The pupal specifier Broad directs progressive morphogenesis in a direct-developing insect

Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2006; 103(18):6925-30. DOI: 10.1073/pnas.0509983103
Source: PubMed


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, we cloned br from the milkweed bug, Oncopeltus fasciatus (Of'br). We find that, 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 prevented 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 caused a repetition of stage-specific pigmentation patterns and prevented 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. Our 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.

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Available from: Lynn M Riddiford
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    • "In insects, BR-C is primarily involved in the signaling pathways of ecdysone and juvenile hormone, two key endocrine hormones that orchestrate the growth and development of insects [3]. Accumulated evidence has demonstrated the critical roles of insect BR-C in numerous developmental processes, including embryogenesis [4], [5], metamorphosis [6], [7], cell death [8], [9], stem cell differentiation [10], wing morphogenesis [11], eye development [12], muscle attachment [13], [14], metamorphic remodeling of the central nervous system [15], [16], and oogenesis [17], [18]. "
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    ABSTRACT: The transcription factor Broad Complex (BR-C) is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB) domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1), a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the PKC phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.
    Full-text · Article · Oct 2014 · PLoS ONE
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    • "in ecdysteroid-signaling pathways in insects. These transcription factors play key roles in specifying the pupal stage in holometabola (Suzuki et al. 2008; Konopova et al. 2011), but are involved in embryogenesis, the growth of wing buds, and wing vein formation in hemimetabola (Erezyilmaz et al. 2006; Piulachs et al. 2010; Huang et al. 2013). All BR-C isoforms have a well-conserved Broad- Tramtrack-Bric-a-brac (BTB) domain in the N-terminus and a variable C 2 H 2 -type zinc finger DNA binding domain in the C-terminus (DiBello et al. 1991). "
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    ABSTRACT: Seven isoforms of Broad-Complex (PhBR-C), in which the sequence of the zinc finger domain differed (referred to as Z1, Z2, Z3, Z2/Z3, Z4, Z5/Z6, and Z6, respectively), were cloned from the yellow-spotted longicorn beetle Psacothea hilaris. The Z1–Z4 sequences were highly conserved among insect species. The Z5/Z6 isoform was aberrant in that it contained a premature stop codon. Z6 had previously only been detected in a hemimetabola, the German cockroach Blattella germanica. The presence of Z6 in P. hilaris, and not in other holometabolous model insects such as Drosophila melanogaster or Tribolium castaneum, suggests that Z6 was lost multiple times in holometabolous insects during the course of evolution. PhBR-C expression levels in the brain, salivary gland, and epidermis of larvae grown under different feeding regimens were subsequently investigated. PhBR-C expression levels increased in every tissue examined after the gut purge, and high expression levels were observed in prepupae. A low level of PhBR-C expression was continuously observed in the brain. An increase was noted in PhBR-C expression levels in the epidermis when 4th instar larvae were starved after 4 days of feeding, which induced precocious pupation. No significant changes were observed in expression levels in any tissues of larvae starved immediately after ecdysis into 4th instar, which did not grow and eventually died. Electronic supplementary material The online version of this article (doi:10.1186/2193-1801-3-539) contains supplementary material, which is available to authorized users.
    Full-text · Article · Sep 2014 · SpringerPlus
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    • "The expression of Br-C, encoding an ecdysone-regulated member of the Bric-à-brac-Tramtrack Broad (BTB)/Pox virus and Zinc finger (POZ) family of transcription factors, is regulated by JH and can be induced by exogenous methoprene [14]. Br-C was demonstrated to act downstream of the antimetamorphic signaling of JH, being critical for embryonic and nymphal development in Hemimetabola [15]–[18] and specifying pupal features in Holometabola [14], [19], [20]. "
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    ABSTRACT: Independent of the design of the life cycle of any insect, their growth and reproduction are highly choreographed through the action of two versatile hormones: ecdysteroids and juvenile hormones (JH). However, the means by which JH can target tissues and exert its pleiotropic physiological effects is currently still not completely elucidated. Although the identity of the one JH receptor is currently still elusive, recent evidence seems to point to the product of the Methoprene-tolerant gene (Met) as the most likely contender in transducing the action of JH. Studies on the role of this transcription factor have mostly been focused on immature insect stages. In this study we used the viviparous cockroach Diploptera punctata, a favorite model in studying JH endocrinology, to examine the role of Met during reproduction. A tissue distribution and developmental profile of transcript levels was determined for Met and its downstream partners during the first gonadotropic cycle of this cockroach. Using RNA interference, our study shows that silencing Met results in an arrest of basal oocyte development; vitellogenin is no longer transcribed in the fat body and no longer taken up by the ovary. Patency is not induced in these animals which fail to produce the characteristic profile of JH biosynthesis typical of the first gonadotropic cycle. Moreover, the ultrastructure of the follicle cells showed conspicuous whorls of rough endoplasmic reticulum and a failure to form chorion. Our study describes the role of Met on a cellular and physiological level during insect reproduction, and confirms the role of Met as a key factor in the JH signaling pathway.
    Full-text · Article · Sep 2014 · PLoS ONE
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