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Broad specifies pupal development and mediates the ‘status quo’ action of juvenile hormone on the pupal-adult transformation in Drosophila and Manduca

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

View larger version (31K): [in a new window]   Fig. 1. JH causes re-expression of BR in Manduca pupal epidermis. (A) Northern analysis of 10 µg total RNA from pupal forewings for the first 10 days after pupal ecdysis, using the Manduca br probe. Either 10 µl cyclohexane (normal) or 10 µg of the JH analog pyriproxifen in 10 µl cyclohexane was applied topically at 19 hours before pupal ecdysis. The Ethidium-stained ribosomal RNA indicates equal loading. (B) BR protein in a JH-treated pupal abdomen 5 days after pupal ecdysis as detected by immunocytochemistry with the Manduca BR core antibody. The JH treatment was as in A. Note the rosettes (R) of cells forming the pock marks of the second pupal cuticle.

View larger version (26K): [in a new window]   Fig. 2. JH causes re-expression of BR mRNA in Manduca pupal wings in vitro. Eighteen-hour-old pupal wings were precultured for 24 hours in either hormone-free medium (no hormone or 20E) or 1 µg/ml (3x10–6 M) JH I (for those being exposed to JH). At time 0 they were transferred to medium without hormone or with 1 µg/ml JH I, 5 µg/ml (10–5 M) 20E, or both, and cultured for various times, then the RNA extracted and analyzed as in Fig. 1.

View larger version (70K): [in a new window]   Fig. 3. JH causes re-expression of mRNAs for BR and a pupal cuticle protein Edg78E and suppression of mRNA for the adult cuticle protein Acp65A in the Drosophila abdomen during adult development. Northern analysis of 10 µg total RNA from separated anterior (head and thorax) and posterior (abdomen) regions after topical application of (A) 0.2 µl acetone (control) or (B) 100 ng pyriproxifen (JH mimic) in 0.2 µl acetone to the white puparium, using the probes described in the Materials and Methods. Note the predominant br transcript re-expressed during 36-72 hours APF is that of the 4.4 kb Z1 isoform (arrow). The Ethidium-stained ribosomal RNA indicates equal loading.

View larger version (80K): [in a new window]   Fig. 4. BR immunostaining of abdominal epidermal cells in control and JH mimic-treated Drosophila that were treated at puparium formation (as in Fig. 3) and at various times thereafter. The large nuclei are in the larval cells, whereas the small nuclei are in the adult cells that are derived from the histoblasts. The wild-type Canton S strain reared at 25°C was used. Scale bar: 50 µm.

View larger version (87K): [in a new window]   Fig. 5. Effects of misexpression of BR on cuticle gene expression during adult development. (A) Northern analysis of mRNAs for BR-Z1, the pupal cuticle protein Edg78E, and the adult cuticle protein Acp65A in the transgenic fly line (w1118; 708-14; 708-1) containing four copies of the BR-Z1 isoform variant (TNT-Q1-Q2-Z1) under a heat shock promoter. Pupae were harvested at various times without heat shock or after a 30 minute heat shock starting at 48 hours APF. (B) Misexpression of each of the BR isoforms has different effects on cuticle gene expression. Each of the BR isoforms was heat-induced as in A at the designated time between 36 and 60 hours APF, then the tissue was collected at 66 hours APF for the pupal cuticle genes and at 72 hours for the adult cuticle gene (the time of its peak expression in both regions; see A). We used transgenic lines with four copies of Z1 as in A and of each of the other Z isoforms as described in the Materials and Methods. (C) Effects of misexpression of BR-Z1 at 64 hours APF on the expression of cuticle genes at 72 hours APF. In all cases, puparia were maintained at 25°C before and after the heat shock. RNA analysis was carried out as in Fig. 3.

View larger version (85K): [in a new window]   Fig. 6. Effects of misexpression of BR-Z1 on the morphology of the resultant pharate adults. All animals were scored about 120 hours APF, about 24 hours after the expected time of eclosion. (A) Pharate adults of the Z1/Z1; Z1/Z1 line (w1118; 527-5; 708-1) that have two copies of the TNT-Q1-Q2 variant and two of the Q1-Q2 formed after expression at various times during adult development. Note the truncated bristles in the head and thorax after misexpression at 32.5 hours, but in the abdomen only later at 35 hours. Note the transparent pupal-like cuticle in the dorsum with little bristle pigmentation except in parts of the abdomen after misexpression at 48 and 52 hours. (B) Whole mounts of abdominal cuticle formed after expression of the same transgene as in A at the designated time APF. Note the truncated bristles after misexpression at 35 and 39 hours and the formation of bristles with little or no pigmentation after misexpression at 45 and 48 hours. (C) Pharate adult after misexpression of Z1/Z1 (line w1118; 708-1) at 36 and again at 48 hours APF. Note the thin transparent pupal-like cuticle over the entire animal. Some bristles are present on the head and dorsal thorax but are not pigmented. The inset shows a pupal-like wing found in some of these animals.

View larger version (45K): [in a new window]   Fig. 7. Thoraces of pharate adults after misexpression of four copies of each of the BR isoforms at 56 hours APF. Note the shiny pupal-like cuticle and thin nonpigmented bristles after expression of the Z1 isoform (the line used in Fig. 5A). The thorax appears normal after misexpression of the Z2 isoform. After misexpression of both Z3 and Z4 isoforms, the cuticle appears normal, but the bristles are nonpigmented with the difference being that the sockets of the macrochaetes are prominent after Z3 but not after Z4 misexpression.

View larger version (30K): [in a new window]   Fig. 8. Premature expression of a pupal cuticle gene Edg78E and suppression of a larval cuticle gene Lcp65A-b by misexpression of BR during the second larval molt. The heat shock was carried out at the time indicated after ecdysis to the second instar (L2) and the RNA extracted at 24 hours L2. RNA analysis was as in Fig. 3. The Z1 transgenic line used was w1118; 708-1; 708-14.

View larger version (18K): [in a new window]   Fig. 9. Summary diagram of the hormonal regulation of Broad (BR) protein expression and its role in the specification of pupal development based on studies of its action on cuticle genes. Juvenile hormone (JH) prevents the pupal molt, by preventing the activation of the br gene by ecdysone and prevents the adult molt by preventing the suppression of br by ecdysone in JH-sensitive tissues. BR is sufficient to activate the pupal program and to suppress both the larval and the adult programs. See text for details.

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