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Endocrine regulation of terminal muscle differentiation
bisect Biochem. Vol. 16, No. I. pp. 203 209, 1986 Printed in Great Britain.All rights reserved
0020-1790/86 $3.00+0.00 Copyright r(- 1986Pergamon Press Ltd
ENDOCRINE REGULATION OF TERMINAL MUSCLE DIFFERENTIATION ATROPHY AND DEGENERATION OF THE INTERSEGMENTAL MUSCLES OF LEPIDOPTERA LAWRENCE M. SCHWARTZ Department of Biology, University of North Carolina, Chapel Hill, NC 27514, U.S.A.
Abstract--The intersegmental muscles of the Lepidoptera pass through three separate, sequential differentiated states during pharate adult development: status quo; atrophy; degeneration. Each of these developmental programs is characterized by a distinct morphology, physiology and endocrine responsiveness. The factors responsible for regulating these differentiative changes are ecdysteroids. In Manduca sexta, the haemolymph ecdysteroid titre declines in a circadian-modified fashion during the last three days of adult development, which parallels the maturation of the intersegmental muscles. Abdomen-ligation, which causes a precipitous decline in the ecdysteroid titre, causes the precocious atrophy and degeneration of these muscles, whereas injection of, or infusion with, 20-hydroxyecdysone greatly delays such changes. While the terminal differentiation of the epidermis and nervous system is also regulated by ecdysteroids, endocrine manipulations have suggested that the development of the intersegmental muscles is independent of these tissues. In the silkmoth Antheraea polyphemus, ecdysteroids are also responsible for regulating intersegmental muscle differentiation, but eclosion hormone (a peptide) acts as the proximal trigger for the activation of the degeneration program. The declining ecdysteroid titre initiates the atrophy program and subsequently determines the timing of both release of eclosion hormone and intersegmental muscle sensitivity to the peptide. Eclosion hormone then acts directly on the muscles, via cGMP, to activate the degeneration program. Ecdysteroids appear to prevent premature muscle degeneration by regulating a biochemical step distal to both the eclosion hormone receptor and the rise in cGMP. Key Word Index: Intersegmental muscles, cell death, eclosion hormone, 20-hydroxyecdysone,cGMP (3',5'-guanosine monophosphate)
INTRODUCTION Metamorphosis in holometabolous insects is marked by the dramatic restructuring of a typically sedentary, feeding larva, into an active gamele dispersing adult. Accompanying these changes in morphology and behaviour, there is a marked restructuring of the muscular system. In most instances this involves the degeneration of embryonically derived skeletal muscles to allow for the development of new, adultspecific muscles (Finlayson, 1975). In lepidopterans, significant exceptions to this general rule are the intersegmental muscles, a wide sheet of flattened fibres attached at the intersegmental boundary of each abdominal segment in the larva. After pupation these muscles degenerate in the first, second, seventh and eighth abdominal segments. The intersegmental muscles of the remaining four segments persist until adult eclosion, at which time they provide the peristaltic waves of abdominal compression which help to extricate the animal from the pupal cuticle and cocoon. After eclosion the intersegmental muscles begin to degenerate and are not detectable 36 hr later (Finlayson, 1956; Lockshin and Williams, 1965a). The intersegmental muscles appear to display three separate but sequential development programs dur-
ing metamorphosis, namely status quo, atrophy and degeneration. Each of these phases is characterized by a distinct ultrastructure, physiology and endocrine responsiveness. STRUCTURE AND FUNCTIONOF THE INTERSEGMENTAL MUSCLES The three developmental states of the intersegmental muscles can be most readily seen by weighing the muscles during adult differentiation (Schwartz and Truman, 1983, 1984a). In the tobacco hornworm Manduca sexta, the mass of the muscle remained constant until day 15 of the normal 18 days development (Fig. i). In the giant silkmoth Antheraea polyphemus, the status quo phase lasted until about day 16 of the normal 17 days development. During the status quo period, the fibres displayed an appearance and physiology typical of arthropod skeletal muscles (Lockshin and Beaulaton, 1974; Schwartz and Truman, 1984a). Three days before eclosion, the intersegmental muscles entered a period of atrophy, which continued until eclosion and is characterized by an approx. 40% loss of weight (Fig. 1). This reduction in mass seems to result from the loss of entire myofibrils
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Fig. I. Change in mass of intersegmental muscles during adult development in intact Manduca (O) and isolated abdomens (C)). Abdomens isolated on day 15. Values are mean dry weight mg/g body wt +SEM (n = 5). E, time of eclosion (from Schwartz and Truman, 1983). rather than from removal of a specific class of contractile proteins (Lockshin and Beaulaton, 1974). While the net strength of the muscle was reduced, several other key physiological parameters remained unchanged, including: (1) the resting potential; (2) the force of tetanic contracture per cross-sectional area; (3) the Ca 2+ concentration required for generation of 50% tension in skinned fibres (PCas0); (4) Hill coefficients (Schwartz, Bolles and Almers, unpublished observations). Atrophy may function to reduce net muscle strength in order to prevent detachment from the pupal cuticle at a time when the latter is being digested in preparation for eclosion. The final period of intersegmental muscle differentiation, namely degeneration, coincided with adult eclosion (Lockshin and Williams, 1965a; Schwartz and Truman, 1982). Ultrastructurally, the process began a few hours after eclosion with the production of lysosomes. The lysosomes persisted through degeneration and ultimately represented the major organelle within dying fibres. The contractile proteins were rapidly digested and became undetectable after 18 20 hr. By 28 hr after eclosion the muscles were reduced to flimsy bags of membranes (Lockshin and Beaulaton, 1974). There were dramatic changes in the physiological properties of the intersegmental muscles as they degenerated (Schwartz, Bolles and Almers, unpublished observations). The concentration of intracellular calcium required for the generation of 50% tension increased by more than three orders of magnitude, rising from 7.77 x 10 7M at eclosion to I. 18 x 10 ~ M 18 hr later. During this period there was a progressive weakening of the muscle, and contraction had ceased by 20 hr after eclosion. Accompanying these changes was a continuous 2.5 mV/hr depolarization of the fibres.
SCHWARTZ
muscle atrophy and degeneration (Schwartz and Truman, 1982, 1983). When day-15 Manduca were ligated between the thorax and abdomen, the intersegmental muscles underwent accelerated differentiation (Fig. 1). The muscles from these preparations entered the atrophy and degeneration phases of development a full day ahead of unligated controls. Accelerated maturation of the intersegmental muscles was not the only effect of abdomen isolation. Several other terminal developmental markers were also observed to appear precociously, including: (1) breakdown of the pupal endocuticle; (2) resorption of the moulting fluid; (3) neuron degeneration; (4) preecdysial behaviour (Schwartz and Truman, 1983; Truman, 1984). In contrast to ligations performed on day 15, abdomen isolation several hours before adult eclosion on day 18 had no impact on either the timing or the rate of intersegmental muscle degeneration. To further characterize these muscles sensitivity to abdomen isolation, ligations were performed systematically at various times during the last 3 days of pharate adult development. By comparing the muscle weight of ligated animals to that of non-ligated controls, the change in developmental rate was determined. As expected from the above data, the extent of precocious muscle degeneration varied with the time of abdomen isolation (Fig. 2). Interestingly, the acceleration of muscle degeneration did not change linearly with the time of ligation, but instead varied in a saltatory fashion. Ligations performed during the scotophase were essentially equipotent, while a shift of a few hours during the photophase resulted in a dramatic change in the rate of muscle development. Presumably the effect of these ligations was to disrupt some influence from the anterior end of the animal. To determine if this signal was neuronal or endocrine in nature, the ventral nerve cords of day 15 animals were severed, much as would have resulted
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THE ROLE OF E C D Y S T E R O I D S
The normal timing of muscle differentiation can be modulated by ligating animals between the thorax and abdomen late in development (Lockshin, 1969). Such isolated abdomens are viable for weeks and allow one to examine the control of intersegmental
~ D A Y 18/ ISOLATION
Fig. 2. Effect of abdomen-ligation on the time-course ol Manduca intersegmental muscle development. Abdomens were isolated at the times indicated and held for varying periods. Muscle mass was compared to that of unmanipulated control insects and the relative advancement o! development was determined. Values are mean advancement +SEM (n = 4~14; from Schwartz and Truman. 1983).
Hormonal control of muscle development
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TIME OF ECDYSTEROID TREATMENT (hours relotive to eedysis) Fig, 3. Progressive inability of 20-OH-ecdysone-injection to delay eclosion behaviour, intersegmental muscle degeneration and death ofmotorneurons D-IV which innervate the interse~mental muscles. Muscle mass was determined 24 hr after normal eclosion, when control animals have a muscle mass of about
0,3 mg/g body wt (from Truman and Schwartz, 1982). from isolation of the abdomen. These animals were then examined one day after the eclosion of unoperated control and were found to have a muscle dry wt of 0.59+ 0.09mg intersegmental muscle/g body wt ( + SEM; n = 5). This compares favourably with the control animals which had a muscle dry wt of 0.68 _ 0.12 mg/g (n = 6). In contrast, animals ligated on day 15 had no detectable intersegmental muscle tissue at this time. When other markers for terminal differentiation were examined, they too were found to be unaffected by nerve-cord transection on day 15 (Schwartz and Truman, 1983; Truman and Schwartz, 1984). These data suggest that the signal from the anterior end of the animal which modulates the timing of terminal differentiative events is hormonal in nature. During the first third of pharate adult development in the Lepidoptera, the circulating titre of ecdysteroids increases (Bollenbacher et al., 1981), which serves to promote adult differentiation (Williams, 1968). During the remaining two thirds of metamorphosis, the ecdysteroid titre declines. It is known that
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injection of 20-hydroxyecdysone can delay eclosion in Tenebrio (Slama, 1980) and Manduca (Truman et al., 1983). It seemed likely therefore, that ecdysteroids could also influence the timing of atrophy and degeneration of the intersegmental muscles, as these too are late differentiative events. To test this hypothesis, Manduca were injected with 25/~g 20-hydroxyecdysone at various times during the last 36 hr of pharate adult development, and the mass of their muscles was measured 16 hr after the eclosion of unmanipulated controls (Fig. 3). Injection of 20-OH-ecdysone prior to lights-off on day 17 prevented degeneration of the intersegmental muscles. Adult eclosion and the death of the D-IV motor neurons, which innervate the intersegmental muscles, were also delayed (Schwartz and Truman, 1983; Truman and Schwartz, 1984). At later times, administration of ecdysteroids was progressively less effective in delaying these terminal events. As Fig. 3 shows, judicious ecdysteroid injection can separate eclosion, intersegmental muscle degeneration, and motor neuron death from one another. This suggests
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Fig. 4. Change in endogenous haemolymph ecdysteroid content during the last 3 days of adult development in Manduca. Insects were catheterized in the thoracic heart and 20 pl blood samples were removed every 3 hr for RIA. Values are ng 20-OH-ecdysone-equivalents/ml haemolymph + SEM (from Schwartz and Truman, 1983).
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LAWRENCE M. SCHWARTZ
that each of these events is directly controlled by the ecdysteroid titre, rather than occurring "dominostyle" as part of a triggered developmental cascade. These data suggest that elevation of the ecdysteroid titre by administration of exogenous hormone may delay the death of the intersegmental muscles. To better understand any relationship between ecdysteorids and muscle development, the circulating titre of ecdysteroids was measured using a radioimmunoassay (RIA) during the last 3 days of adult development in Manduca. Individual insects were catheterized in the thoracic heart, and 201tl haemolymph samples were removed every 3 hr for 24 hr. The pooled data from 40 individuals (283 separate measurements in triplicate) are shown at Fig. 4. As suggested above, the ecdysteroid titre declines during the last 3 days of adult development. The values fell from approx. 1.5,ug/ml 20-OH-ecdysone equivalents on day 15 to <0.15,ug/ml at the time of adult eclosion. This decline in titre was not linear but showed a circadian modulation. While the data displayed variability, there was a tendency for the ecdysteroid titre to decline during the photophases and to remain relatively constant during the scotophases. This pattern is reminiscent of the saltatory efficacy of abdomen ligation on intersegmental muscle development (Fig. 2). If ecdysteroids are responsible for determining the timing of muscle differentiation, then isolation of the abdomen, which causes precocious atrophy and degeneration of the intersegmental muscles, should also cause a reduction in the level of circulating ecdysteroids; and this appears to be the case. On day 15, the haemolymph ecdysteroid titre was 1765 ± 360 ng 20-OH-ecdysone equivalents/ml (#l = 11 animals) in unligated Manduca. This value was reduced in control animals to 590 ± 74ng/ml (n = 8) 48 hr later. In contrast, animals ligated between the thorax and abdomen on day 15 and kept for 48 hr. had an
ecdysteroid titre of only 60 _+ 28 ng/ml (n = 9), Thus abdomen isolation brought about a drastic reduction in the circulating ecdysteroid titre. THE ROLE OF ECLOStON
HORMONE
The three stages of differentiation described above for Manduca intersegmental muscles also occur in the development of these muscles in another moth, Antheraeapoh'phemus (Schwartz and Truman, 1984a). In A. polyphemus, the ecdysteroid titre declines exponentially during the last 5 days ot" adult development. On day 13 of development in this species, lhc haemolymph titre was 474 +- 106 ng 20-OH-ecdysone equivalents/ml (n = 5), while that at the time ol eclosion on day 17 it was 3 0 + 12ng/ml (n =5). When exogenous 20-OH-ecdysone was introduced. either by injection or infusion, atrophy and degeneration of the intersegmental muscles were great b retarded (Fig. 5). A. poh'phemus differed from M. sexta however, in its response to abdomen isolation. As mentioned above, the timing of intcrsegmental muscle degeneration in Manduca was unaffected by abdomen ligation performed early during the last da~ of adult development. In contrast whereas when Antheraea abdomens were ligated at this time, the intersegmental muscles did not degenerate (Schwartz and Truman, 1982; 1984; Fig. 5). Instead, these muscles continued the atrophy program which had already begun earlier in development. In isolated abdomens of A. polyphemus, the intersegmental muscles progressively lost weight for 6 days, after which time they were no longer contractile. Therefore, isolation of the abdomen prior to adult eclosion prevented the muscles from entering the de
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Fig. 5. Changes in intersegmental muscle mass in Antheraea in intact animals and in control and hormone-treated isolated abdomens. Squares: intact, normally-eclosingadults. Circles: abdomens isolated early on day 17. Triangles: isolated abdomens injected with 1 U eclosion hormone. Hexagons: intact animals infused with 20-OH-ecdysone, 30 ng/g per hr, starting 2 days before eclosion. Open symbols indicate contractile muscles, closed symbols non-contractile muscles. Values are mean dry wt _+SEM for 4 7 animals per group.
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Fig. 6. Changes in intersegmental muscle mass in Antheraea in response to injected eclosion hormone (EH) or 20-OHecdysone during the last 36 hr of pharate adult development. Muscle wt were determined 24 hr after the eclosion of intact control animals. Closed circles: muscle wt from intact insects injected with 25#g 20-OH-ecdysone at the times indicated. Open circles: day-17 isolated abdomens injected with I U eclosion hormone. E, time of eclosion (modified from Schwartz and Truman, 1984a).
to release of eclosion hormone, abdominal tissues are denied exposure to this peptide. To determine whether eclosion hormone is responsible for intersegmental muscle degeneration in Antheraea, isolated abdomens were injected on day 17 with 1 U of the peptide (the amount of hormone found in one pair of pharate adult Manduca corpora cardiaca). At various times after injection of eclosion hormone, the intersegmental muscles were removed and their dry weights determined. Hormone injection induced intersegmental muscle degeneration which was qualitatively and quantitatively identical to that seen in normally eclosing adults (Fig. 5). The amount of eclosion hormone released into the haemolymph to initiate adult eclosion is 0,5 U (Truman et al., 1981). When various dosages of eclosion hormone were injected it caused all-or-none cell death at a response threshold of 0.1 U (Schwartz and Truman, 1984a). Since the amount of hormone normally seen by the muscles at eclosion is substantially greater than 0.1 U, eclosion hormone presumably is the proximal trigger for degeneration of the intersegmental muscles in Antheraea. When abdomens were isolated on day 16 of development (24hr before eclosion) and injected with eclosion hormone, degeneration of the intersegmental muscles did not occur. To determine when eclosion hormone sensitivity arose, abdomens were removed at various times during the 24 hr preceding adult eclosion and injected with the hormone. These abdomens were retained for 24 hr and then their muscle mass was determined. As can be seen in Fig. 6, there was a time-dependent increase in intersegmental muscle sensitivity to exogenous eclosion hormone. The acquisition of sensitivity to this hormone corresponded to the loss of muscle ecdysteroid sensitivity. Early injections of 20-OH-ecdysone into intact insects reduced the extent of subsequent degeneration of the muscles. The progressive loss of sensitivity to 20-OH-ecdysone paralleled the normal reduction in the level of circulating ecdysteroids observed late in
207
development. It should be noted that the declining ecdysteroid titre also determines the timing of eclosion hormone release in lepidopterans (Truman et al., 1983). Thus, as the ecdysteroid titre declines, the intersegmental muscles acquire the ability to respond to eclosion hormone at a time just prior to this peptide's gated release. The ability of eclosion hormone to provoke degeneration of the intersegmental muscles could be due either to the direct action of the hormone on the muscles or to indirect action through the effect of the hormone on the nervous system. Earlier reports suggested that the trigger for intersegmental muscle degeneration is the cessation of electrical activity by their innervating motor neurons (Lockshin and Williams, 1965b,c). Since eclosion hormone has been shown to have direct effects on the nervous system (Truman, 1978), this mechanism may seem plausible. It appears however, that eclosion hormone acts directly on the muscles themselves to stimulate their degeneration (Schwartz and Truman, 1984a). Injection of the neurotoxic drug tetrototoxin into isolated abdomens rapidly caused flaccid paralysis due to cessation of neuronal electrical activity. Treated abdomens retained contractile intersegmental muscles of normal mass when examined 24hr later. These muscles from abdomens paralysed in this way still underwent characteristic degeneration when subsequently treated with eclosion hormone. Therefore, neither the patterning nor the complete cessation of electrical activity was responsible for degeneration of the intersegmental muscles. To control for possible trophic maintenance of the muscles by their motor neurons, the ventral nerve cord was removed from abdomens isolated either at the beginning of adult development or just prior to adult eclosion. The intersegmental muscles persisted in these denervated preparations. Again, injection of eclosion hormone resulted in loss of the muscles. These data suggest that this hormone acts directly on the intersegmental muscles to induce degeneration, rather than acting via effects on the nervous system. It is of obvious interest to determine how eclosion hormone precipitates the rapid involution of a viable set of muscles. There are presumably a number of steps between hormone exposure and cell autolysis. Muscle degeneration appears to be sensitive to inhibitors of both RNA and protein synthesis (Lockshin, 1969). Work has recently begun on a molecular analysis of intersegmental muscle degeneration (Schwartz and Bloom, unpublished observations) to answer such basic questions as: how many genes are required to kill a cell, what are the products of these genes, and how are these genes hormonally regulated? While events at the gene level are still unknown, one potential link between eclosion hormone exposure and the activation of the degeneration program has been identified. Several lines of evidence suggest that 3',5'-guanosine monophospahte (cGMP) may serve as a second messenger in this system (Schwartz and Truman, 1984b). Evidence came initially from isolated abdomens injected with inhibitors of the cyclic nucleotide-degradative enzyme phosphodiesterase. Both of the drugs tested, 3-isobutyl-l-methylxanthine (IBMX) and theophylline, induced dose-dependent degeneration of
208
LAWRENCEM. SCHWAR'IZ
the intersegmental muscles, with IBMX displaying about 100-fold greater potency than theophylline. To further characterize the potential involvement of cyclic nucleotides in this process, isolated abdomens were injected with either cAMP or cGMP and the mass of the intersegmental muscles was determined 24 hr later. Injection of cAMP, even at doses of 50~mol/abdomen (haemolymph concentration 50mM) were without effect. Injection of cGMP however, resulted in dose-dependent intersegmental muscle degeneration with a threshold of about 0.5pmol/abdomen (0.5mM in the haemolymph). Maximal degeneration was induced by cGMPinjections greater than 5 ~mol. To determine more directly whether cyclic nucleotides are involved in eclosion hormone action, isolated abdomens were injected with 2 U of the hormone and at various times thereafter, the muscle contents of cGMP and cAMP were measured using RIA. Eclosion hormone provoked only minor fluctuations in muscle cAMP levels, while cGMP levels increased dramatically. They began to rise within 5 rain of hormone injection and then reached a plateau 60min later, at a concentration 22-fold greater than the basal level. The cGMP titre then slowly declined, such that it was still 10-fold higher than basal levels 3.5 hr after hormone exposure. The magnitude of cGMP accumulation was dependent on the dose of eclosion hormone injected. Below 0.1 U of hormone, only minor elevations in muscle cGMP content were detected 60 min later. Above this dose however, there was a marked increase in the accumulation of cGMP. Since the amount of eclosion hormone released at eclosion is 0.5 U, there are dramatic elevations in muscle cGMP content preceding normal degeneration. The ability of eclosion hormone to stimulate an increase in muscle cGMP is independent of the nervous system. When isolated abdomens were denervated and injected with the peptide, the muscles from these preparations displayed the same elevations in cGMP-accumulation as were seen in control isolated abdomens. This observation further strengthens the suggestion of a direct action of eclosion hormone on the intersegmental muscles themselves. As stated above, the intersegmental muscles from day-16 isolated abdomens do not degenerate when injected with eclosion hormone. They also failed to degenerate when injected with IBMX, with exogenous cGMP, or with sodium nitroprusside, a potent stimulator of guanylate cyclase. To determine at what level regulation occurred, day-16 isolated abdomens were injected with 1 U of eclosion hormone. The basal level of c G M P in control muscles was 196 + 46 fmol cGMP/mg protein (n = 5), while peptide-treated muscles had 3044 + 361 fmol cGMP/mg protein (n = 5) 60 rain after hormone exposure. This suggests that receptors for the hormone were present in day-16 muscle membranes, and that some biochemical step distal to the rise in cGMP is regulated by ecdysteroids to allow eclosion hormone to activate the degeneration program. Taken together, these data suggest that ecdysteroids are responsible for regulating the terminal steps in the development of the intersegmental
muscles, as well as other abdominal tissues. During the final days of pharate adult development, the ecdysteroid titre declines. As ecdysleroid activity disappears f,om the haemolymph, tissues are released from developmental inhibition and progress in their differentiation. In the case of thc intersegmental muscles, when the steroid titre drops below a threshold value, the muscles display thc atrophy program. A further reduction in the titre causes the muscles to degenerate. In the case of Manduca. this appears to be a direct effect, i.e. steroid withdrawal activates a developmental program. In Anlheraea too. the ecdysteroid titre controls the timing of degeneration: but here it does so indirectly. The reduction m ecd~steroid titre determines the time of both release of eclosion hormone and muscle sensitivity to this peptide. Eclosion hormone then appears in the haemolymph and acts directly on the muscles, via cGMP, to initiate the degeneration program. Bv manipulating the ecdysteroid titre, it is possible m both species either to accelerate the progression of the intersegmental muscles through these stages or to delay them dramatically. It should be noted, however, that when the muscles enter the degeneration program they become committed to death and cannot be rescued with exogenous steroid. While the development of other tissues, such as the nervous system and the epidermis, is also regulated by the declining ecdysteroid titre, the development of the intersegmental muscles appears to be independent of their development. Since all of these differentiative changes are all regulated by the ecdysteroids late in metamorphic development, the tinting and coordination of such various and complex developmental events can be precisely' controlled (Schwartz and Truman, 1983). Acknowledgements--1 would like to thank Dr C. Bigelow, Dr W. Bollenbacher and Ms D. Rountree for a critical reading of the manuscript. The work was supported by N.I.H. grant number I F32 AG05337-01.
REFERENCES
Bollenbacher W. E., Smith S. k., Goodman W. and Gilbert L. (1981) Ecdysteroid titre during larval pupal adult development of the tobacco hornworm, Manduca scxta. Gen. eomp. Endocr. 44, 302-306. Finlayson L. H. (1956) Normal and induced degeneration o1' abdominal muscles during metamorphosis in the Lepidoptera. Q. J. microsc. Sci. 97, 215 234. Finlayson L. H. (1975) Development and degeneration. In Insect Muscle (Edited by Usherwood P. N. R.), pp. 75-149. Academic Press, New York. Lockshin R. A. (1969) Programmed cell death. Activation of lysis by a mechanism involving the synthesis of protein. J. Insect Physiol. 15, 1505 1516. Lockshin R. A. and Williams C. M. 1t965a) Programmed cell death. I. Cytology of degeneration in the intersegmental muscles of the pernyi moth. J. Insect Physiol. 11, 123-133. Lockshin R. A. and Williams C. M. (1965b) Programmed cell death. IlI. Neural control of the breakdown ot" the intersegmental muscles of silkmoths. J. Insect Physiol. t l, 601 610. Lockshin R. A. and Williams C. M. (1965c) Programmed cell death. IV. The influence of drugs on the breakdown of the intersegmental muscles of silkmoths. J. Insect Physiol. 11, 803-809.
Hormonal control of muscle development Lockshin R. A. and Beaulaton J. (1974) Programmed cell death. Cytochemical evidence for lysosomes during normal breakdown of the intersegmental muscles. J. Ultrastruct. Res. 46, 43-62. Schwartz L. M. and Truman J. W. (1982) Peptide and steroid regulation of muscle degeneration in an insect. Science 215, 1420-1421. Schwartz L. M. and Truman J. W. (1983) Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta. Devl Biol. 99, 103-114. Schwartz L. M. and Truman J. W. (1984a) Hormonal control of muscle atrophy and degeneration in the moth Antheraea polyphemus. J. exp. Biol. 111, 13-30. Schwartz L. M. and Truman J. W. (1984b) Cyclic GMP may serve as a second messenger in peptide-induced muscle degeneration in an insect. Proc. natn. Acad. Sci. U.S.A. 81, 6718-6722. Slama K. (1980) Homeostatic function of ecdysteroids in ecdysis and oviposition. Acta ent. bohem. 77, 145 168. Truman J. W. (1978) Hormonal release of stereotyped motor programmes from the isolated nervous system of the cecropia silkmoth. J. exp. Biol. 74, 151-173.
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Truman J. W. (1980) Eclosion hormone. In Insect Biology in the Future (Edited by Locke M. and Smith D. S.), pp. 385--401. Academic Press, New York. Truman J. W. (1984) The preparatory behavior rhythm of the moth Manduca sexta: an ecdysteroid-triggered circadian rhythm that is independent of the brain. J. comp. Physiol. A 155, 521-528. Truman J. W. and Schwartz L. M. (1982) Insect systems for the study of programmed neuronal death. Neurosci. Commun. 1, 66-72. Truman J. W. and Schwartz L. M. (1984) Steroid regulation of neuronal death in the moth nervous system. J. Neurosci. 4, 274-280. Truman J. W., Taghert P. H., Copenhaver P. F., Tublitz N. J. and Schwartz L. M. (1981) Eclosion hormone may control all ecdyses in insects. Nature 291, 70-71. Truman J. W., Rountree D. B., Reiss S. E. and Schwartz L. M, (1983) Ecdysteroids regulate the release and action of eclosion hormone in the tobacco hornworm Manduca sexta. J. Insect Physiol. 29, 895-900. Williams C. M. (1968) Ecdysone and ecdysone-analogs: Their assay and action on diapausing pupae of the cynthia silkmoth. Biol. Bull. 134, 344-355.
0020-1790/86 $3.00+0.00 Copyright r(- 1986Pergamon Press Ltd
ENDOCRINE REGULATION OF TERMINAL MUSCLE DIFFERENTIATION ATROPHY AND DEGENERATION OF THE INTERSEGMENTAL MUSCLES OF LEPIDOPTERA LAWRENCE M. SCHWARTZ Department of Biology, University of North Carolina, Chapel Hill, NC 27514, U.S.A.
Abstract--The intersegmental muscles of the Lepidoptera pass through three separate, sequential differentiated states during pharate adult development: status quo; atrophy; degeneration. Each of these developmental programs is characterized by a distinct morphology, physiology and endocrine responsiveness. The factors responsible for regulating these differentiative changes are ecdysteroids. In Manduca sexta, the haemolymph ecdysteroid titre declines in a circadian-modified fashion during the last three days of adult development, which parallels the maturation of the intersegmental muscles. Abdomen-ligation, which causes a precipitous decline in the ecdysteroid titre, causes the precocious atrophy and degeneration of these muscles, whereas injection of, or infusion with, 20-hydroxyecdysone greatly delays such changes. While the terminal differentiation of the epidermis and nervous system is also regulated by ecdysteroids, endocrine manipulations have suggested that the development of the intersegmental muscles is independent of these tissues. In the silkmoth Antheraea polyphemus, ecdysteroids are also responsible for regulating intersegmental muscle differentiation, but eclosion hormone (a peptide) acts as the proximal trigger for the activation of the degeneration program. The declining ecdysteroid titre initiates the atrophy program and subsequently determines the timing of both release of eclosion hormone and intersegmental muscle sensitivity to the peptide. Eclosion hormone then acts directly on the muscles, via cGMP, to activate the degeneration program. Ecdysteroids appear to prevent premature muscle degeneration by regulating a biochemical step distal to both the eclosion hormone receptor and the rise in cGMP. Key Word Index: Intersegmental muscles, cell death, eclosion hormone, 20-hydroxyecdysone,cGMP (3',5'-guanosine monophosphate)
INTRODUCTION Metamorphosis in holometabolous insects is marked by the dramatic restructuring of a typically sedentary, feeding larva, into an active gamele dispersing adult. Accompanying these changes in morphology and behaviour, there is a marked restructuring of the muscular system. In most instances this involves the degeneration of embryonically derived skeletal muscles to allow for the development of new, adultspecific muscles (Finlayson, 1975). In lepidopterans, significant exceptions to this general rule are the intersegmental muscles, a wide sheet of flattened fibres attached at the intersegmental boundary of each abdominal segment in the larva. After pupation these muscles degenerate in the first, second, seventh and eighth abdominal segments. The intersegmental muscles of the remaining four segments persist until adult eclosion, at which time they provide the peristaltic waves of abdominal compression which help to extricate the animal from the pupal cuticle and cocoon. After eclosion the intersegmental muscles begin to degenerate and are not detectable 36 hr later (Finlayson, 1956; Lockshin and Williams, 1965a). The intersegmental muscles appear to display three separate but sequential development programs dur-
ing metamorphosis, namely status quo, atrophy and degeneration. Each of these phases is characterized by a distinct ultrastructure, physiology and endocrine responsiveness. STRUCTURE AND FUNCTIONOF THE INTERSEGMENTAL MUSCLES The three developmental states of the intersegmental muscles can be most readily seen by weighing the muscles during adult differentiation (Schwartz and Truman, 1983, 1984a). In the tobacco hornworm Manduca sexta, the mass of the muscle remained constant until day 15 of the normal 18 days development (Fig. i). In the giant silkmoth Antheraea polyphemus, the status quo phase lasted until about day 16 of the normal 17 days development. During the status quo period, the fibres displayed an appearance and physiology typical of arthropod skeletal muscles (Lockshin and Beaulaton, 1974; Schwartz and Truman, 1984a). Three days before eclosion, the intersegmental muscles entered a period of atrophy, which continued until eclosion and is characterized by an approx. 40% loss of weight (Fig. 1). This reduction in mass seems to result from the loss of entire myofibrils
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Fig. I. Change in mass of intersegmental muscles during adult development in intact Manduca (O) and isolated abdomens (C)). Abdomens isolated on day 15. Values are mean dry weight mg/g body wt +SEM (n = 5). E, time of eclosion (from Schwartz and Truman, 1983). rather than from removal of a specific class of contractile proteins (Lockshin and Beaulaton, 1974). While the net strength of the muscle was reduced, several other key physiological parameters remained unchanged, including: (1) the resting potential; (2) the force of tetanic contracture per cross-sectional area; (3) the Ca 2+ concentration required for generation of 50% tension in skinned fibres (PCas0); (4) Hill coefficients (Schwartz, Bolles and Almers, unpublished observations). Atrophy may function to reduce net muscle strength in order to prevent detachment from the pupal cuticle at a time when the latter is being digested in preparation for eclosion. The final period of intersegmental muscle differentiation, namely degeneration, coincided with adult eclosion (Lockshin and Williams, 1965a; Schwartz and Truman, 1982). Ultrastructurally, the process began a few hours after eclosion with the production of lysosomes. The lysosomes persisted through degeneration and ultimately represented the major organelle within dying fibres. The contractile proteins were rapidly digested and became undetectable after 18 20 hr. By 28 hr after eclosion the muscles were reduced to flimsy bags of membranes (Lockshin and Beaulaton, 1974). There were dramatic changes in the physiological properties of the intersegmental muscles as they degenerated (Schwartz, Bolles and Almers, unpublished observations). The concentration of intracellular calcium required for the generation of 50% tension increased by more than three orders of magnitude, rising from 7.77 x 10 7M at eclosion to I. 18 x 10 ~ M 18 hr later. During this period there was a progressive weakening of the muscle, and contraction had ceased by 20 hr after eclosion. Accompanying these changes was a continuous 2.5 mV/hr depolarization of the fibres.
SCHWARTZ
muscle atrophy and degeneration (Schwartz and Truman, 1982, 1983). When day-15 Manduca were ligated between the thorax and abdomen, the intersegmental muscles underwent accelerated differentiation (Fig. 1). The muscles from these preparations entered the atrophy and degeneration phases of development a full day ahead of unligated controls. Accelerated maturation of the intersegmental muscles was not the only effect of abdomen isolation. Several other terminal developmental markers were also observed to appear precociously, including: (1) breakdown of the pupal endocuticle; (2) resorption of the moulting fluid; (3) neuron degeneration; (4) preecdysial behaviour (Schwartz and Truman, 1983; Truman, 1984). In contrast to ligations performed on day 15, abdomen isolation several hours before adult eclosion on day 18 had no impact on either the timing or the rate of intersegmental muscle degeneration. To further characterize these muscles sensitivity to abdomen isolation, ligations were performed systematically at various times during the last 3 days of pharate adult development. By comparing the muscle weight of ligated animals to that of non-ligated controls, the change in developmental rate was determined. As expected from the above data, the extent of precocious muscle degeneration varied with the time of abdomen isolation (Fig. 2). Interestingly, the acceleration of muscle degeneration did not change linearly with the time of ligation, but instead varied in a saltatory fashion. Ligations performed during the scotophase were essentially equipotent, while a shift of a few hours during the photophase resulted in a dramatic change in the rate of muscle development. Presumably the effect of these ligations was to disrupt some influence from the anterior end of the animal. To determine if this signal was neuronal or endocrine in nature, the ventral nerve cords of day 15 animals were severed, much as would have resulted
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~ D A Y 18/ ISOLATION
Fig. 2. Effect of abdomen-ligation on the time-course ol Manduca intersegmental muscle development. Abdomens were isolated at the times indicated and held for varying periods. Muscle mass was compared to that of unmanipulated control insects and the relative advancement o! development was determined. Values are mean advancement +SEM (n = 4~14; from Schwartz and Truman. 1983).
Hormonal control of muscle development
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TIME OF ECDYSTEROID TREATMENT (hours relotive to eedysis) Fig, 3. Progressive inability of 20-OH-ecdysone-injection to delay eclosion behaviour, intersegmental muscle degeneration and death ofmotorneurons D-IV which innervate the interse~mental muscles. Muscle mass was determined 24 hr after normal eclosion, when control animals have a muscle mass of about
0,3 mg/g body wt (from Truman and Schwartz, 1982). from isolation of the abdomen. These animals were then examined one day after the eclosion of unoperated control and were found to have a muscle dry wt of 0.59+ 0.09mg intersegmental muscle/g body wt ( + SEM; n = 5). This compares favourably with the control animals which had a muscle dry wt of 0.68 _ 0.12 mg/g (n = 6). In contrast, animals ligated on day 15 had no detectable intersegmental muscle tissue at this time. When other markers for terminal differentiation were examined, they too were found to be unaffected by nerve-cord transection on day 15 (Schwartz and Truman, 1983; Truman and Schwartz, 1984). These data suggest that the signal from the anterior end of the animal which modulates the timing of terminal differentiative events is hormonal in nature. During the first third of pharate adult development in the Lepidoptera, the circulating titre of ecdysteroids increases (Bollenbacher et al., 1981), which serves to promote adult differentiation (Williams, 1968). During the remaining two thirds of metamorphosis, the ecdysteroid titre declines. It is known that
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injection of 20-hydroxyecdysone can delay eclosion in Tenebrio (Slama, 1980) and Manduca (Truman et al., 1983). It seemed likely therefore, that ecdysteroids could also influence the timing of atrophy and degeneration of the intersegmental muscles, as these too are late differentiative events. To test this hypothesis, Manduca were injected with 25/~g 20-hydroxyecdysone at various times during the last 36 hr of pharate adult development, and the mass of their muscles was measured 16 hr after the eclosion of unmanipulated controls (Fig. 3). Injection of 20-OH-ecdysone prior to lights-off on day 17 prevented degeneration of the intersegmental muscles. Adult eclosion and the death of the D-IV motor neurons, which innervate the intersegmental muscles, were also delayed (Schwartz and Truman, 1983; Truman and Schwartz, 1984). At later times, administration of ecdysteroids was progressively less effective in delaying these terminal events. As Fig. 3 shows, judicious ecdysteroid injection can separate eclosion, intersegmental muscle degeneration, and motor neuron death from one another. This suggests
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LAWRENCE M. SCHWARTZ
that each of these events is directly controlled by the ecdysteroid titre, rather than occurring "dominostyle" as part of a triggered developmental cascade. These data suggest that elevation of the ecdysteroid titre by administration of exogenous hormone may delay the death of the intersegmental muscles. To better understand any relationship between ecdysteorids and muscle development, the circulating titre of ecdysteroids was measured using a radioimmunoassay (RIA) during the last 3 days of adult development in Manduca. Individual insects were catheterized in the thoracic heart, and 201tl haemolymph samples were removed every 3 hr for 24 hr. The pooled data from 40 individuals (283 separate measurements in triplicate) are shown at Fig. 4. As suggested above, the ecdysteroid titre declines during the last 3 days of adult development. The values fell from approx. 1.5,ug/ml 20-OH-ecdysone equivalents on day 15 to <0.15,ug/ml at the time of adult eclosion. This decline in titre was not linear but showed a circadian modulation. While the data displayed variability, there was a tendency for the ecdysteroid titre to decline during the photophases and to remain relatively constant during the scotophases. This pattern is reminiscent of the saltatory efficacy of abdomen ligation on intersegmental muscle development (Fig. 2). If ecdysteroids are responsible for determining the timing of muscle differentiation, then isolation of the abdomen, which causes precocious atrophy and degeneration of the intersegmental muscles, should also cause a reduction in the level of circulating ecdysteroids; and this appears to be the case. On day 15, the haemolymph ecdysteroid titre was 1765 ± 360 ng 20-OH-ecdysone equivalents/ml (#l = 11 animals) in unligated Manduca. This value was reduced in control animals to 590 ± 74ng/ml (n = 8) 48 hr later. In contrast, animals ligated between the thorax and abdomen on day 15 and kept for 48 hr. had an
ecdysteroid titre of only 60 _+ 28 ng/ml (n = 9), Thus abdomen isolation brought about a drastic reduction in the circulating ecdysteroid titre. THE ROLE OF ECLOStON
HORMONE
The three stages of differentiation described above for Manduca intersegmental muscles also occur in the development of these muscles in another moth, Antheraeapoh'phemus (Schwartz and Truman, 1984a). In A. polyphemus, the ecdysteroid titre declines exponentially during the last 5 days ot" adult development. On day 13 of development in this species, lhc haemolymph titre was 474 +- 106 ng 20-OH-ecdysone equivalents/ml (n = 5), while that at the time ol eclosion on day 17 it was 3 0 + 12ng/ml (n =5). When exogenous 20-OH-ecdysone was introduced. either by injection or infusion, atrophy and degeneration of the intersegmental muscles were great b retarded (Fig. 5). A. poh'phemus differed from M. sexta however, in its response to abdomen isolation. As mentioned above, the timing of intcrsegmental muscle degeneration in Manduca was unaffected by abdomen ligation performed early during the last da~ of adult development. In contrast whereas when Antheraea abdomens were ligated at this time, the intersegmental muscles did not degenerate (Schwartz and Truman, 1982; 1984; Fig. 5). Instead, these muscles continued the atrophy program which had already begun earlier in development. In isolated abdomens of A. polyphemus, the intersegmental muscles progressively lost weight for 6 days, after which time they were no longer contractile. Therefore, isolation of the abdomen prior to adult eclosion prevented the muscles from entering the de
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Fig. 5. Changes in intersegmental muscle mass in Antheraea in intact animals and in control and hormone-treated isolated abdomens. Squares: intact, normally-eclosingadults. Circles: abdomens isolated early on day 17. Triangles: isolated abdomens injected with 1 U eclosion hormone. Hexagons: intact animals infused with 20-OH-ecdysone, 30 ng/g per hr, starting 2 days before eclosion. Open symbols indicate contractile muscles, closed symbols non-contractile muscles. Values are mean dry wt _+SEM for 4 7 animals per group.
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Fig. 6. Changes in intersegmental muscle mass in Antheraea in response to injected eclosion hormone (EH) or 20-OHecdysone during the last 36 hr of pharate adult development. Muscle wt were determined 24 hr after the eclosion of intact control animals. Closed circles: muscle wt from intact insects injected with 25#g 20-OH-ecdysone at the times indicated. Open circles: day-17 isolated abdomens injected with I U eclosion hormone. E, time of eclosion (modified from Schwartz and Truman, 1984a).
to release of eclosion hormone, abdominal tissues are denied exposure to this peptide. To determine whether eclosion hormone is responsible for intersegmental muscle degeneration in Antheraea, isolated abdomens were injected on day 17 with 1 U of the peptide (the amount of hormone found in one pair of pharate adult Manduca corpora cardiaca). At various times after injection of eclosion hormone, the intersegmental muscles were removed and their dry weights determined. Hormone injection induced intersegmental muscle degeneration which was qualitatively and quantitatively identical to that seen in normally eclosing adults (Fig. 5). The amount of eclosion hormone released into the haemolymph to initiate adult eclosion is 0,5 U (Truman et al., 1981). When various dosages of eclosion hormone were injected it caused all-or-none cell death at a response threshold of 0.1 U (Schwartz and Truman, 1984a). Since the amount of hormone normally seen by the muscles at eclosion is substantially greater than 0.1 U, eclosion hormone presumably is the proximal trigger for degeneration of the intersegmental muscles in Antheraea. When abdomens were isolated on day 16 of development (24hr before eclosion) and injected with eclosion hormone, degeneration of the intersegmental muscles did not occur. To determine when eclosion hormone sensitivity arose, abdomens were removed at various times during the 24 hr preceding adult eclosion and injected with the hormone. These abdomens were retained for 24 hr and then their muscle mass was determined. As can be seen in Fig. 6, there was a time-dependent increase in intersegmental muscle sensitivity to exogenous eclosion hormone. The acquisition of sensitivity to this hormone corresponded to the loss of muscle ecdysteroid sensitivity. Early injections of 20-OH-ecdysone into intact insects reduced the extent of subsequent degeneration of the muscles. The progressive loss of sensitivity to 20-OH-ecdysone paralleled the normal reduction in the level of circulating ecdysteroids observed late in
207
development. It should be noted that the declining ecdysteroid titre also determines the timing of eclosion hormone release in lepidopterans (Truman et al., 1983). Thus, as the ecdysteroid titre declines, the intersegmental muscles acquire the ability to respond to eclosion hormone at a time just prior to this peptide's gated release. The ability of eclosion hormone to provoke degeneration of the intersegmental muscles could be due either to the direct action of the hormone on the muscles or to indirect action through the effect of the hormone on the nervous system. Earlier reports suggested that the trigger for intersegmental muscle degeneration is the cessation of electrical activity by their innervating motor neurons (Lockshin and Williams, 1965b,c). Since eclosion hormone has been shown to have direct effects on the nervous system (Truman, 1978), this mechanism may seem plausible. It appears however, that eclosion hormone acts directly on the muscles themselves to stimulate their degeneration (Schwartz and Truman, 1984a). Injection of the neurotoxic drug tetrototoxin into isolated abdomens rapidly caused flaccid paralysis due to cessation of neuronal electrical activity. Treated abdomens retained contractile intersegmental muscles of normal mass when examined 24hr later. These muscles from abdomens paralysed in this way still underwent characteristic degeneration when subsequently treated with eclosion hormone. Therefore, neither the patterning nor the complete cessation of electrical activity was responsible for degeneration of the intersegmental muscles. To control for possible trophic maintenance of the muscles by their motor neurons, the ventral nerve cord was removed from abdomens isolated either at the beginning of adult development or just prior to adult eclosion. The intersegmental muscles persisted in these denervated preparations. Again, injection of eclosion hormone resulted in loss of the muscles. These data suggest that this hormone acts directly on the intersegmental muscles to induce degeneration, rather than acting via effects on the nervous system. It is of obvious interest to determine how eclosion hormone precipitates the rapid involution of a viable set of muscles. There are presumably a number of steps between hormone exposure and cell autolysis. Muscle degeneration appears to be sensitive to inhibitors of both RNA and protein synthesis (Lockshin, 1969). Work has recently begun on a molecular analysis of intersegmental muscle degeneration (Schwartz and Bloom, unpublished observations) to answer such basic questions as: how many genes are required to kill a cell, what are the products of these genes, and how are these genes hormonally regulated? While events at the gene level are still unknown, one potential link between eclosion hormone exposure and the activation of the degeneration program has been identified. Several lines of evidence suggest that 3',5'-guanosine monophospahte (cGMP) may serve as a second messenger in this system (Schwartz and Truman, 1984b). Evidence came initially from isolated abdomens injected with inhibitors of the cyclic nucleotide-degradative enzyme phosphodiesterase. Both of the drugs tested, 3-isobutyl-l-methylxanthine (IBMX) and theophylline, induced dose-dependent degeneration of
208
LAWRENCEM. SCHWAR'IZ
the intersegmental muscles, with IBMX displaying about 100-fold greater potency than theophylline. To further characterize the potential involvement of cyclic nucleotides in this process, isolated abdomens were injected with either cAMP or cGMP and the mass of the intersegmental muscles was determined 24 hr later. Injection of cAMP, even at doses of 50~mol/abdomen (haemolymph concentration 50mM) were without effect. Injection of cGMP however, resulted in dose-dependent intersegmental muscle degeneration with a threshold of about 0.5pmol/abdomen (0.5mM in the haemolymph). Maximal degeneration was induced by cGMPinjections greater than 5 ~mol. To determine more directly whether cyclic nucleotides are involved in eclosion hormone action, isolated abdomens were injected with 2 U of the hormone and at various times thereafter, the muscle contents of cGMP and cAMP were measured using RIA. Eclosion hormone provoked only minor fluctuations in muscle cAMP levels, while cGMP levels increased dramatically. They began to rise within 5 rain of hormone injection and then reached a plateau 60min later, at a concentration 22-fold greater than the basal level. The cGMP titre then slowly declined, such that it was still 10-fold higher than basal levels 3.5 hr after hormone exposure. The magnitude of cGMP accumulation was dependent on the dose of eclosion hormone injected. Below 0.1 U of hormone, only minor elevations in muscle cGMP content were detected 60 min later. Above this dose however, there was a marked increase in the accumulation of cGMP. Since the amount of eclosion hormone released at eclosion is 0.5 U, there are dramatic elevations in muscle cGMP content preceding normal degeneration. The ability of eclosion hormone to stimulate an increase in muscle cGMP is independent of the nervous system. When isolated abdomens were denervated and injected with the peptide, the muscles from these preparations displayed the same elevations in cGMP-accumulation as were seen in control isolated abdomens. This observation further strengthens the suggestion of a direct action of eclosion hormone on the intersegmental muscles themselves. As stated above, the intersegmental muscles from day-16 isolated abdomens do not degenerate when injected with eclosion hormone. They also failed to degenerate when injected with IBMX, with exogenous cGMP, or with sodium nitroprusside, a potent stimulator of guanylate cyclase. To determine at what level regulation occurred, day-16 isolated abdomens were injected with 1 U of eclosion hormone. The basal level of c G M P in control muscles was 196 + 46 fmol cGMP/mg protein (n = 5), while peptide-treated muscles had 3044 + 361 fmol cGMP/mg protein (n = 5) 60 rain after hormone exposure. This suggests that receptors for the hormone were present in day-16 muscle membranes, and that some biochemical step distal to the rise in cGMP is regulated by ecdysteroids to allow eclosion hormone to activate the degeneration program. Taken together, these data suggest that ecdysteroids are responsible for regulating the terminal steps in the development of the intersegmental
muscles, as well as other abdominal tissues. During the final days of pharate adult development, the ecdysteroid titre declines. As ecdysleroid activity disappears f,om the haemolymph, tissues are released from developmental inhibition and progress in their differentiation. In the case of thc intersegmental muscles, when the steroid titre drops below a threshold value, the muscles display thc atrophy program. A further reduction in the titre causes the muscles to degenerate. In the case of Manduca. this appears to be a direct effect, i.e. steroid withdrawal activates a developmental program. In Anlheraea too. the ecdysteroid titre controls the timing of degeneration: but here it does so indirectly. The reduction m ecd~steroid titre determines the time of both release of eclosion hormone and muscle sensitivity to this peptide. Eclosion hormone then appears in the haemolymph and acts directly on the muscles, via cGMP, to initiate the degeneration program. Bv manipulating the ecdysteroid titre, it is possible m both species either to accelerate the progression of the intersegmental muscles through these stages or to delay them dramatically. It should be noted, however, that when the muscles enter the degeneration program they become committed to death and cannot be rescued with exogenous steroid. While the development of other tissues, such as the nervous system and the epidermis, is also regulated by the declining ecdysteroid titre, the development of the intersegmental muscles appears to be independent of their development. Since all of these differentiative changes are all regulated by the ecdysteroids late in metamorphic development, the tinting and coordination of such various and complex developmental events can be precisely' controlled (Schwartz and Truman, 1983). Acknowledgements--1 would like to thank Dr C. Bigelow, Dr W. Bollenbacher and Ms D. Rountree for a critical reading of the manuscript. The work was supported by N.I.H. grant number I F32 AG05337-01.
REFERENCES
Bollenbacher W. E., Smith S. k., Goodman W. and Gilbert L. (1981) Ecdysteroid titre during larval pupal adult development of the tobacco hornworm, Manduca scxta. Gen. eomp. Endocr. 44, 302-306. Finlayson L. H. (1956) Normal and induced degeneration o1' abdominal muscles during metamorphosis in the Lepidoptera. Q. J. microsc. Sci. 97, 215 234. Finlayson L. H. (1975) Development and degeneration. In Insect Muscle (Edited by Usherwood P. N. R.), pp. 75-149. Academic Press, New York. Lockshin R. A. (1969) Programmed cell death. Activation of lysis by a mechanism involving the synthesis of protein. J. Insect Physiol. 15, 1505 1516. Lockshin R. A. and Williams C. M. 1t965a) Programmed cell death. I. Cytology of degeneration in the intersegmental muscles of the pernyi moth. J. Insect Physiol. 11, 123-133. Lockshin R. A. and Williams C. M. (1965b) Programmed cell death. IlI. Neural control of the breakdown ot" the intersegmental muscles of silkmoths. J. Insect Physiol. t l, 601 610. Lockshin R. A. and Williams C. M. (1965c) Programmed cell death. IV. The influence of drugs on the breakdown of the intersegmental muscles of silkmoths. J. Insect Physiol. 11, 803-809.
Hormonal control of muscle development Lockshin R. A. and Beaulaton J. (1974) Programmed cell death. Cytochemical evidence for lysosomes during normal breakdown of the intersegmental muscles. J. Ultrastruct. Res. 46, 43-62. Schwartz L. M. and Truman J. W. (1982) Peptide and steroid regulation of muscle degeneration in an insect. Science 215, 1420-1421. Schwartz L. M. and Truman J. W. (1983) Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta. Devl Biol. 99, 103-114. Schwartz L. M. and Truman J. W. (1984a) Hormonal control of muscle atrophy and degeneration in the moth Antheraea polyphemus. J. exp. Biol. 111, 13-30. Schwartz L. M. and Truman J. W. (1984b) Cyclic GMP may serve as a second messenger in peptide-induced muscle degeneration in an insect. Proc. natn. Acad. Sci. U.S.A. 81, 6718-6722. Slama K. (1980) Homeostatic function of ecdysteroids in ecdysis and oviposition. Acta ent. bohem. 77, 145 168. Truman J. W. (1978) Hormonal release of stereotyped motor programmes from the isolated nervous system of the cecropia silkmoth. J. exp. Biol. 74, 151-173.
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Truman J. W. (1980) Eclosion hormone. In Insect Biology in the Future (Edited by Locke M. and Smith D. S.), pp. 385--401. Academic Press, New York. Truman J. W. (1984) The preparatory behavior rhythm of the moth Manduca sexta: an ecdysteroid-triggered circadian rhythm that is independent of the brain. J. comp. Physiol. A 155, 521-528. Truman J. W. and Schwartz L. M. (1982) Insect systems for the study of programmed neuronal death. Neurosci. Commun. 1, 66-72. Truman J. W. and Schwartz L. M. (1984) Steroid regulation of neuronal death in the moth nervous system. J. Neurosci. 4, 274-280. Truman J. W., Taghert P. H., Copenhaver P. F., Tublitz N. J. and Schwartz L. M. (1981) Eclosion hormone may control all ecdyses in insects. Nature 291, 70-71. Truman J. W., Rountree D. B., Reiss S. E. and Schwartz L. M, (1983) Ecdysteroids regulate the release and action of eclosion hormone in the tobacco hornworm Manduca sexta. J. Insect Physiol. 29, 895-900. Williams C. M. (1968) Ecdysone and ecdysone-analogs: Their assay and action on diapausing pupae of the cynthia silkmoth. Biol. Bull. 134, 344-355.