The Dh gene of Drosophila melanogaster encodes a diuretic peptide that acts through cyclic AMP
1 IBLS Division of Molecular Genetics, University of Glasgow, Glasgow G11
6NU, UK
2 Laboratoire de Neuroendocrinologie des Insectes, Université
Bordeaux I, Avenue des Facultés, 33405 Talence Cedex, France
3 Syngenta Biotechnology, Inc., Research Triangle Park, NC 27709,
USA
* Author for correspondence: j.a.t.dow{at}bio.gla.ac.uk
Accepted 20 September 2002
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Summary |
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Key words: neuropeptide, Malpighian tubule, corticotropin-releasing factor (CRF), cyclic AMP, Drosophila melanogaster
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Introduction |
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We describe in this work the Dh gene that encodes Drome-DH44, the first member of the CRF-like family to be found in Drosophila melanogaster. Functional analysis confirms that this peptide is active on the Malpighian (renal) tubule, and acts through cAMP. The peptide is expressed in six neuroendocrine cells in the pars intercerebralis. We also show for the first time that CRF directly activates its cognate phosphodiesterase, so limiting its own signal.
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Materials and methods |
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Drosophila methods
Drosophila melanogaster Meig were maintained in a 12 h: 12 h
light:dark cycle on standard corn meal-yeast-agar medium at 25°C. Strain
Oregon R (wild type), c42-aeq and c710-aeq fly lines were those described
previously (O'Donnell et al.,
1998; Rosay et al.,
1997
; Terhzaz et al.,
1999
). Briefly, c42-aeq and c710-aeq are lines that express an
aequorin gene under the control of the yeast UASG promoter and
appropriate P{Gal4} insertions, which drive expression in principal and
stellate cells of the main segment, respectively
(Sözen et al., 1997
).
Fluid secretion assays
Fluid secretion assays were performed as described previously
(Dow et al., 1994). Malpighian
tubules from adult flies were dissected under Schneider's medium and isolated
into 10 µl drops of a 1:1 mixture of Schneider's medium:Drosophila
saline. All values are means ± S.E.M.
Measurement of intracellular cyclic nucleotide concentrations
Intracellular cAMP and cGMP concentrations were measured by
radioimmunoassay as described previously
(Davies et al., 1995).
Briefly, 20 tubules per sample were dissected under Schneider's medium and
incubated for 10 min in the presence of 10-4moll-1
isobutyl methyl xanthine (IBMX) for cAMP measurements and
10-4moll-1 zaprinast for cGMP measurements, prior to
stimulation with 10-7moll-1 Drome-DH44 for a
further 10 min. The reaction was interrupted with ice-cold ethanol and samples
homogenised, dried and resuspended in 0.05 moll-1 sodium acetate
buffer before being assayed. Samples were assayed for cyclic nucleotide
content by competitive radioimmunoassay following the manufacturer's
instructions (Amersham Pharmacia, plc).
Measurement of intracellular calcium
The effect of Drome-DH44 on intracellular [Ca2+]
levels in Malpighian tubules was assessed using a P{Gal4}/UAS-Aequorin system
as described previously (MacPherson et
al., 2001; O'Donnell et al.,
1998
; Rosay et al.,
1997
; Terhzaz et al.,
1999
). Briefly, Malpighian tubules were dissected and incubated in
Schneider's medium containing 2.5 µmoll-1 coelenterazine for
3-4h. Real-time luminescence was measured with a BertholdWallac
luminometer, using 0.1 s sampling bins. The luminometer was equipped with dual
injectors for addition of drugs. Analysis and back-integration of the results
were performed with a Mac Perl program as described previously
(Rosay et al., 1997
).
Phosphodiesterase assays
For each sample, 50 Oregon R tubules (20-30 µg of protein) were
dissected into 50 µl of PBS (pH7.4) and homogenised. 2 ml of tritiated cAMP
working stock (0.185 kBq ml-1 in 1 mmoll-1 cAMP, 10
mmoll-1 Tris, 5 mmoll-1 MgCl2, pH7.4) were
added to each sample, on ice. Blank samples were prepared using 50 µl of
PBS and 50 µl of working stock; positive controls were made as for blanks
except that PBS was replaced by pCDNA-bovPDE4 transformed cell lysates, to
provide a positive control for phosphodiesterase-4 (PDE4) activity
(Corbin et al., 2000). The
reaction mixtures were incubated at 30°C for 10 min and then terminated by
boiling for 2 min. Samples were chilled on ice and incubated for 10 min with
25 µl of 1 mg ml-1 Ophiophagus hannah
5'-nucleotidase (Sigma) to allow conversion of guanosine monophosphate
to guanosine. 400 µl of resuspended Dowex 1-Cl resin, (Sigma) (1:2 v/v, in
water) was added to each sample and vortexed briefly three times at 5 min
intervals. The tubes were centrifuged at 12 000g for 2 min and 150
µl of the supernatant removed and added to 2 ml Optiflow scintillant. The
samples were counted for 60 s and PDE activities calculated using a standard
formula (Corbin et al., 2000
).
Final activity was expressed per mg protein, by dividing by the amount of
protein in the sample assayed. Protein concentrations were assayed according
to standard protocols (Lowry, 1951).
Assays for tubule cG-PDE activity were carried out as for cA-PDE assays,
except that tritiated cGMP working stock was used (0.185 kBq ml-1
in 1 mmoll-1 cGMP, 10 mmoll-1 Tris, 5 mmoll-1
MgCl2, pH7.4) and positive controls carried out using pCDNA-bovPDE5
transformed cell lysates (Corbin et al.,
2000).
Localisation of expression of the Dh gene
Antibodies
Approximately 2 mg of Drome-DH44 was conjugated to 5 mg of
thyroglobulin using difluorodinitrobenzene as the conjugating agent, as
described elsewhere (Kean et al.,
2002). A single New Zealand white female received a total of four
injections at 5-6 week intervals. The first injection was performed with
complete Freund's adjuvant and for the subsequent injections incomplete
Freund's adjuvant was used. The rabbit was bled 10 days after each booster.
Three antisera to other insect CRF-like diuretic hormones were used, the N-
and C-terminal specific antisera to Manduca sexta diuretic hormone
(Veenstra and Hagedorn, 1991
)
and a previously unpublished antiserum to Culex salinarius CRF-like
diuretic hormone. The latter was produced by J.A.V. while at the University of
Tucson using the same protocol as that described here.
Immunocytochemistry
The protocol used for peptide immunocytochemistry was the same as that
described recently (Kean et al.,
2002). The third and fourth bleedings of the rabbit used to raise
antiserum to Drome-DH44 gave very similar results and were used at
1:2000 dilution. The antisera to the other insect CRF-like diuretic hormones
were used at a dilution of 1:500. For double-labelling experiments the
anti-CRF-like antibodies were purified from the serum by octanoic acid
precipitation, dialyzed, lyophilized and labelled with
carboxytetramethylrhodamine, as described elsewhere
(Veenstra et al., 1995
). For
double labelings the tissues were first incubated with the leucokinin receptor
antibody (Radford et al.,
2002
) at a dilution of 1:1000, followed by a fluorescein-labeled
Fab fragment of goat-anti-rabbit IgG (Jackson Immunologicals) to visualize the
leucokinin receptor antibody, and then the rhodamine-labeled IgGs to CRF-like
diuretic hormone, following a protocol described elsewhere
(Veenstra et al., 1995
).
In situ hybridisation
Adult and larval brains were dissected, fixed for 25 min in 5%
paraformaldehyde in PBT (PBS with 0.1% Tween 20), washed five times in PBT and
digested for 9 min in 1 ml PBT containing 4 µg ml-1 proteinase
K. The digestion was stopped by washing the tissue twice in 1 ml cold 0.2%
glycine in PBT under agitation, and once in PBT. The tissue was then fixed
again for 25 min in 5% paraformaldehyde in PBT, washed five times in PBT, once
with a 1:1 mixture of PBT and hybridisation solution, and thrice in
hybridisation solution. The hybridisation solution consisted of 50% formamide,
5x SSC, 100 µg ml-1 heparin, 100 µg ml-1
denatured salmon sperm DNA and 0.1% Tween 20 in diethyl pyrocarbonate-treated
distilled water (Sambrook and Russell,
2001). After prehybridisation for 1 h at 55°C,
digoxygenin-labelled RNA probes were added and allowed to hybridise overnight.
Digoxygenin-labelled RNA probes were prepared using a commercial kit from
Roche Molecular Biochemicals following the instructions of the manufacturer.
The probe was reduced by alkaline treatment to a size of approximately 300
nucleotides. After washing the tissue five times for 15 min at 55°C in
hybridisation solution, tissues were washed at room temperature, once in 1:1
mixture of hybridisation solution and PBT, and four times in PBT. An
alkaline-phosphatase-labelled digoxygenin antibody was used to localise the
probes.
Immunocytochemistry for cyclic AMP
Slides were treated with 100 µl of 0.1 mg ml-1 poly-L-lysine
solution for 30 min, washed with water and left to dry. Tubules were dissected
in Schneider's Drosophila medium and stuck onto slides in 1x
PBS solution. A solution with final concentration 10-7 mol
l-1 DromeDH44 and 10-5 mol l-1
IBMX (phosphodiesterase inhibitor) in 1x PBS was added for 7 min (the
same solution without peptide was added to control samples). The tubules were
fixed with 4% paraformaldehyde in 1x PBS for 30 min. They were washed 5
times in 1x PBS before permeabilization with 0.2% (v/v) Triton X-100 in
1x PBS for 30 min. Permeabilization solution was changed every 10 min,
after which the tubules were blocked for 3 h in PAT [PBS containing 0.5% (w/v)
Sigma cold fraction V bovine serum albumin and 0.2% (v/v) Triton X-100]. The
tubules were then hybridised overnight in a humidity chamber with the primary
antibody [rabbit anti-cyclic AMP polyclonal antiserum (US Biological C8450)],
at 1:250 dilution in PAT. Tubules were then blocked with PAT containing 2%
(v/v) normal rabbit serum for 2 h. Samples were incubated for 1 h with the
secondary antibody (fluorescein-conjugated anti-rabbit) diluted 1:250 in PAT
with 2% normal rabbit serum in a humidity chamber. The tubules were then
washed 4 times over a period of 2 h in PAT and twice for 10 min in PBS before
being mounted in VectaShield medium (Vector labs). Slides were examined under
epifluorescence (Zeiss) and photographs were taken with an Axiocam HRc (Zeiss)
using appropriate filters, and the same exposure time for all the samples, at
40x/0.75 magnification.
Stastical analysis
Where appropriate, statistical significance of differences was assessed
with Student's t-test (two-tailed) for unpaired samples, taking the
critical level for P=0.05. Significant differences are marked
graphically with an asterisk.
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Results |
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The genomic context of Dh is shown in Fig. 1B. The gene is in a gene-dense area, and sits between Scm, an RNA polymerase II transcription factor, and CG9492, a dynein ATPase, within the genomic sequence. Dh is punctuated by four introns, one large enough to contain an entire gene (frost, encoding a cold-induced gene).
Effect of Drome-DH44 on fluid secretion
Drome-DH44 stimulates fluid secretion rates in Drosophila
melanogaster Malpighian tubules at concentrations equal to or higher than
10-7 moll-1, typically by up to 1.5-2.5 nl
min-1 (Fig. 3A);
this is comparable with stimulation rates observed for other native diuretic
neuropeptides: DLK (Terhzaz et al.,
1999), capa1, capa2 (Kean et
al., 2002
) in Drosophila melanogaster. The
doseresponse curve (Fig.
3B) shows that the stimulation is significant when using
concentrations greater than 10-8 moll-1.
|
Cyclic nucleotide assays
In other species, CRF-like peptides have been shown to elevate
intracellular cAMP levels. No cGMP increase was observed after stimulation
with Drome-DH44 (Fig.
4B). On the other hand, a clear response in cyclic AMP is shown
after treatment, as expected for a CRF-like peptide
(Fig. 4A). A significant
increase, approximately 150% above the basal rate, is observed for
concentrations over 10-8 moll-1, consistent with the
effect on secretion (Fig. 3).
Immunocytochemistry with an antibody against cAMP showed that the levels were
selectively increased in principal cells only
(Fig. 4C,D), consistent with
models for Drosophila tubule function, in which electrogenic cation
transport is performed by the principal cells, while stellate cells provide
the main route for water and chloride fluxes (Dow and Davies,
1999,
2001
; O'Donnell et al.,
1996
,
1998
).
|
Phosphodiesterase assays
For a second messenger to be plausibly implicated in a signalling pathway,
there must be a mechanism for terminating the signal, as well as for
generating it. In the case of cAMP, a phosphodiesterase is likely to be
responsible. Tubules were stimulated with Drome-DH44, and cAMP- and
cGMP-directed phosphodiesterase activity separately measured.
Drome-DH44 treatment doubles cAMP-PDE activity and halves cGMP-PDE activity, respectively, though the latter change is not significant (Fig. 5). Drome-DH44 application thus directly stimulates the activity of the enzyme that breaks down its second messenger, and thus provides a feedback mechanism for limitation of the signal. This is the first time that this breakdown pathway has been shown to be modulated by an insect diuretic hormone.
|
Intracellular calcium
It is conceivable that Drome-DH44 could act indirectly to raise
cAMP levels via intracellular calcium, or that it might act through
calcium in other cell types. Accordingly, the transgenic aequorin system
(Rosay et al., 1997) was used
to seek a calcium correlate of hormone stimulation. The neuropeptides
CAP2b (Davies et al.,
1995
) and DLK (Terhzaz et al.,
1999
) were used as positive controls to generate increases in
calcium levels in principal and stellate cells respectively. As described
previously, CAP2b and DLK produced elevated intracellular
[Ca2+] in principal and stellate cells, respectively, while
Drome-DH44 had no effect (Fig.
6).
|
Localisation of expression of the CRF gene
Both immunocytochemistry (Fig.
7a,b) and in situ hybridisation
(Fig. 7c) techniques mark a
bilateral triplet of cells in the pars intercerebralis. The two antisera
against the Manduca sexta peptide were not immunoreactive in
Drosophila, while the two antibodies against the Dipteran hormones
recognized the same cells, with the Drosophila antibody being clearly
more immunoreactive. In both adults and larvae, CRF-like diuretic hormone
immunoreactivity was found exclusively in three bilateral pairs of cells in
the pars intercerebralis, the axons which could be followed into the
retrocerebral complex. These cells are very similar to the B-cells described
from other flies (Panov,
1976). The morphology of these cells is thus typical of classical
insect neuroendocrine cells. Using in situ hybridization the same
number of cells was found in a closely similar location
(Fig. 7c). No CRF-like
immunoreactive cells were found in the abdominal ganglia, either by
immunocytology or by in situ hybridisation. Interestingly, these
cells also express Leucokinin receptor immunoreactivity, both in the cell
bodies and the neurohaemal axons and axon terminals in the retrocerebral
complex (Fig. 8).
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Discussion |
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Expression of the CRF-like peptide appears to be rather limited, being
confined to only a bilateral triplet of cells in the pars intercerebralis. The
number of CRF-like peptide immunoreactive cells reported here is smaller than
reported previously in the housefly Musca domestica
(Iaboni et al., 1998) using an
antiserum to locust CRF-like DH. Although we cannot exclude the possibility
that the Locusta antiserum is more specific than the one we raised to
the Drosophila hormone, we feel that this is unlikely, as both in
situ hybridisation and immunocytochemistry techniques identify the same
cells. Unlike in the moth Manduca sexta
(Chen et al., 1994
), the locust
Locusta migratoria (Thompson et
al., 1995
) or the bug Rhodnius prolixus
(Te Brugge et al., 2001
),
there are no abdominal neuroendocrine cells in Drosophila producing
CRF-like diuretic hormones. This may be typical of Diptera, as in neither the
mosquito Aedes aegypti (J.A.V., unpublished data) nor the housefly
Musca domestica (Iaboni et al.,
1998
) have such abdominal neuroendocrine cells been found. In
other species, some of these abdominal neuroendocrine cells express both
leucokinins and CRF-like diuretic hormones, and it has been shown for the
locust that these hormones have synergistic effects
(Thompson et al., 1995
).
Although Drosophila has no neuroendocrine cells that produce both
leucokinin and the CRF-like peptide, our observations suggesting expression of
the leucokinin receptor in the CRF-like-peptide-producing neuroendocrine cells
shows an alternative way of interaction between the two peptides; it is
tempting to speculate that release of leucokinin into the haemolymph could
induce and/or facilitate release of the CRF-like peptide.
In Manduca sexta the number of CRF-like-diuretic-hormone-producing
neuroendocrine cells in the pars intercerebralis increases dramatically during
metamorphosis. It had been suggested that this increase might be related to
the very significant loss of water during metamorphosis when a watery
caterpillar is transformed into a much lighter, flying moth
(Veenstra and Hagedorn, 1991).
The transformation of a maggot to a fly is similarly accompanied by a
significant loss of water, but the number of the
CRF-like-diuretic-hormone-producing neuroendocrine cells in
Drosophila does not change during metamorphosis. It will be
interesting to see whether this reflects a physiological functional difference
of these hormones. Overall, it is not yet clear what role CRF-like peptides
play in insect organismal physiology, although in D. melanogaster at
least, a reverse genetic approach is conceivable. It is also interesting that
the cDNA clone that we sequenced came from a testes library; although we have
not addressed here the possibility of expression outside the CNS, it would be
interesting to investigate whether there was any biological significance to
this finding.
It is interesting to note that Drome-DH44 does not act merely to raise cAMP levels, but also to stimulate cAMP-phosphodiesterase twofold. This change would have the effect of terminating the Drome-DH44 signal even faster than would be expected if PDE levels remained constant, and would imply that Drome-DH44 is primarily a short-term modulator of fluid secretion. Technically, we cannot separate the PDE responses of principal and stellate cells in these assays, although as the increase in cAMP concentration is confined to principal cells, it would be reasonable to suppose that the PDE response occurred at least in them. Although statistical significance was not achieved in these experiments (Fig. 5), there was an interesting downward trend in the cGMP-PDE assay, which would have the effect of potentiating stimulation by the capa peptides.
The similarity of action between Drome-DH44 (this work) and
calcitonin-like peptide (Coast et al.,
2001) might at first seem puzzling. However, there is no guarantee
that these peptides act exclusively on Malpighian tubules; it is quite
possible that they have distinct actions elsewhere in the fly. The Malpighian
tubule, as a prime site for homeostatic regulation, is charged with
integrating inputs from a whole range of peptides, many of which may prove not
to be primarily diuretic; and so it is not surprising to find multiple
peptides with similar modes of action on this tissue
(Dow and Davies, 2001
;
O'Donnell and Spring, 2000
;
Skaer et al., 2002
).
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Acknowledgments |
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