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First published online 31 October 2007
doi: 10.1242/dev.010397

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A wave of EGFR signaling determines cell alignment and intercalation in the Drosophila tracheal placode

Mayuko Nishimura^1,2, Yoshiko Inoue^1,* and Shigeo Hayashi^1,2,

¹ Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku Kobe 650-0047, Japan.
² Department of biology, Kobe University Graduate School of Science, Kobe, Japan.

View larger version (50K): [in this window] [in a new window]   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.

View larger version (69K): [in this window] [in a new window]   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.

View larger version (71K): [in this window] [in a new window]   Fig. 3. Cell-row boundary smoothing and cell intercalation prior to invagination. See Movies 1 and 2 in the supplementary material. (A) GFP-Moesin. Intercalating cells are shown in the same color. Shrinking cell boundaries and newly formed cell boundaries are indicated by blue and red arrows, respectively. The position of the tracheal pit is indicated by a red asterisk. Examples of cellular arc formation are indicated by orange and green lines. (B) Analyses of the cell displacement angle. Mean±s.e.m. are shown. Asterisk indicates significant (P<0.05) deviation from random orientation (average 45°). See text for details. (C-E) Transient accumulation of myosin-GFP at the cell-cell interface. (C) Continuous rows of myosin-GFP-enriched cell-row boundaries are indicated by arrows. Cell boundaries at the segment border and dorsal ectodermal margin (green and orange arrows, respectively) stably accumulated myosin-GFP. Myosin-GFP accumulation in the arc-like cell-row boundaries surrounding the invagination site was transient and appeared as temporary waves (blue arrows, see also Movie 2 in the supplementary material). (D) Cell boundary tracing. Each arc-like boundary was formed by the joining of zig-zagging and discontinuous boundaries, which changed with the arrangement of the cells relative to one another (blue, green and magenta lines). The boundary marked with blue lines became discontinuous before entering the tracheal pit. The orange lines indicate cell boundaries in dorsal ectoderm, which were more stable. Red asterisk indicates the invagination site. (E) High accumulation of myosin-GFP in shrinking cell boundaries. For explanation of colored areas, see A. Scale bars: 10 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 4. The dynamic pattern of EGFR activity detected with dp-ERK. (A-C) Spatiotemporal patterns of dp-ERK in three successive stages of invagination. Cells were labeled for dp-ERK (magenta), trh-lacZ (green), GFP-Moesin (green) expression; DNA is shown in gray scale; x-y and y-z sections of the same placode (labeled with a and b, respectively) are shown for each time point. Additional views of B are shown in Bc to Be. dp-ERK expression was initially detected in the nucleus and the cytoplasm of a few cells in the dorsal side of the tracheal placode (A, arrowhead) and subsequently expanded to fill the entire dorsal half of the placode (B). dp-ERK showed a nuclear localization in the peripheral region (arrowhead) and was concentrated in the apical cytoplasm in the central region. Note that Bc is a deeper optical section of Ba and Bd (position of the section is indicated by a thin white line in Bb). Apical constriction had not yet occurred and only a mild degree of cell boundary smoothing was observed at this stage (Bd). The dp-ERK signal was downregulated after invagination (C). (D,E) dp-ERK expression in Egfr+/- (D) and Egfr-/- (E) embryos. D',E' show the dp-ERK signal. (F) rho-lacZ expression was very similar to the pattern of dp-ERK. The tracheal placode (dashed outline) was marked with KNI (Kni). (G) The overlap of KNI and trh-lacZ expression. Scale bars: 10 µm.

View larger version (107K): [in this window] [in a new window]   Fig. 5. Invagination defects in EGFR-related mutants. Tracheal invagination phenotypes in stage 14 embryos. (A) All of the btl-GFP- expressing cells were internal except for the spiracle cells (arrow). (B-D) Many of the btl-GFP- expressing cells remained external in the rho (B), Egfr (C), and pnt (D) mutants. (E,F) The tracheal placode expanded 1.5 fold in the Egfr-/- (F) compared with Egfr+/- (E) embryos. (G) In the Egfr mutants, the invagination position (arrows) was variable and sometimes duplicated. (H-K) Position of invagination sites (% position measured from the dorsal edge) and the number of sites in controls, and in Egfr, Dsor1 and pnt mutants. The incidence of double invagination observed in Egfr and Dsor1 embryos was counted separately, without reference to their position. Scale bars: 10 µm.

View larger version (101K): [in this window] [in a new window]   Fig. 6. EGFR regulates cortical myosin accumulation. (A) Cell boundaries with high myosin-GFP accumulation often corresponded to the outer boundary of dp-ERK. b and b' are higher magnification images of a and a', respectively. Arrows indicate the high myosin-GFP accumulation at the interface of cells with high and low dp-ERK levels. (B,C) MHC accumulated in arc-like patterns in Egfr heterozygous embryos, but these patterns were lost in Egfr homozygotes (see text for quantitative assessment). In B', 8 out of 12 class T junctions were judged to be in arcs. Two examples of arcs are highlighted with yellow curved lines 1 and 2. (D) Two classes of tricellular junctions. In class T junctions, `horizontal' cell boundaries are colored pink, and `vertical' ones, blue. Myosin concentration was more than 50% enriched in horizontal cell junctions compared to the vertical one (mean±s.e.m., arbitrary unit, number of measurement indicated in parentheses, P<0.002, Student's t-test). (E) Expression of sSPI (sSpi) by the prd enhancer in even-numbered parasegments induced the massive activation of ERK and accumulation of MHC at the cell boundaries between cells with high and low dp-ERK expression (arrowheads). sSPI also induced the precocious invagination of the segmental furrow (asterisk) that becomes contiguous with the tracheal pit (tr2). An arrow indicates unaffected segment boundary. (F) Enrichment of junctional myosin by sSPI. The intensity of myosin signal was compared between the border along the stripe of elevated dp-ERK (DV border, red lines) and those intersecting them (AP border, black lines; mean±s.e.m., arbitrary unit). sSpi significantly increased the DV boundary signal compared to AP boundary (*, P<10-5, Student's t-test: two-sample assuming equal variances, two-tail). No such enrichment was observed in the control segments. (G) A model for the EGFR-dependent coordination of cell movement. Left: Myosin accumulates at the boundary of cells with high and low EGFR activity. The contractile force of myosin (blue arrows) helps shrinkage of cell boundary and cell intercalation (juxtaposed cell boundaries shown in green). In addition, the contractile force smoothens other cell-row boundaries. Right: once the cell-row boundaries form a continuous arc, the net force in the convex contractile supracellular actomyosin cable (pink) is oriented toward the inside of the arc (magenta arrows). Red asterisk indicates the invagination site. Scale bars: 10 µm.

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