Fig. 2. Time-lapse analysis of tracheal invagination. Cell boundaries were labeled with GFP-Moesin. The number in the top-right corner is the elapsed time in minutes. In a control embryo (A,B, and see Movie 1 in the supplementary material), time 0 was defined as the start of invagination. Cell division in the dorsal ectoderm outside of the tracheal placode resumed at about 60 minutes (blue double-headed arrow). For mutant embryos, the time of dorsal cell division was defined as 60 minutes. Apically constricted cells (<5 µm²) are colored yellow and the tracheal pit (the space emptied by the internalized cells) is colored pink. Mitotic cells in the tracheal placode are marked with blue asterisks. The region corresponding to the magenta rectangle in A is enlarged in B. In mutant embryos of Egfr (C; see Movie 3 in the supplementary material), rho (D; see Movie 4 in the supplementary material) and pnt (E; see Movie 5 in the supplementary material), the apical constriction was lost or reduced, the onset of invagination was delayed, and precocious mitosis was observed. In Egfr mutants (C), transient pit-like openings (colored orange) due to the ingression of one to two cells were observed multiple times (six times in this example, see Movie 4 in the supplementary material). Scale bars: 10 µm.