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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.
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