Fig. 4. LECs are basally extruded during histoblast nest expansion by a mechanism involving the assembly of an actomyosin apical contractile ring. (A) Transverse snapshots (z-axis) of a 4D confocal reconstruction show the progression of the delamination of an intervening LEC (arrowhead) between the anterior and the posterior dorsal nests. Cells were marked using DE-Cadherin-GFP and the fusion of histoblast nests was monitored by time-lapse confocal microscopy (see Movie 8 in the supplementary material). (B) DAPI staining (green) shows that LECs in the epithelial layer (adjacent to the ventral histoblast nest) have large polyploid nuclei that become condensed (arrowhead) upon extrusion. Apical compartment outlines were visualized using a Fasciclin III antibody (red). A transverse section (lower panel) shows an extruded LEC surrounded by the ECM (arrowhead). The basal lamina was labeled by laminin A (white) and LEC (056-Gal4) membranes were marked using a UAS-Src-GFP (false blue colour). (C) The actomyosin cytoskeleton of histoblasts and LECs was visualized by a myosin light chain GFP fusion protein (Sqh-GFP) under the control of its own promoter (green). Sqh-GFP is present in apical LEC membranes. LECs initiating basal extrusion between the anterior and the posterior dorsal nests (see Movie 9 in the supplementary material) show apical myosin constriction (arrowhead and transverse optical sections, lower panel). (D) Persistant LECs (arrowhead) in a pharate adult clonally expressing GFP (green) and MBSN300, which leads to the constitutive dephosphorylation of MRLC and the inhibition of Myosin contractility. (E) Cuticular abdominal clefts (arrowhead) in an adult escaper clonally expressing MBSN300.