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First published online 20 October 2004
doi: 10.1242/dev.01432

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Caspase inhibition during apoptosis causes abnormal signalling and developmental aberrations in Drosophila

Centro de Biología Molecular CSIC-UAM, Universidad Autónoma de Madrid, Madrid 28049, Spain

View larger version (95K): [in a new window]   Fig. 1. Induction of apoptosis by heat shock and X-rays. (A-C) A set of wing, haltere and leg discs fixed 24 hours after heat shock and doubly stained with TUNEL and Caspase 3. Note that the two stainings are co-extensive. (D-G) X-rayed wing discs fixed different times after irradiation and stained for caspase 3. High apoptotic levels can be observed until 48 hours after the irradiation. After 72 hours (G), the apoptotic levels have dropped to normal.

View larger version (49K): [in a new window]   Fig. 2. Suppression of caspase activity by the baculovirus P35 protein. (A-B) Wing disc of genotype hh-Gal4>UAS-p35 UAS-GFP irradiated 4 hours before fixation. Caspase 3 activity is high in the A compartment and is completely abolished in the P compartment, marked green with GFP fluorescence. (C-D) Disc of the same genotype irradiated 24 hours before fixation. Caspase activity is absent in the P compartment.

View larger version (109K): [in a new window]   Fig. 3. Effects of irradiation and heat shock in wing disc development. (A-C) Untreated disc and adult wing of genotype hh-Gal4>UAS-p35 UAS-GFP. It shows normal wg expression (red) and a normal P compartment (green). The expression of wg is not affected by P35 and the differentiated wing in C is essentially normal, except a slight shortening of vein 5. (D-F) Disc and adult wing from larvae of the same genotype, but irradiated in the first larval period. Note the abnormal shape and bigger size of the P compartment (green) and the numerous foci of ectopic and elevated wg expression. wg expression in the A compartment remains normal. The antero-posterior demarcation line is highly irregular and there are indentations into the A compartment (arrow). The few flies that hatch after this early treatment differentiate wings with very abnormal and overgrown P compartments (F). (G-H) Disc of the same genotype dissected from a larva heat shocked at the first larval period. Note the appearance of numerous dots of ectopic wg expression in the P compartment and the protrusion into the A compartment (arrow). (I-J) Irradiated disc showing ectopic dpp expression in the P compartment. An arrow points to an indentation into the A compartment

View larger version (86K): [in a new window]   Fig. 4. Excess of cell proliferation in irradiated hh-Gal4>UAS-p35 UAS-GFP wing discs. (A-B) BrdU incorporation in a disc irradiated 48 hours before fixation. BrdU levels are clearly higher in the P compartment, but note that there is also an increase of BrdU in the A compartment cells close to the AP border. (C-D) Disc from the same treatment but stained for phospho-histone 3 (PH3), showing a higher number of mitotic cells in the P compartment. (E-F) Discs of the same genotype also showing increased BrdU incorporation in the P compartment and in zones of the A compartment close to the AP border.

View larger version (111K): [in a new window]   Fig. 5. Invasion of A compartments by caspase-inhibited cells in hh-Gal4>UAS-p35 UAS-GFP wing discs. (A-B) Disc showing a group of posterior cells (arrow), labelled green by GFP, invading the A compartment. They express the engrailed (en) gene (red), indicating that their behaviour is not due to change of cell affinities. (C-D,E-F) Two discs showing intrusions into the A compartments (arrows). In each case the caspase-inhibited cells express wg, therefore carrying the secreted Wg product into the A compartment.

View larger version (115K): [in a new window]   Fig. 6. Clones of p35-expressing cells. (A-B) Irradiated wing disc doubly stained for active caspase 3 (red) containing a clone of p35-expressing cells (green). Note that the clone shows no caspase activity (arrow). (C-D) Wing pouch of a wing disc irradiated in the second larval period containing several clones of p35-expressing cells. Note that several of those clones exhibit ectopic or increased expression of wg (arrows). (E-F) Wing disc with several clones of p35-expressing cells. Two of them display gain of dpp activity (red, arrows).

View larger version (136K): [in a new window]   Fig. 7. Clones of p35-expressing cells induce additional proliferation of neighbour cells. (A-B) Disc containing several clones (labelled green), which show increased BrdU incorporation (red) in zones around the clones. (C-D) Region of the wing pouch containing a large p35-expressing clone. The disc was triply stained to label p35-expressing cells (green), wg (blue) and phospho-histone 3 of mitotic cells (red). Note the accumulation of mitotic cells in the vicinity of the clone. Note also (arrow) that the expansion of wg expression occurs inside the clone and its border coincides with that of the clone.

View larger version (110K): [in a new window]   Fig. 8. wg activation in normal apoptotic cells. (A-B) Untreated wing disc of genotype wg-Gal4>UAS-lacZ doubly stained with the anti-Wg antibody (red) and anti-ß-gal (green). The wg and lacZ domains are largely coincident; the relative expansion of the lacZ domain is probably the result of the greater stability of the ß-gal protein. (C-D) Irradiated disc of the same genotype fixed 24 hours after X-rays and stained for caspase 3 and ßgal. The dots of ß-gal staining reflect wg activation in apoptotic cells that induces UAS-lacZ activity.