Spatial Pattern of Constitutive andHeat Shock-Induced Expression of the SmallHeat Shock Protein Gene Family, Hsp30,in Xenopus laevis Tailbud EmbryosLISA LANG, DRAGANA MISKOVIC, PASAN FERNANDO, AND JOHN J. HEIKKILA*Department of Biology, University of Waterloo, Waterloo, Ontario, Canada

ABSTRACT We employed whole-mount in situhybridization and immunohistochemistry to study thespatial pattern of hsp30 gene expression in normal andheatshocked embryos during Xenopus laevis develop-ment. Our findings revealed that hsp30 mRNA accumu-lation was present constitutively only in the cement glandof early and midtailbud embryos, while hsp30 proteinwas detected until at least the early tadpole stage. Heatshock-induced accumulation of hsp30 mRNA and pro-tein was first observed in early and midtailbud embryoswith preferential enrichment in the cement gland, somiticregion, lens placode, and proctodeum. In contrast,cytoskeletal actin mRNA displayed a more generalizedpattern of accumulation which did not change followingheat shock. In heat shocked midtailbud embryos theenrichment of hsp30 mRNA in lens placode and somiticregion was first detectable after 15 min of a 337Cheatshock. The lowest temperature capable of inducingthis pattern was 307C. Placement of embryos at 227Cfollowing a 1-h 337C heat shock resulted in decreasedhsp30 mRNA in all regions with time, although en-hanced hsp30 mRNA accumulation still persisted in thecement gland after 11 h compared to control. In latetailbud embryos the basic midtailbud pattern of hsp30mRNA accumulation was enhanced with additionallocalization to the spinal cord as well as enrichmentacross the embryo surface. These studies demonstratethat hsp30 gene expression can be detected constitu-tively in the cement gland of tailbud embryos and thatheat shock results in a preferential accumulation ofhsp30 mRNA and protein in certain tissues. Dev. Genet.25:365–374, 1999. r 1999 Wiley-Liss, Inc.

Key words: hsp30; heat shock; development; Xeno-pus; in situ hybridization; chaperone; cement gland

INTRODUCTIONThe heat shock response is a rapid and transient

change in cellular activities which ensures cell survivalby protecting essential cellular components against

damage [Atkinson and Walden, 1985; Nover, 1991;Parsell and Lindquist, 1994; Morimoto et al., 1994;Feige et al., 1996]. Transcription of heat shock protein(hsp) genes, which are intimately involved in thisphenomenon, is mediated by the heat shock elementfound in the 58 upstream regions of these genes andinteracts with a transcriptional activating proteinknown as heat shock factor (HSF). Hsps are composedof three major families, the high molecular weight(hsp90) family, the hsp70 family, and the small hspfamily [Morimoto et al., 1994; Feige et al., 1996]. Whilethe hsp90 and hsp70 families are highly conserved in awide range of organisms, the small hsps exhibit a lowdegree of conservation, with the exception of an aminoacid domain that is also found in a-crystallins, themajor protein of the eye lens [Arrigo and Landry, 1994;Waters et al., 1996]. Both small hsps and a-crystallinshave the ability to aggregate into large, highly poly-meric structures [Arrigo and Landry, 1994; Waters etal., 1996]. Small hsps appear to act as molecularchaperones and may be involved in actin capping/decapping activity [Ehrnsperger et al., 1997; Huot etal., 1996], cellular differentiation [Mehlen et al., 1997,1999; Arrigo, 1998] and modulation of redox param-eters [Arrigo, 1998]. Under heat shock conditions, smallhsps protect cellular proteins by preventing their aggre-gation or misfolding and maintaining their solubility[Jakob and Buchner, 1994; Hartl, 1996].

Developmental regulation of hsps has been observedin a number of plant and animal systems [reviewed inHightower and Nover, 1991; Heikkila, 1993a,b]. Ourlaboratory and others have been involved in the exami-nation of developmental regulation of stress-induced

Contract grant sponsor: Natural Sciences and Engineering ResearchCouncil.

*Correspondence to: John J. Heikkila, Ph.D., Department of Biology,University of Waterloo, Waterloo, Ontario, N2L 3G1 Canada.E-mail: heikkila@sciborg.uwaterloo.ca

Received 14 May 1999; Accepted 27 July 1999

DEVELOPMENTAL GENETICS 25:365–374 (1999)

r 1999 WILEY-LISS, INC.

small hsp gene expression during early embryogenesisof the frog, Xenopus laevis [reviewed in Heikkila et al.,1997]. For example, the family of small hsp genes,hsp30, are differentially expressed in a heat-induciblefashion beginning at the tailbud stage of Xenopusdevelopment [Krone and Heikkila, 1988] in contrast tohsp70 and hsp90 which are first heat-inducible afterthe midblastula stage [Heikkila et al., 1985, 1987; Ali etal., 1996]. While Xenopus hsp30A and C genes are firstheat-inducible at early tailbud, hsp30D is not stress-inducible until the late tailbud stage [Krone and Heik-kila, 1988, 1989; Krone et al., 1992; Ohan and Heikkila,1995]. This differential pattern of hsp30 gene expres-sion during development was also observed at the levelof hsp30 synthesis [Tam and Heikkila, 1995].

While the aforementioned studies have examined thedevelopmental stages at which the different small hspgenes were heat-inducible, no information is availablewith respect to the spatial pattern of hsp30 geneexpression during early Xenopus laevis development.In the present study, we used whole-mount in situhybridization and immunohistochemistry to examinethe spatial distribution of Xenopus hsp30 gene expres-sion in normal and heatshock-treated tailbud embryos.

MATERIALS AND METHODS

Maintenance of Xenopus laevis Embryos

Xenopus eggs were collected, fertilized, and dejelledas described by Heikkila et al. [1985]. Embryos weremaintained at 22°C in Steinberg’s solution (60 mMNaCl, 0.7 mM KCl, 0.8 mM MgSO4·7H2O, 0.3 mMCaNO3·4H2O, 1.4 mM Tris base, pH 7.4) and develop-mental stages were determined according to externalcriteria described by Nieuwkoop and Faber [1967].Embryos to be heat-treated were sealed with parafilmin petri dishes containing Steinberg’s solution. Thecontainer was then placed in a water bath at tempera-tures ranging from 26–35°C for 1 h. Control embryoswere maintained at 22°C. Embryos collected for RNAisolation were frozen and stored at 280°C. Embryoscollected for whole mount in situ hybridization werefixed for 2 h in MEMFA (0.1 M MOPS, pH 7.4, 2 mMEDTA, 1 mM MgSO4, 4% paraformaldehyde), rinsedtwice in methanol, and stored at 220°C.

Riboprobe Preparation

In vitro RNAsynthesis generating digoxygenin (DIG)-labeled riboprobes for Northern blot and in situ hybrid-ization was performed according to the manufacturer’sprotocol (Roche Molecular Biochemicals (BoehringerMannheim, Laval, Quebec). To synthesize the hsp30riboprobes, the coding region of hsp30C gene in pUC19[Krone et al., 1992] was isolated using EcoRI andBamHI. The ends were filled in using DNA polymerase(Klenow fragment) and cloned into the SmaI site of thepSP64(poly A) plasmid in both sense and antisense

orientations. Sense and antisense riboprobe was gener-ated by linearization of the appropriate construct withPvuII and transcription with SP6 RNA polymerase. Tosynthesize actin riboprobes, type 8 cytoskeletal actincDNA (pXIcAI) [Mohun et al., 1983] was cut withHindIII and subcloned into the HindIII site of pBlue-script KS (Gibco/BRL, Burlington, Ontario). Actin senseriboprobe was generated by linearization with XhoI andtranscription with T7 RNA polymerase while antisenseriboprobe was generated by linearization with EcoRIand transcription with T3 RNA polymerase.

RNA Isolation and Northern Blot Analysis

Total RNA was isolated from Xenopus embryos usingthe GIT/CsCl centrifugation method of Chirgwin et al.[1979] as modified by Ohan and Heikkila [1995]. Theembryos were homogenized in 10 ml of 4 M guanidineisothiocyanate and layered on 3.3 ml of 5.7 M cesiumchloride solution. A SW-41 Ti rotor (Beckman, Palo Alto,CA) was used to centrifuge the samples at 30 K for 23 h.The RNA pellets were recovered and precipitated twicein ethanol to remove cesium chloride. Concentration,purity, and integrity of the RNA was established byspectrophotometry and formaldehyde agarose gel elec-trophoresis. Three µg of total RNA was electrophoresedin 1.2% formaldehyde agarose gels [Sambrook et al.,1989]. The RNA was then transferred to positivelycharged nylon membrane (Roche Molecular Biochemi-cals, Laval, Quebec) and UV cross-linked with a GS-Gene linker (Bio-Rad, Mississauga, Ontario). The RNAblots were then subjected to rapid reversible stainingwith 0.02% methylene blue prior to hybridization tocheck for equal sample loading [Herrin and Schmidt,1988]. After prehybridization of the membrane in DIG-Easy-Hyb buffer (Roche Molecular Biochemicals, Laval,Quebec) for at least 4 h at 68°C, the buffer was replacedwith the same buffer containing DIG-labeled antisenseriboprobe and incubated at 68°C overnight. Chemilumi-nescence detection was performed in accordance withthe manufacturer’s protocol (Roche Molecular Biochemi-cals) followed by exposure of the blot to Kodak BioMaxfilm.

In Situ Hybridization

Albino Xenopus embryos obtained from the mating ofalbino females and normal males were used in thewhole mount in situ hybridization protocol as describedpreviously [Miskovic and Heikkila, 1999]. The resul-tant embryos did not show the presence of pigmenta-tion until the late tailbud stage. A nutator (VWR;Mississauga, Ontario) was used in all parts of theprocedure requiring shaking. The alkaline phosphatase-conjugated anti-DIG antibody was used at a 1:6,000dilution for hsp30 and a 1:8,000 dilution for cytoskel-etal actin riboprobes. In order to document the results,the embryos were rehydrated with decreasing grada-tions of methanol, counterstained in Bouin’s Fixative(VWR) and cleared for viewing in benzyl benzoate and

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benzyl alcohol at a ratio of 2:1 [Harland, 1991; Drysdaleet al., 1997]. The embryos were photographed using aNikon AFX-11 camera attached to a Nikon dissectingmicroscope utilizing EPT160T Kodak film.

Histological Analysis

Whole-mount in situ hybridized embryos used forhistological serial sectioning were embedded in para-plast, cut in 8–10 µm sections using a rotary microtome(American Optical), and mounted in Permount [Drys-dale et al., 1997]. Photos of histological sections weretaken using a Zeiss Axiophot microscope (Carl Zeiss,Don Mills, Ontario) and employing EPT 200 ASA Kodakfilm.

Whole-Mount Immunohistochemistry

Embryos from selected stages were either heatshocked for 2 h at 33°C or maintained at 22°C. Whole-mount immunohistochemistry of Xenopus embryos wasperformed essentially according to Dent et al. [1989].Fixation of embryos was performed at room tempera-ture for 2 h in 75% methanol / 25% DMSO. Followingrehydration in TBST (137 mM Tris, pH 7.5, 20 mMNaCl, 0.1% Triton X-100), embryos were bleached in15% hydrogen peroxide with TBST for 3 h, washedtwice in TBST, and treated with 5 µg/µl pronase for 15min at 22°C. Embryos were blocked overnight in TBSTwith 2 mg/ml BSA and 10% normal goat serum at 4°Con an orbital shaker. Primary antibody incubationusing an affinity purified polyclonal anti-hsp30 anti-body made against Xenopus recombinant hsp30C (Fern-ando and Heikkila, manuscript in preparation) diluted1:300 in fresh blocking solution was carried out over-night at 4°C. Embryos were washed 5 3 60 min inTBST and then incubated overnight at 4°C with antirab-bit alkaline phosphatase conjugated secondary anti-body (Sigma, St. Louis, MO) diluted 1:500 in blocking

solution. Embryos were washed 4 3 60 min in TBST,3 3 30 min in TBST with 5 mM levamisol and 2 3 10min in 100 mM Tris, pH 9.5, 50 mM MgCl2, 100 mMNaCl, 0.1% Tween 20, 5 mM levamisol. Detection ofbound antibody in whole-mount embryos was per-formed using a prepared NBT/BCIP stock (Boehringer)diluted in the final wash solution.

RESULTS

In Situ Hybridization Analysis of HeatShock-Induced hsp30 mRNA Accumulation

During Xenopus Early Development

As mentioned previously, no information is availableregarding the spatial pattern of hsp30 mRNA accumu-lation during Xenopus embryogenesis. Before carryingout whole-mount in situ hybridization analysis of Xeno-pus embryos the antisense riboprobes complementaryto the above mRNAs were tested for their specificity bymeans of Northern blot analysis. As shown in Figure1A, hsp30 mRNA accumulation was not detected incontrol embryos but was first heat shock-inducible atthe early tailbud stage. The relative levels of heat-induced hsp30 mRNA accumulation then increased atthe midtailbud and late tailbud stage. In Figure 1A, anoverexposed autoradiogram was used to display thepresence of early tailbud hsp30 mRNA such that thesignals for MTB and LTB exceed the linear range of filmdensity. In films given less exposure, we observed morehsp30 mRNA in LTB than in the MTB samples (datanot shown). This is in agreement with previous RNaseprotection analysis data showing an increase in heatshocked-induced hsp30 mRNA accumulation duringdevelopment from the early tailbud to the early tadpolestage [Krone and Heikkila, 1988].

Fig. 1. Relative levels of heat shock-inducible hsp30 mRNAaccumu-lation during early Xenopus laevis development. Total RNA wasisolated from control (c) and heat shocked (h; 1 h at 33°C) embryos atthe cleavage (C; stage 4), early blastula (B; stage 8), gastrula (G; stage11), neurula (N; stage 17/18), early (ETB; stage 22/23), mid- (MTB;stage 28) and late (LTB; stage 35) tailbud stage. Three µg of RNA wassubjected to Northern hybridization analysis employing DIG-labeled

Xenopus hsp30 (A) and cytoskeletal actin (B) antisense riboprobes asoutlined in Materials and Methods. The autoradiogram in A wasoverexposed in order to properly visualize the ETB signal. A slightdistortion in the agarose gel resulted in a slightly higher mobility inthe MTB lane. Arrows indicate the positions of the different mRNAs.Transcript sizes: hsp30, 1.1 kb; cytoskeleton actin, 1.8 and 2.0 kb.

HSP30 GENE EXPRESSION IN XENOPUS EMBRYOS 367

Whole-mount in situ hybridization was used to deter-mine the spatial distribution of hsp30 mRNA duringXenopus development employing DIG-labeled hsp30antisense riboprobe. Hsp30 mRNA, as indicated bydark staining, was not detected in neurula stage control(Fig. 2A) or heat shocked embryos (Fig. 2B). Interest-ingly, at the early (data not shown) and midtailbudstage, embryos displayed constitutive hsp30 mRNAaccumulation in the cement gland (Fig. 2C) which wasundetectable at the late tailbud stage (Fig. 2E). In heatshocked midtailbud embryos (Fig. 2D), hsp30 mRNAwas enriched in the cement gland, lens placode, somites,dorsal tail region, and proctodeum. This pattern ofhsp30 message distribution in heat shocked late tail-

bud embryos was more extensive (Fig. 2F), with addi-tional enrichment across the surface of the embryo aswell as enhanced levels of message in the tissuesassociated with the head region, including the cementgland and lens placode as well as in the somites, spinalcord, and anus. Control tailbud embryos (Fig. 2E)showed no detectable hsp30 mRNA accumulation. How-ever, it should be mentioned that at this stage ofdevelopment there is a natural increase in eye pigmen-tation and melanocyte production. In control experi-ments, we found that DIG-labeled hsp30 sense ribo-probe did not hybridize to any RNA present inmidtailbud control (Fig. 2G) or heat shocked embryos(Fig. 2H). For comparison, we examined the spatialpattern of cytoskeletal actin mRNA accumulation inearly, mid-, and late tailbud stages. Whole-mount insitu hybridization with DIG-labeled Xenopus cytoskel-etal actin antisense riboprobe showed a more general-ized pattern of actin mRNA distribution, with intensivestaining in the head, dorsal, and tail region, with less inthe ventral yolk tissues (Fig. 2I). This pattern was notenhanced by heat shock treatment (Fig. 2J).

The preferential accumulation of heat shock inducedhsp30 mRNA in different tissues of tailbud embryoswas verified by histological analysis of the embryosafter whole-mount in situ hybridization. Hsp30 mRNAwas observed in the cement gland cells of controlmidtailbud embryos (Fig. 3A) which was enhancedfollowing heat shock (Fig. 3B). An enlarged medialregion of a heat shocked in situ hybridized midtailbudembryo demonstrates the presence of hsp30 mRNA inthe somites (Fig. 3D). A cross-section of this area of theembryo confirmed that hsp30 mRNA was enriched inthe somitic region (Fig. 3E). Cross-sections of theheat-induced late tailbud medial region (Fig. 3F) alsorevealed enhanced accumulation of hsp30 mRNA in thesomitic region, as well as in the ectoderm. While hsp30mRNA was not detectable in histological sections of thecontrol midtailbud eye (Fig. 3G), hsp30 mRNA waspresent in the lens placode as well as in the optic cup(Fig. 3H) and proctodeum (Fig. 3C) of heat shockedembryos. Comparable histological analysis of heatshocked mid- and late tailbud stage embryos hybridizedwith DIG-labeled hsp30 sense riboprobe did not revealthe presence of hybridizable RNA (data not shown).

We also characterized the accumulation of hsp30mRNA in time course and recovery experiments. Forexample, heat shock-induced accumulation of hsp30mRNA at 33°C was detectable within 15 min of heatshock in the somites, lens placode, and cement gland(data not shown) reaching maximal levels in theseareas plus the proctodeum at 1 h. The pattern of hsp30mRNA accumulation during recovery conditions at22°C following a heat shock at 33°C for 1 h is shown inFigure 4. After 1 h of recovery there was a significantdrop in the accumulation of hsp30 mRNAin the somites,lens placode, and proctodeum (Fig. 4C). This decline inthe abundance of hsp30 message continued in these

Fig. 2. Spatial pattern of hsp30 mRNA accumulation during earlyXenopus development. Whole-mount in situ hybridization with DIG-labeled hsp30 antisense riboprobe was carried out with control(A,C,E) and heat shocked (B,D,F; 1 h at 33°C) Xenopus embryos atneurula (A,B; stage 17/18), mid- (C,D; stage 28) and late (E,F; stage35) tailbud stages. At the late tailbud stage, control embryos (E)display a natural increase in eye pigmentation and melanocyteproduction. G,H: Control and heat shocked midtailbud stage embryos(stage 28), respectively, hybridized with DIG-labeled hsp30 senseriboprobe. I,J: Control and heat shocked midtailbud stage embryos(stage 28), respectively, hybridized with DIG-labeled actin antisenseriboprobe. LP, lens placode; S, somites; P, proctodeum; CG, cementgland; SC, spinal cord; A, anus; ME, melanocytes.

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tissues after 3 h recovery, although the increase indensity in the lens placode is due to a natural increasein pigmentation at this time of development (Fig. 4D).By 7–11 h of recovery, hsp30 mRNA was not detectablein the somitic regions and proctodeum, while the rela-tive levels in cement gland were still greater thancontrol (Fig. 4E,F). In general, the temporal pattern ofhsp30 mRNA accumulation during heat shock and the

decay of this message during recovery from heat shockis similar to our previous Northern blot hybridizationanalyses with tailbud embryos [Krone and Heikkila,1988].

Effect of Different Temperatures on the SpatialPattern of Hsp30 mRNA Accumulation in

Tailbud Embryos

We examined the effect of a range of heat shocktemperatures from 26–35°C on hsp mRNA accumula-tion (Fig. 5). Elevation of the incubation temperaturefrom 22–26°C for 1 h did not induce the accumulation ofhsp30 mRNA (data not shown). However, placement ofmidtailbud embryos at 30°C resulted in enhancedhsp30 mRNA accumulation in the lens placode andsomites (Fig. 5B). A similar but more enhanced patternwas observed in midtailbud embryos incubated at 33°C,plus an enrichment of hsp30 mRNA in the proctodeumand tail region (Fig. 5C). Compared to the previoustemperatures, the pattern of hsp30 mRNA distributionat 35°C was enhanced in the head and tail regions, withenrichment in the cement gland, lens placode, somites,spinal cord, and proctodeum (Fig. 5D).

Immunohistochemical Analysis of Hsp30 ProteinAccumulation in Xenopus Embryos

In order to examine the accumulation of hsp30protein in control and heat shocked Xenopus late tail-bud and early tadpole embryos before and after heatshock, we carried out immunohistochemistry employ-ing an affinity-purified polyclonal antibody made againstrecombinant Xenopus hsp30 protein. As shown in Fig-ure 6A,C, the antibody reacted with protein in thecement gland of control midtailbud and early tadpoleembryos maintained at 22°C. Following a 2-h heatshock at 33°C, an increase in hsp30 was observed in thelens placode, the somitic region, tail, and the procto-deum of midtailbud embryos (Fig. 6B). In late tailbudembryos, heat shock induced a global accumulation ofhsp30 protein with enrichment in the anterior region(Fig. 6D).

DISCUSSIONWhile our laboratory and others have obtained infor-

mation concerning heat shock-induced accumulation ofhsp30 mRNA during Xenopus laevis development [re-viewed in Heikkila et al., 1997], little is known regard-ing the spatial distribution of these messages duringembryogenesis. To further characterize the expressionpattern of the hsp30 gene family, we employed whole-mount in situ hybridization to examine hsp30 mRNAaccumulation and their spatial distribution duringearly development. Constitutive hsp30 mRNA accumu-lation was detected only in the cement gland of earlyand midtailbud embryos. The cement gland is a mucus-secreting structure found at the anterior end of the frogembryo which permits attachment of the newly hatched

Fig. 3. Histological analysis of hsp30 mRNA accumulation insections from Xenopus tailbud embryos. Whole-mount in situ hybrid-ization with DIG-labeled hsp30 antisense riboprobe was performed oncontrol and heat shocked (1 h at 33°C) mid- (stage 28) and late (stage35) tailbud embryos followed by histological analysis as described inMaterials and Methods. A,B: Anterior sections of control and heatshocked midtailbud embryos, respectively, showing the cement gland.C: Cross-section displaying the proctodeum in heat shocked midtail-bud embryos. D: An enlarged medial region of a heat shocked mid-tailbud whole-mount embryo (stage 28) hybridized with DIG-labeledhsp30 antisense riboprobe and demonstrates the abundance of hsp30mRNA in the somites. E,F: The presence of hsp30 mRNA in the somiticregion of heat shocked mid- and late tailbud embryos, respectively.G,H: Cross-sections of the eye of control and heat shocked midtailbudembryos. CG, cement gland; M, mesencephalon; LP, lens placode; OC,optic cup; N, notochord; S, somites; HG, hindgut; P, proctodeum; E,ectoderm.

HSP30 GENE EXPRESSION IN XENOPUS EMBRYOS 369

embryo to a solid support. It originates from theepithelial layer of frog ectoderm and is the first ectoder-mal organ to differentiate [Jamrich and Sato, 1989;Sive et al., 1989; Drysdale and Elinson, 1993; Sive andBradley, 1996]. The accumulation of hsp30 mRNA inthe cement gland was transient, since it was notevident in late tailbud stage or later embryos. Theaccumulation of this message in the cement gland of

control embryos was presumably below the level ofdetection of Northern blot analysis. These cementgland hsp30 transcripts likely do not include hsp30A,hsp30C, or hsp30D mRNA because these messageswere not detected constitutively at these developmen-tal stages with either RNase protection analysis and/orRT-PCR [Krone and Heikkila, 1989; Krone et al., 1992;Ali et al., 1993; Ohan and Heikkila, 1995]. It is possible

Fig. 4. Pattern of hsp30mRNA accumulation in tailbudembryos during recovery fromheat shock. Whole-mount insitu hybridization was carriedout with DIG-labeled hsp30 an-tisense riboprobe. MidtailbudXenopus embryos (stage 28)were exposed to either 22°C (A)or 33°C for 1 h (B) followed byrecovery at 22°C for either 1 h(C), 3 h (D), 7 h (E) or 11 h (F).CG, cement gland; LP, lens plac-ode; P, proctodeum; S, somites.

Fig. 5. Effect of differenttemperatures on the spatial pat-terns of hsp30 mRNAaccumula-tion in Xenopus midtailbud(stage 28) embryos. Whole-mount in situ hybridization wascarried out with DIG-labeledhsp30 antisense riboprobe. Themidtailbud embryos were ex-posed to 1-h heat treatments ateither 22°C (A), 30°C (B), 33°C(C), or 35°C (D). CG, cementgland; LP, lens placode; S,somites; P, proctodeum; SC, spi-nal cord.

370 LANG ET AL.

that the cement gland hsp30 mRNA is an as-yetunidentified member(s) of the hsp30 family. Whole-mount immunohistochemical analysis indicated thepresence of hsp30 protein in the cement gland of latetailbud and early tadpole stage embryos. Thus, whilehsp30 message accumulated in the early and midtail-bud stages before decaying, hsp30 protein was main-tained until at least early tadpole. Constitutive tissue-specific expression of small hsps has also been describedduring the development of other animals, includingnematode [Stringham et al., 1992], Drosophila [Marinet al., 1993], brine shrimp [Liang and MacRae, 1999],mouse [Gernold et al., 1993; Tanguay et al., 1993;Benjamin et al., 1997], and rat [Wilkinson and Pollard,1993; Mirkes et al., 1996]. For example, in Drosophilathe gene encoding hsp23 was expressed constitutivelyin brain, thoracic ganglion, fat body, and gonads ofyoung flies while hsp26 was expressed primarily inovaries and testes [Marin et al., 1993; Michaud et al.,1997]. The mechanism associated with the constitutiveexpression of hsp30 genes in the cement gland ofXenopus tailbud embryos is unclear at present. It isunlikely that the cement gland undergoes a localizedstress-like response since we have not detected hsp70mRNA in this region under control conditions (Lang,et al., in press). In other organisms, such as Drosophila,transcription of small hsp genes can be inducedby hormones such as ecdysone [Arrigo and Landry,1994]. It is possible that Xenopus cement glandhsp30 genes may be induced by an as-yet uniden-tified hormone or inductive agent. In support of thispossibility, hsp30 gene expression has been observedin the livers of thyroid hormone-treated metamor-

phosing tadpoles of Rana catesbeiana [Helbing et al.,1996].

The function of constitutively synthesized hsp30 inthe cement gland is not known at present. Numerousstudies have shown that small hsps can act as molecu-lar chaperones by binding to partially unfolded proteinsand preventing further aggregation by maintainingthem in a soluble state, as well as enhancing theirrefolding by other molecular chaperones [Arrigo andLandry, 1994; Jakob and Buchner 1994; Hartl, 1996;Arrigo, 1998]. In Xenopus, it is possible that hsp30 mayfunction as a molecular chaperone and interact withcytosolic proteins in the mucus-secreting cells of thecement gland. Another possible role for Xenopus hsp30may be in the prevention of apoptosis of the cementgland, which is a transient organ and eventually lost atthe tadpole stage. It has been shown in mammaliancells that small hsps are transiently expressed duringthe cell division to differentiation transition and appearto be essential for preventing differentiating cells fromundergoing apoptosis [Mehlen et al., 1997, 1999; Arrigo,1998]. Given these previous studies, it is possible thatXenopus hsp30 genes are expressed in cement glandcells to prevent them from undergoing apoptosis. Sincehsp30 mRNA is lost by the late tailbud stage, whereasthe protein is still detectable, it is possible that hsp30could function in this capacity until it is degraded.Further work is required to assess the possible functionof hsp30 as a molecular chaperone or inhibitor ofapoptosis in the cement gland. Since hsp30 proteinappears to be synthesized only in the cement gland intailbud embryos, it could serve as a useful marker instudies of anteriorposterior axis formation, along with

Fig. 6. Whole-mount immu-nohistochemical analysis ofhsp30 protein accumulation inXenopus embryos. Late tailbud(stage 35; A,B) and early tad-pole (stage 41/42) embryos wereincubated at either 22°C (A,C)or 33°C (B,D) for 2 h. The em-bryos were then processed forimmunohistochemical analysisusing an affinity-purified poly-clonal hsp30 antibody as out-lined in Materials and Meth-ods. The outer portion of thetailfin was quite transparent inthe late tailbud stage embryosand is not readily visualized inthe photograph in C. CG, ce-ment gland; LP, lens placode;SR, somitic region; P, procto-deum.

HSP30 GENE EXPRESSION IN XENOPUS EMBRYOS 371

other markers such as the XCG gene, which encodes amucin-like protein whose synthesis is restricted to thecement gland [Sive and Bradley, 1996].

The present study demonstrates that heat shock-induced accumulation of hsp30 mRNA occurred first atthe early tailbud stage and was preferentially enrichedin selected tissues. Two questions arise from thesefindings: Why is heat shock-induced hsp30 mRNAaccumulation not detectable until the early tailbudstage, and Why is there preferential enrichment ofthese messages in specific tissues? The mechanism(s)involved in the heat shock-induced developmental regu-lation of the hsp30 gene family is not known. It ispossible that alterations in chromatin structure may beinvolved in the sequential activation of the Xenopushsp30 gene cluster during embryogenesis [Heikkila etal., 1997]. A similar theory has been put forward toexplain the pattern of expression of a small hsp genecluster in Caenorhabditis elegans [Dixon et al., 1990],as well as the developmental regulation of expression ofthe globin gene family cluster [Lowrey et al., 1992].Regulation of hsp30 gene expression by chromatinstructure would explain previous studies in whichmicroinjected hsp30 gene constructs were prematurelyexpressed at the midblastula stage in a heat-induciblefashion [Krone and Heikkila, 1989; Ali et al., 1993]. Inthese experiments, it was possible that the microin-jected DNA constructs were not assembled into theproper chromatin conformation, and thus were notcorrectly regulated during embryogenesis.

As mentioned above, the present study has demon-strated that heat shock-induced a preferential accumu-lation of hsp30 message in certain tissues of Xenopusmidtailbud embryos. For example, heat shock-induced(33°C) hsp30 message and protein accumulation wasmore abundant in the cement gland, lens placode,somites, and proctodeum than in other tissues of mid-tailbud embryos. In contrast, actin mRNA displayed amore generalized pattern of accumulation which wasnot significantly altered after heat shock. This heat-induced pattern of Xenopus hsp30 gene expression inmidtailbud embryos was first detectable at 30°C,whereas at the higher temperature of 35°C, there was amore widespread pattern of message accumulation.These experiments suggest that the cement gland,somites, lens placode, and proctodeum may be moresensitive in the activation of hsp gene expression thanother tissues in the midtailbud embryo. A similarphenomenon has been observed in other developmentalsystems. For example, preferential heat shock-inducedsmall hsp gene expression was detected in the somiticregions, neuroepithelium, and mesenchymal cells of11-day rat embryos [Mirkes et al., 1996]. Also, duringDrosophila spermatogenesis hsp23 and hsp27 genesare expressed in a cell-specific pattern in the malegonads of heat shocked animals [Michaud et al., 1997].The mechanism involved in the preferential heat shock-

induced accumulation of hsp30 mRNA in selected re-gions of Xenopus midtailbud embryos is unclear. Sinceheat shock-induced hsp30 gene transcription involvesthe activation of heatshock factor (HSF), it is possiblethat these temperature-sensitive tissues of the midtail-bud stage embryo may have a lower temperature setpoint for HSF activation than in other tissues. In adultXenopus, we found that heart tissue has a lower HSFactivation temperature than other tissues examinedsuch as liver [Ali et al., 1997]. A similar finding has beenmade with mouse pachytene spermatocytes [Sarge,1995; Sarge et al., 1995]. In these latter studies, it wassuggested that the temperature of HSF activation maynot necessarily have a fixed value and that it can varyin a tissue-dependent manner. Therefore, it is possiblethat somite, lens placode, cement gland, and procto-deum may have a lower HSF activation temperaturecompared to other tissues. However, it is also possiblethat other Xenopus transcription factors may be in-volved in this phenomenon as well. This possibility hasbeen suggested previously in the cell-specific heatshock induction of hsp23 in the eye and testes ofDrosophila melanogaster [Marin et al., 1996; Michaudet al., 1997].

It is likely that the preferential expression of hsp30genes in somites, lens placode, cement gland, andproctodeum of early and midtailbud embryos after heatshock may be indicative of a protective function. Anumber of studies have shown that the synthesis ofsmall hsps can confer stress resistance presumably viaits role as a molecular chaperone [Arrigo and Landry,1994; Jakob and Buchner, 1994; Hartl, 1996; Arrigo,1998]. Also, the localization of small hsps to the nucleusindicated that they might be involved in protection ofnuclear structure [Arrigo and Landry, 1994]. Finally, itis possible that Xenopus hsp30 may stabilize the actincytoskeleton resulting in prolonged cell survival as hasbeen shown in other systems [Lavoie et al., 1993; Arrigoand Landry, 1994; Huot et al., 1996; Ehrnsperger et al.,1997]. Additional work is required to evaluate thesepossibilities in Xenopus embryos.

ACKNOWLEDGMENTSThis research was supported by a Natural Sciences

and Engineering Research Council grant to J.J.H. Theauthors thank Dr. Tom Drysdale for assistance with insitu hybridization and Dale Weber for technical aid inthe preparation of histological sections.

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