Overexpression of broad: a new insight into its role in the Drosophila prothoracic gland cells
Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA
Author for correspondence (e-mail:
lmr{at}u.washington.edu)
Accepted 28 December 2003
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Summary |
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Key words: broad, critical weight, ecdysteroid, metamorphosis, molt, prothoracic gland, Drosophila
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Introduction |
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The products of the broad gene (br; previously called the
Broad-Complex or BR-C) are ecdysteroid-inducible
transcription factors that are involved in pupal commitment at the onset of
metamorphosis (Zhou et al.,
1998; Zhou and Riddiford,
2001
,
2002
). In Drosophila,
br is among those few `early genes' that respond directly to the
ecdysteroids. These primary response genes, including br, E74 and
E75, regulate several secondary response genes, which in turn direct
appropriate biological responses to each ecdysteroid pulse during development
(Russell and Ashburner, 1996
;
Thummel, 1996
,
2002
;
Baehrecke, 2000
). The proteins
encoded by br are members of the Broad-Tramtrack-Bric-a-brac (BTB)
family of transcription factors that share a common N-terminal domain thought
to be important in proteinprotein interactions
(Zollman et al., 1994
). The
alternately spliced C-terminus contains one of four pairs of
C2H2 zinc fingers that putatively bind DNA
(DiBello et al., 1991
;
von Kalm et al., 1994
;
Bayer et al., 1996
; but see
Mugat et al., 2000
). The
br gene is defined by three genetic functions: broad
(br), reduced bristles on palpus (rbp) and
2Bc (Belyaeva et al.,
1980
; Kiss et al.,
1988
). The rbp genetic function is provided by the BR-Z1
isoform and partially by Z4, the br allele by Z2, and 2Bc by
Z3 (Bayer et al., 1997
).
Mutants devoid of all isoforms develop normally to the final larval instar but
fail to form a normal puparium, indicating that br is required for
metamorphosis (Kiss et al.,
1976
,
1988
).
In Drosophila, all the tissues express Broad (BR) proteins during
metamorphosis, but there is both temporal and tissue specificity as to the
predominant isoform present (Huet et al.,
1993; Emery et al.,
1994
; Bayer et al.,
1996
; Mugat et al.,
2000
; Brennan et al.,
2001
; Ghbeish et al.,
2001
). We have shown previously that the br gene
products, especially the Z1 isoform, can induce the expression of
pupal-specific cuticle genes and suppress both larval and adult cuticle genes
in epidermal cells during a molt, thus causing a pupal molt
(Zhou and Riddiford, 2002
).
Recently, br was also found to be necessary for programmed cell death
in the salivary glands; the maximal expression of the death genes head
involution defective (hid) and reaper (rpr)
require br function (Jiang et
al., 2000
). Additionally, BR proteins directly upregulate the
caspase DRONC, which is an initiator caspase essential for programmed cell
death (Cakouros et al.,
2002
).
During our previous studies, we demonstrated that misexpression of the
different BR isoforms by heat-shock induction of their transgenes during the
late second instar disrupted normal third instar cuticle production during the
molt (Zhou and Riddiford,
2002). Unexpectedly, in these studies we found that premature
expression of the Z3 isoform early in the second instar suppressed the molt to
the third instar and resulted in larvae forming miniature puparia after an
extended feeding period. In the present study, we find that the presence of BR
in the PG cells is responsible for this repression of larval molting. We also
demonstrate, for the first time, that BR proteins are involved in the
degeneration of the PG cells during metamorphosis.
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Materials and methods |
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The following transgenic fly lines carrying one of the BR isoform cDNAs
under the control of the heat-shock promoter were used: w1118;
527-5; 708-1 (four copies of hs-BR-Z1), w1118;
cD5-4C; cD5-1 (four copies of hs-BR-Z2), w1118;
797-3; 797-E8 (four copies of hs-BR-Z3) and
w1118; Z4-13; Z4-1 (four copies of
hs-BR-Z4) (Crossgrove et
al., 1996; Bayer et al.,
1997
). w1118 was used as control. To
overexpress BR proteins, transgenic lines were heat shocked in vials for 30
min at 37°C in a water bath at the beginning of the designated time.
Enhancer trap lines expressing GAL4 in the PG cells of the ring gland were
P0206 and P0163 from Dr W. Janning
(http://FlyView.uni-muenster.de),
and those expressing Gal4 in the putative PTTH-producing cells were Feb 211
and Feb 204 (Siegmund and Korge,
2001). When these lines were used, the embryos were collected at
2-h intervals.
Transgene constructs and germline transformation
To construct UAS-BR isoform transgenic lines, the full-length
cDNAs encoding each of the four Z isoforms were cloned individually into the
P[UAST] vectors between the EcoRI and XhoI sites
(Brand and Perrimon, 1993).
These constructs were used in germline transformation as described
(Rubin and Spradling,
1982
).
Starvation and critical weight
For analysis of the critical feeding period necessary for a second-to-third
instar molt, newly ecdysed second instar larvae were allowed to feed on the
standard cornmealmolasses diet for a designated time at 25°C, then
were moved to a protein-free diet containing only 10% D-glucose and
3% agar. The animals were scored for molting at 2428 h after the
ecdysis into the second instar.
For analysis of the critical weight for pupariation, larvae were weighed individually to the nearest 0.002 mg on a microbalance (Mettler M5). The individual was then starved in a vial with wet tissue, and pupariation was scored.
20E feeding experiments
To provide exogenous ecdysteroid, yeast paste or fly diet containing 1 mg
ml1 20-hydroxyecdysone (Sigma, St Louis, MO, USA) in 5%
ethanol was used.
Immunostaining and microscopy
Drosophila larvae were cut open along the dorsal middle line in
phosphate-buffered saline (PBS) and then fixed in 3.7% formaldehyde and
processed as previously described (Zhou
and Riddiford, 2001). Primary antibodies used were anti-BR core
region monoclonal antibody (mAb) at 1:250 dilution
(Emery et al., 1994
), and
polyclonal antibodies for BR-Z1, -Z2, -Z3 and -Z4 isoforms at 1:3000 dilution
(Mugat et al., 2000
).
Secondary antibodies used were fluorescein isothiocyanate (FITC)-conjugated
donkey anti-rabbit antibody and Texas Red-conjugated donkey anti-rabbit at
1:500 dilution (Jackson ImmunoResearch, West Grove, PA, USA). Fluorescent
visualization was with a BioRad MRC-600 confocal laser scanning microscope and
images were processed with NIH Image and Adobe Photoshop.
Photos of prepupae and pharate adult flies were taken with a Cool Snap camera on a Wild dissecting microscope and processed with RS Image software. Images of whole mounts of mouthhooks were captured with a Sony video camera on a Nikon Optiphot microscope and processed with Adobe Photoshop.
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Results |
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In contrast to the effects of BR-Z3 expression, few or no L2 prepupae were
formed after misexpression of the other BR isoforms during the early second
instar (Table 1).
Overexpression of BR-Z1 at this time caused the death of the animal at
4268 h after the heat shock with no detectable third instar
mouthhooks (N=150). Expression of either BR-Z2 or -Z4 caused a high
rate of death at
26 h of the second instar, the time of ecdysis from the
second to the third instar (N=135 and 107, respectively). Double
mouthhooks were seen in these dead larvae.
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To determine the BR expression pattern after the heat shock, we used a mAb
against the BR protein core region (Emery
et al., 1994). The BR protein appeared globally in all the tissues
up to and including 12 h after the heat shock and then gradually disappeared
(Fig. 2A). At 18 h, only trace
amounts remained in the fat body (Fig.
2B), and by 24 h no BR protein was seen in any tissue except the
central nervous system (CNS; Fig.
2C). The BR staining in the CNS, however, was due to the
endogenous Z3 protein, since in wild-type and w1118
larvae, the CNS normally expresses the Z3 isoform in late embryogenesis
through larval life (Fig. 2F;
Zhou, 2000
; B.Z., D. Williams,
L.M.R. and J.W.T., unpublished). By 36 h, BR began to reappear in the tissues
of these persisting second instar larvae
(Fig. 2D,E). This later round
of expression of br matches the tissue-specific expression seen
during the normal onset of metamorphosis in the mid-to-late third instar
(Crossgrove et al., 1996
;
Mugat et al., 2000
).
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Relationship between the critical feeding period and the effects of BR-Z3 misexpression
Based on the above observations, we hypothesized that misexpression of
BR-Z3 before the synthesis and release of ecdysteroids in the second instar
prevented the later rise of the ecdysteroid titer, so that the larvae did not
molt but continued to eat and grow. Then, once these L2 larvae reached a
critical weight, the metamorphic program was initiated, leading to the
formation of L2 prepupae. To test this hypothesis, we determined the critical
weight necessary for the second-to-third instar larval molt and the period
when misexpression of BR-Z3 could induce the formation of L2 prepupae.
After molting to a given larval instar, many insect larvae must achieve a
certain critical weight before they can initiate the molt to the next instar
(Truman, 1972;
Nijhout, 1981
;
Gilbert et al., 2002
). To
determine whether a similar critical weight exists for the molt from the
second to third instar in Drosophila, we allowed newly ecdysed second
instar larvae to feed on standard cornmealmolasses diet for various
times, then transferred them to a diet containing only glucose. The glucose
diet supported the energetic requirements of the larvae but not further
growth. The animals were then scored for molting at 2428 h after
ecdysis into the second instar. Only
5% of second instar larvae that were
5 h old or younger at the time of the switch to the glucose diet were able to
molt to the third instar (Fig.
3A). By contrast, 71% of 7-h-old larvae and all animals older than
8 h at the time of the switch successfully ecdysed to the third instar at the
normal time. The critical feeding period for the second-to-third instar molt
as defined by the 50% effective time (ET50) was
6.5 h
(Fig. 3A). By that time, the
larvae had attained a weight of 0.15 mg on average (N=46). This
weight then is the critical weight that the larva must achieve before it can
initiate the molt to the third larval instar.
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As seen in Fig. 3B, heat-shock induction of BR-Z3 at any time within the first 5 h of the second instar caused most larvae (>74%, N=55) to continue to eat and grow without entering the third instar, and these larvae eventually formed L2 prepupae. Ectopic expression of BR-Z3 in second instar larvae that were 7 h or older, by contrast, did not prevent forming the pharate third instar, although most did not ecdyse. The ET50 for induction of the formation of L2 prepupae was 5.8 h after entering the second instar (Fig. 3B). This time point is slightly earlier than the critical feeding period for the second-to-third instar molt, which is probably explained by the lag between heat shock and the appearance of the BR-Z3 protein. Our data strongly suggest that ectopic expression of BR-Z3 before the initiation of the endocrine events for the third instar larval molt prevents the normal molting rise of the ecdysteroid titer.
The effect of 20E on the BR-Z3 induced prolonged second instar larvae
If the prolonged second instar caused by misexpression of Z3 was indeed due
to the suppression of the larval ecdysteroid rise, one should be able to
rescue the larval molt by giving exogenous 20E to these animals. We therefore
fed 20E to the second instar larvae expressing BR-Z3 after heat-shock
treatment. In all the subsequent experiments, BR-Z3 was induced by a heat
shock at 5 h after ecdysis to the second instar. After the heat shock, the
larvae were transferred to yeast paste containing 1 mg ml1
20E (referred to as `20E diet' hereafter). All of these larvae subsequently
underwent a larval molt, indicating that 20E could initiate a larval molt
despite the induced BR. This result shows that the second instar larval
feeding period was probably prolonged due to the lack of ecdysteroids.
Interestingly, if larvae were fed on regular diet for more than 20 h after the
induction of BR-Z3 by the heat shock, then transferred to the 20E diet, most
of them continued to eat and formed L2 prepupae after a similar time period as
those continuously fed on regular diet (N=65). Thus, there is a time
after which these larvae can no longer undergo a larval molt, even when
challenged with 20E.
Feeding 20E to the second instar larvae in which BR-Z3 was induced early
will cause them to molt to the third instar, but ecdysis was not successful
and all died with double mouth hooks (N=129). This blockage at
ecdysis is the same as seen when BR-Z3 is induced after the critical
feeding period. The BR-Z3 protein induced by heat shock persisted for 15
h in various tissues (Fig. 2).
To determine if it was the presence of BR-Z3 that caused the lethality during
the ecdysis to the third instar, we placed larvae on a glucose diet for
2024 h after the heat shock. The BR-Z3 protein should completely
disappear during this period (Fig.
2C) when the larvae are unable to grow (i.e. remain below the
critical weight for pupariation). After transfer to the 20E diet, these larvae
formed pharate third instar larvae but still died during ecdysis
(N=91), indicating that the lethality was probably due to the
downstream effects of globally expressed BR-Z3.
Critical weight for L2 pupariation
To determine the critical weight necessary for L2 pupariation, L2 larvae
that had overexpressed BR-Z3 at 5 h after ecdysis were collected at various
times after the heat shock, weighed and held in moist vials without food.
Siblings that had not experienced the heat-shock treatment served as controls.
As seen in Fig. 4, these
BR-Z3-expressing larvae showed a critical weight of 0.45 mg, with those larger
than this size typically forming an L2 puparium. These BR-Z3-expressing larvae
required 32 h after the heat shock [i.e. 85 h after egg laying (AEL)] to
attain this critical weight. The critical weight for L3 pupariation of the
control siblings in this study was 0.80 mg, which was attained by
7 h of
growth in the third instar (i.e. 79 h AEL). A newly ecdysed third instar
w1118 larva weighed 0.52 mg on average
(N=40).
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In view of the different critical weights that are used for L2 and L3
pupariation, we wished to determine whether there is a critical weight for
forming L1 prepupae. When BR-Z3 was induced in early first instar larvae by
heat shock at 25 h, none formed an L1 prepupa (N=60). Instead,
these larvae ecdysed to the second instar but with an 18-h delay at 25°C.
Heat induction of BR-Z3 at 4 h after hatching followed by two heat shocks at
16 h and
28 h prevented 96% of the larvae (N=23) from
molting to the second instar. These permanent first instar larvae continued
feeding and grew for
4 days but were unable either to molt to the second
instar or to form puparia.
As previously noted, transfer of second instar larvae expressing BR-Z3 to the 20E diet caused either a larval molt or formation of L2 prepupae, depending on the time of transfer. As evident in Fig. 4, the switch in response to 20E corresponds to the time that the larvae reached 0.30 mg, which is 0.15 mg less than the critical weight (0.45 mg) determined by starvation. This lower weight (0.30 mg) defines the animal's competency to form a prepupa, since larvae above this weight responded to 20E by initiating metamorphosis. Therefore, we will refer to this weight as the `threshold weight' for metamorphosis. The second critical weight (0.45 mg) determines when the endocrine events involved in the larvalpupal transition are initiated; after attaining this weight, a larva can pupariate without further feeding and on the same time schedule as larvae that continue to feed.
BR-Z3 protein affects functioning of the PG cells, not that of the putative PTTH cells
The data above suggest that the global expression of BR-Z3 may prevent the
ecdysteroid rise in a time-restricted manner. However, these data do not show
which tissue or cells mediate this action of BR-Z3. We therefore used the
GAL4UAS system to target the BR-Z3 expression in selected components of
the neuroendocrine system. The PG cells of the ring gland are the predominant
source of ecdysone, and the synthesis and release of ecdysone is assumed to be
controlled by the brain neuropeptide PTTH
(Gilbert et al., 2002).
Tissue-specific GAL4 enhancer trap lines were used to express BR-Z3 in either
the PG-LP neurons in the brain (the putative PTTH neurons) or the PG cells of
the ring gland.
Two GAL4 enhancer trap lines were used to express BR-Z3 in the PG-LP
neurons. Line Feb 211 has strong expression of GAL4 in the PG-LP neurons and
weak expression in a brain lateral neurosecretory neuron (CC-LP 2), while Feb
204 weakly expresses GAL4 in the PG-LP neurons as well as in a few
neurosecretory neurons of the medial subesophageal ganglion (CC-MS 1 and CC-MS
2) (Siegmund and Korge, 2001).
When the GAL4 line Feb 211 was crossed with a UAS BR-Z3 line, their progeny
underwent normal larval and pupal molts (N=57). However, these
animals failed to eclose completely, with their abdomens remaining in the
puparial cases. Similarly, when the GAL4 Feb 204 line was crossed with the UAS
BR-Z3 line, most of the progeny formed puparia (78%, N=127). All the
prepupae successfully pupated (N=55),
18% of them arrested at
stage P7, 12% arrested as pharate adults, 14% partially eclosed and 56% fully
eclosed. Hence, ectopic expression of BR-Z3 in the putative PTTH-producing
cells has no effect on larval molting or pupariation, although it does
interfere with events late in the metamorphic molt.
To determine if BR-Z3 affects the ecdysteroid titer through effects on the PG cells, we crossed the UAS BR-Z3 line with two lines that express GAL4 in the PG cells and selected other tissues. Line P0206, which has a P element insertion upstream of snail (Eugenio Gutierrez and Alex Gould, personal communication), specifically expresses GAL4 in the ring gland (both the PG cells and the CA cells), salivary gland and oenocytes during later embryonic and larval stages (http://FlyView.uni-muenster.de; data not shown). Approximately 53% of the larvae (N=260) expressing BR-Z3 under control of this driver became permanent first instar larvae surviving for 46 days. Of those larvae that ecdysed to the second instar (N=47), 81% were permanently arrested in the second instar, only 19% ecdysed to the third instar and less than 1% formed L2 prepupae (Fig. 5C). When another enhancer trap line P0163, which also expresses GAL4 in the ring gland (http://FlyView.uni-muenster.de), was crossed with the UAS BR-Z3 line, 63% of the progeny (N=76) remained in the first or second instar (data not shown). These observations strongly suggest that misexpression of BR-Z3 affects the ecdysteroid titer through the ring gland, probably through the PG cells themselves.
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Driving other BR-Z isoforms in the PG cells also blocks molting and can be rescued by feeding 20E
To investigate the effects of the other BR-Z isoforms on PG function, we
used P0206 to drive each of the Z isoforms in the ring gland. Approximately
99% of larvae expressing BR-Z1 and 78% of larvae expressing BR-Z2 in the ring
gland remained in the first instar (Fig.
5A,B). None of the second instar larvae expressing BR-Z2 molted to
the third instar although 4% formed L2 prepupae
(Fig. 5B). When the Z4 isoform
was expressed in the ring gland, all eventually became third instar larvae,
but only 8% pupariated (Fig.
5D).
To determine whether the defects in molting seen after expression of the
BR-Z isoforms in the ring gland are due to the lack of ecdysteroid secretion,
we fed 20E to these animals. Since our starvation experiments suggested that a
critical feeding period of 6.5 h is required for a larval molt
(Fig. 3A), we fed 20E diet to
these BR-expressing animals at
8 h after hatching. These larvae molted to
the second instar (Fig. 5).
Continuing exposure to 20E diet, however, caused high lethality. When the
freshly molted second instar larvae were placed on normal diet for 8 h, then
transferred to 20E diet, most subsequently ecdysed to the third instar. When
20E was given to the `rescued' third instar larvae after one day of feeding on
normal diet, approximately 69%, 52%, 68% and 81% of the larvae misexpressing
the BR-Z1, -Z2, -Z3 and -Z4 isoforms, respectively, pupariated
(Fig. 5). When 20E-rescued
second instar larvae expressing BR-Z1, -Z2 or -Z3 were placed on standard
diet, they grew but remained in the second instar. When transferred to 20E
diet after 2 days on standard diet, most of them formed L2 prepupae rather
than third instar larvae (Fig.
5AC), presumably because they had surpassed the critical
weight for formation of L2 prepupae. Our data suggest that when any of the
BR-Z isoforms are ectopically expressed in the PG cells, various degrees of
ecdysteroid deficiency ensue, resulting in the cessation of a larval molt or
pupariation.
Degeneration of the PG cells after ectopic expression of BR
Tonic expression of BR-Z3 in the ring gland under the control of the P0206
Gal4 driver caused degeneration of the PG portion of the ring gland by 2 days
after ecdysis to the second instar (Fig.
6A, right). The CA were unaffected although the ectopic expression
of BR-Z3 was throughout the ring gland. To visualize the process of
degeneration of the PG portion of the ring gland, we used P0206 to drive each
of the BR isoforms as well as green fluorescent protein (GFP) in the PG cells.
Expressing GFP alone in the PG cells did not affect their growth during larval
life (Fig. 6B, first row). By
contrast, expression of BR-Z1 caused partial degeneration of the PG part of
the ring gland by 24 h after hatching and disappearance by 35 h
(Fig. 6B, second row). The
BR-Z2 isoform caused severe degeneration by 9 h after hatching, whereas
degeneration induced by the BR-Z3 isoform was nearly complete at 48 h
(Fig. 6B, third and fourth
rows). In contrast to the other BR isoforms, BR-Z4 appeared to be rather
ineffectual in causing PG cell degeneration
(Fig. 6B, bottom row). Thus,
the BR isoforms varied in their effectiveness in causing the degeneration of
the PG cells.
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Normal expression pattern of BR in the PG cells
To elucidate the role of BR proteins in the PG cells during metamorphosis,
we determined the normal expression pattern of the four BR isoforms. BR-Z2 and
-Z3 appear in the PG cells 17 h after ecdysis to the third instar (89 h
AEL) and remain at low levels through pupariation and the prepupal period
(Fig. 7), showing little
relationship with the changing ecdysteroid titer through this period. By
contrast, BR-Z4 begins increasing slowly about 96 h AEL, then more rapidly at
the time of wandering. BR-Z1 appears last, beginning at wandering, and also
attains high levels by pupariation. Both of the latter isoforms apparently are
responsive to 20E, which has increased to peak levels at this time
(Riddiford, 1993
).
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Discussion |
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Critical weight and body size
For many insects, especially Lepidoptera, larval size is an important
factor in regulating both larval molting and the onset of metamorphosis.
Within a larval stage there is typically a minimal time that a larva must feed
before it is competent to undertake another larval molt, originally called the
`period of indispensable nutrition'
(Bounhiol, 1938;
Nijhout, 1981
). In addition,
there is a size that the larva must attain before it can begin metamorphosis
(the critical weight for metamorphosis;
Nijhout, 1981
). This size is
thought to be largely independent of the number of larval molts required to
reach it since larvae of the silkworm Bombyx mori undergo a series of
extra larval molts at small sizes when fed ecdysone but eventually initiate
metamorphosis at the appropriate size
(Tanaka and Takeda, 1993
). For
second instar Drosophila larvae, the period of indispensable
nutrition extends through the first 6.5 h of the instar, by which time the
larva has attained a weight of 0.15 mg. By the time that the larva ecdyses to
the third larval stage, its weight has increased to 0.52 mg.
The critical weight for metamorphosis is usually determined by starving
larvae of various weights, then determining the timing of the subsequent onset
of metamorphosis (Nijhout,
1975,
1981
). Using this criterion,
we obtained two values for this critical weight depending on whether the larva
was forming a puparium in the third instar (0.8 mg) or in the second instar
(0.45 mg). The 0.8 mg for the w1118 strain is slightly
lower than the 0.9 mg found by Partridge et al.
(1999
) for their wild-type
strain. It is important to stress that these weights indicate the condition of
the larva when it initiated the metamorphic events but not necessarily its
state when it becomes competent to metamorphose. The latter
`threshold weight' for metamorphosis was established by challenging larvae
with 20E at various times. When this was done for L2 larvae whose molting had
been suppressed by BR-Z3 misexpression, we found that the metamorphic
threshold weight was 0.3 mg. We suggest that this threshold may be assessed
independently of which instar the larva is in. First instar larvae cannot
attain this threshold weight despite experimental manipulations that ensure
prolonged feeding, probably because of cuticular restraints on their growth,
and thus cannot pupariate. Normally growing second instar larvae traverse this
threshold weight only after they have already initiated the molt to the third
larval stage. Since the metamorphic threshold is crossed before the last
instar is begun, higher Diptera such as Drosophila lack the molting
plasticity seen in lepidopteran larvae. Consequently, in higher flies, a
supernumerary fourth larval stage is never produced, irrespective of feeding
conditions in the final larval stage. By interfering with ecdysteroid release
in the second larval stage, however, larvae can attain the metamorphic
threshold before being completely engaged in a larval molt and, hence, they
can switch over into a precocious metamorphic program.
The formation of L2 prepupae is also reported for mutants of dre4,
iptr and E75A (Sliter and
Gilbert, 1992; Venkatesh and
Hasan, 1997
; Bialecki et al.,
2002
). These genes are known to affect the regulation of
ecdysteroidogenesis in the PG cells. Thus, although these larvae may surpass
the critical weight to initiate that larval molt, the onset of the molt is
probably delayed because of the defective production of ecdysteroids. During
the prolonged feeding, they have a chance to surpass the threshold weight for
metamorphosis and begin premetamorphic changes. These larvae then regain the
ability to produce some ecdysteroids, so they can form L2 prepupae. For
example, young second instar larvae of a temperature-sensitive dre4
mutant molt to the third instar after a temperature shift from 31°C to
22°C, while prolonged second instar larvae will form L2 prepupae after
such a temperature shift (Sliter and
Gilbert, 1992
).
Misexpressed BR proteins block the rise of ecdysteroid titer for larval molting
We show here for the first time that br gene products can
interfere with ecdysone synthesis and/or release by the larval PG cells. BR
appears to have two types of effects on the PG cells: short-term effects on
physiology of ecdysteroid synthesis and release, and longer-term effects that
lead to eventual degeneration of the glands. We have concluded that the
suppression of the larval molt caused by ectopic br expression is due
to a suppression of ecdysone synthesis and release. This conclusion is based
on rescue of larval molting in these larvae by supplying them with 20E in the
diet. For the heat-shocked larvae, the 20E caused a larval molt but the
animals were blocked at ecdysis. The latter effect, though, is presumably due
to persistent BR effects in other larval tissues rather than the PG cells. The
same 20E treatment resulted in normal larval molting and ecdysis of larvae
that had larval molting suppressed by BR expression primarily in the ring
gland.
The short-term action of BR-Z3 on the PG cells after heat shock induction
clearly did not cause degeneration in the cells since they became functional
once the induced BR disappeared so that L2 prepupae were formed. Similarly, a
heat-shock-induced pulse of BR-Z1 in the early second instar prevented larvae
from molting into the third instar, indicating that BR-Z1 also prevented the
rise of the ecdysteroid titer. After this treatment, no L2 prepupae were
formed. Instead, the larvae died 50 h after the heat shock, indicating
that some other essential tissues and/or organs had been adversely affected by
the transient global expression of BR-Z1. Interestingly, neither BR-Z2 nor
BR-Z4 had adverse effects on ecdysone biosynthesis or release in this
paradigm, because pharate third instar larvae were formed. These however could
not ecdyse, presumably due to other adverse effects of the presence of the
particular BR isoform.
The manner by which BR-Z1 or -Z3 interferes with the functioning of the
larval PG cells is unclear. During a larval molt there is no BR expression in
PG cells but there is prominent BR expression during the time of the
pupariation peak of ecdysteroid (Fig.
7). Moderate levels of BR-Z2 and -Z3 are evident in the gland
early in the third instar while BR-Z4 becomes prominent early in the
ecdysteroid rise and BR-Z1 appears strongly at the pupariation peak of
ecdysteroid. The late appearance of BR-Z1 would be consistent with its
involvement in the suppression of ecdysone biosynthesis and release that has
been noted at this time (Dai and Gilbert,
1991), and hence in the normal decrease of the ecdysteroid titer
that occurs. Such an explanation is not possible for the effect of BR-Z3 since
it is normally present throughout the metamorphic ecdysteroid peak. An
alternative possibility is that BR-Z3 does not affect ecdysone biosynthesis
directly but changes the sensitivity of the PG cells to growth-related
factors. For example, factors from over-proliferating or regenerating imaginal
discs are able to suppress the metamorphic ecdysone surges and prolong larval
life (Simpson et al., 1980
;
Bryant and Simpson, 1984
).
Such factors would not be expected to interfere with larval molting that
normally occurs as the discs are rapidly proliferating. Expression of BR-Z3 in
the PG cells may be necessary for the glands to become responsive to these
factors.
A recent study on overexpression of BR isoforms during the third instar
further supports the suppressive function of BR on ecdysteroid biosynthesis
(Kuchárová-Mahmood et al.,
2002). Each of the BR isoforms was heat-shock induced before the
rise of the small pulse of ecdysteroid titer that triggers the wandering
behavior of larvae (Riddiford,
1993
). When BR-Z1, -Z3 or -Z4 was ectopically expressed in
9096-h-old feeding larvae, the feeding period was prolonged and
pupariation delayed by 824 h. Ectopic expression of BR-Z2 during the
mid-third instar, however, did not postpone pupariation, consistent with our
observations that heat-shock-induced BR-Z2 in the second instar does not
prevent larval molting. The BR-Z4 protein, whose ectopic expression can delay
pupariation but is unable to prevent larval molting, may have some
stage-specific activities.
In contrast to the effects of BR proteins, the ecdysteroid-induced orphan
nuclear receptor E75A apparently promotes ecdysteroidogenesis
(Bialecki et al., 2002).
Approximately 55% of null E75A mutant larvae either arrest in the
second instar or in the molt to the third instar, but the molt can be rescued
by feeding 20E. An additional 20% form L2 puparia after a prolonged feeding
period of about the same duration as we find after a pulse of BR-Z3 expression
at 5 h after ecdysis. The ecdysteroid titer of these E75A null second instar
larvae remains low for the first 36 h after ecdysis but must rise later in
those undergoing L2 pupariation. Neither E75B nor E75C null mutants show any
problems in larval molting or the onset of metamorphosis, indicating the lack
of effect of these other isoforms on the ecdysteroid titer. Thus, E75A and
BR-Z3 may act as positive and negative regulators, respectively, of
ecdysteroid biosynthesis and release.
BR proteins cause the degeneration of the PG cells
In Drosophila during metamorphosis, the PG portion of the ring
gland gradually degenerates. Its ecdysteroid biosynthetic activity (as
measured by synthesis during a short term culture in vitro)
dramatically decreases at pupariation followed by a more gradual decrease
during the onset of adult development (Dai
and Gilbert, 1991). At 24 h after pupariation (AP), lysosome-like
structures invade the organelles in the PG cells and form giant autophagic
vacuoles by 48 h AP. Only a few remnants of the PG cells are present at adult
eclosion. We show here that BR normally appears in the PG cells
17 h
after ecdysis to the final larval instar, with the Z2 and the Z3 isoforms
appearing first at low levels followed later by high levels of BR-Z1 and -Z4
at the time of pupariation. Since BR-Z1, -Z2 and -Z3 isoforms effectively
cause the gradual degeneration of the PG cells, it is likely that the
appearance of BR proteins at pupariation initiates the progressive involution
of the gland.
Unlike the slow procedure of the degeneration of the PG cells, the larval
salivary glands undergo rapid cell death within a few hours after pupation
(Lee and Baehrecke, 2001). The
larval salivary gland degenerates soon after pupal head eversion presumably in
response to a pulse of ecdysteroid. Steroid-induced BR-Z1, ßFTZ-F1, E74A
and E93 are involved in the regulation of salivary gland programmed cell death
(Jiang et al., 2000
;
Lee and Baehrecke, 2001
;
Lee et al., 2002
). In the
rbp5 mutant, which lacks the BR-Z1 isoform, the
transcription of the cell death genes rpr and hid is
dramatically reduced (Jiang et al.,
2000
; Lee et al.,
2002
). In agreement with these studies, we found that in larvae
misexpressing BR-Z1 in both the PG cells and the salivary glands, the salivary
glands degenerated precociously in larvae rescued to the third instar by 20E
treatment. By contrast, if these larvae were left in standard diet after
hatching and stayed as permanent first instar, their PG cells degenerated
within 35 h (Fig. 6) but their
salivary glands remained intact for at least 72 h (X.Z. and L.M.R.,
unpublished). These observations suggest that in the PG cells, BR is
sufficient to initiate and coordinate the program of degeneration even in the
absence of 20E and molting. For the cell death of the salivary gland, by
contrast, both BR and other 20E-induced factors are required.
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References |
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