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First published online November 3, 2003
doi: 10.1242/10.1242/dev.00806

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Distinct sites in E-cadherin regulate different steps in Drosophila tracheal tube fusion

Mihye Lee^1,2,*, Seungbok Lee^1,3,*, Alireza Dehghani Zadeh^1,* and Peter A. Kolodziej^1,

¹ Department of Cell and Developmental Biology, Center for Molecular Neuroscience, Program in Developmental Biology, Vanderbilt University Medical Center, Nashville TN 37232-2175, USA
² School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea
³ College of Dentistry, Seoul National University, Seoul 110-740, Republic of Korea

View larger version (85K): [in a new window]   Fig. 1. Tube fusion proceeds through the formation and the maturation of a Shot containing track in fusion cells. (A) Schematic of morphological and cytoskeletal changes in fusion cells during fusion in an optical section along the plane of contact. Fusion cells (yellow) meet. They then form a new E-cadherin contact (blue) and initiate the assembly of a cytoskeletal track (red) that contains F-actin, Shot and microtubules (see Fig. 1B-K). This track matures, growing to span the two cells and guiding the invagination of the existing apical surfaces (light green) and associated cytoskeleton (pink). As the branches draw closer, the fusion cells also compress along the anteroposterior axis and elongate along the dorsoventral axis, bringing the existing apical surfaces closer. When the two apical surfaces meet, they fuse, leaving a ring of E-cadherin and associated cytoskeleton at the junction between the fusion cells. The two mature fusion cells are doughnut shaped. Other tracheal cells are wedge shaped. (B-K) Time lapse series of 0.5 µm confocal images of dorsal trunk tracheal cells in a live wild-type embryo expressing Shot L(A)-GFP in tracheal cells. Fusion sites are indicated by arrows, and are enlarged in F-J. Elapsed time is indicated in minutes (upper right corners). (B) At the start of the sequence (late stage 12), the tracheal branch tips are touching, but no track of Shot-GFP is visible at the future fusion site (arrow). (C) At 10 minutes, the anterior fusion cell accumulates Shot-GFP (arrowhead) along one side. (D) At 40 minutes, more Shot has begun to accumulate, and the track is centered more on the fusion cell contact. (D-G) A persistent track of Shot is visible from 40 minutes to 105 minutes. (F-I) The apical surfaces exhibit dynamic behavior (insets; concave arrow indicates an area that accumulates more Shot; the arrowheads indicate an area that loses and then regains Shot). (H) At 110 minutes, the apical surfaces of the two branches come closer together, forming a bottleneck structure. (I) At 115 minutes, a second track of Shot is clearly visible in this cross-section, indicating that the tubes are now joined. (J) The second track becomes darker, and the opening between the branches swells. A ring of Shot is visible (arrowhead, inset). (K) At 145 minutes, the opening assumes a diameter close to that of the adjacent branches. Scale bar: 10 µm. Anterior, upwards; dorsal, rightwards.

View larger version (150K): [in a new window]   Fig. 2. Shot colocalizes with microtubules and the microtubule + end-binding proteins EB1 and CLIP190 in the fusion track. Anterior, leftwards; dorsal, upwards. (A,C,E,G) Before fusion; (B,D,F,H) after fusion. (A-H) Composited 1 µm confocal sections of stage 13 wild-type embryos. (A) Prior to fusion, a track (arrowhead) containing microtubules (red), Shot (green) and EB1 (blue) forms in fusion cells. The same proteins also colocalize apically (arrow). Merge of C, E, G. Scale bar: 10 µm. (B) The fusion track (arrowhead) contains CLIP190 (blue), as well as microtubules (red) and Shot (green, merge of D,F,H). (C,D) Microtubules in the fusion track (arrowhead). (E,F) Shot in the fusion track (arrowhead). (G) EB1 in the fusion track (arrowhead). (H) Clip 190 in the fusion track (arrowhead).

View larger version (147K): [in a new window]   Fig. 3. Shot associates with E-cadherin and is required for adherens junction development in selected cell types. Anterior, leftwards; dorsal, upwards. E-cadherin, red; Shot, green. (A-C) Dorsal trunk tracheal cells in a stage 14 wild-type embryo form adherens junctions associated with Shot. (A) E-cadherin forms a web of junctions that gird the apical surfaces of tracheal cells, joined at segment boundaries (arrowhead). Scale bar: 10 µm in A-C. (B) Shot concentrates at adherens junctions. (C) Merge of A and B. (D-F) Lateral chordotonal (lch) neurons form highly organized adherens junctions with support cells. (D) In a stage 15 wild-type embryo, E-cadherin clusters in two rows of adherens junctions (arrowheads) formed by support cells contacting chordotonal dendrites. The arrow indicates neuronal cell bodies, which do not express E-cadherin. Scale bar: 10 µm in D-L. (E) Shot colocalizes with E-cadherin in the dorsalmost set of adherens junctions associated with lch neurons. (F) Merge of D and E. (G-I) Shot does not appreciably colocalize with epidermal adherens junctions. (G) Cadherin distribution at the dorsal midline (long arrow) of a stage 15 wild-type embryo. This section is taken 1 µm below the section where most E-cadherin is observed, and 1 mm above where most Shot can be detected. (H) Shot distribution in the same optical section. Shot is relatively diffuse, except in spots where it colocalizes with E-cadherin (short arrows). (I) Merge of G and H. E-cadherin and Shot overlap weakly. (J-L) E-cadherin and Shot distribution in shot3 null mutant embryos. (J) Cells in the dorsal tracheal trunk of a stage 14 shot3 mutant embryo. The branches fail to fuse at the segment boundary (arrowhead), and Shot is absent in all cells. E-cadherin appears normal in tracheal cells other than fusion cells. (K) E-cadherin contacts with chordotonal neurons in a stage 15 shot3 mutant embryo are abnormally organized and shaped. (L) E-cadherin contacts between epidermal cells appear normal at the dorsal midline (long arrow) in a stage 15 shot3 mutant embryo, including stereotyped spots where it colocalizes with E-cadherin (short arrows).

View larger version (106K): [in a new window]   Fig. 4. E-cadherin and ß-catenin, but not p120, are required for fusion track assembly. Arrowheads, fusion sites. Anterior, leftwards; dorsal, upwards. (A-C) Stage 14 wild-type embryo. (A) Fusion is complete, and F-actin is concentrated along a continuous apical surface spanning the fusion site. Scale bar in A: 10 µm for A-F. (B) Shot is also concentrated apically. (C) Merge of A and B. F-actin, red; Shot, green. (D-F) Stage 14 shg2 mutant embryo. (D) F-actin does not form a track in fusion cells and the tracheal tubes remain blind-ended. (E) Shot-GFP does not form a track in fusion cells. (F) Merge of D and E. F-actin, red; Shot, green. (G-I) Stage 13 wild-type embryo. (G) E-cadherin contacts form between fusion cells. Scale bar in G: 10 µm for G-L. (H) Tracks of Shot form at the new E-cadherin contacts. (I) Merge of G and H. (J-L) Stage 13 armYD35; P[armS14-C] embryo. (J) New E-cadherin contacts fail to form between fusion cells. K) Tracks of Shot fail to form in fusion cells. L) Merge of J and K. E-cadherin, red; Shot, green. M) A stage 15 p120308 mutant embryo stained with mAb 2A12, which reveals the tracheal lumen (Samakovlis et al., 1996a). Dorsal trunk (arrowheads) and lateral trunk (arrows) fusion appears normal, as does tracheal branching. Scale bar in M: 10 µm.

View larger version (90K): [in a new window]   Fig. 5. A mutation in the E-cadherin juxtamembrane dominantly blocks tracheal tube fusion and disrupts microtubule tracks. Anterior, leftwards; dorsal, upwards (except in P-T where anterior is upwards and dorsal is rightwards. (A-D) Stage 15 embryos stained with mAb 2A12. (A) The lumen in a stage 15 wild-type embryo expressing wild-type E-cadherin in tracheal cells is continuous at fusion sites in the dorsal trunk (arrowheads) and lateral trunk (arrows). Scale bar: 10 µm in A-D. (B) Expression of -arm E-cadherin in tracheal cells does not detectably affect fusion (arrowheads, arrows). (C) Expression of AAA-JXT E-cadherin in tracheal cells blocks fusion in the dorsal (arrowheads) and lateral (arrows), as well as at the dorsal midline (not shown). (D) Expression of E-cadherin mutant in the GGG juxtamembrane sequence in all cells (Pacquelet et al., 2003) has no effect on tracheal fusion or development (arrowheads, arrows). (E) Drosophila S2 cells expressing HA epitope-tagged p120 (red, E') and AAA-JXT mutant E-cadherin (green, E''). E-cadherin and p120 colocalize at cell contacts (arrows). F) Stage 14 embryo expressing wild-type E-cadherin in tracheal cells. Scale bar: 10 µm in F-H,J,K. E-cadherin is localized largely in adherens junctions (arrowhead). (G) Stage 14 embryo expressing arm E-cadherin in tracheal cells. Adherens junctions appear normal (arrowhead). Somewhat more E-cadherin is found outside the adherens junctions than when wild-type is overexpressed in tracheal cells. (H) Stage 14 embryo expressing AAA-JXT E-cadherin in tracheal cells. E-cadherin is delocalized. (I) Western blot with anti-E-cadherin (Oda et al., 1994) revealing the relative amounts of NP40 soluble (S) and pelleted (P) E-cadherin in wild-type Oregon R embryos expressing no additional (OreR) E-cadherin or wild-type E-cadherin (+WT), AAA-JXT E-cadherin, or arm E-cadherin in tracheal cells. (J) Same embryo as in F. Fusion has occurred and F-actin accumulates apically in fusion cells (arrowhead). (K) Same embryo as in H. F-actin accumulates at the fusion site, but in an aggregate (arrowhead). (L-O) Stage 13 embryo expressing AAA-JXT E-cadherin in tracheal cells. Scale bar: 10 µm in L-O. (L) F-actin weakly accumulates at the fusion site (arrowhead). (M) GAP-43 GFP labels the membranes of the fusion cells (arrowhead), which are elongated. (N) A weak track of Shot is visible at the fusion cell contact (arrowhead). (O) Merge of L-N. F-actin, red; GFP, green; Shot, blue. (P-T) Frames from videos of fusion in AAA-JXT mutant embryos. Minutes elapsed, lower left. Scale bar: 10 µm in P-T. (P) A weak Shot fusion track (arrow) is visible at the start of the first sequence. Apical surfaces appear open (arrowheads). (Q,R) The track changes little even after 50 (Q) or 160 (R) minutes, becoming only moderately more intense. The apical surfaces remain open (arrowheads). (S) At 170 minutes in a second sequence, over 1 hour after fusion occurs in wild type, the track persists and the apical surfaces remain open (arrowheads). (T) At 170 minutes in a third sequence, the existing apical surfaces draw closer together after a track forms and shrinks (not shown), but remain blind-ended (arrowheads). (U) Microtubules in dorsal trunk tracheal cells form fusion tracks (arrowheads) in a stage 13 wild-type embryo. Scale bar: 10 µm in U,V. (V) The microtubule track is broken (left arrowhead) or missing in fusion cells (right arrowhead) in a stage 13 embryo expressing AAA-JXT E-cadherin in tracheal cells. (W-Y) A late stage 13 embryo expressing AAA-JXT E-cadherin under the control of hairy-GAL4. Segments expressing the mutant transgene (arrows). (W) Shot fails to accumulate in fusion tracks (arrowheads). (X) E-cadherin contacts fail to form between fusion cells (arrowheads). Adherens junctions appear abnormally arranged in segments expressing the transgene. However, E-cadherin remains largely in adherens junctions (arrows). Scale bar: 10 µm in W-Y. (Y) Merge of W and X. Shot, red; E-cad, green.

View larger version (149K): [in a new window]   Fig. 6. The E-cadherin intracellular domain is required for fusion track assembly and maturation, and maintenance of apical cytoskeletal polarity in tracheal cells. Anterior, leftwards; dorsal, upwards. (A-F) Stage 15 embryos stained with 2A12 to reveal the tracheal lumen (green). In F, the embryo is also stained with anti-E-cadherin (red). Scale bar: 10 µm in A-F. (A) The dorsal trunk is continuous in a wild-type embryo. (B) Numerous breaks and constrictions in the dorsal trunk of a shg2/shgIH embryo. (C) Expression of a wild-type E-cadherin cDNA in tracheal cells restores fusion in a shg2/shgIH embryo. (D) Expression of a arm mutant E-cadherin in tracheal cells enhances fusion defects in a shg2/shgIH embryo. (E) Expression of AAA-JXT mutant E-cadherin in tracheal cells enhances fusion defects in a shg2/shgIH embryo. (F) Expression of both -arm and AAA-JXT mutant E-cadherin in tracheal cells does not rescue fusion defects in a shg2/shgIH embryo. Mutant E-cadherins are delocalized and not organized into adherens junctions. Fusion cells maintain finger-like contacts (arrows) but do not form lumenal connections even as epidermal movements and shape changes pull tracheal branches apart. (I,L,O,S) Shot, red; E, cadherin, green; F-actin, blue (S only). (G-I) A stage 13 shg2/shgIH mutant embryo expressing a arm mutant E-cadherin in tracheal cells. (G) Shot tracks are not detectable at fusion sites (arrows). Scale bar: 10 µm in G-N. (H) E-cadherin is delocalized in tracheal cells. In these embryos, little endogeneous E-cadherin is detectable, so this represents transgene expression. (I) Merge of G and H. (J-L) A stage 13 shg2/shgIH mutant embryo expressing the AAA-JXT mutant E-cadherin in tracheal cells. (J) Shot tracks are weak (left arrow) or absent (right arrow) in fusion cells. Shot is apically concentrated in other tracheal cells (arrowhead). (K) E-cadherin is delocalized in tracheal cells. (L) Merge of J and K. (M-O) Stage 15 shg2/shgIH mutant embryo expressing the AAA-JXT mutant E-cadherin in tracheal cells. (M) Shot is delocalized in tracheal cells. (N) E-cadherin is delocalized in tracheal cells. (O) Merge of M and N. (P-S) A stage 13 shg2/shgIH mutant embryo expressing the AAA-JXT mutant E-cadherin in tracheal cells. (P) Shot tracks are absent from fusion cells (arrows). Scale bar: 10 µm in P-S. (Q) E-cadherin is delocalized. (R) F-actin tracks are absent from fusion cells (arrows). (S) Merge of P-R.

View larger version (20K): [in a new window]   Fig. 7. A model for E-cadherin signaling during tube fusion. E-cadherin signals via ß-catenin (Arm) to initiate track assembly. An unknown factor (X) associated with the juxtamembrane domain is required to assemble or stabilize track-associated microtubules. Microtubules are required for track maturation. Maturation events involve the subsequent reinforcement of initial F-actin/Shot assembly or the disassembly of apical cytoskeletal structures and the track itself prior to fusion. Shot functions to stabilize F-actin and microtubules associated with the track. The track components together stabilize the new E-cadherin contact and coordinate apical surface movements and remodeling.

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