5. Propidium iodide staining reveals timing of trans-epidermal migrations. (A,B,D,E) Projected compilations of confocal z-series, C is a DIC image (anterior is to the right). (A) Pre-competent larvae two hours after hatching; note the lack of any cells outside of the epidermis. Pre-competent larva at five hours after hatching also lack cells within the juvenile tunic (data not shown). (B) A competent larva, seven hours after hatching. Cells that have migrated into the tunic are labeled with an asterisk. (C) Close up DIC image of a cell that has migrated into the tunic in a 7-hour old larva (indicated in B by the white box). Note that the cell clearly lies on the outside of the epidermis but still within the juvenile tunic. The migrated cell is marked by a black dot, an asterisk indicates the epidermis, a black arrowhead indicates the juvenile tunic and a white arrowhead indicates the larval tunic. (D) A competent larva 15 hours after hatching; a regular array of cells can clearly be seen within the juvenile tunic, closely apposed to the outside of the epidermis. (E) Newly settled juvenile, 17 hours after settlement; migrated cells are now embedded within the expanding juvenile tunic, away from the epidermis. The juvenile tunic is indicated by the white arrowheads (at this point the larval tunic has been molted).