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First published online 13 December 2006
doi: 10.1242/dev.02728

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Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis

Nikolay Ninov, Dominic A. Chiarelli^* and Enrique Martín-Blanco

Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Parc Cientific de Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain.

View larger version (70K): [in this window] [in a new window]   Fig. 1. Abdominal metamorphosis: cell proliferation dynamics of histoblasts. (A) During embryonic stages, four nests of abdominal histoblasts (adult epidermal cells precursors) can be distinguished in each hemisegment: anterior dorsal (green mask color), posterior dorsal (red), ventral (blue) and spiracular (yellow). During metamorphosis, histoblasts form the different structures that compose the abdominal adult epidermis, tergites (green), intersegmental membranes (red), pleurites and sternites (blue) and spiracle (yellow) (Roseland and Schneiderman, 1979). (B) In vivo time-lapse observation of histoblast proliferation in prepupal stages (1-6 hours APF) shows a synchronic cadence of three cell cycles leading to cell doubling every 2 hours. The number of cells calculated at the shown time points steadily increase (18, 36, 72 and 128). Cell sizes decrease after each mitosis (856, 605, 396 and 271 arbitrary 2-dimensional units; see Materials and methods). Histoblasts (anterior dorsal nest) expressed UAS-GFP under the control of Esg-Gal4 (see Movie 1 in the supplementary material). (C) Early histoblast cell divisions show planar orientation. Histoblasts (anterior dorsal nest) expressed UAS-Tau-GFP under the control of Esg-Gal4. In the first cell division, spindles orient predominantly along the dorsoventral axis. (D) Doubling times of histoblasts during pupal stages (from 15 hours APF onwards) increase up to 9 hours. Proliferation is stochastic and coupled to cell growth. Cellular outlines (anterior dorsal nest) were highlighted by ubiquitously expressing a DE-Cadherin-GFP fusion. Mitoses of individual cells were followed by in vivo time-lapse (see Movie 2 in the supplementary material). (E) Schematic of histoblast cell cycle dynamics. During prepupal stages (left), histoblasts do not grow between cycles (68% cell size decrease in the first three divisions). By contrast, during the pupal stages, histoblasts undergo intermitotic growth and their sizes remain constant (right). (F) FACS analysis showing cell cycle profiles of dissociated histoblasts from prepupal (black) and pupal stages (red). During prepupal stages, histoblasts lack or have a very reduced G1 phase. In pupal stages, the length of G1 phase increases by 70%.

View larger version (91K): [in this window] [in a new window]   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).

View larger version (71K): [in this window] [in a new window]   Fig. 3. Actin polymerization directs the expansion of histoblast nests. (A) Bristles with morphogenetic defects (yellow arrowheads) and cuticular abdominal clefts (white arrowheads) in an adult escaper of Permanent-Esg-Gal4/UAS-Profilin genotype. (B) In wild-type histoblasts (22 hours APF), most polymerized actin organizes in cortical filaments. Phalloidin-rhodamine staining (red) is shown in the top and bottom panels. UAS-GFP expression (green) under the control of Permanent-Esg-Gal4 is shown in the bottom panel. (C) Actin in histoblasts overexpressing Profilin (22 hours APF) hyperpolymerizes and accumulates in intracellular clumps (arrowheads). Top and bottom panels are as in B. (D) Snapshots from Movie 7 in the supplementary material. Top panel, wild-type dorsal posterior nest (23 hours APF). Arrowheads point to expanding peripheral protrusions. Bottom panel, dorsal posterior nests from pupae (23 hours APF) overexpressing Profilin (Permanent-Esg-Gal4/UAS-GFP; UAS-Profilin). Note the absence of long terminal protrusions and the histoblast expansion delay.

View larger version (85K): [in this window] [in a new window]   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.

View larger version (99K): [in this window] [in a new window]   Fig. 5. Extruded LECs are removed by circulating haemocytes. (A) Macrophage-like haemocytes circulate under the pupal epidermis basal lamina extending and retracting leading lamellipodia (arrowhead). Haemocytes were visualized in vivo through the pupal cuticle by the expression of membrane-bound Src-GFP (green) using a haemocyte-specific driver (Srp-Gal4). (B) Travelling cells in the haemolymph are recruited to the basal surface of LECs. The actomyosin cytoskeleton of LECs was visualized by the expression of Sqh-GFP using confocal XZT acquisition. The apical constriction of LECs (small arrowheads) was followed by the attachment of bright fluorescent bodies to their basal side (large arrowhead). (C) The recruitment of haemocytes to the basolateral surface of LECs occurs sequentially to constriction. Simultaneous in vivo visualization of LECs and histoblasts (ubiquitous expression of Stb-YFP, red) and haemocytes (Srp-Gal4/UAS-GFP, green) (see Movie 10 in the supplementary material). Snapshots show LECs undergoing apical constriction (arrowhead) and being engulfed from their basal surface by haemocytes (asterisk) extending cytoplasmic projections (small arrowheads). Transverse z-projections show that encapsulation initiates before LEC delamination is completed and is extremely fast. The whole process (apical constriction, delamination and engulfment) takes place in approximately 45 minutes.

View larger version (59K): [in this window] [in a new window]   Fig. 6. Histoblast proliferation and LEC death are triggered by Ecdysone signaling. (A) Blocking Ecdysone signaling autonomously in histoblasts abolished their proliferation. The overexpression of an Ecdysone receptor EcR-RNAi construct (Esg-Gal4) results in a prolonged delay in cell division. The left image shows a dorsal anterior nest at the prepupal stage expressing EcR-RNAi. The number of histoblasts in this nest is similar to equivalent nests in wild-type pupae (see Fig. 1B). The middle image shows the same nest 300 minutes later; histoblasts have not divided. In this period, wild-type animals undergo three rounds of division (right image). (B) Clonal autonomous inhibition of Ecdysone signalling in LECs (dominant-negative form of the Ecdysone receptor EcR-DN) blocks their death (see Materials and methods). The left image shows a control animal in which the abdominal epithelial fusion proceeds normally. As a result of the overexpression of EcR-DN (middle image) the expansion of histoblast nests does not succeed, resulting in a dorsal scar phenotype (arrowheads). Simultaneous expression of a GFP marker reveals that LECs (asterisk) have not been eliminated from the abdomen of pharate adult mutant flies (green: right image).

View larger version (106K): [in this window] [in a new window]   Fig. 7. The proliferation of histoblasts and the death of LECs are coordinated by reciprocal interactions. (A) Histoblast early divisions are autonomously blocked by Dacapo. UAS-Dacapo overexpressing clones labelled with GFP (green) were generated using FRT recombination. Dacapo-expressing histoblasts (arrowheads) in the anterior dorsal nest become arrested after two cell cycles and remain enlarged in comparison with wild-type neighbours. Nuclei were labelled by the expression of Histone H2-YFP (red). (B) LECs expressing P35 (induced by FRT recombination-GFP expressing cells) showed impaired cell extrusion due to partial inhibition of their apical constriction. The delamination of LECs expressing P35 (right panel) is strongly delayed and LECs persist in the epithelia for at least 3 hours longer than their wild-type counterparts (arrowheads). Larval cells and histoblasts were labelled using a DE-Cadherin-GFP fusion (wild type) and Stb-YFP, an apical membrane marker. (C) Those few LECs able to undergo extrusion in the presence of P35 remained as viable cells under the epithelial layer (as judged by their nuclear and cellular morphology) and were not engulfed by haemocytes. Cell outlines were visualized using Phalloidin (red) and cell nuclei were labelled with DAPI (blue). Note that the histoblast layer becomes highly pseudo-stratified. (D) The overexpression of Dacapo by heat shock results in the inhibition of cell division in all pupal cells, smaller histoblast nest sizes (DAPI staining) and in the survival of LECs, which remained in the epithelia. Staged heat shocked (right panel) and wild-type (left panel) pupae were dissected at 25 hours APF. Thus, the decreased proliferation rate of histoblasts correlated with a strong reduction in LEC death rate. (E) To exclude indirect anti-apoptotic effects of Dacapo in LECs (see B), Dacapo was exclusively and permanently expressed in histoblasts (see Materials and methods). In this condition (right panel), histoblast nests (green) are smaller than wild-type ones, with fewer cells (left panel) (25 hours APF). LECs (which do not express Dacapo) are not eliminated from the epithelia (asterisks). Thus, the reduction of LEC death rate caused by inhibition of histoblast proliferation is non-autonomous. (F) The delayed elimination of LECs expressing P35 causes a non-autonomous decrease in the number of histoblasts. The average reduction in histoblast numbers (DAPI staining) from ventral nests of animals subjected to FRT recombination (right panel) in comparison with wild-type animals (left panel) at 24 hours APF was approximately 20%. (G) The primary cause of the non-autonomous reduction in histoblast numbers is cell death. P35 expression in LECs (ventral nest, 24 hours APF) results in significant ectopic non-autonomous delamination and death (arrowheads) of histoblasts. Apoptosis was monitored by activated Caspase-3 antibody staining (red). (H) Inhibition of LEC death by P35 does not results in a major change in doubling times for histoblast. Quantification of cell division rates in posterior dorsal nests was performed by time-lapse analysis (from 17 hours APF) and cell counting (see Materials and methods). Cell doubling times are shown in minutes.

View larger version (34K): [in this window] [in a new window]   Fig. 8. Extrinsic and intrinsic signals on the process of generating an epithelial sheet de novo: LEC replacement by histoblasts. During larval stages, histoblasts are arrested in G2 and increase their size. LECs endoreduplicate, become polyploid and secrete the larval cuticle. At the onset of metamorphosis, the histoblasts undergo a series of G1-less synchronous cell divisions and reduce their size. Histoblast nests do not expand and remain confined to their original territories. LECs undergo apolysis, detach from the old larval cuticle and secrete the pupal cuticle. Images show the increment in number and the reduction in size of histoblasts from a ventral nest during pupariation. Histoblasts express GFP under the control of the Esg-Gal4 driver. The cell cytoplasm is marked in red with Propidium Iodide. In pupal stages, histoblasts undertake stochastic cell divisions and nests expand to replace LECs. These extrude from the epithelia, die and are cleared by the action of circulating haemocytes. In the images, histoblasts and LECs can be distinguished by their size (nuclear DAPI staining). Images show in false colour the spreading of nests in the period between 18 and 30 hours APF. Colour coding is as in Fig. 1 (i.e. green, anterior dorsal; red, posterior dorsal; yellow, spiracular; blue, ventral nest). The proliferation and expansion of histoblasts and the death of LECs are very precisely triggered by external (hormonal) inputs. An early Ecdysone peak of expression activates the synchronous divisions of histoblasts in prepupae. A late peak of Ecdysone correlates with histoblast loss of synchrony and it is essential for the initiation of cell replacement. Nonetheless, intrinsic interactive mechanisms involved in the coordination of histoblast proliferation and LEC death are also in place. LECs do not die in the absence of histoblast proliferation; conversely, histoblasts do not expand when LEC death is blocked. Mutual exchange of distinct signals thus appears to be necessary, beyond hormonal triggering events, to implement and harmonize the behaviour of histoblasts and LECs during abdominal epithelial morphogenesis

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