The regulation of expression of insect cuticle protein genes

Insect Biochemistry and Molecular Biology 40 (2010) 205e213

Contents lists available at ScienceDirect

Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb

Review

The regulation of expression of insect cuticle protein genes J.P. Charles* UMR CNRS 5548 Développement-Communication Chimique des Insectes (DCCI), Université de Bourgogne, Faculté des Sciences Gabriel, 6, Bd Gabriel 21000 Dijon, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 November 2009 Received in revised form 9 December 2009 Accepted 11 December 2009

The exoskeleton of insects (cuticle) is an assembly of chitin and cuticle proteins. Its physical properties are determined largely by the proteins it contains, and vary widely with developmental stages and body regions. The genes encoding cuticle proteins are therefore good models to study the molecular mechanisms of signalling by ecdysteroids and juvenile hormones, which regulate molting and metamorphosis in insects. This review summarizes the studies of hormonal regulation of insect cuticle protein genes, and the recent progress in the analysis of the regulatory sequences and transcription factors important for their expression. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Cuticle protein genes Ecdysteroids Juvenile hormones bFTZ-F1 DHR38 Broad Insects Metamorphosis

1. Introduction During the post-embryonic development of insects, epidermal cells synthesize during every molting cycle a novel exoskeleton or cuticle which is sufficiently larger than the previous one, so as to accommodate an increase in body size in relation to the available food resources. This extracellular matrix is a composite material, essentially made of an assembly of chitin fibers and cuticle proteins. It can have very contrasting physical properties (for example: rigid vs. pliant), depending of its protein composition, and also of the degree of cross-linking of its constituents after synthesis (a process known as sclerotization). Cuticles of successive larval stages are typically quite similar, but differ from that of the adult stage, which is characterized by sexual maturation, and the development of functional wings. In holometabolous insects (which undergo metamorphosis), such as butterflies and flies, the epidermis synthesizes three distinct types of cuticle: larval, pupal and adult. In such insects, the difference between larval and adult cuticles is even greater, and is usually associated with a radical change in food source. Dramatically different cuticles are also found in different regions of the same animal, at any developmental stage. For example, hard cuticle is typical of larval or adult mouthparts, while flexible cuticle is

* Fax: þ33 3 80 39 62 89. E-mail address: [email protected] 0965-1748/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2009.12.005

deposited in intersegmental membranes. The properties of the cuticle therefore vary greatly both during development, as well as with the region considered. The genes encoding cuticle proteins are therefore an attractive model for the study of the mechanisms of time- and tissue-specific cell differentiation during the development of animals. This review summarizes the studies dealing with the hormonal regulation of cuticle protein genes (CP genes), as well as those bringing some information on their regulatory sequences, or the transcription factors that might regulate their expression. 2. Temporal regulation: developmental hormones 2.1. Ecdysteroids and molting cycles Molts are triggered and regulated by invertebrate steroid hormones collectively referred to as ecdysteroids (Butenandt and Karlson, 1954; Rees, 1989). Precursors (generally ecdysone or 3-dehydroecdysone) are secreted by prothoracic glands and converted into a more potent hormone, 20-hydroxy-ecdysone (20E) (Hoffmeister et al., 1967; Horn et al., 1968) by peripheral tissues (King et al., 1974). These findings have opened the way for systematic measurements of ecdysteroid titers throughout development in various arthropods. During every molting cycle, a large increase or “peak” in hemolymph ecdysteroid concentrations can be measured (Fig. 1; Steel and Vafopoulou, 1989). Moreover, the successive phases of every ecdysteroid peak (i.e. increasing titer, peak, then decreasing titer), can be correlated with distinct

206

J.P. Charles / Insect Biochemistry and Molecular Biology 40 (2010) 205e213

Fig. 1. Schematic representation of cuticle synthesis and ecdysteroid titers during late larval development and metamorphosis of Drosophila. Top: The bars represent the periods of cuticle synthesis (2,3: cuticles of L2 and L3 larvae; P: pupal cuticle; A: Adult cuticle). Black arrowheads indicate important events of the molting cycles. Ap: Apolysis: the detachment of the old cuticle from the underlying epidermis; E: ecdysis: the shedding of the old cuticle. He: Head eversion: in Drosophila and other brachycera, the formation of the pupa occurs within the last instar cuticle, which becomes the puparium after tanning. Head eversion coincides with larvalepupal ecdysis in other insects, and also marks the transition from a cryptocephalic to a phanerocephalic pupa. The three main parts of each cuticle (from outside to inside: epicuticle, preecdysial cuticle, post-ecdysial cuticle) are respectively represented with black boxes, or with dotted and wavy patterns. Epidermal cells are shown as squares with round nuclei. The five classes of salivary gland puffs described by Ashburner and colleagues (Ashburner et al., 1974) are depicted over the ecdysteroid titers (Riddiford, 1993). Note that cuticle synthesis (top, color bars), is interrupted when ecdysteroid titers are rising, with the exception of the third instar cuticle (see text; Kaznowski et al., 1985; Mitchell et al., 1971). P0 (grey arrowhead) denotes the white prepupal stage (i.e. pupariation), corresponding to the onset of puparium tanning. Grey arrows depict the span of larval and other developmental stages. The small 20E peaks at 8, 20 and 28 hours in the third instar are a schematic representation of those described in Warren et al. (2006). Bottom: RNA levels for CP genes (top), and known or possible transcriptional regulators for CP genes (see text). Expression data from: LCP65Ab1/2 (Charles et al., 1998); L-PCPs, H-PCPs (Doctor et al., 1985), ACP65A (Bruey-Sedano et al., 2005); DHR38 (Kozlova et al., 1998); BrC (Zhou and Riddiford, 2002); L3 and prepupal/early pupal expression of E74A/B, DHR3, bFTZ-F1 (Sullivan and Thummel, 2003); late pupal expression of E74A/B, DHR3, bFTZ-F1 (Bruey-Sedano, 2001). For the sake of clarity, the y- (ecdysteroid titers) and x-axes (time) are not to scale.

activities in epidermal cells (reviewed in Riddiford, 1985). In brief, DNA synthesis or mitoses will occur while ecdysteroid concentrations are rising. Next, the old cuticle will detach from the underlying epidermis (an event known as apolysis, Fig. 1). Epidermal cells next secrete a molting gel containing enzymes that will later be activated to digest the old cuticle. The first layer of the new cuticle, epicuticle, is deposited while ecdysteroids are at about peak levels. It consists of a thin outer epicuticle (also called cuticulin) and a thicker inner epicuticle. Both inner and outer epicuticle are essentially composed of lipoproteins, which are soon stabilized by cross-linking after synthesis. The bulk of the cuticle is deposited while the ecdysteroid titers are declining. These typically much thicker layers are mainly composed of chitin fibers and proteins, and constitute the chitinous cuticle, or procuticle (Locke, 2001; Willis et al., 2005). The old cuticle, partially digested, is shed during ecdysis, once the ecdysteroid concentrations have reached low

(intermolt) levels. The cuticle deposited before ecdysis is referred to as precdysial cuticle or commonly, exocuticle. Conversely, the layers deposited after ecdysis, in the presence of basal concentrations of ecdysteroids, constitute the post-ecdysial cuticle or endocuticle (Fig. 1). Following ecdysis, the cuticle is initially pale and soft. It becomes rapidly hardened, and can also be melanized during the tanning process. The hardening of the cuticle is known as sclerotization, and corresponds to cross-linking of cuticular components (chitin and proteins) by dopamine derivatives (Andersen et al., 1996; Hopkins et al., 2000). The degree of sclerotization varies greatly according to the type of cuticle (hard/rigid vs. soft/pliant) and is essentially limited to precdysial cuticle (Andersen, 2005; Hopkins et al., 2000). Post-ecdysial cuticle can be either less sclerotized or not sclerotized. The stereotyped behavior associated with ecdysis, as well as the control of cuticular melanization and sclerotization, is under the control of peptide hormones

J.P. Charles / Insect Biochemistry and Molecular Biology 40 (2010) 205e213

emanating form the central nervous system or from peripheral peptidergic cells (Honegger et al., 2008; Truman, 2005). The synthesis and release of these peptide hormones is itself under the control of the fluctuating concentrations of ecdysteroids (Zitnan et al., 2007). 2.2. Importance of the ecdysteroid pulse The fact that the bulk of chitinous cuticle (procuticle) is deposited while ecdysteroid titers are declining suggests a particular mode of regulation. Physiological studies have demonstrated that the decline in hemolymph concentrations was required for the normal progression of events during a molting cycle. Injection of 20E into late Tenebrio pupae results in delayed ecdysis (Slama, 1980). Similarly, in the moth Manduca sexta, administration of 20E also delays pigmentation of the newly formed cuticle, resorption of the old endocuticle, as well as ecdysis. Conversely, thoracic ligations, which provoke a rapid drop in 20E concentration in the abdomen, accelerated all these events (Schwartz and Truman, 1983). The importance of the decline in ecdysteroid titers is not limited to epidermis. It is required for the programmed death of abdominal motoneurons that control ecdysis behaviour, and also for the death of abdominal muscles involved in ecdysis (Schwartz and Truman, 1982; Truman, 2005; Truman and Schwartz, 1984). These studies suggested that the progression of the molting cycle needs a “pulse” regimen of ecdysteroids, i.e. an initial stimulation by elevated ecdysteroid concentrations, followed by a decline to basal levels. This paradigm has been tested for cuticle in vitro using Drosophila third larval instar imaginal discs (Fristrom and Yund, 1980; Fristrom et al., 1982). When imaginal discs are cultured in vitro without hormone, no cuticle is formed. In the continuous presence of high (1 mg/ml) concentrations of 20E (equivalent to the late larval peak), cuticle formation is limited to the most external layers (epicuticle). In contrast, a procuticle comparable to that made in vivo is obtained if the discs are cultured for 6 h in the presence of high concentrations of 20E, then either in the absence or in the presence of low levels of 20E (Fristrom et al., 1982). A corollary to this model is that cuticle synthesis should be inhibited when 20E is added back to the tissue after the hormonal wash-out (see Fristrom et al., 1986 for review). As expected, a number of cuticle protein genes were found to be regulated by a pulse of 20E (Table 1). The Drosophila EDG84A and EDG78E both pupal CP genes for example, were isolated in a screen for genes regulated by a 20E pulse (Fechtel et al., 1988). This type of regulation is not limited to Diptera, as the expression of six lepidopteran and one hymenopteran CP genes was clearly shown to require a 20E pulse in vitro (Table 1). The evidence is not as conclusive for all genes. For example, the expression of the Tenebrio molitor ACP20 gene is dramatically reduced (or delayed) in vivo by injecting high doses (10e15 mg/animal) of 20E at the time of the pupal peak (Bouhin et al., 1993), which is consistent with a requirement for declining titers after the peak. Data from in vitro culture experiments, however, only partially fit to the pulse model. In wings explanted at about the time of the endogenous pupal 20E peak, the ACP20 gene can be induced by a wide range of 20E concentrations in vitro (Braquart et al., 1996). This induction requires a 48 h delay, and is dependent on protein synthesis, consistently with regulation by a 20E pulse. However, 20E washout experiments in vitro did not provoke the expected increase in expression. In addition, maintaining the wing in culture in vitro in the presence of a 20E concentration equivalent to about 10 times the value of the pupal peak, could reduce ACP20 expression to only about 50% of its maximal level (Braquart et al., 1996). These results may stem from particularities of the physiology of Coleoptera, such as the ability of some cells (likely epidermal cells or oenocytes) to

207

Table 1 CP genes regulated by developmental hormones. Species are: Am (Apis mellifera); Bm (Bombyx mori); Dm (Drosophila melanogaster); Ms (Manduca sexta), Tm (Tenebrio molitor). Cuticle proteins are classified as RR1 or RR2 according to data retrieved from cuticleDB (Magkrioti et al., 2004). GR: Glycine-rich protein; LCA: Low Complexity, Alanine-rich proteins. Pre/Post refers respectively to precdysial and post-ecdysial cuticle. The stage column indicates to which cuticle the protein contributes (rather than the stage during which it is synthesized); L ¼ larval, P ¼ Pupal, A ¼ Adult, Epi ¼ epicuticle. Question marks denote hypothetical status. 20E: Reported effects of 20E ([ ¼ up regulation, [Y ¼ pulse regulation). JHs: Reported effects of JHs (the [ and Y reflect effects on the differentiation state of the epidermis, and not direct activation or suppression). References are indicated in parenthesis: 1: Apple and Fristrom, 1991; 2: Bouhin et al., 1993; 3: Bouhin et al., 1992a; 4: Bouhin et al., 1992b; 5: Braquart et al., 1996; 6: Charles et al., 1992; 7: Fechtel et al., 1988; 8: Hiruma et al., 1991; 9: Horodyski and Riddiford, 1989; 10: Kawasaki et al., 2002 11: Kimbrell et al., 1988; 12: Lemoine et al., 2004; 13: Murata et al., 1996; 14: Nakato et al., 1992; 15: Nita et al., 2009; 16: Noji et al., 2003; 17: Rondot, 1995; 18: Rondot et al., 1996; 19: Shofuda et al., 1999; 20: Soares et al., 2007; 21: Suzuki et al., 2002; 22: Wang et al., 2009a; 23: Zhong et al., 2006; 24: Zhou and Riddiford, 2002. Gene

Sp.

R&R

Pre/Post

Stage

ACP20 ACP22 ACP65A AmelCPR14 BMCP18 BMCPG1 BMGRP1 BMGRP2 BMGRP3 BMWCP10 BMWCP2 BMWCP5 EDG78E EDG84A EDG91 LCP-14 LCP16/17 LCP2 PCP TMLPCP-22

Tm Tm Dm Am Bm Bm Bm Bm Bm Bm Bm Bm Dm Dm Dm Ms Ms Dm Bm Tm

RR2 RR2 RR1 RR1 RR1 GR GR GR GR RR1 RR2 RR2 RR1 RR2 GR RR1 RR1 RR1 LCA LCA

Pr Pr Pr/Po Pr/Po Pr/Po Pre (Epi?) Pr/Po Pr/Po Pr/Po Pr (Epi?) Pr/Po Pr/Po Pr Pr Pr Pr/Po Po Po Pr ?/Po Pr/Po

A A A P, A L4-5, A L5 L5, P, A L5, P, A L5, P, A P P P, A P P P L4-5 L5 L3 P P

20E [ (5,12)[Y(2) [ (2)

JHs Y (6) Y (3,4) Y (24)

[Y (20) [ (19) [Y [Y [Y [Y [ [Y [Y [Y [Y [Y [Y [ [? [Y [Y

(21) (23) (23) (23) (22, 16) (15) (22) (10, 1, 7) (1, 7,13) (1) (8) (9) (11) (14) (17)

Y (23)

[ (8) Y (9)

[ (18)

generate the haemolymph 20E peaks of the pupal stage (Delbecque et al., 1978; Delbecque and Slama, 1980). An alternative explanation is that this gene, which is mainly expressed during the precdysial period and encodes an RR2 cuticle protein (Charles et al., 1992; Willis et al., 2005), may be a representative of a class of genes that are regulated somewhat differently by ecdysteroids (see below). It is also important to note, however, that pupal procuticle deposition occurs even in the continuous presence of ecdysone in Drosophila (references in Fristrom et al., 1982) see for example (Fristrom et al., 1973; Johnson and Milner, 1990; Milner and Muir, 1987), yet at slower rates than under a pulse regimen of hormone. Similarly, adult sternal cuticle of Tenebrio was synthesized in vitro in the continuous presence of peak levels (4 mg/ml) of 20E, but at a much slower rate than in vivo (Quennedey et al., 1983). Culture parameters may be also important, as for procuticle deposition in vitro, the inhibitory effect of 20E is not as important in the presence of BSA in the culture medium (Fristrom and Yund, 1980). In summary, if a pulse regimen of 20E seems generally required to trigger the expression of procuticle protein genes, further studies may reveal the importance of other mechanisms of regulation. 2.3. CP genes not regulated by a pulse of 20E The cuticles deposited before and after ecdysis may differ ultrastructurally. In soft cuticles, the pre- and post-ecdysial layers are usually similar at the ultrastructural level, and characterized by chitin fibers with a helicoidal organization. In contrast, in hard/rigid

208

J.P. Charles / Insect Biochemistry and Molecular Biology 40 (2010) 205e213

cuticles the chitin filaments often adopt either a preferred orientation, or show alternate layers with helicoidal and preferential orientations (Neville, 1975). In the case of rigid cuticles, the preecdysial layers differ also, in that they are sclerotized after ecdysis. Not surprisingly, large differences in protein composition were found between pre- and post-ecdysial cuticles (see for example, Roberts and Willis, 1980). In addition, even within the pre- or postecdysial phase, cuticle proteins and mRNAs appear with a reproducible and specific schedule (for review, see Willis, 1996). As first recognized by Andersen (2000), cuticle proteins of the RR2 type are mostly expressed before ecdysis (see the review by J.H. Willis, 2010). This is supported by two recent surveys of the developmental expression patterns of many CP genes in Anopheles gambiae (Togawa et al., 2008) and in Bombyx mori (Okamoto et al., 2008). It seems therefore likely that the genes encoding exocuticle protein are not controlled in the same way as those encoding endocuticle proteins, and that a switch in regulation should occur at ecdysis. There is no pupal ecdysis in Drosophila, since the pupa is enclosed within the third instar cuticle, which forms the puparium. There is however a sharp switch in cuticle protein synthesis during head eversion (Fig. 1, He), which can be considered as homologous to the time of pupal ecdysis. Low molecular weight pupal cuticle proteins (L-PCPs) are synthesized before head eversion, and high molecular weight PCPs (H-PCPs) are deposited after head eversion (Doctor et al., 1985). This switch was shown to depend on a small rise in 20E titer, which implies that the genes encoding H-PCPs are regulated somewhat differently by 20E (Doctor et al., 1985). It is unclear if a similar 20E peak occurs at ecdysis in other species (see discussion in Riddiford, 1985 and Dean et al., 1980), so the reasons for the switch between synthesis of exo- and endocuticle remain obscure. The cascade of peptide hormones that regulate ecdysis and tanning of the cuticle (Truman, 2005; Zitnan et al., 2007) may be expected to play a role in this transition, but to this day there is no evidence that these hormones can regulate cuticle gene expression. A few CP genes are clearly expressed independently of the main 20E peaks. The LCP1-4 proteins are major components of the Drosophila third instar cuticle (Chihara and Kimbrell, 1986). This is a very special cuticle that will serve, after tanning, as a protective puparium during metamorphosis. In contrast to other cuticles, it is synthesized until late in the instar, to reach a final thickness of about 25 mM (Fig. 1; Mitchell et al., 1971; Kaznowski et al., 1985). The mRNAs for LCP1 and LCP2 are expressed at in the later part of the instar (Kimbrell et al., 1988), when the 20E titer is high (Fig. 1). Therefore, these genes are likely not controlled by a pulse of 20E. It may be speculated, however, that the small 20E peaks that have been found in early- and mid-third instar (Warren et al., 2006; Fig. 1) may be involved in their regulation. In the last day of feeding, during the last larval instar of M. sexta, a set of new cuticle proteins is synthesized, coincidently with the deposition of very thin lamellae of endocuticle (Wolfgang and Riddiford, 1981, 1986). The onset of expression of the LCP16/17 gene, which encodes one of these proteins, coincides with a small rise in the 20E titer (Horodyski and Riddiford, 1989; Wolfgang and Riddiford, 1986). The LCP16/17 gene is also induced in vitro in the continuous presence of moderate levels of 20E, which suggests that it does not require a typical hormonal pulse (Horodyski and Riddiford, 1989). The outer epicuticle, or cuticulin, is the most external layer of the cuticle, and is deposited when ecdysteroid titers are around peak levels. The regulation of cuticulin formation in Drosophila has been shown to depend, as for the procuticle, of a pulse regimen of 20E but is less sensitive to inhibition by 20E than procuticule (Fristrom and Liebrich, 1986). It may therefore be anticipated that the genes encoding components of the epicuticle are also less sensitive to inhibition by 20E.

The Bombyx BMWCP10 gene encodes a protein of the RR1 type, that has been hypothesized to be a component of the epicuticle. It is directly induced by 20E in vitro, and is expressed concomitantly with the peak that triggers pupal cuticle synthesis (Noji et al., 2003; Table 1). Its regulation by 20E is complicated, since it is initially directly induced by 20E (Noji et al., 2003), but its later expression requires protein synthesis (Wang et al., 2009a; the requirement for protein synthesis is used to distinguish primary response to ecdysteroids from secondary responses). The Tenebrio ACP22 gene encodes a protein that accumulates in the exocuticle layers, but not in the epicuticle (Bouhin et al., 1992a; Lemoine et al., 1989), yet it is stimulated within 3 h by injection of exogenous 20E (Bouhin et al., 1993). As noted above, the ACP20 gene of Tenebrio, also, does not seem to exhibit a typical response to a 20E pulse (Braquart et al., 1996; Lemoine et al., 2004). Taken together, these observations warrant further studies of 20E regulation of genes for exocuticle proteins, as they could possibly hint at novel mechanisms at work early during cuticle synthesis, and acting maybe in parallel with the well demonstrated “pulse” type of regulation.

3. Regulatory sequences and transcription factors involved in expression of CP genes The regulatory sequences of ten CP genes from only 4 insect species have been analyzed in some detail. Their main characteristics and the experimental evidence supporting the existence of response elements for transcription factors are summarized in Table 2. The functional analyses were carried out either in vivo, using transgenic flies, or in transient transfection assays. Transgenic analyses allow one to investigate gene regulation in its normal context, as well as in living animals (using GFP as a reporter, for example). It is also a very practical way to investigate subtle tissuespecific regulation of expression. In contrast, in vitro experiments are best suited to manipulate hormonal concentrations. Five transcription factors have been implicated in the regulation of CP genes, among which two are known to be regulated by 20E.

3.1. The molecular mechanisms of 20E signalling A series of studies on puffing patterns in the salivary glands of Diptera have uncovered the molecular mechanisms underlying the action of ecdysteroids, synthesized in the “Ashburner model” (Ashburner et al., 1974; Thummel, 2002, for reviews). A sequence of five waves of puffing were distinguished in Drosophila, that can be reproduced in vitro by incubating salivary glands in the presence of 20E (Fig. 1). The intermolt puffs are present in mid-third instar larvae, and disappear when the 20E concentrations increase, later in the instar. They are replaced by early puffs, which are directly induced by 20E. The proteins encoded by the early puffs are transcription factors that activate about a hundred of late puffs which appear even in the presence of continuous and elevated concentrations of 20E. The mid-prepupal puffs (Fig. 1), however, are inhibited by high 20E, and appear only if the glands are transferred to a culture medium containing no hormone (Richards, 1976). The mid-prepupal puffs, therefore, are activated by a 20E pulse, much like the L-PCPs and other genes encoding procuticle proteins. Importantly, the puffing sequence observed in salivary glands was shown to occur also in the fat body, although with some differences (Richards, 1982). Furthermore, this finding was confirmed and extended to other tissues (including wing discs, which eventually synthesize the pupal and adult cuticle), when the expression of the transcription factors encoded by these puffs was examined (Huet et al., 1993, 1995; Thummel et al., 1990).

J.P. Charles / Insect Biochemistry and Molecular Biology 40 (2010) 205e213

209

Table 2 Regulatory sequences and transcription factors of insect CP genes. Species are: Bm (Bombyx mori); Dm (Drosophila melanogaster); Hc (Hyalophora cecropia); Tm (Tenebrio molitor). Regulatory sequences: minimal (I) gives the approximate minimal length of regulatory sequences required for expression similar to the endogenous gene; minimal (II) corresponds to the shortest regulatory sequences able to reproduce at least some aspect of the normal expression pattern. Assay: transF ¼ transient transfection assay; transG ¼ analysis in transgenic animals. Position of Response Elements (REs) is given relative to the start of transcription. EMSA: a dot indicates that binding of the putative REs to a putative transcription factor (TF) was checked by gel shift assay. Mut REs: a dot indicates that the importance of REs has been checked by mutagenesis; ? means uncertain. TF co-expressed: indicates if co-expression of a putative regulator and its target cuticle gene is ascertained. Ectopic: induction of expression by forced expression of TF. Mutant: loss of expression was checked in TF mutant background. Intron: intron included in the construct. Tissue: tissue-specific element found. References are indicated in parenthesis: 1: Andres et al., 1993; 2: Bruey-Sedano et al., 2005; 3: Cui et al., 2009; 4: Kawasaki et al., 2002; 5: Kayashima et al., 2005; 6: Kimbrell et al., 1989; 7: Kozlova et al., 2009; 8: Lampe and Willis, 1994; 9: Lemoine et al., 2004; 10: Lestradet et al., 2009; 11: Murata et al., 1996; 12: Nita et al., 2009; 13: Togawa et al., 2001; 14:Wang et al., 2009a; 15: Wang et al., 2009b; 16: Zhou and Riddiford, 2002. Gene

Species Regulatory sequences Minimal (I)

BMWCP2 (12) Bm BMWCP10 (14) Bm BMWCP5 (15) Bm BMCP18 (13) EDG84A (11) EDG78E (4) ACP65A (2)

Bm Dm Dm Dm

ACP20 (9) HCCP66 (8) LCP2 (6) ACP1 (7) CPr92A (7)

Hc Dm Dm Dm

Assay

Minimal (II) w170 bp w410 bp w200 bp

Response elements Position

FTZ-F1: 248/108 BRC-Z2: 423 FTZ-F1: 553/168 BrZ4: 1243/504 SVP: 84 w220 bp transG FTZ-F1: 100 w470 bp w1200 bp transG FTZ-F1: 515/100 w750 bp (2) w180 bp (10) transG DHR38: 61 w 700 bp

transF transF transF

w350 bp

transF

w500 bp

transF

OCT: 81

3.2. bFTZ-F1 One of the mid-prepupal puffs, located at 75CD on the third chromosome, corresponds to the FTZ-F1 gene, which encodes the orphan nuclear receptors (NRs) aFTZ-F1 and bFTZ-F1 (Lavorgna et al., 1993, 1991; Ueda et al., 1990). These two proteins share a common C-terminal part, but have distinct N-termini. In Drosophila, aFTZ-F1 is expressed in blastoderm embryos, while bFTZ-F1 is expressed in late embryos, at the end of larval and prepupal stages (Lavorgna et al., 1993; Ueda et al., 1990), as well as in late pupae (Bruey-Sedano, 2001; see also Fig. 1). An orthologous gene, BmFTZ-F1, was cloned in B. mori (Ueda and Hirose, 1990). Like Drosophila bFTZ-F1, it is expressed after the decline in 20E titers at each molting stage, and is induced by a pulse of 20E in vitro (Sun et al., 1994). BmFTZ-F1 binds to DNA as a monomer on a Nuclear Receptor Element (NRE) half-site with a 50 extension (FTZ-F1RE (RE: Response Element): 50 YCAAGGTCR (Ueda and Hirose, 1991; Ueda et al., 1992)). It was then recognized that a potential binding site for the ecdysone receptor in the 50 flanking region of the Drosophila EDG84A cuticle gene noted in an earlier study (Apple and Fristrom, 1991), was also a potential FTZ-F1RE (Murata et al., 1996). Functional analyses demonstrated unambiguously that bFTZ-F1 is a critical transcriptional activator of EDG84A (Murata et al., 1996; Table 2). Indeed, most criteria required were fulfilled, i.e. (i) overlapping expression domain and timing of EDG84A and bFTZ-F1 (importantly, both genes are induced by a 20E pulse), (ii) presence of a FTZ-F1RE within the region required for expression, (iii) gel shift of the FTZ-F1RE by FTZ-F1 from nuclear extracts of EDG84 expressing tissue (iv) abolition of expression in vivo in constructs with a mutant FTZ-F1RE, and (v) induction of EDG84 expression by forced expression of bFTZ-F1. These findings stimulated further studies, and evidence for the importance of bFTZ-F1 in the activation of three other cuticle genes was obtained (Table 2). Another Drosophila cuticle gene, EDG78E, was originally isolated along with EDG84A, on the basis of its activation by a 20E pulse (Fechtel et al., 1988). Two potential FTZ-F1REs were found in its regulatory sequences, and the most distal (Table 2) is likely more important for EDG78E activation, on the basis of its better affinity for bFTZ-F1 (Kawasaki et al., 2002). In addition, transgenic

Transcription factor EMSA mut REs TF co-expressed Ectopic C C C C C C C

C C C C C C ?

Intron

Tissue

Mutant

BmFTZ-F1 BRC-Z2 BmFTZ-F1; BrZ4 SVP bFTZ-F1 (11,1) bFTZ-F1 (1) DHR38 (3)

C C (5) C C C C DHR38 (2) C (2, 7) C (10) C (2,10) C BrZ1 (16) C

C DHR38 DHR38

constructs with mutated FTZ-F1REs showed reduced expression, and the level of EDG78E RNA was reduced in a mutant bFTZ-F1 background (Kawasaki et al., 2002). An important role of BmFTZ-F1 was also demonstrated for two Bombyx CP genes regulated by a 20E pulse (Nita et al., 2009; Wang et al., 2009a; Tables 1 and 2). The BMWCP2 gene has two FTZ-F1REs, and the proximal element appears to be essential for expression (Nita et al., 2009). The BMWCP5 gene also has two specific binding sites for bFTZ-F1, and in this case both proved to be important for expression (Wang et al., 2009a). Together, these studies demonstrate that bFTZ-F1 is a major transcriptional regulator of CP genes. Likely, many other CP genes expressed during the decline of the 20E titer are also regulated by this transcription factor, because it is expressed in the late part of every developmental stage (Yamada et al., 2000; Bruey-Sedano, 2001) and either its forced expression, or its absence in mutant larvae induced defects in cuticle formation (Yamada et al., 2000). Finally, bFTZ-F1 is not only important for activation of procuticule synthesis, but is broadly implicated in the late part of the molt. It is involved in the regulation of ecdysteroid synthesis (Parvy et al., 2005), and also likely regulates the neuroendocrine cascade occurring in response to declining ecdysteroid titers (see discussion in Zitnan et al., 2007). 3.3. DHR38 DHR38 was first isolated in a screen for heterodimerizing partners of the nuclear receptor USP (Sutherland et al., 1995) and is orthologous to the vertebrate Nerve Growth Factor Induced protein (NGFI-B). Mutational analysis in Drosophila demonstrated that it is required for adult cuticle formation (Kozlova et al., 2009, 1998). Similarly to NGFI-B, it binds to DNA through a nuclear receptor (NR) half-site, with a 50 extension containing A/T base pairs (Wilson et al., 1991). It also binds as a heterodimer with USP to direct repeats of NR half-sites separated by a variable number of base pairs (Crispi et al., 1998). Flies carrying an impaired allele of DHR38 develop normally until the late pharate adult stage, but then display melanized spots predominantly in leg joints and occasionally in the proboscis, antennae and wing hinges. Melanization

210

J.P. Charles / Insect Biochemistry and Molecular Biology 40 (2010) 205e213

occurs as a consequence of rupture of the cuticle, followed by leakage of the haemolymph and melanization within the pupal case (Kozlova et al., 1998; Pokholkova et al., 1999). This phenotype is suggestive, because this apparent cuticle fragility overlaps with the expression domains of the Drosophila ACP65A adult cuticle gene (Bruey-Sedano et al., 2005). ACP65A belongs to a cluster of CP genes mapping at cytological position 65A on the third chromosome (Charles et al., 1997), and encodes a protein of the RR1 type, which is prevalent in soft cuticles (Willis, JH; 2010). Genetic analyses showed that DHR38 is required for normal expression of ACP65A, and conversely, that misexpression of DHR38 leads to overexpression of ACP65A (Bruey-Sedano et al., 2005; Kozlova et al., 2009). A potential DHR38 Response Element for DHR38 was found in the ACP65A promoter region (Table 2), but it is not essential for ACP65A expression (Lestradet et al., 2009). The expression of two additional CP genes (CPr92A and ACP1) also requires DHR38 function (Kozlova et al., 2009), which suggests that this transcription factor may have a broad role during adult cuticle formation. 3.4. Juvenile hormones and stage specificity Juvenile hormones (JHs) are sesquiterpene compounds secreted by the corpora allata of insects, which, together with ecdysteroids, control molting and metamorphosis (Wigglesworth, 1934, 1954). Elevated JH concentrations in larval stages prevent metamorphosis of the cuticle (Willis, 1996). At the end of larval development, the attainment of a critical body weight triggers the cessation of synthesis of JHs, and the activation of JHs degrading enzymes (Nijhout, 1994). In the absence of JHs, ecdysteroids then trigger the reprogramming of larval epidermal cells to produce pupal and adult cuticle. Conversely, exogenous JH disrupts the normal progression of metamorphosis, and cause ecdysteroids to trigger the synthesis of a cuticle identical to that of the previous stage, or combining characters of two developmental stages (Willis et al., 1982). JHs are therefore expected to regulate, either directly or indirectly, the expression of stage-specific CP genes. The CP genes shown to depend on JHs are listed in Table 1. Some genes are inhibited by JHs because they require that epidermis be reprogrammed either to a pupal (BWCP2) or an adult fate (ACP20, ACP22, ACP65A). The others, which are normally shut down during the larvalepupal (BMCP18, LCP1-4) or the pupaleadult transition (TMLPCP-22), are re-activated if JHs are given before the critical period (Table 1). The molecular mechanisms underlying these effects of JHs are now better understood. They involve the Broad proteins, which are members of the Broad-Tramtrack-Bric-a-Brac (BTB) family of transcription factors, and have been shown to be essential for pupal development (Riddiford, 2008 for review). Application of exogenous JHs during the adult development of Manduca or Drosophila provokes the re-expression of the Broad gene, which is normally restricted to the pupal stage. The reexpression of Broad, in turn, triggers the expression of the pupal cuticle gene EDG78E, and suppresses that of the adult cuticle gene ACP65A (Zhou and Riddiford, 2002). Transgenic constructs with ACP65A genomic DNA sequences between 265/þ161 fused to a reporter were suppressed by misexpression of Broad, which indicates that this region encompasses key regulatory elements (Cui et al., 2009). Importantly, overexpression of Broad also suppressed the expression of DHR38 (Cui et al., 2009). Since DHR38 is an activator of ACP65A (Bruey-Sedano et al., 2005; Kozlova et al., 2009), the suppressive effect of Broad is thus mediated, at least in part, by the suppression of DHR38 expression. Consistent with its role as a specifier of the pupal stage, Broad misexpression in the second larval instar suppressed expression of the larval cuticle gene LCP65Ab (Charles et al., 1997, 1998; Zhou and Riddiford, 2002).

Conversely, Broad proteins were shown to be activators for pupal CP genes. The Drosophila pupal CP genes EDG78E, PCP, and in a lesser extent EDG84, can be activated during synthesis of the adult cuticle by overexpression of the Broad BrZ1 isoform (Zhou and Riddiford, 2002). In addition, functional Broad response elements were characterized for two Bombyx pupal CP genes, BMWCP5 and BMWCP10 (Nita et al., 2009; Wang et al., 2009a). Since these two genes are very differently regulated by ecdysteroids (see above), it will be interesting to examine if, and how Broad participates with factors (s) such as bFTZ-F1 to regulate their specific time course of expression during pupal cuticle synthesis. 3.5. Tissue specificity One important problem related to hormone action is how different tissues respond differently to the same hormone. CP genes are well suited to address this question since they have often very restricted expression domains within epidermis, and are targets of ecdysteroids and juvenile hormones. Tissue specificity could be achieved by the spatially-restricted expression of components of an hormonal signalling pathway, as found for the different isoforms encoded by the EcR and Broad early genes (Bayer et al., 1997; Cherbas et al., 2003; Emery et al., 1994; Schubiger et al., 2003; Talbot et al., 1993). Alternatively, tissue-specific transcription factors may exist, that are independent of ecdysteroid signalling. Only a few tissue-specific enhancers, from three Drosophila CP genes have been described so far (Table 2). The EDG84A gene is expressed during pupal cuticle formation in the anterior structures derived from imaginal discs (Murata et al., 1996). One negative element (IE1; Murata et al., 1996) is responsible for repression in abdominal epidermis. Activation in anterior epidermis is driven by a short (ca. 50 bp), yet complex regulatory sequence. It is composed of two independent activating regions, AERI and AERII. AERI encompasses three distinct activating elements, in addition to a repressor element (IE2) that acts upon AERII (Kayashima et al., 2005). These elements lie upstream of, and are therefore clearly distinct from, the bFTZ-F1 response element that is strictly required for EDG84A expression (Murata et al., 1996). The EDG78E gene is expressed in both anterior and abdominal epidermis (Fechtel et al., 1989). In contrast to EDG84A, anterior expression was found to depend on bFTZ-F1 response elements, and consistently, was abolished in anterior epidermis in a FTZ-F1 mutant background (Kawasaki et al., 2002). Tissue-specific elements were also evidenced in the flanking regions of the adult cuticle gene ACP65A (Bruey-Sedano et al., 2005). Sequences located between bp 594 and 256 are required for high level expression in the thorax and the proboscis. A short domain (bp 256/213) contains both an activating element for proximal leg joints, as well as a repressor element for the wing blade and the eyes. When these two domains are deleted however, the timing of expression is unchanged (Bruey-Sedano et al., 2005). Thus, in the case of the ACP65A gene, similarly to EDG84A, the elements regulating time- and tissue-specific expression are at least partly distinct. 3.6. Role of intronic sequences Many CP genes possess an intron that interrupts the signal peptide coding sequence, which suggests a function in gene expression (Binger and Willis, 1994; Charles et al., 1997; Lemoine et al., 2004). In addition, among the 88 Drosophila proteins having a “phase 0” intron localized after the fourth codon, a striking proportion (43%) are annotated as cuticle proteins (Nielsen and Wernersson, 2006). The biological significance of this bias is

J.P. Charles / Insect Biochemistry and Molecular Biology 40 (2010) 205e213

unknown. Introns can alter transcription in two main ways. On one hand, intronic sequences may contain response elements for transcriptional activators or repressors. Alternatively, once transcribed into RNA, intronic sequences can interfere with transcription via components of the RNA splicing machinery (Le Hir et al., 2003; Rose, 2008, for reviews). In the beetle, T. molitor, the promoter-proximal intron of the ACP20 cuticle gene was shown to be required for expression in a transient transfection assay (Lemoine et al., 2004). More recently, the expression of the Drosophila ACP65A gene was shown to be almost completely suppressed in the absence of its intron in vivo (Bruey-Sedano et al., 2005). Importantly, expression was significantly rescued when intronic sequences were included, in transgenic constructs, upstream of the transcription start site (Lestradet et al., 2009). This result strongly suggests that these intronic sequences do not need to be converted into mRNA in order to stimulate transcription. In the case of ACP65A, although transcription was dramatically reduced in intronless constructs, low levels of expression were maintained, with a normal temporal and spatial pattern (Lestradet et al., 2009). It is unclear if this is the rule for other CP genes, as introns were not included in most analyses (Table 2), but it may be interesting to test whether inclusion of intronic sequences can significantly enhance their levels of expression. 4. Concluding remarks There is now a coherent body of evidence demonstrating that many CP genes are activated by a pulse of ecdysteroids, very much like the “mid-prepupal” puffs described in Drosophila salivary glands (Richards, 1976). The evidence is also accumulating for a widespread function of the orphan nuclear receptor bFTZ-F1 in the activation of genes encoding procuticle proteins. The analysis of the transcriptional cascade induced by ecdysteroids has led to the identification of many other transcription factors (Hiruma and Riddiford, 2009; King-Jones and Thummel, 2005; Riddiford et al., 2003), some of which may also regulate CP genes. In addition to bFTZ-F1 Response Elements, consensus binding motifs for E74A and Broad proteins were found in the majority of flanking sequences in a survey on Anopheles cuticle genes (Togawa et al., 2008). The expression periods of these genes (Fig. 1), as well as others (see for example Andres et al., 1993; Sullivan and Thummel, 2003) overlap with the expression of CP genes, so the transcription factors encoded by these genes may prove to be involved in their regulation. As noted above, some CP genes are not expressed in response to a 20E pulse: they are therefore expected to be activated by early or late transcription factors, rather than by transcription factors such as bFTZ-F1. A remarkable feature of the regulatory sequences of CP genes is that they drive expression very strictly in epidermis. Although many transgenic constructs, containing regulatory sequences deleted in various ways, have been established for several genes (Bruey-Sedano et al., 2005; Kawasaki et al., 2002; Kayashima et al., 2005; Lestradet et al., 2009; Murata et al., 1996), none showed expression outside imaginal discs or epidermal cells. In addition, in transgenic flies carrying ACP65A constructs, in which yeast UAS sequences were introduced within regulatory DNA, GAL4 overexpression could stimulate transcription of the reporter gene, but only in cells that normally express ACP65A (Lestradet et al., 2009). Together, these observations suggest that inhibitory mechanisms are at work in most cells that may be relieved by yet unknown epidermis-specific factor(s). It is hoped that the increasing knowledge of CP genes regulatory sequences and their transcriptional regulators will lead to the characterization of the epidermis-specific factor(s), and to a better understanding of the mode of action of JHs at the molecular level.

211

Acknowledgements I thank Pr. J.H. Willis and two anonymous reviewers for helpful comments on the manuscript. This work was supported by the Burgundy council and the Centre National de la Recherche Scientifique (C.N.R.S.). References Andersen, S.O., 2000. Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer. Insect Biochem. Mol. Biol. 30, 569e577. Andersen, S.O., 2005. Cuticular sclerotization and tanning. In: Gilbert, L.I., Iatrou, K., Gill, S.S. (Eds.), Comprehensive Molecular Insect Science, vol. 4. Elsevier Pergamon, Amsterdam, pp. 145e170. Andersen, S.O., Peter, M.G., Roepstorff, P., 1996. Cuticular sclerotization in insects. Comp. Biochem. Physiol. B., Biochem. Mol. Biol. 113B, 689e705. Andres, A.J., Fletcher, J.C., Karim, F.D., Thummel, C.S., 1993. Molecular analysis of the initiation of insect metamorphosis: a comparative study of Drosophila ecdysteroid-regulated transcription. Dev. Biol. 160, 388e404. Apple, R.T., Fristrom, J.W., 1991. 20-Hydroxyecdysone is required for, and negatively regulates, transcription of Drosophila pupal cuticle protein genes. Dev. Biol. 146, 569e582. Ashburner, M., Chihara, C., Meltzer, P., Richards, G., 1974. Temporal control of puffing activity in polytene chromosomes. Cold Spring Harb. Symp. Quant. Biol. 38, 655e662. Bayer, C.A., von Kalm, L., Fristrom, J.W., 1997. Relationships between protein isoforms and genetic functions demonstrate functional redundancy at the BroadComplex during Drosophila metamorphosis. Dev. Biol. 187, 267e282. Binger, L.C., Willis, J.H., 1994. Identification of the cDNA, gene and promoter for a major protein from flexible cuticles of the giant silkmoth Hyalophora cecropia. Insect Biochem. Mol. Biol. 24, 989e1000. Bouhin, H., Braquart, C., Charles, J.P., Quennedey, B., Delachambre, J., 1993. Nucleotide sequence of an adult-specific cuticular protein gene from the beetle Tenebrio molitor: effects of 20-hydroxyecdysone on mRNA accumulation. Insect Mol. Biol. 2, 81e88. Bouhin, H., Charles, J.P., Quennedey, B., Courrent, A., Delachambre, J., 1992a. Characterization of a cDNA clone encoding a glycine-rich cuticular protein of Tenebrio molitor: developmental expression and effect of a juvenile hormone analogue. Insect Mol. Biol. 1, 53e62. Bouhin, H., Charles, J.P., Quennedey, B., Delachambre, J., 1992b. Developmental profiles of epidermal mRNAs during the pupaleadult molt of Tenebrio molitor and isolation of a cDNA clone encoding an adult cuticular protein: effects of a juvenile hormone analogue. Dev. Biol. 149, 112e122. Braquart, C., Bouhin, H., Quennedey, A., Delachambre, J., 1996. Up-regulation of an adult cuticular gene by 20-hydroxyecdysone in insect metamorphosing epidermis cultured in vitro. Eur. J. Biochem. 240, 336e341. Bruey-Sedano, N., 2001. Analyse fonctionnelle des séquences régulatrices du gène cuticulaire ACP65A chez la drosophile. PhD thesis, Université de Bourgogne. Bruey-Sedano, N., Alabouvette, J., Lestradet, M., Hong, L., Girard, A., Gervasio, E., Quennedey, B., Charles, J.P., 2005. The Drosophila ACP65A cuticle gene: deletion scanning analysis of cis-regulatory sequences and regulation by DHR38. Genesis 43, 17e27. Butenandt, A., Karlson, P., 1954. Über die Isolierung eines Metamorphosehormons der Insekten in kristallisierter Form. Z. Naturforsh 9b, 389e391. Charles, J.P., Bouhin, H., Quennedey, B., Courrent, A., Delachambre, J., 1992. cDNA cloning and deduced amino acid sequence of a major, glycine-rich cuticular protein from the coleopteran Tenebrio molitor. Temporal and spatial distribution of the transcript during metamorphosis. Eur. J. Biochem. 206, 813e819. Charles, J.P., Chihara, C., Nejad, S., Riddiford, L.M., 1997. A cluster of cuticle protein genes of Drosophila melanogaster at 65A: sequence, structure and evolution. Genetics 147, 1213e1224. Charles, J.P., Chihara, C., Nejad, S., Riddiford, L.M., 1998. Identification of proteins and developmental expression of RNAs encoded by the 65A cuticle protein gene cluster in Drosophila melanogaster. Insect Biochem. Mol. Biol. 28, 131e138. Cherbas, L., Hu, X., Zhimulev, I., Belyaeva, E., Cherbas, P., 2003. EcR isoforms in Drosophila: testing tissue-specific requirements by targeted blockade and rescue. Development 130, 271e284. Chihara, C.J., Kimbrell, D.A., 1986. The cuticle proteins of Drosophila melanogaster: genetic localization of a second cluster of third-instar genes. Genetics 114, 393e404. Crispi, S., Giordano, E., D'Avino, P.P., Furia, M., 1998. Cross-talking among Drosophila nuclear receptors at the promiscuous response element of the ng-1 and ng-2 intermolt genes. J. Mol. Biol. 275, 561e574. Cui, H.Y., Lestradet, M., Bruey-Sedano, N., Charles, J.P., Riddiford, L.M., 2009. Elucidation of the regulation of an adult cuticle gene Acp65A by the transcription factor Broad. Insect Mol. Biol. 18, 421e429. Dean, R.L., Bollenbacher, W.E., Locke, M., Gilbert, L.I., Smith, S.L., 1980. Haemolymph ecdysteroid levels and cellular events in the intermoult/moult sequence of Calpodes ethilus. J. Insect Physiol. 26, 267e280. Delbecque, J.P., Delachambre, J., Hirn, M., De Reggi, M., 1978. Abdominal production of ecdysterone and pupaleadult development in Tenebrio molitor (Insecta, Coleoptera). Gen. Comp. Endocrinol. 35, 436e444.

212

J.P. Charles / Insect Biochemistry and Molecular Biology 40 (2010) 205e213

Delbecque, J.P., Slama, K., 1980. Ecdysteroid titers during autonomous metamorphosis in a dermestid beetle. Z. Naturforsch. B 35c, 1066e1080. Doctor, J., Fristrom, D., Fristrom, J.W., 1985. The pupal cuticle of Drosophila: biphasic synthesis of pupal cuticle proteins in vivo and in vitro in response to 20-hydroxyecdysone. J. Cell. Biol. 101, 189e200. Emery, I.F., Bedian, V., Guild, G.M., 1994. Differential expression of Broad-Complex transcription factors may forecast tissue-specific developmental fates during Drosophila metamorphosis. Development 120, 3275e3287. Fechtel, K., Fristrom, D.K., Fristrom, J.W., 1989. Prepupal differentiation in Drosophila: distinct cell types elaborate a shared structure, the pupal cuticle, but accumulate transcripts in unique patterns. Development 106, 649e656. Fechtel, K., Natzle, J.E., Brown, E.E., Fristrom, J.W., 1988. Prepupal differentiation of Drosophila imaginal discs: identification of four genes whose transcripts accumulate in response to a pulse of 20-hydroxyecdysone. Genetics 120, 465e474. Fristrom, D., Liebrich, W., 1986. The hormonal coordination of cuticulin deposition and morphogenesis in Drosophila imaginal discs in vivo and in vitro. Dev. Biol. 114, 1e11. Fristrom, D., Yund, M.A., 1980. A comparative analysis of ecdysteroid action in larval and imaginal tissues of Drosophila melanogaster. In: Hoffmann, J.A. (Ed.), Progress in Ecdysone Research. Elsevier/North-Holland Biomedical Press. Fristrom, J.W., Doctor, J., Fristrom, D.K., Logan, W.R., Silvert, D.J., 1982. The formation of the pupal cuticle by Drosophila imaginal discs in vitro. Dev. Biol. 91, 337e350. Fristrom, J.W., Logan, W.R., Murphy, C., 1973. The synthetic and minimal culture requirements for evagination of imaginal discs of Drosophila melanogaster in vitro. Dev. Biol. 33, 441e456. Fristrom, J.W., Sherry, A., Brown, E., Doctor, J., Fechtel, K., Fristrom, D., Kimbrell, D.A., King, D.S., Wolfgang, W.J., 1986. Ecdysone regulation of cuticle protein gene expression in Drosophila. Arch. Insect. Biochem. Physiol. 3, 119e132. Hiruma, K., Hardie, J., Riddiford, L.M., 1991. Hormonal regulation of epidermal metamorphosis in vitro: control of expression of a larval-specific cuticle gene. Dev. Biol. 144, 369e378. Hiruma, K., Riddiford, L.M., 2009. The molecular mechanisms of cuticular melanization: the ecdysone cascade leading to dopa decarboxylase expression in Manduca sexta. Insect. Biochem. Mol. Biol. 39, 245e253. Hoffmeister, H., Grutzmacher, H.F., Dunnebeil, K., 1967. Studies on the structure and biochemical action of ecdysterone. Z. Naturforsch. B. 22, 66e70. Honegger, H.W., Dewey, E.M., Ewer, J., 2008. Bursicon, the tanning hormone of insects: recent advances following the discovery of its molecular identity. J. Comp. Physiol. A. Neuroethol. Sens. Neural. Behav. Physiol. 194, 989e1005. Hopkins, T.L., Krchma, L.J., Ahmad, S.A., Kramer, K.J., 2000. Pupal cuticle proteins of Manduca sexta: characterization and profiles during sclerotization. Insect Biochem. Mol. Biol. 30, 19e27. Horn, D.H., Fabbri, S., Hampshire, F., Lowe, M.E., 1968. Isolation of crustecdysone (20R-hydroxyecdysone) from a crayfish (Jasus lalandei H. Milne-Edwards). Biochem. J. 109, 399e406. Horodyski, F.M., Riddiford, L.M., 1989. Expression and hormonal control of a new larval cuticular multigene family at the onset of metamorphosis of the tobacco hornworm. Dev. Biol. 132, 292e303. Huet, F., Ruiz, C., Richards, G., 1993. Puffs and PCR: the in vivo dynamics of early gene expression during ecdysone responses in Drosophila. Development 118, 613e627. Huet, F., Ruiz, C., Richards, G., 1995. Sequential gene activation by ecdysone in Drosophila melanogaster: the hierarchical equivalence of early and early late genes. Development 121, 1195e1204. Johnson, S.A., Milner, M.J., 1990. Cuticle secretion in Drosophila wing imaginal discs in vitro: parameters of exposure to 20-hydroxy ecdysone. Int. J. Dev. Biol. 34, 299e307. Kawasaki, H., Hirose, S., Ueda, H., 2002. BetaFTZ-F1 dependent and independent activation of Edg78E, a pupal cuticle gene, during the early metamorphic period in Drosophila melanogaster. Dev. Growth Differ. 44, 419e425. Kayashima, Y., Hirose, S., Ueda, H., 2005. Anterior epidermis-specific expression of the cuticle gene EDG84A is controlled by many cis-regulatory elements in Drosophila melanogaster. Dev. Genes. Evol. 215, 545e552. Kaznowski, C.E., Schneiderman, H.A., Bryant, P.J., 1985. Cuticle secretion during larval growth in Drosophila melanogaster. J. Insect. Physiol. 31, 801e813. Kimbrell, D.A., Berger, E., King, D.S., Wolfgang, W.J., Fristrom, J.W., 1988. Cuticle protein gene expression during the third instar of Drosophila melanogaster. Insect Biochem. 18, 229e235. Kimbrell, D.A., Tojo, S.J., Alexander, S., Brown, E.E., Tobin, S.L., Fristrom, J.W., 1989. Regulation of larval cuticle protein gene expression in Drosophila melanogaster. Dev. Genet. 10, 198e209. King, D.S., Bollenbacher, W.E., Borst, D.W., Vedeckis, W.V., O'Connor, J.D., Ittycheriah, P.I., Gilbert, L.I., 1974. The secretion of alpha-ecdysone by the prothoracic glands of Manduca sexta in vitro. Proc. Natl. Acad. Sci. U.S.A. 71, 793e796. King-Jones, K., Thummel, C.S., 2005. Nuclear receptors e a perspective from Drosophila. Nat. Rev. Genet. 6, 311e323. Kozlova, T., Lam, G., Thummel, C.S., 2009. Drosophila DHR38 nuclear receptor is required for adult cuticle integrity at eclosion. Dev. Dyn. 238, 701e707. Kozlova, T., Pokholkova, G.V., Tzertzinis, G., Sutherland, J.D., Zhimulev, I.F., Kafatos, F.C., 1998. Drosophila hormone receptor 38 functions in metamorphosis: a role in adult cuticle formation. Genetics 149, 1465e1475. Lampe, D.J., Willis, J.H., 1994. Characterization of a cDNA and gene encoding a cuticular protein from rigid cuticles of the giant silkmoth, Hyalophora cecropia. Insect Biochem. Mol. Biol. 24, 419e435.

Lavorgna, G., Karim, F.D., Thummel, C.S., Wu, C., 1993. Potential role for a FTZ-F1 steroid receptor superfamily member in the control of Drosophila metamorphosis. Proc. Natl. Acad. Sci. U.S.A. 90, 3004e3008. Lavorgna, G., Ueda, H., Clos, J., Wu, C., 1991. FTZ-F1, a steroid hormone receptor-like protein implicated in the activation of fushi tarazu. Science 252, 848e851. Le Hir, H., Nott, A., Moore, M.J., 2003. How introns influence and enhance eukaryotic gene expression. Trends. Biochem. Sci. 28, 215e220. Lemoine, A., Mathelin, J., Braquart-Varnier, C., Everaerts, C., Delachambre, J., 2004. A functional analysis of ACP-20, an adult-specific cuticular protein gene from the beetle Tenebrio: role of an intronic sequence in transcriptional activation during the late metamorphic period. Insect Mol. Biol. 13, 481e493. Lemoine, A., Millot, C., Curie, G., Delachambre, J., 1989. A monoclonal antibody against an adult-specific cuticular protein of Tenebrio molitor (Insecta, Coleoptera). Dev. Biol. 136, 546e554. Lestradet, M., Gervasio, E., Fraichard, S., Dupas, S., Alabouvette, J., Lemoine, A., Charles, J.P., 2009. The cis-regulatory sequences required for expression of the Drosophila melanogaster adult cuticle gene ACP65A. Insect Mol. Biol. 18, 431e441. Locke, M., 2001. The Wigglesworth Lecture: insects for studying fundamental problems in biology. J. Insect Physiol. 47, 495e507. Magkrioti, C.K., Spyropoulos, I.C., Iconomidou, V.A., Willis, J.H., Hamodrakas, S.J., 2004. cuticleDB: a relational database of Arthropod cuticular proteins. BMC Bioinform. 5, 138. Milner, M.J., Muir, J., 1987. The cell biology of Drosophila wing metamorphosis in vitro. Roux's Arch. Dev. Biol. 196, 191e201. Mitchell, H.K., Weber-Tracy, U.M., Schaar, G., 1971. Aspects of cuticle formation in Drosophila melanogaster. J. Exp. Zool. 176, 429e443. Murata, T., Kageyama, Y., Hirose, S., Ueda, H., 1996. Regulation of the EDG84A gene by FTZ-F1 during metamorphosis in Drosophila melanogaster. Mol. Cell. Biol. 16, 6509e6515. Nakato, H., Izumi, S., Tomino, S., 1992. Structure and expression of gene coding for a pupal cuticle protein of Bombyx mori. Biochim. Biophys. Acta. 1132, 161e167. Neville, A.C., 1975. Biology of the Arthropod Cuticle. Springer, Berlin. Nielsen, H., Wernersson, R., 2006. An overabundance of phase 0 introns immediately after the start codon in eukaryotic genes. BMC Genomics 7, 256. Nijhout, F.N., 1994. Insect Hormones. Princeton University Press, Princeton. Nita, M., Wang, H.B., Zhong, Y.S., Mita, K., Iwanaga, M., Kawasaki, H., 2009. Analysis of ecdysone-pulse responsive region of BMWCP2 in wing disc of Bombyx mori. Comp. Biochem. Physiol. B., Biochem. Mol. Biol. 153, 101e108. Noji, T., Ote, M., Takeda, M., Mita, K., Shimada, T., Kawasaki, H., 2003. Isolation and comparison of different ecdysone-responsive cuticle protein genes in wing discs of Bombyx mori. Insect Biochem. Mol. Biol. 33, 671e679. Okamoto, S., Futahashi, R., Kojima, T., Mita, K., Fujiwara, H., 2008. Catalogue of epidermal genes: genes expressed in the epidermis during larval molt of the silkworm Bombyx mori. BMC Genomics 9, 396. Parvy, J.P., Blais, C., Bernard, F., Warren, J.T., Petryk, A., Gilbert, L.I., O'Connor, M.B., Dauphin-Villemant, C., 2005. A role for betaFTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster. Dev. Biol. 282, 84e94. Pokholkova, G.V., Kozlova, T., Shloma, V.V., Zhimulev, E.F., 1999. Drosophila hormone receptor 38: phenotypic analysis of mutations generated by P-element excision. D.I.S. 82, 56e58. Quennedey, A., Quennedey, B., Delbecque, J.P., Delachambre, J., 1983. The in vitro development of the pupal integument and the effects of ecdysteroids in Tenebrio molitor (Insecta, Coleoptera). Cell Tissue Res. 232, 493e511. Rees, H.H., 1989. Zooecdysteroids: structures and occurrence. In: Koolman, J. (Ed.), Ecdsysone. Georg Thieme Verlag, Stuttgart, New York, pp. 28e38. Richards, G., 1976. Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. IV. The mid prepupal period. Dev. Biol. 54, 256e263. Richards, G., 1982. Sequential gene activation by ecdysone in polyten chromosomes of Drosophila melanogaster. VII. Tissue specific puffing. Whilhelm Roux's Arch. Dev. Biol. 191, 103e111. Riddiford, L.M., 1985. Hormone action at the cellular level. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon Press, Oxford, pp. 37e84. Riddiford, L.M., 1993. Hormones and Drosophila development. In: Bate, M., Arias, A.M. (Eds.), The development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press. Riddiford, L.M., 2008. Juvenile hormone action: a 2007 perspective. J. Insect Physiol. 54, 895e901. Riddiford, L.M., Hiruma, K., Zhou, X., Nelson, C.A., 2003. Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster. Insect Biochem. Mol. Biol. 33, 1327e1338. Roberts, P.E., Willis, J.H., 1980. Effects of juvenile hormone, ecdysterone, actinomycin D, and mitomycin C on the cuticular proteins of Tenebrio molitor. J. Embryol. Exp. Morphol. 56, 107e123. Rondot, I., 1995. Etude de l'expression et de la régulation hormonale d'un gène codant pour une protéine cuticulaire larvo-nymphale spécifique au cours du développement de Tenebrio molitor. Mise en évidence de son appartenance à une batterie de gènes. PhD thesis, Université de Bourgogne. Rondot, I., Quennedey, B., Courrent, A., Lemoine, A., Delachambre, J., 1996. Cloning and sequencing of a cDNA encoding a larvalepupal-specific cuticular protein in Tenebrio molitor (Insecta, Coleoptera). Developmental expression and effect of a juvenile hormone analogue. Eur. J. Biochem. 235, 138e143.

J.P. Charles / Insect Biochemistry and Molecular Biology 40 (2010) 205e213 Rose, A.B., 2008. Intron-mediated regulation of gene expression. Curr. Top. Microbiol. Immunol. 326, 277e290. Schubiger, M., Tomita, S., Sung, C., Robinow, S., Truman, J.W., 2003. Isoform specific control of gene activity in vivo by the Drosophila ecdysone receptor. Mech. Dev. 120, 909e918. Schwartz, L.M., Truman, J.W., 1982. Peptide and steroid regulation of muscle degeneration in an insect. Science 215, 1420e1421. Schwartz, L.M., Truman, J.W., 1983. Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta. Dev. Biol. 99, 103e114. Shofuda, K., Togawa, T., Nakato, H., Tomino, S., Izumi, S., 1999. Molecular cloning and characterization of a cDNA encoding a larval cuticle protein of Bombyx mori. Comp. Biochem. Physiol. B., Biochem. Mol. Biol. 122, 105e109. Slama, K., 1980. Homeostatic function of ecdysteroids in ecdysis and oviposition. Acta Entomol. Bohemos. 77, 145e168. Soares, M.P., Elias-Neto, M., Simoes, Z.L., Bitondi, M.M., 2007. A cuticle protein gene in the honeybee: expression during development and in relation to the ecdysteroid titer. Insect Biochem. Mol. Biol. 37, 1272e1282. Steel, C.G.H., Vafopoulou, X., 1989. Ecdysteroid titer profiles during growth and development of arthropods. In: Koolman, J. (Ed.), Ecdysone. From Chemistry to Mode of Action. Georg Thieme Verlag, Stuttgart, New York, pp. 221e231. Sullivan, A.A., Thummel, C.S., 2003. Temporal profiles of nuclear receptor gene expression reveal coordinate transcriptional responses during Drosophila development. Mol. Endocrinol. 17, 2125e2137. Sun, G.C., Hirose, S., Ueda, H., 1994. Intermittent expression of BmFTZ-F1, a member of the nuclear hormone receptor superfamily during development of the silkworm Bombyx mori. Dev. Biol. 162, 426e437. Sutherland, J.D., Kozlova, T., Tzertzinis, G., Kafatos, F.C., 1995. Drosophila hormone receptor 38: a second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type. Proc. Natl. Acad. Sci. U.S.A. 92, 7966e7970. Suzuki, Y., Matsuoka, T., Iimura, Y., Fujiwara, H., 2002. Ecdysteroid-dependent expression of a novel cuticle protein gene BMCPG1 in the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 32, 599e607. Talbot, W.S., Swyryd, E.A., Hogness, D.S., 1993. Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell 73, 1323e1337. Thummel, C.S., 2002. Ecdysone-regulated puff genes 2000. Insect Biochem. Mol. Biol. 32, 113e120. Thummel, C.S., Burtis, K.C., Hogness, D.S., 1990. Spatial and temporal patterns of E74 transcription during Drosophila development. Cell 61, 101e111. Togawa, T., Dunn, W.A., Emmons, A.C., Nagao, J., Willis, J.H., 2008. Developmental expression patterns of cuticular protein genes with the R&R Consensus from Anopheles gambiae. Insect Biochem. Mol. Biol. 38, 508e519. Togawa, T., Shofuda, K., Yaginuma, T., Tomino, S., Nakato, H., Izumi, S., 2001. Structural analysis of gene encoding cuticle protein BMCP18, and characterization of its putative transcription factor in the silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 31, 611e620. Truman, J.W., 2005. Hormonal control of insect ecdysis: endocrine cascades for coordinating behavior with physiology. Vitam. Horm. 73, 1e30. Truman, J.W., Schwartz, L.M., 1984. Steroid regulation of neuronal death in the moth nervous system. J. Neurosci. 4, 274e280. Ueda, H., Hirose, S., 1990. Identification and purification of a Bombyx mori homologue of FTZ-F1. Nucleic Acids Res. 18, 7229e7234. Ueda, H., Hirose, S., 1991. Defining the sequence recognized with BmFTZ-F1, a sequence specific DNA binding factor in the silkworm, Bombyx mori, as

213

revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis. Nucleic Acids Res. 19, 3689e3693. Ueda, H., Sonoda, S., Brown, J.L., Scott, M.P., Wu, C., 1990. A sequence-specific DNAbinding protein that activates fushi tarazu segmentation gene expression. Genes Dev. 4, 624e635. Ueda, H., Sun, G.C., Murata, T., Hirose, S., 1992. A novel DNA-binding motif abuts the zinc finger domain of insect nuclear hormone receptor FTZ-F1 and mouse embryonal long terminal repeat-binding protein. Mol. Cell. Biol. 12, 5667e5672. Wang, H.B., Iwanaga, M., Kawasaki, H., 2009a. Activation of BMWCP10 promoter and regulation by BR-C Z2 in wing disc of Bombyx mori. Insect. Biochem. Mol. Biol. 39, 615e623. Wang, H.B., Nita, M., Iwanaga, M., Kawasaki, H., 2009b. betaFTZ-F1 and BroadComplex positively regulate the transcription of the wing cuticle protein gene, BMWCP5, in wing discs of Bombyx mori. Insect Biochem. Mol. Biol. 39, 624e633. Warren, J.T., Yerushalmi, Y., Shimell, M.J., O'Connor, M.B., Restifo, L.L., Gilbert, L.I., 2006. Discrete pulses of molting hormone, 20-hydroxyecdysone, during late larval development of Drosophila melanogaster: correlations with changes in gene activity. Dev. Dyn. 235, 315e326. Wigglesworth, V.B.,1934. The physiology of ecdysis in Rhodnius prolixus (Hemiptera) II. Factors controlling moulting and “metamorphosis”. Quart. J. Microsc. Sci. 77, 191e222. Wigglesworth, V.B., 1954. The Physiology of Insect Metamorphosis. Cambridge University Press, Cambridge. Willis, J.H., 1996. Metamorphosis of the cuticle, its proteins, and their genes. In: Gilbert, L.I., Tata, J.R., Atkinson, B.G. (Eds.), Metamorphosis. Postembryonic Reprogramming of Gene Expression in Amphibians and Insect Cells. Academic Press, pp. 253e282. Willis, J.H., 2010. Structural cuticular proteins from arthropods: annotation, nomenclature, and sequence characteristics in the genomics era. Insect Biochem. Mol. Biol. 40, 189e204. Willis, J.H., Iconomidou, V.A., Smith, R.F., Hamodrakas, S.J., 2005. Cuticular proteins. In: Gilbert, L.I., Iatrou, K., Gill, S.S. (Eds.), Comprehensive Molecular Insect Science. Elsevier Pergamon, Amsterdam, pp. 79e110. Willis, J.H., Rezaur, R., Sehnal, F., 1982. Juvenoids cause some insects to form composite cuticles. J. Embryol. Exp. Morphol. 71, 25e40. Wilson, T.E., Fahrner, T.J., Johnston, M., Milbrandt, J., 1991. Identification of the DNA binding site for NGFI-B by genetic selection in yeast. Science 252, 1296e1300. Wolfgang, W.J., Riddiford, L.M., 1981. Cuticular morphogenesis during continuous growth of the final instar larva of a moth. Tissue. Cell. 13, 757e772. Wolfgang, W.J., Riddiford, L.M., 1986. Larval cuticular morphogenesis in the tobacco hornworm, Manduca sexta, and its hormonal regulation. Dev. Biol. 113, 305e316. Yamada, M., Murata, T., Hirose, S., Lavorgna, G., Suzuki, E., Ueda, H., 2000. Temporally restricted expression of transcription factor betaFTZ-F1: significance for embryogenesis, molting and metamorphosis in Drosophila melanogaster. Development 127, 5083e5092. Zhong, Y.S., Mita, K., Shimada, T., Kawasaki, H., 2006. Glycine-rich protein genes, which encode a major component of the cuticle, have different developmental profiles from other cuticle protein genes in Bombyx mori. Insect Biochem. Mol. Biol. 36, 99e110. Zhou, X., Riddiford, L.M., 2002. Broad specifies pupal development and mediates the 'status quo' action of juvenile hormone on the pupaleadult transformation in Drosophila and Manduca. Development 129, 2259e2269. Zitnan, D., Kim, Y.J., Zitnanova, I., Roller, L., Adams, M.E., 2007. Complex steroidpeptide-receptor cascade controls insect ecdysis. Gen. Comp. Endocrinol. 153, 88e96.