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First published online 2 January 2008
doi: 10.1242/dev.016097

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Broad-Complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis

Biology Center, Czech Academy of Sciences and Department of Molecular Biology, University of South Bohemia, Branisovska 31, Ceske Budejovice 37005, Czech Republic.

View larger version (73K): [in this window] [in a new window]   Fig. 1. TcBR-C gene structure and mRNA expression. (A) Exons of the common core region, including the BTB domain, are spliced to one of the five zinc-finger exons (Z1-Z5). Solid and open boxes indicate coding and untranslated mRNA regions, respectively. The open reading frame is preceded by two alternative noncoding exons. The region used for whole gene silencing is shown (RNAi). Mutant line KS342 carries a piggyBac insertion 480 bp upstream of the Z1 exon. (B) Alignment of BR-C BTB domains. From top: Tribolium castaneum, Drosophila melanogaster, Aedes aegypti, Anopheles gambiae, Bombyx mori, Manduca sexta, Apis mellifera, Oncopeltus fasciatus, Acheta domesticus. (C) Total RNA from the indicated stages was subjected to DNase treatment and RT-PCR with primers for the core region or those specifically detecting the Z1-Z5 isoform transcripts. Numbers indicate hours since egg laying or ecdysis. F and M, 10-day old females and males, respectively. The prepupal stage begins at around 72 hours after the last larval ecdysis. Expression of rp49 serves for control.

View larger version (142K): [in this window] [in a new window]   Fig. 2. Loss of TcBR-C blocks Tribolium metamorphosis. (A) A control pupa shows developed and elongated wings (black arrows), legs (white arrow) and gin traps (open arrowheads). (B) A prepupa that had been injected with the common-region TcBR-C dsRNA as a fifth-instar larva developed normally until it died at the end of the final instar. Upon removal of the apolysed larval cuticle the same animal displayed larval shape and rudimentary wings (B', black arrows). (C-G) Essentially the same phenotype was found in dying animals homozygous for the KS342 piggyBac insertion that were freed from the larval cuticle. Note the vestigial wings and short legs (black and white arrows, respectively), defects in compound eyes (black arrowheads), thick antenna (asterisk), short larval urogomphi (F, ur) and missing gin traps (G) above abdominal spiracles (sp). (H) Total RNA from wild-type prepupae and from two sets of arrested TcBR-CKS342 homozygous prepupae (two per sample) was subjected to RT-PCR. Note the reduced Z2 and Z3 mRNA levels in TcBR-CKS342. Expression of rp49 served as a control. (I) Lower doses of the common-region TcBR-C dsRNA allowed ecdysis into imperfect pupae with more developed yet abnormally short wings (black arrows in A through D), partially formed gin traps (white arrowheads), and short legs (white arrow). In B' and C, larval cuticle could not be completely removed from the anterior head and distal legs. Scale bars: in A,1 mm for A-B',I; C, 1 mm; D,F, 500 µm; E,G, 200 µm.

View larger version (97K): [in this window] [in a new window]   Fig. 3. TcBR-C is necessary for proper differentiation of pupal characters. Strong and mild phenotypes in animals that had been injected with TcBR-C dsRNA as fifth to seventh instar larvae are compared with wild-type. (A) Urogomphi in strongly affected animals are short, as in larvae, but elongated and forked, more like in pupae, upon mild TcBR-C RNAi treatment (a-e, yellow arrowheads; adults lack urogomphi). Larval pygopods (black arrow) disappear and genital papillae on the ninth abdominal segment develop (yellow arrows), although the eighth segment is abnormally enlarged after strong TcBR-C RNAi (asterisks). The female genital pore (black arrowheads), visible in its original intersegmental position (d) has migrated between the papillae in a mildly affected female (e). VII, the seventh abdominal sternum. Females shown in b, d and e. (B) Cuticle surface on abdominal sterna is smooth, as in larvae, upon strong TcBR-C RNAi, but shows the pupal-like surface in milder phenotypes (compare a with d, and b with e). The adult cuticle has rounded pits with sensillae (c). (C) Gin traps (gt) form above pupal abdominal spiracles (sp) but are absent in larvae, adults and in strong TcBR-C(RNAi) phenotypes (a-d; arrowheads mark presumptive gin trap positions). Weakly affected animals partially develop gin traps (e). (D) Larval stemmata (a) are replaced by compound eyes that differentiate during pupal stage (b). Ommatidia appear in TcBR-C(RNAi) animals, although they are irregularly spaced (arrows, compare d and e with b). (E) Antennae lose the larval character in TcBR-C(RNAi) animals, but, unlike those in pupae, develop the distal club and divisions between segments. (F) Separations between tarsal segments are clearer in strong TcBR-C(RNAi) than in pupae or mild phenotypes, and sharp double claws (arrowheads), similar to those of adults (c), are visible (d). Larval tarsi are unsegmented and bear a single claw (a). Anterior (A-D) and proximal (E,F) is to the left. Scale bars: 500 µm (Ac,Da,Dc,Fc); 200 µm (Aa,Ab,Ad,Ae,Ca,Db,Dd,De,Eb,Fa,Fb,Fe); 100 µm (Cb-e,Ea,Ec-e,Fd); 50 µm (Ba-e).

View larger version (77K): [in this window] [in a new window]   Fig. 4. TcBR-C isoform-specific effects. (A) Day 1 seventh-instar larvae were injected with indicated isoform-specific dsRNAs and subjected to RT-PCR as prepupae. Knocking down one isoform had no effect on the others. (B-D) Effects of knocking down TcBR-C isoforms. Black arrowheads mark the position to which wings normally extend in wild-type pupae (see Fig. 2A). Depletion of either Z1 (B) or Z2 (C) individually causes shortening of the wings (black arrows) and legs (white arrows), with the Z2 effect being stronger. Also note the aberrant head position (white arrowhead) and the blistered wing (asterisk). Combined double RNAi (D) enhanced the effect of isoform-specific dsRNAs on wings, legs and gin traps (insets), such that the aberrant pupae display the mild phenotypes of common-region TcBR-C RNAi (see Fig. 2I). Scale bars: in B, 1 mm for B-D; in insets, 200 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 5. Loss of TcMet induces upregulation of TcBR-C mRNA in precocious prepupae. RT-PCR shows that low TcBR-C expression in feeding day 1 eighth-instar larvae (L8) rises at the prepupal stage (PP8) 48 hours later (see also Fig. 1C). Injection of TcMet dsRNA to early-fifth instar larvae induced premature sixth-instar prepupae (PP6), which contained reduced TcMet mRNA and strongly elevated TcBR-C expression. Controls injected with egfp dsRNA and analyzed on day 1 of the seventh instar (L7) displayed a low TcBR-C mRNA level, typical for feeding larvae.

View larger version (61K): [in this window] [in a new window]   Fig. 6. TcBR-C upregulation by methoprene requires TcMet. (A) Tribolium pupae aged up to 1 hour after ecdysis were briefly dipped into 0.3 mM methoprene or its solvent (-) and tested (3-4 pupae per sample) for TcBR-C mRNA expression at the indicated times (adults normally emerge after 108-120 hours). Although TcBR-C expression in control pupae was low, similar to that in intact pupae of corresponding age (see Fig. 1C), it was markedly induced by methoprene. (B) Methoprene blocks adult development and its lethal effect is averted by TcMet knockdown (Konopova and Jindra, 2007). Early prepupae were injected either with egfp or TcMet dsRNA, and within four hours after pupal ecdysis were treated with methoprene or acetone. Shown are examples of TcBR-C mRNA expression in individual pupae aged 48 and 96 hours. Note that methoprene did not induce TcBR-C in TcMet(RNAi) pupae. Similar results were obtained with all examined pupae (at least eight for each treatment), aged either 48, 72 or 96 hours.

View larger version (77K): [in this window] [in a new window]   Fig. 7. BR-C knockdown impairs cocoon spinning and pupal differentiation in Chrysopa. (A-C) Normal final (third) instar larva (A) spins a cocoon (C), in which it ecdyses into a pupa (B). (D-F) Injection of CpBR-C dsRNA into larvae kills them as prepupae; some of these larvae are unable to initiate or complete spinning of their cocoons (D). Upon removal of the larval cuticle (E,F), an arrested animal reveals pupal pigmentation, the development of compound eyes (white arrows) and short wings (black arrows), whereas typical larval mouthparts (stylets; A, black arrowhead) and long setae (asterisk) are missing. In contrast to in pupae, antennae are twisted above mouthparts, with their end oriented toward the anterior in CpBR-C(RNAi) animals (compare white arrowheads in panels B,E and F). Scale bars: 1 mm.

View larger version (129K): [in this window] [in a new window]   Fig. 8. CpBR-C RNAi prevents proper differentiation of pupal characters. Chrysopa larvae were injected with CpBR-C dsRNA at the end of the first instar and, after they arrested at the prepupal stage, the larval cuticle was removed. Examples of their heterochronic phenotypes are compared with the normal larval, pupal and adult situation. (A-D) Tarsi (tar) of CpBR-C(RNAi) individuals (D) have distinct borders between segments, as in pupae and adults, whereas their distal tip (pretarsus) retains the larval character and is narrow, with a long arolium (ar). Claws (cl) are large and hooked as in larvae or adults. (E-H) Dorsal abdominal cuticle with short thorns resembles that of the larva. The thorns in CpBR-C(RNAi) (H) are densely packed because the animal had been compressed in its old larval cuticle. (I-L') Instead of larval stemmata (st), CpBR-C(RNAi) animals develop compound eyes, also present in pupae and adults. Compared with pupa, eye differentiation is more advanced, as indicated by the formation, although incomplete, of ommatidial lenses (arrows, compare J' with L'). Also note the perforations (arrowheads) on the CpBR-C(RNAi) eye (L). Scale bars: 500 µm (J); 200 µm (B,C,K); 100 µm (A,D,I,L); 50 µm (E-H,J',L'); 20 µm (K').

View larger version (25K): [in this window] [in a new window]   Fig. 9. Model for functioning of BR-C in Tribolium metamorphosis. In young larvae, naturally occurring JH blocks pupal differentiation by repressing BR-C. JH is absent in early pupae, and its addition blocks adult morphogenesis by causing ectopic BR-C activation and death after a supernumerary pupal cuticle deposition. As both effects of JH on BR-C expression require Met, unknown stage-specific factors must modulate Met function.

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