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Reprogramming in the absence of DNA synthesis in Galleria larval epidermis
Cell Differentiation, 4 (1975) 45 53 © N o r t h - H o l l a n d Publishing C o m p a n y , A m s t e r d a m -- P r i n t e d in The N e t h e r l a n d s
REPROGRAMMING IN THE ABSENCE OF DNA SYNTHESIS IN G A L L E R I A L A R V A L EPIDERMIS
William H. K A S T E R N * and A. K R I S H N A K U M A R A N * *
Department of Biology, Marquelle University, Mihvaukce, Wisconsin 53233, U.S.A. A c c e p t e d 10 D e c e m b e r 1974
Larval e p i d e r m a l cells f r o m a day-1 p e n u l t i m a t e instar Galleria larva on i m p l a n t a t i o n into day-5 last instar larva m e t a m o r p h o s e and d e p o s i t a pupal cuticle at the same time as the h o s t pupates. DNA s y n t h e s i s in the i m p l a n t e d larval cells was m o n i t o r e d with 3 H-thymidine. Various regimens o f 3 H - t h y m i d i n e a p p l i c a t i o n were used and u n d e r no c o n d i t i o n s did t h e larval cells i n c o r p o r a t e label during the period f r o m i m p l a n t a t i o n to d e p o s i t i o n o f pupal cuticle. This suggests t h a t a wax m o t h larval e c t o d e r m cell can r e p r o g r a m its g e n o m e to secrete a pupal cuticle w i t h o u t a p r e c e d e n t cell division.
Following the original observations on chondrogenesis and myogenesis, several studies on chick oviduct (Oka et al., 1969), erythropoiesis (Weintraub et al., 1971; Miura et al., 1971) and pigmented retina cells (Holtzer et al., 1972a) have indicated a need for a critical or 'quantal' mitosis prior to an overt manifestation of the differentiated state of these cells. This evidence led to the hyp:)thesis that 'quantal' mitosis is different from proliferative mitosis in that, during this division cycle, genes hitherto nonfunctional can be turned on (Holtzer et al., 1972b). Furthermore, quantal mitosis has been regarded, on theoretical grounds, as a prerequisite for the expression of hitherto inactive genes (Ebert, 1968). In the course of postembryonic development of saturnid silkmoths, epidermal cells replicated their DNA prior to the metamorphic molts into a pupa and into an adult (Bowers et al., 1964; Krishnakumaran et al., 1967). Similarly, correlations between DNA synthesis and metamorphosis have been observed in Oncopeltus (Lawrence, 1968) and Galleria (Sehnal et al., 1969). And the effects of inhibitors of DNA synthesis and/or cell division on adult differentiation in saturnid moths (FudR, Williams, 1965; Mitomycin C, Krishnakumaran e t m . , 1967; Madhavan et al., 1968) have led to the suggestion that there is a requirement for DNA synthesis and cell divisions in preparation for reprogramming of insect epidermal cells (cf. Krishnakumaran et al., 1967; Schneiderman, 1969). * P r e s e n t address: D e p a r t m e n t o f Biology, Wesleyan University, M i d d l e t o w n , C o n n e c t i c u 06457. ** R e p r i n t r e q u e s t s m a y be addressed to A. K r i s h n a k u m a r a n .
46 However, the occurrence of DNA synthesis immediately preceding reprogramming does n o t establish causal association between the two events. This is particularly the case with insect larval epidermis because cell divisions in this tissue may be associated with growth and not reprogramming. For example, the old cuticle shed at each ecdysis is replaced by a larger one. It is not inconceivable that preparatory to ecdysis the epidermal cells divide and thereby provide a greater surface area. In this case the observed divisions are unrelated to reprogramming. Furthermore, in Rhodtzius Wigglesworth (1964) observed that cell divisions in abdominal epidermal cells are triggered by stretching caused by the blood meal. Hence it is likely that DNA synthesis (and by inference cell division) observed in the epidermal cells prior to molting and metamorphosis in insect larvae may be associated with stretching caused by growth. In order to test the possible causal association between division and reprogramming in insect epidermis we examined whether any experimental conditions would dissociate these two events viz. reprogramming and cell division. For this purpose small pieces of Galleria penultimate instar larval integument were cultured in vivo in the abdomen of a last instar larval host. Depending on the age of the host and of the donor, the implant secreted a larval or pupal cuticle when the host pupated (Kastern et al., 1974). Because of the in vivo culture conditions, the implanted epidermal cells are not subjected to the normal stretching pressures of intact larvae, and we determined the association between DNA synthesis and metamorphosis under these conditions. The results reported in this communication show that even in the absence of DNA synthesis penultimate larval epidermal cells cultured in vivo metamorphose and secrete a pupal cuticle thereby suggesting that the larval epidermal cells can reprogram their genome without prior DNA replication and cell division. Recently Selman et al. (1974a) also reported a similar situation in transdifferentiation of the cuticle-secreting labial glands into salt-secreting cells in silkmoth pupae in the absence of DNA synthesis. Even earlier Wigglesworth (1963) observed that unfed, decapitated 4th instar R h o dn iu s nymphs, when parabiosed to normal 5th instar nymphs, metamorphosed without any cell divisions in the epidermis, thereby suggesting that the same epidermal cells that secrete a nymphal cuticle can deposit an adult cuticle without undergoing cell division. MATERIALS AND METHODS Larvae of the waxmoth, Galleria mellonella, were reared in the laboratory according to procedures described earlier (Krishnakumaran, 1972). Penultimate and last instar larvae, recognized by their pigmentation and size of the head capsule (Beck, 1960) were used in this study and will be referred to in the text as penultimate and last larvae. The day of ecdysis into a stadium is designated as day 1 and after the subsequent 24 hr, day 2, etc. Normally the penultimate stadium lasted about 4 days and the last stadium 9 days. A
47 batch of newly ecdysed, last instar collected at one time usually ecdysed as pupae within 12 hr of one another. Surgical procedures f or excising the integument, its implantation into and retrieval from the host have been described in another c o m m u n i c a t i o n (Kastern et al., 1974). Autoradiographic procedures using Kodak NTB3 liquid emulsion are the same as those used earlier (Krishnakumaran et al., 1967). A cell was regarded as labelled if at least eight silver grains above background were c o n c e n t r a t e d on the nucleus. Control slides treated with DNase were also coated with photographic emulsion and this t r e a t m e n t removed over 60% of the grains in the h e m o c y t e s that were labelled. The schedule o f application of tritiated thymidine (methyl 2c/mM concentration 1 mc/ml New England Nuclear Co) is summarized in Table II. In one series different groups o f host larvae were injected with 3 H thymidine (20 /Jg/g; last larvae weigh between 80 and 250 mg each) on each of the 5 days f r o m implantation to their pupation, and the labelled larvae were sacrificed 6 or 24 hr after receiving the label. In the second series the larvae received repeated injections of 3H t h y m i d i n e at 6- or 24-hr intervals during the entire period from implantation to p~ !)ation. In the 3rd series a different batch of larvae were injected with 3H t hymi di ne at intervals of 3 hr commencing from the time of implantation to the time of pupation. The hosts were sacrificed 6 hr after receiving the isotope. Three animals were used for each o f the points and frequently 5 to 8 animals were used. At the end of the period of exposure to 3H thymidine the hosts were dissected and the implants were retrieved. Following their recovery the implants were fixed in cold Bouin's fluid for 18 hr and processed for autoradiography. At least 3 slides were prepared from each implant and from each slide 6--8 sections were examined. For purposes of comparison some of the host tissues were also processed for autoradiography in a few instances. H e m o c y t e s and some of the ot her host tissues t hat incorporated t hym i di ne exhibited high concentrations of silver grains over the nucleus. RESULTS
Developmental capacity of the penultimate larval epidermis In the first series of experiments we determined the developmental capacity of the day-1 penultimate larval integument when implanted into day 5, 6 or 7 last larvae. For this purpose a 1 m m 2 piece of integument was excised and washed in sterile Ringers solution and placed in the abdomen of an appropriate host. After the host pupated the implant was dissected out and the nature o f the cuticle secreted by it, if any, was determined. The results presented in Table I show that when the host larvae pupated the integument from the day-1 penultimate larva can deposit either a larval cuticle or pupal cuticle depending u p o n the age of the host. Whenever the implant form ed a
48 TABLE I Nature of the cuticle deposited by day-1 p e n u l t i m a t e instar larval epidermis when cultured in vivo in last instar larvae o f d i f f e r e n t ages.
Age of
Number of implants
N u m b e r depositing
the host*
that deposited cuticle
pupal cuticle**
Day-5 Day-6 Day-7
34 50 16
30 (88.2%) 20 (40%) 0 (0%)
* The day of ecdysis into a last larval s t a d i u m is designated as day-1. ** The others deposited a larval cuticle.
pupal cuticle, it rounded up into a closed vesicle with the epidermis on the outer surface and the cuticle facing in. However, when an implant secreted a larval cuticle it did not form a closed vesicle. Presumably the implant and host cells secrete the cuticle at the same time although specific time relations of cuticular syntheses have n o t been determined. The larval cuticle deposited by the implant possessed the characteristic surface plaques and larval bristles and was untanned as in the normal cuticle (Figs. 1 and 2). Similarly the pupal cuticle deposited by the implant resembles the normal pupal cuticle in the presence of a tanned amber colored exocuticle and a characteristic rough
Figs. 1 and 2. Larval cuticle secreted by a n o r m a l larval--larval m o l t ( tg. 1) c o m p a r e d with a larval cuticle deposited by integument from penultimate instar day-1 larva following i m p l a n t a t i o n into day-6 or day-7 last instar larva (Fig. 2). N o t e the similarity in the d i s t r i b u t i o n o f plaques and bristles.
49
Figs. 3 and 4. Pupal cuticle secreted during normal pupation (Fig. 3) compared with a pupal cuticle deposited by penultimate instar larval epidermal cells following implantation into day-4 or day-5 last instar larva (Fig. 4).
surface pattern (Figs. 3 and 4) t h e r e b y suggesting that the cuticle deposited by the implant reflects the state of differentiation of the epidermal cells.
DNA synthesis in the epidermal cells of the implant The fact that the implant can deposit either a larval or pupal cuticle depending on the age of the host afforded an o p p o r t u n i t y to determine whether a critical cell division is needed just prior to expression of the pupal characters. We monitoTed the e x t e n t of DNA synthesis in the epidermal cells of the implant using 3 H thymidine. Day-5 last larvae (host larvae) into which day-1 penultimate larval integument was implanted were injected with 3H t h y mid in e at d if f e r e nt times after implantation. After exposure to label for different lengths of time, the larvae were sacrificed. The implant was retrieved and processed for autoradiography. Details of the time schedules for injection and exposure to isotope and the results of the 3 H thymidine incorporation studies are presented in Table II, and these data show that the epidermal cells o f the implant do not incorporate ~ H t hym i di ne during the entire period from implantation to pupa t i on of the host. Thus, we infer that the epidermal cells of the implant do not engage in DNA synthesis and probably do n o t undergo divisions during this period. However if left in the host till it pupated the implanted integument deposited a pupal cuticle (see Table I). Despite the fact that a large percentage (75% or more) of the implants would have m e t a m o r p h o s e d and secreted a pupal cuticle, n o n e of the cells incorporated 3H thymidine, t hereby suggest-
50 TABLE II Monitoring of 3H thymidine incorporation into penultimate instar Galleria larval integument implanted into day-5 last instar larva and thus programmed to deposit a pupal cuticle.
Experiment
Labelling period
No. of injections
No. of insects used
Incorporation of label
1 2 3 4 5
24 6 6 24* 6*
1 1 1 5 12
12 16 74 7 7
0"* 0 0 0 0
* Interval between injections. The implants were labelled throughout the period from implantation to pupation. ** 0 = less than 5% of the cells were labeled. Exp. 1 and 2. 3H thymidine was injected into host insect on each of the days from the time of implantation till pupation of the host. In experiment I the insects were sacrificed 24 hr after injection of label and in experiment 2, 6 hr after injection. Exp. 3. Larvae into which penultimate larval integument was implanted were injected with 3H thymidine at 3-hr intervals during the first 72 hr after implantation and were sacrificed 6 hr after injection. In view of the fact that the cells in 7 day hosts do not incorporate label, the period beyond 48 hr following implantation was not monitored. Exp. 4 and 5. The same batch of insects received multiple injections at 24-hr (Exp. 4) or 6-hr (Exp. 5) intervals throughout the period from implantation to pupation. These implants did make a pupal cuticle.
ing t h a t even in the absence of D N A synthesis r e p r o g r a m m i n g of the epiderm a l cell g e n o m e c a n o c c u r . In a d d i t i o n to the absence of 3H t h y m i d i n e i n c o r p o r a t i o n into i m p l a n t s t h a t are p u t a t i v e l y p r o g r a m m e d t o s e c r e t e a p u p a l c u t i c l e , t h e d a t a ( T a b l e II, l i n e s 4 a n d 5) s h o w t h a t t h e i m p l a n t s , e x p o s e d t o 3 H t h y m i d i n e t h r o u g h o u t the period f r o m i m p l a n t a t i o n to p u p a t i o n of the host, secreted a p u p a l cuticle a n d y e t did n o t i n c o r p o r a t e isotope. In view of the fact t h a t D N A synthesis a n d r e p r o g r a m m i n g of the g e n o m e has been m o n i t o r e d w i t h i n the same tissue these data clearly suggest that the two events, r e p r o g r a m m i n g and D N A synthesis m a y be dissociated. DISCUSSION T h e r e s u l t s r e p o r t e d a b o v e c l e a r l y d e m o n s t r a t e t h a t t h e e p i d e r m a l cells o f a p e n u l t i m a t e i n s t a r larval Galleria c a n s y n t h e s i z e a p u p a l c u t i c l e w h e n i m p l a n t e d i n t o a n a p p r o p r i a t e h o s t a n d t h a t t h e s a m e cells t h a t s e c r e t e d a larval cuticle at the preceding m o l t d e p o s i t e d a p u p a l cuticle w i t h o u t replicating t h e i r D N A . T h u s t h e d a t a s u g g e s t t h a t t h e Galleria larval e p i d e r m a l cells c a n
51
reprogram their genome to secrete a pupal cuticle without an associated 'quantal mitosis'. Synthesis of a pupal cuticle by the larval epidermis is traditionally recognized as differentiation (see Schneiderman et al., 1964), since a pupal stage normally intervenes during development of an adult insect. Furthermore recent studies indicate that even under different experimental conditions a pupal stage cannot be by-passed at least in som5 of the lepidopterans e.g. the silkworms (Kato, 1973). Undoubtedly initiation of pupal cuticular synthesis requires turning on genes that were previously inactive as is evidenced by the differences in the chemistry and morphology of the pupal and larval cuticles. Unlike in the larval cuticle, the exocuticle constitutes a sizeable part of the pupal cuticle and its chemical composition requires sclerotin and quinones (cf. Hackman, 1964) not present in the larval cuticles. Since formation of the pupal cuticle requires turning on genes hitherto inactive, initiation of pupal syntheses by the larval epidermal cells may be reasonably regarded as reprogramming. In the absence of DNA synthesis during this reprogramming of the genome, it is reasonable to suggest that the insect larval epidermal cells can turn on new genes at any time during a cell cycle and reprogramming does not involve a 'quantal' mitosis. The absence of thymidine incorporation in over 95% of the cells of the implanted integument is not due to the presence of alternate paths of thymidine synthesis and/or high endogenous pools of thymidine because the host cells including epidermal cells do incorporate thymidine (see also Krishnakumaran, 1972). Similarly in view of the monitoring of DNA synthesis during the entire period from implantation into a host until after secretion of a pupal cuticle the failure to find any label is unlikely to be the result of missing the period of DNA synthesis. Furthermore in one of the experiments, 3H thymidine injections were given to different batches of larvae at 3-hr intervals, a period which is shorter than the estimated S period in the epidermal cells of another insect, Oncopeltus (Lawrence, 1968) and hence it is not likely that the methods of study did not permit visualization of DNA synthesis. Although in most of the implants DNA synthesis was not monitored in the same group of epidermal cells whose reprogramming is documented, in one experiment both isotope incorporation into DNA and reprogramming were studied in the same implants. In these experiments 3H thymidine was administered at 24- or 6-hr intervals which theoretically should make the isotope available continuously. In a recent study Selman et al. (1974b) estimated that about 1 0 ~ of the initial level of 3H thymidine persists in the h e m o l y m p h of Polyphemus 18 hr after injection. And our studies (not reported here) show that after injection of even 1/10th the normal dose of isotope, the number of silver grains on labelled nuclei was over 30. Therefore it is reasonable to assume that there was enough 3H thymidine throughout the period from implantation to deposition of new cuticle, and would have been visualized if any DNA synthesis had occurred during this period. Even
52
in this experiment no 3H thymidine was incorporated although the implant secreted a pupal cuticle. Based on these observations we conclude that reprogramming in Galleria larval epidermal cells can take place in the absence of DNA synthesis and cell division. Lastly the results reported here have a bearing on the time of action of juvenile hormone (JH). It is generally assumed that JH acts on the target cells at the time of DNA synthesis and/or cell division (see Schneiderman et al., 1969; Sehnal et al., 1969). The greater sensitivity of injured epidermal cells to JH has been regarded as suggestive evidence in favor of the view that JH acts at the time of cell division. This is based on the fact that injury induces DNA synthesis and cell division in epidermal cells of lepidopteran pupae (see Krishnakumaran et al., 1967). But the present data show that juvenile hormone effects can be overcome even without a cell division thereby suggesting that Jtt action may also be unrelated to cell divi;ion, and the physiological basis of high JH sensitivity in injured epidermal cells remains to be clarified. ACKNOWLEDGMENTS The research was supported by funds from Marquette University. The authors thank Drs. W.F. Millington and K.J. Tautvydas for helpful comments on the manuscript and help with the English.
REFERENCES Beck, S.D.: Trans. Wis. Acad. Sci. Arts Lett. 49, 137--149 (1960). Bowers, B. and C.M. Williams: Biol. Bull. Woods Hole, Mass. 126, 205--219 (1964). Ebert, J.D.: In: Current Topics in Developmental Biology; eds. A.A. Moscona and A. Monroy (Academic Press, New York) Vol. 3, pp. xv--xxv (1968). Hackman, R.H.: In: Insect Physiology; ed. M. Rockstein (Academic Press, New York) Vol. 3, pp. 471--506 (1964). Holtzer, H., H. Weintraub and J. Bell: In: Biochemistry of Cell Differentiation. Proceedings of the 7th Meeting of Federation of European Biochemical Societies; eds. A. Monroy and R. Tsanev (Academic Press, New York) Vol. 24, pp. 41--53 (1972a). Holtzer, H., H. Weintraub, R. Mayne and B. Mochan: In: Current Topics in Developmental Biology; eds. A.A. Moscona and A. Monroy (Academic Press, New York) Vol. 7, pp. 229--256 (1972b). Kastern, W.H. and A. Krishnakumaran: J. Insect Physiol. (in press) (1974). Kato, Y.: J. Insect Physiol. 19, 495--504 (1973). Krishnakumaran, A.: Biol. Bull. Woods Hole, Mass. 142, 281--292 (1972). Krishnakumaran, A., S.J. Berry, H. Oberlander and H.A. Schneiderman: J. Insect Physiol. 13, 1--57 (1967). Lawrence, P.: J. Cell Sci. 3, 391--404 (1968). Madhavan, K. and H.A. Schneiderman: J. Insect Physiol. 14, 777--781 (1968). Miura, Y. and F. Wilt: J. Cell Biol. 48, 523--532 (1971). Oka, T. and R. Schimke: Science, Wash. 163, 83 (1969). Sehneiderman, H.A.: In: Biology and the Physical Sciences; ed. S. Devons (Columbia University Press, New York) pp. 186--208 (1969). Schneiderman, H.A. and L.I. Gilhert: Science, Wash. 143, 325--333 (1964).
53 Schneiderman, H.A., A. Krishnakumaran, P.J. Bryant and F. Sehnal: In: Proceedings, Symposium on Potentials in Crop Protection (New York State Agricultural Experiment Station, Geneva, New York) pp. 14--25 (1969). Sehnal, F. and V.J.A. Novak: Aeta Entomol. Bohemoslov. 66, 137--145 (1969). Selman, K. and F.C. Kafatos: Cell Differentiation 3, 81--94 (1974a). Selman, K. and F.C. Kafatos: J. Insect Physiol. 20, 513--518 (1974b). Weintraub, H., G. Campbell and H. Holtzer: J. Cell Biol. 50, 652--668 (1972) Wigglesworth, V.B.: J. Exp. Biol. 40, 231--245 (1963). Wigglesworth, V.B.: In: Homeostasis and Feedback Mechanisms: Symposium of the Society for Experimental Biology (Academic Press, New York) 18, 265--281 (1964). Williams, C.M.: Science, Wash. 148, 670 (1965).
REPROGRAMMING IN THE ABSENCE OF DNA SYNTHESIS IN G A L L E R I A L A R V A L EPIDERMIS
William H. K A S T E R N * and A. K R I S H N A K U M A R A N * *
Department of Biology, Marquelle University, Mihvaukce, Wisconsin 53233, U.S.A. A c c e p t e d 10 D e c e m b e r 1974
Larval e p i d e r m a l cells f r o m a day-1 p e n u l t i m a t e instar Galleria larva on i m p l a n t a t i o n into day-5 last instar larva m e t a m o r p h o s e and d e p o s i t a pupal cuticle at the same time as the h o s t pupates. DNA s y n t h e s i s in the i m p l a n t e d larval cells was m o n i t o r e d with 3 H-thymidine. Various regimens o f 3 H - t h y m i d i n e a p p l i c a t i o n were used and u n d e r no c o n d i t i o n s did t h e larval cells i n c o r p o r a t e label during the period f r o m i m p l a n t a t i o n to d e p o s i t i o n o f pupal cuticle. This suggests t h a t a wax m o t h larval e c t o d e r m cell can r e p r o g r a m its g e n o m e to secrete a pupal cuticle w i t h o u t a p r e c e d e n t cell division.
Following the original observations on chondrogenesis and myogenesis, several studies on chick oviduct (Oka et al., 1969), erythropoiesis (Weintraub et al., 1971; Miura et al., 1971) and pigmented retina cells (Holtzer et al., 1972a) have indicated a need for a critical or 'quantal' mitosis prior to an overt manifestation of the differentiated state of these cells. This evidence led to the hyp:)thesis that 'quantal' mitosis is different from proliferative mitosis in that, during this division cycle, genes hitherto nonfunctional can be turned on (Holtzer et al., 1972b). Furthermore, quantal mitosis has been regarded, on theoretical grounds, as a prerequisite for the expression of hitherto inactive genes (Ebert, 1968). In the course of postembryonic development of saturnid silkmoths, epidermal cells replicated their DNA prior to the metamorphic molts into a pupa and into an adult (Bowers et al., 1964; Krishnakumaran et al., 1967). Similarly, correlations between DNA synthesis and metamorphosis have been observed in Oncopeltus (Lawrence, 1968) and Galleria (Sehnal et al., 1969). And the effects of inhibitors of DNA synthesis and/or cell division on adult differentiation in saturnid moths (FudR, Williams, 1965; Mitomycin C, Krishnakumaran e t m . , 1967; Madhavan et al., 1968) have led to the suggestion that there is a requirement for DNA synthesis and cell divisions in preparation for reprogramming of insect epidermal cells (cf. Krishnakumaran et al., 1967; Schneiderman, 1969). * P r e s e n t address: D e p a r t m e n t o f Biology, Wesleyan University, M i d d l e t o w n , C o n n e c t i c u 06457. ** R e p r i n t r e q u e s t s m a y be addressed to A. K r i s h n a k u m a r a n .
46 However, the occurrence of DNA synthesis immediately preceding reprogramming does n o t establish causal association between the two events. This is particularly the case with insect larval epidermis because cell divisions in this tissue may be associated with growth and not reprogramming. For example, the old cuticle shed at each ecdysis is replaced by a larger one. It is not inconceivable that preparatory to ecdysis the epidermal cells divide and thereby provide a greater surface area. In this case the observed divisions are unrelated to reprogramming. Furthermore, in Rhodtzius Wigglesworth (1964) observed that cell divisions in abdominal epidermal cells are triggered by stretching caused by the blood meal. Hence it is likely that DNA synthesis (and by inference cell division) observed in the epidermal cells prior to molting and metamorphosis in insect larvae may be associated with stretching caused by growth. In order to test the possible causal association between division and reprogramming in insect epidermis we examined whether any experimental conditions would dissociate these two events viz. reprogramming and cell division. For this purpose small pieces of Galleria penultimate instar larval integument were cultured in vivo in the abdomen of a last instar larval host. Depending on the age of the host and of the donor, the implant secreted a larval or pupal cuticle when the host pupated (Kastern et al., 1974). Because of the in vivo culture conditions, the implanted epidermal cells are not subjected to the normal stretching pressures of intact larvae, and we determined the association between DNA synthesis and metamorphosis under these conditions. The results reported in this communication show that even in the absence of DNA synthesis penultimate larval epidermal cells cultured in vivo metamorphose and secrete a pupal cuticle thereby suggesting that the larval epidermal cells can reprogram their genome without prior DNA replication and cell division. Recently Selman et al. (1974a) also reported a similar situation in transdifferentiation of the cuticle-secreting labial glands into salt-secreting cells in silkmoth pupae in the absence of DNA synthesis. Even earlier Wigglesworth (1963) observed that unfed, decapitated 4th instar R h o dn iu s nymphs, when parabiosed to normal 5th instar nymphs, metamorphosed without any cell divisions in the epidermis, thereby suggesting that the same epidermal cells that secrete a nymphal cuticle can deposit an adult cuticle without undergoing cell division. MATERIALS AND METHODS Larvae of the waxmoth, Galleria mellonella, were reared in the laboratory according to procedures described earlier (Krishnakumaran, 1972). Penultimate and last instar larvae, recognized by their pigmentation and size of the head capsule (Beck, 1960) were used in this study and will be referred to in the text as penultimate and last larvae. The day of ecdysis into a stadium is designated as day 1 and after the subsequent 24 hr, day 2, etc. Normally the penultimate stadium lasted about 4 days and the last stadium 9 days. A
47 batch of newly ecdysed, last instar collected at one time usually ecdysed as pupae within 12 hr of one another. Surgical procedures f or excising the integument, its implantation into and retrieval from the host have been described in another c o m m u n i c a t i o n (Kastern et al., 1974). Autoradiographic procedures using Kodak NTB3 liquid emulsion are the same as those used earlier (Krishnakumaran et al., 1967). A cell was regarded as labelled if at least eight silver grains above background were c o n c e n t r a t e d on the nucleus. Control slides treated with DNase were also coated with photographic emulsion and this t r e a t m e n t removed over 60% of the grains in the h e m o c y t e s that were labelled. The schedule o f application of tritiated thymidine (methyl 2c/mM concentration 1 mc/ml New England Nuclear Co) is summarized in Table II. In one series different groups o f host larvae were injected with 3 H thymidine (20 /Jg/g; last larvae weigh between 80 and 250 mg each) on each of the 5 days f r o m implantation to their pupation, and the labelled larvae were sacrificed 6 or 24 hr after receiving the label. In the second series the larvae received repeated injections of 3H t h y m i d i n e at 6- or 24-hr intervals during the entire period from implantation to p~ !)ation. In the 3rd series a different batch of larvae were injected with 3H t hymi di ne at intervals of 3 hr commencing from the time of implantation to the time of pupation. The hosts were sacrificed 6 hr after receiving the isotope. Three animals were used for each o f the points and frequently 5 to 8 animals were used. At the end of the period of exposure to 3H thymidine the hosts were dissected and the implants were retrieved. Following their recovery the implants were fixed in cold Bouin's fluid for 18 hr and processed for autoradiography. At least 3 slides were prepared from each implant and from each slide 6--8 sections were examined. For purposes of comparison some of the host tissues were also processed for autoradiography in a few instances. H e m o c y t e s and some of the ot her host tissues t hat incorporated t hym i di ne exhibited high concentrations of silver grains over the nucleus. RESULTS
Developmental capacity of the penultimate larval epidermis In the first series of experiments we determined the developmental capacity of the day-1 penultimate larval integument when implanted into day 5, 6 or 7 last larvae. For this purpose a 1 m m 2 piece of integument was excised and washed in sterile Ringers solution and placed in the abdomen of an appropriate host. After the host pupated the implant was dissected out and the nature o f the cuticle secreted by it, if any, was determined. The results presented in Table I show that when the host larvae pupated the integument from the day-1 penultimate larva can deposit either a larval cuticle or pupal cuticle depending u p o n the age of the host. Whenever the implant form ed a
48 TABLE I Nature of the cuticle deposited by day-1 p e n u l t i m a t e instar larval epidermis when cultured in vivo in last instar larvae o f d i f f e r e n t ages.
Age of
Number of implants
N u m b e r depositing
the host*
that deposited cuticle
pupal cuticle**
Day-5 Day-6 Day-7
34 50 16
30 (88.2%) 20 (40%) 0 (0%)
* The day of ecdysis into a last larval s t a d i u m is designated as day-1. ** The others deposited a larval cuticle.
pupal cuticle, it rounded up into a closed vesicle with the epidermis on the outer surface and the cuticle facing in. However, when an implant secreted a larval cuticle it did not form a closed vesicle. Presumably the implant and host cells secrete the cuticle at the same time although specific time relations of cuticular syntheses have n o t been determined. The larval cuticle deposited by the implant possessed the characteristic surface plaques and larval bristles and was untanned as in the normal cuticle (Figs. 1 and 2). Similarly the pupal cuticle deposited by the implant resembles the normal pupal cuticle in the presence of a tanned amber colored exocuticle and a characteristic rough
Figs. 1 and 2. Larval cuticle secreted by a n o r m a l larval--larval m o l t ( tg. 1) c o m p a r e d with a larval cuticle deposited by integument from penultimate instar day-1 larva following i m p l a n t a t i o n into day-6 or day-7 last instar larva (Fig. 2). N o t e the similarity in the d i s t r i b u t i o n o f plaques and bristles.
49
Figs. 3 and 4. Pupal cuticle secreted during normal pupation (Fig. 3) compared with a pupal cuticle deposited by penultimate instar larval epidermal cells following implantation into day-4 or day-5 last instar larva (Fig. 4).
surface pattern (Figs. 3 and 4) t h e r e b y suggesting that the cuticle deposited by the implant reflects the state of differentiation of the epidermal cells.
DNA synthesis in the epidermal cells of the implant The fact that the implant can deposit either a larval or pupal cuticle depending on the age of the host afforded an o p p o r t u n i t y to determine whether a critical cell division is needed just prior to expression of the pupal characters. We monitoTed the e x t e n t of DNA synthesis in the epidermal cells of the implant using 3 H thymidine. Day-5 last larvae (host larvae) into which day-1 penultimate larval integument was implanted were injected with 3H t h y mid in e at d if f e r e nt times after implantation. After exposure to label for different lengths of time, the larvae were sacrificed. The implant was retrieved and processed for autoradiography. Details of the time schedules for injection and exposure to isotope and the results of the 3 H thymidine incorporation studies are presented in Table II, and these data show that the epidermal cells o f the implant do not incorporate ~ H t hym i di ne during the entire period from implantation to pupa t i on of the host. Thus, we infer that the epidermal cells of the implant do not engage in DNA synthesis and probably do n o t undergo divisions during this period. However if left in the host till it pupated the implanted integument deposited a pupal cuticle (see Table I). Despite the fact that a large percentage (75% or more) of the implants would have m e t a m o r p h o s e d and secreted a pupal cuticle, n o n e of the cells incorporated 3H thymidine, t hereby suggest-
50 TABLE II Monitoring of 3H thymidine incorporation into penultimate instar Galleria larval integument implanted into day-5 last instar larva and thus programmed to deposit a pupal cuticle.
Experiment
Labelling period
No. of injections
No. of insects used
Incorporation of label
1 2 3 4 5
24 6 6 24* 6*
1 1 1 5 12
12 16 74 7 7
0"* 0 0 0 0
* Interval between injections. The implants were labelled throughout the period from implantation to pupation. ** 0 = less than 5% of the cells were labeled. Exp. 1 and 2. 3H thymidine was injected into host insect on each of the days from the time of implantation till pupation of the host. In experiment I the insects were sacrificed 24 hr after injection of label and in experiment 2, 6 hr after injection. Exp. 3. Larvae into which penultimate larval integument was implanted were injected with 3H thymidine at 3-hr intervals during the first 72 hr after implantation and were sacrificed 6 hr after injection. In view of the fact that the cells in 7 day hosts do not incorporate label, the period beyond 48 hr following implantation was not monitored. Exp. 4 and 5. The same batch of insects received multiple injections at 24-hr (Exp. 4) or 6-hr (Exp. 5) intervals throughout the period from implantation to pupation. These implants did make a pupal cuticle.
ing t h a t even in the absence of D N A synthesis r e p r o g r a m m i n g of the epiderm a l cell g e n o m e c a n o c c u r . In a d d i t i o n to the absence of 3H t h y m i d i n e i n c o r p o r a t i o n into i m p l a n t s t h a t are p u t a t i v e l y p r o g r a m m e d t o s e c r e t e a p u p a l c u t i c l e , t h e d a t a ( T a b l e II, l i n e s 4 a n d 5) s h o w t h a t t h e i m p l a n t s , e x p o s e d t o 3 H t h y m i d i n e t h r o u g h o u t the period f r o m i m p l a n t a t i o n to p u p a t i o n of the host, secreted a p u p a l cuticle a n d y e t did n o t i n c o r p o r a t e isotope. In view of the fact t h a t D N A synthesis a n d r e p r o g r a m m i n g of the g e n o m e has been m o n i t o r e d w i t h i n the same tissue these data clearly suggest that the two events, r e p r o g r a m m i n g and D N A synthesis m a y be dissociated. DISCUSSION T h e r e s u l t s r e p o r t e d a b o v e c l e a r l y d e m o n s t r a t e t h a t t h e e p i d e r m a l cells o f a p e n u l t i m a t e i n s t a r larval Galleria c a n s y n t h e s i z e a p u p a l c u t i c l e w h e n i m p l a n t e d i n t o a n a p p r o p r i a t e h o s t a n d t h a t t h e s a m e cells t h a t s e c r e t e d a larval cuticle at the preceding m o l t d e p o s i t e d a p u p a l cuticle w i t h o u t replicating t h e i r D N A . T h u s t h e d a t a s u g g e s t t h a t t h e Galleria larval e p i d e r m a l cells c a n
51
reprogram their genome to secrete a pupal cuticle without an associated 'quantal mitosis'. Synthesis of a pupal cuticle by the larval epidermis is traditionally recognized as differentiation (see Schneiderman et al., 1964), since a pupal stage normally intervenes during development of an adult insect. Furthermore recent studies indicate that even under different experimental conditions a pupal stage cannot be by-passed at least in som5 of the lepidopterans e.g. the silkworms (Kato, 1973). Undoubtedly initiation of pupal cuticular synthesis requires turning on genes that were previously inactive as is evidenced by the differences in the chemistry and morphology of the pupal and larval cuticles. Unlike in the larval cuticle, the exocuticle constitutes a sizeable part of the pupal cuticle and its chemical composition requires sclerotin and quinones (cf. Hackman, 1964) not present in the larval cuticles. Since formation of the pupal cuticle requires turning on genes hitherto inactive, initiation of pupal syntheses by the larval epidermal cells may be reasonably regarded as reprogramming. In the absence of DNA synthesis during this reprogramming of the genome, it is reasonable to suggest that the insect larval epidermal cells can turn on new genes at any time during a cell cycle and reprogramming does not involve a 'quantal' mitosis. The absence of thymidine incorporation in over 95% of the cells of the implanted integument is not due to the presence of alternate paths of thymidine synthesis and/or high endogenous pools of thymidine because the host cells including epidermal cells do incorporate thymidine (see also Krishnakumaran, 1972). Similarly in view of the monitoring of DNA synthesis during the entire period from implantation into a host until after secretion of a pupal cuticle the failure to find any label is unlikely to be the result of missing the period of DNA synthesis. Furthermore in one of the experiments, 3H thymidine injections were given to different batches of larvae at 3-hr intervals, a period which is shorter than the estimated S period in the epidermal cells of another insect, Oncopeltus (Lawrence, 1968) and hence it is not likely that the methods of study did not permit visualization of DNA synthesis. Although in most of the implants DNA synthesis was not monitored in the same group of epidermal cells whose reprogramming is documented, in one experiment both isotope incorporation into DNA and reprogramming were studied in the same implants. In these experiments 3H thymidine was administered at 24- or 6-hr intervals which theoretically should make the isotope available continuously. In a recent study Selman et al. (1974b) estimated that about 1 0 ~ of the initial level of 3H thymidine persists in the h e m o l y m p h of Polyphemus 18 hr after injection. And our studies (not reported here) show that after injection of even 1/10th the normal dose of isotope, the number of silver grains on labelled nuclei was over 30. Therefore it is reasonable to assume that there was enough 3H thymidine throughout the period from implantation to deposition of new cuticle, and would have been visualized if any DNA synthesis had occurred during this period. Even
52
in this experiment no 3H thymidine was incorporated although the implant secreted a pupal cuticle. Based on these observations we conclude that reprogramming in Galleria larval epidermal cells can take place in the absence of DNA synthesis and cell division. Lastly the results reported here have a bearing on the time of action of juvenile hormone (JH). It is generally assumed that JH acts on the target cells at the time of DNA synthesis and/or cell division (see Schneiderman et al., 1969; Sehnal et al., 1969). The greater sensitivity of injured epidermal cells to JH has been regarded as suggestive evidence in favor of the view that JH acts at the time of cell division. This is based on the fact that injury induces DNA synthesis and cell division in epidermal cells of lepidopteran pupae (see Krishnakumaran et al., 1967). But the present data show that juvenile hormone effects can be overcome even without a cell division thereby suggesting that Jtt action may also be unrelated to cell divi;ion, and the physiological basis of high JH sensitivity in injured epidermal cells remains to be clarified. ACKNOWLEDGMENTS The research was supported by funds from Marquette University. The authors thank Drs. W.F. Millington and K.J. Tautvydas for helpful comments on the manuscript and help with the English.
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