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First published online 8 April 2004
doi: 10.1242/dev.01095

Development 131, 2183-2194 (2004)
Published by The Company of Biologists 2004
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Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress

Yakov Pesah1, Tuan Pham2, Heather Burgess2, Brooke Middlebrooks2, Patrik Verstreken3, Yi Zhou5, Mark Harding2, Hugo Bellen1,3,4,5 and Graeme Mardon1,2,3,4,5,*

1 Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA
2 Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
3 Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA
4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
5 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA



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  		Fig. 1. Analysis of Parkin amino acid sequence conservation and developmental expression. (A) Schematic of human and Drosophila Parkin and PARK2 proteins. The ubiquitin-homology domain is an N-terminal domain, followed by the first RING finger domain (R1), an in between ring (IBR) domain, and a second RING finger domain (R2). Similarity and identity values for each domain are shown. (B) Multiple sequence alignment of human, mouse and Drosophila Parkin proteins shows a high degree of conservation throughout the lengths of the proteins. Solid boxes indicate identities; shaded boxes represent similarities. (C) Developmental northern analysis of parkin expression. A 1.6 kb parkin transcript is first detected in 0-2 hour embryos but is undetected in subsequent embryonic stages. parkin is again expressed during third instar larval (3L), early (E), mid (M) and late (L) pupal stages, as well as in adults. RP49 was used as a loading control on the same filter.

 


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  		Fig. 2. Genomic map of parkin locus, P-element mutagenesis and eclosion analysis of parkin mutants. (A) Locations of predicted genes and P-elements surrounding the parkin locus are shown. Solid boxes represent Drosophila genes and putative ORFs. The parkin rescue (dpkR) and stop-rescue (dpkSR) transgenes are identical except that dpkSR contains stop codons in all three reading frames of parkin. xxx indicates the position of the stop codons. (B) Generation of a null parkin allele. The dpkP30 element was mobilized to generate the dpkP21 insertion. The resulting double insertion chromosome was then used to delete the entire parkin-coding region while the surrounding genomic DNA remained intact. (C) Eclosion analysis. Progeny of w; dpk{Delta}21/TM3 self cross was analyzed. Although little or no pre-pupal lethality was observed, 68% of parkin mutant pupae fail to eclose (92% of the dead pupae are parkin mutants).

 


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  		Fig. 3. Body size of parkin mutants is significantly reduced at eclosion. (A) parkin mutant animals are visibly smaller than controls 2 days after eclosion. (B) Body mass measurements of 20 individual males and females were performed. In parkin mutants, mass was reduced by 30% in males and 34% in females compared with heterozygous controls. This phenotype shows significant but partial rescue with dpkR but not with dpkSR, confirming the specificity of phenotype to parkin (P<0.001, one-way ANOVA test). (C) parkin mutant wing size is reduced by 23% in females (n=20, P<0.001, one-way ANOVA test).

 


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  		Fig. 4. parkin null flies have reduced longevity and stress resistance. We analyzed life span, and oxidative and cold stress resistance of parkin mutant animals. (A) Longevity profile of w; dpk{Delta}21 as well as w; dpk{Delta}21/Df(3L)pc-MK animals. The Df(3L)pc-MK deficiency uncovers the entire parkin locus. Mean life span for male w; dpk{Delta}21 flies is 13 days and for w; dpk{Delta}21/Df(3)pc-MK flies is 32 days, compared with more than 60 days for heterozygous controls. Similar results were observed with females. (B) Paraquat sensitivity of parkin mutants. Loss of parkin results in rapid mortality when flies are maintained on 2 mM paraquat-containing media. (C) Cold stress resistance test. parkin mutants exhibit poor survival at 4°C. Fifty flies per genotype per sex were analyzed for each assay. Similar results were obtained with w; dpkP23 and w; dpkP24 mutants.

 


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  		Fig. 5. Loss of parkin results in indirect flight muscle degeneration. Light microscopy was used to examine IFM architecture. (A-D) Transverse sections through resin-embedded thoraces at 94 hours of pupation and (E-H) at 2 days after eclosion stained with Toluidine Blue to visualize tissue morphology. (A,E) Heterozygous controls show normal IFM architecture. At 2 days post eclosion (F), but not at the late pupal stage (B), parkin mutants show loss of muscle tissue (asterisks). At 11 days (I-L) the muscle loss becomes much more pronounced (compare J with F and L with H). This phenotype is rescued by the dpkR (C,G,K) but not dpkSR (D,H,L).

 


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  		Fig. 6. TEM analysis of IFM ultrastructural morphology reveals muscle fiber degeneration, mitochondrial defects and apoptosis in parkin mutants 2 days after eclosion. (A) Control animals have well organized uniform muscle architecture and electron dense mitochondria. (B) parkin mutant muscle fibers are fragmented (arrow) and mitochondria show loss of christae (asterisk). These phenotypes are rescued with dpkR (C), but not with dpkSR transgenes (D). (E) Control animals do not show any signs of muscle death; however, parkin mutant IFMs (F) show clear signs of apoptosis, including chromatin condensation and nuclear envelope breakdown (arrows).

 


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  		Fig. 7. parkin mutants do not show dopaminergic neuron loss on whole-mount analysis of dopaminergic dorsomedial cluster (DMC) neurons. (A) Example of a morphologically normal Drosophila brain stained with anti-tyrosine hydroxylase antibodies with a higher magnification of DMC cluster neurons shown in B. (C) Dopaminergic cell counts of parkin mutant animals (w; dpk{Delta}21) at 2 and 21 days after eclosion do not show any reduction in numbers of tyrosine hydroxylase-positive cells in dorsomedial clusters compared with heterozygous controls (w; dpk{Delta}21/+).

 

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© The Company of Biologists Ltd 2004