Molecular Characterization of a Heat Shock CognatecDNA of Zebrafish, hsc70, and DevelopmentalExpression of the Corresponding TranscriptsHELENE SANTACRUZ,1 SOPHIE VRIZ,2 AND NICOLE ANGELIER1*1Groupe Genes et Developpement, Laboratoire de Biologie Moleculaire et Cellulaire du Developpement, UA 1135 CNRS-UPMC, Paris, France2Unite de Genetique des Mammiferes, Institut Pasteur, Paris, France

ABSTRACT To elucidate the potential role of thehsp70 gene family in developmental processes in verte-brates, we chose to study the expression of one of thesegenes in zebrafish. A zebrafish gastrula cDNA librarywas screened with a Pleurodeles waltl hsp70 cDNAprobe. A 2.3-kb cDNA was thus isolated and se-quenced. The predicted amino acid sequence con-tained an open reading frame encoding for a 649-amino acid polypeptide. Sequence analysis showedstrong homology with hsp70-related gene sequences inother species; in particular, the strongest homology wasfound with the cognate members of this family. Tests ofheat inducibility revealed that transcripts were expressedat normal temperature, but the level of transcript expres-sion increased after heat shock. Moreover, experimentsof the neosynthesis of total proteins in heat shockconditions and corresponding immunoblotting assaysshowed that 24-h-stage embryos are able to respond toheat shock. The quantity of 70 kDa proteins, recognizedby a specific antibody of the HSP/C70 protein family, isexpressed in control condition and increased signifi-cantly after heat shock. Furthermore, Northern blotanalysis of transcript expression showed that the corre-sponding mRNAs were detected throughout embryonicdevelopment in the absence of any heat shock. Ourclone, named hsc70, thus corresponded to a cognatemember of the hsp70 gene family, expressed undernormal conditions during development, but also heatinducible. The spatio-temporal pattern of transcripts dur-ing development was determined by in situ hybridizationon wholemount embryos at different stages. As a mater-nal RNA, hsc70 mRNA was uniformly present in theembryo, up to the end of gastrulation. Later, a tissue-specific enrichment of hsc70 transcripts was detected inthe central nervous system (CNS) and in a fraction of thesomites. These results suggest that the hsc70 gene maybe involved in developmental differentiation events. Dev.Genet. 21:223–233, 1997. r 1997 Wiley-Liss, Inc.

Key words: hsc70 transcript expression; zebrafishdevelopment; neurogenesis; somitogenesis

INTRODUCTIONFollowing heat shock or a variety of other stresses,

organisms respond by synthesizing a group of proteinscalled heat shock proteins (HSP). These proteins areamong the most highly conserved throughout evolution[Pelham, 1990; Gupta and Golding, 1993]. In thisfamily, besides heat-inducible genes, a set of heat shockproteins is expressed in unstressed cells under physi-ological conditions. These are called HSCs (heat shockcognate proteins) [for review, see Lindquist and Craig,1988; Craig et al., 1993].

The most abundant and conserved HSPs have amolecular mass of ,70 kDa. Major information abouthsp70 gene family structure and regulation has accumu-lated from studies on bacteria, yeasts, fruit-flies, andmammals [for review, see McKay, 1993]. Bacteria werefound to have only a single stress-70 protein [Georgop-oulos et al., 1979], whereas eukaryotic cells have sev-eral different representatives. In these organisms, dif-ferent forms of HSP70-related proteins have been found,depending on their localization in the cell: these includemitochondrial (and chloroplastic) forms [Craig et al.,1989; Amir-Shapira et al., 1990; Kang et al., 1990;Baker and Schatz, 1991], endoplasmic reticulum spe-cific members (BIP and GRP78 in mammals, e.g.)[Munro and Pelham, 1986; Flynn et al., 1991; Hass,1991; Leustek et al., 1991] and cytosolic forms. Thelatter group includes strictly heat-inducible forms(HSP70) [Ingolia et al., 1980; Bienz, 1984; Leung et al.,

Contract grant sponsor: Centre National de la Recherche Scientifique(CNRS); Contract grant sponsor: Universite Pierre et Marie Curie;Contract grant sponsor: Association pour la Recherche contre leCancer; Contract grant numbers: ARC n°6309, ACC n°4.

*Correspondence to: Nicole Angelier, Groupe Genes et Developpement,Laboratoire de Biologie Moleculaire et Cellulaire du Developpement, UA1135 CNRS-UPMC, Bat. C 6eme et. case 16, 9 quai St. Bernard, 75252 Pariscedex 05, France. E-mail: Genes.Developpement@snv.jussieu.fr

Received 25 February 1997; accepted 6 September 1997.

DEVELOPMENTAL GENETICS 21:223–233 (1997)

r 1997 WILEY-LISS, INC.

1990; Lisowska et al., 1994] and their cognate counter-parts (HSC70) [Chappell et al., 1986; Dworniczak andMirault, 1987; Sorger and Pelham, 1987; Slater andCraig, 1989; Perkins et al., 1990; DeLuca-Flaherty andMcKay, 1990; Zafarullah et al., 1992]. The role ofstress-70 proteins during the heat-shock response hasbeen extensively studied over a number of years [forreviews, see Lindquist, 1986; Craig and Gross, 1991],but their importance in normal cellular processes hasonly more recently been described [Georgopoulos andWelch, 1993; Hartl et al., 1994]. Indeed, HSC70 isessential for cell viability under normal growth condi-tions. It behaves as a molecular chaperone and isinvolved in promoting protein folding, transport, andassembly in both eukaryotic and prokaryotic systems[for reviews, see Ellis and Van der Vies, 1991; Gethingand Sambrook, 1992]. HSP70-related proteins are alsorequired for translocation of nascent proteins throughintracellular membranes [Chirico et al., 1988; Deshaieset al., 1988; Scherer et al., 1990; Vogel et al., 1990;Imamoto et al., 1992; Shi and Thomas, 1992; Terlecky etal., 1992]. These chaperones therefore interfere withother proteins, thus controlling their activity [McKay,1993].

With regard to embryonic development, HSP70 pro-teins have been identified in Drosophila, amphibians,and mammals. In Drosophila, Xenopus, and Pleurode-les, several forms of HSP70 were found to be differen-tially expressed during oogenesis and/or embryogenesis[Bienz and Gurdon, 1982; Craig et al., 1983; Zimmer-man et al., 1983; Bienz, 1984, 1985; Palter et al., 1986;Billoud et al., 1993; Angelier et al., 1996]. Thus in themale germline of mice, two genes belonging to thehsp70 family are constitutively and sequentially ex-pressed during spermatogenesis [Zakeri et al., 1990;Rosario et al., 1992]. Furthermore, in the mammalianembryo, proteins of the HSP70 family were found to besynthesized during the very early stages of develop-ment [Manejwala et al., 1991; Bensaude and Morange,1983; Bensaude et al., 1983]. Moreover, the hsc73 geneis expressed in the absence of heat shock in themammalian embryo [Walsh et al., 1994]. At the presenttime, few results have been reported concerning thespatial distribution of hs(p/c)70 transcripts duringembryogenesis in vertebrates [Walsh et al., 1997] and,consequently, the role of these genes during developmen-tal processes remains unclear.

One means of addressing the question of the role ofhsp70 gene family during development would be toobtain information on the spatial pattern of geneproducts in embryos at different stages correspondingto the onset of critical developmental process, therebyenabling the formulation of hypotheses on the potentialrole of hsp70 gene family expression.

Zebrafish can be considered as a convenient systemfor approaching this problem [Driever et al., 1994;Kimmel et al., 1995; Granato and Nusslein-Volhard,1996]. Indeed, not only does the transparency of their

embryos enable observations in vivo, but it also facili-tates analysis of the gene expression pattern by in situhybridization on wholemount embryos. Recently, Kroneand Sass [1994], Pearson et al. [1996], Sass et al. [1996],and Lele et al. [1997] have addressed the question ofheat shock proteins (HSP90a, HSP90b, and HSP47)during embryonic development in zebrafish. In particu-lar, they have provided evidence for differential regula-tion of hsp90a and hsp90b in developing embryos, andthey have suggested a role for hsp90a during normalmuscle development [Sass et al., 1996; Krone et al.,1997].

In the present study, we isolated a cDNA related tothe hsp70 gene family in zebrafish. Characterization ofthis clone provided evidence for expression of thecorresponding gene in nonstress conditions. We ana-lyzed expression of hsc70 mRNA throughout develop-ment using Northern blot and in situ hybridization onwhole-mount embryos. We were able to show that hsc70RNAs in zebrafish are present from the beginning of thefirst stages of embryogenesis. This expression, which isubiquitous up to the end of gastrulation, exhibits aninteresting enrichment in the forming central nervoussystem and in a fraction of the somites. These resultsare discussed in relation to the potential involvement ofhsc70 during somitogenesis and neurogenesis.

MATERIALS AND METHODS

Animal Accommodations and Embryos

Wild-type zebrafish (Danio rerio) were maintained at28.5°C on a 14-h light/10-h dark cycle as described byWesterfield [1993]. Embryos were obtained by in vitrofertilization and incubated at 28.5°C in ‘‘fish water’’ (60mg/l commercial sea salt in deionized water) [Wargaand Kimmel, 1990]. Developmental stages were definedas hours of postfertilization (hpf) or were determined bymorphological criteria including, e.g., the progressionof epiboly or the number of somites [Hanneman andWesterfield, 1989].

Cloning and Sequencing

Screening of a lZAP cDNA library from zebrafishgastrula embryos [Joly et al., 1993] was performedfollowing standard methods [Sambrook et al., 1988]using a DNA probe obtained from a 534-bp ClaI-EcoRIfragment containing the 38 coding region of the amphib-ian Pleurodeles waltl hsp70 cDNA [Billoud et al., 1993].Three rounds of screening were carried out and sevenpositive clones recovered. A partial restriction map ofeach clone was done. The complete nucleotide sequencewas performed for one of them using the T7 Sequenc-ingy Kit (Pharmacia-Biotech) and synthetic oligonucleo-tides. A 2,306-bp cDNA was thus characterized andcalled hsc70. Clone 11RC was generated from the hsc70clone by eliminating a 1822-bp PstI-digested fragment.Therefore, this clone contained only a 484-bp fragment

224 SANTACRUZ ET AL.

corresponding to the end of the 38 coding region and the38 UTR of hsc70 cDNA (see Fig. 3).

Total RNA Extraction and NorthernBlot Analysis

Total RNA was purified using standard procedures(RNA Insta-Purey-LS System, Eurogentec, Morris-town, NJ) from mature oocytes or whole embryos atdifferent stages of development.

For Northern blot analysis, 20 µg of total RNAsamples from oocytes or selected embryonic stages(from 2-cell stage to 24-h embryos) were loaded on eachlane. Electrophoresis of RNA was performed on agarosegel in phosphate saline buffer [Pelle and Murphy, 1993].Integrity of RNA was controlled by ethidium bromidestaining. RNAs were blotted onto nylon membranes(Nytran-Plus, Schleicher & Schuell, Keene, NH). Aftertransfer, membranes were baked at 80°C for 1 h to fixRNA. Blots were then hybridized in 50% formamide/53SSC/53 Denhardt’s/0.4% SDS overnight at 42°C withhsc70 full-length cDNA probes labeled with [32]P usingthe random priming method. Following hybridization,the blots were washed 3 3 30 min in 23 SSC/0.1% SDSat 42°C and 3 3 20 min in 13 SSC/0.1% SDS at 55°C,and finally exposed to Amersham MP film at 280°C.

Heat Shock Experiments

Inducibility tests were performed using Northernblot analysis in the same conditions mentioned above,but total RNAs were extracted from embryos submittedto heat shock experimental conditions previously de-scribed by Krone and Sass [1994]. After hybridizing tothe 11RC probe, the blot was stripped and reprobedwith a positive control, the hsp90a gene, which is itselfheat inductible [Krone and Sass, 1994].

Wholemount In Situ Hybridization

Plasmid containing the PstI-EcoRI fragment of thehsc70 cDNA (clone 11RC, see Fig. 3) was linearized byEcoRI and PstI, respectively. Sense and antisense RNAprobes were generated using bacteriophage T3 or T7RNA polymerases and were digoxigenin (DIG)-labeledaccording to Boehringer’s specifications. After synthe-sis, RNA probes were purified by two precipitations in 4M lithium chloride and absolute ethanol. Each precipi-tation was followed by three washes in 70% ethanol.

Hybridizations on wholemount control embryos orembryos submitted to 1 h heat shock at 37°C wereperformed according to Schulte-Merker et al. [1992]and Joly et al. [1993]. Detection of RNA probes wascarried out using an anti-DIG antibody (Boehringer,Indianapolis, IN) coupled to alkaline phosphatase (BlueStaining Kit, Boehringer).

Finally, some 24-h embryos processed for whole-mount in situ hybridization were fixed in 4% Parafor-maldehyde (PFA)/phosphate-buffered saline (PBS), de-hydrated through an increasing series of ethanol (from

30–100% of ethanol in H2O), embedded in esterwax,and 10-µm-sectioned. Sections were then observed un-der a Nomarski microscope and photographed.

In Vitro Translation, Western Blot, andNeosynthesis of HSC70 Protein After Heat Shock

In vitro transcription and translation were per-formed from hsc70 cDNA in rabbit reticulocyte lysate(Rabbit Reticulocyte Lysate Systems, Promega, Madi-son, WI) using [35]S-labeled methionine. One-dimen-sional SDS 10% polyacrylamide gel electrophoresis wasperformed for translation products analysis [Laemmli,1970] and the dry gel was autoradiographed. Totalproteins of 24-h or 48-h embryos were purified accord-ing to standard protocols [Westerfield, 1993], and theintegrity of total proteins was tested with Coomassieblue staining. We used the polyclonal antibody N1which was raised against the Pleurodeles waltl HSP70protein [Prudhomme et al., 1997]. The N1 epitope is a15-aa sequence 56 residues upstream of the protein Cterminus. This epitope is common to HSC and HSP70proteins. Western blots with in vitro translated prod-ucts were performed using the polyclonal N1 serum orN1 serum, which had been preadsorbed with the immu-nogen peptide. Western blots with total proteins of 24-hor 48-h embryos were performed using the polyclonalN1 serum. The N1 antibody was revealed with second-ary antirabbit antibodies coupled to peroxidase (Amer-sham Life Science, Buckinghamshire, UK). Immunode-tection was performed with the chemoluminescenceenzymatic system (ECL Kit, Amersham).

For neosynthesis experiments, batches of 50 embryosat the 24-h stage were placed in 200 µl of EmbryoMedium supplemented with 45 µCi of [35]S-methionine(specific activity: 1,000 Ci/mmol, Amersham). One batchof embryos was treated for 1 h at 37°C, and embryoswere allowed to recover for 30 min at 28.5°C. Controlembryos were incubated for 1 h 30 min at 28.5°C. Afterheat shock, embryos were frozen at 280°C, and totalproteins extracted (see above). Samples correspondingto the same total quantity of radioactivity of neosynthe-sized proteins were electrophoresed on SDS PAGE, andimmunodetection on blot with the N1 antibody wasperformed in the conditions previously mentioned.

RESULTS

Isolation and Characterization of Zebrafishhsp70-related cDNA

In order to isolate zebrafish cDNA clones correspond-ing to the hsp70 gene family, a lZAP cDNA library [Jolyet al., 1993] prepared from poly(A)1 RNA of zebrafishembryos at the gastrula stage (100% epiboly) wasscreened at medium stringency using a Pleurodeleshsp70 cDNA probe. This probe corresponds to a 534 bpClaI-EcoRI fragment corresponding to the 38 regioncontaining the translating stop codon of the Pleurodeleswaltl hsp70 cDNA [Billoud et al., 1993].

HSC70 AND ZEBRAFISH DEVELOPMENT 225

Three out of the seven positive clones recovered wereshown, by restriction mapping analysis, to result fromthe same poly(A)1 RNA. One of these three clones wasfurther studied and its cDNA fully sequenced. Analysisof the nucleotide sequence (2306 bp) and its deducedpolypeptide (Fig. 1) provided evidence for an openreading frame (1,950 bp), which encoded a 649-amino

acid polypeptide, the characteristics of which stronglysuggested that it belonged to the HSP70 protein family.Indeed, it exhibited two strictly conserved domainstypical of HSP70 protein family (Fig. 1). These twodomains, called signature 1 (IDLGTTYS) and signature2 (DLGGGTFD), are known to be present in mostHSP70 and HSC70 proteins [Gupta and Golding, 1993].Furthermore, the full-length cDNA deduced polypep-tide sequence was found to be strongly homologous tothe known HSP70 and HSC70 in other species (Table1). For example, we found an identity of 90.1% withrainbow trout HSP70B [Kothary et al., 1984], 82.8%with Xenopus HSP70 [Bienz, 1984], 81.8% withPleurodeles HSP70 [Billoud et al., 1993] and 71.6% withDrosophila HSP70-1 [Ingolia et al., 1980]. Neverthe-less, it is noteworthy that sequence comparison data ofHSC70 and HSP70 in the same species revealed thatour clone-deduced polypeptide showed stronger homol-ogy with the cognate form than with the heat-inducibleform. For example, we found 91.7% identity withhuman HSC71 [Dworniczak and Mirault, 1987] vs.75.7% for HSP70B8 [Leung et al., 1990]. This was alsothe case in the mouse [Giebel et al., 1988; Hunt andCalderwood, 1990], Drosophila [Ingolia and Craig, 1982;Perkins et al., 1990], and rainbow trout [Kothary et al.,1984; Zafarullah et al., 1992] (Table 1). Our clone wasthus named hsc70.

To validate that our clone encoded a protein of theHSP70 family, we synthesized an RNA by in vitrotranscription from this cDNA and used it to produce thecorresponding protein using the rabbit reticulocytelysate. The resulting synthesized protein was thentested with the N1 antibody. This N1 antibody, whichwas raised against an HSP70 protein of the urodelePleurodeles waltl, recognizes proteins of the HSP70

Fig. 1. Nucleotide sequence of zebrafish hsc70 and its deducedamino acid sequence, featuring: IDLGTTYS: signature no. 1 of theHSP70 protein family (aa 9–16); DLGGGTFDV: signature no. 2 of theHSP70 protein family (aa 199–206); TAEREEFEHQQKlEK: epitope ofthe N1 antibody used for Western blot (aa 586–600); PstI: restrictionsite (bases 1822–1827) used for subcloning. (EMBL accession number:Y11413)

TABLE 1. Percent Identity Between HSP70-relatedProtein Deduced From hsp70-related cDNA Cloned in

Zebrafish (Danio rerio) and HSP and/or HSC70Proteins in Other Species*

Species HSP/HSC 70

Proteinssimilarities

(% of identity)Homo sapiens HSC71 91.7%

HSP70B8 75.7%Mus musculus HSC70 91.6%

HSP70-1 83.3%Oncorhynchus mykiss HSC71 91.4%

HSP70A 87.5%HSP70B 90.1%

Xenopus laevis HSP70 82.8%HSC70-1 87.3%

Pleurodeles waltl HSP70 81.8%Drosophila melanogaster HSC70-4 82.2%

HSP70-1 71.6%

*From Ingolia et al., 1980; Bienz, 1984; Kothary et al., 1984;Dworniczak and Mirault, 1987; Giebel et al., 1988; Hunt andCalderwood, 1990; Leung et al., 1990; Perkins et al., 1990;Zafarullah et al., 1992; Billoud et al., 1993; Ali et al., 1996.

226 SANTACRUZ ET AL.

family [Prudhomme et al., 1997]. The in vitro transla-tion product of our RNA gave rise, as expected, to a[35]S-labeled protein of ,70 kDa (Fig. 2, lane 1). Threeother minor translation products were also recovered.However, only the major product of 70 kDa was specifi-cally recognized by the N1 antibody specific to theHSP70 protein family (Fig. 2, lane 3). Furthermore,this major product was no longer detected when anti-body was preabsorbed with immunogen peptide (Fig. 2,lane 4). When tested on total proteins extracted fromembryos, the N1 antibody recognized a protein of ,70kDa, which was likely to be a protein of the HSP70family, present in normal conditions in 24- and 48-hzebrafish embryos (Fig. 2, lanes 5 and 6).

Hsc70 Is a Cognate Member of the hsp70 GeneFamily Expressed Under Normal Conditions and

Inducible Under Stress Conditions

To determine the regulation of hsc70 transcript ex-pression under heat stress conditions, we carried outtest experiments for heat inducibility in embryos usingNorthern blot analysis. In the hsp70-related gene fam-ily, the least conserved part of the sequence was likelyto be gene-specific [Chappell et al., 1987]. Therefore, weused the 11RC subclone (11RC corresponds to the 38UTR and the last 189 bp of the 38 coding region of hsc70(Fig. 3)) as a probe, to perform Northern blot analysis oftotal RNA extracted from 24 h-stage embryos, before

(28.5°C) and after heat shock (34°C or 37°C) (Fig. 4A).After dehybridization, we carried out the control experi-ment on the same blot using part of the hsp90a cDNA,which was shown to be strictly heat-inducible, as aprobe (Fig. 4A). Hsc70 transcripts (2.6 kb) were de-tected in unstressed embryos (28.5°C). Surprisingly,after heat shock, an increase in the amount of tran-scripts was observed. For hsp90a, as described previ-ously by Krone and Sass (1994), an mRNA (3 kb) wasinduced by heat shock, since no transcript was detect-able at control temperature (Fig. 4A).

In order to complete the study on heat-inducibleregulation of the hsc70 gene, we performed experi-ments of HSC70 protein neosynthesis after heat shockin 24-h stage embryos. Thus we investigated whetherthe increase in the amount of hsc70 transcripts afterheat shock is correlated with modifications of theprotein pattern. The immunodetection with the N1antibody showed that the HSC70 protein is clearlypresent in normal conditions (Fig. 5, lane 1). After a 1-hheat shock at 37°C, the amount of the HSC70 proteinnot only was maintained, but also increased (Fig. 5,lane 2).

All these results about expression of hsc70 gene andthe corresponding protein led us to conclude that hsc70gene is expressed under normal conditions and is alsoinducible. Moreover, these results suggest that in bothheat shock and control conditions, the transcripts ex-pression is correlated with the protein expression.

Developmental Expression of hsc70 Transcripts

Characterization of hsc70 transcript expression wasdone by Northern blot hybridizations.

In order to study the expression of the hsc70 tran-script in the process of development, a [32]P-labeledDNA probe corresponding to the full-length sequence ofthe hsc70 clone was used to probe a Northern blot withtotal RNA extracted from oocytes or embryos at differ-ent stages of development. A signal corresponding to atranscript of ,2.6 kb was observed in RNA from bothoocytes and selected embryos (from the 2-cell stage tothe 24-h stage) (Fig. 4B). hsc70 transcripts were there-

Fig. 2. Expression of HSC70 protein. Lane 1: In vitro translationwas performed from hsc70 cDNA in rabbit reticulocyte lysate using[35]S-methionine (see Materials and Methods). Samples of translationproducts were loaded on SDS PAGE and the dry gel was autoradio-graphed. Lane 2: Control tested for possible lysate contamination inwhich no cDNA matrix was added to the translation reaction. Lanes 3and 4: Western blots with in vitro translated products incubated withserum containing polyclonal antibody against N1 peptide fromPleurodeles HSP70 (lane 3) or with N1 serum, which has beenpreadsorbed with the immunogen peptide (lane 4). Lanes 5 and 6:Western blots with extracts of total proteins of 24-h (lane 5) or 48-hembryos (lane 6) incubated with serum containing polyclonal N1antibody.

Fig. 3. Schematic representation of the two clones used. A, hsc70total clone; B, 11RC; `: bluescript vector; w nontranslated se-quence; `: translated sequence; Pst1 restriction site used for subclon-ing.

HSC70 AND ZEBRAFISH DEVELOPMENT 227

fore shown to be expressed in normal conditionsthroughout embryonic development. Furthermore, dur-ing stages of cleavage, the level of hsc70 RNA was lowerthan from the end of gastrulation (Fig. 4B). These dataprovide evidence for expression of hsc70 transcriptsthroughout development under normal conditions.

Spatial Distribution of hsc70 transcripts inembryos during development

In order to analyze the spatial expression of hsc70transcripts during embryogenesis, we carried out insitu hybridizations on wholemount embryos using a434-bp antisense RNA probe labeled with digoxigeninsynthesized from the subclone 11RC (Fig. 3). Someembryos were sectioned after wholemount in situ hy-bridization to determine more accurately the localiza-tion of the signal.

During early embryogenesis, from the two-cell stage(Fig. 6A) up to late gastrula (80% epiboly or 9 hpf; Fig.6B), homogeneous labeling was detected whatever theembryo stage considered (Fig. 6A,B), suggesting thathsc70 transcripts were ubiquitously present.

In contrast, from the beginning of somitogenesis andneurogenesis, an enrichment of hsc70 transcripts wasobserved and superimposed onto a basal level of expres-sion. Thus at the 5–6 somite stage, a more intensivesignal was detected in the cephalic part of the embryos(data not shown). Then, starting from the 22-somitestage (20 hpf), four zones of transcripts enrichmentwere distinguishable. First, strong labeling was ob-served in the developing eye, in the future retina(external and internal layers), and in the forming lens(Fig. 7A). Such labeling was still detected in 24-hembryos (Figs. 7B,F, 8A). The second patch of enriched

Fig. 4. Expression of hsc70 transcripts. A. Northern blot analysis:20 µg of total RNA prepared from 24-h stage embryos without (28.5°C)or after 1-h heat shock (34°C or 37°C). Blot was first hybridized with a[32]P-labeled 11RC probe. Whatever the conditions, a transcript of 2.6kb was then detected. After dehybridization, the same blot washybridized with a [32]P-labeled probe corresponding to hsp90a cDNAand a 3-kb transcript was detected following heat shock. B. Northernblot analysis: 20 µg of total RNA extracted from oocytes or embryos atdifferent stages of development (from 2-cell stage to 24-h stage) wereloaded on each lane and probed with a [32]P-labeled hsc70 completecDNA. For each experiment, as a control for the total amount andintegrity of RNA loaded, photographs of ethidium bromide-stained28S RNA are shown.

Fig. 5. Neosynthesis of HSC70 protein after heat shock and immu-noblotting with the N1 antibody. Embryos at the 24 h-stage have beenimmerged in Embryo Medium supplemented with [35]S-methionine(see Material and Methods) and were, or submitted to heat shock at37°C for 1 h following by 30 mn of recuperation at 28.5°C, or remainedfor 1 h 30 at 28.5°C (control). Total proteins were extracted andsamples corresponding to the same amount of total radioactivity ofheat-shocked or control embryos proteins were loaded on SDS PAGE.The gel was used for immunoblot with the N1 antibody (lane 1: control;lane 2: heat shock). The HSC70 protein, recognized by the N1antibody, is expressed in control condition and increased after heatshock.

228 SANTACRUZ ET AL.

expression was observed at 22 hpf in the midbrainregion, especially in the rostral part (Fig. 7A). In 24-hembryos, such labeling was still detectable in thisregion (Figs. 7B,E,G, 8A). It was localized in the lateralzones of the midbrain (Fig. 8A,B). The forebrain showedno enriched staining (Fig. 7B,E,G). The third regionconcerned by enrichment in transcripts was part of thelateral hindbrain (Figs. 7B, 8B). Furthermore, thetransverse section at the level of the caudal hindbrain(Fig. 8C) displayed more intensive labeling in thecephalic mesenchyme of this region. Finally, the fourthregion of transcripts enrichment corresponded to theventral border of somites in the trunk region (data notshown). Although weaker and more diffuse, such asignal was also detectable at the 24-h stage (Fig. 7B,D).Thus our data showed that hsc70 mRNA expressionpresents a tissue-specific enrichment concerning a partof the CNS and somites.

Finally, in situ hybridization on wholemount heat-shocked embryos at the 24 h-stage revealed that thespatial expression of transcripts was not significantlyincreased in the enriched zones of labeling, but thebasal expression appeared a little stronger, especiallyin the region of somites (Fig. 7C).

DISCUSSION

Identification of a Cognate Form of the hsp70Gene Family in Zebrafish

Screening a zebrafish cDNA library enabled us torecover a clone, the sequence of which is closely relatedto the hsp70 gene family of other species. Indeed,deduced polypeptide sequence analysis revealed thepresence of two protein signatures, which are bothpresent in most members of the HSP70 protein family[Gupta and Golding, 1993]. Furthermore, comparisonbetween our peptidic sequence and those of known HSPor HSC70 in other species points out strong homologywith this family; indeed, in the same species, strongerhomology is found for the constitutive form than for theheat-inducible form. The product of in vitro translationof our clone is a polypeptide of ,70 kDa, specifically

recognized by the N1 antibody. The N1 antibody hasbeen raised against an epitope of the amphibianPleurodeles waltl HSP70. This epitope is also present inthe zebrafish HSP70-related sequence. This antibodyalso recognizes a protein of 70 KDa in extracts of totalproteins from 24- or 48-h stage zebrafish embryos.

Whether a heat shock gene is heat-inducible (hspgene) or a cognate gene (hsc gene) depends on theregulation of this gene in normal somatic cells: aheat-inducible gene is efficiently expressed only afterheat induction, whereas a cognate gene has a highbasal level of expression and is not or is weaklyheat-inducible [McKay, 1993]. In our study in zebrafishembryos, tests of heat inducibility were performed byNorthern blot analysis. Results revealed that the mRNAcorresponding to the cDNA clone that we isolated isclearly detectable whatever the temperature tested,and especially in nonheat shock conditions. However,after heat shock, an increase in the amount of mRNA isobserved. Indeed, in zebrafish, the cognate form of thehsp90 gene, i.e., the b gene, has been shown to beheat-inducible in embryos at different stages of develop-ment [Krone and Sass, 1994]. Furthermore, it has beenreported that in the human hsp70 multigenic genefamily, the level of constitutive expression and thedegree of inducible expression are variable. Thus sev-eral members of the hsp70 gene family are both ex-pressed under normal conditions and are heat-induc-ible [Taviara et al., 1996], which is the case for the genedescribed in the present study. All these results led usto conclude that the clone we isolated corresponds to acognate member of the multigenic hsp70 family, hsc70.

Our results concerning neosynthesis of HSP/C 70provided evidence for the heat shock response of em-bryos at the 24-h stage. Indeed, after heat shock, theHSC 70 protein recognized by the N1 antibody not onlyremained strongly expressed, but also increased. Thismodification in protein pattern after heat shock is ingood agreement with those previously reported in otherspecies, for example Xenopus or Pleurodeles [Heikkila,1993a,b; Prudhomme et al., 1997]. Finally, taken to-gether, results of transcripts expression and results ofneosynthesis strongly suggest that the regulation ofhsc70 mRNA expression, in normal conditions and afterheat shock, is correlated with the expression of corre-sponding protein. However, for the heat shock experi-ment, we cannot exclude that the N1 antibody alsorecognizes the HSP70 protein.

Differential Expression of hsc70 TranscriptsThroughout Embryonic Development

Using the full-length clone hsc70 (2306 bp) as aprobe, we showed by molecular hybridization thathsc70 mRNAexpression takes place throughout embryo-genesis. In our Northern blot analysis, this probehybridizes with a single transcript from total RNA forthe selected embryonic stages. This mRNA correspondsto a 2.6-kb transcript, which suggests that we did not

Fig. 6. In situ hybridization on wholemount embryos. The subclone11RC was used to generate a sense (T3) or antisense (T7) RNAdig-labeled probe. Expression of hsc70 transcripts was ubiquitous inall embryonic structures during the early stages of development. A.Lateral view of an 8-cell stage embryo. B. Lateral view of an 80%epiboly stage embryo. C. Control: lateral view of a 64-cell stage embryoincubated with the sense (T3) dig-labeled probe.

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isolate the complete nontranslating extremities of themRNA, especially in the 58 region. Nevertheless, we canaffirm that such a transcript is expressed throughoutembryogenesis. These results are consistent with thoseobtained in other species, since it was reported that inDrosophila, transcripts of the cognate gene hsc70-4were found throughout embryonic development [Per-kins et al., 1990]. Furthermore, in mammals, an hsc72

gene was also shown to be expressed under normalconditions in embryos [Giebel et al., 1988; Walsh et al.,1994, 1997].

In zebrafish, the midblastula transition (MBT) de-fined by Kane and Kimmel [1993] takes place at the512-cell stage of development. The onset of MBT ismarked by cell cycle lengthening. Independently, pro-gressive asynchrony among blastomere divisions was

Fig. 7. In situ hybridization on wholemount embryos. Molecularhybridization was carried out on whole-mount embryos with a sense orantisense RNA dig-labeled probe generated from 11RC clone. A.Global view of 26 somites-stage embryos: control embryo incubatedwith the sense (T3) dig-labeled probe (left); embryo incubated with theantisense (T7) dig-labeled probe (right). B. Global view of a 24-h-stageembryo. C. Global view of a 24-h-stage heat-shocked embryo. D. Detailof the somites region of 24-h-stage embryo. E,F,G. Details of the head

of 24-h-stage embryos: E, profile view; F, dorsal view; G, dorsal view ofa flatmount embryo. At the beginning of somitogenesis, enrichment oftranscripts was observed in the forming eye (F: arrowhead), in themidbrain (E and G: arrowheads), in the hindbrain (B, arrowhead) andin somites (D, arrowhead). In the 24-h-stage heat-shocked embryo, theexpression of transcripts is not significantly increased in the enrichedzones of labeling, but the basal expression appears a little stronger,especially in the region of somites (C: arrowhead).

Fig. 8. Sections of hybridized wholemount embryos. 24-h stageembryos were sectioned after in situ hybrizations (see Materials andMethods). A and B: parasagittal sections, C: transverse section at thehindbrain level. Labeling could be observed in the forming eye: in theretina (A, arrows) or in the lens (A, arrowhead). In the CNS,expression of hsc70 transcripts could be detected in the midbrain (B,

arrowhead) and in the hindbrain (B, arrow). In the transverse section,labeling in the hindbrain could be noted (C, arrowhead), as well as inthe cephalic mesenchyme (B, arrows). Abbreviations: cm, cephalicmesenchyme; hb: hindbrain; mb, midbrain; n, notocord; nr, neuralretina.

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easily observable. Furthermore, as interphase length-ens, cells become motile and transcription increasesover the background level. The hsc70 mRNA detectedduring early developmental stages in zebrafish have tobe of maternal origin since, as in Xenopus, no zygotictranscription occurs before the midblastula transition.Such a hypothesis is consistent with the fact that thehsc70 gene is strongly expressed in mature oocytes.Furthermore, the level of transcript expression de-creases after fertilization and clearly increases at thestart of somitogenesis.

hsc70 Gene Is Expressed in a SpatiallyTissue-specific Enriched Pattern in Embryo

Our results from in situ hybridization on whole-mount embryos during early development (before theMBT) provided evidence for homogeneous distributionof maternal hsc70 mRNA. Taking into account thechaperone function of HSC70 proteins [Beckmann etal., 1990; Ellis and Van der Vies, 1991], we can assumethat such proteins might play a role during the cleav-age stage. According to this hypothesis, it is presumedthat the ubiquitous maternal expression of hsc70 tran-scripts in early embryos allows HSC70 protein neosyn-thesis to occur in all the blastomeres during these earlysteps of development. The same ubiquitous distributionstill occurs from the MBT to the end of gastrulation, i.e.,when zygotic transcription has already started.

In contrast, from the end of gastrulation, enrichmentof hsc70 gene expression became obvious at the begin-ning of somitogenesis and neurogenesis. Interestingly,this tissue-specific enrichment correlates with an in-crease in the amount of zygotic transcripts observed inNorthern blot analysis. Furthermore, hsc70 transcriptsare more strongly detected in territories covering theCNS (particularly the midbrain), the eye (formingretina and lens), and the differentiating somites. Simi-lar enriched pattern of hsc70 transcripts has beenreported in Drosophila embryo by Perkins et al. [1990]who described an enrichment in hsc70-4 transcripts, inneuroblast, myogenic structures, and embryonic gut.Expression of hsp70 mRNA in the embryonic chickenlens also has been reported by Dash et al. [1994] whosuggest an association of HSP70 with differentiationevents. Furthermore, developmental regulation of acognate mouse mRNA, encoding a 72-kDa heat shock-like protein, was reported by Giebel et al. [1988]. Thetissue-specific enrichment of hsc70 transcripts in ze-brafish thus suggests, in a similar way, an associationof hsc70 gene with events occurring during neurogen-esis and somitogenesis.

Concerning our results of in situ hybridization onheat-shocked embryos, it appeared that the transcriptsexpression did not specifically increase in the enrichedzones of labeling. Taking into account the basal level ofhsc70 expression in most of the cells of the embryo innormal conditions and our observation of in situ pat-

tern of transcripts after heat shock, this result could beexplained by the fact that the transcripts expressionglobally increases, thus attenuating the tissue enrich-ment pattern. Heat-shock conditions would thus pointout the basal ubiquitous expression of hsc70 and not thetissue-specific enrichment of transcripts.

Therefore, under normal conditions, the spatiallyenriched pattern of hsc70 transcripts in zebrafish em-bryos provided by our results suggests that the hsc70gene might be involved in differentiation events thatparticipate in neurogenesis and somitogenesis. An hy-pothesis on the role of the hsc70 gene during control ofdifferentiation processes would be consistent with thewell-known chaperone function of the HSC70 protein[Beckmann et al., 1990; Ellis and Van der Vies, 1991],which might interact with other proteins involved insuch differentiation events. In the mouse, Walsh et al.[1994, 1997] suggested that expression of genes impli-cated in neurogenesis may be modulated by the hsc73gene. The hsc70 gene might be part of the geneticnetwork that controls development of the central ner-vous system and somites.

ACKNOWLEDGMENTSThe authors thank Dr. J.S. Joly for the gift of the

zebrafish cDNA library and Dr. P. Krone for the hsp90aprobe. Francq Borrat is acknowledged for his expertassistance with the Nomarski microscopy. We are verygrateful to Jean Desrosiers and Nadine Peyrieras forhelp in preparing the photographic reproductions, andto Jerri Bram for the English version of the text. Wealso thank all the members of our laboratory for theirhelpful comments and advice during the course of thiswork.

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