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Effects of ecdysone and juvenile hormone on epidermal cell development in Hyalophora cecropia
TISSUE & CELL 1976 8 (4) 649-658 Published by Longman Group Ltd. Printed in Great Britain
BONNIE J. SEDLAK”
and LAWRENCE
I. GILBERT
EFFECTS OF ECDYSONE AND JUVENILE HORMONE ON EPIDERMAL CELL DEVELOPMENT IN HYALOPHORA CECROPlA ABSTRACT. Pupae of Hyalophora cecropia were injected with various doses of fiecdysone (molting hormone) or juvenile hormone and the epidermal cell ultrastructure was then studied with the electron microscope. The hormonal effects were rapidly manifested and appeared to be cell specific and dose dependent. The initial response to both hormones was an outward blebbing of the apical plasma membrane. Large doses of fi-ecdysone elicited both precocious cuticle deposition and premature autophagic vacuole formation. Juvenile hormone prevented the appearance of the autophagic vacuoles which normally preceded cell differentiation into cells capable of adult synthesis. After prolonged exposure to juvenile hormone, there appeared to be an exaggerated separation of the epidermal cells at the basal region suggesting that the juvenile hormone may act at the membrane level.
Introduction
do elicit specific changes in the ultrastructure of the epidermis.
SPECIFIC alterations in cell ultrastructure are evident in the abdominal epidermal cells of Hydophora cecropiu during normal pupaladult development (Sedlak and Gilbert, 1976). Since molting is elicited by ecdysone, modulated by juvenile hormone and is in large measure a reflection of epidermal cell activity (see Gilbert, 1974), it was of interest to determine whether the injection of ecdysone or juvenile hormone would specifically affect epidermal cell development. It is well known that excessive amounts of ecdysone cause abnormal adult development (Kobayashi et al., 1967; Williams, 1970; Judy and Gilbert, 1970) and that the injection of juvenile hormone into pupae results in the production of intermediates possessing both pupal and adult characters (e.g. Gilbert and Schneiderman, 1960). The data presented here indicate that these two growth hormones
Materials and Methods Pupae of Hyulophoru cecropiu were obtained and maintained at 6°C as described previously (Sedlak and Gilbert, 1976). Pupae which were incubated at 6°C were injected with a constant 30 ~1 dose of either /3ecdysone or juvenile hormone through the intersegmental membrane of the third abdominal segment, and were then placed at 25°C until sacrificed. /3-Ecdysone was dissolved in 10% ethanol and injected with a 31 gauge needle in doses of 1, 5 and 50 pg per gram fresh weight. Animals injected with 30 ~1 of 10% ethanol alone were used as controls at all stages tested. Animals were sacrificed 1, 6 and 12 hr and 1, 3 and 5 days later. Juvenile hormone (C~S: mixed isomers) was diluted in peanut oil and injected via a 27 gauge needle in doses of 2, 10 and 20 pg per gram fresh weight and 300 pg per pupa. Peanut oil (30 ~1) was injected as a control at all stages used. Animals were sacrificed 1, 6 and 12 hr and 1, 3, 5, 7, 11 and 21 days later. The epidermis was excised, fixed, stained
Department of Biological Sciences, Northwestern University, Evanston, Illinois 60201. * Present address: Department of the Biological Sciences, Smith College, Northampton, Massachusetts 01060. Received 2 August
1976. 649
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and examined with the electron microscope (Hitachi 11E) as previously described (Sedlak and Gilbert, 1976). Results The ultrastructural modifications elicited by hormone injections are compared to the changes occurring during normal development whenever pertinent (see Sedlak and Gilbert, 1976, for details of normal epidermal cell development). The morphology of epidermal cells from control injected animals was similar to the cell structure of normal uninjected animals. Cellular morphology of b-ecdysone injected animals
The following data reveal what appear to be specific changes in epidermal cell morphology when pupae are injected with doses that approach the physiological range (up to about 1 pg/g; see Bollenbacher et al., 1975). The 50 pg/g of /3-ecdysone exceeds by far the amount needed to elicit ‘normal’ adult development. Membrane blebbing. Within 1-12 hr and up to 3 days after the injection of pupae with 1 pg/g of ,&ecdysone, the epidermal ultrastructure was similar to that of normal pupal cells with the following exceptions. The apical plasma membrane of the p-ecdysone treated animals was either bordered by vacuoles numerous, 2 p, electron-lucent (Fig. 1) or the microvilli of the apical plasma membrane appeared to be greatly distended at their tips. This response will be referred to as ‘membrane blebbing’. By the fifth day following the injection of 1 pg/g of /3-ecdysone the basement membrane (which is typically absent from cells of normal day 5 pharate adults) was extremely thin and membrane
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blebbing was again observed. A similar membrane response was observed within the first 24 hr after the injection of either 5 or 50 pg/g of Becdysone. This abnormal membrane configuration was not observed in any of the cells of the control injected animals. The effect is apparently not an artifact and appears to be cell specific since the vacuoles were often found at the apex of one cell but were absent from the adjacent cell. In some of the cells in which the membrane blebbing occurred, large vacuoles were present and the cisternae of the rough endoplasmic reticulum was dilated. vacuoles. Large myelin figures were observed in cells on the fifth day after the injection of 1 rg/g of Becdysone and giant (5.5-8.0 p) autophagic vacuoles containing myelin figures, mitochondria and rough endoplasmic reticulum were present 1 day after treatment with 5 pg/g of t%ecdysone (Fig. 2). These structures were almost identical to organelles found in normal days 7-9 pharate adults. By the third day after injection of either 5 or 50 pg/g of /I-ecdysone, giant autophagic vacuoles were present. These larger doses elicited the formation of autophagic vacuoles from 3-6 days earlier than in normal animals while with the lowest dose (1 pg/g) the autophagic vacuoles appeared at least 24 days prematurely. Autophagic
Cuticle secretion. Cuticulin deposition which was observed on about day 10 of normal pharate adult development was not seen after injection of 1 pg/g of ,%ecdysone during the 5 days observed. On the fifth day after receiving 5 pg/g of /3-ecdysone the cuticulin appeared in patches adjacent to the microvilli of the plasma membrane and was also noted 3 days following the injection of 50
Fig. 1. Membrane blebbing at the apex of epidermal cells 1 hr after a pupa received 1 pg fi-ecdysonelg fresh weight. mb, membrane bleb. x 3000. Fig. 2. Giant autophagic vacuoles containing cell organelles are present in the epidermis 1 day after the insect was injected with 5 pg /3-ecdysoneig fresh weight. A V, autophagic vacuole; I, lipid; n, nucleus. x 12,000.
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cell evaginate at the apical side forming cone-shaped protrusions. All of the microtubules in such regions appeared to be oriented perpendicular to the apex of the cell. Precisely oriented microtubules were present in the shaft of scale cells of normal animals after 11 days of pharate adult development. Summary.
i
i
b
Days After Injection Fig. 4. Summary diagram indicating major responses to the injection of various doses of molting hormone. q ], membrane blebbing; vacuoles; W, adult endocuticle deposition; cuticulin deposition.
pg/g (Fig. 3). On the fifth day following the largest dose (50 pg/g), endocuticle was deposited at the microvillous apical plasma membrane. Endocuticle is secreted by epidermis of day 11 normal pharate adults. The secretion of cuticulin was observed from 5-7 days prematurely after the injection of large doses of /I-ecdysone and endocuticle deposition was observed 5 days prematurely in the epidermis of animals injected with 50 pg/g of P-ecdysone. Cytoplasmic microtubules. Although microtubules were randomly scattered throughout the epidermal cells at all stages postinjection, these cytoskeletal elements were found oriented perpendicular to the plasma membrane in some of the hormone treated cells. On the third day after the injection of 50 pglg of /3-ecdysone, cuticulin is deposited adjacent to the apical plasma membrane. In some regions of the epidermis small areas of the
Fig. 4 summarizes the characteristic morphology of cells which were exposed to various doses of /3-ecdysone. Membrane blebbing was seen from 1 h-3 days postinjection in the epidermis of animals treated with 1 pg/g fresh weight, and from 1 hr to 1 day in cells of pupae injected with 5 and 50 pg/g fresh weight. Autophagic vacuoles appeared within 1 day in animals injected with 5 pg/g of /3-ecdysone and by day 3 in the epidermis of animals after the injection of 5 or 50 pg/g of p-ecdysone. Cuticulin deposition was apparent on day 5 after the injection of 5 pg/g and on day 3 after the injection of 50 pg/g. Animals receiving the highest dose of hormone secreted endocuticle 5 days later. All effects were dose dependent and adjacent cells are apparently capable of responding independently of their neighbors. Cellular morphology of juvenile hormone injected animals
Pupae injected with juvenile hormone molted into pupal-adult intermediates as expected. For example, a pupa receiving 300 pg of juvenile hormone molted into an intermediate having pupal legs, wings and abdominal cuticle, whereas the entire head and thoracic regions were adult-like (Fig. 5). When a lower dose was injected (i.e. 10 pg/g
Fig. 3. Cuticulin is evident in the epidermis 3 days after the injection fi-ecdysonelg fresh weight. c, cuticulin; m, mitochondrion. x 22,500.
of 50 pg
Fig. 5. A pupal-adult intermediate 21 days after the pupa was injected with 300 pg juvenile hormone. Note adult eyes and pupal abdomen. Fig. 6. Membrane blebbing seen as swollen tips of microvilli in cells from animals injected with 20 pg juvenile hormone 12 hr previously. mb, membrane bleb; mvb, multivesicular body; v, vacuole. x 21,000. 42
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fresh weight), the anterior regions were more adult-like but the abdomen was still entirely pupal. It should be noted that the isomeric mixture of Cl8 juvenile hormone used in these studies has about one-tenth the juvenile hormone activity of the pure f,l,c isomer. The dose of 300 pg of juvenile hormone per pupa is equivalent to about 70 pg/g fresh weight or 7 pg of the t,t,c isomer per gram fresh weight. Membrane b/ebbing. As early as 1 hr and as late as 3 days after the injection of either 2 or 10 pg/g juvenile hormone, cell specific membrane blebbing was discerned and scattered vacuoles plus rough endoplasmic reticulum with distended cisternae were observed in some of the epidermal cells. When 20 pg/g was injected, membrane blebbing was present for the first 12 hr (Fig. 6) and following the injection of 300 pg per pupa the membrane response was evident for only the first hour. This membrane blebbing and accompanying dilation of the cisternae of the rough endoplasmic reticulum was identical to the response seen in the epidermis of P-ecdysone injected animals but was never observed in the cells of the control injected animals. Autophagic vacuoles. The giant autophagic
vacuoles pharate animals receiving
characteristic of normal days 7-9 adults and /3-ecdysone injected were never observed in any insects juvenile hormone.
Cuticle deposition. By the seventh day following the injection of either 2 or 10 pg/g juvenile hormone, a layer of cuticulin and several layers of fibrous endocuticle were observed adjacent to the microvillous apical plasma membrane. This deposition of the
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cuticle layers was evident from 3-4 days earlier than in the normally developing animals, i.e. cuticulin is deposited on day IO and endocuticle is secreted by day 11 in normal pharate adults. Endocuticle adherent to the microvillous apical plasma membrane was deposited by day 5 in animals injected with 20 pg/g of juvenile hormone. By day 21 the endocuticle immediately adjacent to the cell apex appeared to be much less regularly layered than the endocuticle layers more distal to the cell apex (Fig. 7). On the first day following the injection of 300 pg of juvenile hormone per pupa, a fibrous material was evident and by the third day several layers of endocuticle were deposited. This cuticle deposition occurred at least 8 days prematurely in animals injected with this very large dose of hormone. Glycogen-like material. The large accumula-
tion of normal creased pharate dermal animals
glycogen-like inclusions seen in animals by day 16 and which into a maximum in day 21 normal adults, was not found in the epicells of juvenile hormone treated (Fig. 7).
Large intercellular spaces. The lateral plasma membrane of epidermal cells from some of the juvenile hormone injected animals appeared to be separated by large intercellular spaces at the basal region. In the epidermis of animals injected with 2 or 10 pg/g of juvenile hormone, these large intercellular spaces were noted by day 21, and after the injection of either 20 pg/g or 300 pg per pupa, the response was evident by day 1I (Fig. 8). Summary
Membrane
blebbing
was seen
Fig. 7. Survey micrograph of epidermis from an animal injected with IO pg juvenile hormone/g fresh weight 21 days previously. Note disorganized layers of endocuticle at the cell apex. PC, endocuticle; m, mitochondrion; mv, microvilli; n, nucleus. x 15,000. Fig. 8. Wide extracellular spaces between epidermal cells in the basal region 1I days after the pupa was injected with 300 pg juvenile hormone. d, desmosome; m, mitochondrion. x 43,000.
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to respond to the hormones. The significance of these observations is not known although the blebbing may represent an attempt to eliminate excess membrane stimulated by exogenously introduced hormones. Alternatively, blebbing may be the visual manifestation of excess molting fluid secretion, which normally occurs from days l-14 in H. cecropia (Lensky et al., 1970).
0
= s zi
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2
3
5
7
9
Dayr After
,‘1
Jm 21
Injection
Fig. 9. Summary diagram indicating major responses to the injection of various doses of juvenile hormone. [m, membrane blebbing; [g, cuticulin deposition; W, endocuticle deposition; [XI, spaces appear between cells at the basal region.
from 1 hr to 3 days after the injection of 2 or 10 pg/g of juvenile hormone; from 1 hr to 1 day after 20 pg/g has been injected; and 1 hr after the injection of 300 pg per pupa. Cuticle was deposited by the seventh day after either 2 or 10 pg/g of juvenile hormone was injected; by day 5 following the injection of 20 pg/g; and at least by day 3 in animals injected with 300 pg of juvenile hormone. The giant autophagic vacuoles and large accumulations of glycogen-like material which characterized the epidermis in the latter stages of normal development were not observed following the introduction of juvenile hormone. Prolonged exposure to the hormone resulted in abnormally wide intercellular spaces between cells in the basal regions from days 11-21 postinjection. These cellular responses to juvenile hormone are summarized in Fig. 9.
Initial responses to the hormones. The initial response to the injection of either /I-ecdysone or juvenile hormone was ‘membrane blebbing’; a response of the apical plasma membrane which was apparent as either balloon-like distensions of the microvillous apical plasma membrane or as vacuoles adjacent to the cell apex. This response was observed within the first hour following the introduction of the hormone and was dose dependent. In several cases the abnormal membrane morphology was characteristic of one cell but absent from the neighboring cell, suggesting some cell specificity in the ability
Cuticle secretion. Dose-dependent precocious deposition of adult cuticle was observed in the epidermis of animals injected with either /3-ecdysone or juvenile hormone. Cuticle was deposited 5-7 days precociously by the epidermis of animals after the injection of ,Gecdysone and from 3-8 days prematurely by epidermal cells of insects following the injection of juvenile hormone. As noted in the introduction, excessive doses of ecdysone injected into a pupa can result in ‘hyperecdysonism’ characterized by the production of an abnormal, scaleless adult. It has been suggested that this syndrome results from precocious cuticle deposition which might lock the cells into an early developmental stage (Williams, 1970). The present study confirms this suggestion and offers ultrastructural evidence to support it. The lowest dose of /3-ecdysone ( 1 pg/g) did not result in cuticle secretion during the time that the animals were observed, suggesting that this may be a subthreshold quantity. With the largest dose of juvenile hormone (300 pg per pupa), fibrous endocuticle-like material was seen at the cell apex within the first 24 hr after injection. Since cuticulin was not observed adjacent to this fibrous layer, the sample may either have been damaged in preparation or the very large dose of juvenile hormone may have specifically stimulated the deposition of endocuticle while omitting cuticulin secretion.
Cellular remodelling. The giant autophagic vacuoles observed in normal pharate adults from days 7-9 were present 2-6 days earlier following ,&ecdysone injection. Similarly, the premature appearance of ecdysone stimulated lysosomes has been reported for the midgut of the fly, Sarcophaga bullata (Radford and Misch, 1971). Giant autophagic vacuoles have been associated with cellular restructuring in the normal pharate adult epidermis of H. cecropia (Sedlak and Gilbert,
ECDYSONE
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1976) and their presence precedes the differentiation of general epidermal tissue into scale and socket cells. The microtubules which are also normally associated with cell remodelling and the maintenance of cell shape (Lawrence, 1966; Locke, 1966; Overton, 1966) are found 6 days prematurely in the epidermis of animals injected with P-ecdysone (e.g. on day 5 after the injection of 5 pg/g). The precocious appearance of these cytoskeletal support elements and the associated changes in cell shape may delineate the onset of scale formation in these hormone stimulated animals. The premature cuticle deposition already noted, in turn probably prevents scale development and results in the scaleless adults typical of ‘hyperecdysonism’. Autophagic vacuole formation was never observed in the epidermis of animals following the injection of juvenile hormone suggesting the absence of cellular remodelling in juvenile hormone affected epidermis. /3-Ecdysone stimulated cell responses. The premature formation of giant autophagic vacuoles can be attributed to the presence of exogenously introduced S-ecdysone since these organelles were never seen in the juvenile hormone injected animals. Both the initial membrane blebbing and premature cuticle deposition on the other hand were responses observed after the injection of either /3-ecdysone or juvenile hormone. Since juvenile hormone can stimulate pupal prothoracic glands to secrete endogenous ecdysone (e.g. Gilbert and Schneiderman, 1959) both of these cellular responses may be the result of ‘hyperecdysonism’. Juvenile hormone stimulated cell responses.
The injection of juvenile hormone resulted in the formation of pupal-adult intermediates lacking abdominal scales. Scale formation is presumably a consequence of cellular differentiation and giant autophagic vacuoles were noted in the epidermis prior to the appearance of scale cells in normal pharate adults (Sedlak and Gilbert, 1976). As noted above, juvenile hormone may prevent normal cell remodelling by the suppression of giant autophagic vacuole formation. This suggestion is consistent with other observations suggesting that juvenile
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hormone prevents a number of naturally occurring histolytic processes that occur at metamorphosis (see Gilbert and King, 1973). The accumulation of glycogen-like inclusions, which is characteristic of normal adult epidermis, is also apparently prevented by juvenile hormone injections. The absence of these energy stores may account for the disrupted appearance of the endocuticle layers deposited late in development (e.g. the endocuticle of day 21 animals injected with 10 pg/g of juvenile hormone). Following juvenile hormone injection, large intercellular spaces are seen in the basal regions of epidermal cells during the latter part of development. Although this occurs to a much lesser degree in normal pharate adults, it may reflect a physiological change which facilitates the transport of macromolecules between cells, and may be analogous to the spacing observed between the ovarian follicle cells of Rhodnius after juvenile hormone has acted as a gonadotropic hormone (Huebner and Davy, 1973). Summary. The appearance of cell markers through normal development (Sedlak and Gilbert, 1976) were used to assess specific epidermal responses elicited by the injection of the insect hormones controlling development. Ecdysone initially affects the apical plasma membrane and results in the membrane blebbing response, which in turn may result from the excessive production and elimination of membrane. In normal development, excess membrane is geometrically organized as microvilli. Subsequently, ecdysane speeds up normal development by initiating the precocious appearance of giant autophagic vacuoles which may be necessary for the cell remodelling preceding scale formation. Injection of ecdysone also causes the premature secretion of cuticle, thereby locking the cells into an early developmental program and thus prevents normal scale formation. Juvenile hormone prevents differentiation and continued development to the adult, perhaps in part by inhibiting the production of giant autophagic vacuoles. The accumulation in the epidermis of the glycogen-like inclusions, a possible energy supply associated with cuticle deposition in the mature adult, is also prevented. An additional response to the juvenile hormone is noted late
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in development and is manifested as the appearance of large intercellular spaces at the basal portions of epidermal cells. These large basal spaces may reflect an increased accessibility to a variety of molecules at this time. Although the actions of ecdysone and juvenile hormone are probably ultimately expressed at the level of gene transcription (see Gilbert et al., 1976) and can only be analyzed in a quantitative sense by utilizing the techniques of biochemistry and molecular biology, the morphological effects noted
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above do help explain many of the gross observations noted after administration of the hormones (see Gilbert and King, 1973) and provide a temporal base for future studies at the molecular level.
Acknowledgements This work was supported by grants AM02818 from N.I.A.M.D.D. of the National Institutes of Health and PCM76-03620 from the National Science Foundation.
BOLLENBACHER,W., VEDECKIS, W., GILBERT, L. 1. and O’CONNOR, J. D. 1975. Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta. Devl. Biol., 44, 46. GILBERT, L. I. 1974. Endocrine action during insect growth. Recent Prog. Harm. Res., 30, 347. GILBERT, L. I. and KING, D. S. 1973. Physiology of growth and development: endocrine aspects. The Physiology ofZnsecfa (ed. M. Rockstein), 2nd edn, Vol. I, p. 249. Academic Press, New York and London. GILBERT, L. I. and SCHNEIDERMAN, H. A. 1959. Prothoracic gland stimulation by juvenile hormone extracts of insects. Narura, Lond., 184, 171. GILBERT, L. I. and SCHNEIDERMAN, H. A. 1960. The development of a bioassay for the juvenile hormone of insects. Trans. Am. microsc. Sot., 79, 38. GILBERT, L. I., GOODMAN, W. and NOWOCK, J. 1976. The possible roles of binding proteins in juvenile hormone metabolism and action. ActualitPs sur Ies Hormones d’Znvertebrt5. Int. Colloquium of the CNRS, no. 251, p. 413. Paris. HUEBNER, E. and DAVEY, K. G. 1973. An antigonodotropin from the ovaries of the insect Rhodnius prolixus Stal. Can. J. Zool., 51, 113. JUDY, K. and GILBERT, L. I. 1970. Effects of juvenile hormone and molting hormone on rectal pad development in Hyaiophoru cecropia. J. Morplr., 131, 301. KOBAYASHI, M., TAKEMOTO, T., OGAWA, S. and NISHIMOTO, N. 1967. The moulting hormone activity ot ecdysterone and inokosterone isolated from Achyranthis radix. J. Insect Physiol., 13, 1395. LAWRENCE,P. A. 1966. Development and determination of hairs and bristles in the milkweed bug, Oncopelt~s fasciatus (Lygaedae, Hemiptera). J. Cell Sci., 1, 475. LENSKY, Y., COHEN, C. and SCHNEIDERMAN,H. A. 1970. The origin, distribution and fate of the molting fluid proteins of the Cecropia silkworm. Biol. Bull. mar. biol. Lab., Woods Hole, 139, 277. LOCKE, M. 1966. The structure and formation of the cuticulin layer in the epicuticle of an insect, Calpo&.~ ethlius (Lepidoptera, Hesperiidae). J. Morph., 118,461. OVERTON, J. 1966. Microtubules and microfibrils in morphogenesis of the scale cells of EphPstia kuhniella. J. Celi Biol., 29, 293.
RADFORD, S. V. and MISCH, D. W. 1971. The cytological effect of flesh-fly Sarcophaga bullata. J. Cell Biol., 49, 702. SEDLAK, B. J. and GILBERT, L. I. 1976. Epidermal cell development of Hyalophora cecropia. Tissue & Cell, 8, 637. WILLIAMS, C. M. 1970. Hormonal interactions between plants and Sondheimer and J. B. Simeone), pp. 103-132. Academic Press,
ecdysterone during
on the midgut
the pupal-adult
cells of the
metamorphosis
insects. In Chemical Ecology (eds. E. New York and London.
BONNIE J. SEDLAK”
and LAWRENCE
I. GILBERT
EFFECTS OF ECDYSONE AND JUVENILE HORMONE ON EPIDERMAL CELL DEVELOPMENT IN HYALOPHORA CECROPlA ABSTRACT. Pupae of Hyalophora cecropia were injected with various doses of fiecdysone (molting hormone) or juvenile hormone and the epidermal cell ultrastructure was then studied with the electron microscope. The hormonal effects were rapidly manifested and appeared to be cell specific and dose dependent. The initial response to both hormones was an outward blebbing of the apical plasma membrane. Large doses of fi-ecdysone elicited both precocious cuticle deposition and premature autophagic vacuole formation. Juvenile hormone prevented the appearance of the autophagic vacuoles which normally preceded cell differentiation into cells capable of adult synthesis. After prolonged exposure to juvenile hormone, there appeared to be an exaggerated separation of the epidermal cells at the basal region suggesting that the juvenile hormone may act at the membrane level.
Introduction
do elicit specific changes in the ultrastructure of the epidermis.
SPECIFIC alterations in cell ultrastructure are evident in the abdominal epidermal cells of Hydophora cecropiu during normal pupaladult development (Sedlak and Gilbert, 1976). Since molting is elicited by ecdysone, modulated by juvenile hormone and is in large measure a reflection of epidermal cell activity (see Gilbert, 1974), it was of interest to determine whether the injection of ecdysone or juvenile hormone would specifically affect epidermal cell development. It is well known that excessive amounts of ecdysone cause abnormal adult development (Kobayashi et al., 1967; Williams, 1970; Judy and Gilbert, 1970) and that the injection of juvenile hormone into pupae results in the production of intermediates possessing both pupal and adult characters (e.g. Gilbert and Schneiderman, 1960). The data presented here indicate that these two growth hormones
Materials and Methods Pupae of Hyulophoru cecropiu were obtained and maintained at 6°C as described previously (Sedlak and Gilbert, 1976). Pupae which were incubated at 6°C were injected with a constant 30 ~1 dose of either /3ecdysone or juvenile hormone through the intersegmental membrane of the third abdominal segment, and were then placed at 25°C until sacrificed. /3-Ecdysone was dissolved in 10% ethanol and injected with a 31 gauge needle in doses of 1, 5 and 50 pg per gram fresh weight. Animals injected with 30 ~1 of 10% ethanol alone were used as controls at all stages tested. Animals were sacrificed 1, 6 and 12 hr and 1, 3 and 5 days later. Juvenile hormone (C~S: mixed isomers) was diluted in peanut oil and injected via a 27 gauge needle in doses of 2, 10 and 20 pg per gram fresh weight and 300 pg per pupa. Peanut oil (30 ~1) was injected as a control at all stages used. Animals were sacrificed 1, 6 and 12 hr and 1, 3, 5, 7, 11 and 21 days later. The epidermis was excised, fixed, stained
Department of Biological Sciences, Northwestern University, Evanston, Illinois 60201. * Present address: Department of the Biological Sciences, Smith College, Northampton, Massachusetts 01060. Received 2 August
1976. 649
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and examined with the electron microscope (Hitachi 11E) as previously described (Sedlak and Gilbert, 1976). Results The ultrastructural modifications elicited by hormone injections are compared to the changes occurring during normal development whenever pertinent (see Sedlak and Gilbert, 1976, for details of normal epidermal cell development). The morphology of epidermal cells from control injected animals was similar to the cell structure of normal uninjected animals. Cellular morphology of b-ecdysone injected animals
The following data reveal what appear to be specific changes in epidermal cell morphology when pupae are injected with doses that approach the physiological range (up to about 1 pg/g; see Bollenbacher et al., 1975). The 50 pg/g of /3-ecdysone exceeds by far the amount needed to elicit ‘normal’ adult development. Membrane blebbing. Within 1-12 hr and up to 3 days after the injection of pupae with 1 pg/g of ,&ecdysone, the epidermal ultrastructure was similar to that of normal pupal cells with the following exceptions. The apical plasma membrane of the p-ecdysone treated animals was either bordered by vacuoles numerous, 2 p, electron-lucent (Fig. 1) or the microvilli of the apical plasma membrane appeared to be greatly distended at their tips. This response will be referred to as ‘membrane blebbing’. By the fifth day following the injection of 1 pg/g of /3-ecdysone the basement membrane (which is typically absent from cells of normal day 5 pharate adults) was extremely thin and membrane
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blebbing was again observed. A similar membrane response was observed within the first 24 hr after the injection of either 5 or 50 pg/g of Becdysone. This abnormal membrane configuration was not observed in any of the cells of the control injected animals. The effect is apparently not an artifact and appears to be cell specific since the vacuoles were often found at the apex of one cell but were absent from the adjacent cell. In some of the cells in which the membrane blebbing occurred, large vacuoles were present and the cisternae of the rough endoplasmic reticulum was dilated. vacuoles. Large myelin figures were observed in cells on the fifth day after the injection of 1 rg/g of Becdysone and giant (5.5-8.0 p) autophagic vacuoles containing myelin figures, mitochondria and rough endoplasmic reticulum were present 1 day after treatment with 5 pg/g of t%ecdysone (Fig. 2). These structures were almost identical to organelles found in normal days 7-9 pharate adults. By the third day after injection of either 5 or 50 pg/g of /I-ecdysone, giant autophagic vacuoles were present. These larger doses elicited the formation of autophagic vacuoles from 3-6 days earlier than in normal animals while with the lowest dose (1 pg/g) the autophagic vacuoles appeared at least 24 days prematurely. Autophagic
Cuticle secretion. Cuticulin deposition which was observed on about day 10 of normal pharate adult development was not seen after injection of 1 pg/g of ,%ecdysone during the 5 days observed. On the fifth day after receiving 5 pg/g of /3-ecdysone the cuticulin appeared in patches adjacent to the microvilli of the plasma membrane and was also noted 3 days following the injection of 50
Fig. 1. Membrane blebbing at the apex of epidermal cells 1 hr after a pupa received 1 pg fi-ecdysonelg fresh weight. mb, membrane bleb. x 3000. Fig. 2. Giant autophagic vacuoles containing cell organelles are present in the epidermis 1 day after the insect was injected with 5 pg /3-ecdysoneig fresh weight. A V, autophagic vacuole; I, lipid; n, nucleus. x 12,000.
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cell evaginate at the apical side forming cone-shaped protrusions. All of the microtubules in such regions appeared to be oriented perpendicular to the apex of the cell. Precisely oriented microtubules were present in the shaft of scale cells of normal animals after 11 days of pharate adult development. Summary.
i
i
b
Days After Injection Fig. 4. Summary diagram indicating major responses to the injection of various doses of molting hormone. q ], membrane blebbing; vacuoles; W, adult endocuticle deposition; cuticulin deposition.
pg/g (Fig. 3). On the fifth day following the largest dose (50 pg/g), endocuticle was deposited at the microvillous apical plasma membrane. Endocuticle is secreted by epidermis of day 11 normal pharate adults. The secretion of cuticulin was observed from 5-7 days prematurely after the injection of large doses of /I-ecdysone and endocuticle deposition was observed 5 days prematurely in the epidermis of animals injected with 50 pg/g of P-ecdysone. Cytoplasmic microtubules. Although microtubules were randomly scattered throughout the epidermal cells at all stages postinjection, these cytoskeletal elements were found oriented perpendicular to the plasma membrane in some of the hormone treated cells. On the third day after the injection of 50 pglg of /3-ecdysone, cuticulin is deposited adjacent to the apical plasma membrane. In some regions of the epidermis small areas of the
Fig. 4 summarizes the characteristic morphology of cells which were exposed to various doses of /3-ecdysone. Membrane blebbing was seen from 1 h-3 days postinjection in the epidermis of animals treated with 1 pg/g fresh weight, and from 1 hr to 1 day in cells of pupae injected with 5 and 50 pg/g fresh weight. Autophagic vacuoles appeared within 1 day in animals injected with 5 pg/g of /3-ecdysone and by day 3 in the epidermis of animals after the injection of 5 or 50 pg/g of p-ecdysone. Cuticulin deposition was apparent on day 5 after the injection of 5 pg/g and on day 3 after the injection of 50 pg/g. Animals receiving the highest dose of hormone secreted endocuticle 5 days later. All effects were dose dependent and adjacent cells are apparently capable of responding independently of their neighbors. Cellular morphology of juvenile hormone injected animals
Pupae injected with juvenile hormone molted into pupal-adult intermediates as expected. For example, a pupa receiving 300 pg of juvenile hormone molted into an intermediate having pupal legs, wings and abdominal cuticle, whereas the entire head and thoracic regions were adult-like (Fig. 5). When a lower dose was injected (i.e. 10 pg/g
Fig. 3. Cuticulin is evident in the epidermis 3 days after the injection fi-ecdysonelg fresh weight. c, cuticulin; m, mitochondrion. x 22,500.
of 50 pg
Fig. 5. A pupal-adult intermediate 21 days after the pupa was injected with 300 pg juvenile hormone. Note adult eyes and pupal abdomen. Fig. 6. Membrane blebbing seen as swollen tips of microvilli in cells from animals injected with 20 pg juvenile hormone 12 hr previously. mb, membrane bleb; mvb, multivesicular body; v, vacuole. x 21,000. 42
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654
fresh weight), the anterior regions were more adult-like but the abdomen was still entirely pupal. It should be noted that the isomeric mixture of Cl8 juvenile hormone used in these studies has about one-tenth the juvenile hormone activity of the pure f,l,c isomer. The dose of 300 pg of juvenile hormone per pupa is equivalent to about 70 pg/g fresh weight or 7 pg of the t,t,c isomer per gram fresh weight. Membrane b/ebbing. As early as 1 hr and as late as 3 days after the injection of either 2 or 10 pg/g juvenile hormone, cell specific membrane blebbing was discerned and scattered vacuoles plus rough endoplasmic reticulum with distended cisternae were observed in some of the epidermal cells. When 20 pg/g was injected, membrane blebbing was present for the first 12 hr (Fig. 6) and following the injection of 300 pg per pupa the membrane response was evident for only the first hour. This membrane blebbing and accompanying dilation of the cisternae of the rough endoplasmic reticulum was identical to the response seen in the epidermis of P-ecdysone injected animals but was never observed in the cells of the control injected animals. Autophagic vacuoles. The giant autophagic
vacuoles pharate animals receiving
characteristic of normal days 7-9 adults and /3-ecdysone injected were never observed in any insects juvenile hormone.
Cuticle deposition. By the seventh day following the injection of either 2 or 10 pg/g juvenile hormone, a layer of cuticulin and several layers of fibrous endocuticle were observed adjacent to the microvillous apical plasma membrane. This deposition of the
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cuticle layers was evident from 3-4 days earlier than in the normally developing animals, i.e. cuticulin is deposited on day IO and endocuticle is secreted by day 11 in normal pharate adults. Endocuticle adherent to the microvillous apical plasma membrane was deposited by day 5 in animals injected with 20 pg/g of juvenile hormone. By day 21 the endocuticle immediately adjacent to the cell apex appeared to be much less regularly layered than the endocuticle layers more distal to the cell apex (Fig. 7). On the first day following the injection of 300 pg of juvenile hormone per pupa, a fibrous material was evident and by the third day several layers of endocuticle were deposited. This cuticle deposition occurred at least 8 days prematurely in animals injected with this very large dose of hormone. Glycogen-like material. The large accumula-
tion of normal creased pharate dermal animals
glycogen-like inclusions seen in animals by day 16 and which into a maximum in day 21 normal adults, was not found in the epicells of juvenile hormone treated (Fig. 7).
Large intercellular spaces. The lateral plasma membrane of epidermal cells from some of the juvenile hormone injected animals appeared to be separated by large intercellular spaces at the basal region. In the epidermis of animals injected with 2 or 10 pg/g of juvenile hormone, these large intercellular spaces were noted by day 21, and after the injection of either 20 pg/g or 300 pg per pupa, the response was evident by day 1I (Fig. 8). Summary
Membrane
blebbing
was seen
Fig. 7. Survey micrograph of epidermis from an animal injected with IO pg juvenile hormone/g fresh weight 21 days previously. Note disorganized layers of endocuticle at the cell apex. PC, endocuticle; m, mitochondrion; mv, microvilli; n, nucleus. x 15,000. Fig. 8. Wide extracellular spaces between epidermal cells in the basal region 1I days after the pupa was injected with 300 pg juvenile hormone. d, desmosome; m, mitochondrion. x 43,000.
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9I
GILBERT
to respond to the hormones. The significance of these observations is not known although the blebbing may represent an attempt to eliminate excess membrane stimulated by exogenously introduced hormones. Alternatively, blebbing may be the visual manifestation of excess molting fluid secretion, which normally occurs from days l-14 in H. cecropia (Lensky et al., 1970).
0
= s zi
AND
2
3
5
7
9
Dayr After
,‘1
Jm 21
Injection
Fig. 9. Summary diagram indicating major responses to the injection of various doses of juvenile hormone. [m, membrane blebbing; [g, cuticulin deposition; W, endocuticle deposition; [XI, spaces appear between cells at the basal region.
from 1 hr to 3 days after the injection of 2 or 10 pg/g of juvenile hormone; from 1 hr to 1 day after 20 pg/g has been injected; and 1 hr after the injection of 300 pg per pupa. Cuticle was deposited by the seventh day after either 2 or 10 pg/g of juvenile hormone was injected; by day 5 following the injection of 20 pg/g; and at least by day 3 in animals injected with 300 pg of juvenile hormone. The giant autophagic vacuoles and large accumulations of glycogen-like material which characterized the epidermis in the latter stages of normal development were not observed following the introduction of juvenile hormone. Prolonged exposure to the hormone resulted in abnormally wide intercellular spaces between cells in the basal regions from days 11-21 postinjection. These cellular responses to juvenile hormone are summarized in Fig. 9.
Initial responses to the hormones. The initial response to the injection of either /I-ecdysone or juvenile hormone was ‘membrane blebbing’; a response of the apical plasma membrane which was apparent as either balloon-like distensions of the microvillous apical plasma membrane or as vacuoles adjacent to the cell apex. This response was observed within the first hour following the introduction of the hormone and was dose dependent. In several cases the abnormal membrane morphology was characteristic of one cell but absent from the neighboring cell, suggesting some cell specificity in the ability
Cuticle secretion. Dose-dependent precocious deposition of adult cuticle was observed in the epidermis of animals injected with either /3-ecdysone or juvenile hormone. Cuticle was deposited 5-7 days precociously by the epidermis of animals after the injection of ,Gecdysone and from 3-8 days prematurely by epidermal cells of insects following the injection of juvenile hormone. As noted in the introduction, excessive doses of ecdysone injected into a pupa can result in ‘hyperecdysonism’ characterized by the production of an abnormal, scaleless adult. It has been suggested that this syndrome results from precocious cuticle deposition which might lock the cells into an early developmental stage (Williams, 1970). The present study confirms this suggestion and offers ultrastructural evidence to support it. The lowest dose of /3-ecdysone ( 1 pg/g) did not result in cuticle secretion during the time that the animals were observed, suggesting that this may be a subthreshold quantity. With the largest dose of juvenile hormone (300 pg per pupa), fibrous endocuticle-like material was seen at the cell apex within the first 24 hr after injection. Since cuticulin was not observed adjacent to this fibrous layer, the sample may either have been damaged in preparation or the very large dose of juvenile hormone may have specifically stimulated the deposition of endocuticle while omitting cuticulin secretion.
Cellular remodelling. The giant autophagic vacuoles observed in normal pharate adults from days 7-9 were present 2-6 days earlier following ,&ecdysone injection. Similarly, the premature appearance of ecdysone stimulated lysosomes has been reported for the midgut of the fly, Sarcophaga bullata (Radford and Misch, 1971). Giant autophagic vacuoles have been associated with cellular restructuring in the normal pharate adult epidermis of H. cecropia (Sedlak and Gilbert,
ECDYSONE
AND
JUVENILE
HORMONE
1976) and their presence precedes the differentiation of general epidermal tissue into scale and socket cells. The microtubules which are also normally associated with cell remodelling and the maintenance of cell shape (Lawrence, 1966; Locke, 1966; Overton, 1966) are found 6 days prematurely in the epidermis of animals injected with P-ecdysone (e.g. on day 5 after the injection of 5 pg/g). The precocious appearance of these cytoskeletal support elements and the associated changes in cell shape may delineate the onset of scale formation in these hormone stimulated animals. The premature cuticle deposition already noted, in turn probably prevents scale development and results in the scaleless adults typical of ‘hyperecdysonism’. Autophagic vacuole formation was never observed in the epidermis of animals following the injection of juvenile hormone suggesting the absence of cellular remodelling in juvenile hormone affected epidermis. /3-Ecdysone stimulated cell responses. The premature formation of giant autophagic vacuoles can be attributed to the presence of exogenously introduced S-ecdysone since these organelles were never seen in the juvenile hormone injected animals. Both the initial membrane blebbing and premature cuticle deposition on the other hand were responses observed after the injection of either /3-ecdysone or juvenile hormone. Since juvenile hormone can stimulate pupal prothoracic glands to secrete endogenous ecdysone (e.g. Gilbert and Schneiderman, 1959) both of these cellular responses may be the result of ‘hyperecdysonism’. Juvenile hormone stimulated cell responses.
The injection of juvenile hormone resulted in the formation of pupal-adult intermediates lacking abdominal scales. Scale formation is presumably a consequence of cellular differentiation and giant autophagic vacuoles were noted in the epidermis prior to the appearance of scale cells in normal pharate adults (Sedlak and Gilbert, 1976). As noted above, juvenile hormone may prevent normal cell remodelling by the suppression of giant autophagic vacuole formation. This suggestion is consistent with other observations suggesting that juvenile
EFFECTS
651
hormone prevents a number of naturally occurring histolytic processes that occur at metamorphosis (see Gilbert and King, 1973). The accumulation of glycogen-like inclusions, which is characteristic of normal adult epidermis, is also apparently prevented by juvenile hormone injections. The absence of these energy stores may account for the disrupted appearance of the endocuticle layers deposited late in development (e.g. the endocuticle of day 21 animals injected with 10 pg/g of juvenile hormone). Following juvenile hormone injection, large intercellular spaces are seen in the basal regions of epidermal cells during the latter part of development. Although this occurs to a much lesser degree in normal pharate adults, it may reflect a physiological change which facilitates the transport of macromolecules between cells, and may be analogous to the spacing observed between the ovarian follicle cells of Rhodnius after juvenile hormone has acted as a gonadotropic hormone (Huebner and Davy, 1973). Summary. The appearance of cell markers through normal development (Sedlak and Gilbert, 1976) were used to assess specific epidermal responses elicited by the injection of the insect hormones controlling development. Ecdysone initially affects the apical plasma membrane and results in the membrane blebbing response, which in turn may result from the excessive production and elimination of membrane. In normal development, excess membrane is geometrically organized as microvilli. Subsequently, ecdysane speeds up normal development by initiating the precocious appearance of giant autophagic vacuoles which may be necessary for the cell remodelling preceding scale formation. Injection of ecdysone also causes the premature secretion of cuticle, thereby locking the cells into an early developmental program and thus prevents normal scale formation. Juvenile hormone prevents differentiation and continued development to the adult, perhaps in part by inhibiting the production of giant autophagic vacuoles. The accumulation in the epidermis of the glycogen-like inclusions, a possible energy supply associated with cuticle deposition in the mature adult, is also prevented. An additional response to the juvenile hormone is noted late
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in development and is manifested as the appearance of large intercellular spaces at the basal portions of epidermal cells. These large basal spaces may reflect an increased accessibility to a variety of molecules at this time. Although the actions of ecdysone and juvenile hormone are probably ultimately expressed at the level of gene transcription (see Gilbert et al., 1976) and can only be analyzed in a quantitative sense by utilizing the techniques of biochemistry and molecular biology, the morphological effects noted
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above do help explain many of the gross observations noted after administration of the hormones (see Gilbert and King, 1973) and provide a temporal base for future studies at the molecular level.
Acknowledgements This work was supported by grants AM02818 from N.I.A.M.D.D. of the National Institutes of Health and PCM76-03620 from the National Science Foundation.
BOLLENBACHER,W., VEDECKIS, W., GILBERT, L. 1. and O’CONNOR, J. D. 1975. Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta. Devl. Biol., 44, 46. GILBERT, L. I. 1974. Endocrine action during insect growth. Recent Prog. Harm. Res., 30, 347. GILBERT, L. I. and KING, D. S. 1973. Physiology of growth and development: endocrine aspects. The Physiology ofZnsecfa (ed. M. Rockstein), 2nd edn, Vol. I, p. 249. Academic Press, New York and London. GILBERT, L. I. and SCHNEIDERMAN, H. A. 1959. Prothoracic gland stimulation by juvenile hormone extracts of insects. Narura, Lond., 184, 171. GILBERT, L. I. and SCHNEIDERMAN, H. A. 1960. The development of a bioassay for the juvenile hormone of insects. Trans. Am. microsc. Sot., 79, 38. GILBERT, L. I., GOODMAN, W. and NOWOCK, J. 1976. The possible roles of binding proteins in juvenile hormone metabolism and action. ActualitPs sur Ies Hormones d’Znvertebrt5. Int. Colloquium of the CNRS, no. 251, p. 413. Paris. HUEBNER, E. and DAVEY, K. G. 1973. An antigonodotropin from the ovaries of the insect Rhodnius prolixus Stal. Can. J. Zool., 51, 113. JUDY, K. and GILBERT, L. I. 1970. Effects of juvenile hormone and molting hormone on rectal pad development in Hyaiophoru cecropia. J. Morplr., 131, 301. KOBAYASHI, M., TAKEMOTO, T., OGAWA, S. and NISHIMOTO, N. 1967. The moulting hormone activity ot ecdysterone and inokosterone isolated from Achyranthis radix. J. Insect Physiol., 13, 1395. LAWRENCE,P. A. 1966. Development and determination of hairs and bristles in the milkweed bug, Oncopelt~s fasciatus (Lygaedae, Hemiptera). J. Cell Sci., 1, 475. LENSKY, Y., COHEN, C. and SCHNEIDERMAN,H. A. 1970. The origin, distribution and fate of the molting fluid proteins of the Cecropia silkworm. Biol. Bull. mar. biol. Lab., Woods Hole, 139, 277. LOCKE, M. 1966. The structure and formation of the cuticulin layer in the epicuticle of an insect, Calpo&.~ ethlius (Lepidoptera, Hesperiidae). J. Morph., 118,461. OVERTON, J. 1966. Microtubules and microfibrils in morphogenesis of the scale cells of EphPstia kuhniella. J. Celi Biol., 29, 293.
RADFORD, S. V. and MISCH, D. W. 1971. The cytological effect of flesh-fly Sarcophaga bullata. J. Cell Biol., 49, 702. SEDLAK, B. J. and GILBERT, L. I. 1976. Epidermal cell development of Hyalophora cecropia. Tissue & Cell, 8, 637. WILLIAMS, C. M. 1970. Hormonal interactions between plants and Sondheimer and J. B. Simeone), pp. 103-132. Academic Press,
ecdysterone during
on the midgut
the pupal-adult
cells of the
metamorphosis
insects. In Chemical Ecology (eds. E. New York and London.