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First published online 2 January 2008
doi: 10.1242/dev.016097
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Biology Center, Czech Academy of Sciences and Department of Molecular Biology, University of South Bohemia, Branisovska 31, Ceske Budejovice 37005, Czech Republic.
* Author for correspondence (e-mail: jindra{at}entu.cas.cz)
Accepted 6 November 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Metamorphosis, Juvenile hormone, Broad-Complex, Methoprene-tolerant, Tribolium castaneum
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Current models propose that two types of lipophilic hormones regulate the
entry to metamorphosis. In the absence of the sesquiterpenoid juvenile hormone
(JH), ecdysteroids initiate the pupal program, in which larval structures are
transformed into imaginal ones and new pupal cuticle is deposited
(Riddiford, 1994
;
Buszczak and Segraves, 2000;
Gilbert et al., 2000;
Thummel, 2001;
Dubrovsky, 2005;
Truman and Riddiford, 2007).
The role of JH is anti-metamorphic, as in the presence of JH the larva cannot
pupate but molts to another larva. Our knowledge about the molecular events
underlying metamorphosis is primarily based on the Drosophila model.
Unfortunately, fly development has largely lost dependence on JH, as constant
exposure to JH cannot prevent entry to the pupal program
(Riddiford and Ashburner,
1991; Restifo and Wilson,
1998). Thus, in response to an elevated ecdysteroid titer,
Drosophila larval epidermis dies and the adult head and thorax, with
appendages, develop from imaginal discs, while the abdominal epidermis
proliferates from histoblasts (Madhavan
and Schneiderman, 1977; Bayer
et al., 1996b). Only at this later stage does ectopic JH interfere
with Drosophila metamorphosis, most notably with the making of the
adult abdomen (Postlethwait,
1974; Riddiford and Ashburner,
1991; Restifo and Wilson,
1998; Zhou and Riddiford,
2002). By contrast, both ecdysteroid and JH signals are necessary
to coordinate metamorphosis in typical holometabolous larvae, whose
polymorphic epidermal cells sequentially produce first larval, then pupal and
finally adult structures (Riddiford,
1994), while internally growing imaginal discs, if any occur, give
rise to appendages only, particularly the wings
(Svacha, 1992).
Seminal studies in Drosophila have defined the ecdysteroid
signaling pathway. The heterodimeric nuclear receptor consisting of EcR and
Usp (Thomas et al., 1993;
Yao et al., 1993) regulates
primary ecdysteroid-response genes that encode transcription factors
(Buszczak and Segraves, 2000).
Of these transcription factors, E74
(Fletcher et al., 1995) and
Broad-Complex (BR-C) are specifically required for metamorphosis.
(BR-C has been designated as br by FlyBase, but, in keeping
with nomenclature commonly used in the literature and to avoid confusion with
the BR-C complementation group br, we use the original
name.)
The BR-C gene encodes a Broad-Complex-Tramtrack-Bric-a-brac (BTB)
domain with one of four alternatively spliced C2H2 zinc-finger motifs Z1-Z4
(DiBello et al., 1991;
Bayer et al., 1996a). Mutants
in the isoform-specific regions form three complementation groups (br,
rbp and 2Bc), and display both specific and partly overlapping
defects in the differentiation of adult tissues and the death of larval ones;
loss of the entire gene in nonpupariating (npr1) mutants
blocks metamorphosis completely (Belyaeva
et al., 1980; Kiss et al.,
1988; Restifo and White,
1992; Fletcher and Thummel,
1995; Bayer et al.,
1997). The function of BR-C is conserved at least in
lepidopterans, as BR-C Z4 from Manduca sexta partially
rescues the rbp mutants (Bayer et
al., 2003), and corresponding metamorphic defects result from
BR-C RNAi in the silkmoth Bombyx mori
(Uhlirova et al., 2003).
However, because Diptera and Lepidoptera represent advanced and related insect
orders, it is of interest to examine the role of BR-C in other
holometabolans.
BR-C is an attractive target of the as yet poorly characterized JH
signaling. Studies in Manduca
(Zhou et al., 1998) and
Bombyx (Reza et al.,
2004) have shown that removal of the JH-producing corpora allata
glands causes ectopic BR-C induction as well as precocious pupation,
whereas exposure of larvae to JH prevents both BR-C transcription and
pupation. BR-C is therefore thought of as the JH-dependent switch
between larval and pupal programs (Zhou
and Riddiford, 2002;
Dubrovsky, 2005), but causal
genetic evidence for this idea is missing. In the absence of a bona fide JH
receptor, it is also unclear how JH might influence BR-C
expression.
An excellent model with which to address these problems is the red flour
beetle Tribolium castaneum, in which JH exerts its classical
anti-metamorphic effect. We have recently shown that perturbed function of the
Tribolium ortholog of the Drosophila Methoprene-tolerant
(Met) gene causes larvae to metamorphose prematurely, before reaching
their final instar (Konopova and Jindra,
2007). This phenotype is compatible with Drosophila Met
mutation conferring resistance to JH and its mimic methoprene
(Wilson and Fabian, 1986;
Ashok et al., 1998), and this,
together with the high-affinity binding of Met to JH
(Shemshedini and Wilson, 1990;
Miura et al., 2005), makes Met
currently the best candidate for a JH receptor. Interestingly, Met
and BR-C mutations interact genetically during Drosophila
development (Wilson et al.,
2006).
In this study, we use Tribolium and a neuropteran, lacewing Chrysopa perla, to show that the primary role of BR-C in directing the larval-pupal transition may have been present in primitive holometaboly. We propose that this role is a temporal coordination of tissues during the metamorphic process, which is at least partly achieved by Met-dependent JH regulation of BR-C expression.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), with primers mapping to the BTB and Z2 zinc-finger domains.
PCR with a degenerate primer set 5'-AARWSIACICCITGYAARCAYCC and
5'-CKISWRCARTAIACICKYTCRCA was followed by PCR with nested primers
5'-TAYCAYGGIGARGTIAAYGTNCA and 5'-TAYTCYTCYTGICKIWCNGCRTG.
BR-C cDNA sequences from both species are available from GenBank
(accession numbers EU200752-EU200758).
Animals, RNAi and mutant analysis
Wild-type Tribolium castaneum, strain San Bernardino (obtained
from G. Bucher, Georg August University, Goettingen) was reared at 32°C
and staged as described (Konopova and
Jindra, 2007). Under constant conditions, pupation takes place
after seven or eight larval instars. Line KS342 carrying a
piggyBac insertion within the TcBR-C gene produced by the
GEKU screen was kindly provided by S. Brown (Kansas State University,
Manhattan, KS). An enhancer-trap line pu11 that marks developing
wings (Lorenzen et al., 2003;
Tomoyasu and Denell, 2004) was
a gift from Y. Tomoyasu (Kansas State University, Manhattan, KS). Chrysopa
perla larvae were maintained at 24°C on a culture of live aphids
(Acyrthosiphon pisum).
dsRNA of the indicated lengths (see
Table 2) was prepared by using
T3 and T7 MEGAscript kits (Ambion, Austin, TX). dsRNA concentrated up to 5
µg/µl was injected into the abdomen of CO2 anesthetized
Tribolium or Chrysopa larvae as described
(Tomoyasu and Denell, 2004;
Konopova and Jindra,
2007).
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) with
primers 5'-GCAAAATTGCATCCGAGAAC and 5'-GTTTGCTTCACCGATATGAC
flanking the site of piggyBac insertion.
Methoprene treatment
Early Tribolium prepupae were injected with TcMet or
egfp dsRNA and allowed to pupate. These RNAi pupae or intact pupae
0-4 hours after ecdysis were briefly dipped into 0.3 mM methoprene (VUOS,
Pardubice, Czech Republic) in acetone, or into acetone alone
(Konopova and Jindra, 2007).
At the desired stage the pupae were subjected to mRNA expression analysis.
mRNA expression analysis
cDNA was prepared from 2 µg of total RNA isolated with TRIzol reagent
(Invitrogen, Carlsbad, CA) and treated with DNase (Roche, Mannheim, Germany)
as described (Konopova and Jindra,
2007). cDNA samples diluted 5-fold with water were subjected to
standard PCR reactions with Taq DNA polymerase (Unis, Top-Bio, Prague, Czech
Republic); the GC-rich Z3 cDNA was amplified with Phusion DNA polymerase
(Finnzymes, Espoo, Finland). For primer sequences and cycle numbers see
Table 1.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Bayer et al.,
1996a; Ijiro et al.,
2004; Nishita and Takiya,
2004; Reza et al.,
2004). Although in these species and in mosquitoes
(Chen et al., 2004) no more
than four isoforms have been found, an additional exon in Tribolium
(Fig. 1A) predicts a fifth
zinc-finger domain (Z5), whose sequence does not match any of the known BR-C
proteins. All five isoforms produce transcripts with both of the alternatively
spliced 5' untranslated exons. The sequence homology together with the
conserved microsynteny of the zinc finger-encoding exons demonstrates that the
gene we have isolated is a Tribolium BR-C ortholog.
Continuous TcBR-C expression peaks during the larva-pupa transition
To identify stages at which TcBR-C is expressed and potentially
required, we analyzed cDNA samples prepared from embryos, fifth and eighth
(final) instar larvae, pupae and adults. The transcript region common to all
TcBR-C isoforms was detected continuously at moderate levels, with a
strong peak rising at the onset of the prepupal (i.e. pharate pupal) stage and
declining soon after ecdysis in early pupae
(Fig. 1C, top row). To see
whether there was any stage specificity in the presence or predominance of
individual isoform mRNAs, we repeated RT-PCR with primer sets, each of which
was able to amplify only one of the five zinc finger-encoding exons.
Figure 1C shows that isoforms
Z1, Z4 and Z5 were mainly responsible for the sharp rise of total
TcBR-C mRNA at 72 hours of the eighth instar, whereas isoforms Z2 and
Z3 showed a more gradual increase. Nevertheless, all isoform-specific mRNAs
could be detected at all examined stages. These data suggest that
TcBR-C may play a role throughout development, although its
upregulation during the onset of metamorphosis indicates its requirement for
pupal differentiation.
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A prepupa is a pharate pupa in which larval cuticle has been apolysed and the newly deposited cuticle has attained pupal characters. However, removal of the apolysed larval cuticle from the arrested TcBR-C(RNAi) animals revealed recurrence of some larval features and disruption of the normal pupal morphogenesis (Figs 2, 3). These severely affected animals had an overall larval appearance with only rudimentary pupal wings (Fig. 2B'). The same lethal phenotype (Fig. 2C) was observed in 23% (n=260) of progeny produced by crossing beetles heterozygous for a piggyBac insertion KS342 within TcBR-C (Fig. 1A). Similar to the effect of RNAi, the mutant prepupae possessed larval urogomphi and vestigial wings, and they lacked the pupal-specific cuticular structures called gin traps (Fig. 2C-G). As would be expected from the almost 1:3 ratio of dying to normally developing animals, the arrested prepupae were homozygous for the KS342 mutation (see Materials and methods). TcBR-CKS342 homozygotes had reduced levels of mRNAs encoding isoforms Z2 and Z3 (Fig. 2H), whose exons resided farther from the piggyBac insertion than exons Z1 and Z4 (Fig. 1A). We speculate that splicing of the Z2 and Z3 mRNA products might be compromised by the insertion, but further analysis is necessary. These results show that BR-C is required for pupal development.
Closer examination revealed that although cuticle in severely affected TcBR-C(RNAi) animals lacked the long larval setae, it remained smooth, without the microsculpture typical for pupae (Fig. 3A, part d; Fig. 3B, part d). The urogomphi retained their larval shape (Fig. 3A) and the gin traps were missing (Fig. 3C) in strongly affected TcBR-C(RNAi) prepupae. Other structures appeared to be less dependent on normal TcBR-C function and continued the pupal or adult program in arrested animals. For instance, distal abdominal segments differentiated pupal characters such as the genital papillae, but abnormalities were clearly visible (Fig. 3A, part d). The compound eyes developed several rows of ommatidia, although these were not evenly spaced as in normal pupae (Fig. 3D; Fig. 2D,E). Although the antennae lacked sensillae, they apparently developed towards the adult fate, with their club shape and clear separation of segments (Fig. 3E). Finally, the legs of TcBR-C(RNAi) animals lost the larval character as they possessed the double claws that normally develop in pupae and are typical for the adult leg (Fig. 3F). Slightly accelerated development was suggested by more distinctly separated tarsal segments and sharper claws in TcBR-C(RNAi) animals relative to normal pupae (Fig. 3F).
For a better insight into how TcBR-C effects the larval-pupal transition, we elicited a milder phenotypic response by diluting the dsRNA up to a 1000-fold. These lowered dsRNA doses were still lethal but allowed prepupae to ecdyse. All such treated animals had noticeably shortened and blistered wings and legs relative to control pupae (Fig. 2A,I). Compared with strong RNAi phenotypes, some pupal characters became more prominent. The animals developed gin traps, albeit aberrant (Fig. 2I; Fig. 3C, part e), their urogomphi were elongated, genital segments were nearly perfectly differentiated (Fig. 3A, part e), and the cuticle surface had a pupal-like microsculpture (Fig. 3B, part e). Except for the absence of sensory bristles, the antennae resembled the adult ones (Fig. 3E, part e). Conversely, legs with less prominent claws and segment borders suggested a weaker acceleration of the adult program than that observed with the strong RNAi effect (Fig. 3F).
Effects of TcBR-C isoforms
In Drosophila, some functions are shared by all BR-C proteins,
whereas others are fulfilled by a specific isoform
(Bayer et al., 1997). To see
whether any unique functions apply to Tribolium BR-C isoforms, we
injected larvae with dsRNA against each of the five zinc finger domains
separately. Probably due to the limited sequence lengths, transcript levels of
the targeted isoforms declined only partially
(Fig. 4A). All larvae treated
for a single isoform ecdysed into pupae displaying a degree of aberrancies
increasing in the order of isoforms: Z5<Z1<Z4<Z3<Z2, with the most
visible effect being the shortening of the wings and legs
(Fig. 4; see also Fig. S1 in
the supplementary material). These pupae developed into adults that either
eclosed normally or died unable to ecdyse. Targeting of BR-C Z5 had no obvious
effect. Interestingly, a simultaneous knockdown of Z1 and Z2 isoforms enhanced
the short wing and leg phenotype, such that it resembled the mild effect of
the common-region RNAi, including imperfect development of the gin traps
(compare Fig. 2I with
Fig. 4D). Thus, Z1 and Z2 might
together be indispensable for wing elongation. Similarly, the apparent loss of
Z2 and Z3 mRNAs in the TcBR-CKS342 mutants could not be
compensated for by the remaining BR-C products
(Fig. 2H). Taken together,
these results suggest at least some specific roles for the TcBR-C
isoforms.
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;
Wigglesworth, 1954;
Nijhout, 1994). Precocious
pupation of early-stage Tribolium larvae can be achieved by silencing
of the Methoprene-tolerant gene (TcMet), which is necessary
for JH signaling (Konopova and Jindra,
2007). If TcBR-C is indeed essential for the pupal program as
suggested by our results, then TcBR-C mRNA should be upregulated in
such untimely TcMet(RNAi) prepupae. To test this hypothesis, we
injected early-fifth instar larvae with TcMet dsRNA and examined
TcBR-C expression in resulting sixth-instar precocious prepupae.
Figure 5 shows that the
TcBR-C mRNA level in these TcMet(RNAi) prepupae was high
relative to in control early-seventh instar larvae. The increase was
comparable to that normally seen during the onset of metamorphosis
(Fig. 5 and
Fig. 1C). This result links
precocious pupal development that ensues from a deficiency in JH signaling
with upregulation of TcBR-C. It also supports the premise that
TcBR-C is required for pupation, even if premature.
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). In Manduca and Drosophila this ectopic JH
treatment induces BR-C expression
(Zhou and Riddiford, 2002). We
have used this effect to provide evidence that TcBR-C acts in JH signaling
downstream of TcMet. First, we established that methoprene treatment indeed
caused overexpression of TcBR-C in pupae.
Figure 6A shows that, whereas
in controls TcBR-C mRNA declines to its basal level after day 1 of
normal pupal development, it is highly abundant throughout the pupal stage
after methoprene application. As was shown previously
(Konopova and Jindra, 2007),
these methoprene-treated animals died as second-stage pupae.
As the above effect of methoprene can be averted by the silencing of
TcMet (Konopova and Jindra,
2007), we next checked whether TcMet RNAi also prevented
the upregulation of TcBR-C. Early prepupae were injected either with
TcMet or with control dsRNA and, after pupation, the animals were
treated with methoprene. Pupae aged 24, 48, 72 and 96 hours were then
subjected to RT-PCR. As shown by the 48-hour- and 96-hour-old pupae
(Fig. 6B), TcMet
depletion specifically prevented the induction of TcBR-C by
methoprene. Consistent with our previous data
(Konopova and Jindra, 2007),
the methoprene-treated TcMet(RNAi) pupae produced adult beetles. This
experiment demonstrates that TcMet is required for JH-induced upregulation of
TcBR-C mRNA in Tribolium pupae. It also suggests that
TcBR-C is a target of TcMet during JH-induced formation of the
ectopic pupal stage.
BR-C is required for metamorphosis in the lacewing Chrysopa perla
To explore whether the essential role of BR-C in pupal development
might be common to other orders with less derived holometaboly, we chose to
study the lacewing Chrysopa perla (Neuroptera). Chrysopa
develops via three larval instars that are, unlike in Tribolium,
easily discernible by distinct cuticle pigmentation and sensillation. Before
pupation, Chrysopa larvae spin a cocoon from Malpighian tubules. We
have isolated a cDNA fragment of a putative Chrysopa perla BR-C
ortholog, hereafter referred to as CpBR-C. At its N terminus, this
sequence shows 90% amino acid identity with the last 23 residues of the BR-C
BTB domain; the C-terminal 23 amino acids match the Drosophila BR-C
Z2 zinc-finger domain with 82% identity. The region between the two conserved
domains shows little homology. Like in Tribolium, moderate
CpBR-C mRNA levels were detected throughout embryogenesis (data not
shown) and all three larval instars, with a marked expression peak before
cocoon spinning at the end of final larval instar (see Fig. S2 in the
supplementary material).
When first- and second-instar Chrysopa larvae were injected with CpBR-C dsRNA, no developmental defects were observed until the larval-pupal transition. Then, 95% (n=146) of the injected larvae arrested after the third instar at the prepupal stage, and 41% of them failed to complete or even initiate spinning their cocoons (see Table S2 in the supplementary material). The CpBR-C(RNAi) animals looked like imperfect pupae with very short wings and smaller compound eyes (Fig. 7). Although their cuticle pigmentation resembled that of pupae and lacked the long larval setae (Fig. 7), the cuticle showed typical larval thorns and was neither smooth, as in normal pupae, nor did it carry the long bristles seen in adults (Fig. 8A-D). The tarsi in CpBR-C(RNAi) animals became segmented as in pupae and adults, but pretarsi retained the larval character: compared with the pupal leg, they were narrow with hooked claws and ended with a long arolium similar to that found in larvae (Fig. 8E-H). In wild-type pupae, the long antennae pointed dorsally and were coiled on lateral sides (Fig. 7B), whereas in CpBR-C(RNAi) animals they were directed proximally and were twisted above the pupa-like mouthparts (Fig. 7F). Interestingly, differentiation of the compound eye in CpBR-C(RNAi) prepupae was more advanced relative to the smooth eye of control pupae, because it showed development of ommatidial lenses, although defective (Fig. 8I-L,J'-L'). Such premature eye differentiation probably resulted in holes instead of normal lenses (Fig. 8L). These results show that in Chrysopa, like in Tribolium, BR-C is required for pupation and plays important roles in the morphogenesis of certain pupal characters.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Kiss et al., 1988), and with
recent data on Tribolium (Suzuki
et al., 2008). However, as neither RNAi nor the likely hypomorphic
TcBR-CKS342 allele present a complete loss-of-function
situation, we cannot exclude a possibility that BR-C plays some
additional role, not visualized by our phenotypes. Importantly, the lethal
phase correlates with a strong upregulation of BR-C expression. At
least in beetles, this stage coincides with a peak of ecdysteroid titer that
causes larvae to initiate prepupal development
(Hirashima et al., 1995;
Aribi et al., 1997).
In contrast to Drosophila npr1 mutants, metamorphosis was not
completely blocked by BR-C deficiency in Tribolium or
Chrysopa. Instead the arrested prepupae showed a blend of larval,
pupal, and partially even adult features. Based on the absence of the
pupal-specific gin traps in Tribolium and on the surface
microsculpture, the cuticle was apparently larval in both species, thus
confirming the requirement of BR-C for the pupal commitment of the
epidermis (Zhou et al., 1998;
Zhou and Riddiford, 2002).
Interestingly, although the thorny cuticle in Chrysopa BR-C(RNAi)
animals was distinctly larval, similar to in Tribolium, the body
pigmentation resembled that of pupae. We cannot be sure whether this mixed
character of the epidermis might be due to persisting CpBR-C
function, or might be because CpBR-C is not necessary for the pupal
pigmentation.
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).
BR-C silencing prevented the gradual wing enlargement even in larvae
of the hemimetabolous milkweed bug Oncopeltus fasciatus
(Erezyilmaz et al., 2006).
Imaginal discs fail to elongate in Drosophila br mutants with
disrupted BR-C Z2 function (Kiss et al.,
1988; DiBello et al.,
1991; Bayer et al.,
1997). The short legs and wings are not due to insufficient
proliferation of the disc cells but are due to their inability to change shape
in response to the ecdysteroid (von Kalm
et al., 1995). This cell shape change requires cytoskeletal
components whose mutations enhance the effect of br
(Gotwals and Fristrom, 1991;
Ward et al., 2003). The
rudimentary wings, present even in animals most severely affected by
TcBR-CKS342 mutation or by RNAi, suggest that cell shape
changes, rather than cell proliferation may be disrupted by the loss of BR-C
in Tribolium as well. Growing wings marked by EGFP in arrested beetle
prepupae (see Fig. S3 in the supplementary material) support this idea. The
legs in Tribolium BR-C(RNAi) animals were short also but were
distally specified as pupal with two tarsal claws. By contrast, the arrested
Chrysopa prepupae retained pretarsi with the larval-specific
elongated arolium, thus suggesting a stronger requirement for BR-C
function in the Chrysopa leg.
Except for small deviations, gross morphology of Tribolium genital
segments with the pupal genital papillae was pupal in BR-C(RNAi)
animals (see Fig. 3A). In
addition, the larval-pupal transformation of the visual system was initiated,
as larval stemmata were replaced with ommatidia of the compound eyes. However,
as in Drosophila (Brennan et al.,
2001), TcBR-C was important for compound eye
differentiation. These observations suggested that not all aspects of pupal
development were completely blocked by BR-C depletion.
While the above described structures were retarded in their development in
BR-C(RNAi) animals, others appeared accelerated in their development
towards the adult state, although none could be unambiguously defined as
adult. For instance, the antennae in Tribolium or the compound eyes
in Chrysopa resembled their adult counterparts, but in fact were
intermediates between pupal and adult organs. These heterochronic phenotypes
suggest that BR-C may not only be a pupal specifier
(Zhou and Riddiford, 2002),
but rather a temporal coordinator of the extensive morphogenesis in diverse
tissues during metamorphosis.
Drosophila organs require a temporally regulated balance between
both inductive and repressive BR-C functions, represented by the individual
isoforms (Karim et al., 1993;
Crossgrove et al., 1996;
Mugat et al., 2000). We
therefore see two alternative explanations for the heterochronically advanced
phenotypes. First, these structures may require BR-C to repress precocious
adult morphogenesis in them, but the inductive BR-C function is dispensable
for development beyond larval state. Consequently, loss of BR-C accelerates
their development. Second, if both functions are required but the repressive
one is more sensitive to reduced BR-C dose, then the inductive function will
prevail under an incomplete BR-C knockdown. We favor the first alternative,
because progression beyond the pupal stage seems to depend on BR-C
downregulation (Zhou and Riddiford,
2002) (this work).
Regulation of BR-C by Met-dependent JH signaling
Periods of JH absence are required first in larvae to initiate the pupal
program, and later in pupae to exit it. BR-C in both cases promotes the pupal
fate (Zhou and Riddiford,
2002), and therefore JH must regulate BR-C differently in
larvae and in pupae. In lepidopteran (Zhou
et al., 1998; Reza et al.,
2004), as well as in Tribolium
(Suzuki et al., 2008) larvae,
JH prevents BR-C expression until the onset of metamorphosis, and
presumably that is how JH prevents pupal differentiation. Conversely, removal
of the JH source (allatectomy) causes both BR-C misexpression and
precocious pupal development. In pupae, ectopic JH induces BR-C
(Zhou et al., 1998;
Zhou and Riddiford, 2002;
Reza et al., 2004;
Wu et al., 2006), and in many
insects, including Tribolium
(Konopova and Jindra, 2007),
such JH application causes reiteration of the pupal stage. In Drosophila,
BR-C misexpression alone is sufficient to inhibit adult cuticle formation
(Zhou and Riddiford, 2002).
BR-C is therefore a prime target of JH signaling, but how JH
regulates BR-C expression is unknown.
We showed here that precocious pupation, triggered by interference with the
putative JH receptor Met, coincided with precocious TcBR-C mRNA
increase in the sixth instar. Thus, disrupted JH signaling induced
TcBR-C similarly to allatectomy in lepidopteran larvae
(Zhou et al,. 1998;
Reza et al., 2004). As
expected, TcBR-C not only marked but also was necessary for the
untimely pupation, as TcMet; TcBR-C double-RNAi resulted in
a phenotype similar to TcBR-C RNAi alone, i.e. entry to a lethal
prepupal stage, except one or two instars too early (data not shown).
Therefore, although the metamorphic program could be prematurely induced by
silencing of TcMet, it could not be completed without
TcBR-C. However, loss of Met has been shown to worsen the
effect of BR-C mutations in Drosophila, without altering
BR-C expression (Wilson et al.,
2006). This again might reflect the different response to JH in
the fly.
The evidence that TcMet is required for regulation of
TcBR-C came from pupae, where the JH mimic methoprene induced
TcBR-C mRNA, but not after TcMet knockdown. This result
places TcBR-C downstream of TcMet in JH signaling.
Importantly, the averting of ectopic TcBR-C expression by
TcMet RNAi also rescued the methoprene-treated animals from repeating
the pupal stage and allowed them to become adult
(Konopova and Jindra, 2007).
Together, these findings suggest that, similar to in Drosophila
(Zhou and Riddiford, 2002),
downregulation of BR-C is required to exit the pupal state in
Tribolium.
We propose the following model for BR-C function in holometabolan metamorphosis (Fig. 9). In larvae, JH acts through Met to prevent BR-C induction until the final instar, when JH decline relieves the repression, and BR-C coordinates pupal morphogenesis. Loss of BR-C function causes both retardation and acceleration of development in diverse epidermal tissues, thus producing a mix of larval-, pupal- and adult-like features. In early pupae, low JH titer normally allows BR-C expression to drop, which is necessary for proper adult differentiation. Exogenous JH, again acting via Met, causes BR-C misexpression, which in turn promotes another round of pupal, instead of adult, development. Whether Met regulates BR-C expression directly, and what determines whether BR-C will be repressed or activated requires further work.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/3/559/DC1
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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