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First published online 14 November 2007
doi: 10.1242/dev.009902
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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.
* Author for correspondence (e-mail: ross.hodgetts{at}ualberta.ca)
Accepted 17 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila melanogaster, Tyrosine hydroxylase, Tanning, Eclosion
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Ecdysone is
released before the larval moults, pupation and eclosion, and initiates a
hormonal cascade that ultimately leads to shedding of the cuticle, a process
known as ecdysis.
The precise timing of ecdysis occurs in response to the neuronal release of
peptide hormones, which act on ecdysone-primed tissues
(Fig. 1). In Manduca
sexta, if ecdysone titres do not decline, ecdysis is blocked
(Curtis et al., 1984),
suggesting that a decline in steroid hormone titre signifies a readiness to
shed the cuticle (Truman,
1996). The first hormone to be released in response to decreasing
ecdysone levels is ecdysis-triggering hormone (ETH)
(Park et al., 2002). This
triggers an increase in eclosion hormone (EH) levels
(Clark et al., 2004;
Horodyski et al., 1993) and,
in fact, EH acts in a positive-feedback loop to cause further release of ETH
(Ewer et al., 1997;
Kingan and Adams, 2000;
Kingan et al., 2001). Together
ETH and EH are responsible for the preparatory behaviour called pre-ecdysis
(Baker et al., 1999). EH causes
the release of crustacean cardioactive peptide (CCAP)
(Ewer and Truman, 1996;
Gammie and Truman, 1999). CCAP
is thought to control the ecdysis motor response by shutting off pre-ecdysis
and turning on the ecdysis motor program
(Gammie and Truman, 1997;
Horodyski et al., 1993;
Park et al., 2003). In fact,
the timing and organisation of ecdysis are severely disrupted in organisms
carrying CCAP ablation knockouts (Park et
al., 2003). CCAP causes activation of protein kinase A (PKA) to
initiate ecdysis behaviour (Luan et al.,
2006). The post-eclosion hormone, bursicon, a heterodimeric
neuropeptide whose 
and β subunits are encoded by the
bursicon (burs) and CG15284 genes respectively, colocalises
with most CCAP neurons (Dewey et al.,
2004; Luan et al.,
2006; Luo et al.,
2005; Mendive et al.,
2005). Bursicon is released into the haemolymph following eclosion
and acts through its receptor, encoded by rickets (rk)
(Baker and Truman, 2002;
Dewey et al., 2004;
Luo et al., 2005;
Mendive et al., 2005). RK is a
G-coupled glycoprotein hormone receptor and its activation causes an increase
in cAMP (Kimura et al., 2004).
Wing expansion, and probably cuticular expansion and tanning, occur in
response to cAMP activation of PKA.
Wing and cuticle expansion are complete within 20 minutes of eclosion,
whereas tanning of the cuticle takes 3 hours. Neck-ligation of flies at
eclosion prevents tanning, whereas flies neck-ligated 30 minutes after
eclosion tan normally (Fraenkel and Hsiao,
1962). Bursicon is released within 20 minutes of eclosion, and
this process is disrupted in flies neck-ligated at eclosion
(Fraenkel et al., 1966).
Tanning in such flies is rescued by injection of 8-Br-cAMP or haemolymph
extracted from flies 20 minutes after eclosion
(Baker and Truman, 2002;
Fraenkel et al., 1966;
Luo et al., 2005;
Mendive et al., 2005). We are
interested in how the precise timing of the developmental events following
eclosion is controlled by the ecdysis neuropeptide cascade.
Flies without EH or CCAP (EH-KO and CCAP-KO, respectively) can be generated
by ablating cells that produce these neuropeptides. This is achieved by
triggering cell-specific apoptosis using the Gal4-UAS system to express the
gene, reaper, under the control of an EH or CCAP driver. EH-KO and
CCAP-KO flies have a similar adult mutant phenotype to burs and
rk mutants (Baker and Truman,
2002; Dewey et al.,
2004; McNabb et al.,
1997; Park et al.,
2003). Such adults fail to expand their wings and exhibit a delay
in tanning of their cuticle. This phenotype is mirrored in organisms that
express a dominant negative form of the ecdysone receptor under the control of
an EH driver, demonstrating the importance of ecdysone as a trigger for this
hormonal cascade (Cherbas et al.,
2003). Unlike burs and rk mutants, EH-KO and
CCAP-KO flies also have severe defects at the larval and pupal ecdyses that
result in reduced viability (Baker and
Truman, 2002; Gammie and
Truman, 1997; McNabb et al.,
1997; Park et al.,
2003).
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). The first step
in the synthesis of DA is the rate limiting conversion of tyrosine to
dihydroxyphenylalanine (dopa) by tyrosine hydroxylase (TH), which is encoded
by the pale (ple) locus
(Fig. 2A)
(Levitt et al., 1965;
Nagatsu et al., 1964;
Neckameyer and White, 1993).
The conversion of dopa to DA is catalysed by dopa decarboxylase (DDC)
(Livingstone and Tempel,
1983), encoded by Ddc
(Hirsh and Davidson, 1981).
Similar to its role in vertebrates, dopamine plays a critical role in the
Drosophila CNS; however, only 6% of DDC activity is present in the
brain of third instar larvae (Scholnick et
al., 1983). The remaining activity is present in the epidermis,
demonstrating the importance of epidermal DA production. In addition to the
requirement for DA following eclosion, DA is needed for tanning of the
puparium at pupariation and for pigmentation of the pharate adult bristles and
epidermis.
Both Ddc and ple mRNAs are alternatively spliced to
produce neural- and epidermal-specific transcripts in Drosophila
(Fig. 2B)
(Birman et al., 1994;
Morgan et al., 1986). The
Ddc epidermal transcript lacks the neural-specific second exon,
whereas the ple epidermal transcript contains the third and fourth
exons, which are not included in the mRNA for the neural form of the enzyme.
Both Ddc and ple null mutants are homozygous lethal, and
rare Ddc mutant `escaper' flies exhibit defects in cuticular tanning
and die within 24 hours of eclosion
(Neckameyer and White, 1993;
Wright et al., 1976). Using
transgenes to supply the respective epidermal, but not neural, forms of the
enzymes to Ddc mutants (Morgan et
al., 1986) or to ple mutants
(Friggi-Grelin et al., 2003)
rescues lethality, illustrating the importance of epidermal DA production. In
vertebrates, TH activity is regulated at a variety of levels, including
transcriptional regulation, alternative splicing, RNA stability, DA feedback
inhibition and protein kinase activation
(Dunkley et al., 2004;
Hufton et al., 1995;
Kumer and Vrana, 1996;
Zigmond et al., 1989). Both
transcriptional regulation and alternative splicing regulate TH activity in
Drosophila (Birman et al.,
1994). The additional exons included in the epidermal ple
transcript encode a very acidic segment of 71 amino acids within the
regulatory domain of this enzyme that reduces the sensitivity of TH to DA
inhibition in vitro (Vie et al.,
1999). Furthermore, Drosophila TH is activated in vitro
by phosphorylation at Ser32, a site homologous to vertebrate Ser40
(Vie et al., 1999), the major
site of PKA phosphorylation and activation
(Dunkley et al., 2004;
Kumer and Vrana, 1996).
Phosphorylation of Ser32 has been proposed to increase the activity of the
epidermal isoform of TH by reducing the ability of the protein to bind to
inhibitory DA, and by increasing the rate of binding of the essential cofactor
tetrahydrobiopterin (Vie et al.,
1999). Interestingly, both CCAP and bursicon activate PKA by
elevating cAMP levels (Baker and Truman,
2002; Luan et al.,
2006), suggesting that either of these neuropeptides may be
involved in activation of TH to regulate the onset of tanning.
In this paper, we set out to determine how the ecdysis neuropeptide hormone cascade regulates the precise timing of developmental events following eclosion. To achieve this, we focused on tanning because its biochemical basis is known, and it is a readily scored marker of post-eclosion development. We find that the onset of tanning does not depend on the regulation of Ddc. Rather, it is the regulation of TH protein accumulation and activity, elicited by the ecdysis neuropeptide hormone cascade that precisely controls tanning. Specifically, post-eclosion accumulation of TH from the ple transcripts that are already present in pharate adults is regulated by CCAP, whereas TH is activated by PKA as a result of bursicon signalling through RK. Regulation of TH transcription plays no part in the switch, effected by the ecdysis hormone cascade, that triggers post-eclosion tanning.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
mutations in rk1 and rk4 flies
introduce a premature stop codon in the transmembrane domain of this receptor
(Baker and Truman, 2002). To
obtain EH-KO flies, we crossed w UAS-rpr females to EH-Gal4
males. CCAP-KO flies have severe defects at both the larval and pupal ecdyses,
rarely yielding viable adult flies. To obtain a sufficient number of viable
CCAP-KO adult flies for analysis, we crossed yw;CCAP-Gal4 females to
w;UAS-rpr males. In each experiment, yw flies were used as a
wild-type control.
Neck ligations and CCAP and 8-Br-cAMP injections
Pupae that were within 6 hours of eclosion, according to previously defined
cues (Kimura and Truman,
1990), were mounted on double-sided tape on a slide, and the
operculum was removed. As each fly emerged from the pupal case, it was
immobilised on an ice-cooled Petri dish and was neck-ligated using a strand of
hair. When injected, approximately 300 nl of 0.1 mM CCAP (Bachem) or 0.1 M
8-Br-cAMP was delivered into the thorax through a fine glass capillary needle.
Flies were kept in a humidified chamber until 2.5 hours after eclosion and
frozen at -80°C for subsequent analysis.
RNA manipulations and RT-PCR
RNA was extracted from pools of organisms using TRIzol, treated with
amplification grade DNaseI, and reverse transcribed using Superscript II
according to the manufacturer's instructions (Invitrogen).
Table 1 shows the sequence of
all primers used in these experiments. Synthesis of the Ddc- and
ple-specific cDNAs was initiated using the DDC1 and PLE1 primers,
respectively, on 150 ng isolated RNA. First-strand reactions were co-reverse
transcribed with RPL32-1, a primer specific for the ribosomal gene
RpL32, which serves as a loading control. A 3 µl aliquot of the
resulting cDNA mixture was combined either with ple-specific primers
(PLE-F and PLE-R) and amplified for five cycles or with Ddc-specific
primers (DDC-F and DDC-R) and amplified for three cycles. The program was then
stopped, RpL32 gene-specific primers were added, and the program was
allowed to continue for 23 more cycles. After an initial denaturing step of 2
minutes, the final PCR conditions for ple consisted of 28 cycles of
95°C for 1 minute, 60°C for 1 minute and 73°C for 1 minute. The
conditions were the same for Ddc, except the reaction ran for a total
of 26 cycles.
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).
Western blots
Organisms were macerated in running buffer (0.125 M Tris-HCl pH 6.8, 12.5%
glycerol, 2.5% SDS, 1.25% β-mercaptoethanol and 0.025% Bromophenol Blue),
boiled for 10 minutes, and 0.5 (TH detection), 1 (phospho-Ser32 TH detection),
or 0.25 (actin detection) organism equivalents were loaded and separated on 8%
denaturing acrylamide gels. Proteins were transferred to a nitrocellulose
membrane (Amersham Biosciences Hybond-ECL). Membranes were blocked in
Tris-buffered saline with 0.1% Tween (TBS-T) and 1% skimmed milk powder.
Membranes were then incubated with either rabbit anti-TH antibody (1:3000
dilution, 3 hours, room temperature), rabbit anti-phospho-TH (Ser40) (1:2000
dilution, overnight, 4°C), or mouse anti-actin (1:10,000 dilution, 1 hour,
room temperature). The rabbit anti-TH antibody was a gift from Wendi
Neckameyer (St Louis University, St Louis, MO), the rabbit anti-phospho-TH
(Ser40) antibody was purchased from Zymed Laboratories, and the mouse
anti-actin (JLA-20) antibody developed by Jim Jung-Ching Lin was obtained from
the Developmental Studies Hybridoma Bank developed under the auspices of the
NICHD and maintained by The University of Iowa, Department of Biological
Sciences. The membranes were then washed with TBS-T and incubated with either
a goat anti-rabbit IgG (H + L)-HRP Conjugate (Bio-Rad) or an ECL anti-rabbit
IgG HRP-linked species-specific whole antibody (from donkey) (Amersham
Biosciences), or a goat anti-mouse IgG (H+L)-HRP conjugate (Invitrogen)
(1:1000 dilution, 1 hour, room temperature). Membranes were washed with TBS-T
and developed using Supersignal West Pico Chemiluminescent Substrate (Pierce)
according to the manufacturer's instructions.
Tyrosine hydroxylase activity assays
Except where indicated, all reagents were obtained from Sigma. Pools of
organisms were macerated in 1x phosphate buffered saline containing
1x Complete, Mini, EDTA-free Protease Inhibitor Cocktail (Roche) (25
µl per organism). The extract was cleared by centrifugation, and a 25 µl
aliquot of the supernatant was added to the reaction mixture (50 mM PIPES, pH
8.0, 25 µM L-tyrosine, 5 mM DTT, 10 µM
Fe(NH4)2(SO4)2, 40 µg/ml
catalase and 1 µCi L-[3,5-3H]tyrosine (Amersham), in a total
volume of 49 µl. The reaction mixture was equilibrated to 27°C for 3
minutes, and then 1 µl of 100 µM tetrahydrobiopterin or H2O
(for blanks) was added to start the reaction. The reaction was incubated at
27°C for 15 minutes. Residual L-[3,5-3H]tyrosine was removed
with 500 µl of 7.5% activated charcoal in 1.M HCl. Finally, 100 µl
supernatant was combined with 10 ml scintillation fluid and counted.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TH protein and activity levels are high in pharate adults, drop before eclosion and rise thereafter
Transcriptional control of ple and Ddc cannot regulate
the precise timing of tanning following eclosion because transcripts of both
genes accumulate before eclosion. DDC has never been shown to be regulated at
a translational or post-translational level, and high levels of the active
enzyme are present at eclosion (Kraminsky
et al., 1980). However, TH is known to be under translational and
activational control in mammalian systems
(Dunkley et al., 2004;
Kumer and Vrana, 1996). For
this reason, we looked at TH protein accumulation from pupariation until after
eclosion (Fig. 4A). We staged
flies as WPP and sampled the collection every 12 hours. We also separated the
108-hour APF organisms into pharate adult and eclosed adults, to examine the
differences in these distinct samples. The TH antibody was not epidermal
specific; however, identification of the epidermal isoform of TH was easily
facilitated as it migrated much more slowly than the neural isoform. To
simplify, only the epidermal isoform of TH is shown. The high level of TH in
WPP decreased to an undetectable amount by 12 hours APF, and rose again 84
hours APF (Fig. 4A). Protein
levels dropped before eclosion but rose sharply thereafter, remaining high for
about 12 hours.
We also examined the activity profile of TH in yw organisms (Fig. 4B). We found that TH activity (expressed as a percentage of WPP activity) closely parallels the protein profile of TH. Enzyme activity could still be detected 132 hours APF although protein levels are below the limits of detection.
TH protein and activity levels are reduced after eclosion in CCAP-KO flies
EH-KO, CCAP-KO, burs and rk flies show defects in tanning
after eclosion. To determine whether the ecdysis neuropeptide-signalling
pathway (Fig. 1) plays a role
in the regulation of TH, we examined the transcriptional, translational and
activity profiles of TH in these flies
(Fig. 4C,D). Organisms were
staged as WPP and sampled every 24 hours until 144 hours APF. Again, the
108-hour time point was separated into pharate adults and eclosed adults. No
CCAP-KO flies eclose by 108 hours, so this time point is absent in
Fig. 4C. In the original
description of EH-KO flies, it was noted that the proportion of flies that
failed to expand their wings varied depending on the UAS-rpr line
used (McNabb et al., 1997).
Under our conditions, 58% (n=746) of the flies failed to expand their
wings. For the experiments shown, only flies that failed to expand their wings
were included. For most of our analysis, we used bursZ1091
and rk4 homozygous flies, although we did some
complementary heteroallelic studies using the bursZ5569
and rk1 alleles. Although rk1 and
rk4 mutations are null mutants because of a truncation of
the receptor in the critical transmembrane domain
(Baker and Truman, 2002), both
bursZ1091 and bursZ5569 mutations are
probably hypomorphic owing to single amino acid conversions that fail to
completely eliminate protein function
(Dewey et al., 2004).
ple transcript levels in the EH-KO, CCAP-KO,
bursZ1091 and rk4 flies mimicked those
exhibited by the yw flies (data not shown). Similar to control
organisms (compare Fig. 4C with
4A), the mutants and ablation knockout flies had high levels of TH
protein in WPP, which decreased to undetectable levels in the next 24-48
hours. Protein accumulated again at 96 hours but dropped in all four lines
before eclosion (see Fig. 4C,
108 hour pharate adults). Protein appeared following eclosion in EH-KO,
bursZ1091 and rk4 flies and detectable
levels persisted for longer than in the control flies. Importantly, TH protein
failed to accumulate in CCAP-KO flies following eclosion.
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TH protein is translated within an hour of eclosion and transiently phosphorylated and activated thereafter
Two lines of evidence suggest that TH protein accumulation following
eclosion is regulated: (1) TH protein levels dropped near eclosion in control
flies (Fig. 4A) and rose again
following eclosion, whereas ple transcript levels did not fluctuate
during this period (Fig. 3A);
(2) TH protein was absent after eclosion in CCAP-KO flies
(Fig. 4C), although
ple was transcribed normally (data not shown), indicating that CCAP
may be required for TH accumulation. To determine precisely when TH protein
first appears, western blotting was carried out on organisms collected at
eclosion and every 30 minutes thereafter (top line,
Fig. 5A). TH was absent in
flies at eclosion, and was first detectable 1 hour later. Protein levels
reached a maximum 2 hours after eclosion, and remained elevated until at least
12 hours after eclosion (Fig.
4A). We attribute the presence of TH in the 108-hour eclosed flies
sample in Fig. 4A to organisms
older than 1 hour.
The major site of activational control of Drosophila TH is the PKA
phosphorylation site at Ser32 (Vie et al.,
1999), and both CCAP and RK signalling lead to activation of PKA
(Kimura et al., 2004;
Luan et al., 2006). The Ser32
residue is homologous to the rat TH Ser40, so we were able to use a monoclonal
antibody specific to rat phospho-Ser40-TH to determine the phosphorylation
state of Ser32 (bottom line, Fig.
5A). Phosphorylated TH was detected only during a very short
interval 1.5-3 hours after eclosion.
TH activity was low in flies at eclosion, and began to increase within 1.5 hours, peaking at 2.5 hours. This peak of activity corresponds to the time of phosphorylation and tanning.
TH phosphorylation and activity are eliminated in flies neck-ligated at eclosion
Flies neck-ligated at eclosion fail to tan, whereas those neck-ligated 30
minutes after eclosion tan normally
(Fraenkel and Hsiao, 1962).
The lack of tanning is due to the prevention of bursicon release
(Baker and Truman, 2002;
Fraenkel et al., 1966;
Luo et al., 2005;
Mendive et al., 2005) and can
be rescued by injection of 8-Br-cAMP (Baker
and Truman, 2002), a membrane permeable analog of cAMP. We sought
to determine whether the rescue by 8-Br-cAMP injection was a result of the
activation of the epidermal form of TH. Flies were neck-ligated at eclosion or
30 minutes following eclosion, and those treated with 8-Br-cAMP were injected
immediately. All flies were aged 2.5 hours before TH western blots and
activity measurements were carried out
(Fig. 6A). We found that no TH
protein was present at eclosion in control flies, but protein was detectable
2.5 hours later, in agreement with Fig.
5A. In the flies ligated at eclosion, less protein was apparent
than in control flies, and phosphorylation of Ser32 does not occur
(Fig. 6A). When flies are
neck-ligated 30 minutes after eclosion, they accumulate and phosphorylate TH.
TH phosphorylation is restored in flies neck-ligated at eclosion by injection
of 8-Br-cAMP.
Neck-ligation of flies at eclosion reduces TH activity to 6% of the level in control flies (Fig. 6B). Flies neck-ligated 30 minutes after eclosion had double the activity of those neck-ligated at eclosion, and flies injected with 8-Br-cAMP had nearly sixfold higher activity than the flies neck-ligated at eclosion.
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) (data
not shown). We found that TH is absent in EH-KO and CCAP-KO flies, and
consequently no phosphorylated protein was detected
(Fig. 7A, lanes 2 and 4). This
absence of TH in EH-KO flies appears to contradict the accumulation of TH
evident in the 108-hour time point shown in
Fig. 4C. We assume the 108-hour
sample included organisms older than 2.5 hours. TH levels and phosphorylation
were restored in both EH-KO and CCAP-KO flies injected with 8-Br-cAMP
(Fig. 7A, lanes 3 and 5), and
injection of CCAP into CCAP-KO flies caused accumulation, but not
phosphorylation, of TH (see Fig. S1 in the supplementary material). Both
bursZ1091 and rk4 flies have normal
levels of TH protein (Fig. 7A,
lanes 6 and 8), but Ser32 phosphorylation was reduced in
bursZ0191 and absent in rk4 flies. We
ruled out second-site mutation effects in the homozygous lines by showing that
TH protein levels were normal in
bursZ1091/bursZ5569 and
rk1/rk4 heterozygotes, although Ser32
phosphorylation was absent (see Fig. S2 in the supplementary material). The
phosphorylation defect in bursZ1091 and
rk4 mutants was rescued by 8-Br-cAMP injection
(Fig. 7A, lanes 7 and 9). Loss of TH protein in EH-KO and CCAP-KO flies corresponded with a loss of activity (Fig. 7B). Injection of 8-Br-cAMP restored TH activity. Activity is only slightly reduced in bursZ1091 mutants and unchanged by injection of 8-Br-cAMP. The reduced activity in rk4 flies is restored by 8-Br-cAMP injection.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Tanning at pupariation is probably controlled by the release of tyrosine from
tyr-P. No appreciable accumulation of tyr-P occurs before eclosion
(Bel et al., 1992;
Lunan and Mitchell, 1969),
suggesting that post-eclosion tanning is switched on by a different
mechanism.
We established a role for the ecdysis neuropeptide cascade in post-eclosion
tanning by examining the regulation of two genes, ple and
Ddc, which encode two enzymes with critical roles in tanning. We used
semi-quantitative RT-PCR to examine the profile of transcription after
puparium formation. Levels of both transcripts are high in WPP
(Fig. 3), but they drop and
then rise again before eclosion. Ddc levels begin to increase 60
hours APF, reach their peak 84 hours APF, and decline thereafter. DDC enzyme
activity is required before eclosion for pigmentation of the pharate adult
bristles and epidermis and after eclosion for tanning of the adult cuticle,
and reaches a peak at eclosion (Kraminsky
et al., 1980). This indicates that Ddc is transcribed and
translated before eclosion to ensure enzyme activity is present when substrate
becomes available. We investigated whether the control of substrate
availability, and therefore the control of tanning, was effected by the
transcriptional, translational, or post-translational regulation of TH.
Levels of ple transcripts are high during the 24 hour period spanning eclosion (Fig. 3A). The early appearance of ple transcripts is not surprising, because pigmentation of the pharate adult bristles and epidermis occurs between 84 and 96 hours APF. Both ple (and Ddc) transcription are normal in EH-KO, CCAP-KO, bursZ1091 and rk4 flies (data not shown). The accumulation of ple transcripts before eclosion, the maintenance of high levels of TH transcription until 12 hours after eclosion and the fact that neuropeptide mutant and ablation knockout flies exhibit normal ple transcription, led us to conclude that the precise onset of tanning following eclosion is not due to regulation of ple transcription.
TH protein and activity levels are high before eclosion when pigmentation
of the pharate adult bristles and epidermis occurs
(Fig. 4). Levels fall rapidly
just before eclosion and rise thereafter. During this entire time,
ple transcripts are present, suggesting that protein levels are being
regulated. The drop in TH protein levels may occur through repression of
translation from ple transcripts and/or increased turnover of the
protein. The complete failure of CCAP-KO flies to accumulate TH protein
following eclosion (Fig. 4C),
although they transcribe ple normally (data not shown), indicates a
role for CCAP in this process. This could occur at the level of translation;
alternatively, CCAP signalling may alter TH protein stabilisation. Since PKA
signalling has been shown to regulate proteins involved in translational
control (Denman et al., 2004),
it is more likely that CCAP signalling activates PKA to cause translation, not
stabilisation, of TH following eclosion.
EH-KO, bursZ1091 and rk4 flies all appear to have relatively normal TH protein and activity profiles (compare Fig. 4C with 4A and 4D with 4B). Although all three exhibit a considerable range of activity in WPP, the pupal cases of these organisms tan normally. Despite the initial delay in TH accumulation in EH-KO flies following eclosion (Fig. 7A), these flies, and bursZ1091 and rk4 mutants, maintain high levels of TH until 144 hours APF, a time when TH is undetectable in control flies (Fig. 4A). This persistence of TH indicates a delay in the execution of the neuropeptide hormone cascade. Interestingly, rk4 flies also show a delay in degradation of TH following pupariation (Fig. 4C). Perhaps there is a requirement for RK signalling to trigger TH degradation following tanning of the puparium.
Neck-ligation of flies at eclosion prevents tanning, whereas flies ligated
30 minutes after eclosion tan normally
(Fraenkel and Hsiao, 1962).
Furthermore, tanning of flies neck-ligated at eclosion is rescued by injection
of 8-Br-cAMP (Baker and Truman,
2002). TH protein begins to accumulate 1 hour after eclosion in
control flies (Fig. 5A).
Phosphorylation of the protein by PKA at Ser32 leads to enzyme activity rising
between 1.5 and 3 hours after eclosion
(Fig. 5). We conclude that the
translational and activational state of TH is responsible for controlling
tanning following eclosion. We found that TH protein accumulates, but is not
phosphorylated (Fig. 6A) in
flies neck-ligated at eclosion resulting in reduced TH activity
(Fig. 6B). Interrupting
neuropeptide signalling after eclosion reveals that the element that controls
TH translation is released before eclosion. The loss of TH accumulation in
CCAP-KO flies (Fig. 4C and Fig.
8A) and the restoration of TH accumulation upon injection of CCAP (see Fig. S1
in the supplementary material) suggests that CCAP is responsible for inducing
TH translation.
Flies neck-ligated 30 minutes after eclosion, translate and phosphorylate TH normally (Fig. 6A). By allowing neuropeptide signalling after eclosion, we have demonstrated that a factor is released within 30 minutes of eclosion that causes phosphorylation and therefore activation of TH. The reduced phosphorylation of Ser32 in bursZ1091 flies, and complete loss of phosphorylation in rk4, bursZ1091/bursZ5569 and rk1/rk4 flies (Fig. 7A and see Fig. S2 in the supplementary material) suggests that bursicon signalling through RK controls this process. Activity levels of TH are significantly reduced in flies neck-ligated at eclosion compared with control flies (Fig. 6B). Flies ligated 30 minutes after eclosion show twofold higher levels than flies neck-ligated at eclosion and this difference probably accounts for the presence or absence of tanning. This suggests that a critical threshold of TH activity exists that is surpassed in the flies ligated at 30 minutes. Thus, although the activity present in these flies is significantly less than that in control flies, the organisms have sufficient TH activity to tan, whereas flies ligated at eclosion do not attain the threshold of activity required for tanning. Injection of 8-Br-cAMP into flies neck-ligated at eclosion rescues tanning by restoring phosphorylation and therefore activation of TH (Fig. 6). Although injection of 8-Br-cAMP does not restore TH activity to control levels, it increases activity nearly sixfold (Fig. 6B), achieving the threshold of activity required for tanning following eclosion.
These results, taken together, suggest that at least two factors control
the precise timing of tanning after eclosion. One, released before eclosion,
causes translation of TH; the other, released after eclosion, causes
phosphorylation and activation of TH. Both EH and CCAP are released before
eclosion to control pre-ecdysis and ecdysis, respectively
(Clark et al., 2004;
Ewer and Truman, 1996;
Gammie and Truman, 1999;
Horodyski et al., 1993). EH-KO
and CCAP-KO flies both exhibit extreme post-eclosion tanning defects. EH-KO
flies take more than 9 hours to tan and CCAP-KO flies fail to tan (M.M.D.,
unpublished results). TH protein is undetectable in EH-KO flies immediately
following eclosion (Fig. 7A),
but these flies do eventually accumulate TH
(Fig. 4C) and tan. The complete
failure of CCAP-KO flies to tan, combined with the fact that CCAP-KO flies
fail to accumulate TH (Fig. 5A
and Fig. 8A) from the ple transcripts that are present at eclosion
(data not shown), suggest that CCAP is responsible for inducing TH
translation. The initial failure of EH-KO flies to accumulate TH
(Fig. 7A) is probably caused by
a failure to trigger the rapid release of CCAP
(Fig. 1). Presumably, enough
CCAP is eventually released in these flies to effect the translation of TH and
eventually tanning, because the EH genetic ablation is leaky. Consistent with
this prediction, EH-KO flies that expand their wings accumulate TH normally
(data not shown), suggesting that CCAP is released normally in these flies. TH
translation is restored in CCAP-KO flies injected with CCAP (see Fig. S1 in
the supplementary material) and rescue of TH accumulation and phosphorylation
occurs when EH-KO and CCAP-KO flies are injected with 8-Br-cAMP
(Fig. 7). Rescue of both
defects probably occurs because injection of 8-Br-cAMP activates PKA in CCAP
target cells, thus circumventing the need for CCAP release
(Fig. 1), and also activates
PKA in TH-expressing cells, leading to phosphorylation and activation of
TH.
Our data suggest that the post-eclosion factor causing the phosphorylation
of Ser32 is the heterodimeric hormone bursicon. It is responsible for tanning
and wing expansion (Dewey et al.,
2004; Fraenkel et al.,
1966; Luo et al.,
2005; Mendive et al.,
2005) and acts through its receptor RK
(Baker and Truman, 2002).
Consistent with the role of bursicon in the phosphorylation of TH,
rk4 flies fail to phosphorylate TH and have reduced
activity (Fig. 7). These flies
show a delay in tanning, taking up to 9 hours to tan
(Baker and Truman, 2002).
Injection of 8-Br-cAMP rescues tanning by restoring phosphorylation and
therefore activation of TH (Fig.
7).
Two mutants in the subunit of bursicon have been identified, of
which one - bursZ5569 - shows a delay in tanning in 40% of
the progeny, whereas a delay is present in 82% of
bursZ1091/bursZ5569 flies
(Dewey et al., 2004). The
bursZ1091 mutant does not show a delay in tanning,
although phosphorylation of TH is reduced in these flies
(Fig. 7A). Phosphorylation of
Ser32 is undetectable in
bursZ1091/bursZ5569 flies (see Fig. S2
in the supplementary material), probably causing the more severe tanning
defect seen in these flies. The reduced phosphorylation of TH in
bursZ1091 flies corresponds to a minor loss of TH activity
(Fig. 7B). Thus, it seems that
the threshold TH activity required for proper tanning is achieved in
bursZ1091 flies, although they do not have wild-type
levels of TH phosphorylation or activity. Normal tanning in these flies cannot
be attributed to residual activity of the β subunit of bursicon, CG15284,
recently proposed to be encoded by pu (S. McNabb and J. Truman,
personal communication), because neither subunit independently confers
bursicon activity (Luo et al.,
2005; Mendive et al.,
2005). The bursZ1091 allele is probably a
hypomorph, and creation of a null allele would be useful. Additional studies
on the activational state of TH in pu or burs null mutants
will help to elucidate why tanning is not delayed in
bursZ1091 flies.
Our data indicate that CCAP is responsible for initiating TH translation
following eclosion. In Drosophila, translational regulation often
occurs through microRNA (miRNA)-dependent RNAi-mediated repression through
binding sites in the 3'UTR of transcripts
(Wilhelm and Smibert, 2005).
Three miRNAs - let-7, mir-iab-4-3p and mir-iab-4-5p - have been predicted to
regulate TH translation in Drosophila
(Enright et al., 2003). It is
conceivable that one or more of these miRNAs, in association with the RISC
complex, could bind to ple transcripts to cause the repression of
translation through a miRNA-dependent RNAi-mediated mechanism. It is also
plausible that PKA, activated by CCAP signalling, might relieve repression of
TH translation by phosphorylation of one of the subunits of the RISC complex
or associated proteins. Future work will establish whether there is a role for
these miRNAs in the repression of TH translation before eclosion.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/24/4395/DC1
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ACKNOWLEDGMENTS |
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