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First published online 3 August 2005
doi: 10.1242/dev.01949

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Puckered, a Drosophila MAPK phosphatase, ensures cell viability by antagonizing JNK-induced apoptosis

Donald G. McEwen^1,*, and Mark Peifer^1,2

¹ Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA
² Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA

View larger version (73K): [in a new window]   Fig. 1. Loss of Puc function triggers apoptosis in embryonic epithelia. (A-D) Cuticles, ventral view, anterior upwards. (A) Wild-type; (B) pucA251.1 M/Z; (C) pucA251.1 maternal-only (arrowheads indicate denticle belts); (D) pucA251.1 zygotic mutant. pucA251.1 M/Z mutants secrete fragmented cuticle; this is suppressed by paternal Puc. (E-N) Embryos, anterior leftwards. Dorsal is upwards unless indicated otherwise. (E-J) Lateral views, TUNEL-label (green). Anti-phosphotyrosine (red) outlines cells. (E,F) Wild type stage 10 (E) and stage 13 (F). Arrowhead in F indicates leading edge cells. (G,H) Stage 10 (G) and stage 13 (H) presumptive pucA251.1 M/Z mutants undergoing massive apoptosis. (I,J) Stage 10 (I) and stage 13 (J) presumptive pucA251.1 maternal-only mutants, with fewer TUNEL-positive cells. (K,L) Wild type (K) and pucA251.1 maternal mutants (L). Engrailed is in red. Arm-eGFP (green) indicates cell boundaries. (M,N) TUNEL-labeled (green) pucA251.1 zygotic mutants. Mutants identified by ectopic JNKREP activity (blue). Cell boundaries are indicated with DE-cad (red). (M) Ventrolateral view, stage 14. Arrowheads indicate TUNEL-positive cells. (N) Lateral view, stage 13. Arrowhead-leading edge cells with elevated JNKREP activity. (O) UAS-rpr;en-Gal4;pucA251.1/+, TUNEL-labeled (green). (P) JNKREP activity from O (red). puc heterozygotes identified by JNKREP activity at leading edge. Apoptosis is induced in segmental stripes, without induction of JNKREP.

View larger version (75K): [in a new window]   Fig. 2. JNK signaling promotes the loss of puc mutant imaginal tissues. (A-D) Third-instar wing discs. GFP-expressing (green) cells are clonal patches of cells homozygous for: (A) wild-type control, (B) pucA251.1, (C) pucR10, (D) UAS-myc-Puc; pucA251.1. Actin (purple). puc mutant clones are not recovered unless Puc+ function is trangenically restored (D). The GFP-signal in B is from clones outside the disc proper in the parapodial membrane. (E-I) Adult eyes. All eye cells homozygous for: (E) wild-type control, (F) pucA251.1, (G) pucR10, (H) bsk2/+; pucA251.1 and (I) pucA251.1 p53[5A-1-4]. Eye tissue reappears in bsk heterozygotes or p53 homozygotes.

View larger version (94K): [in a new window]   Fig. 3. p53-dependent activation of the JNK signaling pathway is required for -radiation-induced apoptosis. Third-instar wing discs. (A) DIC. en-Gal4 (schematically overlaid in red) is expressed in the posterior compartment. (B-E) Wing discs oriented as in A. JNKREP (red), actin (blue). (B) Untreated pucA251.1/+. (C-E) Four hours after -radiation (4000 rads here and below). (C) pucA251.1/+, (D) en-Gal4/UAS-myc-Puc; pucA251.1/+ and (E) en-Gal4/UAS-myc-PucDEAD; pucA251.1/+. (F-H) TUNEL (green), Myc-Puc (red) and DNA (blue). Untreated (F) or -irradiated (G,H) discs expressing Myc-Puc (G) or Myc-PucDEAD (H) in the posterior compartment. (G',H') TUNEL alone. (I,J) TUNEL (green), JNKREP (red) and DNA (blue). (I) -irradiated pucA251.1 p53[5A-1-4]/+ p53[5A-1-4] disc. (J) Untreated en-Gal4/UAS-p53GUS2.1; pucA251.1/+ disc. (J',J'') Posterior compartment of J: JNKREP (J') and TUNEL (J''). (K) en-Gal4 UAS-myc-Puc /UAS-p53GUS2.1 mutant. TUNEL (green, K''), Myc-Puc (red, K') and DNA (blue).

View larger version (76K): [in a new window]   Fig. 4. JNK signaling functions upstream of Rpr and Hid. (A-D) Control (A,C) or irradiated (B,D) wing discs from rpr-11-lacZ (A,B) or en-Gal4 UAS-myc-Puc; rpr-11-lacZ (C,D) larvae. ß-Gal (red) and DNA (blue). rpr-reporter is blocked by Myc-Puc expression. (E-H) Discs, pucA251.1/+ (E,F) or pucA251.1/hs-hid (G,H) four hours after heat shock. (E,G) JNKREP (red) and DNA (blue). (F,H) JNKREP alone from E,G, respectively. Neither heat shock alone nor heat shock-hid induction strongly induces JNKREP.

View larger version (59K): [in a new window]   Fig. 5. JNK-dependent cell death is required for normal development. (A-C) Male external genitalia. (A) Wild type, (B) en-Gal4/UAS-p35 and (C) en-Gal4/UAS-myc-Puc. Arrow in lower right-hand corner, penis-to-anus orientation. Mis-expression of either p35 or Myc-Puc disrupts genital orientation. (D) Quantitation. (E-G) Adult thorax. (E) Wild type, (F) ptc-Gal4/UAS-p35 and (G) ptc-Gal4/UAS-myc-Puc. (Insets) Higher magnification of the scutellar bristles; arrowheads indicate extra macrochaetae. (H) Quantitation.

View larger version (114K): [in a new window]   Fig. 6. Elevating JNK signaling while blocking apoptosis promotes ectopic overgrowth of imaginal tissues. Adult wings (A-G). Foreleg (H). (A) Wild type, (B) en-Gal4/UAS-p35, (C-H) en-Gal4/UAS-p35;pucA251.1/+. (E,F) Higher magnification of the overgrowths from C,D, respectively. Overgrowths occur only in the posterior compartment. Cells adopt appropriate fates. (G) Additional wing defects (disruption of polarity, extra wing vein material). (I-L) en-Gal4/UAS-p35;pucA251.1/+. (I) DNA. (J,L) JNKREP (red) and DNA (blue). (J) TUNEL label (green). Ectopic JNKREP activity is associated with regions of overgrowth. (J) Less severe example. (L) More severe example. (K) Higher magnification of J. (M,N) Irradiated en-Gal4/UAS-p35;pucA251.1/+. (M) JNKREP (red) and DNA (blue). p35 does not inhibit JNKREP activation in the posterior compartment. (N) DNA (blue). TUNEL label (green). p35 blocks radiation-induced apoptosis. (O,P) JNKREP (red), Wg (green) and DNA (blue). (O) pucA251.1/+ control. Normal pattern of Wg. (P) en-Gal4/UAS-p35;pucA251.1/+. Wg is ectopically expressed by a subset of cells that activate the JNKREP (arrowheads). (Inset) Higher magnification of P.