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First published online 9 January 2008
doi: 10.1242/dev.014001

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Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure

Departments of Biochemistry and Physiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1DA, UK.

View larger version (105K): [in this window] [in a new window]   Fig. 1. Expression pattern of en-RFP-Moesin and ptc-GFP-Moesin in the dorsal epithelium. (A) Schematic illustrating construction of the fly line, transgene expression pattern and dorsal trichome pattern. (B,C,F) Images of embryos expressing en-RFP-Moesin (red) and ptc-GFP-Moesin (green). (Bi-iii) Dorsal view of embryos at the start (i), midway through (ii), and shortly after completion (iii) of DC. (Ci-iii) Time course of the latter stages of DC in a live embryo. Misplaced ptc-GFP-Moesin-expressing cells (asterisks in i) are present at a conserved position at the leading edge of the en domains of all segments shown. (D) Bar chart showing proportion of embryos with misplaced ptc-GFP-Moesin-expressing cells in each segment. (E) Dorsal view of stage 16 embryo expressing GFP-Moesin (green) constitutively and RFP-Moesin (red) under control of en-Gal4. Pairs of cells in the P compartment not expressing RFP-Moesin are indicated by arrowheads. (Fi-iv) Images from Movie 1 (see Movie 1 in the supplementary material) showing cell rearrangements during DC that lead to the presence of the misplaced ptc cell (asterisk) in the en domain. Scale bars: 10 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Cell matching and realignment during DC is mediated by filopodia. Zippering in Drosophila embryos expressing en-RFP-Moesin (red) and ptc-GFP-Moesin (green), as shown merged (top row) and in isolation (middle and bottom rows, respectively). (Ai-iv'') Images from Movie 2 (see Movie 2 in the supplementary material) showing filopodial matching. (i) Red and green filopodia protrude from leading edge cells. (ii) Contacts are made between red filopodia from opposing epithelia, while at the same time separate contacts are made between green filopodia. (iii) Further contacts are made between red filopodia; however, green filopodia in close proximity to these red filopodial contacts do not interact. (iv) Green filopodia transiently form contacts between ptc-GFP-Moesin cells over the top of the fused red cells. (Bi-v'') Images from Movie 3 (see Movie 3 in the supplementary material) showing realignment of misaligned epithelial sheets by filopodial searching and pulling. (i) The two epithelial sheets are initially poorly aligned. A filopodial tether transiently exists between green cells but later breaks. (ii) Contacts form between filopodia from en-RFP-Moesin-expressing cells. (iii,iv) Green filopodia in the lower sheet do not interact with nearby red filopodia and ultimately form contacts with green cells some distance away in the opposing sheet. (v) The tethers that result from the contacts made by red and green filopodia pull the sheets into alignment. The described filopodial interactions are indicated by arrowheads. Scale bars: 10 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Matching in embryos with asymmetries between the opposing epithelial sheets. DC zippering in Drosophila embryos with asymmetries as revealed by en-RFP-Moesin (red) and ptc-GFP-Moesin (green) expression, shown merged (top row) and in isolation (middle and bottom rows, respectively). (Ai-iv'') Images from Movie 4 (see Movie 4 in the supplementary material) showing zippering in an embryo with a spontaneous asymmetry. (i) The upper epithelial sheet has two rather than one misplaced ptc cell in the en domain and one of these has associated with the A compartment of the lower sheet blocking matching of en cells. (ii) As a result, the blocked en cells make filopodial contacts with en cells of the neighbouring segment. (iii) These develop into permanent contacts. (iv) Cells that are not associated with matching partners continue to produce filopodia. (Bi-iii'') Zippering in an embryo with a laser-induced asymmetry. (i) An en stripe has been removed from the leading edge of the lower epithelial sheet by laser ablation (asterisk), whereas the opposing en stripe (arrow) is normal. (ii,iii) On contact with the opposing leading edge, the unpartnered en stripe constricts and then withdraws completely from the leading edge. The described filopodial interactions are indicated by arrowheads. Scale bars: 10 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Filopodial matching occurs during healing of a wound in the ventral epithelium. Images from Movie 5 (see Movie 5 in the supplementary material) showing healing of a wound across an en stripe in the epithelium of an en-RFP-Moesin (red), ptc-GFP-Moesin (green) Drosophila embryo. (A-A'') Filopodia are produced by wound edge cells. (B-C'') Interactions occur between ptc-GFP-Moesin cells across the wound. (D-E'') Eventually a filopodial tether forms between en-RFP-Moesin cells on either side of the wound and this tether rapidly leads to fusion of the two en cells. Notably, the last part of the wound to heal is at the junction between en and ptc cells. The described filopodial interactions are indicated by arrowheads. Scale bar: 10 µm.