Molecular and Cellular Endocrinology 1 (1974) 249-257. 0 North-Holland Publ. Comp.

GENE ACTIVATION FOLLOWING MICROINJECTION OF

j3-ECDYSONE INTO NUCLEI AND CYTOPLASM OF LARVAL

SALIVARY GLAND CELLS OF DROSOPHILA

T. BRADY, H. D. BERENDES and A. M. C. KUIJPERS

Department of Genetics, University of Nijmegen, The Netherlands

Received 14 February 1974 Accepted 4 April 1974

A comparison of the activity of chromosome region 478B (puff size) following (3-ecdysone injection into nuclei or cytoplasm of larval salivary glands of Drosophila hydei revealed that direct injection of the hormone into the nucleus resulted in a significantly smaller puff size (lower activity) than injection into the cytoplasm. Control experiments indicated that this lower response is not a consequence of damage of the nuclear membrane by the injection.

Following nuclear and cytoplasmic injection of 8-ecdysone into apical glands cells, region 4-78B becomes active in all cells of the gland. However, puff size decreases with increasing distance between the injected and measured cell nuclei.

The results are interpreted in terms of the necessity of a carrier to transport the steroid from the cytoplasm to the nucleus.

Keywords: puff induction; hormone action; ecdysone; Dipteran polytene chromosomes.

The steroid molting hormone ecdysone induces in vivo as well as in vitro

definite changes in the chromosomal puffing pattern of Dipteran polytenic

tissues (Clever and Karlson, 1960; Clever, 1966; Berendes, 1967; Ashburner,

1970, 1971). In spite of several hypotheses as to mechanism of action of ecdy-

sone, resulting in the initiation as well as in inhibition of transcription at certain

chromosome loci (Kroeger and Lezzi, 1966; Sekeris et al., 1965; Berendes, 1972;

Ashburner, 1973), there is little convincing evidence in support of any of these

hypotheses.

At certain stages of development, the larval salivary gland cells of Drosophila

incorporate labeled ecdysone and accumulate the hormone within the nucleus

(Emmerich, 1969; Claycomb et al., 1971).

Recently, indications have been obtained, suggesting that ecdysone is, at the

cytoplasmic and nuclear level, associated with protein(s) (Emmerich, 1970,

1972; Butterworth and Berendes, 1974). Moreover, the response of the genome

250 T. Brady et al.

to ecdysone is accompanied by migration of a particular polypeptide species

into the nucleus (Helmsing and Berendes, 1971; Helmsing 1972). On the basis

of these findings, it has been speculated that the steroid ecdysone may be

similar in its mechanism of action to mammalian steroid hormones in their

respective target cells (see Jensen and De Sombre, 1973). In other words, ecdy-

sone would require a protein transport vehicle to enter the nucleus and exert its

effect on the transcriptional control of certain genes. As such, this idea is

supported by the observation that isolated salivary gland nuclei of Drosophila,

when incubated in medium containing ecdysone, do not respond with pulf

formation at the hormone-specific loci, even though net nuclear RNA synthesis

is increased (Berendes and Boyd, 1969; Alonso, 1972). However, because these

experiments leave some doubt as to whether or not the isolated nuclei are still

able to respond adequately to the hormone, it seemed appropriate to rein-

vestigate this question by comparing the effect of direct injection of the hormone

into the nucleus and into the cytoplasm of intact salivary gland cells of Droso-

phila. At the same time, this approach allows the investigation of the inter-

cellular transport of the hormone.

MATERIAL AND METHODS

All experiments were performed with isolated mid third instar salivary glands

of Drosophila hydei raised under standardized conditions as described pre-

viously (Berendes, 1965).

Microinjection : injection needles were prepared from Pyrex capillary tubes

(interior diameter, f 1 mm) with a Narishighe microelectrode puller and

mounted on a deFonbrune micromanipulator. The tip of the needle (external

diameter: 3-4 u; interior diameter: 0.9 f 0.2 u) was, under microscopic obser-

vation, broken to provide a sharp point. The needle was connected with a

stationary, 2 ml glass syringe. The injection volume was controlled, using a

calibrated ocular micrometer. The volume injected into the nucleus or cyto-

plasm of a salivary gland cell varied between 6 and 12 x 1O-6 l.tl.

Following the dissection of the salivary glands from mid instar larvae, one

gland was fixed in ethanol-acetic acid (3 : 1) stained and squashed (see Berendes,

1965). This gland was used as a control to measure the diameter of region

4-78B, one of the chromosome sites which responds with formation of a puff

to ecdysone (Berendes, 1967). The other gland was immediately transferred to

a drop of Mg-Ringer (pH 7.2; Leenders et al., 1970) and fixed to a siliconized

microscope slide with plasticine. Following the injection of ecdysone into the

apical cells, the Ringer solution surrounding the gland was changed several

Gene activation by microinjection of ecdysone 251

times in order to remove ecdysone which might have been present on the out-

side of the needle.

In all instances two or three apical gland cells were injected. In order to

distinguish injected from control nuclei in squash preparations, a piece of one

of the chromosomes was pulled through the nuclear.membrane. This piece can

easily be recognized in the squashed chromosome preparations (fig. 1).

Fig. 1. Squash preparation of an injected nucleus showing a piece of chromosome (arrow) which was pulled through the nuclear membrane in order to enable recognition of the injected

nucleus. (Bar = 5 p)

When only the response of injected cells with that of cells of the control gland

had to be compared, a small apical section of the gland was processed (fixed,

stained and squashed).

For the study of cell-to-cell diffusion of the hormone, following injection of

the apical cells and subsequent incubation, the glands were divided into three

regions (the apical, central and proximal part) before processing.

The response of the genome to ecdysone was determinded by measuring the

diameter of region 4-78B in acetic-orcein stained and squashed chromosomes

(Berendes, 1967) and comparing the values obtained with those of the controls.

All experiments were performed with a solution of 100 ug/ml P-ecdysone in

Mg-Ringer (pH 7.2). Before injection the activity of the solution was tested by

incubating some mid third instar glands and assaying for the appearance of the

ecdysone specific puffs within a l-h incubation period.

T. Brady et a:. 252

RESULTS

As has been reported previously, in vitro incubation of Drosophilu hydei

salivary glands in salt media results in changes in the puffing pattern. However,

the chromosome sites at which these changes occur (Berendes, 1967) are not

identical with those responding to ecdysone.

The results of injections of ecdysone into the cytoplasm and nucleus of apical

RELATIVE

PUFF SIZE

P N=7

/ ” CVTOPLASM /

/

N=7

NUCLEUS

N=15

‘N-25 I I

, P ( CONTROL B

10 20 30 10 50

INCUBATION TIME IN MIN

Fig. 2. Puff size of region 4-78B in relation to the time of incubation following the injection of P-ecdysone into the nucleus or into the cytoplasm of apical gland cells. Control A and B:

see text. N = number of experiments performed; vertical bars: S.E.M.

salivary gland cells and subsequent incubation of the glands for lo,20 or 45 min

respectively, are presented in fig. 2. From the injections performed, only those

which gave a clear-cut and measurable response are included (fig. 3). In many

instances, the response of region 4-78B could not be measured exactly because

this area was situated under or above other chromosome regions in the squash

Gene activation by microinjection of ecdysone 253

Fig. 3. &Ecdysone injection into the cytoplasm of an apical salivary gland cell (a), with as a result the activation of region 4-78B (c). Region 4-78B was inactive in the sister gland (b).

The size of puff 4-788 as shown in c was attained 45 min after injection. (Bar = 5 p)

preparation. Only those measurements which were made on well stretched

chromosomes are included (fig. 3).

As can be seen, injection into both the nucleus and the cytoplasm results in

the activation of region 4-788. However, the response following nuclear injec-

254 T. Brady et al.

tion is at all time intervals after injection significantly smaller (P > 0.001) than

following injection of the hormone into the cytoplasm. Both, the ultimate puff

size and the kinetics of increase in puff size after injection of the hormone into

the cytoplasm are similar to that observed when whole glands are incubated in

a medium containing 8-ecdysone.

It could be argued that the smaller response of region 4-78B after injection

into the nucleus is due to damage caused by the microneedle. In order to test

whether or not the injection into the nucleus as such impairs the response at the

genome level, the following experiments were performed. Nuclei were injected

with 6-12 x 1O-6 ul Mg-Ringer and a similar quantity of Mg-Ringer containing

ecdysone was injected into the cytoplasm. At 20 min after the injection, the

relative puff size of region 478B was similar to that observed 20 min after

injection of cytoplasm without damaging the nuclear membrane (fig. 2, control

A). An essentially similar control experiment in which certain puffs were induced

by a temperature shock after injection of a Ringer solution into the nucleus,

gave the same result: the agent-specific puff sites became active (Berendes,

1971). It should be pointed out that the mere injection of Ringer solution into

the nucleus does not have any effect at region 4-78B, the relative diameter remain-

ing unaltered at 1.06 rt 0.04 (fig. 2, control B).

Although the results of these experiments render it unlikely that the smaller

response of puff 4-78B after injection of ecdysone into the nucleus as compared

with injection into the cytoplasm is due to damage of the nuclear membrane,

they do not exclude the possibility that the difference observed is due to an

‘overdose’ of steroid which may cause a suboptimal response (Poels, 1970).

However, injection of cytoplasm and nucleus of the same cell with the same

amount of ecdysone (6-12 x IF6 ul of the standard solution) did result in a

relative size of 1.64 for puff 4-78B after 20 min (1 experiment).

Because the question of whether or not there is a free exchange of the small

steroid molecule (M.W. 481) between the nucleus and the cytoplasm seemed to

be irucial for the interpretation of the data presented above, an attempt was

made to obtain some information about this exchange.

This information could be obtained indirectly by measuring the response of

cells adjacent to those injected provided that there is an efficient cell to cell

exchange of the hormone. In order to test this, two apical cells received an

ecdysone injection into the cytoplasm and the response of adjacent and more

distant cells was measured at 10, 20 and 45 min after injection. It was found

that after 45 min cells in different sections of the gland display almost identical

puff diameters of puff 4-78B. With shorter intervals between injection and

measurements of puff size (10 and 20 min), the responses observed in cells of

central and proximal gland sections are significantly smaller than in the distal

Gene activation by microinjection of ecdysone 2.55

part. Following a lo-min interval between injection and puff size measurement,

no significant increase of puff size could be observed in the proximal part of the

gland, whereas in the central section puff size was larger than in the controls

but smaller than in cells adjacent or close to the injected ones (apical section)

(fig. 4a).

REI

2,Ol

I,5

1,O

LA1 ‘IVE PUFF SIZE (INJECTION IN CVT~PLASM 1

i 65 min.

c

T

+ ?

T

f

t I

Z 20 min. I

t I

i

10 min.

I I I I DISTAL PART MIDDLE PART PROXIMAL PART

e.

[iNJEclI0N IN NUCLEUS]

, ’ , iOmin.1 ,

DISTAL PART MIDDLE PART PROXIMAL PART

LOCATION IN THE GLAND

Fig. 4. Puff sizes of region 4-78B in relation to the distance of the measured nuclei from the injected cell. Ecdysone was injected either into the nucleus or the cytoplasm of a cell of the distal (= apical) region. Following 10, 20 or 45 min of incubation, the gland was subdivided into three areas. The diameter of region 4-78B was measured in 4-5 nuclei of each gland area. The values shown are means of at least 20 nuclei (pooled values of 5 separate experiments).

Vertical bars: S.E.M.; A diameter of region 4-78B before injection.

A comparison of these data with those obtained following injection of the

nuclei of two apical cells reveals that the response of adjacent cells (apical part),

even after a 45min interval, is significantly lower if compared with the effect of

hormone injection into the cytoplasm (fig. 4b). Since it seems unlikely that cell

to cell exchange should be impaired, the finding of a lower response may

256 T. Brady et al.

indicate that the cytoplasmic concentration of ecdysone following injection

into the nucleus is lower than following injection into the cytoplasm. As such,

these data indicate that the nuclear-cytoplasmic exchange within a cell is less

efficient than cell to cell exchange.

DISCUSSION

From the data presented, it is obvious that injection of the steroid ecdysone

directly into the cell nucleus does induce the activity of the hormone-responsive

region 4-78B. However, the level of response is significantly smaller than after

injection of an equal quantity of hormone into the cytoplasm. This finding

together with the fact that injection of the nucleus as such does not impair the

genome response demonstrates that the injection of hormone into the cytoplasm

is more effective with regard to gene activation than a direct administration of

the steroid to the nucleus. These observations support the idea that a cyto-

plasmic factor may be required in order to elicit an adequate response at the

genome level. In fact, it cannot be excluded that the response of region 4-78B

observed after injection of ecdysone into the nucleus occurs as a consequence

of ecdysone molecules which have been lost from the nucleus after its injection,

become associated with a necessary cytoplasmic factor and entered the nucleus

again in a form appropriate for the induction of a specific response at the genome

level. As already outlined in the introduction the cytoplasmic factor may be

a protein required not only as a transport vehicle but also for the induction of

the specific response. So far, however, only circumstantial evidence for the

occurrence and necessity of such a protein factor(s) has been presented.

The significantly lower response of region 4-78B in adjacent and more

distant cells, when the effect of injection into the cytoplasm and into the nucleus

of two apical gland cells is compared indicates that the nuclear-cytoplasmic

exchange of the steroid is less efficient than the cell-to-cell exchange. The

obviously lower exchange rate could result from an association of the steroid

with nuclear protein(s) in a complex which is unable to pass the nuclear mem-

brane. On the other hand, the steroid could undergo chemical modification

within the nucleus such that its gene activating properties are lost. An efficient

exchange of these modified molecules would not be recognized by the method

applied because the assay is based on the effects at the genome level. If the

latter suggestion were correct, however, it would be difficult to explain why as a

result of increasing the time interval between injection of the nucleus and

observation of the response, the size of puff 4-78B increases even in the most

distant part of the gland. This finding at least indicates an increase with time in

Gene activation by microinjection of ecdysone 257

the availability of active steroid molecules for cell-to-cell exchange, even though

the level is by far not as high as after injection of the steroid into the cytoplasm.

Thus, it seems more likely that the steroid when injected directly into the nucleus

is reversibly fixed without losing its inductive properties. A non-specific fixation

of the steroid within the nucleus could inhibit its release from the nucleus. A

mechanism as such could also be operative when the combination steroid-

cytoplasmic (protein) factor enters the nucleus and becomes dissociated after

or during the completion of its function. The subsequent non-specific binding of

the free steroid would in this case inhibit its recycling.

Studies of the properties of ecdysone-binding proteins and of the steroid-

protein complexes at the cytoplasmic and nuclear level of dipteran salivary

gland cells should provide more detailed information as to the mechanisms of

ecdysone action proposed here.

REFERENCES

Alonso, C. (1972) Develop. Biol. 28, 372. Ashburner, M. (1970) Chromosoma 31, 356. Ashburner, M. (1971) Nature New Biol. 230, 222. Ashburner, M. (1973) Cold Spring Harbor Symp. Quant. Biol. 38 (in press). Berendes, H. D. (1965) Chromosoma 17, 35. Berendes, H. D. (1967) Chromosoma 22, 274. Berendes, H. D. (1971) In: Society of Experimental Biology Symposium, Vol. 25 (Cambridge

University Press) p. 145. Berendes, H. D. (1972) In: Results and Problems in Cell Differentiation, Ed.: W. Beermann

(Springer Verlag, Berlin, Heidelberg, New York) p. 181. Berendes, H. D. and Boyd, J. B. (1969) J. Cell Biol. 41, 591. Butterworth, F. M. and Berendes, H. D. (1974) J. Insect Physiol. (in press). Claycomb, W. C., LaFond, R. A. and Villee, C. A. (1971) Nature 234, 302. Clever, U. (1966) Develop. Biol. 14, 421. Clever, U. and Karlson, P. (1960) Exptl. Cell Res. 20, 623. Emmerich, H. (1969) Exptl. Cell Res. 58, 261. Emmerich, H. (1970) Z. Vergl. Physiol. 68, 385. Emmerich, H. (1972) J. Gen. Comp. Endocrinol. 19, 543. Helmsing, P. J. (1972) Cell Differentiation 1, 19. Helmsing, P. J. and Berendes, H. D. (1971) J. Cell Biol. 50, 893. Jensen, E. V. and DeSombre, E. R. (1973) Science 182, 126. Kroeger, H. and Lezzi, M. (1966) Ann. Rev. Entomol. 11, 64. Leenders, H. J., Wullems, G. J. and Berendes, H. D. (1970) Exptl. Cell Res. 63, 159. Poels, C. L. M. (1970) Develop. Biol. 23, 210. Sekeris, C. E., Dukes, P. P. and Schmidt, W. (1965) Hoppe Seyler’s Z. Physiol. Chemie 341,

152.