Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Chromosoma (Berl.) 68, 205 227 (1978) CHROMOSOMA �9 by Springer-Verlag 1978

Patterns of Puffing Activity in the Salivary Gland Chromosomes of Drosophila IX. The Salivary and Prothoracic Gland Chromosomes of a Dominant Temperature Sensitive Lethal of D. melanogaster

Jeanette J. Holden 1 and Michael Ashburner

Department of Genetics, University of Cambridge, Cambridge CB2 3EH, England ; 1 present address: Department of Biology, Queen's University, Kingston, Ontario, Canada

Abstract.Heterozygotes for the dominant temperature sensitive mutant DTS- 3 of Drosophila melanogaster die as third instar larvae when grown at 29 ~ C. These larvae have grossly hypertrophied ring glands due to the growth of the prothoracic gland cells. The nuclei of the prothoracic glands possess polytene chromosomes which are as large as those of salivary gland nuclei. The banding pattern of these chromosomes is similar, if not identical, to that of salivary gland polytene chromosomes but the puffing patterns are quite different. The salivary gland chromosomes of DTS-3/+ larvae also hypertrophy and lack the normal ecdysone induced puffs. Both the prothoracic gland and salivary gland chromosomes of these mutant larvae react to ecdysone in vitro by the induction of similar puffs.

Introduction

Many larval tissues of Drosophila melanogaster possess polytene nuclei. Under normal circumstances the level of polyteny is sufficient only in the salivary glands of third instar larvae to permit detailed study of the polytene chromosomes, their banding and puffing patterns. The level of polyteny is normally a characteristic for each tissue type, though just how this control of chromosome replication is achieved is unknown. Both environmental and genetic factors may, however, affect the level of polyteny. The environmental factors include starvation and allowing extra time for development, by the transplantation of a tissue into an adult host (Hadorn et al., 1963). Genetic factors that influence the degree of polyteny include alleles at the giant (gt) locus, which increase it (Kaufman, 1972), and mutations at the lethal-giant larvae (l(2)gl) locus, which decrease it (Welch, 1957). One of us (J.H.) discovered that heterozygotes for the dominant temperature sensitive lethal DTS-3 of D.melanogaster possess, when grown at high temperature, a hypertrophied ring gland, the prothoracic

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206 J.J. Hotden and M. Ashburner

g l a n d cel ls o f w h i c h h a v e p o l y t e n e c h r o m o s o m e s w h i c h m a y b e as l a r g e as

t h o s e f o u n d i n wi ld t y p e s a l i v a r y g l a n d s . T h e p o l y t e n e c h r o m o s o m e s o f wi ld

t y p e p r o t h o r a c i c g l a n d s a re v e r y smal l . T h e l a r g e p o l y t e n e c h r o m o s o m e s o f t h e p r o t h o r a c i c g l a n d s o f D T S - 3 l a r v a e

o f f e r t h e o p p o r t u n i t y to c o m p a r e b o t h t h e b a n d i n g a n d p u f f i n g p a t t e r n s o f

t h e s e c h r o m o s o m e s w i t h t h o s e o f t h e s a l i v a r y g l a n d s . T h e p r o t h o r a c i c g l a n d

is t h e s i te o f s y n t h e s i s o f ~ - e c d y s o n e ( B o l l e n b a c h e r e t al . , 1976), t h e i m m e d i a t e

p r e c u r s o r o f t h e g r o w t h a n d m o u l t i n g h o r m o n e o f Drosoph i la , 2 0 - h y d r o x y e c d y -

sone . S ince t h e pu f f s o f t h e s a l i v a r y g l a n d s n o r m a l l y r e s p o n d v e r y d r a m a t i c a l l y

t o e x o g e n o u s e c d y s o n e (eg A s h b u r n e r 1972) i t is o f o b v i o u s i n t e r e s t t o see

w h e t h e r or n o t t h e c h r o m o s o m e s o f t h e t i s sue t h a t s y n t h e s i s e s t h i s h o r m o n e

c a n a l so r e s p o n d to it, a n d , i f so, in w h a t m a n n e r .

Materials and Methods

Stocks. The stock of l(3)3 ms (abbreviated DTS-3) was one in which the lethal was balanced against In(3LR)TM2, Ubx:3~ It was maintained, at 22~ on an " ins tant" Drosophila medium (Philip- Harris, Weston-Super-Mare, U.K.). To provide experimentaI material the DTS-3 stock was out- crossed to virgin females homozygous for an allele at the ebony locus (e H) and the cultures maintained on yeast-glucose medium at either 22~ or 29 ~ C. The DTS-3/e ~: larvae could be distinguished from their sibs heterozygous for the TM2 balancer chromosome by the colour of their spiracles (those TM2/e :~ being dark due to the e ~ allele of TM2).

The Canton-S stock used in some control experiments was from the Oak Ridge National Labo- ratory, the Oregon-R-369 from Dr. D. Suzuki's laboratory, University of British Columbia, Van- couver and the e tl stock from Dr. P.A. Lawrence, Laboratory for Molecular Biology, Cambridge.

Cytology. Temporary squash preparations of the salivary gland chromosomes were made by staining with lacto-orcein after hydrolysis in I N HC1 according to the schedule of Yoon et al., (1973). For the prothoracic gland chromosomes neither this method nor squashing in propionic- orcein after fixation in 3:1 ethanol:propionic acid gave satisfactory results. To obtain good preparations of the ring gland chromosomes it was necessary to change the lacto-acetic-orcein technique by increasing both the times of fixation and hydrolysis according to whether or not the tissues had been freshly dissected in 45% acetic acid or cultured in vitro. For fresh material fixation was for 15 see and hydrolysis for between 1 and 5 min; for in vitro cultured material these times were extended to I min and 15 mln respectively.

In vitro Culture. Both salivary glands and ring glands of third instar larvae were cultured in modified Grace's medium (Ashburner, 1972). All in vitro cultures of organs were done at 25 ~ C. 20-hydroxyecdysone was purchased from Rohto (Osaka) and used at a final concentration of 7.5 • 10-6 M: control cultures contained an equivalent amount of solvent (10% ethanol).

As a control for salivary gland cultures one lobe was fixed at the time of dissection and its sister lobe, together with the ring gland from the same larva, cultured. Ring glands were dissected free from the brain and cultured simultaneously with, but separately from, the salivary glands of the same animal. Salivary glands from 5 d old Canton-S larvae and both salivary glands and ring glands from 6 and 8 d old DTS-3/e ~: larvae were cultured for either 1 or 2.5 h. All larval ages are from the day of egg laying.

The DTS-3 Mutation. DTS-3 is an ethyl methane sulphonate induced dominant temperature sensitive mutation mapping to approximately 84.5 on chromosome arm 3R (Holden and Suzuki 1973). Homozygous DTS-3/DTS-3 animals die at all temperatures between 17 ~ and 30.5 ~ C. Heterozygotes, DTS-3/+, are fully viable at 22 ~ C, or below, but completely lethal at the restrictive temperature of 27 ~ C, or above. The time of death of the heterozygotes varies with the culture temperature:

Puffing Patterns in Salivary Gland Chromosomes of Drosophila. IX 207

at 29~ many survive until the third larval instar and may live for as long as 2 to 3 weeks after their non DTS-3 sibs have pupariated. Most heterozygotes that survive to the third larval instar die as larvae though a few (less than 1%) do form abnormal puparia, externally similar to those described as "pseudopupae" by Hadorn (1937) for homozygotes of the recessive l(2)gl.

Results

1. The Salivary Gland Chromosomes o f DTS-3 / e 11

At the permissive temperature of 22 ~ C the salivary gland polytene chromosomes of DTS-3 /e 11 larvae appear to be normal in both their general morphology and puffing patterns. When the larvae are grown at the restrictive temperature of 29 ~ C, however, both the morphology and puffing patterns of these chromosomes are very abnormal.

We examined the salivary gland chromosomes of 125 D TS-3 / e 1 ~ larvae grown at 29~ and aged between 6 and 14 d. In the 6 d old larvae chromosome morphology was not conspicuously abnormal. With increasing age there was a progressive increase in chromosome width and decrease in chromosome length so that, by 10 d or so, the chromosomes had a very abnormal appearance (Fig. 1 a, b). The X chromosome of the male larvae showed a different behaviour from the autosomes in that its contraction was excessive and it lost almost all indication of its normal banding pattern (Fig. 1 c).

When DTS-3 / e ~ larvae were grown at 29 ~ C their salivary gland polytene chromosomes failed to show the complex changes in puffing activity that characterise wild type larvae prior to, and after, pupar ium formation (see Becker, 1959; Ashburner, 1967; Zhimulev, 1974). In only a minority (24/125) of the larvae studied, was any sign of puffing visible at those sites which are normally the first to be active in the wild type ecdysone induced puffing sequence (i.e. 23E, 74EF, 75B). The few larvae which did possess small puffs at these sites were all aged 8 d or younger. Only one larva (aged 6 d) showed a pattern of puffs that had progressed beyond a very early puff stage: this exceptional animal had a pattern of puffing activity similar to that described as Puff Stage 6 by Becker (1959), that is to say had puffs active at 62E and 78D on chromosome arm 3L in addition to those at 74EF and 75B.

Before the release of ecdysone initiates the late larval puffing sequence the salivary gland chromosomes of wild type third instar larvae have large puffs at few sites. The most promiment of these " in t e rmou l t " puffs are at 3C, 25AC, 68C and 90BC and two of these, at least, code for polypeptides of the salivary gland secretion (Korge, 1975, 1977; Akam et al., 1978). All of these puffs, except that at 25AC, were as active in 6-8 d old D TS-3 / e 1~ larvae as in the wild type. As the mutant larvae age these puffs gradually decrease in size. The salivary glands of the mutant larvae contain secretion granules, as judged by their appearance after staining with periodic acid-Schiff.

With increasing age the polytene chromosomes of the mutant larvae show puffs at " a b n o r m a l " sites (Fig. la , b). The most prominent of these are at 25D and 97D. In contrast to the situation seen in some other late larval lethals

208 J.J. Holden and M. AshburneI

Fig. I a-e. The salivary gland chromosomes of DTS-3/e 11 larvae (at 29~ a The X-chromosome of a 13d old male larva b chromosome arm 2L, (13d); c chromosome arm 3R, (14d); the large abnormal puffs in 25D and 97D are arrowed in b and e respectively. (a • 1000; b, c • 1800)


(e.g. l(3)tl, van Breugel and Bos, 1969) puffs are not seen at the so called 'heat shock' sites (Ashburner, 1969) in D TS-3/e 11 larvae.

2. The Ring Gland Chromosomes of Wild Type Larvae

The ring gland of the higher Diptera is an organ composed of at least three quite different cell types. These are the cells of the corpus cardiacum, corpus allatum and of the prothoracic (or ecdysial) gland. The prothoracic gland and corpus allatum cells have polytene nuclei, the nuclei of the corpus cardiacum are presumed to be diploid. The degree of polyteny in the wild type prothoracic gland was estimated to be 64C by Welch (1957), certainly these chromosomes are too small in either Canton-S or Oregon-R-369 larvae (5-6 d) for any detailed analysis. In Figure 2a we illustrate a chromosome from a wild type prothoracic gland nucleus which can be identified as the end of chromosome arm 3R. However, we must emphasise that this nucleus was the "bes t " , cytologically speaking, we observed in the ring glands from 200 wild type larvae. The only facts that we could determine with any confidence concerning the puffing activities of this nucleus were the presence of a large puff on chromosome 4 and of puffs in region 94-95 of 3R. Growing wild type larvae at 18~ or 29~ does not improve their prothoracic gland polytene chromosomes.

3. The Prothoracic Gland Chromosomes of DTS-3 Larvae

The ring gland itself and the polytene chromosomes of the prothoracic gland cells of DTS-3/e 11 larvae grown at 22~ appear identical to those of wild type glands.

At a restrictive temperature of 29 ~ C, however, the ring gland of the mutant larvae is seen to be grossly hypertrophied. By 6 d the ring glands are as large as a hemisphere of the brain and distinctly yellowish in colour. Cell counts, from Feulgen stained whole mounts of mutant and wild type ring glands, show that the cell number of the prothoracic gland region is the same, about 40 in each case (in agreement with the cell number estimated by Korochkina and Nazarova, 1977). The ring gland hypertrophy appears to result entirely from an increase in size of the prothoracic cells, although abnormalities of the cardiacum and allatum cannot yet be ruled out (ultrastructural studies of the mutant ring gland are being carried out by A. Parducz and J. Gausz).

The hypertrophied prothoracic cells possess polytene chromosomes which, at their largest, rival those of a wild type salivary gland in size (Fig. 3). Even in those mutant larvae with t h e " best" chromosomes there is always considerable variation in chromosome size between cells. As judged by the degree of chromosome polyteny the prothoracic cells have attained their maximum size by 6d, no further increase in chromosome size occurs subsequently (at least to 14d). We would estimate that the largest mutant prothoracic cell chromosomes have undergone 3 or 4 replications of the 64C chromosomes that characterise the wild type.

210 J J . HoIden and M. Ashburner

Fig. 2a and b, The distal end of chromosome arm 3R in a prothoracic cell chromosomes of a wild type (Canton-S) larva (5 d at 25 ~ C) and b prothoracic gland cells of a D722-3/e 11 larva (8 d at 29 ~ C)


Fig. 3a and b. Whole polytene nuclei from a salivary gland, b prothoracic gland of 14 and 8d old DTS-3/e ~1 larvae (at 29~ respectively

The banding pattern of the mutant prothoracic cell chromosomes is, in its general features, the same as that of the salivary gland chromosomes and we have used the standard salivary gland chromosome maps (Bridges, 1935; Lefevre, 1976) to interpret these chromosomes. As we will discuss in detail below considerable differences in puffing activity are seen from a comparison of prothoracic gland and salivary gland chromosomes. These hamper any very detailed comparison of the banding patterns of these chromosomes, at least over long chromosome regions. However, whenever we have made detailed

212 J.J. Holden and M. Ashburner

]Fig. 4a and b. A detailed comparison of the banding patterns of the tip of chromosome arm 2L in a prothoracic gland and b salivary gland of 8 d old DTS-3/e 11 larvae

Puf f i ng P a t t e r n s in Sa l iva ry G l a n d C h r o m o s o m e s o f Drosophila. IX 213

T a b l e 1. A tist o f t he m a j o r pu f f s o f c h r o m o s o m e a r m 3L in wi ld t ype sa l iva ry g l a n d s (Sg) a n d D T S - 3 / e 1l p r o t h o r a c i c g l a n d (Ptg) c h r o m o s o m e s

L o c u s Sg P t g

62A3 .8 + + 62E + + 63A4 .7 + +

63E1.3 + + 63F + - -

6 5 D 1 . 3 - - + 66B + -

66E1.3 + + 6 7 A 5 . 9 - +

67B + - - 67F1 .2 + + 68C1 .4 - + 68C5 .7 + -

68F1 .3 - +

69A + -

69C - + 71B + - 7 1 D E + .-~-

7 2 D + ---

7 3 F / 7 4 A 1.5 - + 7 4 E F + +

75B + + 7 5 C D + +

7 8 D + -

Fig . 5 a and b. C o m p a f i son o f the puffs o f p o l y t e n e c h r o m o s o m e s o f D TS-3 /e 1 ~ (at 29 ~ C) l a rvae - d is ta l 2L. a Sa l iva ry g l a n d ( 6 d ) ; b p r o t h o r a c i c g l a n d (Sd) t r e a t ed wi th 2 0 - h y d r o x y e c d y s o n e f o r l h

in v i t ro

214 J.J. Holden and M. Ashburnei

Fig. 6a and b. Comparison of the puffs of polytene chromosomes of DTS-3/e la (at 29 ~ C) i a rvae - proximal 2R. a Salivary gland (7 d); b prothoracic gland (8 d)


Fig. 7 a and b. Comparison of the puffs of polytene chromosomes of DTS-3/e ~ ~ (at 29 ~ C) larva -distal 2R. a Salivary gland (6d); b prothoracic gland (6d), (a and b same larva)

banding pattern comparisons, such as that for the tip of 2L shown in Figure 4, no differences have been found and we can say that the banding pattern of the prothoracic gland chromosomes is similar, if not identical, to that of the salivary gland chromosomes.

The similarity, if not identity, of banding patterns of the prothoracic gland and salivary gland chromosomes contrasts strikingly with the quite different patterns of puffs active in these two tissues. We have studied the puffs of 90 D T S - 3 / e 11 larvae (grown at 29 ~ C) and aged between 6 and 14 d. As a general rule the pattern of puffs active in the prothoracic gland chromosomes of these larvae is constant over this period.

We will restrict our detailed comparison of these puffs to chromosome arm 3L since this will illustrate the general features of the pattern. The most prominent prothoracic gland chromosome puffs active on chromosome arm 3L of the mutant larvae are listed in Table 1. This table also indicates whether or not the "same' puff is active in the salivary gland chromosome of wild type larvae. We must mention one caution in interpreting this table f rom the point of view of tissue specific puffing activity. Whilst it is easy to establish non-identity of puffs, to unequivocally establish identity of two puffs located in the same general chromosome region is difficult. Ideally to do so would require that we know that the two puffs code for the same product when active in the two tissues. We are far from meeting this ideal and have to rely on the cytological evidence alone. This could be misleading, even were we always certain that the puffs were formed from the same band (since there may be more than one " g e n e " per band). In practice the degree of cytological resolution is not that precise. Puffs of the salivary and ring glands that we have given the same name appear to be f rom the same bands and have the same morphology. These are our operational criteria for " ident i ty" .

216 J,J. HoIden and M. Ashburner

Fig. 8a and b. Comparison of the puffs ofpolytene chromosomes of DTS-3/e 11 (at 29 ~ C) larvae --mid- dle of 3L. a Salivary gland (6 d), b prothoracic gland (8 d)

Three features of the prothoracic gland chromosome puffing patterns are evident. The first is that there are several puffs active in the prothoracic gland that are also active, at some time during development, in the salivary gland chromosomes of wild type larvae. The second is that even when this is so the combination of puffs active simultaneously in one tissue is quite unlike that seen in the other. The third feature is that many of the puffs are tissue specific, either to the prothoracic gland or to the salivary gland.

The tissue specific puffs are indicated in Table 1. The uniqueness of the pat tern of puffs active in the prothoracic gland can perhaps be best illustrated


Fig. 9a and b. Comparison of the puffs of polytene chromosomes of DTS-3/e :I (at 29 ~ C) larvae-proximal 3R. a Salivary gland (6 d), b prothoracic gland (8 d)


Fig. 10a and b. Comparison of the puffs of polytene chromosomes of DTS-3/e 1~ (at 29 ~ C) larvae- chromosome 4. a Salivary gland (6 d); b prothoracic gland (12 d). Note the nucleus, presumably diploid and perhaps from the corpus cardiacum in b and the c~-heterochromatin of the chromocentre

by considering only those puffs common to the salivary gland: In the prothoracic gland chromosomes large puffs are simultaneously active at, for example, 63E, 66E, 67F, 74EF, 75B and 75CD. There is no period during normal development when these puffs are simultaneously active in wild type salivary glands. Indeed there is good evidence to suggest that the factors that control the activity of some are mutually exclusive: for example in prepupae 63EF and 75CD are only active in the absence of ecdysone, whilst in both larvae and prepupae the activity of 74EF and 75B is absolutely dependant upon ecdysone being present (for review: Ashburner and Richards, 1976).

The puffing activities of chromosome regions in the prothoracic glands other than chromosome arm 3L are illustrated by Figures 5 to 10. Many large puffs are active in the prothoracic gland chromosomes and the general features, with respect to tissue specificity are similar to those described for 3L. We draw attention to the very large puff active on the 4th chromosome (Fig. 10) and to the fact, mentioned above, that this puff is also active in the prothoracic gland chromosomes of wild type larvae.

4. The Induction of Puffs by Ecdysone : the Salivary Glands

When wild type salivary glands, explanted from third instar larvae prior to the time of ecdysone release, are cultured in the presence of ecdysone the


Fig. l l a c. a and b Proximal chromosome arm 3L from salivary glands of DTS-3/e 11 larvae (at 29 ~ C) before a and after b in vitro culture in the presence of 20-hydroxyecdysone for 2.5 h. Note induction of puffs at 74EF, 75B and 78D by the hormone. For comparison, c is an ecdysone induced wild type (Canton-S} salivary gland chromosome a 8 d, b 6 d


Fig. 12a and b. The proximal region of chromosome arm 3L from prothoracic gIand cells of DTS-3/e 11 larvae (at 29 ~ C) before a and after b in vitro culture for 2.5 h in the presence of 20-hydroxyecdysone. Compare with Figure 11. See text. a 8 d, b 6 d

entire sequence of puffing activity that, during normal development, character- ises the period from 5-6 h before to 3-4 h after puparium formation is induced (Ashburner, 1972). Since, when grown at 29 ~ C, the salivary glands of DTS-3/e 11 larvae show only very slight signs of the early stages of this sequence we cultured these glands in the presence of ecdysone to determine whether or not they were capable of showing a full ecdysone response.

The salivary glands of 35 mutant animals were cultured for 1 or 2.5 h in the presence of ecdysone at 25 ~ C. The response of their chromosomes to the hormone was, with respect to the early ecdysone puffs, very similar to that of wild type chromosomes (Fig. 11 and 13). That is to say puffs were induced at 2B, 23E, 63F, 74EF and 75B. We conclude that the failure of the salivary gland chromosomes of DTS-3/e ~ larvae to show activity of these puffs in situ is not a consequence of either an abnormal salivary gland or of an abnormal chromosomal response to ecdysone. On the other contrary it would seem that the concentration of ecdysone is too low, in the mutant larvae, to elicit a normal puffing response.

5. The Induction of Pufjs by Ecdysone : The Prothoracic Glands

Ring glands, freed from the adhering brain and imaginal discs, of 35 6 or 8 d old DTS-3/e 11 (at 29 ~ C) animals were cultured with ecdysone for 1 or 2.5 h in order to determine the response of the prothoracic gland chromosomes to the hormone. Ring glands from 30 8 d old animals were cultured in the absence of ecdysone as a control of the culture conditions.

The prothoracic gland chromosomes show a dramatic response to ecdysone (Figures 12, 13 and 14). In general terms the effect of the hormone is to make


Fig. 13a-e. The distal end of chromosome arm 3L in prothoracic gland cells and salivary gland cells of DTS-3/e 11 larvae (at 29 ~ C) showing the effects of ecdysone, a and b prothoracic gland chromosomes of 12 and 8 d old larvae respectively, control; c the same region after 2.5 h in vitro culture in the presence of 20-hydroxyecdysone (6d old larva), d salivary gland chromosomes of 6 d old larva alter 2.5 h in vitro culture in the presence of 20-hydroxyecdysone and e the same region, control

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the puffing pattern of these chromosomes more similar to that of the salivary glands. That is to say some (63E, 67A, 65D, 73F/74A) but not all (66A, 67F, 69C) of the prothoracic gland puffs regress, either partially or fully, and puffs are induced, or enlarged, in the gland at the same sites as are induced by the hormone in salivary glands, including 2B, 23E, 62E, 63F, 74EF, 75B and 78D. The prothoracic gland chromosome puffs that regress under the influence of ecdysone include both puffs specific to this tissue and puffs active in common wild type salivary glands.

The chromosomes of control incubated ring glands do not show these changes in puffing activity.

Discussion

The salivary gland chromosomes of DTS-3 heterozygotes grown at 29 ~ C are not dissimilar in overall morphology to those seen in other late larval lethals of Drosophila (e.g. l(3)tl) or in the salivary glands of wild type larvae subjected to such environmental treatments as prolonged culture in an adult abdomen (Hadorn et at., 1963; van Breugel and Bos, 1969; Zhimulev et al., 1976). This syndrome, a progressive shortening and thickening of the chromosomes, is itself not a necessary consequence of a prolonged larval life since it is not seen in homozygotes for the mutation l(2)gl, which may live as long as the DTS-3/+ heterozygotes, nor in homozygous rosy larvae grown in the presence of toxic concentrations of purine (Ashburner, unpublished observations, t971), We do not know what factors are responsible for the hypertrophy of the chromosomes although Zhimulev et al. (1976) speculate that they are '~ with the drastic impairment of cell metabolic functions". We presume that, in the broad sense, this is true.

The reason why DTS-3/+ larvae fail to pupariate and continue development at the restrictive temperature is c l e a r - t h e y have insufficient ecdysone to do so. There are two lines of evidence that this is so. On the one hand, measurements of the amount of ecdysone extractable from DTS-3/+ larvae (aged 4 to 7 d), by radioimmune assay, show the level of 20-hydroxyecdysone to be only 5-10% that of wild type larvae at puparium formation (P. Maroy, personal communica- tion). On the other hand, if wild type ring glands are transplanted into DTS-3/+ larvae then the mutant larvae are corrected and will develop to adults even at 29 ~ C (Holden et al., in preparation). The low level of ecdysone in DTS-3/+ larvae is also indicated by the fact that only small ecdysone puffs are ever seen in them, indeed the size of 74EF and 75B in these larvae is fully consistent with an effective 20-hydroxyecdysone concentration of the order 5 x 10 ̀ -9 M, that is some 5% of the normal maximum in wild type (Ashburner, 1973; Borst et al., 1974; Hodgetts et al., 1977). This concentration is well below that necessary for the puffing patterns to progress beyond that stage characterised by small early puffs (Ashburner, 1973).

The DTS-3/+ larvae we examined for puffing activity were rather variable, the majority showed no sign of puffing at the early ecdysone puff sites, a


minority showing small early ecdysone puffs and one animal had late ecdysone puffs. These data suggest that the DTS-3 mutation is leaky at 29~ and that the mutation is not an amorph but a hypomorph.

The puffing patterns of the DTS-3/+ salivary gland chromosomes pose no great problems for interpretation. Except for a rather small puff at 25AC they are, in 6 to 8 d old animals, quite normal. That the intermoult puffs fail to regress and the ecdysone sensitive puffs fail to be induced (at least fully) are consequences of the lack, or low concentration, of ecdysone in the larvae. If the hormone is supplied to the glands exogenously then their chromosomes show a normal response. The appearance of puffs at abnormal sites in old DTS-3/+ larvae is no suprise in view of what must be very abnormal cellular conditions at that time.

The prothoracic gland chromosomes are much more interesting. It is to be emphasized that the banding patterns of these chromosomes are similar, if not identical, to those of wild type salivary gland chromosomes, confirming that, when comparisons of banding are made between larval tissues of the Diptera, at least, the pattern is constant (Beermann, 1950; Pavan and Breuer, 1952). In terms of their puffing activities, however, the prothoracic gland chromosomes are quite novel and the question arises as to whether or not they reflect the puffing activities of prothoracic gland chromosomes in wild type larvae. It is difficult to answer this question with any degree of confidence, although our limited observations of wild type prothoracic gland chromosomes suggest that the mutant and wild type are not too dissimilar. One possible way to overcome the difficult analysis of the wild type prothoracic gland chromosomes would be to assay, by in situ hybridisation to salivary gland chromosomes, the RNAs being made in the wild type glands, and to compare this pattern with that of the puffs in the mutant prothoracic gland. This would be a valuable experiment since it might give clues for a future analysis of the genetic control of ecdysone biosynthesis by the ring gland.

The puffs active in the mutant prothoracic gland suggest, on the whole, that the gland is not accumulating ecdysone or, if it is, then the hormone must be sequestered in a physiologically inactive way. The data relating to this statement are not entirely unequivocal since some of the puffs active in the prothoracic gland (e.g. 74EF) are known to be dependant upon ecdysone for their activity, at least in salivary glands. It is possible, of course, that the puff we score as 74EF in prothoracic glands is not the same as that we call 74EF in salivary glands.

It is the response of the prothoracic gland puffs to exogenous ecdysone, however, which most strongly leads us to the conclusion that the endogenous hormone levels cannnot be high in the mutant prothoracic gland. This response involves both the regression of specific puffs, such as 63E and 73F/74A and the activation (e.g. 2B, 23E, 62E, 63F, 78D) or enlargement (e.g. 74EF, 75B) of others. Overall, the general feature of the response of the prothoracic gland chromosomes to ecdysone is to make their puffing patterns rather similar to those of ecdysone stimulated salivary gland chromosomes.

There is an interesting parallel between the response of the prothoracic gland chromosomes and of prepupal salivary gland chromosomes to ecdysone


in the behaviour of the puff at 63E. In untreated prothoracic glands this is a prominent puff and it regresses when the glands are exposed to the hormone. Similarly in the 6-8 h prepupa the activity of this puff is repressed by ecdysone (Richards 1976a).

This comparison of puffing in prothoracic gland and salivary gland chromosomes has again shown that the puffs of two different tissues are overlapping sets, that is to say some puffs are common to both whilst others are tissue specific. In fact we have found a rather higher proport ion of tissue specific puffs than the only other detailed study of Drosophila tissues, that of Berendes (1965, 1966) of the salivary glands, Malpighian tubules and gut of D.hydei.

That the prothoracic gland responds to ecdysone, at the level of the puffs, as well as making at least the pro-hormone ~-ecdysone, is not great suprise. The ring gland of the Diptera and its homologue in the Hemiptera and Lepi- doptera is a larval tissue par excellance and histolyses at metamorphosis; this is a consequence of exposure to ecdysone at a time when the level of juvenile hormone is low (Wigglesworth, 1955). Indeed the similarity in puffing response to ecdysone of the salivary glands and ring glands may be a reflection of the similar developmental fates of these two tissues.

The prothoracic gland cells of DTS-3/+ larvae (at 29 ~ C) are the only cell type which is obviously hypertrophied. This must be, in general terms, a consequence of this tissue "escaping" the normal mechanisms that control its growth. This cannot be simply a matter of a low ecdysone titre acting on the gland itself since the prothoracic gland cells of l(2)gl are very small; (Scharrer and Hadorn, 1938; Aggarwal and King, 1969; Korochkina and Naza- rova, 1977). l(2)gl is presumed, on the basis of its partial correction by exogenous ecdysone (Karlson and Hanser, 1952; Richards, 1976b)0 also to be ecdysone deficient.

The simplest model is that prothoracic gland growth is controlled, via the brain, by the circulating ecdysone concentration. Sensing a low ecdysone concentration growth of the gland would be stimulated by the brain, perhaps by the same pathway as is used by the brain to bring about the release of hormone by the gland. In wild type larvae this stimulation would be inhibited by a feed back to the brain from ecdysone in the blood, in DTS-3/+ larvae, the absence of this negative feed back due to the inability of the gland to secrete ecdysone, could result in prothoracic gland hypertrophy.

Why should the prothoracic gland of I(2)gl homozygotes be so small? The phenotype of l(2)gl includes an extensive proliferation of the optic centres of the brain (Gateff and Schneiderman, 1974). This invasive proliferation could either destroy the brain's ability to respond to the haemolymph ecdysone or destroy the ability of the brain to signal growth of the prothoracic gland. On either model l(2)gl is expected to be epistatic to DTS-3/+ in the double mutant.

Acknowledgements. This work was supported by Grants, B/RG/74297 and GR/Al8993 from the Science Research Council, London to M.A. We thank Trevor Littlewood and Anne Tomsett for maintenance of Drosophila stocks and G. Richards and Jan Eeken for helpful discussions and comments on the manuscript of this paper.


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Received December 5, 1977/Accepted March 15, 1978 by W. Beermann Ready for press March 29, 1978

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

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