Developmental expression of Drosophila melanogaster small heat-shock

proteins

CHRISTIAN HAASS, ULRIKE KLEIN and PETER-M. KLOETZEL*

ZMBH/Molekulare Genetik, University of Heidelberg, Im Neuenheimer Feld 282, D-6900 Heidelberg, FRG

* Author for correspondence

Summary

We have investigated the developmental expressionof the small heat-shock proteins (hsps) during em-bryogenesis and in adult flies by immunocytologyusing an antibody that specifically identifies thesmall hsps. Antibody staining of unstressed earlyembryos reveals a predominantly cytoplasmic,homogeneous distribution of the small hsps through-out the embryo. At 6 h of development small hspexpression can be identified in large, neuroblast-likecells within the extended germ band and in the brainof the embryo. During germ band contraction thesecells appear to migrate to the midline where theyalign pairwise in a segmental pattern. After germband contraction is complete a high level of small hspexpression can be observed in the midline glia(MECs) and in a cluster of six non-neuronal cellswithin the midline. In contrast to several other genes

that are known to be important for embyogenesisand are expressed in the central nervous system(CNS) of embryos, CNS-specific expression of thesmall hsps is not restricted to the embryo but is alsoobserved in the adult fly. In adult flies strong smallhsp expression is observed in the brain, the thoracicganglion and the leg nerves. Since the small hspsseem to be expressed predominantly in the glia of thenervous system, our data suggest a protective orstabilizing function of the small hsps within thenervous system during normal fly development,which is independent of the stress response.

Key words: Drosophila, developmental expression, small heat-shock proteins, immunolocalization.

Introduction

The small heat-shock genes of Drosophila melanogasterthat encode the small heat-shock proteins, i.e. hsp27,hsp26, hsp23 and hsp22, belong to a particularly interest-ing gene family, since their activation involves complexregulatory mechanisms. The synthesis of the small hsps isinduced by environmental stress, by the molting hormone

For ecdysone induction cells were grown in the presence of 1 /(M a-ecdysone.

Isolation of16 S hsp complexesEcdysone-treated S3 culture cells were collected by centrifugationat 700 g for 5 min and washed twice in wash buffer (100 IDM NaCl,10 mM CaCl2, 10 mM Tris-HCl, pH7.2). For isolation of 16 Scomplexes the cells were broken by a 15 s cold shock at -20°C inmethanol/ice and subsequent resuspension on ice in a detergent-free extraction buffer containing 80 mM potassium acetate, 20 HIMHepes, pH7.5, 5mM magnesium acetate. The broken cells werecentrifuged in a Sorvall SS34 rotor at 0°C and 40 000gfor 10 min.The resulting 'cytoplasmic' sol-80 (Schuldt and Kloetzel, 1985)supernatant contains the small hsps in the form of 16 S com-plexes.

Purification of the small hsps and antibody productionSol-80 supernatant of S3 cells that were grown in the presence of1 /

Results

Pi-eparation of small hsp-specific antiserumPreviously, we (Haass et al. 1989) and others (Arrigo andPauli, 1988) have shown that in Drosophila the small hspsform globular 16 S complexes throughout developmentand that the formation of these complexes is independentof the mode of induction of the small hsps. For productionof antibodies directed against the small hsps we tookadvantage of the fact that the synthesis of the small hspsand hence the formation of 16 S complexes is stronglyinduced by or-ecdysone in Schneider's S3 tissue culturecells. The presence of cr-ecdysone for 48 h results in a largeincrease in hsp23 synthesis relative to the induction ofhsp27/26 (Fig. 1A). 16 S hsp complexes were isolated fromhormone-induced Schneider's S3 cells by sucrose-gradientcentrifugation followed by further purification on non-denaturing polyacrylamide gels (Haass et al. 1989; Haassand Kloetzel, unpublished). For the production of anti-bodies small hsp 16 S complexes, which containedhsp27/26 and hsp23, were electroeluted and the undena-tured proteins used for injection. The antiserum obtained(M16) was affinity purified against over-expressed hsp23.Its specificity was tested on total protein extracts ofhormone-treated S3 tissue culture cells and by immuno-fluorescence analysis (Fig. 1A,B,C). Although the proteingel is strongly overloaded, only hsp27, hsp26 and hsp23are identified by antibody M16 (Fig. 1A). The finding thatM16 identifies hsp27 and hsp26 as well as hsp23 is notsurprising, since the three small hsps are highly homolo-gous (Ingolia and Craig, 1982). On the other hand, anti-body M16 did not show any detectable cross-reaction withhsp22.

When antibody M16 is used for immunofluorescenceanalysis, a bright cytoplasmic fluorescence is seen in a-ecdysone-treated S3 cells while control cells give only aweak signal (Fig. 1B,C), which most likely is the result ofa small constitutive synthesis of the small hsps in S3 cells(Schuldt and Kloetzel, 1985). The immunoreaction couldbe competed with excess amounts of isolated small hsp,and in controls using preirnmune sera or secondary anti-body alone no staining was observed.

Expression ofhsp27/26 in embryonic tissuesImmunoblotting on embryonic protein extracts shows thatantibody M16 also identifies, besides hsp27 and hsp26,small amounts of hsp23 (Fig. ID). This in contrast to anearlier report that states that only the genes encodinghsp27 and hsp26 are transcribed during embryogenesis(Mason et al. 1984). Since the small hsps are organized andassembled as 16 S complexes throughout development,imrnunolocalization identifies the expression of hsp27/26and hsp23. Immunostaining in whole mounts was in allcases completely blocked when M16 antiserum was pre-incubated with excess amounts of isolated small hsps,supporting the specificity of the observed immunoreaction.

Immunocytological analysis of unstressed early em-bryos that are still transcriptionally inactive reveal ahomogeneous distribution of the small hsp proteinsthroughout the embryo (Fig. 2A). As already observed inS3 culture cells, the staining is almost exclusively cyto-plasmic. Upon cellularization the intensity of the immuno-signal is strongly reduced and restricted to the cortex ofthe embryo (Fig. 2B). Notably, no staining is observed inpole cells of the embryo. Towards the end of gastrulationthe small hsps can again be identified in the cytoplasm ofthe embryonic cells. No small hsps can be detected withinthe cells of the various morphogenetic furrows. In polecells, which during the blastula stage exhibit no small hspexpression, the small hsps can now be localized in thecytoplasm (Fig. 2C,D). Small hsp expression in pole cellsceases again once the pole cells have reached the pocket ofthe posterior midgut primordium (Fig. 2E). With continu-ing development, small hsp expression increases in theelongating germ band. At 6h of development small hspexpression can be identified in large neuroblast-like cellswithin the extended germ band (Fig. 2E). Correspondingto the number of body segments, there are 14 such cells,some of which appear to be in a dividing state. In additiona small number of neuroblast-like cells can be identified inthe brain of the embryo (Fig. 2E,F). During germ bandcontraction these cells appear to migrate to the midlinewhere they align pairwise in a segmental pattern(Fig. 2G). In fully developed embryos small hsp expressionis strictly CNS-specific (Figs 21, 3A). The expression of the

M 1 2A Mr

X10 - 3

* -27/26• • - 2 3

J3IJJ

DHx10 - 3

27/26-23-

Ecdysone Control

Fig. 1. Characterization of antibody M16. A. The affinity-purified antiserum M16 was tested by immunoblotting on total proteinextracts of 40000g supernatants of 48h a^-ecdysone-induced S3 tissue culture cells. M, molecular weight markers (xlO~31. Lane 1,Ponceau stain of a nitrocellulose filter with 100 ^g of total protein extract; lane 2, immunoblot with antiserum M16 on nitrocellulosefilter shown in lane 1. Only the a--ecdysone-induced small hsps are identified. B,C. Immunofluorescence analysis of 48 h a^ecdysone-induced S3 tissue culture cells (B) and control S3 tissue culture cells (C) grown under standard conditions using antibody M16. M16reveals a predominantly cytoplasmic localization of the small hsps in a--ecdysone-induced S3 cells. X4O0. D. The affinity-purifiedantiserum M16 was tested by immunoblotting on total protein extracts of 0-12 h old embryos prepared. M16 identifies hsp27/26 andinterestingly also small amounts of hsp23.

Drosophila heat-shock proteins 415

small hsps follows a segmental pattern and is exclusivelycytoplasmic. Within the CNS two clusters of cells in themidline show elevated small hsp expression (Fig. 3A-C).One cluster is a stretch of six cells that are alignedpairwise along the midline. As judged from comparisonwith data published by Jacobs et al. (1989) these cells mostlikely are identical with the midline glia (MECs). Theidentity of the other more rosette-like cell cluster, which islocated slightly posterior, is not entirely clear. These cellsmay represent other non-neuronal cells of the midline.

In situ hybridization of stage 14 embryos using hsp27and hsp26 gene probes also indicates a strong expression ofboth small hsp genes in cells of the nervous system,showing that the presence of the small hsps is due totranscriptional activity in these cells (Fig. 2J). At thisstage of development there is still some residual small hspexpression in other parts of the embryo (Fig. 2H).

To analyse whether the cell-specific expression of thesmall hsps is preserved under stress conditions embryoswere heat-shocked for l h at 35 °C and returned to 23 °C,growth temperature, for 2h to allow for maximal stress-induced synthesis of the small hsps. As shown in Fig. 4B,heat shock induces small hsp synthesis in the wholeembryo. Interestingly, there seems to be almost no markedincrease in small hsp expression in those tissues, i.e. thenervous system of the embryo, which express the smallhsps at elevated levels at normal growth temperature.

Expression of the small hsps in the adult flyThe experiments described above have shown that duringearly fly development the small hsps are strongly ex-pressed in cells of the CNS. To determine whether thisCNS-specific expression is restricted to embryogenesis,frozen sections of adult flies were analysed by immuno-cytology. Immunostaining of adult flies demonstrates thatthe nervous system-specific expression observed in theembryo also persists in the adult fly (Fig. 5A). In para-sagittal sections a strong expression of the small hsps canbe seen in the brain, the connective and the thoracicganglion, while the cardia, flight muscles and gut showonly low expression. Strong expression of the small hspsalso takes place in the leg nerve as revealed by immuno-staining and ire situ hybridization (Fig. 5B,C). Consideringthat axons have only an extremely low rate of transcrip-tional activity, if any, it seems reasonable to assume thatsmall hsp expression in adult flies, as in the embryo,predominantly takes place in non-neuronal support cellsor in the glia surrounding the neuronal cells. In agreementwith data on the transcription of the small hsp genes(Mason et al. 1984), a strong immunosignal is obtained inthe ovaries (Fig. 5A). Upon stress induction of small hspsynthesis most tissues that under normal growth con-ditions show only limited small hsp synthesis now expressthe small hsps to high levels (Fig. 5C). This is in particulartrue for the cardia, the flight muscles and the gut. On theother hand there seems to be no marked increase in smallhsp expression in the brain and the thoracic ganglion, i.e.tissues that express the small hsps at elevated levels atnormal growth temperature.

Discussion

With the aim of gaining more insight into the unresolvedfunction(s) of the small hsps we have determined theirsites of expression during embryogenesis and in adult fliesof Drosophila melanogaster. During early embryogenesis

Fig. 2. Expression of the small hsps during Drosophilaembryogenesis. Small hsp expression was analysed byimmunostaining using the affinity-purified antiserum M16. Theanterior end of the embryos is to the left, the posterior to theright. A. Stage 3 embryo: in syncytial blastoderm small hspsare located with the cytoplasm, no nuclear staining is observed.B. Stage 4 embryo: during cellularization small hsp expressionis strongly reduced. No staining is observed within the polecells (P) of the embryo. C. Stage 6 embryo: during earlygastrulation small hsps are again exclusively localized withinthe cytoplasm, cf, cephalic furrow. D. Stage 7 embryo: the polecells start to express the small hsps. atf, anterior transversalfold; ptf, posterior transversal fold. E. Stage 11 embryo: aftermigration of the pole cells (p) into the posterior midgutprimordium no small hsp expression can be observed. A strongexpression of small hsps can be observed within segmentallyorganized neuroblast like cells. F. Stage 12 embryo:identification of a neuroblast like cell cluster in the brain of theembryo (marked by an arrow). G. Stage 12 embryo (ventralview): small hsp-expressing neuroblast-like cells (arrows)migrate towards the ventral midline where they align pairwiseand segmentally repeated. H. Stage 14 embryo: small hspexpression becomes predominant in cells of the central nervoussystem (ens), b, brain. I. Stage 17 embryo: small hsp expressionis now restricted exclusively to the CNS; two clusters of cellsexpressing the hsps can be visualized. J. Stage 14 embryo: insitu hybridization using a probe specific for hsp27 mRNAtranscripts; transcription is predominant in the CNS; identicalresults were obtained using probes specific for hsp27 and hsp23transcripts (data not shown).

no cell or tissue specificity in small hsp expression can beobserved and as in a--ecdysone-induced Schneider's S-3cells, the small hsps are almost exclusively localized in thecytoplasm. The first cell-specific expression of the smallhsps can be observed after 6h of development in largeneuroblast-like cells within the nerve cord. These cellsmigrate medially during germ band shortening and alignin a segmental pattern along the midline. These cells mostlikely represent the precursors of the non-neuronal mid-line glia (MEC) (Jacobs et al. 1989). Within the midline acluster of six cells, located slightly posterior to the MECs,also exhibits expression of small hsps. The identity ofthese cells is not clear and they may in fact representanother type of non-neuronal support cell.

However, in contrast to many of the proteins that arefound to be expressed within the embryonic CNS, andwhich are essential for early development, small hspexpression is not restricted to the CNS of the embryo butalso takes place in cells of the nervous system of adultflies. In fact, together with the ovaries, the adult nervoussystem is the tissue that reveals the highest level of smallhsp expression. In situ hybridization indicates strongtranscriptional activity in the nervous system. Sinceaxons usually do not exhibit high levels of mRNA, thesmall hsps are most likely synthesized in the glia sur-rounding the neuronal cells. This conclusion is also com-patible with the observation that the vertebrate homol-ogues of the developmentally regulated Drosophila smallhsps, the afi-crystallins (Ingolia and Craig, 1982; Piati-gorsky and Wistow, 1989) are found to be expressed inastrocytes of the vertebrate brain (Iwaki et al. 1989). Thusthe expression of the thermodynamically stable class ofproteins within the nervous system seems to be a generalphenomenon. In brains of scrapie-infected hamsters(Duguid et al. 1988) and patients with Alexander's disease(Iwaki etal. 1989) a-B-crystallin synthesis is in factincreased, leading to the formation of aggregates of so-called afi-crystallin fibers. This is reminiscent of the stress

416 C. Haass et al.

Fig. 3. Expression of the small haps in the CNS. A. Stage 16embryo ventral view: small hsps are expressed in the CNS;strong expression is seen in MECs and cells of the midline(arrows). B. Stage 16 embryo ventral view: same as in A butdifferent level of focus, showing that the small hsps areexpressed in the MECs (upper arrow) and in a cluster ofseparate cells slightly posterior to the MECs (lower arrow).C. Stage 17 embryo: magnification of stage 17 embryo shown inFig. 21, showing the hsp expression within the two cells clustersof the CNS and their cytoplasmic localization.

Fig. 5. Analysis of small hsp expression in adult flies.A. Parasagittal section of an adult female. Immunostainingreveals a strong hsp expression in the brain, the thoracicganglion (tg) and the ovaries (ov). Small arrows indicate thespecific expression of the small hsps in the leg nerves.B. Freeze-fracture section through-the leg of an adult fly. In situhybridization on hsp27 mRNA using an in vitro transcribedantisense RNA probe. In, leg nerve; car, cardie; fm, flightmuscles; con, connective; st, stomach. C. Parasagittal section ofa heat-shocked adult female fly. Flies were heat shocked for 1 hat 35 °C and allowed to recover for 2h at 23 °C. Immunostainingindicates a strong expression of the small hsps in the hsps inthe cardia, the flight muscles and the gut.

*&>• • i ...

4A y*»..-

B

Fig. 4. Immunolocalization of small hsps in frozen tissue thinsections of stage 16 Drosophila melanogaster embryos withantiserum M16. Immunostaining reveals a strong expression ofthe small hsps in the central nervous system. A. Parasagittalsection of a non heat-shocked stage 16 embryo, phm,pharyngeal musculature; ph, pharynx; pv, proventriculus.B. Parasagittal section of a heat-shocked embryo. Embryos wereheat-shocked at 35 °C for 1 h and allowed to recover for 2 h at23°C. After heat shock the small hsps are strongly expressed inthe entire embryo.

situation in Drosophila, chicken and man (Haass et al.1989; Collier etal. 1988; Arrigo etal. 1988), where so-called stress granules (Nover et al. 1989) are formed by thesmall hsps.

As one would expect, heat shock results in a strongincrease in the immunosignal for the small hsps inembryos as well in adult flies, thus underlining the

specificity of the immunosignal obtained with antibodyM16. It is interesting to note, however, that under theheat-shock conditions used, which favour small hsp syn-thesis, certain tissues that under normal growth con-ditions possess high levels of small hsp synthesis reveal nodetectable increase in immunoreactivity. These data arein agreement with the observation that in pupae, wheredue to a^ecdysone induction all four hsps are synthesized,heat shock has little effect on the toal amount of smallhsps (Arrigo, 1987). Thus, besides a spatial and temporalregulation of small hsp expression during normal flydevelopment there also seems to exist a differential,tissue-specific response in small hsp gene activation dur-ing exposure to environmental stress. Since, independentof their mode of induction, the small hsps of Drosophilapossess identical molecular characteristics (Arrigo, 1987;Haass et al. 1989), it appears that the small hsps possesssimilar functions that are crucial for normal development,but which at an enhanced level are also required for stressresponse. Although there exists to date only limitedinsight into the function of the small hsps during normaldevelopment, there is some, though not entirely unequivo-cal, evidence (Petko and Lindquist, 1986; Hallberg et al.1985) from a variety of species, including Drosophila, thatthe small hsps may be involved in the acquisition ofthermal tolerance after heat shock and during normaldevelopment (Loomis and Wheeler, 1980; Li and Werb,1982; Nover etal. 1983; Berger and Woodward, 1983).Thus the expression of small hsps in non-neuronal cellssurrounding the nerve cells and the observation that theirvertebrate developmental homologues, the ofi-crystallinsare expressed in astrocytes indicate that the small hspfamily may serve supporting or protective functions thatare crucial for certain cells during normal development,but which at an enhanced level are also required forgeneral protection of the organism against environmentalstress.

The authors thank Professor E. K. F. Bautz for providingexcellent working conditions and for his interest in this work. Weare also indebted to Dr S. Hoffmeister and Dr I. Hermans-Borgmeyer for introduction to the freeze-sectioning technique andProfessor C. H. Schaller for permission to use the Leitz Cryostate.We thank Dr G. Petersen for helpful advice and discussion duringthe course of the work and Dr Mestril for the clone hsp27 XN-blue.This work was supported by the Deutsche Forschungsgemein-schaft SFB 229 (K1/C4) and the BMFT.

References

ARRIGO, A. P. (1987). Cellular localization of hap23 during Drosophiladevelopment and following subsequent heat shock. Devi Biol. 122,39-48.

ARRIGO, A. P. AND PAULI, D. (1988). Characterisation of hsp27 and threeimmunologically related polypeptides during Drosophila developmentExpl Cell Res. 175, 169-183.

ARRIOO, A. P., SUHAN, J. P. AND WELCH, W. L. (1988). Dynamic changesin the structure and intracellular locale of the mammalian lowmolecular weight heat shock protein. Molec. cell. Biol. 12, 6059-5071.

AYME, A. AND TISSIKRES, A. (1985). Locus 67B of Drosophiia melanogastercontains seven, not four, closely related heat shock genes. EMBO J. 4,2949-2954.

BKRGKR, E M. AND WOODWARD, M. P. (1983). Small heat shock proteinsin Drosophila may confer thermal tolerance. Expl Cell Res. 147,437-442.

CAMPOS-OETSGA, J. A. AND HARTENSTEIN, V. (1985). The EmbryonicDevelopment of Drosophila melanogaster. Springer-Verlag, Berlin,Heidelberg, New York.

COLLIER, N. C, HEUSER, J., AACH LEVY, M., SCHLESINGIR, M. (1988). Thedynamic stage of heat shock proteins in chicken fibroblasts. J. CellBiol. 106, 1131-1139.

Drosophila heat-shock proteins 417

DUGUID, J. R., ROHWER, R. G. AND SEED, B. (1988). Isolation of cDNAs ofscrapie-modulated RNAs by subtractive hybridization of a cDNAlibrary. Proc. natn. Acad. Sci. U.S.A. 85, 5738-5742.

GLASER, R. L., WOLFNER, M. F. AND LIS, J. T. (1986). Spatial andtemporal pattern of hsp26 expression during normal development.EMBO J. 5, 747-754.

HAFEN, E., LEVINE, R., GARBER, R. L. AND GEHRINO, W. J (1983). Animproved in situ hybridization method for the detection of cellularRNAs in Drosophila tissue sections and its application for localizingtranscripts of the homeotic antennapedia gene complex. EMBO J. 4,617-623.

HALLBERO, R. L., KRAUS, K. W. AND HALLBERG, E. M. (1985). Induction ofacquired thermotolerance in Tetrahymena thermophila: effects ofprotein synthesis inhibitor. Molec. cell. Biol. 5, 2061-2069.

HAASS, CH., FALKENBURG, P. E. AND KLOETZEL, P.-M. (1989). Themolecular organization of the small heat shock proteins in Drosophilamelanogaster. In Stress-induced Proteins UCLA Symposium onMolecular and Cellular Biology: New Series (ed. M. L. Pardue, J. R.Feramisco and S. Lindquist), vol. 96, pp. 175-185. Alan R. Liss, NewYork.

HAASS, CH. AND KLOETZEL, P.-M. (1989). The Drosophila proteasomeundergoes changes in its subunit pattern during development ExplCell Res. 180, 243-252.

INGHAM, P. W., HOWARD, K. R. AND ISH-HOROWITZ, D. (1985).Transcription pattern of Drosophila segmentation gene hairy. Nature318, 439-445.

INGOLIA, T D AND CRAIG, E. A. (1982). Four Drosophila small heatshock proteins are related to each other and to mammalian acrystallin. Proc. natn. Acad. Sci. U.S.A. 79, 2360-2364.

IRELAND, R. C. AND BERGER, E. (1982). Synthesis of the low molecularweight heat shock peptides stimulated by ecdysterone in a culturedDrosophila cell line. Proc. natn. Acad. Sci. U.S.A. 79, 855-859.

IWAKI, T., KUME-IWAKI, A., LlEM, R. K. H. AND GOLDMAN, J. E. (1989).aB-crystallin is expressed in non-lenticular tissues and accumulates inAlexander's disease brain. Cell 57, 71-78.

JACOBS, J. R., HIROMI, Y., PATEL, N. H. AND GOODMAN, C. S. (1989).Lineage, migration, and morphogenesis of longitudinal glia in theDrosophila CNS as revealed by a molecular lineage marker. Neuron 2,1625-1631.

LAEMMLI, U. K. (1970). Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 122, 680-685.

Li, G. C. AND WERB, Z. (1982). Correlation between synthesis of heatshock proteins and development of thermotolerance in Chinesehamster fibroblasts. Proc. natn. Acad. Sci. U.S.A. 79, 3218-3222.

LOOMIS, W. F. AND WHEELER, S. (1980). Heat shock response inDictyostelium. Devi Biol. 79, 399-408.

MASON, P. J., HALL, L. M. C. AND GAUSZ, J. (1984). The expression ofheat shock genes during normal development in Drosophilamelanogaster. Mol. Gen. Genet. 194, 73-78.

NOVER, L., SCHARF, K. D. AND NEUMANN, D. (1983). Formation ofcytoplasmic heat shock granules in tomato cells. Molec. cell. Biol. 3,1648-1655.

NOVER, L., SCHARF, K. D. AND NEUMANN, D. (1989). Cytoplasmic heatshock granules are formed from precursor particles and are associatedwith a specific set of mRNAs. Molec. cell. Biol. 9, 1298-1308.

PETKO, L. AND LINDQUIST, S. (1986). Hsp 26 is not required for growth athigh temperature, nor for thermotolerance, spore development andgermination. Cell 45, 885-894.

PIATIGORSKI, J. AND WISTOW, G. J. (1989). Enzyme/crystallins: genesharing as an evolutionary strategy. Cell 57, 197-199.

RUBIN, G. M. (1986). The Rubin Lab. Methods Book. Berkeley, CA.SCHNEIDER, E. (1972). Cell lines derived from late embryonic stages of

Drosophila melanogaster. J. Embryol. exp. Morph. 27, 353-365.SCHULDT, C. AND KLOETZEL, P.-M. (1985). Analysis of 19s ring-type

particles in Drosophila melanogaster which contain hsp23 at normalgrowth temperature. Devi Biol. 110, 65-74.

SHIELDS, G. AND SANG, J. H. (1977). Improved medium for culture ofDrosophila embryonic cells. Drosoph. Inf. Serv. 252, 161.

SIROTKIN, K. AND DAVIDSON, N. (1982). Developmentally regulatedtranscription from Drosophila melanogaster site 67B. Devi Biol. 89,196-210.

SIWICKI, K. K., EASTMAN, C, PETERSEN, G., ROSBASH, M. AND HALL, J. C.(1988). Antibodies to the period gene product of Drosophila revealdiverse tissue distribution and rhythmic changes in the visual system.Neuron 1, 141-150.

SOUTHGATE, R., AYME, A. AND VOELLMY, R. (1983). Nucleotide sequenceanalysis of the Drosophila small heat shock gene cluster at locus 67B.J. molec. Biol. 165, 35-57.

TAUTZ, D. AND PFEIFLE, C. (1989). A non-radioactive in situ hybridizationmethod for the localization of specific RNA's in Drosophila embryosreveals a translational control of the segmentation gene hunchback.Chromosoma 98, 81-85.

ZIMMERMAN, J. L., PETRI, W. AND MESELSON, M. (1983). Accumulation of

a specific subset of Drosophila melanogaster heat shock mRNA innormal development without heat shock. Cell 32, 1161-1170.

(.Received 16 February 1990 - Accepted 3 April 1990)

418 C. Haass et al.