Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program
Department of Zoology, University of Washington, Box 351800 Seattle, WA 91895, USA
* Author for correspondence at present address: Department of Neurobiology and Behavior, SUNY at Stony Brook, Stony Brook, NY 11794, USA (e-mail: jabaker{at}ms.cc.sunysb.edu)
Accepted 30 May 2002
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Summary |
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Key words: Receptor, bursicon, neuroethology, behavior, hormone, tanning, eclosion, ecdysis triggering hormone
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Introduction |
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Drosophila and blowflies are higher flies in the suborder of
Cyclorapha. These insects show a delay between the emergence of the adult from
the puparium and the onset of wing expansion with its accompanying cuticular
tanning and melanization. Flies that were decapitated by neck ligation
immediately after emergence failed to show normal post-ecdysis cuticular
tanning, but tanning could be induced with injections of blood collected from
intact flies a few minutes after their emergence
(Cottrell, 1962a;
Fraenkel and Hsiao, 1962
). The
blood borne tanning factor, bursicon, is a 33 kDa peptide hormone
(Kaltenhauser et al., 1995
;
Kostron et al., 1995
) made by
neurosecretory cells in the nervous system
(Garcia-Scheible and Honegger,
1989
; Honegger et al.,
1992
; Kostron et al.,
1996
; Taghert and Truman,
1982
). It appears in the blood at or after ecdysis
(Reynolds, 1980
;
Truman, 1981
) and induces a
transient elevation of cAMP in target tissues. Although the cuticular actions
of bursicon have been examined, its possible behavioral actions are poorly
understood because most assays for bursicon involve decapitation of the
insect. Furthermore, the scope of comparative studies on bursicon action is
constrained by the fact that, in many insects, the ecdysis and expansional
phases of emergence behavior overlap considerably.
We chose a molecular genetic approach to understand the changes that
characterize the final phase of adult eclosion, studying the behavior in
wild-type Drosophila and in rickets (rk) mutants
that do not complete the post-ecdysial expansion behaviors. The rk
gene encodes a member of the glycoprotein hormone receptor family of
G-protein-coupled receptors (Ashburner et
al., 1999; Eriksen et al.,
2000
), which appears to be an integral component of the bursicon
signaling pathway. Experiments using the rk mutants give a novel
insight into the complex regulatory pathway that controls wing expansion
programs in insects and suggest a physiological role for a glycoprotein
hormone receptor in insects.
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Materials and methods |
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Stocks
The two strong rk alleles we used, rk4 and
rk1, were maintained as homozygous stocks. The two
deficiencies that have been genetically defined to break in the rk
gene are Df(2)b-L and Df(2)A376
(Ashburner et al., 1982). Both
deficiencies are lethal when homozygous so each was maintained as a balanced
stock over CyO. The progeny of a cross between these two deficiency
stocks were viable and fertile but displayed a severe rickets phenotype. We
used Canton S as our wild-type stock of Drosophila melanogaster.
Extracts
A hormone extract that contained bursicon was made from the abdominal
ganglia of Sarcophaga bullata (Pkr.). The fused ventral ganglia of
pharate adults were dissected and homogenized in saline
(Ephrussi and Beadle, 1936)
(127.7 mmoll-1 NaCl, 4.7 mmoll-1 KCl) at a concentration
of 1 CNS/5 µl. The extract was centrifuged for 5 min, and the supernatant
was divided into portions and frozen until use. For cAMP experiments a 1
mmoll-1 solution of 8-Br-cAMP (Sigma) was prepared in saline
(Ephrussi and Beadle, 1936
).
Injections were performed as described below.
Ligations and injections
Newly eclosed adults were collected from bottles onto an ice-chilled Petri
plate at 3 min intervals. When enough animals were collected for an
experiment, the flies were neck ligated using a very fine nylon monofilament
line. They were maintained at room temperature in a humid chamber for 1 h and
ligated flies that tanned or spread their wings during this interval were
discarded. The remaining flies were injected with the hormone extract, saline,
or cAMP, using a picospritzer II (General valve, corp.) and glass
micropipettes. Flies were injected with between 180 and 300 nl of solution.
The volume was estimated by measuring the volume of a drop injected into oil.
Flies that bled excessively following injection were discarded. The cuticular
darkening induced by bursicon was evident within 2 h of injection.
For injections of hemolymph, blood samples were collected by removing a wing and compressing the fly between two glass microscope slides. A drop of blood was produced at the site of the injury and was collected in a micropipette injection needle. Blood collected in this way was relatively free of cellular debris and could be injected without clogging the needle. The contents of a needle were injected into a single fly. Blood from wild type was collected after wing expansion had begun. Blood from mutants was collected 15 min after adult eclosion because rk mutants do not expand their wings.
Cuticle preparations
Abdomens of flies were opened along the ventral midline, pinned flat in a
sylgard (Dow Corning) dish and fixed in 4% formaldehyde in phosphate buffered
saline (PBS). The abdomens were dehydrated through an ethanol series, cleared
in Xylene, and mounted in DPX (Fluka Biochimika). Pictures were collected on a
Macintosh PowerPC 7600/120 from a Sony CCD camera connected to a Nikon
Optiphot microscope. An average of 15-20 focal planes of each portion of the
cuticle were collected and assembled into a montage using layers in Adobe
PhotoShop 3.0.
Immunocytochemistry
Nervous systems of rk4 homozygotes were dissected in
cold saline (Ephrussi and Beadle,
1936) from either developing adults at the smooth to smooth-grainy
stage (approximately 5-9 h before adult eclosion) or from newly eclosed
adults. They were fixed in 4% formaldehyde overnight at 4°C. After
fixation the nervous systems were washed three times in PBS with 0.3% Triton
X-100 (PBS-TX) for 1-3 h. A rabbit anti-EH (eclosion hormone) antiserum was
diluted 1:100 in PBS-TX with 1% normal donkey serum (NDS) (Jackson labs) and
0.005% sodium azide. The tissue was incubated in the primary antibody
overnight at 4°C. After washing in 5 ml of PBS-TX, 3x, for 1-3 h,
the tissues were incubated in a donkey anti-rabbit IgG antibody conjugated to
Texas Red (Jackson Labs). Secondary antibodies were used at a dilution of
1:1000 in PBS-TX with 1 % NDS and 0.005 % sodium azide for either 8 h at room
temperature or overnight at 4 °C. The nervous systems were then washed 3
times in PBS-TX for 1-3 h, mounted on poly-lysine coated coverslips and
dehydrated, cleared and mounted as described above. Images were collected
using a BioRAD MRC-600 confocal microscope.
Behavioral observations
Pharate pupae were collected in small Petri dishes ringed with Whatman
paper and observed periodically for the initiation of ecdysis. When ecdysis
was completed, a timer was started and observations were made either
continuously or at 1 min intervals.
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Results |
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Eriksen et al. (2000)
reported that a p-element insertion into the first exon of the DLGR2
gene produced an embryonic lethal phenotype. They concluded that DLGR2
performed some vital function during embryogenesis. The embryonic lethality of
this allele is at odds with the phenotype of the other 21 described alleles of
rk and with the phenotype of the overlapping deficiencies described
above. Consequently, we wondered whether the lethality was associated with the
disruption of the rk gene or with a second, unmapped lethal mutation.
Therefore, we complementation tested rkw11p
against the two mapped deficiencies, Df(2L)A376 and
Df(2L)b-l. We found a rk phenotype in 29 % (67/229) of the
eclosing flies from the Df(2L)A376/CyO x
rkw11p/CyO cross and in 26 % (31/119) of
the Df(2L)b-L/Cyo x rkw11p/CyO
cross. These results are close to the expected value of one third of the flies
being mutant, indicating that there is a very low level of lethality.
Complementation testing against two strong alleles,
rk1 and rk4, gave
similar results, indicating that the embryonic lethality in
rkw11p is not associated with the rk
region. To test whether the rkw11p p-element is
separable from the lethality by recombination,
Ww1118/Ww1118;
rkw11p/rk4 flies
were maintained as a red-eyed rk stock for several generations. 10
red-eyed males were isolated from this stock and individually crossed to
Ww1118/Ww1118;
Sp/CyO females.
Ww1118/Ww1118;
rkw11p/Cyo flies were backcrossed and tested for
the rk phenotype and for retention of the p-element by eye color. One
line had red eyes and a strong rk phenotype. In a test cross, 31 %
(91/203) of progeny showed the rk phenotype, consistent with the loss
of the embryonic lethality from this chromosome. Flies homozygous for this
recombinant chromosome are viable and fertile enough to be maintained as a
stock.
The phenotype of Df(2L)b-L,Df(2L)A376 transheterozygotes is
essentially identical to the two available strong alleles of rk,
rk1 and rk4.
Consequently, we hypothesized that rk1 and
rk4 might be null alleles. We sequenced the
rk coding region from flies homozygous for
rk1 and for rk4. The
gene consists of 15 exons, 14 of which code for protein
(Eriksen et al., 2000). The
first 13 exons comprise the leucine-rich extracellular domain while the 14th
exon encodes the seven transmembrane domains and the intracellular portion of
the protein. In both mutants we found a mutation resulting in a premature
termination codon in the transmembrane domain. In
rk1 the mutation is C to A, converting tyrosine
698 to a stop codon that would terminate translation prior to the first
transmembrane domain (Fig. 1D).
In rk4 the mutation is a G to A change that
converts tryptophan 954 to a stop codon, which would truncate the protein
between transmembrane domains 5 and 6 (Fig.
1E). Both mutations should prevent the production of a functional
membrane receptor.
rk and the endocrinology of the ecdysis sequence
In Lepidoptera and Diptera, one steroid (20-Hydroxyecdysone) and four
peptide [EH (eclosion hormone), PETH (pre-ecdysis triggering hormone), ETH
(ecdysis triggering hormone) and CCAP (crustacean cardioactive peptide)]
hormones regulate the ecdysis sequence
(Gammie and Truman, 1997;
Truman, 1981
;
Zitnan et al., 1996
;
Baker et al., 1999
;
Park et al., 1999
).
Collectively, these hormones coordinate the pre-ecdysis and ecdysis behaviors.
By contrast, a single peptide hormone, bursicon, is known to play a role in
the post-ecdysial phase of development
(Cottrell, 1962a
; Frankel and
Hsiao, 1962). Bursicon has been shown to hasten the tanning reaction that
normally begins after ecdysis, serving to harden the newly expanded cuticle.
The common link between strong and weak alleles of rk is a failure to
fully expand their wings and thoracic cuticle, giving rise to adults with
crossed post-scutellar bristles and the kinked femurs that are the basis of
the gene name (Edmondsen,
1948
). In addition to these deficits, we observed that strong
alleles of rk lacked the rapid tanning response characteristic of
wild type. By 3 h after eclosion the cuticle of wild-type flies is tanned and
the abdominal tergites are fully darkened
(Fig. 2A,B). By comparison,
flies homozygous for rk4 have not begun to darken
at this time (Fig. 2C,D). This
difference is no longer apparent by 6-9 h after eclosion.
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Pre-eclosion and eclosion in wild-type and rk flies
We studied rk4 homozygotes to see if the
events prior to adult eclosion were normal. Mutations in the rk gene
had no effect on the time course of adult development. For example, wild-type
and rk4 homozygote flies were collected at the
white puparium stage and maintained together in the same humid chamber through
metamorphosis (N=10 each). Under these conditions all flies of both
genotypes eclosed within 1 h of each other. In addition, during the staging of
pharate adult rk4 homozygotes, we observed all of
the stages that characterize the last 12 h of adult development of wild-type
flies (Kimura and Truman,
1990), suggesting that the events that immediately precede ecdysis
are normal in rk mutants. Significantly, the only defect that is
evident prior to eclosion is the flattening of the tarsi in severe alleles and
deficiency combinations (Ashburner et al.,
1982
). This defect arises immediately following head eversion,
which is the equivalent of pupal ecdysis in other insects. Hence, this tarsal
phenotype is likely also a post-ecdysial defect, albeit of pupal ecdysis
rather than adult ecdysis. Based on this phenotype, a recent review suggested
that rk might be involved in pupal limb development
(Brody and Cravchik, 2000
).
Such an involvement, however, is probably indirect and due to a failure of the
pupal cuticle to expand properly. The penetrance of the tarsal phenotype is
relatively low in rk4 homozygotes (17%, N=60) and
rk4/rkw11p heterozygotes (44%, N=55),
whereas the penetrance of the failure to expand the wings is 100%.
Flies that have their eclosion hormone (EH) neurons genetically ablated
undergo adult emergence but often show a folded wings phenotype similar to
rk (McNabb et al.,
1997). This similarity suggests that EH production or release and
rk may be involved in the same pathway. We used immunocytochemistry
to study the accumulation and release of EH in rk mutants. Abundant
EH-immunoreactivity was present in the EH neurons of pharate adult
rk4 homozygotes. As in wild type, the EH staining was then
depleted during adult ecdysis (Fig.
3A,B). Interestingly, rk4 homozygotes still
showed some residual EH-immunoreactivity in the axons after ecdysis, a result
that was seen only occasionally in Canton S flies
(Baker et al., 1999
). Closer
inspection of the axons before and after ecdysis (insets,
Fig. 3) shows the loss of the
brightly staining varicosities that are characteristically seen in the EH
axons prior to ecdysis. It is not clear whether the small amount of residual
EH is due to a subtle effect of the disruption of rk or due to the
genetic background of the rk4 homozygotes.
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In wild-type Drosophila, injection of ecdysis-triggering hormone
(EH) from Manduca sexta (MasETH) into sensitive pharate adults leads
to the release of endogenous EH stores and to precocious ecdysis
(Baker et al., 1999;
Park et al., 1999
). Injection
of comparably staged rk4 homozygotes with MasETH also
induced premature ecdysis with a latency of about 50 min
(Fig. 3C), the same latency as
wild-type flies (Baker et al.,
1999
).
The post-ecdysial phase in wild-type and rk flies
An obvious link between rk and post-ecdysial processes is evident
from the failure of mutant flies to expand their wings or to show the normal
time-course of tanning and melanization. If rk were associated with
the bursicon pathway, it could influence the pathway either upstream or
downstream of bursicon release. If the defect in rk mutants is
upstream of the bursicon receptor, then rk flies may lack bursicon or
fail to release it. To examine this possibility we used newly eclosed,
neck-ligatured Drosophila (see Materials and methods) to test the
blood for bursicon activity (the ability to induce cuticular tanning and
melanization). As seen in Fig.
4, blood taken from flies immediately after eclosion showed no
tanning activity, but blood taken from flies 20 min later, when they were
actively expanding their wings, had strong tanning activity. Similarly, the
blood from the rk4 flies lacked tanning activity
immediately after ecdysis but showed substantial activity 20 min later.
Interestingly, these same flies did not show wing expansion behavior despite
the appearance of bursicon activity in their blood (see below). In summary,
the rk flies appear to release bursicon but show none of the
physiological or behavioral changes that are normally associated with that
release.
|
Bursicon has yet to be sequenced and synthesized, and so we could not
examine the response of flies to a synthetic peptide. We did, however, examine
the ability of rk flies to respond to bursicon-containing extracts.
We neck-ligatured flies within 3 min of eclosion and then injected them with
extracts prepared from the ventral nervous system of the flesh fly
Sarcophaga bullata. These extracts caused a normal melanization
response in wild-type flies. By contrast, the same extract elicited no
response from neck-ligatured rk4 flies
(Fig. 5,
Table 1). Ligated
rk4 flies also failed to respond to blood from wild-type
Drosophila that were actively expanding their wings
(Fig. 4). The inability of
rk mutants to melanize in response to bursicon could occur by
disrupting any one of the many components of the receptor-signaling pathway.
Studies on blowflies have implicated an increase in cAMP in the action of
bursicon (Seligman and Doy,
1972,
1973
;
Von Knorre et al., 1972
;
reviewed in Reynolds, 1980
).
As seen in Fig. 5E and
Table 1, injection of
rk4 flies with 8Br-cAMP resulted in strong melanization of
the abdominal tergites showing that at least this part of the response
machinery is functional in the mutants. Hence, the epidermis is capable of
melanization although not in response to bursicon. It should be noted that
cAMP injections do not evoke components of the wing expansion program in
either rk or wild-type flies (data not shown).
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Fig. 6 shows the timing of behaviors that occur immediately following eclosion in normal and mutant flies. The post-ecdysial behavior can be separated into two phases. The first is a perch selection phase, which is quite variable in duration, depending on environmental conditions, and in the types of behaviors performed. The second is the expansional phase, a stereotyped motor program that is relatively insensitive to environmental stimuli. Perch selection was complete in all flies by 2 min (N=6). Having settled on a perch, the flies then use their legs to clean their head, antennae, legs, wings and abdomen. The expansional phase begins with the ingestion of air, when the flies extend their proboscis and the cibarium begins pumping. Pumping is followed by a tonic contraction of the lateral tergosternal muscles of the abdomen. The expansional behaviors last for 7.6±0.3 min (N=11), and force blood into the thorax and wings, stretching the newly plasticized cuticle in preparation for tanning. If unmolested, the average time to the onset of wing expansion for wild-type flies is 16.6±0.6 min (N=16). Once expansion is completed, wild-type flies remain in place for more than 1 h, cleaning and testing the state of their wings. In contrast to wild-type flies, rk4 flies are much more active over the first 30 min after emergence, repeatedly alternating between cleaning and walking (Fig. 6). They do not show air swallowing behavior (N=30), nor do they exhibit the sustained abdominal contraction characteristic of wing expansion (N=17).
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The effects of decapitation on wing expansion behavior and
tanning
In a number of higher flies [Calliphora
(Cottrell, 1962a);
Sarcophaga (Fraenkel and Hsaio, 1962); Drosophila
(Kimura and Truman, 1990
)],
and in the moth, Manduca sexta
(Truman and Endo, 1974
)
decapitation experiments have been used to define the timing of the signal
that initiates wing expansion. In Drosophila, flies decapitated by
neck ligation immediately after adult emergence do not subsequently tan or
expand their wings. If the decapitation is delayed until about 10 min
post-ecdysis, though, the flies can then initiate and complete wing expansion
despite the removal of their head (Kimura
and Truman, 1990
). Thus, there appears to be a descending signal
that occurs about 10 min after ecdysis that is needed to induce both bursicon
release and wing expansion. A similar requirement for the head is seen in
pharate adult flies that are in the extended ptilinum stage, a stage that
begins about 40 min before ecdysis. These flies have just undergone their
major release of EH and, when decapitated, they show rapid eclosion but never
show wing expansion (Baker et al.,
1999
).
The requirement for the head in initiating tanning and wing expansion is different, however, in the period prior to EH release. There is a sensitive period between 3 h before ecdysis and the time of extension of the ptilinum (40 min before eclosion), when decapitation is still followed by ecdysis but only after a long and variable latency. Many of these decapitated flies also showed tanning and wing expansion with the percentage increasing as flies got closer to the extended ptilinum stage. For example, for flies decapitated 3 h before ecdysis, 25 % of the decapitated flies that ecdysed also tanned (N=20). For flies decapitated 55-48 min prior to ecdysis, 64 % of the ecdysing flies tanned and 36 % spread their wings after ligature (N=33) (Fig. 7A). No decapitated flies spread their wings without also tanning. Importantly, those flies that were decapitated early began tanning and wing expansion during or immediately following ecdysis and, thus, did not show the normal 10-15 min delay between eclosion and wing expansion. Thus, relative to ecdysis and wing expansion, the decapitation experiments identify two windows: an early window during which decapitation results in some flies showing a delayed ecdysis along with tanning and wing expansion and a late window during which decapitation is followed by immediate ecdysis but no tanning or expansion.
|
The involvement of EH in these windows was examined by repeating the
ligature experiments on EH cell knockouts, flies that had their EH cells
genetically ablated (McNabb et al.,
1997). These flies were extremely reluctant to ecdyse after neck
ligation, regardless of when the ligation was performed
(Baker et al., 1999
). Of the
few that ecdysed, none tanned or spread their wings after ligation, regardless
of when the ligature was performed (N= 14)
(Fig. 7A). The involvement of
rk in this phenomenon was examined by ligaturing
rk4 homozygotes at various times before their expected
ecdysis. Flies mutant for rk ligated during the late window never
showed either tanning or wing expansion. Surprisingly though, 54 % of ecdysing
rk homozygotes decapitated during the first window (N=11)
showed wing expansion behavior, although none of them tanned
(Fig. 7A). The wing spreading
in the decapitated rk flies was accomplished by sustained contraction
of the lateral tergosternal muscles as in wild-type flies
(Fig. 7B), but, because the
cuticle did not tan, the wings refolded when the abdominal contraction
terminated.
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Discussion |
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A point of controversy, however, comes from an analysis of the phenotype
that arises from the removal of the rk product. Eriksen et al.
(2000) described the sequence
and expression profile of DLGR2 and showed that flies carrying a
p-element inserted into the 5'-untranslated region of DLGR2 had
an embryonic lethal phenotype. Their paper did not, however, address the
synonymy of DLGR2 and rk or the fact that out of 22 reported
rk alleles, rkw11p is the only one that is
lethal. The data we present here indicate that complete removal of the
rk function is not lethal. Flies that carry deficiencies that
truncate DLGR2 from telomeric and centromeric directions
(Fig. 1) are unlikely to make
functional protein, but are viable and fertile with only a failure of tanning
and post-ecdysial expansion. Two strong alleles, rk1 and
rk4, are shown to contain nonsense mutations in the
transmembrane containing exon and these also show the classic rk
phenotype. Moreover, the phenotype is the same when these mutations are over
deficiencies for the region. The embryonic lethality observed in the
rkw11p stock seems more likely to be due to the presence
of an unidentified lethal mutation elsewhere on the second chromosome, or to
an effect of misexpressing DLGR2 in embryonic tissues in a way that
is lethal. Our observation that the lethality and the p-element insert are
separable by recombination supports the second-site lethal interpretation for
the embryonic lethality. Also, crosses of rkw11p with
either Df(2L)b-L or Df(2L)A376 give a rk phenotype
but no embryonic lethality. From the loss of function phenotypes, we have
concluded that normal DLGR2 function is associated only with
signaling processes that occur around the time of ecdysis.
The rk mutations specifically interfere with the last phase (the
expansional phase) of the ecdysis sequence. These flies fail to expand their
wings and thorax and they delay the onset of tanning and melanization. This
phenotype is also observed in flies that have had their EH cells genetically
killed (McNabb et al., 1997),
implicating EH in the regulation of post-ecdysial expansional behavior as well
as of ecdysis itself. Our analysis of the EH system in rk flies,
however, shows that they respond normally by initiating ecdysis when
challenged with MasETH, a response that requires the activity of the EH
neurons (McNabb et al., 1997
),
and that they show EH depletion from the nervous system following eclosion.
These results indicate that rk is probably downstream of EH release
and action.
In blowflies and Drosophila decapitation immediately after ecdysis
results in a delay in tanning that phenocopies rk mutations. As with
the rk flies, such decapitated flies gradually darken over a period
of several hours after decapitation
(Cottrell, 1962a; J.D.B.,
unpublished). The lack of rapid pigmentation has been ascribed to the lack of
the tanning hormone bursicon. The assay of blood from rk flies
shortly after emergence shows that these flies contain bursicon activity and
that they release it on schedule. The quantitative difference in bursicon
activity between rk and wild type is probably due to differences in
the timing; we have no behavioral assay for the time of hormone release in the
mutants and, therefore, had to rely on time after eclosion. However,
rk flies do not respond to injection of bursicon-containing material;
neither the blood from normal Drosophila that are expanding their
wings nor CNS extracts from the fly Sarcophaga provoked a response.
Although these flies do not melanize in response to bursicon extracts, they
show prominent melanization in response to injection of cAMP, the suggested
second messenger mediating bursicon action
(Seligman and Doy, 1972
). This
result shows that the cuticle of rk flies has all of the machinery
needed for melanization, but that it simply cannot activate this machinery in
response to bursicon. It also argues that the lesion caused by rk
probably occurs in the bursicon signaling pathway between the reception of
bursicon and the production of cAMP. From the nature of the DLGR2
product, we think that the most likely possibility is that it is the receptor
for bursicon. The large extracellular domain of this family of receptors is
used for binding large, glycoprotein hormones
(Dufau, 1998
). Intriguingly,
bursicon is one of the largest of the insect hormones, with an estimated
molecular mass of 33 kDa (Kostron et al.,
1995
). Because of its size, however, this hormone has yet to be
isolated and sequenced. Therefore, we cannot yet test directly whether DLGR2
can bind bursicon.
The regulation of wing expansion behavior
Hormonal and behavioral control over wing expansion in Drosophila
is time dependant and appears to involve both EH and rk (and, by
inference, bursicon). In wild-type individuals, decapitation within 10 min of
ecdysis prevents both the release of bursicon (shown by their lack of rapid
tanning) and wing expansion (Kimura and
Truman, 1990). During a transition period starting at around 10
min post-ecdysis, decapitated flies would occasionally tan without wing
expansion, but not vice versa. Shortly thereafter, both tanning and
wing expansion consistently followed decapitation. This timing suggests that
bursicon release and wing expansion are activated at about 10 min post-ecdysis
in a stereotypical sequence, with bursicon release being followed by
activation of the wing expansion program. This pattern is consistent with a
model in which bursicon release triggers the wing expansion motor programs. We
found that in rk mutants, the release of bursicon occurs on schedule
(Fig. 4) but the expansional
behaviors fail to occur (Fig.
6). If rk does indeed encode the bursicon receptor, then
this is the first direct evidence that bursicon activates the wing expansion
program.
In Drosophila, the effects of post-ecdysial ligatures mirror those
seen in the blowflies Sarcophaga and Calliphora
(Cottrell, 1962a; Fraenkel and
Hsaio, 1962; Kimura and Truman,
1990
). Surprisingly, we have identified a pre-ecdysial period
during which ligation activates the post-ecdysial behavior and melanization
rather than inhibiting it. There is an early response window that begins when
the animals reach the `grainy stage' approximately 3 h before ecdysis. Pharate
adults never eclose if decapitated prior to this time but routinely eclose if
decapitated after they start the grainy stage. The latencies from decapitation
to ecdysis ranged from 20 min to 5 h, with some flies emerging earlier than
expected, based on their developmental stage, and other flies emerging later.
None of the late emerging flies were seen to then tan or spread their wings,
but, by contrast, most of the prematurely ecdysing individuals showed normal
tanning and wing expansion despite lacking their head. Interestingly, these
flies began wing expansion during or immediately after ecdysis rather than
waiting the 10-15 min that is typical for intact animals. The significance of
this difference in timing will be discussed below. During this early window,
between 3 h and 50 min before ecdysis (the start of the `extended ptilinum
stage'; Kimura and Truman,
1990
), the fraction of flies showing precocious ecdysis and wing
expansion gradually increased with time. A different set of responses were
observed starting at about 50 min before ecdysis. During this second window,
the latency period before ecdysis, after decapitation, was abruptly reduced
down to about a minute or less (Baker et
al., 1999
), but tanning and wing expansion never occurred.
Therefore, in terms of wing expansion, flies decapitated during this second
window behaved in the same way as flies decapitated immediately after
eclosion.
Two pieces of evidence link these response windows to EH. First, the
transitions in the response of developing flies to decapitation correlate with
immunocytochemical changes in the EH-expressing cells
(Baker et al., 1999). At 4-7 h
before ecdysis there is a shift in EH immunostaining that represents either an
initial release of EH or a redistribution of EH to the axon terminals. At
around 50 min before ecdysis the major depletion of the EH neurons occurs.
Second, flies that lack EH neurons do not show premature ecdysis in response
to decapitation (Baker et al.,
1999
), and they do not subsequently tan or expand their wings. In
contrast, experiments with rk flies suggest that bursicon is not
involved in the early window. When decapitated during the initial window,
early ecdysing mutant flies also expand their wings, although the wings
subsequently collapse because tanning does not occur. Hence, the elements
driving the wing expansion program are present in the mutant but cannot be
activated in response to bursicon after eclosion.
These data, coupled with our findings that flies decapitated prior to adult
ecdysis often spread their wings, suggest that an inhibitory component
descending from the brain/SEG suppresses these ecdysial and post-ecdysial
motor programs. At about 3 h before ecdysis developmental changes make the
ecdysis and post-ecdysis motor programs competent to be expressed. The data
from the EH cell knockout flies suggest that the EH neurons are involved with
this competence, and it may be a low-level release of EH that initially
activates the motor programs. Although expansion occurs along with a response
to bursicon in this case, it does not depend on it as shown by the ability of
rk flies to expand their wings if decapitated at this early time. The
major release of EH at 50 min before ecdysis causes a strong activation of the
ecdysis program but suppresses both bursicon secretion and the wing expansion
program. Decapitation after this EH release is rapidly followed by ecdysis but
wing expansion is never displayed (Baker et
al., 1999). Subsequent descending commands from the head that
occur after ecdysis bring about bursicon release and this, in turn, is
required to activate the expansional program.
This seemingly complex control may have arisen from elaboration of a
simpler control system evident in most other insects. In many insects
(Carlson, 1977;
Hughes, 1980
; Mills,
1967
,
1966
;
Reynolds, 1980
;
Srivavista and Hopkins, 1975
)
ecdysis and tanning are closely linked and expansion begins as the insect is
escaping from the old cuticle. By contrast, insects that pupate underground or
in confined sites delay the expansion of delicate wings until the insect has
dug its way to freedom. In these insects, the fixed relationship between the
ecdysial and expansional phases has been replaced by a period of behavioral
flexibility that allows escape from a buried pupation chamber before the
initiation of cuticular expansion and hardening. This separation is found in
the Cycloraphous Diptera and also in some months such as the tobacco hornworm,
Manduca sexta (Truman and Endo,
1974
).
Intriguingly, during larval ecdysis of Manduca and Drosophila, expansion is already underway at the start of ecdysis. Hence, in these early stages both programs may be under the direct activation of EH. This link between ecdysis and expansion must be reconfigured during adult development to give the delay between ecdysis and expansion that is characteristic of adult behavior. It appears that in Drosophila, the larval relationships may remain intact but are masked by a strong inhibition that is imposed at the time of the major release of EH, about 50 min before ecdysis. Decapitation prior to this time may reveal the persisting larval circuit that links the ecdysial and post-ecdysial phases under common control of EH.
In most insects, the activation of ecdysis and tanning are linked to limit the risk of desiccation and predation to the soft, flexible post-ecdysis cuticle. In Drosophila and Manduca, evolution has replaced this program with a period of behavioral flexibility that permits the adult insect to take advantage of sensory information to escape its pupal confinement and to control the time and place in which the expansional behaviors are initiated.
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