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First published online 14 November 2007
doi: 10.1242/dev.009902

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A neuropeptide hormone cascade controls the precise onset of post-eclosion cuticular tanning in Drosophila melanogaster

¹ Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada.
² Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2H7, Canada.

View larger version (15K): [in this window] [in a new window]   Fig. 1. The neuropeptide signalling pathway at eclosion. In response to decreasing levels of ecdysone, ecdysis-triggering hormone (ETH) is released, triggering the release of eclosion hormone (EH). These hormones act together in a positive-feedback loop, increasing the release of one another and regulating pre-ecdysis behaviour. EH causes the release of crustacean cardioactive peptide (CCAP), which shuts off pre-ecdysis and turns on the ecdysis motor program. At eclosion, CCAP causes release of bursicon, which binds to its receptor RK, and induces post-eclosion events by elevating levels of cAMP and causing tanning. This figure is modified from Clark et al. (Clark et al., 2004) and McNabb et al. (McNabb et al., 1997).

View larger version (11K): [in this window] [in a new window]   Fig. 2. Critical steps in the tanning pathway. (A) Metabolites of dopamine are required for tanning. Tyrosine hydroxylase (encoded by ple) converts tyrosine into dopa and dopa decarboxylase (encoded by Ddc) catalyses the conversion of dopa to dopamine. (B) Alternative splicing of ple and Ddc. Both ple and Ddc are alternatively spliced to produce neural-specific (shown above exon boxes) and epidermal-specific (shown below exon boxes) transcripts. The coding sequence is shaded in grey.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Quantitative RT-PCR of ple and Ddc transcripts following pupariation. (A) ple or (B) Ddc transcripts are shown with an RpL32 loading control. Bands from at least four different extracts for each time point were quantified and averaged, and expressed as a percentage of the loading control band to produce the graphs. Error bars indicate s.e.m.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Tyrosine hydroxylase protein and activity profiles in yw, EH-KO, CCAP-KO, bursZ1091 and rk4 flies following puparium formation. Times are measured from pupariation. (A) Western blot of epidermal tyrosine hydroxylase (TH) from yw flies. An actin loading control is shown below the TH blot in this and subsequent figures. (B) TH activity in yw organisms. TH activity in this and subsequent figures is expressed as a percentage of the yw white pre-pupae (WPP) value. (C) Western blots of epidermal TH from EH-KO, CCAP-KO, bursZ1091 and rk4 flies. (D) TH activity in the same organisms as C. In B and D, black circles represent organisms within the pupal case, white circles represent adult flies. Values are means±s.e.m. of a minimum of three separate experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 5. TH protein and activity in yw flies after eclosion. (A) Western blots of epidermal TH protein and phospho-Ser32-TH. (B) TH activity in the same organisms. Values are means±s.e.m. of a minimum of three independent trials.

View larger version (16K): [in this window] [in a new window]   Fig. 6. TH protein, phosphorylation and activity in neck-ligated flies. (A) Western blots showing TH and phospho-Ser32-TH in yw flies neck-ligated at eclosion or 30 minutes thereafter and rescue by injection of 8-Br-cAMP. (B) TH activity in the same organisms. Each value is the mean+s.e.m. of a minimum of three independent experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Effects of 8-Br-cAMP on TH protein accumulation, phosphorylation and activity in peptide hormone mutants. (A) Western blots showing epidermal TH and phospho-Ser32-TH levels 2.5 hours after eclosion in untreated flies or flies injected with 8-Br-cAMP at eclosion. (B) TH activity in the same organisms. Each value is the mean+s.e.m. of a minimum of three independent experiments.