Fig. 2. The process of histoblast nest expansion is associated with cell shape changes and active cytoskeleton dynamics. (A) Snapshots of the process of histoblast expansion (ubiquitous DE-Cadherin-GFP; see Movie 3 in the supplementary material) show that at early stages of nest spreading (anterior dorsal nest), leading cells intercalate within and disjoin LECs. A leading histoblast (red) successively intercalates in between individual LECS (yellow). Overall, leading histoblasts move over a distance of 40 µm in 3 hours. (B) Leading histoblasts (Esg-Gal4/UAS-GFP, posterior dorsal nest) extend invasive dynamic protrusions (arrowheads), which promote the forward movement of histoblast cell bodies (red arrows) (see Movie 4 in the supplementary material). (C) The long cellular protrusions of histoblasts are enriched with actin at their tips (arrowheads). UAS-Actin-GFP was expressed in histoblasts (Esg-Gal4) and visualized with anti-GFP antibodies (green). Cell morphology was revealed by Phalloidin staining. Nuclei are in blue (DAPI). (D) The protruding structures of leading histoblasts grow by distal actin filament polymerization (arrowhead). Actin dynamics in vivo were monitored with Actin-GFP (Esg-Gal4) by confocal time-lapse microscopy (anterior dorsal nest) (see Movie 5 in the supplementary material). (E) Spreading histoblasts (Esg-Gal4/UAS-GFP, posterior dorsal nest) crawl over the larval epithelia and send out apical cellular projections in the form of lamellipodia and small filopodia (arrowheads). (F) Long filopodia (arrowhead) are observed in the basolateral membrane of peripheral histoblasts (Esg-Gal4/UAS-GFP, anterior dorsal nest). These structures are enriched in filamentous actin and are highly motile. (G) During the fusion of neighbouring histoblast nests, the apical domains of adjacent histoblasts become organized in a purse string (arrowhead). Histoblasts (Esg-Gal4/UAS-Src-GFP, spiracular and ventral nest) were monitored by time-lapse confocal microscopy (see Movie 6 in the supplementary material).