- Email: [email protected]
Nucleic acid synthesis in Rhynchosciara hollaenderi polytene chromosomes: I. Dose response and temporal sequence after injection of 20-hydroxy-ecdysone
Insect Biochem., Vol. 9, pp. 517 to 523. © Pergamon Press Ltd. 1979. Printed in Great Britain.
0020-1700/79/0901-0517
$02.00/0
NUCLEIC ACID SYNTHESIS IN RHYNCHOSCIARA HOLLAENDERI POLYTENE CHROMOSOMES: I. DOSE RESPONSE AND TEMPORAL SEQUENCE AFTER INJECTION OF 20-HYDROXY-ECDYSONE CATALINA LOURDES FRESQUEZ C.C.V.I. Incarnate Word College, San Antonio, TX 78209, U.S.A. (Received 20 March 1978; revised 19 March 1979)
Abstract--Injection of various doses of 20-hydroxyecdysone into 30-day old Rhynchosciara hollaenderi female larvae resulted in enhancement of DNA synthesis in salivary gland chromosomes with doses 0.002 /tg/mg or greater and in the induction of DNA puffs in the same tissue with doses of 0.01 #g/rag or greater. Higher incorporation of label at the DNA puff sites was observed only with doses 0.02/~g/mg or greater. In all cases, a lag period of 6-10 hr was observed before the response to the injected 20-hydroxyecdysone was detected. The number of cells responding and the length of time spent in synthesis were dose dependent. Malpighian tubules were refractory to 20-hydroxyecdysone. Key Word Index: 20-hydroxyecdysone, Rhynchosciara hollaenderi, salivary glands, Malpighian tubules, DNA synthesis, DNA puffs, dose dependent, temporal sequence
INTRODUCTION STUDIES on D N A synthesis in polytene chromosomes date back to the early sixties (GAY, 1963; KEYL and PELLING, 1963; PLAUT, 1963; GABRUSEWYCZ-GARClA, 1964); for others, see FRESQUEZ, 1976; BOSTOCK and SUMNER, 1978). In these investigations, synchronization of nuclear activity was assumed to have been achieved by using larvae of a specified chronological age. The observations of CROUSE (1968), CLARK (1970), STOCKER et al. (1973), and STOCRER and PAVAN (1974) that ecdysone or its derivative, 20-hydroxyecdysone, induced general D N A synthesis provided a new approach to the study of D N A synthesis in polytene chromosomes. The results presented in this ~vork deal with the temporal response of Rhynchosciara hollaenderi polytene chromosomes to several doses of injected 20hydroxyecdysone, noting the number of nuclei responding at various times after hormone treatment, and the effect on D N A puff formation. As has been shown by CLARK (1970) and STOCKER and PAVAN (1974), injection of 20-hydroxyecdysone into young fourth instar R. hollaenderii larvae induces the formation of puffs normally observed later in development. One of these, the puff in region 2 of chromosome B (BREUER and PAVAN,1955; STOCKER and PAVAN, 1974) is a D N A puff, i.e. a site of accumulation of D N A (FIcQ and PAVAN, 1957; RUDKIN and CORLETTE, 1957), and is the puff considered in this study.
MATERIALS AND METHODS Thirty-one day old (___ 2 days) Rhynchosciara hollaenderi female larvae were used. Larvae of this age normally have low levels of DNA synthesis (SIM6ES, 1967, 1970). The treated 517
larvae were given a 2 pl injection of 20-hydroxyecdysone and the control larvae were sham injected with an equal volume of the solvent (59/0ethanol). At various times after the sham or 20-hydroxyecdysone treatment, two to five larvae were each injected with 2 pl of tritiated thymidine and sacrificed 15 min later (see FRESQUEZ,1976 for injection method). Larvae were dissected in cold fixative (3:1, ethanol/acetic acid, v/v). Squash preparations were made of the salivary glands and Malpighian tubules. Only the proximal (S 1) and middle ($2) sections of the salivary glands (PAVAN, 1965; STOCKERand PAVAN, 1974) were used. The slides were later processed for autoradiography by coating them with a 1:1 solution of emulsion (NTB-2: distilled water). The emulsion was exposed to the radioactive tisstte for 5 days at 4°C and then developed for 2 rain in half-strength Dektol, rinsed in distilled water, fixed for 6 rain, and washed in running tap water for 15 rain. After this processing, the wet slides were stained for 30-60 sec with Unna's polychrome methylene blue and air-dried. Excessive stain was removed by passage through an alcoholto-water series. 20-Hydroxyecdysone (~-ecdysone), purchased from Rohto (Osaka, Japan), was dissolved in 5~ ethanol to make a stock solution. A Beckman Acta III spectrophotometer was used to measure the absorbance of the stock solution. Its exact concentration was determined from its absorbance at 242 nm, the absorbance maximum for 20-hydroxyecdysone, assuming an extinction coefficient of 12,400 and a molecular weight of 480 (HOFFMEISTER et al., 1965; HOCKS and WmCHERT, 1966). The desired dilutions were made from this concentrated solution (5.5/~g/pl, STOCKER,personal comm.). The effective concentration of a solution depends on the weight of the animal to be injected. Hence, rather than stating the concentration of the solution, we have reported the dose of the injected 20-hydroxyecdysone in terms of micrograms of 20-hydroxyecdysone injected per milligram of wet larval weight. [Methyl-H3]thymidine was purchased from Schwarz/Mann. Three different specific activities were used: 11 Ci/mmole; 16.7 Ci/mmole, and 21 Ci/mmole. The specific activity used in each experiment is given in the legend of the corresponding figure. NTB-2 emulsion, Dektol and fixer
518
CATALINA
LOURDES
were obtained from Eastman Kodak. Unna's polychrome methylene blue was purchased from Carolina Biological Supply Company. With the tritiated thymidine and the autoradiographic procedure used, labelling of chromosomes (nuclei) was intense. For the temporal sequences, label over the whole nuclei was scored. In chromosomes which were lightly labelled, close to a thousand grains could be counted in a single chromosome. The grain density observed in most of the chromosomes was such that individual grains were not discernible. Because of this high grain density, it was easy to score the chromosomes or nuclei as labelled or unlabelled without having to count grains. In chromosomes labelled extremely lightly and in the detailed analysis of B-2 puff formation and/or DNA synthesis, the criteria of HOWARD and PLAUT(1968) was applied,.i.e, the site was scored as being labelled if any band in the region had at least four grains above it in a tight cluster. The cases in which this criterion had to be applied occurred in less than 1~ of the chromosomes analyzed.
FRESQUEZ
/
~00
90
80
m J
m
7O
.o o
60
E 50 o
E o 2:
40
u
~ 3o
RESULTS
A. Dose response--temporal sequence In stydying the response of salivary gland chromosomes to different concentrations of injected 20-hydroxyecdysone in the haemolymph, several experiments were conducted. In one experiment, the larvae were 29 days old and had an average weight of 38 mg. Ninety-four larvae were injected with 0.0005 #g/mg of 20-hydroxyecdysone and 85 larvae were injected with 0.01 gg/mg; a comparable number of siblings were sham injected. At each sample point three to four larvae were each injected with 2 ~1 of tritiated thymidine and sacrificed 15 min later. As can be seen in Fig. 1 0.0005 #g/mg of 20hydroxyecdysone had no detectable effect on D N A synthesis in chromosomes from the proximal region of 80
7O
6O
~o 5o
~° ~ 3o
2oI ~0
0
tO
20
50
70
80
T i m e a f t e r treatment,
40
50
60
hr
90
I00
I I0
Fig. 1. Results of [methyl-H3]-thymidine incorporation in chromosomes from S] sections of salivary glands. Twentynine day old female larvae: average wet weight, 38 mg; sham or hormone treatment at 0 hr. Tritiated thymidine (spec. act. 21 Ci/mmole), 15 min, at times indicated (see Methods); three to four larvae/sample point, emulsion exposed to radioactivity for 5 days, 4°C. Sample points whose standard error (S.E.) is less than or equal to one, are shown as symbols without bar lines. Controls, © ©; 0.0005 gg/mg, I I - - I l l l ; 0.01 #g/mgA A.
I
I0
20
30
Time
40 offer
50
60
treatment,
i
I
I
70
80
90
hr
Fig. 3. Results of tritiated thymidine incorporation in chromosomes from S 1 sections of salivary glands. Thirty-day old larvae: average larval weight, 41 rag; sham or hormone treatment at 0 hr; DNA precursor (spec. act. I 1 Ci/mmole). 15 min, at times indicated; 4-5 larvae/sample point; emulsion exposed to radioactivity for 5 days, 4°C. Standard error not shown if less than or equal to one (See Fig. 1). Controls, O O; 0.002/~g/mg, A - - A ; 0.02 l~g/mg, I I 1 - - 1 1 . the salivary gland (S1). The results obtained with this dose were very similar to those of the control larvae. No effect different from the control was observable at either the 2 or 8 hr sample point after injection with the higher dose (0.01/~g/mg) but a very evident effect was observed in the 24 hr sample in which 56~o of the chromosomes showed label. This was in marked contrast to the samples taken at this time from larvae injected with the lower dose (0.0005/~g/mg) or sham injected, which showed only 12~o of their chromosomes labelled. The number of labelled chromosomes in larvae receiving the higher dose (0.01 #g/mg) decreased by 48 hr, but it was still higher than that shown by the other two samples. By 60 hr, and up to the termination of the experiment, chromosomes from all groups had less than 10~o label. The B-2 puffwas observed only at 36 hr (Fig. 2). The results of another experiment in which the treated larvae received either 0.002 or 0.02/~g/mg of 20-hydroxyecdysone are shown in Fig. 3. The sham injected larvae had less than 5~o of the chromosomes labelled throughout the period of observation. The lower dose, 0.002 #g/mg, induced a response in 2 8 ~ of the chromosomes by 18 hr which increased slightly after that, and dropped to I l ~ by 53 hr. The puff in B-2 was not observed at any of the sample points. The percentage of cells responding to the higher dose, 0.02 gg/mg, was 81% by 18 hr, 979/o by 30 hr, and maintained at this high frequency until 62 hr at which time it dropped down to 77%. By 72 hr the labelling in
Fig. 2. Labelhng in chromosomes B and X (S,) 36 hr after injection of tritiated thymidine. Conditions same as Fig. 1. Simple arrow indicates DNA puff in B-2. Double-barred arrow indicates intercalary heterochromatin. Exposure to emulsion, 5 days at 4°C. Stained with Unna’s polychrome methylene blue. Scale: 50 p. Fig. 4. Labelling in S, salivary gland chromosomes. Conditions same as Fig. 3. Double-barred arrow indicates B-2 puff. Simple arrows indicate two other DNA puff sites (B-3, C-3). Only part of chromosome A shown. Scale: 50 p.
521
DNA synthesis in Rhynchosciara
all three groups was less than 10%. Besides causing a greater increase in the per cent of chromosomes labelled, 0.02 pg/mg was also effective for a longer period of time. In addition, the B-2 puff was observed at 30 and 40 hr. Additional experiments (not shown) supported the observations reported here and also showed that the S, and S, sections of the salivary glands respond in a similar manner (FRESQUEZ, 1976). Contrary to what CROUSE(1968) observed in Sciara coprophifa, R. hollaenderi salivary gland chromosomes showed high incorporation of tritiated thymidine in the DNA puff regions only with doses 0.02 pg/mg or greater (Fig. 4). In all other cases, the grain density over the DNA puff site was equal to or less than that of the rest of the chromosome. B. Salivary glands compared with Malpighian tubules
In addition to the study of 20-hydroxyecdysoneinduced DNA synthesis in salivary gland chromosomes, a comparable study was carried out with Malpighian tubule chromosomes. In normal development at late fourth instar, the salivary gland chromosomes respond by forming DNA puffs. These puffs have never been observed in Malpighian tubule chromosomes. At this state of larval development, the titre of 20-hydroxyecdysone in the haemolymph is very high (WEIRICH, personal comm.). For this analysis, a dose of 0.03 pg/mg was used. This dose .is sufficient to induce intense DNA synthesis and the formation of the B-2 puff in salivary gland toor
a0 -
chromosomes of young fourth instar larvae. At the time of 20-hydroxyecdysone treatment, the larval group was 33 days old and the average larval weight was 42 mg. A total of 68 larvae were injected with 20hydroxyecdysone. Forty-five siblings were given a sham injection. For each of the sample points, four treated larvae and four nontreated larvae were injected with 2 ~1 of tritiated thymidine and sacrificed 15 min later.,The response of the S, section of the salivary glands and of the Malpighian tubules to this injection is shown in Figs. 5a and b. The S, section of the gland was chosen for the comparison because it responded earlier and more noticeably to the injected hormone than did the proximal region, S,. As mentioned previously the temporal sequence and degree of labelling observed in S, and S, after hormone treatment is very similar. As shown in Fig. Sa, the salivary gland nuclei had responded to the exogenous hormone by 6 hr (26% nuclei labelled) and a steady increase in the number of nuclei engaged in DNA synthesis was observed in the next five sample points. By 72 hr, 90°%labelling was observed. This was in sharp contrast to the 98 hr sample where synthesis had terminated in all but 4% of the nuclei. The number of nuclei incorporating the radioactive precursor in nontreated larvae ranged from 2 to 18% in the first 48 hr. This amount of variation is common during normal development @I&ES, 1967, 1970). The rise detected at 98 hr correlates with the increase observed during normal development in untreated 37-day old larvae. No detectable effect of 20-hydroxyecdysone on DNA synthesis in nuclei from Malpighian tubules was observed as indicated in Fig. Sb. The variation observed in nontreated and treated larvae was similar (5-21x compared with 4-18x). DNA synthesis and DNA puff formation were observed in salivary gland chromosomes, but neither response was observed in the Malpighian tubule chromosomes.
70 -
-0
2
60-
$ :
50-
25
0 4 z d0
40-
30
i
0
IO
20
30
40
Time after
50
60
treatment,
70
60
90
100
hr
Fig. 5a. Per cent labelling observed in S, nuclei. Thirty-three old larvae; average larval weight 42 mg; sham or hormone treatment at 0 hr. Tritiated thymidine (spec. act. 21 Ci/mmole), 15 min, at times indicated; 4-5 larvae/sample point. Emulsion exposed to radioactivity, 5 days, at 4°C. SE. not shown if less than or equal to one (see Fig. 1). Controls, A-A; 0.03 &ng, A-A.
0
IO
20
30
Time
40
ofter
50
60
treotment.
70
80
90
100
hr
Fig. 5b. Per cent labelling in Malpighian tubule nuclei. Conditions same as Fig. 5a. Data of Figs. 5a and 5b based on same larvae. Control, A-A; 0.03 pg/mg, A-&
522
CATALINA LOURDESFRESQUEZ
DISCUSSION Enhancement of DNA synthesis in insect cells in the presence of ecdysone was first observed by KRISHNAKUMARAN (1962). KRISHNAKUMARANet al. (1965) concluded from their studies on DNA synthesis in a moth that "an immediate response of most insect tissues to ecdysone is DN A synthesis..." DANIELIand RODINO (1967) working with Drosophila hydei questioned the assumption that the effect of ecdysone on DNA synthesis is primary and immediate. In their experiment, they showed that in Drosophila salivary glands, a rapid decrease in the synthetic activity of DNA occurred when the concentration of hormone in the haemolymph was high, although DNA synthesis did occur later. Our results in R. hollaenderi also indicated that the effect of 20-hydroxyecdysone on DNA synthesis was not immediate. This is not to say, however, that there is no immediate effect on nuclear activities. WYATT(1972), studying wing development of Hyalophora cactopia, observed a rise in both RNA and protein synthesis approximately 6 hr after injection of ecdysone. The 6-10 hr delay observed in R. hollaenderi between the. time of injection and the first indication of DNA synthesis suggests that other events precede the initiation of DNA synthesis. The system of hormone-induced DNA synthesis in salivary gland chromosomes of R. hollaenderi is presently being used to determine cytologically the relationship between DNA, RNA and protein synthesis. CROUSE (1968), after injecting ecdysone in Sciara coprophila, a member of the Sciaridae as is R. hollaenderi, concluded that the chromosomes showed high incorporation of DNA precursors mainly in the DNA puff regions. This high incorporation of tritiated thymidine may be observed also in R. hollaenderi. However, the results presented here showed that a separation can be made between induction of the puff and the extra synthesis of DNA. The puff in B-2, as well as general DNA synthesis, was induced in many cells by 0.01 and 0.02 pg/mg of hormone, but high incorporation of tritiated thymidine indicating extra synthesis of DNA at the specific chromosomal site was obtained only with doses of 0.02 #g/mg or greater. This dose dependency has not been demonstrated previously (CROUSE, 1968; STOCKERand PAVAN,1974). The data of KRISHNAKUMARANet al. (1965) suggest tissue specificity to ecdysone induced DNA synthesis in moths. We found that R. hollaenderi showed definite tissue specificity. The Malpighian tubule cells of R. hollaenderi showed no increase in DNA syntheis. This was in marked contrast to the response of the salivary gland chromosomes of the same larvae in which over 90~o of the chromosomes were labelled during a 4 day observation period. The tissue specificity observed in 20hydroxyecdysone-induced DNA synthesis in R. hollaenderi could be the result of specific membrane receptors in the cells of the salivary gland comparable to those found in target cells in mammals. However, the presence of a more efficient system for metabolizing 20-hydroxyecdysone in the Malpighian tubules cannot be excluded. If the latter is true, very high doses of exogenous hormone might be able to induce DNA synthesis and DNA puff formation in Malpighian tubule chromosomes. Alternately, the
Malpighian tubules may have been in their last replication cycle at the developmental stage which was used and were therefore incapable of responding to the hormone. The latter two possibilities will be the basis of future experiments. Acknowledgements--I would like to express my thanks to Professor C. PAVANof the University of S~o Paulo, Brazil, for reviewingthe manuscript, and to R. SANCHEZfor typing the manuscript. Research was made possible by a National Institutes of Health grant, PHS-GM 19331-03, to C. PAVAN and by a National Institute of General Medical Sciences grant, PHS 5T01 GM00337, and a fellowshipfrom the Ford Foundation to C. L. FRESQUEZ.This work was presented in partial fulfilment of Ph.D. requirements. During the preparation of the manuscript, the author was supported by a National Institutes of Health grant No. 1-S06-RR-08170-01.
REFERENCES BOSTOCKC. J. and SUMNERA. T. (1978) The Eukaryotic Chromosome. Ch. 7, North Holland, New York. BREUERM. E. and PAVANC. (1955) Behavior of polytene chromosomes of Rhynchosciara angelae at different stages of development. Chromosoma 7, 371-386. CLARKF. M. (1970) DNA synthesisin the fourth larval instar of Rhynchosciara and how it is affected by ecdysterone. Ph.D. dissertation, University of Tennessee. CROUSE H. V. (1968) The role of ecdysone in DNA puff formation and DNA synthesis in the polytene chromosomes of Sciara coprophila. Proc. natn. Acad. Sci., U.S.A. 61, 971-978. DANIELIG. A. and RODINOE. (1967) Larval moulting cycle and DNA synthesis in Drosophila hydei salivary glands. Nature, Lond. 213, 424-425. FICQA. and PAVANC. (1957) Autoradiography of polytene chromosomes of Rhynchosciara angelae at different stages of larval development. Nature, Lond. 180, 983-984. FRESQUEZ C. L. (1976) Nucleic acid synthesis in Rhynchosciara polytene chromosomes (Effect of ecdysterone). Ph.D. dissertation, The University of Texas at Austin. GABRUSEWYCZ-GARCIA N. (1964) Cytological and autoradiographic studies in Sciara coprophila salivary gland chromosomes. Chromosoma 15, 312-344. GAY H. (1963) Chromosomal structure and function. Carnegie Inst. Wash. Yearbook 62, 501-510. HOCKS P. and WIECHERTR. (1966) 20-Hydroxyecdyson, isoliert aus Insekten. Tetrahedron Lett. 26, 2989-2993. HOEFMEISTERH., RUFERC., KELLERH. H., SCHAIRERH. and
KARLSON P. (1965) Zur chemie des Ecdysons. III. Vergleichende Spektometrische Untersuchungen an ct, flunges/ittigten Steroidketonen. Chem. Bcr. 98, 2361-2375. HOWARD E. F. and PLAUT W. 0968) Chromosomal DNA synthesis in Drosophila melanogaster. J. Cell Biol. 39, 415-429. KEYL H. G. and PEELING C. (1963) Differentielle DNSReplikation in den Speicheldr/isen yon Chironomus thummi. Chromosoma 14, 347-359. KRISHNAKUMARAN A. (1962) Endocrine control of metabolism in arthropods. Ergebn. Biol. 79-92; 246-7. KRISHNAKUMARANA., OBERLANDERH. and SCHNEIDERMAN
H. A. (1965) Rates of DNA and RNA synthesis in various tissues during a larval moult cycle of Samia cynthia ricini (Lepidoptera). Nature, Lond. 205, 1131-1133. PAVANC. (1965) Synthesis.In Genetics Today, Ed. by GEERTS S. J. Vol. 2, pp. 335-342. Proceedings of the llth International Congress of Genetics, Pergamon Press, Oxford. PLAUTW. (1963) On the replicativeorganization of DNA in the polytene chromosome of Drosophila melanogaster. J. molec. Biol. 7, 632-635.
DNA synthesis in Rhynchosciara RUDKIN G. T. and CORLETTES. U (1957) Disproportionate synthesis of DNA in a polytene chromosome region. Proc. natn. Acad. Sci. U.S.A. 43, 964-968. SIM6ES L. C, G. (1967) Sintese de DNA durante o desenvolvimento larval de Rhynchosciara sp. Tese de Doutoramento apresentada ~ Cadeira de Biologia Geral do Instituto Central de Biologia da Universidade Federal de Minas Gerais, Beio Horizonte. S1M6ES L. C. G. (1970) Studies on DNA synthesis during larval development of Rhynchosciara sp. Revta. bras. Biol. 30, 191-199,
523
STOCKER A. J., FRESQUEZ C. and PAVAN C. (1973) Ecdysterone-induced gene amplification and general DNA synthesis in Rhynchosciara. Genetics. (Suppl.) 74, part 2, s266. STOCKER A. J. and PAVAN C. (1974) The influence of ecdysterone on gene amplification, DNA synthesis, and puff formation in the salivary gland chromosomes of Rhychosciara hollaenderi. Chromosoma 45, 295-319. WYATTG. R. (1972) Insect hormones. In Biochemical Actions of Hormones, Ed. by LITWACKG. Vol. III, pp. 386-490. Academic Press, New York.
0020-1700/79/0901-0517
$02.00/0
NUCLEIC ACID SYNTHESIS IN RHYNCHOSCIARA HOLLAENDERI POLYTENE CHROMOSOMES: I. DOSE RESPONSE AND TEMPORAL SEQUENCE AFTER INJECTION OF 20-HYDROXY-ECDYSONE CATALINA LOURDES FRESQUEZ C.C.V.I. Incarnate Word College, San Antonio, TX 78209, U.S.A. (Received 20 March 1978; revised 19 March 1979)
Abstract--Injection of various doses of 20-hydroxyecdysone into 30-day old Rhynchosciara hollaenderi female larvae resulted in enhancement of DNA synthesis in salivary gland chromosomes with doses 0.002 /tg/mg or greater and in the induction of DNA puffs in the same tissue with doses of 0.01 #g/rag or greater. Higher incorporation of label at the DNA puff sites was observed only with doses 0.02/~g/mg or greater. In all cases, a lag period of 6-10 hr was observed before the response to the injected 20-hydroxyecdysone was detected. The number of cells responding and the length of time spent in synthesis were dose dependent. Malpighian tubules were refractory to 20-hydroxyecdysone. Key Word Index: 20-hydroxyecdysone, Rhynchosciara hollaenderi, salivary glands, Malpighian tubules, DNA synthesis, DNA puffs, dose dependent, temporal sequence
INTRODUCTION STUDIES on D N A synthesis in polytene chromosomes date back to the early sixties (GAY, 1963; KEYL and PELLING, 1963; PLAUT, 1963; GABRUSEWYCZ-GARClA, 1964); for others, see FRESQUEZ, 1976; BOSTOCK and SUMNER, 1978). In these investigations, synchronization of nuclear activity was assumed to have been achieved by using larvae of a specified chronological age. The observations of CROUSE (1968), CLARK (1970), STOCKER et al. (1973), and STOCRER and PAVAN (1974) that ecdysone or its derivative, 20-hydroxyecdysone, induced general D N A synthesis provided a new approach to the study of D N A synthesis in polytene chromosomes. The results presented in this ~vork deal with the temporal response of Rhynchosciara hollaenderi polytene chromosomes to several doses of injected 20hydroxyecdysone, noting the number of nuclei responding at various times after hormone treatment, and the effect on D N A puff formation. As has been shown by CLARK (1970) and STOCKER and PAVAN (1974), injection of 20-hydroxyecdysone into young fourth instar R. hollaenderii larvae induces the formation of puffs normally observed later in development. One of these, the puff in region 2 of chromosome B (BREUER and PAVAN,1955; STOCKER and PAVAN, 1974) is a D N A puff, i.e. a site of accumulation of D N A (FIcQ and PAVAN, 1957; RUDKIN and CORLETTE, 1957), and is the puff considered in this study.
MATERIALS AND METHODS Thirty-one day old (___ 2 days) Rhynchosciara hollaenderi female larvae were used. Larvae of this age normally have low levels of DNA synthesis (SIM6ES, 1967, 1970). The treated 517
larvae were given a 2 pl injection of 20-hydroxyecdysone and the control larvae were sham injected with an equal volume of the solvent (59/0ethanol). At various times after the sham or 20-hydroxyecdysone treatment, two to five larvae were each injected with 2 pl of tritiated thymidine and sacrificed 15 min later (see FRESQUEZ,1976 for injection method). Larvae were dissected in cold fixative (3:1, ethanol/acetic acid, v/v). Squash preparations were made of the salivary glands and Malpighian tubules. Only the proximal (S 1) and middle ($2) sections of the salivary glands (PAVAN, 1965; STOCKERand PAVAN, 1974) were used. The slides were later processed for autoradiography by coating them with a 1:1 solution of emulsion (NTB-2: distilled water). The emulsion was exposed to the radioactive tisstte for 5 days at 4°C and then developed for 2 rain in half-strength Dektol, rinsed in distilled water, fixed for 6 rain, and washed in running tap water for 15 rain. After this processing, the wet slides were stained for 30-60 sec with Unna's polychrome methylene blue and air-dried. Excessive stain was removed by passage through an alcoholto-water series. 20-Hydroxyecdysone (~-ecdysone), purchased from Rohto (Osaka, Japan), was dissolved in 5~ ethanol to make a stock solution. A Beckman Acta III spectrophotometer was used to measure the absorbance of the stock solution. Its exact concentration was determined from its absorbance at 242 nm, the absorbance maximum for 20-hydroxyecdysone, assuming an extinction coefficient of 12,400 and a molecular weight of 480 (HOFFMEISTER et al., 1965; HOCKS and WmCHERT, 1966). The desired dilutions were made from this concentrated solution (5.5/~g/pl, STOCKER,personal comm.). The effective concentration of a solution depends on the weight of the animal to be injected. Hence, rather than stating the concentration of the solution, we have reported the dose of the injected 20-hydroxyecdysone in terms of micrograms of 20-hydroxyecdysone injected per milligram of wet larval weight. [Methyl-H3]thymidine was purchased from Schwarz/Mann. Three different specific activities were used: 11 Ci/mmole; 16.7 Ci/mmole, and 21 Ci/mmole. The specific activity used in each experiment is given in the legend of the corresponding figure. NTB-2 emulsion, Dektol and fixer
518
CATALINA
LOURDES
were obtained from Eastman Kodak. Unna's polychrome methylene blue was purchased from Carolina Biological Supply Company. With the tritiated thymidine and the autoradiographic procedure used, labelling of chromosomes (nuclei) was intense. For the temporal sequences, label over the whole nuclei was scored. In chromosomes which were lightly labelled, close to a thousand grains could be counted in a single chromosome. The grain density observed in most of the chromosomes was such that individual grains were not discernible. Because of this high grain density, it was easy to score the chromosomes or nuclei as labelled or unlabelled without having to count grains. In chromosomes labelled extremely lightly and in the detailed analysis of B-2 puff formation and/or DNA synthesis, the criteria of HOWARD and PLAUT(1968) was applied,.i.e, the site was scored as being labelled if any band in the region had at least four grains above it in a tight cluster. The cases in which this criterion had to be applied occurred in less than 1~ of the chromosomes analyzed.
FRESQUEZ
/
~00
90
80
m J
m
7O
.o o
60
E 50 o
E o 2:
40
u
~ 3o
RESULTS
A. Dose response--temporal sequence In stydying the response of salivary gland chromosomes to different concentrations of injected 20-hydroxyecdysone in the haemolymph, several experiments were conducted. In one experiment, the larvae were 29 days old and had an average weight of 38 mg. Ninety-four larvae were injected with 0.0005 #g/mg of 20-hydroxyecdysone and 85 larvae were injected with 0.01 gg/mg; a comparable number of siblings were sham injected. At each sample point three to four larvae were each injected with 2 ~1 of tritiated thymidine and sacrificed 15 min later. As can be seen in Fig. 1 0.0005 #g/mg of 20hydroxyecdysone had no detectable effect on D N A synthesis in chromosomes from the proximal region of 80
7O
6O
~o 5o
~° ~ 3o
2oI ~0
0
tO
20
50
70
80
T i m e a f t e r treatment,
40
50
60
hr
90
I00
I I0
Fig. 1. Results of [methyl-H3]-thymidine incorporation in chromosomes from S] sections of salivary glands. Twentynine day old female larvae: average wet weight, 38 mg; sham or hormone treatment at 0 hr. Tritiated thymidine (spec. act. 21 Ci/mmole), 15 min, at times indicated (see Methods); three to four larvae/sample point, emulsion exposed to radioactivity for 5 days, 4°C. Sample points whose standard error (S.E.) is less than or equal to one, are shown as symbols without bar lines. Controls, © ©; 0.0005 gg/mg, I I - - I l l l ; 0.01 #g/mgA A.
I
I0
20
30
Time
40 offer
50
60
treatment,
i
I
I
70
80
90
hr
Fig. 3. Results of tritiated thymidine incorporation in chromosomes from S 1 sections of salivary glands. Thirty-day old larvae: average larval weight, 41 rag; sham or hormone treatment at 0 hr; DNA precursor (spec. act. I 1 Ci/mmole). 15 min, at times indicated; 4-5 larvae/sample point; emulsion exposed to radioactivity for 5 days, 4°C. Standard error not shown if less than or equal to one (See Fig. 1). Controls, O O; 0.002/~g/mg, A - - A ; 0.02 l~g/mg, I I 1 - - 1 1 . the salivary gland (S1). The results obtained with this dose were very similar to those of the control larvae. No effect different from the control was observable at either the 2 or 8 hr sample point after injection with the higher dose (0.01/~g/mg) but a very evident effect was observed in the 24 hr sample in which 56~o of the chromosomes showed label. This was in marked contrast to the samples taken at this time from larvae injected with the lower dose (0.0005/~g/mg) or sham injected, which showed only 12~o of their chromosomes labelled. The number of labelled chromosomes in larvae receiving the higher dose (0.01 #g/mg) decreased by 48 hr, but it was still higher than that shown by the other two samples. By 60 hr, and up to the termination of the experiment, chromosomes from all groups had less than 10~o label. The B-2 puffwas observed only at 36 hr (Fig. 2). The results of another experiment in which the treated larvae received either 0.002 or 0.02/~g/mg of 20-hydroxyecdysone are shown in Fig. 3. The sham injected larvae had less than 5~o of the chromosomes labelled throughout the period of observation. The lower dose, 0.002 #g/mg, induced a response in 2 8 ~ of the chromosomes by 18 hr which increased slightly after that, and dropped to I l ~ by 53 hr. The puff in B-2 was not observed at any of the sample points. The percentage of cells responding to the higher dose, 0.02 gg/mg, was 81% by 18 hr, 979/o by 30 hr, and maintained at this high frequency until 62 hr at which time it dropped down to 77%. By 72 hr the labelling in
Fig. 2. Labelhng in chromosomes B and X (S,) 36 hr after injection of tritiated thymidine. Conditions same as Fig. 1. Simple arrow indicates DNA puff in B-2. Double-barred arrow indicates intercalary heterochromatin. Exposure to emulsion, 5 days at 4°C. Stained with Unna’s polychrome methylene blue. Scale: 50 p. Fig. 4. Labelling in S, salivary gland chromosomes. Conditions same as Fig. 3. Double-barred arrow indicates B-2 puff. Simple arrows indicate two other DNA puff sites (B-3, C-3). Only part of chromosome A shown. Scale: 50 p.
521
DNA synthesis in Rhynchosciara
all three groups was less than 10%. Besides causing a greater increase in the per cent of chromosomes labelled, 0.02 pg/mg was also effective for a longer period of time. In addition, the B-2 puff was observed at 30 and 40 hr. Additional experiments (not shown) supported the observations reported here and also showed that the S, and S, sections of the salivary glands respond in a similar manner (FRESQUEZ, 1976). Contrary to what CROUSE(1968) observed in Sciara coprophifa, R. hollaenderi salivary gland chromosomes showed high incorporation of tritiated thymidine in the DNA puff regions only with doses 0.02 pg/mg or greater (Fig. 4). In all other cases, the grain density over the DNA puff site was equal to or less than that of the rest of the chromosome. B. Salivary glands compared with Malpighian tubules
In addition to the study of 20-hydroxyecdysoneinduced DNA synthesis in salivary gland chromosomes, a comparable study was carried out with Malpighian tubule chromosomes. In normal development at late fourth instar, the salivary gland chromosomes respond by forming DNA puffs. These puffs have never been observed in Malpighian tubule chromosomes. At this state of larval development, the titre of 20-hydroxyecdysone in the haemolymph is very high (WEIRICH, personal comm.). For this analysis, a dose of 0.03 pg/mg was used. This dose .is sufficient to induce intense DNA synthesis and the formation of the B-2 puff in salivary gland toor
a0 -
chromosomes of young fourth instar larvae. At the time of 20-hydroxyecdysone treatment, the larval group was 33 days old and the average larval weight was 42 mg. A total of 68 larvae were injected with 20hydroxyecdysone. Forty-five siblings were given a sham injection. For each of the sample points, four treated larvae and four nontreated larvae were injected with 2 ~1 of tritiated thymidine and sacrificed 15 min later.,The response of the S, section of the salivary glands and of the Malpighian tubules to this injection is shown in Figs. 5a and b. The S, section of the gland was chosen for the comparison because it responded earlier and more noticeably to the injected hormone than did the proximal region, S,. As mentioned previously the temporal sequence and degree of labelling observed in S, and S, after hormone treatment is very similar. As shown in Fig. Sa, the salivary gland nuclei had responded to the exogenous hormone by 6 hr (26% nuclei labelled) and a steady increase in the number of nuclei engaged in DNA synthesis was observed in the next five sample points. By 72 hr, 90°%labelling was observed. This was in sharp contrast to the 98 hr sample where synthesis had terminated in all but 4% of the nuclei. The number of nuclei incorporating the radioactive precursor in nontreated larvae ranged from 2 to 18% in the first 48 hr. This amount of variation is common during normal development @I&ES, 1967, 1970). The rise detected at 98 hr correlates with the increase observed during normal development in untreated 37-day old larvae. No detectable effect of 20-hydroxyecdysone on DNA synthesis in nuclei from Malpighian tubules was observed as indicated in Fig. Sb. The variation observed in nontreated and treated larvae was similar (5-21x compared with 4-18x). DNA synthesis and DNA puff formation were observed in salivary gland chromosomes, but neither response was observed in the Malpighian tubule chromosomes.
70 -
-0
2
60-
$ :
50-
25
0 4 z d0
40-
30
i
0
IO
20
30
40
Time after
50
60
treatment,
70
60
90
100
hr
Fig. 5a. Per cent labelling observed in S, nuclei. Thirty-three old larvae; average larval weight 42 mg; sham or hormone treatment at 0 hr. Tritiated thymidine (spec. act. 21 Ci/mmole), 15 min, at times indicated; 4-5 larvae/sample point. Emulsion exposed to radioactivity, 5 days, at 4°C. SE. not shown if less than or equal to one (see Fig. 1). Controls, A-A; 0.03 &ng, A-A.
0
IO
20
30
Time
40
ofter
50
60
treotment.
70
80
90
100
hr
Fig. 5b. Per cent labelling in Malpighian tubule nuclei. Conditions same as Fig. 5a. Data of Figs. 5a and 5b based on same larvae. Control, A-A; 0.03 pg/mg, A-&
522
CATALINA LOURDESFRESQUEZ
DISCUSSION Enhancement of DNA synthesis in insect cells in the presence of ecdysone was first observed by KRISHNAKUMARAN (1962). KRISHNAKUMARANet al. (1965) concluded from their studies on DNA synthesis in a moth that "an immediate response of most insect tissues to ecdysone is DN A synthesis..." DANIELIand RODINO (1967) working with Drosophila hydei questioned the assumption that the effect of ecdysone on DNA synthesis is primary and immediate. In their experiment, they showed that in Drosophila salivary glands, a rapid decrease in the synthetic activity of DNA occurred when the concentration of hormone in the haemolymph was high, although DNA synthesis did occur later. Our results in R. hollaenderi also indicated that the effect of 20-hydroxyecdysone on DNA synthesis was not immediate. This is not to say, however, that there is no immediate effect on nuclear activities. WYATT(1972), studying wing development of Hyalophora cactopia, observed a rise in both RNA and protein synthesis approximately 6 hr after injection of ecdysone. The 6-10 hr delay observed in R. hollaenderi between the. time of injection and the first indication of DNA synthesis suggests that other events precede the initiation of DNA synthesis. The system of hormone-induced DNA synthesis in salivary gland chromosomes of R. hollaenderi is presently being used to determine cytologically the relationship between DNA, RNA and protein synthesis. CROUSE (1968), after injecting ecdysone in Sciara coprophila, a member of the Sciaridae as is R. hollaenderi, concluded that the chromosomes showed high incorporation of DNA precursors mainly in the DNA puff regions. This high incorporation of tritiated thymidine may be observed also in R. hollaenderi. However, the results presented here showed that a separation can be made between induction of the puff and the extra synthesis of DNA. The puff in B-2, as well as general DNA synthesis, was induced in many cells by 0.01 and 0.02 pg/mg of hormone, but high incorporation of tritiated thymidine indicating extra synthesis of DNA at the specific chromosomal site was obtained only with doses of 0.02 #g/mg or greater. This dose dependency has not been demonstrated previously (CROUSE, 1968; STOCKERand PAVAN,1974). The data of KRISHNAKUMARANet al. (1965) suggest tissue specificity to ecdysone induced DNA synthesis in moths. We found that R. hollaenderi showed definite tissue specificity. The Malpighian tubule cells of R. hollaenderi showed no increase in DNA syntheis. This was in marked contrast to the response of the salivary gland chromosomes of the same larvae in which over 90~o of the chromosomes were labelled during a 4 day observation period. The tissue specificity observed in 20hydroxyecdysone-induced DNA synthesis in R. hollaenderi could be the result of specific membrane receptors in the cells of the salivary gland comparable to those found in target cells in mammals. However, the presence of a more efficient system for metabolizing 20-hydroxyecdysone in the Malpighian tubules cannot be excluded. If the latter is true, very high doses of exogenous hormone might be able to induce DNA synthesis and DNA puff formation in Malpighian tubule chromosomes. Alternately, the
Malpighian tubules may have been in their last replication cycle at the developmental stage which was used and were therefore incapable of responding to the hormone. The latter two possibilities will be the basis of future experiments. Acknowledgements--I would like to express my thanks to Professor C. PAVANof the University of S~o Paulo, Brazil, for reviewingthe manuscript, and to R. SANCHEZfor typing the manuscript. Research was made possible by a National Institutes of Health grant, PHS-GM 19331-03, to C. PAVAN and by a National Institute of General Medical Sciences grant, PHS 5T01 GM00337, and a fellowshipfrom the Ford Foundation to C. L. FRESQUEZ.This work was presented in partial fulfilment of Ph.D. requirements. During the preparation of the manuscript, the author was supported by a National Institutes of Health grant No. 1-S06-RR-08170-01.
REFERENCES BOSTOCKC. J. and SUMNERA. T. (1978) The Eukaryotic Chromosome. Ch. 7, North Holland, New York. BREUERM. E. and PAVANC. (1955) Behavior of polytene chromosomes of Rhynchosciara angelae at different stages of development. Chromosoma 7, 371-386. CLARKF. M. (1970) DNA synthesisin the fourth larval instar of Rhynchosciara and how it is affected by ecdysterone. Ph.D. dissertation, University of Tennessee. CROUSE H. V. (1968) The role of ecdysone in DNA puff formation and DNA synthesis in the polytene chromosomes of Sciara coprophila. Proc. natn. Acad. Sci., U.S.A. 61, 971-978. DANIELIG. A. and RODINOE. (1967) Larval moulting cycle and DNA synthesis in Drosophila hydei salivary glands. Nature, Lond. 213, 424-425. FICQA. and PAVANC. (1957) Autoradiography of polytene chromosomes of Rhynchosciara angelae at different stages of larval development. Nature, Lond. 180, 983-984. FRESQUEZ C. L. (1976) Nucleic acid synthesis in Rhynchosciara polytene chromosomes (Effect of ecdysterone). Ph.D. dissertation, The University of Texas at Austin. GABRUSEWYCZ-GARCIA N. (1964) Cytological and autoradiographic studies in Sciara coprophila salivary gland chromosomes. Chromosoma 15, 312-344. GAY H. (1963) Chromosomal structure and function. Carnegie Inst. Wash. Yearbook 62, 501-510. HOCKS P. and WIECHERTR. (1966) 20-Hydroxyecdyson, isoliert aus Insekten. Tetrahedron Lett. 26, 2989-2993. HOEFMEISTERH., RUFERC., KELLERH. H., SCHAIRERH. and
KARLSON P. (1965) Zur chemie des Ecdysons. III. Vergleichende Spektometrische Untersuchungen an ct, flunges/ittigten Steroidketonen. Chem. Bcr. 98, 2361-2375. HOWARD E. F. and PLAUT W. 0968) Chromosomal DNA synthesis in Drosophila melanogaster. J. Cell Biol. 39, 415-429. KEYL H. G. and PEELING C. (1963) Differentielle DNSReplikation in den Speicheldr/isen yon Chironomus thummi. Chromosoma 14, 347-359. KRISHNAKUMARAN A. (1962) Endocrine control of metabolism in arthropods. Ergebn. Biol. 79-92; 246-7. KRISHNAKUMARANA., OBERLANDERH. and SCHNEIDERMAN
H. A. (1965) Rates of DNA and RNA synthesis in various tissues during a larval moult cycle of Samia cynthia ricini (Lepidoptera). Nature, Lond. 205, 1131-1133. PAVANC. (1965) Synthesis.In Genetics Today, Ed. by GEERTS S. J. Vol. 2, pp. 335-342. Proceedings of the llth International Congress of Genetics, Pergamon Press, Oxford. PLAUTW. (1963) On the replicativeorganization of DNA in the polytene chromosome of Drosophila melanogaster. J. molec. Biol. 7, 632-635.
DNA synthesis in Rhynchosciara RUDKIN G. T. and CORLETTES. U (1957) Disproportionate synthesis of DNA in a polytene chromosome region. Proc. natn. Acad. Sci. U.S.A. 43, 964-968. SIM6ES L. C, G. (1967) Sintese de DNA durante o desenvolvimento larval de Rhynchosciara sp. Tese de Doutoramento apresentada ~ Cadeira de Biologia Geral do Instituto Central de Biologia da Universidade Federal de Minas Gerais, Beio Horizonte. S1M6ES L. C. G. (1970) Studies on DNA synthesis during larval development of Rhynchosciara sp. Revta. bras. Biol. 30, 191-199,
523
STOCKER A. J., FRESQUEZ C. and PAVAN C. (1973) Ecdysterone-induced gene amplification and general DNA synthesis in Rhynchosciara. Genetics. (Suppl.) 74, part 2, s266. STOCKER A. J. and PAVAN C. (1974) The influence of ecdysterone on gene amplification, DNA synthesis, and puff formation in the salivary gland chromosomes of Rhychosciara hollaenderi. Chromosoma 45, 295-319. WYATTG. R. (1972) Insect hormones. In Biochemical Actions of Hormones, Ed. by LITWACKG. Vol. III, pp. 386-490. Academic Press, New York.