Hoch, M.

Pflanz, R.

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Gene expression patterndtrap-1 encodes a novel member of the heat shock super family of proteinsand is expressed in derivatives of all three germlayers during Drosophila embryogenesisRalf P¯anz, Michael Hoch1,*Max-Planck-Institut fuÈr Biophysikalische Chemie, Abteilung Molekulare Entwicklungsbiologie, Am Fassberg 11, D-37077 GoÈttingen, GermanyReceived 10 March 2000; received in revised form 9 May 2000; accepted 26 June 2000AbstractHeat shock proteins (Hsps) comprise a highly conserved superfamily of proteins that are required for stress tolerance in living cells. Atphysiological conditions, these proteins act as chaperones during protein folding and protein assembly processes (Parsell and Lindquist,1993. Annu. Rev. Genet. 27, 427±496). Members of the subfamily of Hsp90 proteins were shown to be additionally involved in thepresentation and structural modi®cation of components of diverse cellular signal transduction pathways including steroid hormone receptionand regulatory kinase activities (Pratt, 1993. J. Biol. Chem. 268, 21455±21458); Cutforth et al., 1994. Cell 77, 1027±1036; van der Straten etal., 1997. EMBO J. 16, 1961±1997; Hunter and Poon, 1997. Trends Cell Biol. 7, 157±161). We have identi®ed a Drosophila gene, calleddtrap-1, which encodes a Hsp of a novel subfamily that is related to the Hsp90 family of proteins. During oogenesis dtrap-1 is expressed innurse cells and its transcripts accumulate in the oocyte. The maternal transcripts remain in the egg but rapidly degrade during earlyembryogenesis, except in the posterior pole region. Zygotic expression is initiated after the onset of gastrulation showing dynamic patternsof transcripts in the developing mid- and hindgut as well as a subset of mesoderm derivatives. q 2000 Elsevier Science Ireland Ltd. All rightsreserved.Keywords: Hsp90; Chaperones; Organogenesis; dtrap-11. ResultsMembers of the Hsp 90 family of proteins are evolution-ary conserved from bacteria to humans (Parsell and Lind-quist, 1993). They act as abundant molecular chaperonesboth in the cytosol and in the endoplasmatic reticulum.Human Hsp90A, the prototype member of this family,was shown to facilitate structural changes and presentationof key regulatory components of certain signal transductionpathways such as steroid receptors, kinases and p53, actingin concert with a number of speci®c partner proteins(Prodromou et al., 1997a,b). Chaperone function is ATP-dependent, mediated by an ATP binding site within thehighly conserved aminoterminal region of the Hsp (Panar-etou et al., 1998). Further analysis has shown that Hsp90possesses two chaperone sites located in the amino- andcarboxyterminal regions, respectively (Scheibel et al.,1998). The aminoterminal fragment binds preferentially tonon-native peptides and prevents aggregation whereas thecarboxyterminal region recognizes structured substrates andcochaperones with tetratricopeptide repeats (Owens-Grilloet al., 1996). These functional domains of human Hsp90 areconserved in its Drosophila homolog Hsp83 (Cutforth andRubin, 1994).During the analysis of a lethal P-element insertion,l(2)04535 (Karpen and Spradling, 1992), which resideswithin the second intron of the Vha16 (also called ductin;Finbow et al., 1994) transcript in region 42C on the rightarm of the second chromosome, we noticed an open readingframe 5 0 to the Vha16 transcription unit. We used a genomicDNA fragment of the newly identi®ed open reading frameas a molecular probe to isolate a total of 9 overlappingcDNA clones from an embryonic cDNA library. Sequenceanalysis of these clones and the corresponding genomicDNA fragment revealed a transcription unit composed ofthree exons. The physical map of the transcription unit, theP-element insertion site as well as the Vha16 transcriptionunit are shown in Fig. 1A.The newly identi®ed transcription unit contains a singleopen reading frame, also represented in the LD14417 cDNAMechanisms of Development 96 (2000) 219±2220925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved.PII: S0925-4773(00)00403-2www.elsevier.com/locate/modo* Corresponding author.E-mail address: m.hoch@uni-bonn.de (M. Hoch)1 Present address: Institut fuÈr Zoologie, Abteilung Entwicklungsbiologie,UniversitaÈt Bonn, RoÈmerstrasse 164, 53117 Bonn, Germany.obtained from the Berkeley Drosophila Genome Project.Sequencing revealed that this single open reading framecould be conceptually translated into a polypeptide of 691amino acid residues which shows a high degree of similarityto the human Trap-1 protein (Song et al., 1995), establishingthat we have identi®ed the putative Drosophila homologreferred to as dTrap-1. The dtrap-1 gene is identical to therecently identi®ed Trap1dr isolated by Felts and coworkers(Felts et al., 2000), that gives rise to a protein (accessionnumber AF115775) for which chaperone-like propertiescould not be shown. However, it accumulates in mitochon-dria and has putatively functions different from those ofHsp83 (Felts et al., 2000). Nevertheless, the degree ofsequence similarity between human Trap-1, dTrap-1 andthe founding member of the Hsp 90 family of proteins isstriking (Fig. 2A in Felts et al., 2000 and alignment under-lying Fig. 1B). Furthermore, phylogenetic analysis indicatesthat dTrap1, human Trap-1 and a hypothetical proteindeduced from a Caenorhabditis elegans genomic sequencecomprise a new family of the superfamily of heat shockproteins (Fig. 1B). This analysis implies in addition thatthe founding member of this subfamily has diverged earlyfrom the large body of Hsp90 family members during eukar-yotic evolution.In order to determine dtrap-1 expression during Droso-phila embryogenesis, we used a RNA antisense transcriptprobe for in situ hybridization of whole-mount preparationsof staged embryos. Fig. 2B shows that transcripts are evenlydistributed in the preblastoderm stage embryo in the absenceof nuclear transcription. Thus, these transcripts must be ofmaternal origin consistent with the ®nding that dtrap-1 isstrongly expressed during oogenesis and that transcripts aretransported from nurse cells into the growing oocyte (Fig.2A and inset). As development proceeds, and nuclei appearin the periphery of the embryo (late syncytial stage; seeCampos-Ortega and Hartenstein, 1997) transcripts aredegraded throughout the embryo. However, they remain inthe posterior pole region of the embryo (Fig. 2C) up until thetransition between syncytial and cellular blastoderm stage.A similar pattern of transcript degradation and remaining oftranscripts in the posterior region of the embryo has beenreported for the transcripts of Drosophila hsp83 (Bashirul-lah et al., 1999).First zygotic expression of the gene is initiated aftergastrulation has started, weakly in the mesoderm primor-dium and more prominently in the developing endoderm(white arrows, Fig. 2D). Transcripts continue to accumulatein the developing muscles, remain prominent in the midgutR. P¯anz, M. Hoch / Mechanisms of Development 96 (2000) 219±222220Fig. 1. Physical map of the dtrap-1 transcription unit. (A) Genomic organization of dtrap-1 (black) and Vha-16 (gray). Untranslated leaders and trailers areshaded lighter. Diagnostic restriction sites are indicated; EcoRI (R), HindIII (H), SalI (S) and XbaI (X). The insertion site of P l(2)04535 is marked by a triangle.Exon-intron boundaries were determined by sequence comparison of cDNA versus genomic sequence. (B) Unrooted tree of several Hsp83/90 proteins, dTrap-1, Trap-1 and a C. elegans genomic sequence homologous to dTrap-1. It was constructed from the full peptide sequences using the pileup program from theWisconsin Package (Genetics Computer Group; version 8.0) with gap creation and extension penalties of 12 and 4, respectively. Abbreviations (accessionnumber): hTrap-1 human Trap-1 (A55877), dTrap-1Drosophila melanogaster Trap-1 (AF115775), cTrap-1 Caenorhabditis elegans Trap-1 fromCosmid R151 (U00036), H.a. Haemophilus actinomyceticomites HtpG (U26968), E.c. Escherichia coli HtpG (P10413), H.v. Hordeum vulgareHsp90 (X67960), C.r. Catharanthus roseus Hsp90 (L14594), C.a. Candida albicans Hsp90 (X81025), S.c. Saccharomyces cereviseae Hsp82(P02829), D.m. Drosophila melanogaster Hsp83 (P02828), A.a. Anopheles albimanus Hsp82 (L47285), S.s Sus scrofa Hsp90 (U94395),H.s. Homo sapiens Hsp90A (P07900), G.g.Gallus gallus Hsp90 (X70101), O.t.Oncorhynchus tshawytscha Hsp90 (U89945), D.d.Dictyosteliumdiscoideum Hsc90 (L43591), L.i. Leishmania infantum Hsp83 (P87770), L.a. Leishmania amazonensis Hsp83 (P27741), T.b. Trypanosoma bruceiHsp83 (X14176), T.c. Trypanosoma cruci Hsp85 (P06660) and T.p. Trypanosoma parva Hsp90 (M57386).and in addition in the hindgut (arrow, Fig. 2E). During laterstages of development, dtrap-1 is maintained in the midgutbut splits into three expression domains where the tran-scripts are expressed at high levels (arrows, Fig. 2F).Towards the end of embryogenesis, transcripts are foundin a highly dynamic pattern in muscles (Fig. 2G,H).Among these, the pharynx muscles are the most prominentsites of transcript accumulation (Fig. 2G) and the only oneswhere expression is continuos from the precursors onward(black arrowheads in Fig. 2D,E,G). In conclusion, the dtrap-1 gene encodes a novel member of a subfamily of Hsp 90-related Hsps and is transcribed in distinct expressiondomains in all three germ layers during Drosophila embry-ogenesis.2. Materials and methodsCloning of the dtrap-1 gene was initiated by plasmidrescue (Wilson et al., 1989) using the l(2)04535 P element.The resulting fragment was used to screen a genomic phagelibrary (Stratagene). Sequence comparison of subclonesR. P¯anz, M. Hoch / Mechanisms of Development 96 (2000) 219±222 221Fig. 2. Transcript pattern of the dtrap-1 gene visualized by whole-mount in situ hybridization. Follicles are shown at stages 4 and 13 (A), embryos at embryonicstages 2 (B), 4 (C), 10 (D), 13 (E), 15 (F), 17 (G) and a blow up of body wall muscles at stage 16 (H). Labeled antisense RNA probe was derived from theXS800-fragment (see Fig. 1; stages according to Campos-Ortega and Hartenstein, 1997). The arrow in (A) points to the nurse cell remnants and the arrowheadmarks the oocyte nucleus. The inset shows the start of prominent germ cell expression in a stage 4 follicle. Whereas the freshly laid egg has an uniformdistribution of transcript (B), the cellularizing embryo shows rapid degradation of dtrap-1-RNA throughout its whole length with an exception at the veryposterior pole (C). After gastrulation (stage 10, D) new transcripts are synthesized in the endoderm (white arrows) and in the mesoderm (arrowheads). At stage14 (E) a prominent domain in the developing hindgut (arrow, ectoderm) complements the persisting expression in derivates of the endoderm (fusing midgutprimordia) and mesoderm (fatbody and muscle precursors; e.g. arrowhead: pharynx muscles precursers). During organogenesis the endodermal expressionsplits into three domains (F; arrows) and at the end of embryogenesis (G; stage 17) the most prominent expression domains reside in some muscles and in themid- and hindgut. In the precluding stages expression in muscles was extremely short timed with the exception of the primordia and the thereof resulting pharyxmuscles, in which the expression of dtrap-1 persisted (black arrowheads in (D,E,G)). The arrowheads in (H) point to ephemeral expression in laterallongitudinal muscles, where most prominent staining is detected next to the nuclei within the fused muscles. Embryos are shown in lateral (A±C,F),dorso-lateral (D) or dorsal view (E). In all images anterior is left and in (A±D,F) dorsal is up.identi®ed the Vha16 locus and an additional putative openreading frame (pORF) 5 0 to Vha16. With a genomic frag-ment corresponding to this pORF (XS800) an embryoniccDNA library was screened (Clontech) and 9 independentclones were obtained. They were assembled to a cDNA with2235 bp that was fully sequenced as cDNA and genomicDNA. This cDNA overlaps in the ORF completely with theclone LD14417 from the Berkley Drosophila GenomeProject and was con®rmed by 5 0-RACE with mRNAprepared from CantonS embryos (protocol according tothe manufacturers manual, Clontech). The screening probeXS800 was additionally used to prepare digoxygenin-labeled sense and antisense RNA probes as outlined bythe manufacturer (Boehringer, Mannheim). In situ hybridi-zations were performed as described by Hauptmann andGerster (1996).AcknowledgementsThis work was supported by the Graduiertenkolleg`Molekulare Genetik der Entwicklung', the SFB 271 (B4)and the Max-Planck-Society (Abt. Molekulare Entwick-lungsbiologie). We thank G. Dowe for sequencing and G.Dowe and H. JaÈckle for critically reading the manuscript.ReferencesBashirullah, A., Halsell, S.R., Cooperstock, R.L., Kloc, M., Karaiskakis,A., Fisher, W.W., Fu, W., Hamilton, J.K., Etkin, L.D., Lipshitz, H.D.,1999. Joint action of two RNA degradation pathways controls thetiming of maternal transcript elimination at the midblastula transitionin Drosophila melanogaster. EMBO J. 18 (9), 2610±2620.Campos-Ortega, J.A., Hartenstein, V., 1997. The Embryonic Developmentof Drosophila melanogaster, Springer-Verlag, Berlin, p. 227.Cutforth, T., Rubin, G.M., 1994. Mutations in Hsp83 and cdc37 impairsignaling by the Sevenless receptor tyrosine kinase in Drosophila.Cell 77, 1027±1036.Felts, S.J., Owen, B.A., Nguyen, P., Trepel, J., Donner, D.B., Toft, D.O.,2000. The hsp90-related protein TRAP1 is a mitochondrial protein withdistinct functional properties. J. Biol. Chem. 275, 3305±3312.Finbow, M.E., Goodwin, S.F., Meagher, L., Lane, N.J., Keen, J., Findlay,J.B.C., Kaiser, K., 1994. Evidence that the 16 kDa proteolipid (subunitc) of the vacuolar H 1 -ATPase and ductin from gap junctions are thesame polypeptide in Drosophila and Manduca: molecular cloning of theVha16k gene from Drosophila. J. Cell Sci. 107, 1817±1824.Hauptmann, G., Gerster, T., 1996. Multicolour whole mount in situ hybri-disation to Drosophila embryos. Genes Dev. Evol. 206, 292±295.Karpen, G.H., Spradling, A.C., 1992. Analysis of subtelomeric heterochro-matin in the Drosophila minichromosome Dp1187 by single P-elementinsertional mutagenesis. 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A molecularclamp in the crystal structure of the N-terminal domain of the yeastHsp90 chaperone. Nat. Struc. Biol. 4, 477±483.Scheibel, T., Weikl, T., Buchner, J., 1998. Two chaperone sites in Hsp90differing in substrate speci®city and ATP dependence. Proc. Natl. Acad.Sci. USA 95, 1495±1499.Song, H.Y., Dunbar, J.D., Zhang, Y.X., Guo, D., Donner, D.B., 1995.Identi®cation of a protein with homology to hsp90 that binds the type1 tumor necrosis factor receptor. J. Biol. Chem. 279 (8), 3574±3581.Wilson, C., Pearson, R.K., Bellen, H.J., O'Kane, C.J., Grossniklaus, U.,Gehring, W.J., 1989. P-element-mediated enhancer detection: an ef®-cient method for isolating and characterizing developmentally regulatedgenes in Drosophila. Genes Dev. 3, 1301±1313.R. P¯anz, M. Hoch / Mechanisms of Development 96 (2000) 219±222222

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dtrap-1 encodes a novel member of the heat-shock super family of proteins and is expressed in derivatives of all three germ layers during Drosophila embryogenesis.

dtrap-1 encodes a novel member of the heat-shock super family of proteins and is expressed in derivatives of all three germ layers during Drosophila embryogenesis.

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