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Files in this Data Supplement:
Fig. S1. Epithelial expansion involves the motile activity of histoblasts. (A) Analysis of cytoskeleton (Actin) and cell adhesion (DE-Cadherin) markers reveal that before the onset of active expansion (15 hours APF), histoblasts (anterior dorsal nest) are organized in a pseudostratified epithelial monolayer of columnar cells. Lower panel shows a transverse section throughout the epithelium. Actin (white) was visualized using Phalloidin; DE-Cadherin (green) was labeled by DE-Cadherin-GFP; nuclei (DAPI-stained) are shown in blue. (B) At later stages (30 hours APF), histoblast nests become organized in an unstratified epithelia in which cells are flattened along their apico-basal axis. Markers are as in A. (C) Histoblast nests and LECs constitute adjacent bounded epithelia. The anterior and posterior dorsal histoblast nests are shown as they expand to fuse into a single hemitergite nest between 15 and 18 hours APF (ubiquitous expression of a DE-Cadherin GFP fusion protein) (see Movie 2). De- Cadherin is slightly down regulated in peripheral cells (arrowheads).
Fig. S2. Histoblasts and LECs express both Ecdysone Receptor A and B1 isoforms during pupal stages. (A) Histoblasts and LECs show nuclear expression (green antibody staining) of the A isoform of the Ecdysone Receptor in the pupal period (20 hours APF), but not in larval stages. Nuclei were labeled with DAPI (blue). (B) The Ecdysone Receptor isoform B1 is also expressed in histoblasts and LECs (green antibody staining) during the pupal period (20 hours APF). This isoform is also present in larvae. Nuclei were labeled with DAPI (blue).
Movie 1. Onset of histoblast proliferation: synchronous cell divisions (1-7 hours APF). Histoblast proliferation was monitored in vivo using confocal time-lapse microscopy of Esg-Gal4-driven GFP-expressing pupae (anterior dorsal histoblast nest). Histoblast proliferation begins with a rapid cadence of synchronous and oriented cell divisions and during the first 6 hours of pupariation almost all cells in the nest divide three times (cell doubling time of around 2 hours). Synchronous mitoses are highlighted.
Movie 2. Expansion and fusion of histoblast nest and replacement of LECs (15-27 hours APF). Histoblasts and LECs were monitored during the pupal stage. Cells were marked by the ubiquitous expression of a DE-Cadherin GFP fusion protein. Time-lapse analysis shows that histoblast divisions slow down and become asynchronous and stochastically distributed. Beginning at 15 hours APF, histoblasts initiate epithelial expansion while pre-existing LECs are cleared from the epithelia (fusion of anterior and posterior dorsal histoblast nests).
Movie 3. Planar intercalation of histoblasts (15-21 hours APF). Cells were marked by the ubiquitous expression of a DE-Cadherin GFP fusion protein (anterior dorsal nest). Time-lapse analysis shows the planar intercalation of histoblasts through the larval epidermal palisade. Individual cells (a leading histoblast and three larval cells) have been marked in different colors) to help following of the intercalation process.
Movie 4. Intercalation of histoblasts cell bodies (22 hours APF). Histoblasts were marked by the expression of GFP under the control of Esg-Gal4 (posterior dorsal nest). A time-lapse recording shows the intercalation of a leading histoblast cell body in between LECs following the sequential extension and contraction of a long terminal protrusion.
Movie 5. Leading histoblasts send out actin-rich comet-like projections at the onset of epithelial expansion (17-18 hours APF). Actin was visualized in vivo by expressing an Actin-GFP fusion protein in histoblasts (anterior dorsal nest) using an Esg-Gal4 driver. Arrowheads point to the growth of comet-like extensions of leading histoblast. Note the predominant polymerization of actin filaments at the distal-most end of the projections.
Movie 6. Cell dynamics at the fusion of histoblast nests (24-25 hours APF). Histoblasts were labeled by expressing a Src-GFP fusion protein under the control of the EsgGal4 driver and the fusion of two neighbouring nests (spiracular and ventral) was followed using confocal microscopy. During nest fusion, histoblasts form at their apical surface a supracellular purse string.
Movie 7. Long terminal extensions of histoblast are inhibited by actin hyperpolymerization (18-27 hours APF). Histoblasts were labeled by the expression of GFP under the control of a permanent Esg-Gal4 driver. Comparison of the expansion of posterior dorsal nests (top panel, a wild-type control; bottom panel, animals expressing Profilin) shows that the long terminal protrusions emitted by wild-type histoblasts are absent from Profilin-expressing histoblasts, which only emit few short filopodia.
Movie 8. Apoptotic LECs extrude basally (16-22 hours APF). Histoblasts and LECs were labeled by ubiquitous expression of a DE-Cadherin-GFP. This movie shows an apico-basal 3D reconstruction from a confocal xyz stack of images. A larval cell (arrowhead) between two adjacent histoblast nests (anterior and posterior dorsal) condenses and delaminates basally from the epithelial layer. The extrusion of the cell is synchronic with the fusion of the two adjacent histoblast nests to avoid discontinuities and perforation of the epithelial layer.
Movie 9. Apical constriction and delamination of LECs is mediated by an acto-myosin contractile ring (20-24 hours APF). Pupae carrying a spaghetti-squash-GFP fusion protein (Sqh-GFP) under the control of the sqh endogenous promoter show prominent localized apical expression of GFP in LECs. The extrusion of dying LECs (arrows) during the fusion of the anterior dorsal and posterior dorsal histoblast nests is preceded by the assembly of apical myosin rings and their sequential contraction. Although most of the LECs delaminate at the periphery of histoblast nests (red), note that some LECs extrude at quite long distance (yellow arrows).
Movie 10. The extrusion of epithelial cells involves a coordinated process of apical constriction and corpse removal by circulating haemocytes (23-24 hours APF). Simultaneous in vivo visualization of LECs, histoblasts and haemocytes was monitored by time-lapse confocal microscopy. The apical domains of larval epithelial cells and histoblasts were labeled by the ubiquitous expression of the apical membrane protein marker Stb-YFP (red), while haemocytes were visualized using a Srp-Gal4 driver and a UAS-Src-GFP reporter (green). The central panel presents an xyz projection of the most apical region of the epidermis where Stb is enriched (the area within the double yellow lines marked in the xz and yz projections). The bottom and right panels show transverse sections trough the epidermis along the xz and yz axes. The coordinates of the transverse sections are indicated by thick yellow lines. A larval epidermal cell rounds up and initiates the process of apical constriction (white arrowheads). At the late stages of constriction, an underlying haemocyte intimately adheres to the constricting cell. The haemocyte extends long cytoplasmic projections (yellow arrowheads) to encapsulate the constricted cell and executes the final clearance of the corpse.
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