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First published online February 20, 2004
Journal of Experimental Biology 207, 1151-1161 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00855
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Overexpression of broad: a new insight into its role in the Drosophila prothoracic gland cells

Xiaofeng Zhou, Baohua Zhou*, James W. Truman and Lynn M. Riddiford{dagger}

Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA



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  		Fig. 1. Effects of overexpression of BR-Z3 in w1118; hs-Z3; hs-Z3 larvae. (A) Induction of BR-Z3 at 11 h after ecdysis to the second instar. Left: blocked pharate third instar larva showing both the second instar (arrow heads) and the third instar (arrows) mouthhooks. Right: double spiracles in a blocked pharate third instar larva (arrow head points to the second instar spiracle, and arrow points to that of the third instar). (B) Induction of BR-Z3 at 5 h after ecdysis to the second instar. Left: normal third instar puparium. Middle: second instar puparium after experimental treatment. Note the uneverted anterior spiracles (arrow head) in the L2 puparium as compared with the everted multi club-shaped third instar spiracles (arrow). The L2 puparium is about two-thirds the size of the w1118 normal puparium. Most of the L2 pupae developed up to stage P8 and, in very rare cases, they formed pharate adults (right).

 


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  		Fig. 2. Broad (BR) protein expression pattern after heat induction of BR-Z3 at 5 h of the second instar. (A) BR proteins were detected in all tissues by an anti-BR core region antibody at 12 h after the heat shock. (B) At 18 h only trace amounts of BR protein remain in the fat body. BR is also found in certain cells in the CNS, as in normal second instar larvae (F). (C) By 24 h after the heat shock, BR proteins were only detected in the CNS, as in the normal second instar larva (F), indicating that all heat-induced BR proteins were catabolized by that time. (D) A new round of br expression began around 36 h after the heat shock, and (E) high levels of BR were seen in the imaginal discs at 72 h. Abbreviations: ad, antenna disc; br, brain; cns, central nervous system; ed, eye disc; epd, epidermis; fb, fat body; ld, leg disc; sg, salivary gland; wd, wing disc.

 


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  		Fig. 3. Overexpression of BR-Z3 during the critical feeding period for the second-to-third instar molt causes a prolonged second instar and the formation of L2 prepupae. (A) Second instar larvae were allowed to feed on standard diet for a designated period and were then transferred to glucose diet (protein starvation). Ecdysis to the third instar was scored. Each point (circle) represents a mean of four repetitions (N=16, 8, 8, 8) ± standard deviation (S.D.). Note the ET50 for the critical feeding period is ~6.5 h. (B) BR-Z3 was overexpressed by a heat shock at a designated time in the second instar, and the animals were maintained on the standard diet. The L2 prepupae were scored. Each point (diamond) represents a mean of four repetitions (N=25, 10, 10, 10) ± S.D. Note that the ET50 of the induction of L2 prepupae is ~5.8 h after ecdysis into the second instar.

 


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  		Fig. 4. Critical weights for pupariation for w1118; hs-Z3; hs-Z3 larvae. Larvae were raised on standard diet, then given (or not given) a heat shock at 5 h after ecdysis into the second instar. Individuals of various ages (16–35 h after the heat shock) were weighed, then were either starved or fed 20-hydroxyecdysone (20E) diet. Pupariation was scored for each group of treatment. The percentage of pupariation was calculated from groups of larvae whose weight was the designated point ± 0.025 mg. The number of animals used for measuring the critical weights for normal L3 pupariation (triangles), L2 prepupae (squares) and competence to form L2 prepupae (circles) was 80, 117 and 99, respectively.

 


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  		Fig. 5. The effect of ectopic expression of BR-Z isoforms in the prothoracic gland (PG) cells on Drosophila molts. (A–D) Larvae with ectopic expression of one of the BR isoforms in the PG cells under the control of the P0206 Gal4 driver were fed either standard diet or 20-hydroxyecdysone (20E) diet as described in Materials and methods. Each bar represents an average of at least three repetitions of the percentage of successful molting (mean ± S.D.). The total number of animals for each observation is shown in the bar.

 


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  		Fig. 6. Ectopic expression of Broad (BR) proteins in the prothoracic gland cells of the ring gland causes degeneration. (A) Expression of BR-Z3 in the ring gland driven by P0206 caused degeneration of the entire PG portion of the ring gland in a prolonged second instar larva (48 h after ecdysis into the second instar; right). Only part of the corpora allata (CA) remained intact (arrowhead). Compare to an intact ring gland of a second instar control larva (left). (B) The process of degeneration of the prothoracic gland (PG) cells was visualized by expressing both green fluorescent protein (GFP) and BR isoforms via P0206. The ring glands are outlined by broken lines, and the arrows point to the CA. Scale bar, 25 µm. Abbreviations: br, brain; ca, corpora allata; ed, eye disc; pg, prothoracic gland cells.

 


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  		Fig. 7. Normal expression pattern of Broad (BR) proteins in the prothoracic gland (PG) cells. Third instar larvae or white puparia were dissected at designated times (hours after egg laying). Each ring gland was double stained with antibodies against BR core region and one of the BR isoforms as described in Materials and methods. Note that the anti-BR-Z2 antibody also nonspecifically stained the nucleoli.

 

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© The Company of Biologists Ltd 2004