Fig. 1. Overview of tracheal invagination. (A) A stage 10 embryo expressing trh-lacZ (green). (B) Cell shape changes in the tracheal placode in four successive stages of invagination. x-y (Ba-Bd) and y-z (Ba'-Bd') confocal sections of fixed embryos expressing trh-lacZ (green) and DLG (Dlg; magenta) are shown. The bracket indicates internalizing cells with apical constriction (phase 1). Asterisks indicate internalizing cells without apical constriction (phase 2). The thin red and green lines indicate the position where the confocal sections were made. Ba" is a gray-scale image of the DLG signal. (C) Spatiotemporal order of the cell internalization events in a control sqh-GFP-Moe embryo. Tracheal placode cells at time -48 minutes are labeled with a number indicating their time of internalization, which was measured by tracing individual cells in time-lapse movie (see Movie 1 in the supplementary material). The cells internalized with a constricted cell apex (<1 µm²) are marked by a red line. (D) The orientation of cell divisions in the tracheal placode of a control sqh-GFP-Moe embryo after the onset of invagination. The asterisk at the center indicates the invagination site. (E) Summary of the time course of apical constriction, cell intercalation and internalization, and mitosis in the tracheal placode. The time when the first cell disappeared from the apical plane of the tracheal placode was set as time 0. Scale bar: 10 µm.