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First published online 25 February 2009
doi: 10.1242/dev.034017

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The role of Dpp and Wg in compensatory proliferation and in the formation of hyperplastic overgrowths caused by apoptotic cells in the Drosophila wing disc

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

View larger version (71K): [in this window] [in a new window]   Fig. 1. Effect of radiation-induced apoptosis on the size of mutant posterior wing compartments. Posterior (P) wing compartments were mutant for dpp (A-C), wg (D-F) or for dpp and wg (G-I). In all experiments, P compartments are labelled by loss of the GFP marker (green; see Materials and methods). The discs in C, F and I were irradiated in the second instar and fixed when the larvae reached the wandering or prepupal stage. (A) Non-irradiated wing disc in which the P compartment is homozygous for dppd12. The small GFP patches represent the few remaining M/+ cells. (B) Disc of the same genotype as in A, fixed and stained for caspase activity (red) 3 hours after irradiation. (C) Irradiated disc of the same genotype as in A, fixed at the prepupal stage. The size of the P compartment is not significantly altered. (D) Non-irradiated wing disc in which the P compartment is mutant for wgRF, double stained for GFP and Wg (blue). Note the small size of the P compartment and that it lacks Wg protein. (E) Disc of the same genotype as in D, showing caspase activity (red) 4 hours after irradiation. (F) Prepupal disc of the same genotype as in D, fixed and doubly stained for GFP and Wg. The disc was irradiated in the second instar and fixed at the prepupal stage. The irradiation does not affect the size of the P compartment. (G) Non-irradiated disc in which the P compartment is doubly mutant for dppd12 and wgRF, double stained for GFP and Wg. (H) Disc of the same genotype as in G, fixed and stained for caspase activity (red) 4 hours after irradiation. (I) Prepupal disc of the same genotype as in G, fixed and stained for GFP and Wg to show the lack of effect of irradiation on the size of the P compartment. The disc was irradiated in the second instar and fixed at the prepupal stage. (J) Posterior:anterior (P:A) size ratio in the various phenotypes. Non-irradiated discs; mauve; irradiated discs, blue. The left bar shows the P:A ratio of a wild-type disc and, from left to right, the various combinations of discs in which the P compartment is mutant for dppd12, contains the UAS-shmi Dpp2 construct, is mutant for wgRF, or is mutant for dppd12 wgRF. Note that there is no significant difference in irradiated versus non-irradiated discs in any of these combinations.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Role of wg and dpp in the hyperplastic overgrowths caused by undead apoptotic cells. (A) Irradiated disc of genotype hh-Gal4>UAS-p35 UAS-GFP fixed and stained for GFP and Hid (red in inset). In this, as in the following cases, the disc was irradiated in the second larval period and then fixed at the prepupal stage. Note the two groups of undead cells of posterior origin (arrows) that have penetrated into the anterior compartment. (B) Irradiated prepupal hh-Gal4>UAS-p35 disc in which the posterior compartment is entirely mutant for dppd12 (see Materials and methods). There are numerous undead cells because they contain the activated form of caspase (although functionally blocked by P35), shown in red. However, the posterior compartment is not enlarged in size. The few green spots are remaining M/+ cells. (C) Prepupal irradiated hh-Gal4>UAS-p35 disc in which the posterior compartment is mutant for wgRF. The disc was stained with anti-Wg to show there is no wg activity in the posterior compartment. It is also stained with anti-Dronc to reveal the presence of undead cells. As in the disc in B, the posterior compartment is not enlarged. (D) P:A size ratio in discs of genotypes as in A, B and C. The P:A ratio in hh>p35 is significantly higher than in hh>p35 dppd12 and hh>p35 wgRF discs. The P:A ratio of the latter is similar to that of normal discs (compare with Fig. 1J).

View larger version (118K): [in this window] [in a new window]   Fig. 3. Expression of wg and dpp in undead cells lacking dpp and wg, respectively. (A) Control hh>p35 UAS-GFP irradiated wing disc doubly marked for GFP (green) and wg (blue; white in the inset) showing ectopic wg expression in the posterior compartment. (B) hh>p35 disc in which the posterior compartment is mutant for dppd12 (see Materials and methods for details). wg expression is labelled blue (white in the inset); the lack of dpp is marked by loss of GFP label. Note that there is no ectopic wg expression. The small green patches in the posterior compartment correspond to the remaining dpp+ cells in the compartment. (C) Control hh>p35 irradiated disc showing ectopic dpp expression in the posterior compartment (arrows) after in situ hybridization with a general dpp probe. (D) In situ hybridization with the same probe in an hh>p35 disc in which the posterior compartment is wgRF. Note that there is no ectopic dpp expression.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Response of the JNK pathway to irradiation. (A) Non-irradiated wing stained to reveal JNK activity (red, monitored by puc-LacZ). JNK expression is restricted to a few cells in the proximal region of the disc. (B) Irradiated wing disc stained for puc-LacZ 8 hours after irradiation, showing overall activation of the JNK pathway. (C) The same disc as is portrayed in B displaying high levels of caspase activity (blue). (D-F) Irradiated disc of genotype hh-Gal4>UAS-p35 UAS-GFP; puc-LacZ, fixed 96 hours after irradiation and stained for lacZ (red) and wg (blue). Note the high levels of JNK activity in the posterior compartment (E) associated with ectopic wg expression (F).

View larger version (34K): [in this window] [in a new window]   Fig. 5. Induction of wg and dpp by JNK activity in absence of apoptosis. (A) Wing disc of genotype spalt-Gal4>UAS-GFP showing the spalt domain (green). The disc is also stained for wg activity (blue). (B) Wild-type disc with double in situ/antibody fluorescent staining for wg (green) and dpp (red). (C-E) Similar staining of a disc of genotype spalt-Gal4>UAS-hepAct;dronc-. There is ectopic wg (D) and dpp (E) activity in the region corresponding to the spalt domain.

View larger version (40K): [in this window] [in a new window]   Fig. 6. Induction of hyperplastic overgrowths by JNK activity. (A) A spalt>GFP droncl19 mutant disc showing the normal spalt domain (green) in the wing pouch. (B) Forced activity of JNK, using the activated form of hemipterous (spalt>hepActGFP droncl19), produces hyperplastic overgrowth in the spalt-expressing region. (C) Ratio of the normal spalt domain relative to the total size of the disc in dronc mutant discs in the absence (left bar) or presence (right bar) of JNK activity. The size of the spalt domain in the B is enlarged and also shows abnormal morphology, when compared with that of the disc in A.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Compensatory growth versus hyperplastic overgrowths after massive apoptosis in the wing disc. Our view of the events that occur after X-ray-induced massive apoptosis in an early disc. (Top) In an hh-Gal4>UAS-GFP wing disc, cells entering apoptosis are not protected by P35; (bottom) cells entering apoptosis in a hh>UAS-p35 UAS-GFP wing disc contain P35. In the disc in which cells are not protected by P35, many cells acquire JNK pathway activity, which triggers apoptosis and other JNK-related functions, such as Dpp/Wg signalling and the ability to migrate. However, the latter functions are normally inconsequential because apoptotic cells die very quickly. After 24-48 hours all apoptotic cells have disappeared. This probably causes a transient diminution of size, but eventually the surviving cells proliferate to achieve the stereotyped size of each compartment. In the hh-Gal4>UAS-p35 UAS-GFP disc, the irradiation causes JNK-mediated cell death in the anterior and posterior compartment. The anterior compartment behaves as in the disc above, but in the posterior one the cells acquiring JNK/Dpp/Wg signalling cannot be eliminated because of the presence of P35. They remain in the compartment, persistently emitting the Dpp and Wg mitogens during the rest of the growth phase of the disc, in effect causing a hyperplastic overgrowth. Some of the undead cells in the posterior compartment can penetrate into the anterior one, a property that possibly derives from their JNK activity.

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