Expression and function of small heat shock protein genes during Xenopus development

Seminars in Cell & Developmental Biology 14 (2003) 259–266

Expression and function of small heat shock protein genes during Xenopus development John J. Heikkila∗ Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1

Abstract The hsp30 small heat shock protein family is a stress-inducible group of molecular chaperones in the frog, Xenopus laevis. Hsp30 genes are intronless and present in clusters. Expression of these genes are developmentally regulated likely at the level of chromatin structure. Also heat-induced hsp30 transcripts and protein are enriched in selected embryonic tissues. In vitro studies revealed that multimeric hsp30 binds to heat denatured target protein, inhibits their aggregation and maintains them in a folding-competent state until reactivated by other cellular chaperones. Finally optimal chaperone activity and secondary structure of hsp30 can be inhibited by phosphorylation or mutagenesis of the C-terminal end. © 2003 Elsevier Ltd. All rights reserved. Keywords: Heat shock; Development; mRNA; Transcription; Chaperone

1. Introduction The heat shock response is a rapid and transient change in cellular activities that ensures cell survival by protecting essential cellular components against damage [1–4]. Transcription of heat shock protein (hsp) genes, which are intimately involved in this phenomenon, is mediated by the heat shock element (HSE) found in the 5 upstream regions of these genes and interacts with a transcription activating protein known as heat shock factor (HSF). HSF preexists within the cell as an inactive monomer that is converted into an active trimer upon heat shock that is capable of binding to the HSE and facilitating transcription of hsp genes. One of the triggers for HSF activation is the accumulation of unfolded protein. Hsps are composed of three major families, the high molecular weight (hsp90) family, the hsp70 family, and the small hsp (shsp) family [1–4]. The shsps range in size from 12 to 43 kDa and include the lens protein ␣-crystallin since they are inducible by stress and possess many of the same physical and functional properties found in other shsps [5–8]. The shsps exhibit a low degree of conservation with the exception of an 80–100 amino acid domain that is also found in ␣-crystallins [9,10]. Both shsps and ␣-crystallins have the ability to aggregate into large, highly polymeric structures that appear to be necessary for function within the

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cell [9,10]. Various in vivo functions have been proposed for shsps including chaperone activity, actin capping/decapping activity, cellular differentiation and modulation of redox parameters [5,7,8]. It has been demonstrated that overexpression of shsps can confer thermotolerance [5,11]. This is likely due to the molecular chaperone properties of shsps in protecting cellular proteins by preventing their aggregation or misfolding and maintaining their solubility [12,13]. Finally shsp synthesis or mutations have been associated with various diseases such as cancer, multiple sclerosis, a range of neuropathologies and muscle myopathy [14–16]. While much is known about shsps in cultured cells less is known about the expression and function of these molecular chaperones during early animal development. Our laboratory has been involved in the examination of the expression and function of the shsp gene family, hsp30, during early embryogenesis of the South African Clawed frog, Xenopus laevis. Xenopus is a unique model system that has been used for many decades. Their eggs are easily obtainable in large quantities and can be fertilized in vitro and the large size of the oocytes and eggs make them suitable for microinjection studies [17]. A large amount of information is available regarding Xenopus early embryonic development at both the cellular and molecular level. Early development is characterized by a relatively rapid series of cleavages following fertilization during which the zygotic genome is essentially transcriptionally quiescent [18]. Synthesis of new protein during this time utilizes pre-exisiting maternal mRNA. At the midblastula stage the embryonic genome is activated and

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results in the transcription of selected genes. In response to a series of inductive events the embryos then develop through gastrula, neurula, tailbud and tadpole stages. The following report will be an overview of hsp30 gene structure and expression as well as the ability of the hsp30 to act as a molecular chaperone.

2. Hsp30 gene structure Two clusters of Xenopus laevis hsp30 genes (hsp30A–E) have been isolated to date. The first cluster containing hsp30A and hsp30B are not representative of the hsp30 family since hsp30A contains a 21 bp insertion in the coding region while hsp30B appears to be a pseudogene [19,20]. The second gene cluster isolated in our laboratory contains two complete hsp30 genes, hsp30C and hsp30D as well as a portion of a third gene, hsp30E [21]. The DNA sequence of hsp30C and hsp30A share a high degree of similarity (97%) while hsp30D gene was only 75% similar to the aforementioned hsp30 genes. Both the hsp30C and hsp30D genes are intronless and encode 24 kDa proteins. An examination of the amino acid sequence of hsp30C and hsp30D revealed the presence of a conserved ␣-crystallin domain in the C-terminal region that has similarity to other eukaryotic shsps [21,22]. The 5 regulatory region of the hsp30C gene contains two TATA boxes, three HSEs (one single and two present as an overlapping doublet), and a CCAAT box. The 3 untranslated portion of the hsp30C gene is AT-rich, contains a polyadenylation element and regulatory region as well as a mRNA instability sequence [21]. It is likely that there are additional hsp30 genes since early work by Bienz [19] found 2 unique hsp30 cDNAs. Furthermore, a large hsp30 gene family is suggested by the numerous hsp30 isoforms detected by two-dimensional (2D) polyacrylamide gel electrophoresis (2D-PAGE) and immunoblot analysis [23,24].

3. Developmental regulation of stress-inducible hsp30 gene expression Early studies by Bienz [19,20] found that hsp30A was not constitutively expressed during early embryogenesis and that it was not heat-inducible until the tadpole stage. Subsequent experiments summarized in Table 1, employing RNase protection analysis and reverse transcription–polymerase chain reaction (RT–PCR) assays were able to narrow down the developmental stage at which hsp30A and hsp30C genes were first heat-inducible to the late neurula/early tailbud stage [25–28]. Interestingly, an analysis of hsp30D gene expression determined that it was first heat-inducible at the midtailbud stage which is approximately 1 day later in development than found for hsp30A and hsp30C [29]. Thus, the developmental pattern of hsp30 gene expression is in contrast to other hsp genes including hsp70 and hsp90 that

Table 1 Differential heat-induced expression of the hsp30 gene family during Xenopus laevis developmenta MrNA

Cleav.

MB

Gast.

Neur.

Early TB

MidTB

TP

Hsp30A Hsp30C Hsp30D Hsp70 Hsp90

− − − − −

− − − + +

− − − + +

± ± − + +

+ + − + +

+ + + + +

+ + + + +

Cleav.: cleavage; MB: midblastula; Gast.: gastrula; Neur.: neurula; Early TB: early tailbud; MidTB: midtailbud; TP: tadpole. a All embryos were heat shocked and the isolated RNA was analyzed by either RNase protection analysis or RT–PCR.

are first heat-inducible after the midblastula stage that coincides with the activation of the embryonic genome [30–32]. Whole mount in situ hybridization and immunocytochemical analysis of heat shock-treated embryos revealed a preferential accumulation of hsp30 mRNA and protein in selected tissues of Xenopus midtailbud embryos [28]. For example, heat shock-induced hsp30 mRNA and protein accumulation was more abundant in the cement gland, lens placode, somites, and proctodeum than in other tissues of midtailbud embryos. However actin mRNA displayed a more generalized pattern of accumulation which was not significantly altered after heat shock. These experiments suggested that certain embryonic tissues were more sensitive in the activation of hsp gene expression than other tissues in the midtailbud embryo (Fig. 1A and B). A similar phenomenon has been observed in other developmental systems. For example, preferential heat shock-induced shsp gene expression was found in the somitic regions, neuroepithelium and mesenchymal cells of 11-day rat embryos [33]. Also, during Drosophila spermatogenesis, hsp23 and hsp27 genes are expressed in a cell-specific pattern in the male gonads of heat-shocked animals [34]. The preceding studies demonstrated that heat-inducible expression of hsp30 genes are developmentally regulated in Xenopus embryos. In an effort to determine the mechanism(s) involved in the developmental stage-dependent expression of hsp30 genes initial investigations employed microinjection technology. However, microinjection of hsp30A promoter/test gene constructs or tagged hsp30 gene constructs with extensive 5 and 3 flanking regions of the gene displayed correct heat-inducibility but were not developmentally regulated after the midblastula stage [26,27]. It was suggested that the premature expression of microinjected hsp30 gene constructs reflected the inability of these constructs to enter a chromatin state that was analogous to the endogenous hsp30 genes. Recently we examined the role of histone hyperacetylation on heat shock-induced expression of hsp30 genes [35]. Embryos were treated with the histone deacetylase inhibitors (HDIs), trichostatin A, sodium butyrate and valproic acid (Fig. 1C). These agents have been shown to enhance the acetylation of lysine residues of histone tails by reducing their positive charge, weakening

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Fig. 1. Pattern of hsp30 mRNA accumulation during early Xenopus development. Whole mount in situ hybridization with DIG-labeled hsp30 antisense riboprobe was carried out with control (A) and heat shocked (B) (1 h at 33 ◦ C) Xenopus midtailbud stage embryos. Preferential heat-induced accumulation of hsp30 mRNA was observed in lens placode (LP), somites (S), proctodeum (P) and cement gland (CG). Constitutive hsp30 mRNA accumulation was also observed in cement gland. (C) Treatment of Xenopus embryos with HDIs resulted in premature heat-induced accumulation of hsp30 mRNA at the gastrula stage rather than at the late neurula–early tailbud stages as found in non-HDI-treated embryos. HDI treatment enhanced heat-induced hsp30 mRNA accumulation in neurula and tailbud embryos. These experiments suggest that hsp30 gene expression during development is regulated at the level of chromatin structure. Xenopus embryos were incubated with (+) or without (−) 30 nM trichostatin A (TSA), 5 mM valproic acid (VPA), or 5 mM sodium butyrate (NaB). Total RNA was isolated from control (C; 22 ◦ C) and heat shocked (H; 1 h at 33 ◦ C) embryos and subjected to Northern blot analysis using an hsp30 antisense riboprobe. Arrows indicate the positions of hsp30 mRNA.

electrostatic interactions with DNA and other nucleosomes resulting in a looser chromatin conformation [36,37]. HDI treatment not only enhanced heat-induced hsp30 mRNA accumulation but also caused it to first occur at the gastrula stage of development rather than late neurula/early tailbud in non-HDI-treated embryos. These findings are supported by studies showing that the repression of Drosophila hsp26 gene expression in a reconstituted chromatin cell-free system was removed when the chromatin was acetylated [38]. Furthermore, other studies examining a variety of developmentally regulated including genes encoding ␥-globin, Xenopus histone H1, telomerase, and ␤-interferon [39–42], exhibited premature or enhanced expression in the presence of HDIs. The mechanism responsible for the HDI associated premature expression and enhancement of heat shock-induced hsp30 mRNA accumulation in Xenopus embryos is not known. It has been suggested that histone acetylation in the presence of HDIs results in the relief of repressive chromatin domains that contain deacetylated histone [43]. If this is the case then HDIs may alter the conformational state of hsp30 chromatin such that it is more accessible to transcription factors. Future studies will be required to determine whether the putative repressed state of hsp30 chromatin in

early embryos is associated with histone deacetylase and co-repressor complexes such as Rpd3 and Sin3 that are present during early Xenopus development [44,45]. Also once the state of hsp30 chromatin is relieved from repression it is not known whether their transcription is associated with histone acetylation. The involvement of histone acetylation in the activation of transcription is complex and may be promoter-specific. For example, analysis of yeast HSF-1 promoters revealed that in response to heat shock some hsp gene promoters such as SSA3 and CUP1 show increased histone acetylation while others such as SSA4, hsp104, and hsp82 displayed a decrease [43]. While it is possible that the transcriptional activation of some Xenopus shsp genes may require chromatin acetylation the possibility that other types of modification such as phosphorylation cannot be ruled out. The mechanism involved in the preferential heat shock-induced accumulation of hsp30 mRNA in selected regions of Xenopus midtailbud embryos is unclear. HDI treatment did not alter the spatial pattern of heat-induced hsp30 mRNA accumulation in neurula and tailbud embryos [35]. Thus it is likely that this aspect of hsp30 gene expression is not governed by chromatin conformation. Since heat shock-induced hsp30 gene transcription involves the

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activation of HSF, it is possible that these temperature sensitive tissues of the midtailbud stage embryo may have a lower temperature set point for HSF activation than in other tissues. In previous work we found that adult heart tissue had a lower HSF activation temperature than other tissues examined such as liver [46]. Comparable results have been reported with mouse pachytene spermatocytes [47,48]. In these latter studies it was suggested that the temperature of HSF activation may have varied in a tissue-dependent manner. Therefore it is possible that in Xenopus the somite, lens placode, cement gland and proctodeum may have a lower HSF activation temperature compared to other tissues. However one cannot exclude the possibility that other Xenopus transcription factors are involved in this phenomenon.

4. Constitutive Hsp30 gene expression in cement gland In whole mount in situ hybridization analyses constitutive hsp30 mRNA accumulation was detected only in the cement gland of early and midtailbud embryos (Fig. 1A) [28]. The cement gland, which is a mucus-secreting structure found at the anterior end of the frog embryo, permits attachment of the newly hatched embryo to a solid support. This structure is derived from the epithelial layer of frog ectoderm and is the first ectodermal organ to differentiate [49,50]. Hsp30 mRNA accumulation in the cement gland was transient since it was not evident in late tailbud or later stage embryos. These cement gland hsp30 transcripts do not include hsp30A, hsp30C or hsp30D mRNA because these messages were not detected constitutively at these developmental stages with either S1 nuclease, RNase protection analysis and/or RT–PCR [19,21,25,27,29]. It is likely that the cement gland hsp30 mRNA is an as yet unidentified member(s) of the hsp30 family. Whole mount immunohistochemical analysis revealed the presence of hsp30 protein in the cement gland of late tailbud and early tadpole stage embryos. Thus, while hsp30 message accumulated in the early and midtailbud stages before decaying, hsp30 protein was present until at least early tadpole. Constitutive tissue-specific shsp gene expression has also been described during in other animal developmental systems including nematode, Drosophila, brine shrimp, mouse, and rat [33,51–54]. The mechanism associated with the constitutive expression of hsp30 genes in the cement gland of Xenopus tailbud embryos is not known. It is unlikely that the cement gland undergoes a localized stress-like response since we did not detect hsp70 mRNA in this region under control conditions [55]. In other organisms, such as Drosophila, shsp genes are inducible by hormones such as ecdysone [9]. It is possible that hsp30 genes in Xenopus cement gland was induced by an as yet unidentified hormone or inductive agent. In support of this possibility, hsp30 gene expression has been observed in the livers of thyroid hormone-treated metamorphosing tadpoles of Rana catesbeiana [56].

It is possible that hsp30 may function as a molecular chaperone and interact with cytosolic proteins in the mucus-secreting cells of the cement gland. Another possible role for Xenopus hsp30 may be in the prevention of apoptosis of the cement gland, which is a transient organ and eventually lost at the tadpole stage. In mammalian cells shsps are transiently expressed during the cell division to differentiation transition and appear to be essential for preventing differentiating cells from undergoing apoptosis [5]. Therefore, it is possible that Xenopus hsp30 genes are expressed in cement gland cells to prevent them from undergoing apoptosis. Since hsp30 mRNA is lost by the late tailbud stage whereas the protein is still detectable, it is possible that hsp30 could function in this capacity until it is degraded. Further work is required to assess the possible function of hsp30 as a molecular chaperone or inhibitor of apoptosis in the cement gland.

5. Pre-tailbud Hsp30-like mRNAs We have also detected by Northern blot analysis a unique group of heat-inducible hsp30-like mRNAs in late blastula and gastrula Xenopus embryos [29]. These hsp30-like transcripts originally escaped attention due to the fact that they were present in very low amounts in heat shock-treated embryos (approximately 100-fold less than heat shocked tailbud embryos). Furthermore, these transcripts were not detected with specific probes designed for hsp30A, hsp30C and hsp30D transcripts in RNase protection and RT–PCR assays. The exact identity of these pre-tailbud (PTB) hsp30-like transcripts will require the cloning of their cDNAs. The PTB hsp30-like mRNAs appear to be unstable transcripts since translation products were not detected and treatment of late blastula and gastrula embryos with cycloheximide, an agent known to stabilize unstable messages, greatly enhanced their levels after heat shock.

6. Hsp30 and Basic Shsp gene expression in somatic cells Analysis of hsp30 gene expression in Xenopus laevis A6 kidney epithelial cells has been used for comparative purposes with early embryos to examine the effect of various stressors. In addition to heat shock other stressors can induce hsp30 gene expression including sodium arsenite and herbimycin A [23,57,58]. Continuous exposure of cells to either heat shock or sodium arsenite resulted in transient but markedly different temporal patterns of hsp30 mRNA accumulation and hsp30 protein synthesis [23]. For example maximal hsp30 mRNA accumulation occurred after 1 h in response to a 33 ◦ C heat shock whereas hsp30 message levels after sodium arsenite treatment (50 ␮M) peaked at 12 h. Interestingly, treatment of A6 cells with a combination of stressors resulted in a synergistic effect on hsp30 gene ex-

J.J. Heikkila / Seminars in Cell & Developmental Biology 14 (2003) 259–266

pression. Treatment of cells with low heat shock temperatures or sodium arsenite concentrations produced in a very minor accumulation of hsp30 mRNA [57]. However, combining the stresses caused a very dramatic increase in hsp30 mRNA levels that is similar to that found with higher heat shock temperatures. Similar findings have been made with herbimycin A and heat shock [58]. The effect of multiple stressors is of importance given the fact that aquatic organisms are particularly at risk for this type of situation. Thus, it will be important to extend these analyses to early embryogenesis. In addition to the hsp30 gene family we have also detected a new group of 5 stress-inducible basic 30 kDa shsps (Bshsps) in Xenopus cultured cells by means of 2D non-equilibrium pH gradient gel electrophoresis [59]. These Bshsps were distinct from the more acidic hsp30 family based on charge and immunoreactivity. Also two of the five Bshsps were present constitutively. Heat shock-inducibility of Bshsp synthesis was regulated at the level of transcription and coordinately expressed with the hsp30 gene family. The presence of unique acidic and basic families of shsps has also been described in desert topminnows [60]. Isolation of the genes for the Bshsps will assist in determining their regulation and function in somatic cells and developing embryos as well as determining their evolutionary relationship with respect to other eukaryotic shsps.

7. Hsp30 synthesis and chaperone function Protein labeling and immunoblot studies revealed that neither constitutive nor heat-inducible synthesis of hsp30 was detected in cleavage, blastula, gastrula or neurula stage embryos [24]. During development the first heat shock-induced hsp30 family member to be synthesized was hsp30C at the early tailbud stage [24]. Furthermore, 2D-PAGE/immunoblot analysis revealed 5 additional heat-inducible hsp30 polypeptides in late tailbud embryos and a total of 13 shsps at the early tadpole stage. Approximately 16 members of the hsp30 family were detected in Xenopus A6 kidney epithelial cells. A comparison of the early tadpole and A6 cell hsp30 pattern revealed a number of common and unique proteins. Given the large number of shsps in Xenopus tadpoles it is likely that some of the spots on 2D-PAGE gels were the result of post-translational modifications such as phosphorylation that has been observed recently with Xenopus hsp30 [61]. It is possible that multiple hsp30 genes are responsible for some of the isoforms. Multiple shsps are not unique to Xenopus since they have been detected in other organisms such as Drosophila, desert topminnow and Caenorhabditis elegans [60,62,63]. Once hsp30 is synthesized in response to heat shock these proteins assemble into high molecular weight complexes as detected by rate zonal centrifugation and pore exclusion limit electrophoresis [64]. In A6 kidney epithelial cells these aggregates contain essentially all of the differ-

263

ent hsp30 isoforms synthesized in these cells. Additionally, heat-inducible hsp70 was present but it could not be determined whether it was part of the multimeric complex. Future studies of heat shock-induced hsp30 multimeric complexes during development will be of interest given the fact that different hsp30 isoforms are synthesized at different stages of embryogenesis [24]. In an attempt to understand their functional role the chaperone activity of recombinant hsp30C and hsp30D was examined [65,66]. In these studies we found that synthesis of Xenopus hsp30C in bacteria conferred thermal resistance and that recombinant hsp30C and D functioned as molecular chaperones in an ATP-independent fashion by inhibiting heat-induced aggregation of citrate synthase (CS) and luciferase (LUC) by maintaining them in a soluble form (Fig. 2A) [65,66]. The question of whether hsp30C and hsp30D could maintain heat- or chemically-denatured proteins in a folding-competent state was addressed using an in vitro rabbit reticulocyte lysate (RRL) refolding assay system as well as a novel in vivo Xenopus oocyte microinjection assay [66]. Both systems contain molecular chaperones needed for refolding. While LUC heat denatured alone or with BSA or IgG did not regain significant enzyme activity in these assays, denaturation of LUC with hsp30C resulted in enzyme reactivation up to 80–100% (Fig. 2B). Therefore hsp30C maintains its target protein in a folding competent state. Recent mutagenesis studies revealed that deletion of the last 25 amino acids from the C-terminal end of hsp30C or reduction of the net negative charge of the C-terminal tail by converting two aspartic acid residues to glycines inhibited its chaperone activity [65,66]. Also circular dichroism studies revealed that the ␣-helical and ␤-sheet structure of hsp30C was reduced in the double mutant and essentially lost in the C-terminal deletion mutant [66]. Therefore the C-terminal tail of hsp30C appears to be required for optimal chaperone activity and secondary structure. The inhibited chaperone activity of these mutants is likely due to their reduced ability to maintain target protein in a soluble state. In a recent study, we found that heat shock-induced hsp30 can be phosphorylated in vivo in A6 kidney epithelial cells primarily during recovery from stress [61]. Furthermore, it was determined that hsp30 was a substrate for mitogen-activated kinase-activated protein kinase-2 (MAPKAPK-2). This phosphorylation involved the p38 MAP kinase pathway since treatment of A6 cells with SB203580, an inhibitor of the p38 MAP kinase pathway, resulted in a loss of hsp30 phosphorylation. Phosphorylation induced the formation of smaller multimeric hsp30 complexes as well as a significant loss of hsp30 secondary structure. Furthermore, phosphorylation of hsp30 was associated with reduced binding to denatured proteins. Consequently phosphorylated hsp30 had a reduced ability to inhibit heat-induced aggregation of CS and LUC in vitro compared to unphosphorylated hsp30C. Xenopus hsp30 and mammalian hsp25/27 perform similar functions as molecular chaperones [5,7,8]. Additionally, all of these shsps can be

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(A) 120

(B) 120

100

% Luciferase Activity

100

% Aggregation

80

60

40

20

80

60

40

20

0

0

0

10

20

30

40

50

60

0

20

Time (min)

40

60

90

Time (min)

Fig. 2. Molecular chaperone activity of Xenopus hsp30. (A) The presence of hsp30D inhibited heat-induced aggregation of citrate synthase (CS). CS at a 150 nM monomer concentration was mixed with various molar amounts of recombinant hsp30D protein or incubated alone in a 50 mM HEPES–KOH buffer, pH 7.5 and heated at 42 ◦ C. Light scattering was determined at 10 min intervals in a Beckman DU7 spectrophotometer at 320 nm. An increase in absorbance was indicative of protein aggregation. Data were calculated as percentage of the maximum aggregation of CS after 60 min and were expressed as the mean ± S.E. CS was heat-treated alone (䉬, 0.1 ␮M) or in the presence of either hsp30D (䉱, 0.1 ␮M; 䊏, 0.5 ␮M) or IgG (×, 0.5 ␮M). (B) Luciferase (LUC) heat denatured in the presence of hsp30C was refolded in vivo after microinjection into Xenopus oocytes. However, LUC heat-treated alone or with BSA did not regain enzyme activity after injection. In these experiments LUC (0.2 ␮M) was incubated at 22 ◦ C (䉬) or heat denatured alone at 42 ◦ C (×) or in the presence of either 6 ␮M BSA (䊉) or hsp30C at hsp30C:LUC molar ratios of 1:1 (䊐), 10:1 (䉱) or 30:1 () for 15 min. Mixtures (containing 1.38 fmol of LUC in 26.7 nl) were microinjected into oocytes and LUC activity in the oocytes was monitored over time. Data are representative of three to five trials and shown as the mean ± S.E.

phosphorylated by MAPKAPK-2. Unlike hsp25/27, hsp30 is not found constitutively in the cell (with the exception of the cement gland of tailbud embryos) but appears following heat or other stress-related cellular insults. It is possible that phosphorylation during recovery from stress may accelerate the release of tightly bound Xenopus hsp30 particles from its target proteins by inducing a change in the oligomeric assembly of hsp30. The release of target proteins permits them to undergo refolding and reactivation that is assisted by other molecular chaperones including hsp/hsc70. Shsps have been shown to play an essential role in maintaining the integrity of actin and the intermediate filaments [67,68]. It has also been suggested that phosphorylated shsps present in small oligomers interact directly or indirectly to protect microfilaments against the further disruption by inhibiting the action of actin-severing proteins activated by the stress response and then promoting their recovery [68]. It will be interesting to determine if hsp30 is involved in this process in Xenopus embryos and cells.

Clearly, a great deal of valuable research can be accomplished examining shsps in Xenopus laevis. However, this species may not be ideal for carrying out key genetic experiments since these animals normally take 1–2 years to reach sexual maturity. Furthermore, the fact that they are tetraploid may complicate the generation of knockout mutants. Future research using Xenopus tropicalis may circumvent these problems since this species is diploid and has a generation time of less than 5 months [69,70]. Also transgenic technology has been developed for this organism as well as being the subject of a major effort with respect to genomics and proteomics.

Acknowledgements This research was supported by Natural Science and Engineering Research Council grants to J.J.H. who is also the recipient of a Canada Research Chair in Stress Protein Gene Research.

8. Future directions As evident from this review there are a great many unanswered questions regarding the hsp30 and Bshsp gene families with respect to their regulation of expression and function during frog development and in somatic cells.

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