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First published online February 29, 2008
Journal of Experimental Biology 211, 890-899 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.014837

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A new role for a classical gene: White transports cyclic GMP

Jennifer M. Evans, Jonathan P. Day, Pablo Cabrero, Julian A. T. Dow and Shireen-Anne Davies^*

Division of Molecular Genetics, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, UK

View larger version (41K): [in this window] [in a new window]   Fig. 1. Phenotypes associated with the w gene. (A) The eye colour phenotype: w null (w1118; bottom left), and Canton S (top right) adult D. melanogaster. Female w null is 3 mm long. (B) Tubule phenotype: Malpighian tubules from Canton S (top) and w1118 (bottom) adults. Anterior tubules are on the left, and posterior tubules on the right of the picture. Scale bar, 110 µm.

View larger version (8K): [in this window] [in a new window]   Fig. 2. cGMP is transported by tubules. (A) Effect of depolarisation on the cGMP transport ratio. Leucokinin (100 µmol l–1) was used to depolarise the Malpighian tubule 30 min prior to the addition of cGMP (100 µmol l–1; values are means ± s.e.m.; N10; ***P<0.0001, Student's t-test). (B) cGMP-dependent kinase (cGK) activity in tubules is stimulated by fluid secreted from cGMP-treated Malpighian tubules. Data are expressed as mean (± s.e.m.) cGK activity, in pmol ATP min–1 mg–1 protein (N=3). cGK activity in tubule extracts is significantly stimulated by secreted drops from cGMP-treated tubules or by 100 µmol l–1 cGMP, but not by secreted fluid from non-treated tubules (***P<0.0001, Student's t-test).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Transport of cAMP and cGMP by the tubule are distinct processes. (A) cGMP and/or cAMP transport assays were performed on wild-type tubules in the absence (controls=100%) or presence of cyclic nucleotide (cAMP for cGMP transport; cGMP for cAMP transport) Percentage changes in the ratio of cGMP or cAMP transport compared to the controls are shown as mean ± s.e.m. (N5). Statistical significance was verified by Student's t-test compared with control (no added drugs), taking P<0.05 as the critical level. (B) cGMP and cAMP transport ratios from tubules assayed in either normal Drosophila saline (normal DS, unshaded bars) or minimal saline (shaded bars). (C) cAMP transport ratio from tubules assayed in either normal Drosophila saline (normal DS, unshaded bars); in minimal saline (shaded bar); or in minimal saline with additions of specific amino acids or other compounds as indicated (hatched bars). Data are mean ± s.e.m. (N5). ***Data significantly different (P<0.001; Student's t-test) from cAMP transport ratio in normal DS.

View larger version (31K): [in this window] [in a new window]   Fig. 4. w does not affect fluid transport but has a direct effect on cGMP transport. (A) Fluid transport rates by wild-type (Canton S, black circles) and w1118 (Bloomington stock, red squares and `cantonised' stock, red triangles). Malpighian tubules were stimulated by cGMP (100 µmol l–1, added at 30 min, indicated by arrow). Data are expressed as mean (± s.e.m.) fluid transport rate in nl–1 min (N=7). (B) Ratio of cGMP and cAMP transport by Canton S and w1118 Malpighian tubules. Data are mean ratios ± s.e.m. (N5). (C) Ratio of cGMP (100 µmol l–1) transport by Canton S tubules and by tubules from cantonised w1118 and wH lines. Data are mean ± s.e.m. (N5). (D) Ratio of cGMP (100 µmol l–1) transport in three independently derived parental transgenic w::eYFP lines (4D, 5E, 8H, unshaded bars) and offspring from crosses to a GAL4 driver, c42 (c42/w::eYFP; grey bars). Data expressed as a ratio of cGMP transport ± s.e.m. (N5). cGMP transport for the c42 GAL4 driver was: 1.9±0.17 (N=7). Significance of data between progeny compared to parental controls of each line is indicated by **P<0.01; *P<0.05, Student's t-test. (E) Q-PCR of w mRNA levels in untreated (control) tubules; and in tubules treated with 100 µmol l–1 cGMP. w expression was normalised against a cDNA standard (rp49) as previously described. Data are expressed as mean (± s.e.m.) fold-difference of w expression of cGMP-treated tubules compared to control tubules (N=4). ***Data significantly different from control (P<0.001; Student's t-test).

View larger version (100K): [in this window] [in a new window]   Fig. 5. Expression of White in the Drosophila Malpighian tubule. Staining of White expression in c42/w::eYFP adult tubules by immunocytochemistry with anti-GFP antibody. Intact tubules from adult progeny of crosses between c42 and two independently derived UAS-w::eYFP lines (E5 and H8) were used. (A) Main segment of c42/w::eYFP (UAS line E5) tubule showing exclusion of staining in stellate cells, yellow arrows. (B) Main segment of c42/w::eYFP (UAS line H8) showing exclusion of staining in stellate cells, yellow arrows. (C) Canton S tubules (high magnification) stained with anti-GFP antibody. Only background fluorescence is observed. (D) Principal cell in c42/w::eYFP (UAS line E5) tubule at high magnification showing vesicular staining. Scale bars: A,B 10 µm; C,D 5 µm.