The morphostatic actions of juvenile hormone

ARTICLE IN PRESS Insect Biochemistry and Molecular Biology Insect Biochemistry and Molecular Biology 37 (2007) 761–770 www.elsevier.com/locate/ibmb

Review

The morphostatic actions of juvenile hormone James W. Truman,1, Lynn M. Riddiford1 Department of Biology, University of Washington, Seattle, WA 98195, USA Received 21 January 2007; received in revised form 14 May 2007; accepted 15 May 2007

Abstract The maintenance of ‘‘status quo’’ in larvae by juvenile hormone (JH) involves both the programming of ecdysteroid-dependent synthesis during the molt and the suppression of morphogenetic growth during the intermolt. The latter morphostatic action does not require ecdysteroids, and has been studied in the formation of imaginal discs in Manduca sexta. Preultimate larval instars have both invaginated discs and imaginal primordia, both of which grow isomorphically with the larva. In the last instar, the young discs/primordia initiate the morphogenesis and patterning that results in a mature disc. JH suppresses both the initiation and progression of the signaling that transforms immature discs or primordia into a fully patterned imaginal disc. This transformation normally occurs in the context of the rapid growth of the last larval stage, and nutrient-dependent factors appear to be able to override the JH suppression. The morphostatic action of JH may have been important for the evolution of the larval stage. Studies on embryos of basal, hemimetabolous insects show that their premature exposure to JH can truncate patterning programs and cause precocious tissue maturation, factors essential for organizing a novel larval form. This suppression of embryonic patterning then results in embryonic fields that remain dormant as long as JH is present. These are the primordia that can transform into imaginal discs once JH disappears in preparation for metamorphosis. r 2007 Published by Elsevier Ltd. Keywords: Juvenile hormone; Morphostasis; Metamorphosis; Embryogenesis; Imaginal discs

1. Introduction There is nothing more captivating in insects than metamorphosis, with the dramatic transformation of a larva into an adult, through the ‘‘half-way house’’ of the pupa. The hormonal key to the regulation of metamorphosis is juvenile hormone (JH), with its classic ‘‘status quo’’ action (Williams, 1961). In the presence of JH, ecdysone and its metabolite 20-hydroxyecdysone (20E) induce a molt that results in a repeat of the previous stage. This recapitulation involves the production of a cuticle whose structure and appearance is similar to that of the stage that initiated the molt, e.g., the molting larva makes another larval cuticle (see Williams, 1961). This repeat of Corresponding author. Tel.: +1 206 543 6513.

E-mail address: [email protected] (J.W. Truman). Current address: Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA. 1

0965-1748/$ - see front matter r 2007 Published by Elsevier Ltd. doi:10.1016/j.ibmb.2007.05.011

cuticular structure has been nowhere better characterized than in the abdominal epidermis of Lepidoptera such as Manduca by Lynn Riddiford and her students (reviews in Riddiford, 1994, 1996). It requires that JH is present prior to the molting ecdysone surge to direct the pattern of ecdysone-induced gene expression. This maintenance of the status quo, however, requires more than just the conservation of cuticle structure. It also requires ‘‘morphostasis’’— the conservation of form. While the selection of a program of cuticle synthesis can be made rapidly, the regulation of form is more complex because it entails selective growth. While we know much about the role of JH in directing ecdysone action (Riddiford, 1994; Dubrovsky, 2005; Berger and Dubrovsky, 2005), relatively little is known about how it controls growth and form. The dramatic changes in body form seen at metamorphosis result from both epidermal proliferation and programmed cell death. Cell death is used to remove structures that are characteristic of the previous stage while

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proliferation is required for the generation of new, stagespecific structures (review, Fristrom and Fristrom, 1993). Cell death is typically linked to the 20E surge that drives molting. In Drosophila, for example, many larval tissues degenerate following pupariation (e.g., Jiang et al., 1997) as 20E directs development through the prepupal period. By head eversion (equivalent to pupal ecdysis), the larval thoracic epidermis has degenerated and has been replaced by the spreading and fusion of the thoracic imaginal discs, but in the abdomen, larval epidermal cells are still present and make the cuticle for the pupal stage. The larval epidermis in the abdomen is then replaced early in adult differentiation by imaginal epidermal cells spreading out from the histoblast nests. For both the anterior and posterior epidermis, though, the death of the larval cells occurs during relatively brief windows that are locked to the surges of 20E. Although the degeneration events associated with metamorphosis can be rapid, the growth required for metamorphosis is typically spread over much longer spans of time. In higher Diptera like Drosophila, the cells that will make pupal structures, such as the legs, wings, and head, are set aside as imaginal discs in the embryo and begin to proliferate in the late first or early second larval instar (Cohen, 1993), so that sufficient cells have accumulated and been patterned prior to the metamorphic surges of 20E. Consequently, for higher Diptera, the growth and death processes that are the basis of the larval–pupal transformation have very different relationships to the surges of 20E that drive this transformation. The degenerative processes typically follow the appearance of 20E whereas proliferation anticipates the steroid rise. In its role in controlling morphology, then, JH regulates the steroidinitiated processes underlying cuticle synthesis and cell death, but also growth processes that may be independent of steroid signaling. This brief review will concentrate on the role of JH in the proliferation aspects of ‘‘status quo’’. 2. The growth and morphogenesis of imaginal discs The dynamics of the growth that produces pupal structures are best understood for the early forming imaginal discs of higher Diptera as described above. Their growth occurs in parallel with that of the larva so that larval growth and imaginal growth occur simultaneously through successive larval instars. Along with those in the higher Diptera, early disc formation and growth is also characteristic of the wing discs of Lepidoptera. These ‘‘classic’’ imaginal discs do not contribute to larval function and are only externalized when they evert during the larval–pupal transformation. Although internalized for much of their growth, the discs remain connected to the epidermal monolayer, albeit sometimes through tenuous stalks, and make a thin cuticle during the larval molts (Sva´cha, 1992). Such early-forming discs are a derived feature in insects with complete metamorphosis (Sva´cha, 1992; Truman and Riddiford, 1999) and an adaptation for

rapid life cycles. The ancestral condition in the Holometabola is for all of the epidermis to form larval cuticle. Some regions, however, have the capacity to later transform into imaginal discs during the last larval instar. These regions, the primordia, are comprised of diploid cells and do not make specialized cellular structures, such as dermal glands or bristles. After the molt to the last larval stage, the cells in the primordia become columnar and start proliferating, transforming into infolded discs that will produce the pupal structures. Lepidoptera, like Manduca sexta, have both early- and late-forming discs, with the wings arising from an early-forming disc and the legs, antennae and eyes coming from late-forming discs. This diversity provides an excellent opportunity for dissecting the role of JH in directing disc formation and morphogenesis. In the Lepidoptera, the primordia for the adult legs (Kim, 1959; Tanaka and Truman, 2005) and eyes (Monsma and Booker, 1996; MacWhinnie et al., 2005) are associated with their larval counterparts. The eye primordium is located just anterior to the simple larval eyes, the stemmata, while the leg primordia are part of the larval leg (Fig. 1A–C). The transformation of a primordium into a disc begins at a few stereotyped sites where cells become columnar and begin to divide. Recruitment of other cells into the forming disc then spreads from these foci and eventually includes the entire primordium. The eye has two initiating foci, representing the dorsal and ventral halves of the adult eye. The complex ‘‘disc’’ of the leg arises from about a half dozen of such foci. The transformation of primordia into discs requires both that the larva is in its last instar and that it feeds. When larvae are starved from the outset of the last instar, the wing discs stop growing and the primordia for the lateforming discs remain dormant (Fig. 1D). This failure of starved larvae to form their discs, however, is not due to the lack of nutrient-related growth factors but results from an active suppression of morphogenesis by JH (Truman et al., 2006). As in other insects, the JH titers in Manduca begin to drop in the last larval instar (Bergot et al., 1981), but if these larvae are starved, the JH titer then remains high (Cymborowski et al., 1982). As seen in Fig. 1E, the suppression of disc formation caused by starvation does not occur in larvae that are allatectomized (CAX). These larvae show disc formation and growth despite the fact that the larva itself is starving and losing weight. In starved CAX larvae that are treated with the JH mimic (JHM), pyriproxifen, however, the disc formation and growth is inhibited (Fig. 1F), showing that JH is responsible for the suppression. Both in vivo and in vitro studies suggest that JH acts directly on the primordium to suppress disc formation in starved larvae (Truman et al., 2006). For example, in starved, CAX larvae, the topical application of pyriproxifen formulated in wax to a single leg locally suppresses disc formation in the treated leg while the discs in the other legs form normally. Likewise, primordia removed from early

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Fig. 1. Interaction of hormone and nutrient manipulations on imaginal disc organization and growth in larval Manduca sexta. (A) Drawings showing the location of the eye and leg primordia (red) and wing imaginal discs (green) at the onset of the last larval stage. Blue shows the sites that start the disc transformation. (B)–(F) Propidium iodide-stained projections of confocal Z-stacks showing the state of the primordia and discs after different treatments. (B) At the start of the last instar the primordia were quiescent and the wing disc was small. (C) After 4 days of feeding, the primordia formed invaginated discs (white arrows) and the wing disc grew and established the wing veins (inset, arrowheads). (D) Larvae starved for 4 days showed neither eye or leg disc formation nor wing disc growth. (E) Larvae lacking their corpora allata (CAX) and starved for 4 days showed wing disc growth and vein formation (inset, arrowhead), and both the eye and leg discs formed (arrows). (F) Treatment of CAX-starved larvae with the juvenile hormone mimic (JHM), pyriproxifen, (10 mg/larva) suppressed disc formation and wing disc growth and vein formation (inset). The red dashed line in the legs shows the muscle insertion on the dorsal distal tibia. From Truman et al., 2006.

last stage larvae and maintained in culture show discassociated proliferation when JH is absent but proliferation is suppressed in a dose-dependent fashion by treatment with pyriproxifen. Unlike the actions of JH in directing the nature of the molt and in suppressing cell death, its ability to suppress proliferation and morphogenetic growth appears to require neither ecdysone nor 20E. This is best illustrated in newly ecdysed last stage larvae that are decapitated by neck ligation. Such decapitated larvae show the same pattern of leg disc organization and growth as seen in starved, CAX larvae and this growth is inhibited by treatment with JHM. A more posterior placement of the ligature between the 2nd and 3rd thoracic segments removes the prothoracic glands in T1 as well as the cephalic endocrine centers. These reduced preparations still show leg imaginal disc formation in T3, despite the absence of the prothoracic glands (Truman et al., 2006). These results, and those from the culture experiments described above, show that the organization of primordia into discs does not require the participation of the prothoracic glands and its secretion of ecdysteroids. The above experiments show that the removal of JH is crucial for the organization of late-forming imaginal discs, but what about early-forming discs such as the wing discs? The wing discs are already identifiable in the embryo as

simple sac-like structures and they maintain this form through the early larval stages. During the final larval stage, however, they transform from a simple sac to a folded, mature disc that has channels that prefigure the location of the future veins [Fig. 1, insets]. As seen in Fig. 1 (bottom), the growth of the wing disc depends on larval feeding, and in starved larvae proliferation stopped and the size of the wing discs remained constant. In starved CAX larvae, by contrast, proliferation was maintained and the discs had grown markedly by the 4th day of starvation. In addition, they had undergone the morphological changes associated with the mature disc. Hence, treatment with JHM prevents both the growth and the morphogenesis seen in starved CAX larvae. Therefore, as illustrated in Fig. 2, from the perspective of growth, morphogenesis and their endocrine control, the early- and late-forming discs are actually quite similar. In both cases growth can be divided into an early ‘‘isomorphic phase’’ that occurs during the preultimate instars, and a ‘‘morphogenic phase’’ that is confined to the last larval stage. For the wing, the isomorphic phase involves the gradual growth of a discrete invaginated disc. For late-forming discs, like the eye, however, the isomorphic growth is more cryptic because it involves an increase in the size of a primordium that is an integral part of the larval body. In both instances, though, the

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Fig. 2. Comparison of the two phases of growth for early- and lateforming imaginal discs. The gray cells show the region of the epidermis that will produce the imaginal disc, which is already invaginated in the early-forming disc but part of the larval epidermis, as a persisting primordium, for the late-forming disc. Both increase in size isomorphically with the body as a whole under the influence of JH in the preultimate larval instars. The removal of JH in the last larval stage allows the morphogenetic growth that produces the mature form of the disc.

isomorphic phase is characterized by the disc/primordium expanding through the early instars by increasing cell number but without a significant change in shape. For both types of discs, the removal of JH in the last instar then allows the morphogenetic patterning that transforms the simple sac/epidermal sheet into a complexly patterned mature disc. Although invaginated discs and primordia share some features in common during the early larval instars, they also have obvious differences. One difference is in the type of cuticle that they secrete, with that of an invaginated disc being very thin and unpigmented, whereas the cuticle secreted by the primordium is typical larval cuticle. This thin disc cuticle, however, includes a subset of normal larval cuticle proteins (Gu and Willis, 2003). Another difference is in their pattern of proliferation. The invaginated disc proliferates through the intermolt period (e.g., Bryant, 1970); the early division patterns have not been well characterized in the primordia, although one would expect that they are confined to early in the molting period along with the other larval epidermis. 3. JH and the spread of induction The removal of JH allows the initiation of morphogenetic signaling in a primordium or a young disc, but its

continued absence is required for that induction to spread across the disc or the primordium. Such a relationship is evident in the study by Kremen and Nijhout (1998) who examined the effects of the timing of JHM treatment to last instar larvae of Precis coenia on the extent of pupal cuticle made in the eye, wing, leg and antennae of the resultant larval–pupal intermediates. They found that the later the time of JHM treatment, the larger the areas of pupal cuticle in the discs, and that this ability to make pupal cuticle spread across each structure in a stereotyped fashion. They concluded that commitment of the disc cells to make pupal cuticle was a progressive process that spread over the forming disc and that JH had to be absent for this spread to occur. A more direct assessment of the effects of JHM treatment on the spread of morphogenetic induction was performed in decapitated Manduca larvae that had just started to form their leg discs (Truman et al., 2006). Larvae were treated with JHM and their subsequent development compared to control decapitated larvae. The pattern and level of proliferation in the primordia began to differ between the two groups by 12 h after treatment with JHM. In the treated group, proliferation continued in the region that had already been induced, but the recruitment of new cells into the forming disc was suppressed. For the maintenance of the larval form, therefore, JH must be present during the intermolt to suppress the morphogen systems that would transform the primordia into imaginal discs. It is not clear, though, when in larval life the primordia become competent to begin this morphogenesis. In Manduca, the JH titer is high through the latter half of embryogenesis but then drops prior to hatching so that the newly hatched larva begins growth with little circulating JH (Bergot et al., 1981). In many insects, the treatment of embryos with allatocidal agents such as precocene fails to evoke premature metamorphosis until after the second larval/nymphal instar (AboulafiaBaginsky et al., 1984; Bru¨ning et al., 1985; Pener et al., 1988) suggesting that the very young tissues may be refractory to the lack of JH. Similarly, in Bombyx the depression of circulating JH by expression of a transgene for JH esterase does not impact larval molting until the 3rd instar (Tan et al., 2005), suggesting that the early larval instars may be insensitive to the lack of JH. Possibly the primordia/discs must achieve a minimal size before they are competent to begin the signaling that will drive morphogenesis and that this critical size (number of cells) is not achieved until the 2nd or 3rd larval stage. Until the morphogenetic systems are competent, the presence or absence of JH would be inconsequential. An unresolved question is the identity of the morphogen systems that are the targets of the JH suppression. Studies on the morphogenesis of imaginal discs in Drosophila have identified a number of morphogens that are involved in patterning the diverse discs of the fly, the major ones being the wingless, decapentaplegic [transforming growth factor b], hedgehog, and epidermal growth factor systems

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(e.g., Kojima, 2004). At this time, we do not know which one(s) of these is the target for JH suppression, and whether this suppression occurs at the level of transcription of the morphogen or of its receptor or at a more distal step in the response pathway. 4. Interaction of JH suppression with nutrition-related signaling Disc formation and morphogenesis are restricted to the last larval stage and also require that the larva feeds. Early starvation results in elevated JH titers (Cymborowski et al., 1982) but also in the lack of nutrition-related hormones, such as the insulin-like peptides [the bombyxins (Adachi et al., 1989)]. In starved larvae of Manduca, JH is a potent suppressor of the primordium to disc transformation, but in larvae feeding on normal diet, the discs can form and initiate morphogenesis, even when faced with high doses of JHM (pyriproxifen or methoprene). Likewise, topical application of JHM formulated in wax to leg primordia of feeding larvae is unable to locally block disc formation although this same treatment effectively suppresses disc formation in starved larvae that are allatectomized or neckligatured (Truman et al., 2006). The latter results indicate that the insensitivity of the primordia to JH seen in feeding larvae cannot be explained by systemic clearance of applied hormone or mimic. Rather, they suggest that nutritionrelated factors can override the JH suppression at the level of the target tissues. Since the ability of feeding to override of JH suppression is only evident in the last instar, it suggests that feeding causes the release of a metamorphosis initiating factor (MIF) that is unique to the last larval stage (MacWhinnie et al., 2005; Truman et al., 2006). There is precedent for nutrition-related hormones to change in the last larval stage. For example, in Drosophila, the spectrum of insulin-like peptides differs between the second and third (last) larval stage (Ikeya et al., 2002). Whether MIF is an insulin-like peptide, however, is unknown at this time. To this point, attempts to explain the observation that nutrition can override JH suppression of morphogeneis in the last larval stage have involved the hypothesis that there are nutrition-related factors that are unique to this instar (MacWhinnie et al., 2005; Truman et al., 2006). Another possibility, however, is that nutrition-related growth factors are the same throughout the larval instars, but the nature of the target tissue, the primordium, changes during the molt to the last larval stage to alter its response to nutrition-based factors. Obviously, the nature of the difference in the last instar relative to JH suppression and to nutrition-related factors needs to be resolved. In Manduca, growth during the last larval instar accounts for approximately 90% of the total weight increase since hatching. At the end of this growth period disc size needs to match the overall size of the larva, the latter being a function of how much nutrition the larva had acquired. The disc growth during the last larval stage comes about through two different pathways, one asso-

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ciated with morphogenesis and the other associated with nutrient intake. Normally, these two pathways act together, but the use of starved CAX larvae allowed the temporal separation of these two processes (Truman et al., 2006). Two results were notable. Firstly, when the spread of induction through the primordium in starved CAX larvae was compared with normally fed individuals, we found that recruitment of cells into the disc occurred faster in the fed animals, so that the entire primordium was eventually recruited into the mature disc. Without the nutritional input, only a fraction of the primordium cells become part of the final disc. Presumably, nutritional signals act synergistically with the absence of JH to enhance the level of morphogen signaling and recruitment into the nascent disc. Secondly, when starved CAX larvae were finally fed, the unrecruited cells at the margins of the primordium were subsequently omitted from the final disc. It appears that once signaling begins to form the disc, there is a restricted window during which this signaling could then occur. Once this window closed, additional primordium cells could not be incorporated into the disc despite the presence of abundant food. Thus, nutritional input at the start of disc formation was essential for the disc to attain maximal size. Failure to feed at this time could not be compensated for by feeding later in the instar. 5. The origins of imaginal primordia: relationship to embryonic development of basal insects JH exerts its morphostatic action during the intermolt periods by suppressing the transformation of the primordia in the growing larva. The probable origins of these primordia and their relationship to JH can be seen in the embryonic development of more basal insect groups. The most primitive insects, such as the ametabolous silverfish and bristletails, undergo direct development and the newly hatched juvenile is essentially a miniature version of the adult. The growth that occurs after hatching is isomorphic, with structures undergoing a constant ratio of increase with each molt as the juvenile gradually approaches adult size. The first winged insects, such as the Paleodictyoptera, were apparently also ametabolous; their immature stages had articulated winglets that grew proportionately with the growth of the body (Kukalova-Peck, 1978). Hence, in these basal insects morphogenetic signaling was confined to embryogenesis. With the shift to a hemimetabolous pattern of development, the goal of embryogenesis is the production of a nymph, which is basically a miniature version of the adult form. However, in the nymph, some of the adult structures, such as the wings and the genitalia, are suppressed and these initially appear as small buds or pads that are rigidly fixed to the body. During the subsequent growth of the nymph, the buds show disproportionate growth in order to reach a size necessary to differentiate into appropriately sized wings or genitalia for the adult. Morphogenesis of most of the body, then, is still confined to embryogenesis,

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with only restricted regions having the capacity for morphogenetic growth during postembryonic life. Unlike the somewhat piecemeal shift seen in hemimetabolous orders, the shift of morphogenetic processes into postembryonic life is pervasive in the Holometabola, affecting virtually every tissue in the animal. An alteration in embryonic morphogenesis results in the hatching of a larva which bears no resemblance to the adult. Larval structures are typically simpler than their adult counterparts, and likely originated by truncation and modification of the developmental programs which, in the ancestral condition, had gone on to make a miniature, adult-like nymph. The role of developmental truncation in making a larva is especially evident in the case of the nervous system (Truman, 1996), but it is also seen in the development of larval versus adult versions of epidermal structures such as the eye (Friedrich, 2006) and the leg (Tanaka and Truman, 2007). The relationship of the larval to the adult eyes is relatively straightforward (Friedrich, 2006). In most insects, the compound eye forms progressively, starting in the embryo with the first ommatidia forming on the posterior border of the eye primordium and then new rows of ommatidia are added as a wave of induction moves anteriorly across the eye primordium. Typically in hemimetabolous insects, a substantial compound eye, with many rows of ommatidia, is produced during embryogenesis, and additional rows are added during nymphal molts from a persisting primordium at the anterior margin of the eye (Anderson, 1978). With the notable exception of the eyes of scorpionflies, holometabolous larvae do not have compound eyes, but rather have a series of 5–6 simple eyes, the stemmata. The latter are highly modified ommatidia that originally formed the posterior border of the eye. The remaining eye primordium, anterior to the stemmata, is dormant and does not add any new larval photoreceptors, but in the last larval stage it grows and transforms into the eye disc that will make the adult compound eye. Thus, in comparison to hemimetabolous embryos, those of the Holometabola show a heterochronic shift in development with a very early arrest of photoreceptor production during embryogenesis and the arrested embryonic primordium then persisting through most of larval life until its transformation into an imaginal disc in preparation for metamorphosis. The basic story for the insect leg is similar, although the anatomy is more complex than the eye. In the leg, the patterning processes that generate the larval leg are also consistent with a truncation of a patterning program that ancestrally gave rise to a nymphal/adult leg (Tanaka and Truman, 2007). For example, in the patterning of the distal regions of an adult-like leg, bric-a-brac is initially expressed in a single broad domain that later resolves into discrete subdomains associated with the tarsal segmentation (Chu et al., 2002). This broad domain of bric-a-brac expression arises during the embryonic phase of leg formation in Manduca, but then remains stable as the larval leg

differentiates. The progression to discrete subdomains is delayed until the onset of metamorphosis and the organization of the adult version of the leg. Consequently, for the appendages, the primordia that will transform into imaginal discs have a persisting embryonic capacity because they likely are derived from the premature arrest of embryonic signaling in the first place. When the removal of JH allows the resumption of signaling in these centers, they must now pattern large fields of cells that had expanded considerably during larval growth. There are some cases, however, where such morphogenetic centers appear to have arisen de novo and are not likely related to structures in ancestral embryos. One such example is the horns that adorn the head or thorax of dung beetles (see Emlen et al., 2006). These horns form late in the larval stage from imaginal disc-like structures and are not likely related to arrested patterning fields in ancestral embryos. 6. Suppression of embryonic morphogenesis by JH How, then, did the embryonic patterning centers become suppressed in the ancestor of the Holometabola? The fact that holometabolous larvae appear to have a wide-spread suppression/arrest of developmental processes suggests that a global signal, such as that supplied by a hormone, might have been involved. We think that JH played a major role in this suppression (Truman and Riddiford, 1999, 2002), and that the truncation of morphogenesis resulted from an advancement in the appearance of JH during embryogenesis. Embryonic development typically involves a morphogenesis phase, during which rapid growth and changes in cell shape bring about the final body form, followed by a tissue maturation phase as cells differentiate to form functional tissues. In embryos of hemimetabolous species such as grasshoppers (Temin et al., 1986) and cockroaches (Imboden et al., 1978), JH appears relatively late in development, after morphogenesis is completed and while the embryo is undergoing tissue maturation. Treatment of cricket (Erezyilmaz et al., 2004) and locust (Nova´k, 1969; Truman and Riddiford, 1999) embryos with a JHM during the morphogenesis phase suppresses growth and causes premature tissue maturation. This effect of early exposure to JHM is best seen for the development of the eye in embryonic grasshoppers. As seen in Fig. 3, treatment of embryos with JHM at progressively later times in development results in a progressively larger eye at the end of embryogenesis. When JH-treated embryos were examined during the time of eye formation, we found that the furrow formed on time and began to move over the eye primordium, but it then stalled after making only a few rows of ommatidia (J.W. Truman and L.M. Riddiford, unpublished). The ommatidia that had formed behind the furrow underwent precocious maturation but the region of the primordium anterior to the furrow just made head cuticle. The gradual increase in final

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Fig. 3. The effects of the timing of treatment with the JH mimic pyriproxifen on the growth of the eye of embryos of Schistocerca gregaria. (A)–(E) pictures showing the final size of the eye of embryos that were treated with pyriproxifen at: (A) 5%, (B) 18%, (C) 30%, (D) 50%, and (E) 70% of embryonic development. (F) Quantification of the relative size of the eye (in arbitrary units) made after treatment at the indicated times. Each was based on measurements on at least 5 embryos. (J.W.Truman, L.M.Riddiford, & M.Akam, unpublished).

eye size (with more rows of ommatidia) seen with progressively later treatments with JHM suggest that once the furrow is initiated, the appearance of JH can arrest it at any time as it moves across the primordium. Importantly, in the JHM-treated nymphs, the unpatterned region of the eye primordium anterior to the furrow provides a region that could resume its ‘‘embryonic’’ growth if JH is removed at a later time. An unresolved question is why do locust embryos produce any eyes at all when JHM is applied at the start of embryogenesis? A hint of an answer is provided by treating embryos of the firebrat, Thermobia domesticus with JHM (Rohdendorf and Sehnal, 1973; J.W.Truman and L.M.Riddiford, unpublished). Embryos of Thermobia are apparently more sensitive to JHM as compared to those of grasshoppers (Truman and Riddiford, 1999) or crickets (Erezyilmaz et al., 2004). When treated with JHM at the start of embryogenesis, the resulting embryos are eyeless and form limb buds that then regress. Despite the striking difference in the effects of early JHM treatment in grasshoppers and silverfish, JHM-treated embryos of both species show essentially normal development until just before the start of katatrepsis, when the embryo starts moving through the yolk to assume its final position in the egg. JHM-treated embryos do not complete katatrepsis and they show precocious differentiation, such as the premature deposition of rigid cuticle. The important difference between the two species, then, is in how far embryonic development has progressed when katatrepsis begins. In grasshoppers, morphogenesis is almost complete by katatrepsis and eye formation is well underway. In Thermobia, by contrast, eye formation has not yet started and embryonic limb patterning is at a very early stage. Therefore, these two embryos are at very different stages of

development when JH is first able to drive tissue differentiation. The events that make the tissues competent to respond to JH just prior to katatrepsis are unknown. The extent that tissue patterning and morphogenesis have progressed by the onset of competence, though, determines how much of patterning program is available for making the larval body versus the portion which can be suppressed and deferred for making the imaginal discs to produce the adult form. Embryos also provide a different insight into the role of JH in directing the character of the ecdysone-induced molt. In postembryonic stages of both hemimetabolous and holometabolous insects, the presence of JH results in the reformation of the cuticle that was characteristic of the molting insect—the classic ‘‘status-quo’’ molt. In hemimetabolous embryos, however, JH promotes a progressive molt rather than a status-quo molt. In grasshoppers, the hatching stage (the vermiform larva, or the pronymph) has a highly sculptured cuticle, that aids in digging, and is devoid of hairs, whereas the first nymphal cuticle is smooth but bears bristles and hairs (Bernays, 1971; Konopova´ and Zrzavy, 2005). Treatment with JHM prior to the pronymphal molt in grasshoppers results in the production of a nymphal, rather than a pronymphal, cuticle (Nova´k, 1969; Truman and Riddiford, 1999). Likewise in the cricket, Achaeta, JH treatment before the pronymphal molt results in the failure to form the labral hatching ‘‘teeth’’ of the pronymph and the premature appearance of the sclerotized mandibles of the nymph (Erezyilmaz et al., 2004). Thus, in hemimetabolous embryos, JH promotes the production of nymphal characters and its continuing presence during successive postembryonic molts then maintains the nymphal state.

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7. Concluding considerations Juvenile hormone remains as one of the most fascinating and enigmatic hormones in animals. Its actions are fundamental to the maintenance of growth and form, but its mode of action still eludes our understanding, perhaps because JH acts via a number of different receptor systems (see Wheeler and Nijhout, 2003). Our understanding of the actions of JH in regulating cuticle morphology and the nature of a molt is complicated by its intimate relationship to ecdysone and 20E in this process. The action of JH in regulating morphogenesis, though, may be simpler to understand because this action occurs during the intermolt, when ecdysone titers are low and the removal of JH results in a rapid onset of morphogenetic signaling. This ability of JH to suppress morphogenesis is an ancient function of JH that is already evident in the embryos of the most basal insect orders, such as the Thysanura. The intimate linkage of JH action with morphogen systems is not a new idea, but was originally proposed by Nova´k (1975) in terms of the interaction of JH with his ‘‘gradient factor’’ that was associated with morphogenesis. His early ideas were formulated prior to the molecular understanding of morphogen signaling, and were therefore somewhat nebulous. Nevertheless, they were quite insightful in closely linking the action of JH to the developmental pathways that control growth and form and in trying to devise a unified hypothesis that linked together JH actions on insect embryos with those on postembryonic stages. The key issue at present, though, is determining which one(s) of these morphogen systems is the target for JH suppression. It is also important to realize that the landscape of signaling molecules changes when morphogenesis is moved from the embryo and out into postembryonic life. The embryo is a closed system in which growth is primarily the product of morphogenesis, without the need to adjust final organ size based on external nutrients. When shifted into the postembryonic realm, morphogenesis now occurs in the context of tissue growth, that is very flexible and a function of nutrient input. We know very little of how JH-sensitive and nutrient-sensitive pathways converge and interact to regulate growth and form. The question, then, is how does JH action intersect with the morphogen systems? A key gene involved in mediating at least some of the actions of JH is the transcription factor broad. Broad was first shown in Drosophila to be essential for the larval–pupal transformation (Kiss et al., 1978; Bayer et al., 1996 for a review). In Manduca it is a marker of cellular commitment to the pupal fate (Zhou and Riddiford, 2001) and in Drosophila ectopic expression of broad can cause cells initiating a larval or an adult molt to switch to making pupal products (Zhou and Riddiford, 2002). The appearance of broad expression is one of the earliest molecular events associated with pupal commitment seen in the primordia (Allee et al., 2006; Truman

et al., 2006), a timing that would make it a possible controller of morphogen pathways. In the context of complete metamorphosis, broad expression occurs as morphogenesis starts to make the adult form (as evidenced by the pupal ‘‘mold’’), but then is absent during the pupa–adult transition when tissue maturation then occurs. A similar association of broad with morphogenetic changes is also evident in the milkweed bug, Oncopeltus fasciatus, which undergoes incomplete metamorphosis (Erezyilmaz et al., 2006). Broad expression begins during embryogenesis and is prominent during the nymphal molts, but disappears during the nymph–adult molt, as adult structures like wings and genitalia differentiate. Although the nymph–adult transition is associated with the cessation of broad expression, the lack of broad is not sufficient to cause metamorphosis, as shown by the result that knock-down of broad expression by RNA interference in either the third or fourth nymphal instar, does not result in a precocious adult molt. In these nymphs that lack broad, however, tissues like the wing buds that normally show a disproportionate growth to achieve their normal size at the adult molt now show an isomorphic growth that is locked to the growth of the body in general. The resulting adults are of normal size but their wings are severely undersized (Erezyilmaz et al., 2006). Hence, broad is associated with morphogenesis in both wing discs and wing buds, but in the buds of hemimetabolous insects this morphgenetic action is carried through a number of nymphal instars, rather than being confined to the last larval stage.

Acknowledgments We thank Drs. T. Koyama and Y. Suzuki for helpful discussion during preparation of this manuscript. Unpublished studies were supported by grants from NSF (IOB 0344933 to LMR and IBN 9904959 to JWT). The embryonic studies were carried out in the laboratory of Prof. Michael Akam, Museum of Zoology, University of Cambridge.

References Aboulafia-Baginsky, N., Pener, M.P., Staal, G.B., 1984. Chemical allatectomy of late Locusta embryos by a synthetic precocene and its effect on hopper morphogenesis. J. Insect Physiol. 11, 839–852. Adachi, T., Takiya, S., Suzuki, Y., Iwami, M., Kawakami, A., Takahashi, S.Y., Ishizaki, H., Nagasawa, H., Suzuki, A., 1989. cDNA structure and expression of bombyxin, an insulin-like brain secretory peptide of the silkmoth Bombyx mori. J. Biol. Chem. 264, 7681–7685. Allee, J.P., Pelletier, C.L., Fergusson, E.K., Champlin, D.T., 2006. Early events in adult eye development of the moth, Manduca sexta. J. Insect Physiol. 52, 450–460. Anderson, H., 1978. Postembryonic development of the visual system of the locust, Schistocerca gregaria. I. Patterns of growth and developmental interactions in the retina and optic lobe. J. Embryol. Exp. Morphol. 45, 55–83.

ARTICLE IN PRESS J.W. Truman, L.M. Riddiford / Insect Biochemistry and Molecular Biology 37 (2007) 761–770 Bayer, C.A., von Kalm, L., Fristrom, J.W., 1996. Gene regulation in imaginal disc and salivary gland development during Drosophila metamorphosis. in: Gilbert, L.I., Tata, J.R., Atkinson, B.G. (Eds.), Metamorphosis: Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells. Academic Press, San Diego, pp. 321–361. Berger, E.M., Dubrovsky, E.B., 2005. Juvenile hormone molecular actions and interactions during development of Drosophila melanogaster. Vitam. Horm. 73, 175–215. Bergot, B.J., Baker, F.C., Cerf, D.C., Jamieson, G., Schooley, D.A., 1981. Qualitative and quantitative aspects of juvenile hormone titers in developing embryos of several insect species: discovery of a new JHlike substance extracted from eggs of Manduca sexta. In: Pratt, G.E., Brooks, G.T. (Eds.), Juvenile Hormone Biochemistry. Elsevier/NorthHolland, Amsterdam, pp. 33–45. Bernays, E.A., 1971. The vermiform larva of Schistocerca gregaria (Forska˚l): form and activity (Insects, Orthoptera). Z. Morphol. Tiere 70, 183–200. Bru¨ning, E., Saxer, A., Lanzrein, B., 1985. Methyl farnesoate and juvenile hormone III in normal and precocene-treated embryos of the ovoviviparous cockroach Nauphoeta cinerea. Int. J. Invertebr. Reprod. Dev. 8, 269–278. Bryant, P.J., 1970. Cell lineage relationships in the imaginal wing disc of Drosophila melanogaster. Dev. Biol. 22, 389–411. Chu, J., Dong, P.D., Panganiban, G., 2002. Limb type-specific regulation of bric-a-brac contributes to morphological diversity. Development 129, 695–704. Cohen, S.M., 1993. Imaginal disc development. In: Bate, M., Martinez-Arias, A. (Eds.), The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Plainview, pp. 747–841. Cymborowski, B., Bogus, M., Beckage, N.E., Williams, C.M., Riddiford, L.M., 1982. Juvenile-hormone titers and metabolism during starvation-induced supernumerary larval molting of the tobacco hornworm, Manduca sexta L. J. Insect Physiol. 28, 129–135. Dubrovsky, E.B., 2005. Hormonal cross talk in insect development. Trends Endocrinol. Metab. 16, 6–11. Emlen, D.J., Szafran, Q., Corley, L.S., Dworkin, I., 2006. Insulin signaling and limb-patterning: candidate pathways for the origin and evolutionary diversification of beetle ‘horns’. Heredity 97, 179–191. Erezyilmaz, D.F., Riddiford, L.M., Truman, J.W., 2004. Juvenile hormone acts at embryonic molts and induces the nymphal cuticle in the direct-developing cricket. Dev. Genes Evol. 214, 313–323. Erezyilmaz, D.F., Riddiford, L.M., Truman, J.W., 2006. The pupal specifier broad directs progressive morphogenesis in a direct-developing insect. Proc. Natl. Acad. Sci. USA 103, 6925–6930. Friedrich, M., 2006. Continuity versus split and reconstitution: exploring the molecular developmental corollaries of insect eye primordium evolution. Dev. Biol. 299, 310–329. Fristrom, D., Fristrom, J.W., 1993. The metamorphic development of the adult epidermis. In: Bate, M., Martinez-Arias, A. (Eds.), The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 843–887. Gu, S., Willis, J.H., 2003. Distribution of cuticular protein mRNAs in silk moth integuement and imaginal discs. Insect Biochem. Mol. Biol. 33, 1177–1188. Ikeya, T., Galic, M., Belawat, P., Nairz, K., Hafen, E., 2002. Nutrientdependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila. Curr. Biol. 12, 1293–1300. Imboden, H., Lanzrein, B., Delbecque, J.P., Lu¨scher, M., 1978. Ecdysteroids and juvenile hormone during embryogenesis in the ovoviviparous cockroach Nauphoeta cinerea. Gen. Comp. Endocrinol. 36, 628–635. Jiang, C., Baehrecke, E.H., Thummel, C.S., 1997. Steroid regulated programmed cell death during Drosophila metamorphosis. Development 124, 4673–4683.

769

Kim, C.-W., 1959. The differentiation centre inducing development from larval to adult leg in Pieris brassicae (Lepidoptera). J. Embryol. Exp. Morphol. 7, 572–582. Kiss, I., Szabad, J., Major, J., 1978. Genetic and developmental analysis of puparium formation in Drosophila. Mol. Gen. Genet. 164, 77–83. Kojima, T., 2004. The mechanism of Drosophila leg development along the proximodistal axis. Dev. Growth Differ. 46, 115–129. Konopova´, B., Zrzavy, J., 2005. Ultrastructure, development, and homology of insect embryonic cuticles. J. Morphol. 264, 339–362. Kremen, C., Nijhout, H.F., 1998. Control of pupal commitment in the imaginal discs of Precis coenia (Lepidoptera: Nymphalidae). J. Insect Physiol. 44, 287–298. Kukalova-Peck, J., 1978. Origin and evolution of insect wings and their relation to metamorphosis, as documented by the fossil record. J. Morphol. 156, 53–126. MacWhinnie, S.G., Allee, J.P., Nelson, C.A., Riddiford, L.M., Truman, J.W., Champlin, D.T., 2005. The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta. Dev. Biol. 285, 285–297. Monsma, S.A., Booker, R., 1996. Genesis of the adult retina and outer optic lobes of the moth, Manduca sexta. I. Patterns of proliferation and cell death. J. Comp. Neurol. 367, 10–20. Nova´k, V.J.A., 1969. Morphological analysis of the effects of juvenile hormone analogues and other morphologically active substances on embryos of Schistocerca gregaria Forsk. J. Embryol. Exp. Morphol. 21, 1–21. Nova´k, V.J.A., 1975. Insect Hormones, 2nd English En. Chapman and Hall, London. Pener, P.M., Tobe, S.S., Stay, B., 1988. Effects of a synthetic precocene on late embryos and subsequent development of the cockroach Diploptera punctata. Can. J. Zool. 66, 2295–2300. Riddiford, L.M., 1994. Cellular and molecular actions of juvenile hormone. I. General considerations and premetamorphic actions. Adv. Insect Physiol. 24, 213–274. Riddiford, L.M., 1996. Juvenile hormone: the status of its ‘‘status quo’’ action. Arch. Insect Biochem. Physiol. 32, 271–286. Rohdendorf, E.B., Sehnal, F., 1973. Inhibition of reproduction and embryogenesis in the firebrat, Thermobia domestica. J. Insect Physiol. 19, 37–56. Sva´cha, P., 1992. What are and what are not imaginal discs: reevaluation of some basic concepts (Insects, Holometabola). Dev. Biol. 154, 101–117. Tan, A., Tanaka, H., Tamura, T., Shiotsuki, T., 2005. Precocious metamorphosis in transgenic silkworms overexpressing juvenile hormone esterase. Proc. Natl. Acad. Sci. USA 102, 11751–11756. Tanaka, K., Truman, J.W., 2005. Development of the adult leg epidermis in Manduca sexta: contribution of different larval cell populations. Dev. Genes Evol. 215, 78–89. Tanaka, K., Truman, J.W., 2007. Molecular patterning mechanism underlying metamorphosis of the thoracic leg Manduca sexta. Dev. Biol. 305, 539–550. Temin, G., Zander, M., Roussel, J.P., 1986. Physico–chemical (GC-MS) measurements of juvenile hormone III titres during embryogenesis of Locusta migratoria. Int. J. Invertebr. Reprod. 9, 105–112. Truman, J.W., 1996. Insect nervous system metamorphosis. In: Gilbert, L.I., Tata, J.R., Atkinson, B.G. (Eds.), Metamorphosis: Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells, Academic Press, San Diego, pp. 283–320. Truman, J.W., Riddiford, L.M., 1999. The origins of insect metamorphosis. Nature 410, 447–452. Truman, J.W., Riddiford, L.M., 2002. Endocrine insights into the evolution of metamorphosis in insects. Annu. Rev. Entomol. 47, 467–500. Truman, J.W., Hiruma, K., Allee, J.P., MacWhinnie, S.G., Champlin, D.T., Riddiford, L.M., 2006. Juvenile hormone is required to couple imaginal disc formation with nutrition in insects. Science 312, 1385–1388.

ARTICLE IN PRESS 770

J.W. Truman, L.M. Riddiford / Insect Biochemistry and Molecular Biology 37 (2007) 761–770

Wheeler, D.E., Nijhout, H.F., 2003. A perspective for understanding the modes of juvenile hormone action as a lipid signaling system. Bioessays 25, 994–1001. Williams, C.M., 1961. The juvenile hormone. II. Its role in the endocrine control of molting, pupation, and adult development in the cecropia silkworm. Biol. Bull. 121, 572–585.

Zhou, B.H., Riddiford, L.M., 2001. Hormonal regulation and patterning of the Broad-Complex in the epidermis and wing discs of the tobacco hornworm, Manduca sexta. Dev. Biol. 231, 125–137. Zhou, X., Riddiford, L.M., 2002. Broad specifies pupal development and mediates the ‘status quo’ action of juvenile hormone on the pupal–adult transformation in Drosophila and Manduca. Development 129, 2259–2269.