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First published online June 27, 2008
Journal of Experimental Biology 211, 2263-2274 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.015818
Light and peptidergic eclosion hormone neurons stimulate a rapid eclosion response that masks circadian emergence in Drosophila
Department of Zoology, Box 351800, University of Washington, Seattle, WA 98195-1800, USA
* Author for correspondence (e-mail: smcnabb{at}u.washington.edu)
Accepted 12 May 2008
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Summary |
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Mrosovsky,
1999). Classic examples of masking by changes in light include the
initiation of singing in sparrows at lights-on
(Binkley et al., 1983) and the
onset of hamster running behavior at lights-off
(Redlin and Mrosovsky, 1999).
Although numerous examples of masking have been identified, their underlying
cellular and molecular mechanisms and how they interact with circadian systems
are not well understood.
For many insects, the emergence of the adult (eclosion) is under circadian
regulation (Helfrich-Förster,
2006; Saunders,
1976; Saunders,
1982; Saunders,
2002). In Drosophila melanogaster Meigen, the circadian
rhythm of eclosion is based on a central clock that depends on the interaction
of a number of proteins encoded by genes including Clock, cycle,
period and timeless (reviewed by
Helfrich-Förster, 2006).
The circadian clock restricts the timing of Drosophila eclosion to a
`gate' that occurs during the early part of the day
(Engelmann and Honegger, 1966;
Jackson, 1983;
Lorenz et al., 1989). Gating
results in a discontinuous eclosion pattern in which entrained pharate adults
that complete adult development at night wait for the opening of the gate at
around dawn in order to eclose
(Pittendrigh and Skopik,
1970). Release of the neuropeptide eclosion hormone (EH), a key
regulator of ecdysis (reviewed by Truman,
2005), is gated in the moth Manduca sexta, suggesting
that eclosion gating may result from circadian regulation of EH release. This
could occur directly as the result of declining ecdysone titers that are
associated with the activation of ecdysis
(Hewes and Truman, 1991).
Alternatively, it could result from the ecdysone-responsive release of ecdysis
triggering hormone (ETH) (Kingan and
Adams, 2000; Zitnan et al.,
1999; Zitnanova et al.,
2001), which stimulates EH release
(Ewer et al., 1997;
Kingan et al., 1997;
Zitnan et al., 1996). Pigment
dispersing factor (PDF) produced by the lateral neurons may influence the gate
by modulating ecdysteroid release from the prothoracic gland
(Myers et al., 2003).
The circadian pattern of Drosophila eclosion can be masked by
light. When administered close to the eclosion gate, a lights-on (LOn) signal
shifts the distribution of flies emerging within the gate. This is manifest as
a burst of eclosion soon after the LOn signal. The pathway of light reception
for this LOn response is distinct from those utilized for circadian
entrainment. Drosophila mutants that lack both the ocelli and
compound eyes lack the lights-on response but nevertheless show normal
circadian entrainment of their ecdysis clock
(Engelmann and Honegger,
1966). For circadian regulation of locomotion and eclosion, the
central clock resides in lateral neurons of the brain that express central
clock proteins, the neuropeptide pigment-dispersing factor (PDF) and a
cryptochrome photoreceptor (reviewed by
Helfrich-Förster, 2006;
Nitabach and Taghert,
2008).
Eclosion is regulated by a cascade of peptide hormones. These peptides
include EH from the brain, pre-ecdysis triggering hormone (PETH) and ecdysis
triggering hormone (ETH) from the epitracheal glands
(Park et al., 2002;
Zitnan et al., 1996), and
crustacean cardioactive peptide (CCAP) from the ventral central nervous system
(CNS) (reviewed by Truman,
2005). To test the requirement for EH in eclosion, molecular
genetic tools were used to target the ablation of the EH-expressing neurons in
Drosophila (McNabb et al.,
1997). Surprisingly, these experiments showed that EH is not
strictly required for eclosion. However, the EH cell knockouts had significant
defects. A third died at larval ecdyses with defects in tracheal filling, and
the two thirds that eclosed as adults had defects in eclosion and
post-eclosion behaviors. Interestingly, the EH cell knockouts had normal
circadian eclosion rhythms but lacked the LOn response. Thus, like the retinal
photoreceptors, the EH neurons appear to be components of the LOn pathway. In
this paper, we define the basic characteristics of the LOn response and
examine the way this signal interacts with EH release, eclosion, and
post-ecdysial wing spreading.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The
w1118 strain was the recipient strain for the transposon
that contains the GAL4 transcription factor under the regulation of
EH gene upstream sequences (see below). When crossed to the UAS-rpr
strain, it is the control for the
EHups-Gal4xUAS-rpr flies. Since the
UAS-rpr insertion is carried on a y w67
c23 X chromosome, the hemizygous male progeny of this cross
have yellow cuticle and pale apricot eyes. Female progeny are heterozygous
w1118/y w67 c2 UAS-rpr, with
wild-type cuticle and eyes that are slightly paler than those of the males.
Despite these differences, no difference in LOn responsiveness or wing
spreading latency (see below) was detected between these males and
females.
The EH cell knockout flies (McNabb et
al., 1997) were generated by using the GAL4-UAS system
(Brand and Perrimon, 1993). EH
gene upstream sequences (EHups) fused to Gal4
(EHups-Gal4;P{GAL4-Eh2.4}) were used to drive expression of the cell
death gene reaper (rpr) in the neurons that produce EH. Male
flies from the C21 EHups-Gal4 strain were crossed to females of the
UAS-rpr strain. Their progeny are referred to as
EHupsxUAS-rpr throughout this paper. Since the C21
transposon is located on the second chromosome, the progeny possess the X
chromosome genotype described for the
w1118xUAS-rpr strain above, which results
in male progeny with yellow cuticle and females with wild-type cuticle. All
knockouts were heterozygous for the second chromosome, i.e. C21/+. The
EHups transposon conferred bright orange-red eyes on all progeny,
although the eyes of females were slightly paler than those of the males.
Despite differences in cuticle and eye pigmentation, no differences between
males and females were detected in the LOn response or wing spreading
latency.
Canton-S (CS), a standard wild-type lab strain, was used as a control for
the ocelliless and eyeless strains. Although the progenitor strains for these
mutants is unknown, it is likely to be CS. Strains that lacked ocelli were
ocelliless (oc1)
(Flybase, 1999;
Lindsley and Zimm, 1992) and
sine oculis (so+2). Strains that lacked compound
eyes were alleles of eyes absent [eya2; also
called clifteya-2
(Bonini et al., 1993)] and
eya1 [also called clieya-1
(Eissenberg and Ryerse, 1991;
Sved, 1986)]. To ensure that
the lack of a LOn response observed for the ocelliless and eyeless strains
were not due to locomotor defects, they were tested for geotaxis using a
countercurrent assay (Benzer,
1967). All exhibited positive geotaxis. These strains were also
tested by immunocytochemistry for normal levels and release of EH. The CNS of
flies that were staged at approximately 6 h prior to eclosion and those that
had just eclosed were labeled with anti-EH and analyzed as described below.
These strains appeared to synthesize normal levels of EH and to release it at
eclosion as expected.
The w1118xUAS-TNT-L and
EHupsxUAS-TNT-L flies were generated by crossing males
from the w1118 strain or the C21 EHups-Gal4
strain, respectively, to females of the UAS-TNT-L strain
(Sweeney et al., 1995). We
detected no sex-specific differences in cuticle or eye pigmentation, LOn
response or wing spreading latency.
Lights-on (LOn) assays
Flies were cultured in half-pint culture bottles containing cornmeal agar
food, at 25°C. Cultures were raised continuously under a 14 h:10 h
light:dark (14L:10D) cycle at approximately 750 lx. Embryos were collected
over 24 h to an experimentally determined optimum density that depended on
both parental strain fecundity and mortality of the progeny. Food was removed
from the bottles on the afternoon prior to assay. Five bottles of flies were
reared for each assay condition and the eclosing flies for each treatment were
pooled upon collection. Flies that emerged before the day of assay were
removed just prior to lights-off of the preceding night. Emerging flies were
collected at 10 min intervals, except as noted, and subsequently counted. One
final collection of adults was made just prior to the end of the photoperiod
and the total number of flies that emerged between the first and last
collection periods used to generate eclosion rates as a percentage of the
day's total eclosion. Each treatment group was reared and tested in
parallel.
For the LOn shift paradigm, entrained adults were subjected to normal lighting or to a LOn that was shifted either earlier (–1 h or –2 h) or later (+2 h) than normal. Each strain was tested at least twice and each test yielded qualitatively similar results.
Immunocytochemistry
Fly CNSs were dissected into Ca2+-free Ikeda's saline
(Ashburner, 1989) then fixed in
3.7% formaldehyde in phosphate-buffered saline (PBS) overnight at 4°C.
Samples were washed extensively (5x10 min) with PBS containing 0.3%
Triton X-100 (PBST) and blocked in 1% normal donkey serum (Jackson
ImmunoResearch Laboratories, Inc., West Grove, Pennsylvania, USA). They were
then incubated overnight at 4°C in a rabbit anti-EH antiserum
(Copenhaver and Truman, 1986)
at 1:100 in PBST. Samples were washed extensively in PBST, and then incubated
in donkey anti-rabbit Cy5 (Jackson ImmunoResearch Laboratories, Inc.) at
1:1000 overnight at 4°C. Samples were then washed extensively, mounted on
poly-lysine-coated coverslips, passed through an ethanol dehydration series,
cleared in xylene, and mounted in DPX histological mountant (Fluka BioChemika,
Sigma Aldrich Chemie, Steinheim, Germany). Preparations were examined on a
Bio-Rad MRC-600 (Hercules, California, USA) confocal microscope. Image stacks
were scored visually and representative z-series were collapsed to
provide two-dimensional images.
To examine EH release in w1118xUAS-TNT-L
and EHupsxUAS-TNT-L strains, CNSs were collected
either 8–11 h prior to eclosion before meconium transport
(Kimura and Truman, 1990) or
within 1 min post-eclosion.
To examine the effects of light on EH release, we used w1118xUAS-rpr flies. On the night before eclosion, 0–2 h before lights-off, late pharate adults with anterior meconia were removed from their puparial cases. One group was dissected immediately as a no-treatment control and the rest were returned to 14L:10D conditions. One group was dissected 1h before normal LOn to verify that EH release had not occurred; another was dissected 10–20 min after LOn. Two groups served as controls: one was maintained in darkness and dissected at the same time as the post-LOn set, the other was observed for time of eclosion. The latter control showed that the flies selected at this stage emerged approximately 1 h after the normal LOn signal. The CNSs of flies in the no-treatment control group were dissected into saline and fixed overnight. All other groups were dissected directly into fix to preserve their in vivo release state and maintained in fix overnight at 4°C. They were subsequently treated as described above. Samples were examined on a confocal microscope (as above) and graded according to intensity of labeling, on a scale of 0–4. Z-series were collapsed to provide two-dimensional images.
Wing spreading assay
To determine if light influences the interval between eclosion and the
completion of wing spreading (referred to as the wing spreading latency; WSL),
newly emerged flies from LOn shift experiments were collected, placed four to
a vial, held in the light at 25°C and scored for wing spreading at 10 min
intervals. It was important to avoid disturbing the flies during this time as
physical agitation delayed the wing spreading process. For assessing the
effect of the LOn signal, we examined only the flies that eclosed within the
first 20 min after LOn. Each experiment was performed at least twice.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Eggs
collected over a 24 h period and subsequently maintained under a 14L:10D cycle
emerged as adults over a 3–4 day period. Since previous experiments
demonstrated that the largest peak of the day's eclosion takes place within 1
h of LOn, we focused on the distribution of eclosion around the beginning of
the LOn signal, collecting newly emerged flies at 10 min intervals. We also
counted the total number of flies that emerged each day and have expressed the
emergence observed within a particular time bin as a percentage of the day's
total eclosion. This experiment was repeated three times and yielded very
similar results each time. Very few flies eclosed during the 9 h of darkness
between lights-off and –1 h, on average 7±2% of the day's
eclosion whereas 42±4% of the day's eclosion took place within 2 h of
light onset on each day sampled.
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The effects of a light pulse on stimulating eclosion
To separate the effects of light as a LOn signal from its role as an
entraining signal, we used a pulse as a LOn signal. Entrained
w1118xUAS-rpr flies were subjected in
parallel to either light at the normal time, extended darkness or extended
darkness except for a brief (20 min) pulse of light.
Fig. 2 summarizes the results
of four independent replicate experiments for which sample sizes were between
993 and 1316. For flies that received light at the normal LOn time, 14% of the
day's total eclosion occurred within 20 min of the signal
(Fig. 2A). This `burst' of
eclosion was not seen for flies maintained in darkness
(Fig. 2B). Instead, those flies
showed a normal distribution of eclosion that ranged up to a maximum of 5% of
the daily emergence per 10 min collection window. When a 20 min light pulse
was delayed to 1 h after the expected LOn signal
(Fig. 2C), it also resulted in
a massive eclosion burst (25% of the day's eclosions in 20 min) that rapidly
tailed off. Despite the differences in eclosion distributions, the proportion
of the day's total eclosion that ensued in the first 4h after normal LOn was
very similar under each of the three conditions: 60% for flies that received
the normal LOn, 62% for flies that received the pulse, and 59% for flies held
in the dark.
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Effects of varying the time of the LOn signal on control and EH cell knockout flies
Experiments were performed to determine the effects of early or late LOn
signals on eclosion in w1118xUAS-rpr
controls and in the eclosion hormone (EH) cell knockout strain. Entrained
adults were subjected to normal lighting or to a LOn signal that was either
advanced (–1 h or –2 h) or delayed (+2 h) relative to the expected
LOn signal. Representative results are shown in
Fig. 3. For the
w1118xUAS-rpr flies, the normal LOn signal
resulted in a robust burst of eclosion, with 29% of the day's emergence
occurring during the first 20 min (Fig.
3A). Delaying the light signal by 2 h resulted in a corresponding
delay in the abrupt eclosion burst, with about 14% of the flies emerging
during the 20 min following LOn (Fig.
3B). This delayed LOn peak was smaller than that observed at
normal LOn because the majority of flies that would eclose during this gate
had already emerged. However, this peak represented about 41% of the flies
that had not yet eclosed at the time of the shifted LOn signal. This
percentage is similar to that seen in Fig.
3A, where 36% of the flies emerged within 20 min of the LOn
signal. When the LOn signal was advanced by 1 h there was no immediate burst
of eclosion (Fig. 3C), but a
substantial increase in eclosion was observed during the next hour as compared
to flies that were still in the dark (compare with
Fig. 3B). Similar results were
obtained when the LOn signal was advanced by 2 h
(Fig. 3D).
|
The LOn response observed for the
w1118xUAS-rpr controls was absent in the EH
cell knockout (KO) flies (Fig.
3E–H). The EH cell KOs did not show an eclosion burst in
response to any of the conditions tested. Advancing the LOn signal failed to
swell the leading edge of the eclosion distribution (compare Fig.
3G,H with dark region of
Fig. 3F). Overall, the phase of
eclosion relative to the L:D cycle is the same in controls and the EH cell KOs
(McNabb et al., 1997). Hence,
entrainment of the circadian clock for eclosion is normal. It is only the
masking of the LOn signal that is missing upon removal of the EH neurons.
The role of the compound eyes and ocelli in the LOn response
A previous report showed that flies that lack both compound eyes and ocelli
due to a mutant allele of the sine oculis (so) locus,
so1, fail to show a LOn response
(Engelmann and Honegger,
1966). To determine if the LOn response requires only one or both
of the sets of optic photoreceptors, we tested mutant strains that lack either
the compound eyes or the ocelli. The strains that had no ocelli were mutant
for the oc1 allele of ocelliless
(Flybase, 1999;
Lindsley and Zimm, 1992) and
the so+2 (Heitzler et
al., 1993) allele of sine oculis. The strains that lacked
compound eyes were mutant for alleles of eyes absent, eya2
(clieya-2; Bonini et
al., 1993) and eya1
(clieya-1; Eissenberg
and Ryerse, 1991; Sved,
1986). The Canton-S (CS) wild-type strain was used as a control.
Preliminary results obtained with oc1 and
so+2 were essentially identical. Similarly, results
obtained with eya2 and eya1 were
essentially identical to each other. It was difficult to obtain large cultures
of each of these mutant strains because of their reduced viability, so we
focused on oc1 and eya2 for the
detailed studies described below.
The eclosion patterns of the CS and
w1118xUAS-rpr strains differed in a few
aspects. First, for CS, the normal entrained eclosion distribution started
about an hour earlier and a substantial number emerged prior to LOn. Secondly,
the CS LOn response was not as robust (Fig.
4A–D). In addition, the phase of the CS eclosion gate was
much broader; by 3 h after normal LOn, only 32% of the flies had eclosed
vs 69% of w1118xUAS-rpr flies
(
2-test, P<0.0001). The observation that the CS
strain showed a definite LOn response when the LOn signal was advanced to
–1 h and –2 h suggests that the CS strain is competent to respond
to light earlier in the day than is the
w1118xUAS-rpr strain. We assume that this
difference is due to the phase of the eclosion gate being earlier for the CS
than the w1118xUAS-rpr strain (compare
Fig. 3B with
Fig. 4B).
|
) and
geotaxis. For both the oc1 and the
eya2 strains, no significant increases in eclosion rate
were observed regardless of when the flies were exposed to a LOn signal
(2-test, P<0.0001). The apparent increase seen in
Fig. 4F was not seen upon
repetition of this test and thus appears to be due to random fluctuation, much
like the variation observed throughout the day. In both mutant strains,
particularly oc1, the eclosion rate was variable and
substantial eclosion preceded the LOn signal. However, overall
oc1 eclosion appeared circadian. Few flies emerged during
the night (7% for oc1 vs 8% for CS) and the proportion of
the day's eclosion that took place within the first 3 h of LOn (28%) was
similar to the CS strain (32%).
Effects of targeted expression of tetanus toxin light chain in the EH neurons
Targeted ablation of the EH cells eliminated the LOn response (above) but
did not distinguish whether the loss of the LOn response was due to the lack
of eclosion hormone itself or to some other function of the EH cells. One
potential way to leave the EH neurons intact but block EH release was by
targeting expression of an intracellular form of tetanus toxin light chain
(TeTxLC) in the EH neurons. To produce these flies, we crossed the
EHups-Gal4 strain to flies that carried a UAS-TNT-L
transgene (Sweeney et al.,
1995). The control flies were progeny of the
w1118xUAS-TNT-L cross. The LOn shift
paradigm was used to determine if the flies showed a LOn response.
The w1118xUAS-TNT-L strain demonstrated a strong LOn response when the LOn signal occurred at its normal time (Fig. 5A,B), similar to that observed for the w1118xUAS-rpr strain. However, moderate increases in eclosion were also detected when the LOn signal was advanced to –1 and –2 h (Fig. 5C,D), similar to the CS strain. EHupsxUAS-TNT-L flies lacked the LOn response (Fig. 5E–H) irrespective of the phase of the LOn signal. In a replicate experiment, the small increase in eclosion rate at the onset of light shown in Fig. 5F was not seen.
|
;
Horodyski et al., 1993). We
harvested the CNS of control
w1118xUAS-TNT-L and
EHupsxUAS-TNT-L flies the night before eclosion or
within 1 min post-eclosion and immunostained them for EH. The CNS of
w1118xUAS-TNT-L flies prior to eclosion
showed strong immunolabeling of the cell bodies and processes of the EH
neurons (N=4; Fig.
5I). By contrast, after eclosion these neurons had little or no EH
in descending axons in the ventral ganglion and reduced levels in the cell
bodies and processes in the brain (N=12;
Fig. 5J). In the
EHupsxUAS-TNT-L flies, the EH neurons also showed
strong immunostaining prior to eclosion (N=5;
Fig. 5K), but after eclosion
the neurons still showed abundant EH (N=14;
Fig. 5L). The level of EH in
the CNSs from post-eclosion EHupsxUAS-TNT-L flies was
substantially higher than in CNSs from either control or pre-eclosion
EHupsxUAS-TNT-L flies. This suggests that the peptide
continues to accumulate in the EH cells during the final 8–11h before
ecdysis and that TeTxLC suppresses its release.
As an additional means of assessing whether the effects of expressing
TeTxLC in the EH cells was equivalent to ablating them, we compared other
ecdysial and post-ecdysial effects in EHupsxUAS-TNT-L
and EHupsxUAS-rpr (EH cell knockout) flies. The EH
cell knockout flies showed extensive larval mortality due to incomplete
shedding and inflation of the trachea
(McNabb et al., 1997). By
contrast, the EHupsxUAS-TNT-L strain exhibited no
larval mortality. The principal post-ecdysial defect of the EH cell knockout
is the failure to spread the wings, a phenotype observed in a large proportion
(76%) of adult flies (McNabb et al.,
1997). Expression of TeTxLC in the EH cells had a less severe
effect on wing spreading; only 4±1% of all
EHupsxUAS-TNT-L adults failed to inflate their wings
(three experiments). Although the magnitude of this effect is much less than
that seen for the EHupsxUAS-rpr strain, it is
significantly elevated (two-tailed t-test, P=0.01) above the
levels seen for w1118xUAS-TNT-L controls
(0.3±0.2%, two experiments).
Effects of light on wing spreading latency
Wing expansion follows eclosion and is the last behavioral component of the
adult eclosion sequence (Fraenkel,
1935). It is regulated by Bursicon
(Dewey et al., 2004) and EH
(McNabb et al., 1997;
Truman and Riddiford, 1974).
To determine if the LOn signal influences wing spreading latency (WSL), we
compared the interval between eclosion and the completion of wing spreading
for flies that eclosed in response to the LOn signal to flies that eclosed in
the dark. To focus on flies that eclosed in response to the LOn signal, we
monitored only those that eclosed within 20 min of LOn. We compared rates of
wing spreading between strains that exhibited the LOn response and their
counterparts that did not.
For the w1118xUAS-rpr strain, the WSL was dramatically different for flies that eclosed in response to the LOn signal vs flies that eclosed in the dark (Fig. 6). The flies that eclosed in response to LOn took an average of 86±6 min to spread their wings (Fig. 6A). Flies that eclosed in the dark spread their wings in less than half the time, with an average of 41±4 min. For flies that eclosed in the dark, 41% spread their wings within the first 10 min after eclosion, strongly skewing the distribution to the left (Fig. 6B). By comparison, only 17% of flies that eclosed at LOn spread their wings within the first 10 min.
|
). In
current experiments, only four of the 45 that eclosed within 20 min of LOn
expanded their wings. Although both EHupsxUAS-rpr and
EHupsxUAS-TNT-L flies lacked the LOn response, the
latter were better for assessing the relationship of the LOn signal to wing
spreading behavior because more of them spread their wings. The
w1118xUAS-TNT-L control strain showed a
similar WSL to that of the w1118xUAS-rpr
strain (Fig. 6C) – flies
that eclosed at LOn took much longer to spread their wings than those that
eclosed in the dark (2-test, P=0.0001). By contrast,
the EHupsxUAS-TNT-L flies that eclosed at LOn showed a
very similar distribution to those that eclosed in the dark
(Fig. 6D). The proportion of
flies that spread their wings within the first 10 min after eclosing was also
similar under the two conditions (2-test, P>0.1).
These results suggest that for strains that exhibit the LOn effect, such as
the w1118xUAS-rpr and
w1118xUAS-TNT-L strains, the WSLs differ
substantially for flies that eclose in response to the LOn signal vs
those that eclose in the dark. In the absence of a LOn effect, as in the case
of the EHupsxUAS-TNT-L strain, there is little
difference under the two conditions.
The correlation between the LOn response and the WSL was further tested
using the ocelliless oc1 strain, the eyeless
(eya2) strain and a CS control
(Fig. 6E–G). The
differences in WSL for CS flies that eclosed in response to a LOn signal and
those that eclosed in the dark were modest when compared with
w1118xUAS-TNT-L and
w1118xUAS-rpr strains but followed the same
trends. For CS flies, the distribution of WSLs seen for flies that eclosed in
the dark is slightly more skewed to the left than for flies that emerged after
LOn (Fig. 6E). On average, CS
flies that eclosed just after LOn completed wing spreading in
33±4.7min, compared to 23±3.0min for those that emerged in the
dark, a statistically significant difference (2-test,
P<0.01). In both cases, almost half of the CS flies spread their
wings within the first 10min of eclosion. For both the oc1
and eya2 strains (Fig.
6F,G), flies that eclosed at LOn and those that eclosed in the
dark showed similar WSLs and proportions of flies that spread their wings
within 10min of eclosion. These results are consistent with those of the two
previous sets of experiments. Strains such as CS that show a LOn response
complete wing expansion more rapidly when they emerge in the dark than after a
LOn signal. By contrast, those that lack a LOn response,
oc1 and eya2, show no difference under
the two conditions.
|
). In
addition to suppressing descending inhibition, another mechanism by which the
LOn signal could cause a shift in emergence time is by directly stimulating EH
release. To determine if the LOn signal could induce EH release, we examined
levels of EH immunostaining in CNSs from pharate adult
w1118xUAS-rpr flies exposed to different
light treatments. These pharate adults were selected carefully to ensure that
they were not destined to eclose within an hour after the time of normal LOn
(see Materials and methods) and a large number of CNSs (see below) were tested
for each condition to ensure that developmental stage did not influence the
differences observed. The night before eclosion, the EH cell bodies and their
projections showed strong EH immunoreactivity, scoring 3.7 out of 4 (see
Methods) (Fig. 7A,E). At
–1 h, EH levels were approximately as high as they were the night
before, demonstrating that the flies had not yet released EH in preparation to
eclose (score 3.5; Fig. 7B,F).
Some of the pharate adults were subjected to the normal LOn signal, while
others were maintained in darkness. Nervous systems from flies that were
collected within the first 20 min of the LOn signal showed a dramatic decrease
in EH levels (score 0.5; Fig.
7C,G), with 14 of 36 nervous systems showing a score of 1 or
lower. By contrast, CNSs from flies that had been maintained in the dark and
then sacrificed at the same time as the above flies continued to show strong
EH staining (score 2.9; Fig.
7D,H), with only one of 25 showing a score of 1 or below. The
observation that the CNSs from flies maintained in the dark scored lower than
the groups from before the LOn signal is consistent with the notion that some
of the flies held in the dark are within the eclosion gate and have initiated
EH release. |
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The second pathway involves the masking effect of the LOn
signal that causes a pronounced skewing of the eclosion peak within this gate.
We have shown that the eclosion response to the LOn signal is rapid and
strong, typically occurring within 10 min and resulting in up to 22% of the
day's eclosion within that 10 min interval. The LOn response can occur either
earlier or later in the eclosion gate but it can only take place after the
normal gate has opened. The response is exhibited in the absence of a normal
circadian rhythm in per and tim mutants (S.L.M., unpublished
data). A temperature transition may also exert a masking effect on eclosion
via the EH neurons (Jackson et
al., 2005).
|
) or
tetanus toxin light chain to suppress synaptic release
(Fig. 5). How do the EH neurons
mediate the LOn response? As seen in Fig.
7, one effect of the LOn signal appears to be the stimulation of
EH release. Our immunocytochemical studies show that the LOn signal caused a
rapid depletion of EH in pharate adult flies that were expected to release EH
later in the day.
A second effect of the LOn signal appears to be in decreasing the latency
of eclosion relative to EH action. In Drosophila, the normal latency
from EH release to eclosion is 40–60 min
(Baker et al., 1999) but the
LOn response occurs within 10 min (e.g.
Fig. 1). Experiments in both
Manduca and Drosophila suggest that the delay between EH
release and ecdysis is due to a descending inhibition that is set up as a
consequence of EH release. EH release normally takes place well in advance of
ecdysis. In Manduca, the delay period is about 20–25 min for
larval ecdysis (Ewer et al.,
1994), and 2–3 h for adult eclosion
(Ewer and Truman, 1996).
Decapitation of Manduca pharate adults during the delay period leads
to the rapid onset of eclosion behavior
(Ewer et al., 1997;
Reynolds, 1980), suggesting
that the delay is due to descending inhibition from the head. Transection of
the CNS at different levels shows that neurons from the subesophageal and
thoracic ganglia contribute to this descending inhibition
(Fuse and Truman, 2002;
Zitnan and Adams, 2000).
Similarly in Drosophila, decapitating pharate adults that have
released EH results in their rapid eclosion
(Baker et al., 1999). This
rapid behavioral response to decapitation is not seen in EH cell knockout
flies (Baker et al., 1999),
suggesting that EH release is required for the inhibition to be set in
place.
A LOn signal appears to be another way to elicit early eclosion in flies
that have released EH. The simplest interpretation is that the LOn signal acts
by suppressing the descending inhibition. This mechanism is favored by the
rapid nature of the LOn response and by evidence from the pulse experiment
with the w1118xUAS-rpr strain. If the LOn
response results from the removal of the descending inhibition, flies that
eclose during the LOn peak should include all of the pharate adults that have
released EH and are in the delay period at the time of the pulse. This can be
seen by comparing the results of the pulse and dark experiments
(Fig. 8A). In the
dark-maintained group, about 21% of the flies that eclosed during the day
emerged between 1 and 2 h after the time of normal LOn. Given a latency of
40–60 min between EH release and ecdysis
(Baker et al., 1999), most of
these flies would have been expected to have released EH by the time the LOn
pulse was given at 1 h. In this example, in the group that received the pulse,
the LOn signal was followed by 20% of the flies emerging within the first 10
min, similar to the 21% predicted to be in the waiting period. This is
consistent with the interpretation that flies that demonstrate the LOn
response have already released EH, are competent to respond to the LOn signal,
and are rapidly released from the inhibition that causes the normal delay
(Fig. 8B). If the LOn signal
acted only to suppress the descending inhibition and hence reduce the eclosion
latency in pharate adults that had released EH, we would expect eclosion to
drop down to zero for 40–60 min after the LOn peak. The fact that we do
not observe this severe depression is most likely explained by our observation
that the LOn signal can induce premature EH release in addition to reducing
the eclosion latency.
|
). Using mutations that eliminate either the compound eyes or
the ocelli, we have shown that both sets of photoreceptors are required for
the response under our assay conditions. The reason that both sets of
photoreceptors are required is unknown, but one possibility is that they act,
either in series or in parallel, to amplify the response. We also cannot rule
out the possibility that the mutants we tested had defects in other components
of the ecdysis pathway that may mediate the LOn response. For example,
eyes absent mutations affect the development of additional regions of
the brain (Bonini et al., 1998;
Boyle et al., 1997), and
ocelliless and sine oculis also affect other developing
tissues (Finkelstein et al.,
1990; Serikaku and O'Tousa,
1994).
We postulate a mechanism in which a signal from the photoreceptors acts on
the eclosion inhibitory neurons to suppress the descending inhibition
(Fig. 9). Before the cellular
pathway and the mechanism of the release of the inhibition can be elucidated,
the source of the inhibition must be identified. Based on the results of head
ligations described above, it must reside in the head. In Manduca,
neurons of the cell 27/704 group are EH targets
(Ewer et al., 1994;
Ewer et al., 1997) and some
that are located in the subesophagheal ganglion are candidate sources of the
inhibition (Fuse and Truman,
2002; Zitnan and Adams,
2000). The identity of the Drosophila inhibitory neurons
is less clear. Efforts to identify the Drosophila homologs of the
cell 27/704 group as EH targets have been unsuccessful because they do not
show a cGMP response following EH release
(Ewer and Truman, 1996).
Currently, the function of the Drosophila CCAP-expressing neurons in
eclosion is uncertain, as their ablation does not prevent eclosion
(Park et al., 2003). However,
several other sets of neurons demonstrate calcium transients in response to
ETH, suggesting candidate neuropeptide-expressing neurons as downstream
activators (Kim et al., 2006).
The pathway by which the optic photoreceptors signal the ecdysis inhibitory
neurons also remains to be identified.
Differences in magnitude of the LOn response were observed between the
control strains. The w1118xUAS-rpr flies
have pale apricot eyes and are highly responsive to the LOn signal. The
Canton-S and w1118xUAS-TNT-L flies have red
eyes and show a weaker LOn response. The magnitude of the response may be a
function of light sensitivity and thus be higher in strains with less
pigmentation since screening pigments are associated with decreased
sensitivity of the eyes to light (Ostroy
and Pak, 1974; Zimmerman and
Ives, 1971).
Expression of tetanus toxin light chain in the EH neurons suppresses the LOn response and EH release
We have demonstrated three different ways of eliminating the LOn response:
(1) removal of the EH neurons by targeted expression of the cell death gene
rpr (i.e. making an EH cell knockout), (2) removal of either ocelli
or compound eyes by the use of previously identified mutants, and (3)
alteration of EH cell function by targeting expression of an intracellular
form of tetanus toxin light chain (TeTxLC) to the EH cells. The ability of EH
neuron-targeted TeTxLC to block the LOn response shows that the elimination of
this response in the EH cell knockouts is not due to death of EH cells, but
rather to loss of EH cell function. Since TeTxLC, which acts by inhibiting
synaptobrevin-mediated docking (reviewed by
Humeau et al., 2000), inhibits
the release of EH (Fig. 5K,L),
synaptobrevin appears to play a role in the release of neuropeptides. Flies
that express TeTxLC in their EH neurons also show a partial failure to spread
their wings, a post-ecdysial effect that is characteristic of the EH cell
knockouts (McNabb et al.,
1997). We do not yet understand the difference in penetrance of
this phenotype between these two strains. It may reflect a low level of EH
release by the flies that express targeted TeTxLC, interstrain variation (as
seen previously in McNabb et al.,
1997) or a combination of these factors.
Wing spreading, a post-ecdysial behavior that is regulated by EH, is not accelerated by the LOn signal
For strains that exhibited a strong LOn response, flies that eclosed in
response to LOn took substantially longer to spread their wings than flies
that eclosed in the dark. For w1118xUAS-rpr
flies, it took an average of 47 min longer; for
w1118xUAS-TNT-L flies, about 40 min longer.
This time difference is within the 40–60 min latency range between EH
release and eclosion (Baker et al.,
1999). Our findings are consistent with EH playing roles in
initiating both eclosion and wing spreading, and with these two behaviors
having separate downstream pathways with distinct latencies. The LOn signal
interacts with the ecdysis delay pathway to result in early ecdysis, but does
not affect the wing spreading circuit. Hence, the LOn signal causes early
ecdysis and a corresponding increase in latency between eclosion and wing
expansion.
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Acknowledgments |
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