There are ample structural homologs of Broad protein. The Drosophila Bric à brac protein and the transcriptional regulators Tramtrack, and Trithorax-like, as well as Fruitless, Abrupt, Longitudinals lacking, and Suppressor of Hairy wing: all contain a highly conserved domain of approximately 115 amino acids termed the BTB (for BR-C, TTK and BAB) domain. Six additional Drosophila genes have been identified that encode this domain. Five of these genes are developmentally regulated, and one of them appears to be functionally related to bric a brac. The BTB domain defines a gene family with an estimated 40 members in Drosophila. This domain is found primarily at the N terminus of zinc finger proteins and is evolutionarily conserved from Drosophila to mammals. It is likely BTB serves as a protein interaction domain (Zollman, 1994).

The primary processes of metamorphosis in Drosophila involve the differentiation of imaginal discs and other imaginal precursor cells and the wholesale death of most larval tissues except for remodeling of the central nervous system and Malpighian tubules. Juvenile hormone (JH) appears to have little effect on the initiation of methamorphosis, but disrupts some aspects of adult differentiation. In contrast to Drosophila, the polymorphic larval epidermis of the tobacco hornworm, Manduca sexta, becomes reprogrammed (committed) by ecdysone for pupal differentiation at the onset of metamorphosis, and this reprogramming can be prevented by JH. Thus, Manduca serves as a model organism for studying the role of JH in the prevention of insect metamorphosis. A cDNA homolog of the Drosophila melanogaster Broad Complex (BrC) gene was isolated from Manduca, that shows a predicted 88% amino acid identity with Drosophila BrC protein in the N-terminal BTB domain. Three zinc finger domains encoding homologs of the Drosophila Z2, Z3, and Z4 domains (93%, 100%, and 85% identity, respectively) were obtained by RT-PCR. In Manduca dorsal abdominal epidermis, BRC mRNAs were not observed during the larval molt. Three BRC transcripts (6.0, 7.0, and 9.0 kb) first appear at the end of the feeding stage of the fifth (final) instar when the epidermis is exposed to ecdysteroids in the absence of juvenile hormone and becomes committed to pupal differentiation. These RNAs were induced in vitro in day 2 fifth larval epidermis by 20-hydroxyecdysone (20E) in the absence of JH, with dose-response and time courses similar to the induction of pupal commitment. This induction by 20E in vitro is prevented by the presence of JH I at levels seen in vivo during the larval molt. In the wing discs, the BRC mRNAs appear shortly after ecdysis to the fifth instar and coincide with the onset of metamorphic competence in these discs. Application of a JH analog pyriproxifen during the fourth instar molt delays and reduces the levels of BRC mRNAs seen in the wing discs in the early fifth instar, but does not completely prevent their appearance in this tissue, which first differentiates at metamorphosis. Drosophila imaginal discs also become competent to metamorphose during the late second (penultimate) instar. At this time, imaginal disc metamorphosis becomes completely insensitive to JH. The expression of the BrC transcription factors thus appears to be one of the first molecular indications of the genetic reprogramming of the epidermis necessary for insect metamorphosis. How JH prevents BrC expression in this epidermis may provide the key to understanding how this hormone controls metamorphosis. In Drosophila, with its fixed number of larval instars and early wholesale jettisoning of learval tissues for new ones at metamorphosis, the dependence on JH to regulate the initial events of metamorphosis, such as expression of BrC, appears to have been lost (Zhou, 1998).

Broad (br), a transcription factor containing the Broad-Tramtrack-Bric-a-brac (BTB) and zinc finger domains mediate 20-hydroxyecdysone (20E) action and pupal development in Drosophila melanogaster and Manduca sexta. This study determined the key roles of br during larval-pupal metamorphosis using RNA interference (RNAi) in a coleopteran insect, Tribolium castaneum. Two major peaks of T. castaneum broad (Tcbr) mRNA, one peak at the end of feeding stage prior to the larvae entering the quiescent stage and another peak during the quiescent stage were detected in the whole body and midgut tissue dissected from staged insects. Expression of br during the final instar larval stage is essential for successful larval-pupal metamorphosis, because, RNAi-mediated knock-down of Tcbr during this stage derailed larval-pupal metamorphosis and produced insects that showed larval, pupal and adult structures. Tcbr dsRNA injected into the final instar larvae caused reduction in the mRNA levels of genes known to be involved in 20E action (EcRA, E74 and E75B). Tcbr dsRNA injected into the final instar larvae also caused an increase in the mRNA levels of JH-response genes (JHE and Kr-h1b). Knock-down of Tcbr expression also affected 20E-mediated remodeling of midgut during larval-pupal metamorphosis. These data suggest that the expression of Tcbr during the final instar larval stage promotes pupal program while suppressing the larval and adult programs ensuring a transitory pupal stage in holometabolous insects (Parthasarathy, 2008).

The BTB/POZ domain defines a conserved region of about 120 residues; it has been found in over 40 proteins to date. It is located predominantly at the N terminus of Zn-finger DNA-binding proteins, where it may function as a repression domain, and less frequently in actin-binding and poxvirus-encoded proteins, where it may function as a protein-protein interaction interface. A prototypic human BTB/POZ protein, PLZF (promyelocytic leukemia zinc finger) is fused to RARalpha (retinoic acid receptor alpha) in a subset of acute promyelocytic leukemias (APLs), where it acts as a potent oncogene. The exact role of the BTB/POZ domain in protein-protein interactions and/or transcriptional regulation is unknown. The BTB/POZ domain from PLZF (PLZF-BTB/POZ) has been overexpressed, purified, characterized, and crystallized. Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that PLZF-BTB/POZ forms a homodimer with a Kd below 200 nM. Differential scanning calorimetry and equilibrium denaturation experiments are consistent with the PLZF-BTB/POZ dimer undergoing a two-state unfolding transition. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% beta-sheet. Crystals of the PLZF-BTB/POZ have been prepared that are suitable for a high resolution structure determination using x-ray crystallography. The data support the hypothesis that the BTB/POZ domain mediates a functionally relevant dimerization function in vivo. The crystal structure of the PLZF-BTB/POZ domain will provide a paradigm for understanding the structural basis underlying BTB/POZ domain function (Li, 1997).

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein-protein interaction motif found at the N terminus of 5%-10% of C2H2-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism for linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 A resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and it is expected that the full-length PLZF protein exists as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins (Ahmad, 1998).

The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-a-brac tramtrack broad complex/pox viruses and zinc fingers). The LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation (Dhordain, 1997).

The bcl-6 proto-oncogene encodes a POZ/zinc finger transcriptional repressor expressed in germinal center (GC) B and T cells and required for GC formation and antibody affinity maturation. Deregulation of bcl-6 expression by chromosomal rearrangements and point mutations of the bcl-6 promoter region are implicated in the pathogenesis of B-cell lymphoma. The signals regulating bcl-6 expression are not known. Antigen receptor activation leads to BCL-6 phosphorylation by mitogen-activated protein kinase (MAPK). Phosphorylation, in turn, targets BCL-6 for rapid degradation by the ubiquitin/proteasome pathway. These findings indicate that BCL-6 expression is directly controlled by the antigen receptor via MAPK activation (Niu, 1998).

MAPK is a ubiquitous, evolutionarily conserved signal transducer that is activated by heterogeneous signals that originate from the cell membrane and are transduced to MAPK via RAS proteins. Accordingly, POZ/zinc finger proteins represent a large family of highly conserved transcription factors, including Drosophila cell fate regulators such as Tramtrack and Broad-complex, as well as human cancer-associated proteins such as BCL-6 and PLZF. These molecules have strong structural (POZ and ZF domains), as well as functional homologies since they are transcriptional repressors that control cell differentiation. Most notably, POZ/zinc finger proteins also carry possible MAPK phosphorylation sites and PEST sequences in approximately the same position as those carried by BCL-6. In Drosophila, degradation of TTK88, a POZ/zinc finger inhibitor of neural-cell differentiation, has been shown to be mediated by MAPK. Thus, degradation of POZ/zinc finger transcription factors may represent a general mechanism by which the RAS/MAPK pathway controls cell function and differentiation (Niu, 1998 and references).

Virtually all diffuse large cell lymphomas and a significant fraction of follicular lymphomas contain translocations and/or point mutations in the 5' non-coding region of the putative oncogene BCL-6, that are presumed to deregulate the expression of BCL-6. BCL-6 encodes a Cys2-His2 zinc finger transcriptional repressor with a POZ domain at its amino-terminus. The POZ (or BTB) domain, a 120-amino-acid motif, mediates homomeric and, in some proteins, heteromeric POZ-POZ interactions. In addition, the POZ domain is required for transcriptional repression of several proteins, including BCL-6. Using a yeast two-hybrid screen, N-CoR and SMRT have been identified as BCL-6 interacting proteins. Both N-CoR and SMRT, which were originally identified as co-repressors for the unliganded nuclear thyroid hormone and retinoic acid receptors, are components of large complexes containing histone deacetylases. The interaction between BCL-6 and these co-repressors is also detected in the more physiologically relevant mammalian two-hybrid assay. The POZ domain is necessary and sufficient for interaction with these co-repressors. BCL-6 and N-CoR co-localize to punctate regions of the nucleus. Furthermore, when BCL-6 is bound to its consensus recognition sequence in vivo, it can interact with N-CoR and SMRT. In vitro POZ domains from a variety of other POZ domain-containing proteins (including the transcriptional repressor PLZF, as well as ZID, GAGA and a vaccinia virus protein, SalF17R) also interact with varying affinities with N-CoR and SMRT. BCL-6 POZ domain mutations that disrupt the interaction with N-CoR and SMRT no longer repress transcription. In addition, these mutations no longer self associate, suggesting that self interaction is required for interaction with the co-repressors and for repression. More recently N-CoR has also been implicated in transcriptional repression by the Mad/Mxi proteins. The demonstration that N-CoR and SMRT interact with the POZ domain containing proteins indicates that these co-repressors are likely involved in the mediation of repression by multiple classes of repressors and may explain, in part, how POZ domain containing repressors mediate transcriptional repression (Huynh, 1998).

A novel zinc finger protein, ZID (standing for zinc finger protein with interaction domain) was isolated from humans. ZID has four zinc finger domains and a BTB domain, also know ans a POZ (standing for poxvirus and zinc finger) domain. At its amino terminus, ZID contains the conserved POZ or BTB motif present in a large family of proteins that include otherwise unrelated zinc fingers, such as Drosophila Abrupt, Bric-a-brac, Broad, Fruitless, Longitudinals lacking, Pipsqueak, Tramtrack, and Trithorax-like (GAGA). The POZ domains of ZID, TTK and TRL act to inhibit the interaction of their associated finger regions with DNA. This inhibitory effect is not dependent on interactions with other proteins and does not appear dependent on specific interactions between the POZ domain and the zinc finger region. The POZ domain acts as a specific protein-protein interaction domain: The POZ domains of ZID and TTK can interact with themselves but not with each other, or POZ domains from ZF5, or the viral protein SalF17R. However, the POZ domain of TRL can interact efficiently with the POZ domain of TTK. In transfection experiments, the ZID POZ domain inhibits DNA binding in NIH-3T3 cells and appears to localize the protein to discrete regions of the nucleus (Bardwell, 1994).

The pupal specifier broad directs progressive morphogenesis in a direct-developing insect: Evidence from Oncopeltus

A key regulatory gene in metamorphosing (holometabolous) insect life histories is the transcription factor broad (br), which specifies pupal development. To determine the role of br in a direct-developing (hemimetabolous) insect that lacks a pupal stage, br was cloned from the milkweed bug, Oncopeltus fasciatus (Of’br). Unlike metamorphosing insects, in which br expression is restricted to the larval–pupal transition, Of’br mRNA is expressed during embryonic development and is maintained at each nymphal molt but then disappears at the molt to the adult. Induction of a supernumerary nymphal stage with a juvenile hormone (JH) mimic prevents the disappearance of br mRNA. In contrast, induction of a precocious adult molt by application of precocene II to third-stage nymphs caused a loss of br mRNA at the precocious adult molt. Thus, JH is necessary to maintain br expression during the nymphal stages. Injection of Of’br dsRNA into either early third- or fourth-stage nymphs causes a repetition of stage-specific pigmentation patterns and prevents the normal anisometric growth of the wing pads without affecting isometric growth or molting. Therefore, br is necessary for the mutable (heteromorphic) changes that occur during hemimetabolous development. These results suggest that metamorphosis in insects arose as expression of br, which conveys competence for change, became restricted to one postembryonic instar. After this shift in br expression, the progressive changes that occur within the nymphal series in basal insects became compressed to the one short period of morphogenesis seen in the larva-to-pupa transition of holometabolous insects (Erezyilmaz, 2006).

Life history strategies are highly plastic within animal phyla; some groups develop directly, whereas related taxa pass through a metamorphosis. Regulation of stage-specific differences may be under either environmental or hormonal control, but relatively little is known of the molecular switches involved or how changes in the timing of these switches can lead to evolutionary change. In insects, metamorphosis arose once from a direct-developing ancestor ~300 million years ago. A key regulatory gene in metamorphosing (holometabolous) insect life histories is the transcription factor broad. In both moths and flies, epidermal expression of br is restricted to the larval–pupal transition, and its expression at this time is required for activation of pupal-specific gene expression, as well as suppression of larval- and adult-specific gene expression. Accordingly, Drosophila null mutants never enter the pupal stage; instead, they remain in a prolonged larval state. In addition, gynander larvae mosaic for br null and br+ tissue produce mosaic larval and pupal tissue, respectively, at the larval–pupal transition. Loss of br also prevents the larval–pupal transition in the silkmoth; tissues that were transformed with a vector driving br RNA interference are unable to produce adult structures, and transformed larval organs are not destroyed at metamorphosis (Erezyilmaz, 2006).

The restriction of br expression at the larval–pupal transition of holometabolous insects occurs through the action of two hormones: the steroid 20-hydroxyecdysone (20E) and the sesquiterpenoid juvenile hormone (JH). Peaks of 20E trigger molts between stages, whereas the presence or absence of JH determines the type of cuticle that is produced and whether br is expressed. During larval life, the presence of JH suppresses metamorphosis and br expression. As JH titers disappear in the last larval stage, a small peak of 20E triggers 'pupal commitment' as it induces br. Although JH levels again rise at the pupal molt, when they suppress precocious adult development, JH does not suppress br at this stage. In fact, topical application of JH during the adult molt, which normally occurs in the absence of JH and br, causes the reinduction of br and the production of a second pupal cuticle (Erezyilmaz, 2006).

To determine the role of br in a nonholometabolous, direct-developing insect, br was isolated from the milkweed bug, Oncopeltus fasciatus. As in metamorphosing insects, br is required for morphogenesis and its expression is regulated by JH at molts. In this insect, however, br is expressed at each nymphal molt, and its expression is required for progressive changes in proportions and pattern from instar to instar. These results suggest that metamorphosis in insects arose as expression of this factor, which conveys competence for change, became restricted to one postembryonic instar (Erezyilmaz, 2006).

After knock-down of br expression in the nymph, wing pad growth continues but becomes more isometric, and its proportions from the previous stage are repeated. This aspect of br function appears to be retained during metamorphosis of the fly, because the wings of a br allele (lacking the Z2 isoform of br) are defective in their morphology and are shortened and 'broad', a phenotype similar to that seen in the wings of Oncopeltus that lack Of’br during the third or fourth nymphal stages. In contrast to the progressive role that Of’br plays through successive nymphal molts, the function of br in the Drosophila wing disc is restricted to the final larval instar as the wing disc translates patterning information to produce the pupal wing. Therefore, the ancestral function of br, to support progressive anisometric growth of the developing wing pad over a number of instars, has been restricted to the premetamorphic period in the last larval instar of holometabolous insects (Erezyilmaz, 2006).

The homology of the pupal stage to a developmental stage of hemimetabolous insects has been a recurring issue among naturalists. During the latter half of the 20th century, the prevailing theory considered the pupal state to be derived from the final nymphal stage. An older idea considered pupal development to be more akin to the events that occur during embryonic development of direct developers, which has recently been expanded into the pronymph hypothesis. The current data support this older idea. In both crickets and milkweed bugs, br mRNA is present during the latter half of embryonic development. In these hemimetabolous embryos, this period is characterized by differential growth as the embryo progresses from the phylotypic germ band stage to a miniature version of the adult. In contrast, br mRNA is not present in the epidermis of holometabolous embryos, and the growth during the corresponding phase of embryogenesis is more isometric. This isometric growth then persists through postembryonic development until br reappears at the larval–pupal transition to help direct the differential growth needed to generate the adult form. Because br is required for postembryonic differential growth in the hemimetabolous insect Oncopeltus, it is suggested that metamorphosis emerged in insects as br expression and its regulation of differential growth became transposed from late embryonic development to the penultimate postembryonic molt (Erezyilmaz, 2006).

br may confer mutability to insect life history stages through its BTB/POZ domain, a motif implicated in the establishment and maintenance of complex differentiated states. Many BTB-containing proteins regulate complex states through chromatin deacetylation, thereby affecting the access of subsequent transcription factors to response elements. In the context of nymphal changes in Oncopeltus, the loss of Br may prevent the access of transcription factors to response elements that are needed for change from one stage to the next (Erezyilmaz, 2006).

The role of Broad in the development of Tribolium castaneum: implications for the evolution of the holometabolous insect pupa

The evolution of complete metamorphosis in insects is a key innovation that has led to the successful diversification of holometabolous insects, yet the origin of the pupa remains an enigma. This study analyzed the expression of the pupal specifier gene broad (br), and the effect on br of isoform-specific, double-stranded RNA-mediated silencing, in a basal holometabolous insect, the beetle Tribolium castaneum. All five isoforms are weakly expressed during the penultimate instar and highly expressed during the prepupal period of the final instar. Application of hydroprene, a juvenile hormone analog, during the penultimate instar caused a repeat of the penultimate br expression patterns, and the formation of supernumerary larvae. Use of dsRNA against the br core region, or against a pair of either the br-Z2 or br-Z3 isoform with the br-Z1 or br-Z4 isoform, produced mobile animals with well-differentiated adult-like appendages, but which retained larval-like urogomphi and epidermis. Disruption of either the br-Z2 or the br-Z3 isoform caused the formation of shorter wings. Disruption of both br-Z1 and br-Z4 caused the appearance of pupal traits in the adults, but disruption of br-Z5 had no morphological effect. These findings show that the br isoform functions are broadly conserved within the Holometabola and suggest that evolution of br isoform expression may have played an important role in the evolution of the pupa in holometabolous insects (Suzuki, 2008).

Krüppel homolog 1, an early juvenile hormone-response gene downstream of Methoprene-tolerant, mediates its anti-metamorphic action in the red flour beetle Tribolium castaneum

Juvenile hormone (JH) prevents ecdysone-induced metamorphosis in insects. However, knowledge of the molecular mechanisms of JH action is still fragmented. Krüppel homolog 1 (Kr-h1) is a JH-inducible transcription factor in Drosophila melanogaster. Analysis of expression of the homologous gene (TcKr-h1) in the beetle Tribolium castaneum showed that its transcript was continuously present in the larval stage but absent in the pupal stage. Artificial suppression of JH biosynthesis in the larval stage caused a precocious larval-pupal transition and a down-regulation of TcKr-h1 mRNA. RNAi-mediated knockdown of TcKr-h1 in the larval stage induced a precocious larval-pupal transition. In the early pupal stage, treatment with an exogenous JH mimic (JHM) caused formation of a second pupa, and a rapid and large induction of TcKr-h1 transcription. JHM-induced formation of a second pupa was counteracted by the knockdown of TcKr-h1. RNAi experiments in combination with JHM treatment demonstrated that in the larval stage TcKr-h1 works downstream of the putative JH receptor Methoprene-tolerant (TcMet), and in the pupal stage it works downstream of TcMet and upstream of the pupal specifier broad (Tcbr). Therefore, TcKr-h1 is an early JH-response gene that mediates JH action linking TcMet and Tcbr (Minakuchi, 2009).

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

Metamorphosis of holometabolous insects, an elaborate change of form between larval, pupal and adult stages, offers an ideal system to study the regulation of morphogenetic processes by hormonal signals. Metamorphosis involves growth and differentiation, tissue remodeling and death, all of which are orchestrated by the morphogenesis-promoting ecdysteroids and the antagonistically acting juvenile hormone (JH), whose presence precludes the metamorphic changes. How target tissues interpret this combinatorial effect of the two hormonal cues is poorly understood, mainly because JH does not prevent larval-pupal transformation in the derived Drosophila model, and because the JH receptor is unknown. The red flour beetle Tribolium castaneum has been used to show that JH controls entry to metamorphosis via its putative receptor Methoprene-tolerant (Met). This study demonstrates that Met mediates JH effects on the expression of the ecdysteroid-response gene Broad-Complex (BR-C). Using RNAi and a classical mutant, it has been show that Tribolium BR-C is necessary for differentiation of pupal characters. Furthermore, heterochronic combinations of retarded and accelerated phenotypes caused by impaired BR-C function suggest that besides specifying the pupal fate, BR-C operates as a temporal coordinator of hormonally regulated morphogenetic events across epidermal tissues. Similar results were also obtained when using the lacewing Chrysopa perla (Neuroptera), a member of another holometabolous group with a primitive type of metamorphosis. The tissue coordination role of BR-C may therefore be a part of the Holometabola groundplan (Konopova, 2008).

In both Tribolium and Chrysopa, BR-C RNAi compromises the larval-pupal transition without affecting earlier development, regardless of the time of dsRNA injection. The TcBR-CKS342 homozygotes die at the same stage. These data suggest that the moderate levels of BR-C mRNAs, detectable during premetamorphic stages in both species, has no essential role. This scenario would agree with the fact that zygotic BR-C function is not required in Drosophila BR-C null nonpupariating mutants until the onset of metamorphosis. However, as neither RNAi nor the likely hypomorphic TcBR-CKS342 allele present a complete loss-of-function situation, a possibility that BR-C plays some additional role, not visualized by the phenotypes cannot be excluded. 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 (Konopova, 2008).

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. 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. It is not certain 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 (Konopova, 2008).

Pupal characters in BR-C(RNAi) animals included rudimentary wings. In particular, the weak phenotypes in Tribolium (produced with either isoform-specific or diluted common-core dsRNAs) revealed that wing elongation was highly sensitive to BR-C depletion. A similar effect of BR-C RNAi was described for pupal appendages in Bombyx mori. BR-C silencing prevented the gradual wing enlargement even in larvae of the hemimetabolous milkweed bug Oncopeltus fasciatus. Imaginal discs fail to elongate in Drosophila br mutants with disrupted BR-C Z2 function. 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. This cell shape change requires cytoskeletal components whose mutations enhance the effect of br. 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 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 (Konopova, 2008).

Except for small deviations, gross morphology of Tribolium genital segments with the pupal genital papillae was pupal in BR-C(RNAi) animals. 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, TcBR-C was important for compound eye differentiation. These observations suggest that not all aspects of pupal development are completely blocked by BR-C depletion (Konopova, 2008).

While the above described structures are 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, but rather a temporal coordinator of the extensive morphogenesis in diverse tissues during metamorphosis (Konopova, 2008). Drosophila organs require a temporally regulated balance between both inductive and repressive BR-C functions, represented by the individual isoforms. Two alternative explanations are seen 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. The first alternative alternative is favored, because progression beyond the pupal stage seems to depend on BR-C downregulation (Konopova, 2008).

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, and therefore JH must regulate BR-C differently in larvae and in pupae. In lepidopteran, as well as in Tribolium 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, and in many insects, including Tribolium, such JH application causes reiteration of the pupal stage. In Drosophila, BR-C misexpression alone is sufficient to inhibit adult cuticle formation. BR-C is therefore a prime target of JH signaling, but how JH regulates BR-C expression is unknown (Konopova, 2008).

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. 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. 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. This again might reflect the different response to JH in the fly (Konopova, 2008).

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. Together, these findings suggest that, similar to in Drosophila, downregulation of BR-C is required to exit the pupal state in Tribolium (Konopova, 2008).

The following model for BR-C function in holometabolan metamorphosis. 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 (Konopova, 2008).

broad: Biological Overview | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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