Stress Protein HSP70 in Fish

Michiaki Yamashita,* Takeshi Yabu and Nobuhiko Ojima

National Research Institute of Fisheries Science 2-12-4 Fukuura, Yokohama 236-8648, Japan

Abstract

Stress proteins (heat-shock proteins, HSPs), which comprise an evolutionally well- conserved protein family, are induced in response to a variety of stress conditions and metabolic insults. When cells are subjected to sudden environmental changes, stress proteins are induced and play a central role in cellular homeostasis. A response to sudden adverse environmental changes is referred to as the heat-shock or stress response and is accompanied by a rapid increase in the synthesis of stress proteins. Given the importance of stress proteins in thermal adaptation at the cellular level, we have studied the expression, regulation, and protective functions of the members of the HSP70 stress protein family under normal and stress conditions in a variety of fish species. HSP70/heat-shock cognate protein-70 (HSC70) plays essential roles in the receptor complex formation and activation of Activin/Nodal/transforming growth factor-β and bone morphogenetic protein receptors and facilitates Nodal signaling. In addition, chaperone-mediated autophagy assisted by HSP70/heat-shock cognate (HSC)70 may be responsible for the stress responses in fish cells. HSP70 and HSC70 translocated into the lysosomes were found to accelerate protein degradation and catabolism under both stressed and normal conditions.

Keywords

activin, development, HSP70, HSC70, heat-shock transcription factor HSF, molecular chaperone, rainbow trout, Nodal, stress protein TGF-β, transgenic zebrafish


1. Introduction

Stress responses, such as the temperature-dependent regulation of gene expression, enable marine organisms to successfully adapt or acclimate to new environments. As such, they are critical for the growth and survival of ectothermic animals living in marine environments with variable temperatures (Mosser et al. 1986; Hightower and Renfro 1988; Iwama et al. 1998). The temperature range to which a fish species can adapt is dependent upon adaptive cellular functions and stress responses. For example, the adaptable temperature ranges from 20 to 32°C for the experimental laboratory fish platyfish Xiphophorus maculatus and zebrafish Danio rerio, and cultured cells of these species are also maintained in a similar temperature range (Yamashita et al. 2004). Stress proteins, which are a evolutionally well-conserved family of proteins, are induced in response to a variety of stress conditions and metabolic insults. Most stress proteins perform essential biological roles as molecular chaperones that facilitate the synthesis and folding of proteins and participate in protein assembly, secretion, trafficking, and protein degradation (Pelham 1982; Lindquist 1986; Lindquist and Craig 1988; Morimoto et al. 1990; Georgopoulos and Welch 1993; Wu 1995; Hartl 1996; Hartl and Hayer-Hartl 2002). When cells are subjected to rapid changes in their environment, induced stress proteins play a central role in the maintenance of cellular homeostasis. A response to sudden adverse environmental changes is referred to as the heat-shock or stress response and is accompanied by a rapid increase in the synthesis of stress proteins. Given the importance of stress proteins in thermal adaptation at the cellular level, we focused on the expression, regulation, and protective functions of the heat-shock protein 70 (HSP70) protein family under normal and stressed conditions in a variety of fish species.

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2. HSP70 in fish

HSP70 is widely distributed group of HSPs found in numerous organisms from bacteria to mammals, and its expression is markedly induced in response to environmental stresses, such as heat shock, UV and γ-irradiation, and chemical exposure (Pelham 1982; Lindquist 1986; Lindquist and Craig 1988; Morimoto et al. 1990; Georgopoulos and Welch 1993; Wu 1995). Transgenic and mutant zebrafish strains have been used as model animals for stress research (Fig. 1) (Yamashita 1999; Yamashita et al. 2003; Yamashita and Hojo 2004). HSP70 is thought to have a molecular chaperone function such that it transiently binds to nascent polypeptides and unfolded proteins, thereby preventing intramolecular and intermolecular interactions that can result in misfolding or aggregation of these substrate proteins (Welch and Feramisco 1985) (Fig. 2). Many studies have examined gain- or loss-of- functions to elucidate the biological roles of HSP70 as a molecular chaperone in animal cells in vitro and in vivo. The chaperone functions of HSP70 appear to be closely related to stress tolerance in animal cells, and overexpression of HSP70 enhances anti-apoptotic activity against cellular stress (Feder et al. 1996; Kim et al. 1997; Kondo et al. 1997; Ravagnan et al. 1997; Li et al. 2000; Mosser et al. 2000a, b). The heat- inducible gene expression and transcriptional regulation of the hsp70 gene has also been characterized in ectothermic animals, such as rainbow trout and zebrafish (Yamashita et al. 2004; Ojima et al. 2005a).


Fig. 1. Expression of HSP70 in the heat-shocked zebrafish embryo. Embryos maintained at 28.5°C were transferred to 37°C for 1 h. The embryos were fixed with 10% formalin in PBS, and HSP70 was stained with a specific monoclonal antibody (Embiotech Laboratories, Tokyo, Japan) vs. heat-inducible HSP70 raised against the C-terminal region of zebrafish HSP70-a by detection of signals with BM Blue POD substrate (Roche Japan, Tokyo, Japan). (A) Whole mount immunostaining showed HSP70 expressed in tissue specific manners. (B) Western blotting.

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Fig. 2. Model of molecular chaperone functions of the HSP70 family. HSP70 and HSC70 function as intracellular chaperones for other proteins, regulating protein-protein interactions, such as protein folding, establishment of protein disassembly and rearrangement, prevention of protein aggregation, transport of target proteins into the intracellular compartments, and translocation of proteins across membranes (Welch and Feramisco 1985). MtHSP70 and GRP78 are localized to the mitochondria and the endoplasmic reticulum, respectively (Yamashita et al. 2004).

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Two distinct isoforms of HSP70 cDNA have been identified from both rainbow trout and zebrafish (Yamashita et al. 2004; Ojima et al. 2005a). The amino acid sequences of fish HSP70 are highly homologous to heat-inducible-type HSP70 proteins in other vertebrates, such as human HSP70-1 and HSP70B¢ (Fig. 3) (Voellmy et al. 1985), with a very high identity (75–80%), indicating that fish HSP70 proteins belong to the heat-inducible HSP70 family. Molecular phylogeny studies on all HSP70 proteins that have been identified in fish reveal two clusters, namely, "fish HSP70-1" or "fish HSP70-2" (Fig. 4), suggesting that these diverged during vertebrate evolution (Yabu et al. unpublished). The two distinct HSP70 isoforms identified in rainbow trout, zebrafish, and platyfish are thought to have subsequently evolved in fish.


Fig. 3. Amino acid sequences of deduced proteins encoded in the heat-inducible hsp70 genes identified in the zebrafish genome. Comparisons of the predicted amino acid sequences. Residues identical to the amino acid in the zebrafish HSP70-a sequence are indicated by dots. Amino acids that are present in zebrafish HSP70-a, but not in other HSP70s, are marked by dashes. The zebrafish cDNA sequence for HSP70-a has been deposited in the DDBJ database with the accession number AB062116. The genes encoding HSP70-b and HSP70-ctg9500 are present on zebrafish chromosome 16 and 8, respectively (Yamashita et al. 2004).

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Fig. 4. Molecular phylogenic tree of the HSP70 family members. Amino acid sequences of the vertebrate HSP70s were compared by the neighbor-joining method with the CLUSTAL W program (version 1.83). Bootstrap confidence values for the sequence groupings are indicated in the tree (n = 1000). The scale indicates the evolutionary distance of one amino acid substitution per site. Sequence database accession numbers in GenBank or genomic contig scaffold numbers in the zebrafish and Takifugu Ensenbl Genome Servers are indicated in parentheses.

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2-1. HSP70 and other HSPs in rainbow trout

Stress responses have been well characterized in rainbow trout and its cultured cells. Currie and Tufts (1997) observed that erythrocytes synthesized HSP70 both constitutively and in response to an increase in temperature. Airaksinen et al. (1998) examined the effects of heat stress (from 18 to 26°C) and low oxygen tension (1% O2 = 1 kPa) on protein synthesis in primary cultures of hepatocytes, gill epithelial cells, and RTG-2 cells of rainbow trout. All of these cells displayed elevated levels of 67-, 69-, and 92-kDa proteins, whereas a 104-kDa protein was induced only in RTG-2 cells. Hypoxia induced a cell-type-specific response, increasing the synthesis of 36-, 39-, and 51-kDa proteins in the gill epithelial cells. When juvenile trout reared in freshwater were transferred from freshwater at 13.5°C to freshwater at 25.5°C, held for there for 2 h, returned to freshwater at 13.5°C for 12 h, and then transferred to 32 ppt seawater at 13.5°C, the level of branchial HSP70 increased approximately tenfold in the heat-shocked fish relative to the control. Such a mild temperature shock had only modest effects on the ability of rainbow trout to resist osmotic stress during fresh- to seawater transfer (Niu et al. 2008). HSP70 expression differs among rainbow trout clonal lines, suggesting the genetic control of differences in HSP70 expression (Heredia-Middleton et al. 2008). Trout cells have also been used in bioassays of heavy metal exposure in which the expression of HSP70 and metallothioneins was the indicator of exposure (Kothary and Candido 1982; Misra et al. 1989). In these studies, HSP70 accumulated in juvenile trout tissues, including the gill and liver, in response to exposure to metal (Cd2+, Cu2+, Pb2+, Zn2+)-contaminated water and diet. However, HSP70 levels in juvenile rainbow trout did not increase significantly when the dissected tissues were exposed individually to environmentally relevant Cd2+ or Cr2+ levels.

HSP70a and HSP70b have 98.1% identity in their deduced amino acid sequences (Ojima et al. 2005a, b). Southern blot analysis indicated that the two HSP70s are encoded by distinct genes in the trout genome, and northern blot analysis showed that heat stress of RTG-2 cells resulted in each of HSP70a and HSP70b expressing two mRNA species of different sizes (Ojima et al. 2005a). The induction levels of total HSP70b mRNAs were observed to be consistently higher than those of their HSP70a counterparts during heat stress, although the expression profiles of the two genes were similar to one another in temperature-shift and time-course experiments. Moreover, a mRNA species with a larger molecular size was expressed only under severe heat stress (i.e., not less than 28°C) irrespective of HSP70a and HSP70b. Ojima et al. (2005b) isolated multiple HSPs, including HSP90βa, HSP90βb, glucose-regulated protein (GRP)78, HSP70a, heat-shock cognate (HSC)70a, HSC70b, CCT8, HSP47, and DnaJ homolog, from RTG-2 cells. Quantitative reversetranscription (RT)-PCR analyses showed that the mRNA accumulation levels of HSP70a, HSP70b, HSC70a, HSC70b, and HSP47 were significantly elevated after heat shock, with those of two HSP70s, HSP70a, and HSP70b, showing the greatest increase. HSC70b showed a greater increase than HSC70a.

Ojima et al. (2005b) cloned two splice variants of HSPB1 from the rainbow trout and found that the C-terminus of the deduced proteins had a polyglutamic acid (polyE) stretch not found in other vertebrate HSPB1s. These two splice variants, HSPB1_tv1 and HSPB1_tv2, were identified in fish exposed to a continuous heat shock, with the mRNA level of HSPB1_tv1 increasing in response to this stress while that of HSPB1_tv2 decreased. Northern blot and RT-PCR analyses showed that under normal physiological conditions, HSPB1_tv1 mRNA is predominantly expressed in muscle tissues, although it is present in all organs. In contrast, HSPB1_tv2 mRNA is selectively expressed in muscle tissues, particularly in the heart. Distinctive features of rainbow trout HSPB1, such as having two splice variants and a polyE stretch, may contribute to the function of the protein under the typical low-temperature habitat of cold-water fish.

2-2. Zebrafish HSP70

We isolated cDNA clones encoding heat-inducible HSP70 from a cDNA library of 1-day-old zebrafish embryos heat-shocked at 37°C for 1 h. The complete cDNA sequence of a heat-inducible hsp70 in zebrafish embryos has 77–79% homology to heat-inducible hsp70 genes in platyfish, rainbow trout, and humans. The zebrafish hsp70 gene exhibited heat-induced expression, as measured by Northern blotting and in situ hybridization analyses, following a temperature shift from 28.5 to 37°C. Krone et al. (1997) and Sueltmann et al. (2000) reported a partial nucleotide sequence of the 5'-flanking region of the zebrafish hsp70 and a 1,977-bp open reading frame (ORF), respectively. Neither of the clones were full-length cDNA, and mismatches in nucleotide sequences occurred between them. The sequence reported by Sueltmann et al. (2000) carries the same ORF as our isolated sequence up to the point of heterogeneity, P-632, where a deletion of a G at base 2557 occurs, after which the predicted amino acid sequence diverges until a stop codon after residue P-632; this sequence also lacks the C-terminal conserved acidic amino acid sequence "EEVD." Since no apparent homology existed in the 3'-noncoding sequences, the sequence variations are probably due to a distinct gene product. Thus, the zebrafish genome appears to contain another hsp70 gene, whose structure and expression profile are different, as described in Subsection 2-3.

Amino acid sequence homology analysis revealed that the zebrafish HSP70 is a member of the heat- inducible HSP70 family in vertebrates (Fig. 3) and that it shares extensive homology with the heat-inducible HSP70s from other fish species, such as rainbow trout and platyfish (Fig. 3) (Yamashita et al. 2004). On the other hand, two distinct mammalian groups have been identified, namely, a Major Histocompatibility Complex (MHC)-linked group consisting of human HSP70-1 and HSP70-2 and mouse HSP70.1 (Hunt and Morimoto 1985; Wu et al. 1985; Milner and Campbell 1990) and a group containing the human and pig HSP70B' (Voellmy et al. 1985; Gunther and Walter 1994). Thus, the heat-inducible HSP70s may have diverged into several distinct groups during vertebrate evolution.

2-3. Gene structure of the hsp70 gene in zebrafish

Zebrafish and the tiger pufferfish Takifugu rubripes, whose draft genomic sequences have been well documented, each have several copies of hsp70 genes (Figs. 5, 6). In zebrafish, a BLAST search of the Ensenbl Zebrafish Genome Server localized the hsp70 genes to two distinct loci on chromosomes 8 and 16 (Sanger Institute, UK; Yamashita et al. 2004), with two tandemly linked hsp70 genes, i.e., hsp70-a and -b, on chromosome 16 (Fig. 5). The hsp70-a gene could be induced in the cultured zebrafish cells and embryos under heat-shock conditions (Fig. 6), whereas the hsp70-b gene was weakly expressed constitutively, based on RT-PCR results, suggesting that it may be a pseudogene. In addition, an intron-less hsp70-like gene on chromosome 8 showed constitutive expression in zebrafish embryos under heat-shock conditions, indicating that this may also be a pseudogene. Thus, thehsp70-a gene is a heat-inducible HSP70 isoform among the HSP70 family proteins in the zebrafish genome.


Fig. 5. Gene structure of the hsp70 genes in zebrafish and human genomes. (A) The zebrafish genome contains three copies of the hsp70 genes, and HSP70-a was the only heat-inducible member of the HSP70 family. The gene encoding HSP70-b is tandemly linked to the gene encoding HSP70-a on the zebrafish chromosome 16, and the gene encoding HSP70-ctg9500 is present on zebrafish chromosome 8 (Yamashita et al. 2004). Arrows indicate the heat-inducibility of these genes measured by RT-PCR (Yabu et al. unpublished). (B) The human genome contains five copies of the hsp70 and its related genes; hsp70-1, hsp70-2, and hsp70B' are heat-inducible, whereas Hsp70HOM and Hst70 are testis-specific.

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Fig. 6. The nucleotide sequence of the 5'-upstream region of the zebrafish hsp70-a gene. The transciption start site was determined by primer extension. The nucleotide sequences in exons are underlined. The zebrafish hsp70 gene contains only one intron in the 5'-noncoding region. The translation start ATG codon is in italics. The heat-inducible expression of the zebrafish hsp70 gene is suggested to be regulated by a dual promoter system, i.e., a heat-shock promoter containing HSE upstream of the transcription start site and another constitutive promoter in the first intron (Yabu et al. unpublished).

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2-4. Genomic analysis of fish hsp70 genes

Postlethwait et al. (1998) compared the zebrafish genome organization with that of humans and found that large chromosome segments have been conserved for the 430 million years since the divergence of the two lineages. These authors suggested that two large-scale gene duplication episodes—possibly whole- genome duplication events—occurred prior to the divergence of the fish and mammal lineages. Analysis of the zebrafish hox gene clusters revealed that the zebrafish has two copies of human chromosome segments, suggesting that an additional genome duplication occurred in the zebrafish genome (Amores et al. 1998). A relationship between the divergence of hsp70 genes and a chromosomal duplication event was characterized by comparisons with zebrafish chromosomes and mammalian orthologs. The genetic map of the hsp70 genes in the zebrafish genome revealed a striking conservation of synteny with a region of human and mouse chromosomes (Yamashita et al. 2004). The almost orthologous genes in zebrafish chromosome 8, which contains the hsp70 gene, were well conserved on human chromosome 1 and mouse chromosome 3 (Table 1). This finding indicates that zebrafish hsp70 (Chr. 8) is orthologous to human Hsp70B', which localized to human chromosome 1q23.1. Therefore, the fish hsp70-1 group, containing platyfish hsp70-1, zebrafish hsp70 (Chr. 8), and Takifugu hsp70 (ctg1502), is orthologous to the mammalian Hsp70B'. In contrast, the orthologous genes of zebrafish hsp70-a on chromosome 16 could not be identified on human and mouse chromosomes. The mammalian orthologous genes corresponding to the fish genes on zebrafish chromosome 16 were widely distributed on human chromosomes 3, 12, 16, and 19 and mouse chromosomes 5, 6, 7, 8, 9, and 11 (Table 1). Thus, the mammalian ortholog of the fish hsp70-2 group appears not to exist in the mammalian genome. The fish hsp70-2 group may have arisen by an ancient chromosomal duplication event from the fish hsp70-1 group.


Table 1. Zebrafish genes neighboring the HSP70 genes.

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2-5. Stress-inducible HSC70 isoforms

In an earlier study, we established a cell line derived from the tail fin of the yellowtail Seriola quinqueradiata, a marine teleost fish, and isolated cytosolic HSP70 and two HSC70 isoforms (Yabu et al. unpublished). We observed that the heat-shock regulation of these two hsc70 genes is quite distinct, and named them hsc70-1, which is constitutively expressed, and hsc70-2, which is only induced by heat shock (Yabu et al. unpublished). In addition, HSP70/HSC70 was translocated into the lysosomes, and chaperone- mediated autophagy (CMA) assisted by HSP70/HSC70 was induced in the heat-shocked cells (Yabu et al. unpublished). The hsp70 gene possesses a cis-acting heat-shock element (HSE) responsive to heat-induced transcriptional activation (Amin et al. 1988; Xiao and Lis 1988; Wu 1995). Thus, under stress conditions, both hsp70 and hsc70-2 genes may be regulated by a heat-shock factor via a HSE in the promoter. In rainbow trout cells, the promoter region of the hsc70 gene possesses potential HSE sequences, but its stress-inducibility is unclear (Zafarullah et al. 1992). HSE sequences have also been found in the promoter regions of mammalian hsc70 genes that lack significant heat-shock inducibility (Sorger and Pelham 1988). Two hsc70 genes, hsc70-1 and hsc70-2, were expressed in the hepatopancreas and muscle of carp under unstressed conditions in a characteristic tissue-specific manner (Ali et al. 2003). These were insensitive to or only weakly induced by the stressors, with two exceptions: cadmium treatment and cold-shock-induced hsc70-1 in the liver and enhanced induction of hsc70-2 in the muscle (Ali et al. 2003). Thus, the fish genome possesses two genetically distinct hsc70 genes, the hsc70-1 and hsc70-2 groups.

When we compared gene synteny of hsc70 genes in the zebrafish genome, the hsc70-1 gene was found to be orthologous to the human hsc70 gene, showing similar gene synteny, whereas the hsc70-2 gene was different from the human orthologs (Yabu et al. unpublished). This finding suggests that two distinct hsc70 genes showing different expression patterns under heat stress and other stress conditions might be present in the fish genomes. In zebrafish, the hsc70-1 gene is constitutively expressed in cultured cells as well as expressed in a tissue-specific manner, particularly in the brain and yolk sac (Graser et al. 1996; Yamashita et al. 2003), whereas the hsc70-2 gene is not expressed in the cultured cells, suggesting that the hsc70-2 gene may be silent. On the other hand, members of the hsc70-2 group, such as platyfish HSC70 (Yamashita et al. 2004), medaka HSC70 isoforms [NP_001098384], Seriola HSC70-2, and carp HSC70-2 (Ali et al. 2003), are expressed in the respective fish tissues. Therefore, both hsc70-1 and hsc70-2 genes may act as molecular chaperones in different tissues and cell types under normal and/or stressed conditions. The vertebrate HSP70/HSC70 proteins, including the heat-inducible HSP70, testis-specific protein, the MHC-linked HSP70, and the constitutively expressed HSC70, are postulated to have evolved from four distinct groups by gene duplication and translocation in the vertebrate genome as potential adaptations to various environmental stresses (Yamashita et al. 2004). Among these proteins, teleost fish possess two copies of HSC70, which was duplicated during fish evolution. The cytosolic HSP70/HSC70 may be responsible for CMA under both normal and stressed conditions (Yabu et al. unpubilished).

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3. Heat-shock transcription factor in fish

Heat-inducible transcriptional regulation is mediated by the heat-shock transcription factor (HSF) that binds to HSEs found upstream of all stress protein genes (Morimoto et al. 1990; Wu 1995). The human, chicken, and tomato genomes contain multiple distinct hsf genes (Scharf et al. 1990; Rabindran et al. 1991; Sarge et al. 1991; Schuetz et al. 1991; Nakai and Morimoto 1993), whereas the genomes of yeast, Drosophila, and Xenopus each contain only a single hsf gene (Sorger and Pelham 1988; Wiederrecht et al. 1988; Mercier et al. 1997). In mammals and chickens, HSF1 and HSF3 were identified as rapidly activated, stress-responsive factors (Rabindran et al. 1991; Sarge et al. 1991; Nakai and Morimoto 1993; Nakai et al. 1995). HSF2 responds to erythrocyte differentiation and preimplantation embryonic development (Hensold et al. 1990; Schuetz et al. 1991; Sistonen et al. 1992), and HSF4 acts as both an activator and a repressor of tissue-specific heat-shock genes through alternative splicing (Nakai et al. 1997; Tanabe et al. 1999). The HSF proteins have highly conserved amino-terminal regions that contain the DNA-binding domain for HSE as well as hydrophobic heptad repeats that form coiled-coil structures. In mammals and Drosophila, the inactive monomeric form of HSF is converted to a trimer in response to heat and other stresses, possibly through a switch from intramolecular to intermolecular coiled-coil interactions (Westwood et al. 1991; Rabindran et al. 1993; Zuo et al. 1994). HSF binds to the promoter region and activates transcription through a potent heat- activation domain in its C-terminus, which is negatively regulated in the absence of stress (Green et al. 1995; Shi et al. 1995; Zuo et al. 1995; Newton et al. 1996; Wisniewski et al. 1996; Kline and Morimoto 1997).

3-1. Zebrafish HSF

The nucleotide sequence of the isolated zebrafish HSF cDNA was predicted to encode a full-length HSF of 497 amino acids (accession No. BAB72171) (Fig. 7). Comparison of this amino acid sequence to other previously characterized members of the HSF family revealed that the zebrafish HSF belongs to the HSF1 group consisting of human, mouse, and chicken HSF1. These proteins regulate the heat-inducible transactivation of the stress genes. The amino acid sequence of zebrafish HSF is 57, 55, and 57% identical to human, mouse, and chicken HSF1, respectively. The DNA-binding domain of the zebrafish HSF is well conserved with previously reported HSF1s (Fig. 7). The amino acid sequence of the DNA-binding domain of the zebrafish HSF1 is 92, 92, and 94% identical to the corresponding region in human, mouse, and chicken HSF1, respectively. Other conserved motifs found in the zebrafish HSF include the hydrophobic repeat that functions as the oligomerization motif, which is 70% identical to the corresponding region of human HSF1. The carboxyl-terminal heptad repeat, which plays a role in intramolecular negative regulation of DNA-binding activity in HSF1, is also conserved in zebrafish HSF.


Fig. 7. Comparisons of the predicted amino acid sequence of zebrafish HSF with those of human HSF1, mouse HSF1, chicken HSF1, and rainbow trout HSF1a proteins. Residues identical to the amino acid in the zebrafish HSF (accession number AB062117) are indicated by dots. Amino acids that are present in zebrafish HSF, but not in other HSFs, are marked by dashes. The regions boxed with a solid line or broken line indicate the predicted DNA binding domains and hydrophobic heptad repeats, respectively. The potent heat-activation domain boxed in red is suggested to regulate temperature ranges for HSF activation and conformational changes.

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The zebrafish HSF shares extensive homology with the rainbow trout HSF (Fig. 7). Components found in other vertebrate HSF1s, such as the DNA-binding domain, predicted nuclear localization signal, hydrophobic heptad repeat (HR)-A/B and HR-C, are conserved in the zebrafish HSF. Thus, HSF transactivation of stress genes is a common phenomenon in fish and mammals. However, the question as to whether other types of HSF, such as HSF2, HSF3, and HSF4, which have been reported in humans and chickens, are also present in fish, remains to be resolved.

A recent study by Rabergh et al. (2000) reported the nucleotide sequences of two splice variants of the zebrafish hsf gene. There are several differences between sequence reported by us and that reported by these authors. In particular, we noted the following amino acid substitutions: P-252 to S, P-254 to S, SALTPP (342–347) to FRPDSA, L-430 to Val, IVLPDPL (446–452) to SFSPIPF, and A-478 to T. These substitutions are due to nucleotide heterogeneity and frameshifts caused by nucleotide insertions and deletions. The C-terminal amino acid sequence, KLS (496–498), was well conserved in the known HSFs, while in the sequence of Rabergh et al. (2000) it was replaced with SRTRIGDPCFKLKKESKR by an insertion of the unknown nucleotide sequence after the nucleotide at position 1631, which is suggested to be due to a splicing variant containing introns. Although these structural differences in HSF may affect DNA- binding and transactivation properties, as previously pointed out in studies on Drosophila (Wu 1995), their functional properties have not been examined (Rabergh et al. 2000).

3-2. Rainbow trout HSF1

Two distinct cDNA clones encoding HSFs have been isolated from RTG-2 cells of rainbow trout and subsequently denoted HSF1a and HSF1b (Ojima and Yamashita 2004). The predicted amino acid sequence of HSF1a shows 86.4% identity to that of HSF1b. The two proteins contained the general structural motifs of HSF1, i.e., a DNA-binding domain, hydrophobic heptad repeats, and nuclear localization signals. Southern blot analysis showed that each HSF1 is encoded by a distinct gene. The two HSF1 mRNAs were co- expressed in unstressed rainbow trout RTG-2 cells as well as in a variety of tissues. An electrophoretic mobility shift assay revealed that each in vitro translated HSF1 binds to the HSE, and a chemical cross-linking and immunoprecipitation analysis showed that HSF1a and HSF1b form heterotrimers as well as homotrimers. Based on these results, two distinct HSF1 isoforms that can form heterotrimers are present in rainbow trout cells, suggesting that a unique molecular mechanism regulated by a combination of distinct HSF1 isoforms underlies the stress response in rainbow trout. These HSF1 isoforms may have diverged during the evolution of tetraploid fish.

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4. Transcriptional control of fish hsp70

The expression of the hsp70 and hsf genes in zebrafish embryos at six different life-cycle stages was examined by Northern blot analysis (Fig. 8). hsp70 mRNA levels were found to be very low in control embryos incubated at 28.5°C under non-stress conditions; however, these dramatically increased at each stage following heat shock, with the exception of the 3-h embryos (blastula stage), in which no hsp70 expression was observed. hsf mRNA was not found in 3-h embryos, but appeared as both 2-kb and 6-kb molecules in the embryos after 6 h and thereafter. In zebrafish embryos, hsp70 mRNA was expressed after the gastrula period under heat-shock conditions, as identified by Northern blot analysis. The results of the in situ hybridization experiment indicated that the hsp70 gene was expressed in a stage- and tissue- specific manner. The hsp70 mRNA was localized in the brain, eye, otic vesicle, and yolk sac under heat stress conditions. Given that hsf mRNA was expressed in the embryos after the gastrula period, the hsf gene is thought to be induced by zygotic expression during early development, which is similar to most other housekeeping genes. Based on the observation that hsf mRNA expression paralleled the heat-inducible expression of hsp70, hsf expression after the gastrula period may accompany the heat induction of the zebrafish hsp70 gene in a stage- and tissue-specific manner.


Fig. 8. Northern blot analysis of hsp70 and hsf mRNAs from zebrafish embryos. Total RNA was isolated from embryos incubated at 28.5°C and heat-shocked embryos that had undergone a 1-h temperature shift to 37°C, and then subjected to Northern analysis. (A) hsp70 mRNA, (B) hsf mRNA. The hsf gene was expressed as a primary transcript of 6 kb and a mature form of 2 kb after the gastrula stage, which coincided with the expression pattern of the hsf gene. Embryonic staging was according to Kimmel et al. (1995).

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hsp70 expression patterns were examined in vivo by whole-mount in situ hybridization (Fig. 9). During the cleavage and blastula periods, no apparent expression of hsp70 mRNA occurred even in heat-shocked 3-h embryos. However, heat-shock induction of hsp70 mRNA was found in the embryos 6 h after fertilization. A temperature elevation from 28.5 to 37°C for 1 h induced hsp70 mRNA expression throughout the entire embryo during the gastrula and segmentation periods. Most notably, strong expression was observed in the brain, notochord, and yolk sac of heat-shocked 1- to 2-day-old embryos. In the 2-day heat-shocked embryos, hsp70 mRNA expression was elevated in the forebrain, hindbrain, notochord, otic vesicle, yolk sac, and skeletal muscle in the middle part of the body (Fig. 10). Conversely, control embryos cultured at 28.5°C showed no apparent expression of hsp70 mRNA in any of the stages tested in this study (Fig. 9).


Fig. 9. Heat-inducible expression of the hsp70 gene detected by in situ hybridization. The fish hsp70 gene showed stress-inducible gene expression. In zebrafish embryos, hsp70 mRNA was expressed at the segmentation stage after the gastrula period under heat-shock conditions. During the cleavage and blastula periods, no apparent expression of hsp70 mRNA was seen, even in the heat-shocked 3-h embryos (B). However, heat-shock induction of hsp70 mRNA was found in the embryos 6 h after fertilization (D). A temperature shift from 28.5°C to 37°C for 1 h induced hsp70 mRNA expression throughout the entire embryo during the gastrula and segmentation periods (B), (D). Conversely, control embryos cultured at 28.5°C showed no apparent expression of hsp70 mRNA in any of the stages tested in this study (A), (C).

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Fig. 10. Heat-inducible expression of the hsp70 gene detected by in situ hybridization. Most notably, strong expression of the hsp70 gene was observed in the brain, notochord, and yolk sac of heat-shocked 1- to 2-day-old embryos. In the case of the 2-day-old heat-shocked embryos, hsp70 mRNA expression was elevated in the forebrain, hindbrain, notochord (nt), otic vesicle (ov), yolk sac (yc), and skeletal muscle (m) in the middle part of the body. Thus, the hsp70 gene was expressed in stage- and tissue-specific manners.

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The expression levels of hsf mRNA were also examined (Fig. 11). hsf mRNA was not observed in embryos within 3 h of fertilization, as shown by the Northern blot analysis (see Fig. 8), but it was present throughout all developmental stages of zebrafish embryos after the gastrula period (Fig. 11). In 2-day-old embryos, hsf mRNA was highly expressed in the brain, notochord, otic vesicle, and yolk sac. Thus, the spatiotemporal expression of zebrafish hsf mRNA accounts for the tissue-specific heat-inducibility of HSP70 under stress conditions.


Fig. 11. Expression of hsf mRNA by in situ hybridization. The hsf gene was expressed after the gastrula period. The hsf gene is induced by zygotic expression during early development, similar to most other housekeeping genes. hsf mRNA was not observed in embryos within 3 h of fertilization (A) as shown by Northern blot analysis (see Fig. 8). After the gastrula period, hsf mRNA was observed throughout the developmental stages of zebrafish embryos (B)–(D). In 2-day-old embryos, HSF mRNA was highly expressed in the brain, notochord (nt), otic vesicle (ov), and yolk sac (yo) (D). Thus, the spatiotemporal expression of zebrafish hsf mRNA accounts for the tissue-specific heat-inducibility of HSP70 under stress conditions.

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4-1. Stress responses in embryos

hsp70 mRNA is expressed after the gastrula period under heat-shock conditions, as reported in previous studies (Bienz 1986). Since hsf mRNA is expressed in embryos after the gastrula period, the hsf gene is probably induced by zygotic expression during early development in a manner similar to that of other housekeeping genes (Newport and Kirschner 1982). hsf mRNA expression also paralleled the heat-inducible expression of hsp70, which is mainly regulated by HSF. Therefore, hsf expression after the gastrula period is likely accompanied by heat induction of the hsp70 gene in a stage-specific manner.

In mice, the MHC-linked heat-inducible Hsp70.1 gene exhibits complex transcriptional regulation in response to serum factors, infection, development, and stress (Milner and Campbell 1990; Morimoto et al. 1990). This gene is transcribed during the cleavage period and also after gastrulation in response to cell cycling, but not to stress induction (Bevilacqua and Mangia 1993; Christians et al. 1997). The mouse and human Hsp70 genes located in the MHC locus are expressed by transcription factors other than HSF1 (Morimoto et al. 1990; Wu 1995). Conversely, given that the zebrafish hsp70 shows strict heat-inducible expression, it appears to undergo a simple transcriptional regulation that may involve only HSF, a system that is similar to that observed for the Drosophila and human HSP70B' (Craig et al. 1979; Voellmy et al. 1985; Morimoto et al. 1990; Gunther and Walter 1994).

The overexpression of HSF constructs lacking the regulatory domain result in the expression of hsp70 and hsp47 in embryos, even under non-stress conditions. This finding indicates that the deleted regulatory domain plays an important role in repressing the conversion of the inactive monomer to an active trimer. The zebrafish HSF is considered to play a major role in the stress-inducible expression of hsp70 and other hsp genes in zebrafish embryos. HSF monomers associate with each other, resulting in the formation of homotrimers in response to physiological stress and ultimately activating target hsp genes by acquiring DNA-binding ability (Wu 1995; Zhong et al. 1998). Phosphorylation of serine and threonine residues in HSF has also been reported to be involved in a signal transduction system, including mitogen-activated protein kinase (MAPK) pathways (Knauf et al. 1996). A number of authors have reported that hsf expression regulates in vivo thermotolerance in Drosophila and mice (Jedlicka et al. 1997; Xiao et al. 1999) and cultured mammalian cells (Mivechi et al. 1995; McMillan et al. 1998). Thus, the spatiotemporal expression of hsf in embryos reported here may determine the heat-inducible expression of hsp genes during zebrafish development, thus causing the embryos to be resistant against environmental stresses, such as changes in temperature.

4-2. Transactivation by the cloned zebrafish HSF

We investigated the transcriptional activities of the zebrafish hsf to confirm that the cloned hsf regulates the heat-inducible expression of zebrafish hsp70. The hsf cDNAs that encode the protein with a fused His-tag at the C-terminal and its truncated forms were subcloned downstream of a T7 promoter and their cRNAs subsequently synthesized using in vitro transcription.

Expression vectors encoding HSF or mutant HSFs were introduced into zebrafish embryos, and the induced levels of the stress genes were assayed by RT-PCR to examine whether the zebrafish HSF functions as a transcriptional activator (Fig. 12). Embryos containing the deletion mutants HSF-Del1 and HSF-Del2, in which the heat-activation domain between HR-A/B and HR-C, respectively, of HSF is deleted, expressed high levels of mRNA encoding hsp70 and hsp47 in a pattern similar to that seen with the heat-induced expression of hsp70 and hsp47. Conversely, the full-length HSF clone did not induce the expression of hsp70 and hsp47 when introduced into embryos. As a control experiment, the zebrafish hsc70 gene was constitutively expressed in every embryo tested in this study. These findings indicate that the zebrafish HSF without the heat-activation domain can transactivate hsp70 and hsp47 under non-stress conditions and that the full-length hsf is negatively regulated under non-stress conditions. When the cRNA encoding zebrafish HSF or the truncated mutants, HSF-Del1 (Del1) and HSF-Del2 (Del2), was microinjected into zebrafish embryos at the one-cell stage, along with the hsp70-GFP gene construct, the induction of GFP expression under the control of the hsp70 promoter was observed in 12-h embryos by fluorescence microscopy (Fig. 12). The introduction of the truncated HSF (e.g., Del1 and Del2) induced green fluorescence, indicating that the truncated sequence negatively regulated the transcription of the hsp70 promoter. In contrast, the introduction of original HSF did not induce green fluorescent protein (GFP) expression under the control of the hsp70 promoter.


Fig. 12. Transactivation activities of zebrafish HSF. (A) Gene expression induced by overexpression of HSF. The cRNA encoding zebrafish HSF (HSF, lane 2) and the HSF mutants, HSF-Del1 (Del1, lane 3) and HSF-Del2 (Del2, lane 4), was introduced into zebrafish embryos at the one-cell stage. The embryos were then incubated at 28.5°C for 12 h. The control embryos were maintained at 28.5°C (control, lane 1), and the heat-shocked embryos were incubated at 37°C from the 10 h post-fertilization (hpf) to the 12 hpf (heat shock, lane 5). In the 12-h embryos, the mRNA expression levels of hsp70-a, hsp47, hsc70-1, and hsf were examined by RT-PCR. (B) The cRNA encoding zebrafish HSF or that of the truncated mutants, HSF-Del1 (Del1) and HSF-Del2 (Del2), was microinjected into zebrafish embryos at the one-cell stage, along with the hsp70-GFP gene construct. The induction of GFP expression under the control of the hsp70 promoter was observed in 12-h embryos by fluorescence microscopy.

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5. Characterization of the HSP70 family members in vertebrates

The HSP70 family consists of several members, some of which are heat inducible and others that are constitutively expressed (Morimoto et al. 1990). Both HSP70 and HSC70 are cytosolic members of the HSP70 family. HSP70 is markedly induced under a variety of stresses, including heat shock, UV irradiation, and treatment with heavy metals or arsenite, while HSC70 is expressed under normal growth conditions (Morimoto et al. 1990). GRP78 is located in the endoplasmic reticulum and induced under the inhibitory condition of glycosylation (Morimoto et al. 1990). A testis-specific HSP70-related protein found in the mammalian testis has been shown to be involved in spermatogenesis (Allen et al. 1988; Matsumoto and Fujimoto 1990). These HSP70 family members have evolutionarily diverged, and they have been classified by phylogenetic analysis into four distinct clusters corresponding to their intracellular localization, i.e., the cytoplasm, endoplasmic reticulum, mitochondria, or chloroplasts (Morimoto et al. 1990; Boorstein et al. 1994).

In addition, multiple HSP70 isoforms have been found in various animal cell types by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis (Welch and Feramisco 1985; Allen et al. 1988; Matsumoto and Fujimoto 1990; Gutierrez and Guerriero 1995). These isoforms are assumed to be due to the presence of the multiple Hsp70 gene copies that have been identified in humans (Wu et al. 1985; Gunther and Walter 1994), mice (Hunt et al. 1993; Gunther and Walter 1994), and Drosophila (Craig et al. 1979). Alternatively, posttranslational modifications, such as methylation, ribosylation, and phosphorylation, of HSP70 might account for isoform diversity.

5-1. The HSP70 family members in fish cells

Yamashita et al. (2004) characterized the expression of HSP70 family proteins in the platyfish fibroblast cell line EHS and isolated three distinct cDNA clones that encoded two isoforms of HSP70 and HSC70. A phylogenetic analysis of the heat-inducible members of the HSP70 family in vertebrates revealed four distinct groups. This study demonstrates that a shift in the incubation temperature of platyfish EHS cells from 28 to 37°C apparently induces a set of stress proteins, including HSP28, HSP70, HSP90, GRP78, and GRP94, in a manner similar to that seen in mammalian and Drosophila cells (Morimoto et al. 1990). Several additional spots of 40, 35, 32, and 25 kDa, indicating small HSPs induced by the stress treatments, have also been found, but these have never been characterized in fish cells (Yamashita et al. 2004).

The anti-HSP70 antibody shows a broad specificity against HSC70 and HSP70 in animal cells (BRM-22; Sigma-Aldrich, St. Louis, MO), and a specific rabbit antibody has been raised against the N-terminal sequences derived from the platyfish cDNA clones. Both of these results indicate the stress-inducible expression of the HSP70-1 and HSP70-2 isoforms. The antibody against the constitutively expressed HSC70 also cross-reacted with the two immunologically cross- reactive proteins by differential phosphorylation of a single gene product (Yamashita et al. 2004). Phosphorylated isoforms of HSP70 have been also found in bovine cells (Leustek et al. 1992). The kinase and the locations of the phosphorylated amino acid residues in HSC70 remain unknown and should be characterized.

5-2. Phylogenetic analysis of fish HSP70

Phylogenetic analysis has demonstrated that the eukaryotic HSP70 can be classified into four distinct clusters, such as HSP70/HSC70, GRP78, mitochondrial HSP70, and plastid HSP70, according to their intracellular localization in the cytoplasm, endoplasmic reticulum, mitochondria, or chloroplasts, respectively (Boorstein et al. 1994). In addition, the Hsp70 genes in humans and mice constitute a multigene family (Wu et al. 1985; Hunt et al. 1993; Gunther and Walter 1994). The HSP70 family protein genes are distinguished by at least three different expression patterns: strictly heat-inducible Hsp70 (e.g., human Hsp70B; Voellmy et al. 1985), cell cycle-dependent, heat-inducible Hsp70 (e.g., human Hsp70-1 and Hsp70-2; Hunt and Morimoto 1985; Milner and Campbell 1990), and constitutively expressed, less stress-dependent Hsp70 genes (e.g., Hsc70; Ali et al. 1996; Hsp70Hom; Milner and Campbell 1990). The Hsp70 genes are distributed on at least five different human chromosomes (Gunther and Walter 1994; Yamashita et al. 2004). The amino acid homology analysis showed that the heat- inducible members of the vertebrate HSP70 family can be classified by adding new members of fish HSP70 family into six clusters, tentatively named "fish HSP70," "mammalian testis-specific HST70," "mammalian HSP70B'," "mammalian MHC-linked HSP70," "bird HSP70," and "amphibian HSP70", respectively (Fig. 13).


Fig. 13. The model of divergence of the HSP70 family members during vertebrate evolution. The hsp70 genes may have diverged during vertebrate evolution several times. The duplication of the hsp70 genes may have occurred during fish evolution (open circle) or mammalian evolution (close circle).

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Platyfish HSP70-1 and HSP70-2, rainbow trout HSP70, and zebrafish HSP70 belong to the "fish HSP70" cluster. Since the expression of these fish HSP70 is inducible at high levels in fish cells, the HSP70 proteins in this group are considered to be the major heat-inducible forms. According to the phylogenetic analysis in Fig. 4, fish HSP70 can be classified into two genetically distinct groups, "fish HSP70-1" and "fish HSP70-2." The presence of two HSP70 isoforms belonging to these groups suggests the duplication of the heat-inducible hsp70 gene during evolutionary divergence in fish.

5-3. Mammalian HST70

The second cluster "mammalian HST70" contains the testis-specific, constitutively expressed mouse hsp70.2 as well as its human and rat orthologs. To date, no member of this cluster has ever been identified in lower vertebrates (Yamashita et al. 2004). Both the mouse homolog hsp70.2 gene and the other testis-specific hsp70-related hsc70t gene regulate testicular formation and spermatogenesis (Allen et al. 1988; Matsumoto and Fujimoto 1990). These mammalian Hst70 genes have an intron in the 5'-flanking sequence similar to the other heat-inducible fish hsp70s, but they are expressed in a testis-specific manner. Thus, mammalian HST70s may have diverged structurally and functionally from the heat-inducible hsp70 by the replacement of a testis-specific promoter (Gunther and Walter 1994). These findings suggest that mammalian Hst70s are important in testicular function specific to homeotherms, but not in the lower vertebrates.

5-4. Mammalian MHC-linked HSP70

Two heat-inducible Hsp70s encoded in the mammalian MHC class III locus are classified into the "mammalian MHC-linked HSP70" cluster. In the human genome, three copies of Hsp70-related genes are tandemly localized in the MHC system locus on chromosome 6p21 (Gunther and Walter 1994). The first and second of these genes encode heat-inducible Hsp70, whereas the third gene in the sequence encodes the constitutively expressed testis-specific HSP70-related protein in the opposite orientation to the neighboring Hsp70 genes (Hunt and Morimoto 1985; Milner and Campbell 1990; Gunther and Walter 1994). This gene organization is also conserved in the mouse and rat (Gunther and Walter 1994). Conversely, the MHC system locus in the zebrafish genome does not contain the homologous gene to the mammalian MHC-linked Hsp70, suggesting that three copies of the MHC-linked Hsp70 genes may have been duplicated from an ancestral MHC-linked Hsp70 gene during mammalian evolution. In addition, the third gene (i.e., human Hsp70Hom (Milner and Campbell 1990), mouse hsc70t (Matsumoto and Fujimoto 1990), and rat hsp70.3 (Walter et al. 1994)] of the three tandemly linked hsp70 genes in the mammalian MHC locus is testis-specific. Their promoter regions lack HSEs and display testis-specific expression in mammalian spermatogenesis (Allen et al. 1988; Matsumoto and Fujimoto 1990).

5-5. Mammalian HSP70B'

The syntenic analysis of the zebrafish genome revealed that the fish HSP70-1 group containing zebrafish hsp70 (Chr. 8) is orthologous to the human Hsp70B' on human chromosome 1q23.1. The pig genome contains a similar Hsp70B' gene, whereas the mouse and rat genomes have lost this gene during mammalian divergence. Comparison of the promoter functions of the two distinct human Hsp70 gene promoters (Morimoto et al. 1990) revealed that the gene encoding Hsp70B' exhibits simple transcriptional regulation, which is similar to that found in yeast and Drosophila. Conversely, the MHC-linked heat-inducible Hsp70 genes exhibit complex transcriptional regulation and respond to diverse cellular signals, such as serum factors, viral activation, developmental regulation, and stress induction (Hunt and Morimoto 1985; Milner and Campbell 1990). Both human promoters have conserved HSEs that can bind the heat-shock transcription factor (Hunt and Morimoto 1985; Wu et al. 1985; Milner and Campbell 1990; Morimoto et al. 1990; Gunther and Walter 1994).

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6. Stress response in transgenic zebrafish models

Transgenic zebrafish models expressing stress proteins, HSF, and reporter genes under the control of the heat-shock promoter have been generated and used for stress research in fish with the aim to observe the stress response and temperature regulation in vivo (Yamashita et al. 2003). The minimum hsp70 promoter containing a single HSE and showing high heat-inducible transcriptional activity was used to visualize the stress response occurring in intact embryos using the GFP gene expression system. In addition, transgenic zebrafish overexpressing HSF showed enhanced stress tolerance. The expression of stress proteins induced by HSF activation may determine the stress response and stress tolerance in vivo.

6-1. Transgenic zebrafish as a biosensor for stresses

The gene expression of hsp70 is regulated by a heat-shock promoter. Its transcription is activated by changes in body temperature as well as a variety of other stresses. For this reason, HSP70 is considered to be a useful marker protein of the stress conditions of animals. The molecular basis for this induction has been attributed to its regulatory regions, which effectively direct the production of heterologous proteins under stress conditions. The transgenic nematode, which possesses lacZ driven by the hsp16-1 gene promoter, shows different tissue-specific effects following heat-shock and chemical stresses (Stringham et al. 1992). Heat shock was observed to affect the entire body, whereas chemical stressors affected only specific organs, such as the pharynx and nerve ring. The transgenic mouse with an introduced hsp68-lacZ gene construct shows notable heat-shock responses of the transgene in the amnion, epidermis, heart, and neural tissues in a tissue-specific manner (Kothary et al. 1989). In addition, a transgenic mouse with a human growth hormone under the control of the human Hsp70 promoter was found to release growth hormone in the plasma following intraperitoneal injection with a toxic compound (Sacco et al. 1997). The results of these studies support the concept that such transgenic animals make useful models for examining the conditions causing stress responses and cellular damage. The organic basis for this induction has been identified to the regulatory regions, which effectively direct the production of heterologous proteins under stress conditions.

Induction of the hsp70 gene in a stress-inducible manner is essentially regulated by a transcriptional mechanism. Previous attempts to introduce a mouse and human Hsp70 promoter into fish (Liu et al. 1990; Bayer and Campos-Ortega 1992) suggest that mammalian promoters have only low transcription activity. Since the HSE contains three mismatched nucleotides, in comparison to the trout, and the consensus HSE sequences, we used the minimum hsp70 promoter containing the consensus HSE sequence and generated a consensus sequence by base substitution of the three mismatched nucleotides and then fused the construct to the bacterial lacZ (Fig. 14). Transgenic zebrafish were produced by microinjecting a one-cell stage embryo with the circular form of the plasmid DNA of the lacZ construct (Yamashita 1999). DNA from the tail fins of the transgenic individuals of the F0 generation was then screened by PCR. The integrated transgene was detected in 32 F0 individuals among the 120 F0 fish screened, and germ line transmission was found in 15 F0 individuals. Among the transgenic fish, an F1 transgenic founder, tentatively named the P7 strain, showed high β-galactosidase activity only under stress conditions. Fish embryos derived from a cross between an F1 female and a wild type were fixed and stained using X-gal before and after being exposed to heat shock. Following a temperature increase from 28.5 to 37°C for 6 h, high levels of lacZ expression were observed in every tissue of the embryo. In comparison, treatment with a chemical stressor, sodium arsenite, affected lacZ expression in a tissue-specific manner. Specifically, the eyes and intestinal organs showed high β-galactosidase activity. These results show that heat-shock stress affects all tissues in the body, whereas the chemical stressor affects only the specific target tissues and cells. A state of acute arsenite toxicity was reached within 24 h in the transgenic zebrafish, at an estimated 560 μM; in contrast, the chemical stressor-specific transgene expression in the eye and the intestine was observed at 5 μM of sodium arsenite (Fig. 15). This experiment demonstrates that an approach using transgenic fish carrying the hsp70-lacZ gene construct provides an easy procedure for in vivo toxicity monitoring in aquatic environments. Therefore, the hsp70 promoter used here is considered to be one of the most active and useful promoters in fish transgenesis.


Fig. 14. Expression of the β-galactosidase gene under the control of the hsp70 promoter. Each 72-h-stage transgenic zebrafish of the F1 generation was fixed and stained with X-gal (Yamashita 1999). The control transgenic fish (control) was cultured at 28.5°C for 72 h after fertilization. The DNA construct of the hsp70 promoter-lacZ gene was microinjected into the cytoplasm of the one-cell-stage embryo. The injected embryos were cultured for 5 days in sterilized tap water in an incubator at 28.5°C, and juvenile fish were then cultivated to adult fish. The transgenic F1 generation was treated with 100 μM sodium arsenite for the last 8 h (As) or heat-shocked in warm water at 37°C for the last 6 h (HS). The tissue-specific expression of the transgene was induced in the lens and yolk extension by sodium arsenite.

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Fig. 15. Dose response of the β-galactosidase gene expression under the control of the hsp70 promoter to sodium arsenite. The transgenic embryos showing β-galactosidase activity in the eyes were counted, and 50% of the fish positive for β-galactosidase gene expression could be determined at 5 μM sodium arsenite (filled triangles), whereas lethality (50% lethal dose, LD50) following a 24-h arsenite treatment was observed at 560 μM (filled circles). This finding indicates that transgene expression is a more sensitive marker than lethality by approximately 100-fold.

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In the case of transgenic mice carrying a growth hormone gene driven by the human Hsp70 promoter, transgene expression, as determined by the plasma growth hormone level, occurred in mice injected with 2.5 mg/kg of sodium arsenite as well as in the culture cells treated with 10–50 μM sodium arsenite or other inorganic compounds (Sacco et al. 1997). The promoter region of the Hsp70 gene regulates heat-inducible expression through HSE, corresponding to inverted repeats of GAA and TTC. The heat induction of the transgene was found in every tissue in the body, with especially strong expression occurring in the yolk sac and eye, whereas chemical induction by arsenite was restricted to the eye and part of the intestine. Given that no sequences other than the HSE were found in the minimum promoter used for the expression system in the present study, the tissue-specific expression by arsenite might have been due to tissue-specific uptake and/or unknown chemical responsive transactivation mechanisms mediated by HSF.

Yamashita et al. (2003) observed that the stress- inducible expression of the transgene was easily detected in situ by induced fluorescence in GFP transgenic fish, enabling analysis of stress responses in fish in their aquatic environment. The specific GFP expression by the transgene has also been observed in living cells and tissues in embryos (Fig. 16). Such an inducible gene expression system can be used as a bioassay tool for measuring responses to aquatic stress stimuli, such as hyper- and hypoosmolarity, temperature, environmental contaminants, radiation, and UV-irradiation.


Fig. 16. Transgenic zebrafish expressing green fluorescent protein (GFP) under the control of the heat-shock promoter. Heat shock (A) and γ-irradiation (B) induced the expression of the hsp70-GFP transgene in some surface cells in the head and yolk sac in the living transgenic embryos in comparison with no apparent GFP expression in the control embryo (C). Photo shows fluorescent view of the transgenic embryos at 36 hpf. Panel D indicates the gene construct of the GFP gene under the control of the rainbow trout heat shock promoter (Yamashita 1999).

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In addition to its application as a stress-inducible promoter, the hsc70 promoter can be used for fish transgenesis. HSC70 is a constitutive expressing member of the HSP70 family in animal cells. This protein is considered to play a chaperone function, supporting protein folding and the transmembrane under normal growth conditions. Yamashita et al. (2003) examined the expression pattern of the hsc70 gene in transgenic zebrafish carrying the bacterial β-galactosidase lacZ reporter gene under the control of the trout hsc70 promoter. The hsc70 genes have also been isolated and characterized from rainbow trout and zebrafish (Zafarullah et al. 1992; Graser et al. 1996). hsc70 was expressed in a tissue-specific manner in the brain, retina, lens, spinal cord, and yolk sac in zebrafish embryos during the hatching period. Thus, this promoter is available for examining overexpression of a specific gene under non-stress conditions for the generation of transgenic fish.

6-2. Enhanced stress tolerance via the expression of active HSF

Environmental stresses, such as heat shock, UV- irradiation, infection, and toxic chemicals, affect the growth and physiological conditions of fish in aquatic environments. Biotechnology offers the possibility to modify and improve fish physiological properties. However, an important challenge remains—that of identifying the genes that can improve stress tolerance.

Heat-inducible transcriptional regulation is known to be mediated by HSF, which binds to HSE present upstream of every type of stress protein gene. The known HSF proteins share high sequence conservation in an N-terminal region comprising the DNA-binding domain, which recognizes HSE in the heat-shock gene, and hydrophobic heptad repeats that form coiled-coil structures (Wu 1995; Xiao et al. 1999). Inactive HSF is monomeric under normal physiological conditions and converted to a trimer in response to heat and other stresses, possibly through a switch from intramolecular to intermolecular coiled-coil interactions (Fig. 17) (Wu 1995; Xiao et al. 1999). Once bound to the promoter, HSF activates transcription through a potent heat-activation domain in the C-terminus, which is negatively regulated in the absence of stress (Jedlicka et al. 1997; Nakai et al. 2000). We isolated a cDNA for a zebrafish HSF1 homolog that was predicted to encode a full-length HSF of 497 amino acids. When the cRNA encoding a mutant HSF lacking the possible heat activation domain was transiently introduced into zebrafish embryos, the overexpression resulted in the expression of hsp70 and hsp47 in embryos even under non-stress conditions (Fig. 12). Thus, the expression of HSF mutants lacking a heat-activation domain can induce various types of the stress protein genes regulated by the heat-shock promoter and HSF even under non-stress conditions.


Fig. 17. Activation of HSF. (A) Model of HSF activation under stress conditions as described in Yamashita et al. (2003). (B) The HSF constructs lacking a heat-activation domain used to obtain stress-resistant transgenic lines. The nucleotide sequences of the gene constructs have been deposited to the DDBJ/EMBL/GenBank DNA Database (AB062118-AB062120).

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Nakai et al. (2000) observed that, in transgenic mice in which an active HSF mutant was expressed, many HSP genes were overexpressed under normal growth conditions. These transgenic mice showed growth arrest of testis tissue, but the relationship between the overexpression of HSF and stress tolerance has never been characterized in mice in vivo. Drosophila mutants showing tolerance against heat stress have also been established (Tanabe et al. 1998). These mutants possess point mutations in the DNA-binding domain in HSF and express high levels of HSPs under non-stress conditions. The mutants showed apparent tolerance against heat-shock stress during developmental stages. In contrast, an in vitro study using mouse and chicken cultured cells reported that disruption of the HSF gene reduced stress tolerance. Additionally, in an in vivo assay, a knockout mouse showed multiple phenotypes, such as defects of the chorioallantoic placenta and prenatal lethality, growth retardation, female infertility, elimination of the classical heat-shock response, and exaggerated tumor necrosis factor alpha production (Xiao et al. 1999). These reports suggest that HSF1 regulates critical physiological events during extraembryonic development and under pathological conditions both in vivo and in vitro. However, the relationship between the expression of HSPs regulated by HSF and the stress tolerance remains to be elucidated in vertebrates in vivo.

To establish zebrafish cell lines expressing HSF mutants, we generated two kinds of expression plasmids containing the zebrafish HSF cDNA lacking the heat-activation domain under the control of the CMV promoter (Fig. 17). The two transgenic lines, Del1 and Del2, carrying the plasmids, pCMV-HSFdel1 and pCMV-HSFdel2, respectively, were generated by microinjection of plasmids into zebrafish embryos. The transgene product mutant HSF with a His-tag sequence in the C-terminus end of the protein was detected by Western blotting in all transgenic fish lines of homozygous F2 progenies (Fig. 18). We selected the two lines (i.e., Del1 and Del2) that exhibited the highest expression levels of mutant HSF in embryos for further evaluation.


Fig. 18. Transgene expression in zebrafish. Induced expression of HSP70/HSC70 was assayed in the embryos overexpressing HSF mutants Del1 and Del2. Transgenic embryos (lanes 1, 2: Del1 individual; lanes 3, 4: Del2 individual; lane 5: wild-type embryo) at 48 hpf were used for Western blotting with anti-His-Tag (Roche) and anti-bovine HSC70 (Sigma-Aldrich) antibodies.

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The overexpression of the active form of the HSF mutant was confirmed by Western blotting and a gel-mobility shift assay. The integration of the transgene was confirmed by PCR amplification. The transgenic fish was found to express both HSFs by Western blotting (Fig. 18). Both transgenic lines, Del1 and Del2, showed higher levels of the transgene product than the wild-type fish.

To provide an understanding of the relationship between cellular HSF levels and stress tolerance in vivo, we examined the survival rates of transgenic zebrafish that overexpressed HSF under stressed conditions using UV-irradiation as a model. When transgenic fish embryos were exposed to UV irradiation at 12 hpf (hour post-fertilization), the HSF-expressing transgenic fish had higher survival rates in a dose-dependent manner than wild-type fish (Fig. 19). These findings show that HSF overexpression determines stress tolerance.


Fig. 19. Survival of transgenic embryos following UV irradiation. The embryos were maintained at 28.5°C after UV-irradiation at 60 mJ/cm2 and 12 hpf. Survival rate was assayed for wild-type zebrafish (open circle) and transgenic strains with Del1 mutant (filled triangle) or Del2 mutant (filled circle). Each value represents the mean of three independent experiments. Error bars represent one standard deviation (n = 30).

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The transgenic expression of the active form of HSF in zebrafish embryos effectively enhanced stress tolerance in vivo. Given that the transgenic embryos expressed stress proteins, the enhanced stress tolerance in the fish embryo was likely due to overexpression of stress genes, such as hsp70, hsp47, and hsp28, which are upregulated by the active form of HSF.

Conversely, the disruption of the hsf1 gene in mouse cells and the hsf3 gene in chicken cells leads to a loss of the heat-shock response (Tanabe et al. 1998). In addition to previous studies that support HSF being closely related to the stress response in vivo, our data clearly demonstrate that the active HSF determines stress tolerance in vertebrates at the whole organism level in vivo, indicating that the transition of HSF from the inactive form to the active trimer is critical for the improvement of stress tolerance. Given that the amino acid substitution of the DNA-binding domain and heat-activation domain in HSF caused the activation of HSF1 by a conformation change even under normal non-stress conditions, such a sequence substitution in the hsf gene may modulate stress tolerance in fish and other animals in vivo.

Engineered stress tolerance in fish has not been previously reported. The transgenesis of the active HSF form could be extended to other fish and animals as well as cultured cells, thereby providing tolerance to broad ranges of environmental stresses, such as heat, UV-irradiation, hyperosmolarity, heavy metals, toxic chemicals, and infection. Moreover, other stress- responsive transcription factors, such as heavy metal-responsive transcription regulator (MTF) and interferon regulatory transcription factor (IRF), could be used such that gene transfer of their active forms is mutated into transactivation domains of transgenic fish and animals to improve other types of stress tolerance (Radtke et al. 1995).

6-3. Overexpression of HSP70

Numerous studies have examined gain- or loss-of-function to elucidate the biological roles of HSP70 as a molecular chaperone in animal cells in vitro and in vivo. Overexpression of HSP70 in mammalian cultured cells has been shown to enhance anti-apoptotic activity against cellular stress (Kim et al. 1997; Kondo et al. 1997; Ravagnan et al. 1997; Li et al. 2000; Mosser et al. 2000a, b). HSP70 blocks apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome (Apaf-1/cytochrome C/caspase-9 activation complex) (Beere et al. 2000; Saleh et al. 2000; Xanthoudakis and Nicholson 2001). In contrast, TCR/CD3- and Fas- mediated apoptosis is enhanced by HSP70 overexpression in vitro (Liossis et al. 1997). In transgenic mice and rats, HSP70 overexpression in the heart was found to improve survival after the animals were subjected to ischemic heart injury (Marber et al. 1995; Hutter et al. 1996). Extra copies of the HSP70 gene resulted in increased inducible thermotolerance in transgenic Drosophila larvae (Feder et al. 1996). Thus, the chaperone functions of HSP70 appear to closely regulate thermotolerance in animal cells. However, whether the enhanced HSP70 levels improve stress tolerance in vertebrates at a whole-organism level in vivo has never been elucidated.

During the early stages of development, heat- inducible HSP70 can be induced by heat shock as well as other kinds of stress after the gastrula stage (Bensaude et al. 1983; Christians et al. 1995; Krone et al. 1997). In comparison, HSC70, a constitutively expressed member of the HSP70 family, is present as a maternal protein in oocytes and early embryos before gastrulation (Bensaude et al. 1983). Although the HSP70 and HSC70 proteins found in embryos are thought to be associated with target proteins as molecular chaperones under both normal and stressed developmental conditions, the physiological role of HSP70/HSC70 proteins in vivo remains unclear.

To investigate the biological significance of the enhanced HSP70 levels in early development, we generated and characterized transgenic zebrafish lines that overexpressed HSP70 (Yamashita and Hojo 2004). Unexpectedly, constitutive expression of HSP70 resulted in extensive apoptosis in the zebrafish embryos. We report here several unique features of the transgenic lines that are apparently associated with stress tolerance and the proapoptotic mechanism regulated by the chaperone function of HSP70.

Yamashita and Hojo (2004) generated HSP70-overexpressing transgenic zebrafish that stably expressed platyfish HSP70 cDNA driven by the rainbow trout hsc70 promoter. This gene construct was linked to the GFP gene under the control of the CMV promoter. The use of the GFP reporter gene as a marker enabled easy selection of transgenic embryos. The plasmid DNA containing the hsc70 promoter-HSP70 cDNA linked to the CMV promoter-GFP construct was introduced by microinjection into single-cell zebrafish embryos, and transgenic fish were obtained in the F1 generation. Since the transgenic embryos displayed bright-green GFP fluorescence, F1 embryos stably expressing the transgene were easily selected. Two distinct transgenic lines were generated, tentatively named SG1 and SG2, respectively. The integration of the transgene was confirmed by PCR amplification. Western blotting revealed that the transgenic fish expressed both HSP70 and GFP. The transgenic lines SG1 and SG2 showed different levels of transgene HSP70 product, with the SG1 line having a higher expression of the introduced HSP70 protein than the SG2 line.

Transgene expression was verified by western blotting and RT-PCR of DNA extracted from the tail fin of F1 transgenic fish (Yamashita and Hojo 2004). Detection with anti-bovine HSP70 monoclonal antibody showed that the transgenic lines expressed approximately 2.4-fold higher levels of HSP70 than wild-type fish. Because of its broad specificity against HSP70/HSC70 proteins, this antibody cross-reacted to both the transgene product platyfish HSP70 protein and the endogenous zebrafish HSP70/HSC70 proteins in the wild-type fish. RT-PCR analysis also revealed the specific expression of the transgene product, namely, platyfish HSP70 mRNA.

During early development, transgenic embryos showed extensive apoptosis that induced abnormal morphogenesis (Yamashita and Hojo 2004), and by 5 days postfertilization, 12–25% of the embryos were dead. Fluorescent staining of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) revealed the presence of fluorescent-labeled apoptotic cells throughout the embryos, particularly in the eyes, brain, and spinal cord. In addition, when HSP70 cRNA was microinjected into single-cell-stage zebrafish embryos, the HSP70 cRNA also induced extensive apoptosis and abnormal morphogenesis. The degeneration of the whole embryo and notochord was indicated by HSP70 overexpression. When the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) was co-microinjected with HSP70 cRNA into zebrafish embryos, the inhibitor prevented the apoptosis induced by HSP70 overexpression. However, when an HSP70 mutant lacking the C-terminal peptide binding domain was microinjected, no induction of abnormal morphogenesis was observed. These findings indicate that HSP70 overexpression induced a caspase-mediated apoptotic pathway by a chaperone function through the C-terminal peptide-binding domain.

The spatial and tissue-specific nature of the observed apoptosis was most likely relevant to the expression pattern of the transgene under the control of the HSC70 promoter (Yamashita and Hojo 2004). These results suggest that HSP70 is able to induce extensive apoptosis in zebrafish embryos. Although the surviving embryos developed into adults, the transgenic fish showed defects in the jaw, had small eyes, and lacked a tail fin (Yamashita and Hojo 2004). The transgenic adult fish overexpressing HSP70 showed abnormal morphogenetic development and a defect of the upper jaw.

We have successfully generated transgenic zebrafish that overexpress HSP70 (Yamashita and Hojo 2004). However, the fish show extensive apoptosis and abnormal morphogenesis in early development. Feder et al. (1996) reported that the expression of a higher number of HSP70 transgene copies resulted in en hanced heat tolerance in transgenic Drosophila, whereas the protective role of HSP70 has been demonstrated in transgenic mice with heart injury (Marber et al. 1995; Hutter et al. 1996). The transient overexpression of HSP70 in cultured cells is considered to be protective against stress-inducible apoptosis (Kim et al. 1997; Kondo et al. 1997; Ravagnan et al. 1997; Li et al. 2000; Mosser et al. 2000a, b). In contrast, our findings indicate that HSP70 overexpression during zebrafish development brings about stress sensitivity. As HSP70 is believed to exhibit chaperone functions under cellular stress conditions, the HSP70 overexpression in early embryos may regulate the expression pattern of various target proteins that bind to HSP70. Also, bone morphogenetic protein-4 (BMP4) mRNA level has been found to be higher in transgenic embryos than in wild-type embryos, suggesting that the enhanced HSP70 levels regulate the BMP4- mediated apoptotic signaling in early development. The transgenic embryos overexpressing HSP70 showed enhanced levels of phosphorylated Smad2 (Fig. 20). Smad2 is phosphorylated by the activation of activin-like kinases, including BMP4 and Nodal receptors, via ligand binding. This finding suggests that the receptor may be activated by the overexpression of HSP70.


Fig. 20. Induction of Smad2 phosphorylation in the transgenic zebrafish embryos at 9 hpf by whole-mount staining with antibodies vs. phosphorylated Smad2 and HSP70. The embryos overexpressing platyfish HSP70 under the control of the HSC70 promoter (Yamashita and Hojo 2004) were fixed with 10% formalin in PBS, and phosphorylated Smad2 and HSP70/HSC70 were stained with anti-phosphorylated Smad2 rabbit monoclonal antibody (Cell Signaling) and anti-bovine HSC70 (Sigma-Aldrich) by detection of signals with BM Blue POD substrate (Roche Japan).

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BMP4 is known to negatively regulate head and somite formation (Graham et al. 1994; Furuta et al. 1997; Trousse et al. 2001). Given that high HSP70 levels were found to induce BMP4 signaling and repress head formation in this study, we propose that in order for morphogenesis to occur in early development, HSP70 is required to manifest BMP4-mediated apoptosis-inducing activity by regulating downstream genes and/or serving as one component of multiple inductive signals. As BMPs have been shown to regulate interdigital cell death in the avian embryo, the undifferentiated distal mesodermal cells may undergo chondrogenic differentiation or apoptosis depending on whether they are incorporated into the future digital rays or into the interdigital spaces (Merino et al. 1999). Both chondrogenesis and apoptosis are induced by local BMPs. BMP pro-apoptotic activity is reported to regulate the expression of members of the msx family of closely related homeobox-containing genes and is finally mediated by caspase activation (Merino et al. 1999). These findings indicate that HSP70 may modulate the ability of BMPs to induce apoptosis through an upstream signaling factor present in the early embryos, such as members of the fibroblast growth factor (FGF) and Transforming growth factor beta (TGF-β) families.

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7. Molecular chaperone function of HSP70 in zebrafish embryos

7-1. Control of TGF-β receptor activation

Several gain- or loss-of-function studies have been performed to elucidate the biological roles of HSP70 as a molecular chaperone in animal cells in vitro and in vivo (Marber et al. 1995; Feder et al. 1996; Hutter et al. 1996; Kondo et al. 1997; Liossis et al. 1997; Ravagnan et al. 1997; Beere et al. 2000; Mosser et al. 2000a, b; Saleh et al. 2000; Xanthoudakis and Nicholson 2001). We recently demonstrated that the overexpression of HSP70 in zebrafish embryos induces BMP4 expression and extensive apoptosis during early development, which results in ventralized phenotypes (Yamashita and Hojo 2004). Therefore, our current focus is on the molecular chaperone functions of the HSP70 family proteins in Activin/Nodal/TGF-β signaling mechanisms responsible for vertebrate dors oventral formation.

Members of the TGF-β superfamily are involved in many biological activities, including growth, differentiation, migration, cell survival, and adhesion in both the diseased and normal states (Whitman 2001; Schier 2003). They are classified into two major groups: Activin/Nodal/TGF-β and BMP/GDF. TGF-β ligand binding induces the formation of a receptor complex that consists of receptor type II and type I, both of which are required for signal transduction (Luo and Lodish 1996; Renucci et al. 1996; Lawler et al. 1997; Garg et al. 1999; Nagaso et al. 1999). Both type II and type I receptors contain serine/threonine kinase domains in their intracellular portions. Type II receptor kinases are constitutively active and, upon ligand binding, hetero-tetrameric complexes, composed of two molecules each of the type II and type I receptors, are formed (Luo and Lodish 1996; Lawler et al. 1997). In the tetrameric receptor complexes, type II receptor kinases transphosphorylate type I receptors, which are thereby activated and phosphorylate intracellular substrates, i.e., transcription factor Smad proteins (Derynck et al. 1998; Muèllera et al. 1999; Miyazawa et al. 2002). To date, several cofactors, including extracellular proteins, Lefty proteins, Cerberus, Tomoregulin-1, and the transmembrane protein Dpr2, have been found to be associated with the receptors and to inhibit Nodal signaling during early embryogenesis (Whitman 2001; Schier 2003; Zhang et al. 2004). However, intracellular cofactors have been poorly characterized with respect to the receptors for Nodal signaling. In this study, we characterized the activation of Nodal receptors by HSP70/HSC70 with the aim of investigating the molecular chaperone function and the target protein of HSP70/HSC70 in early development.

Zebrafish hsc70 is expressed as a maternal protein, and mRNA in the fertilized eggs and its zygotic gene expression occur after gastrulation, especially in the brain and yolk sac (Graser et al. 1996; Santacruz et al. 1997). When segmentation starts, hsc70 is expressed in the dorsal trunk neural tube, the lateral plate mesoderm, and the tail bud. This expression pattern of hsc70 suggests a role in early mesoderm induction.

Since HSC70 deficiency reduces the expression of the gsc and lim1 genes, genetic interactions likely occur between HSC70 and Nodal signaling. Activin and Nodal have mesoderm-inducing abilities producing a variety of mesoderm derivatives in a dose- dependent manner (Whitman 2001; Schier 2003). In zebrafish, the type I receptors consist of Activin receptor-like kinase (ALK)2 and ALK4, while type II receptors comprise ActRIIB (Luo and Lodish 1996; Renucci et al. 1996; Lawler et al. 1997; Garg et al. 1999; Nagaso et al. 1999). Nodal signaling can be promoted by phosphorylation of the transcription factor Smad2, which is mediated by Nodal/Activin/TGF-β receptors. The HSC70 deficiency caused by microinjection of the Morpholino antisense oligo, HSC-MO, which is a synthetic inhibitor against translation of the hsc70 gene, is accompanied by an apparent reduction in Smad2 phosphorylation at the shield stage (Yamashita et al. unpublished).

The phosphorylation of Smad2 was observed to be inhibited in embryos treated with the type I receptor kinase inhibitor SB-431542 (Muèllera et al. 1999; Ho et al. 2006). This inhibitor did not affect the expression of HSC70. In addition, Smad2 phosphorylation was induced following the microinjection of HSP70 cRNA into zebrafish one-cell-stage zebrafish embryos (Yamashita et al. unpublished). It can therefore be concluded that Smad2 phosphorylation by Activin-like receptor kinase is regulated by the expression levels of HSC70. The detection of phosphorylated Smad2 is an important marker for Nodal signaling enhanced by HSC70 in zebrafish embryos. To characterize the molecular mechanism of enhanced Nodal signaling by HSC70, we examined the relationship between the type I and type II receptors and stress proteins (Yamashita et al. unpublished).

A constitutively active form of the type I serine/threonine kinase receptor Taram-A* type I receptor shows kinase activity in the absence of any ligand-receptor complex formation with the type II receptor (Renucci et al. 1996). This reaction is not inhibited by HSC-MO injection (Yamashita et al. unpublished). Therefore, Smad2 phosphorylation is mediated by the type I receptor alone and is not regulated by HSC70.

During the activation process of tetrameric receptor complexes, the ActRIIB kinase acts as an upstream component of the type I receptor in the signaling pathways (Luo and Lodish 1996; Renucci et al. 1996; Lawler et al. 1997; Garg et al. 1999; Nagaso et al. 1999). Overexpression of ActRIIB kinase following the injection of cRNA into the zebrafish embryos induced the phosphorylation of Smad2. In addition, co- injection of ActRIIB kinase cRNA and HSC-MO inhibited Smad2 phosphorylation (Yamashita et al. unpublished). Therefore, the activation of the type II receptor in Nodal signaling is regulated by HSC70. The expression patterns of the constitutive HSC70 and heat-inducible hsp70 genes may affect the complex pattern of Nodal expression during early embryogenesis. Since HSP70 is induced and accumulated under stress conditions, these protein levels may affect the patterning of morphogenesis mediated by Nodal and other TGF-β family ligands (Yamashita et al. unpublished).

In this study, we propose a novel molecular chaperone function of HSP70/HSC70 on the kinase activation of type II receptor, i.e., ActRIIB kinase (Fig. 21). First, the serine residue around the catalytic center, which is the primary autophosphorylation site for kinase activation of the type II receptor, is phosphorylated by an intermolecular mechanism assisted by HSP70/HSC70. Second, autophosphorylation of other serine and tyrosine residues is accompanied by homodimerization of the type II receptor. Furthermore, the type II receptor kinase phosphorylates the cytoplasmic domain of the type I receptor at serine and threonine, and the phosphorylation of both types of cytoplasmic domain contributes to the stability of the heteromeric complex. Therefore, as a cytosolic molecular chaperone, the HSC70 protein plays essential roles in the formation and activation of Activin/Nodal/TGF-β and BMP receptors.


Fig. 21. Model for the formation and activation of Activin/Nodal/TGF-β and BMP receptors by HSP70/HSC70. HSP70/HSC70 binds directly to the type II receptor kinases of the receptors and facilitates Nodal signaling.

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7-2. CMA in fish

Starvation is an important stress condition that causes a variety of physiological dysfunctions and deteriorates the eating quality of fish. Recent studies have identified biomarkers to detect the state of stress and/or starvation in fish and cultured cells. In particular, an autophagic pathway that forms autophagic vacuoles (i.e., autophagosomes) is responsible for the survival of the heat-shocked and/or starved cells by the withdrawal of amino acids from culture medium. The autophagy has been identified as an apoptotic pathway induced by stress conditions, such as starvation, heat shock, and hypoxia, followed by intracellular protein degradation (bulk degradation), and differs from the ubiquitin-proteasome system (Yabu et al. unpublished). We characterized the induction processes of autophagic pathways in cultured fish cells as a model to evaluate the influence and the degree of biochemical changes by heat stress and amino acid starvation (Yabu et al. unpublished). A microtubule-binding protein that is localized in autophagosome membranes can be used as a molecular marker for induction of autophagy. We established stable transformants of a zebrafish ZE cell line introduced with a fusion protein of GFP and microtubule-associated protein 1-light chain 3 (MAP1-LC3) to detect fluorescent autophagosomes associated with the GFP-MAP1-LC3 fusion protein (Yabu et al. unpublished). The induction of autophagy was examined under amino acid starvation and heat-stress conditions. The high temperature and amino acid withdrawal in the ZE cells were shown to induce autophagy in a time-dependent manner, as evidenced by the number of fluorescent particles localized with the GFP-MAP1-LC3 fusion protein in the cell. The autophagy induced by the amino acid withdrawal was suppressed in the presence of the phosphatidylinositol 3-kinase inhibitor, 3-methyladenine in the culture medium and by overexpression of the bcl-2 gene. A phosphatidylinositol 3-kinase, a target of rapamycin (TOR), mediates the autophagic signaling pathway that regulates the starvation-induced autophagy (Yabu et al. unpublished). These results show that this is a new biological model of the autophagic signaling pathway in cultured fish cells under heat-stress and amino acid-starvation conditions.

The cytosolic members of the HSP70 family participate in CMA (Cuervo et al. 1995; Reggiori and Klionsky 2002; Dice et al. 2003). The lysosomal pro teolytic system is important in the removal of oxidized and abnormal proteins produced under stressed conditions. In mammalian cells, three main, but different, mechanisms contribute to the degradation of intracellular components inside lysosomes (Cuervo et al. 1995; Reggiori and Klionsky 2002; Dice et al. 2003). HSC70 selects substrate cytosolic proteins degraded through this pathway (Cuervo et al. 1995, 1998, 1999, 2003). The cytosolic proteins contain a targeting motif biochemically related to the pentapeptide KFERQ (Chiang and Dice 1988; Dice et al. 2003). This motif, present in about 30% of the proteins in the cytosol, is recognized by HSC70. The interaction with HSC70 and with the co-chaperones targets the substrates and leads them to the lysosomal membrane (Agarraberes and Dice 2001). Substrates must be unfolded before translocation into the lysosomal lumen, and several cytosolic chaperones associated with the lysosomal membrane have been proposed to assist in the unfolding (Agarraberes and Dice 2001). Although some basal level of CMA is probably present in most cells, nutritional stress has been shown to maximally activate this pathway (Cuervo et al. 1995). Activation during nutrient deprivation is associated with higher levels of HSC70 in the lysosomal lumen (Chiang and Dice 1988; Cuervo et al. 1995; Agarraberes et al. 1997). In addition to starvation, activation of CMA has also been observed in rat liver and kidney following exposure to gasoline derivatives (Cuervo et al. 1999), in fibroblasts from patients with galactosialidosis, which lack the protective protein/cathepsin A, and in cells overexpressing lamp2a (Chiang and Dice 1988; Dice et al. 2003). In contrast, CMA activity is reduced during renal tubular cell growth (Franch et al. 2001) and in aging. The decrease in CMA activity in old cells may be associated with their known tendency to accumulate oxidized proteins (Cuervo and Dice 2000).

The cytosolic HSP70/HSC70 might be responsible for CMA under both normal and stressed conditions (Yabu et al. unpublished). This effect may be depend ent of the synthesis of heat-inducible HSC70 and HSP70, which requires a minimum of 1 h under stress conditions. The selective proteolysis of cytosolic protein substrates by CMA produces amino acids as an energy source. The fish cells appear to utilize CMA and to survive under the heat-shocked conditions mediated by the heat-inducible members of the HSP70 family. In mammalian cells, CMA is activated during oxidative stress and toxic exposure resulting in the selective degradation of chemically modified abnormal proteins altered under the stress conditions (Cuervo et al. 1999). In goldfish GTFe-2 cells, Sato et al. (1990) reported that a variety of stress proteins are synthesized constitutively at 37°C, with HSP70 and HSP30 being the dominantly synthesized proteins of the goldfish cells at 40°C. Therefore, enhanced CMA mediated by HSP70/HSC70 and other stress proteins may be responsible for the tolerance to stress conditions. HSP70/HSC70 may be used as the source of protein degradation and thus effectively function as an energy source under heat and other stress conditions. Therefore, in fish, HSP70 and HSC70 may play important roles in protein degradation/catabolism by autophagy in both stressed and non-stressed environments (Figs. 22, 23) (Yabu et al. unpublished).


Fig. 22. Induction of autophagy. Two pathways, i.e., CMA and macroautophagy, are characterized in fish cells (Yamashita 2010; Yabu et al. unpubilished). The selective proteolysis of cytosolic protein substrates by CMA produces amino acids as an energy source under the heat-shocked conditions mediated by the heat-inducible members of the HSP70 family. In addition, macroautophagy that forms autophagic vacuoles (i.e., autophagosomes) is responsible for the survival of starved cells by intracellular protein bulk degradation.

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Fig. 23. Induction of CMA in the muscles of bluefin tuna. The burnt tuna meat showed induction of CMA and extensive proteolysis (Yamashita 2010). Three individuals with normal, burnt, and severely (heavy) burnt muscles were used for the assay. Upper panel: SDS-PAGE on the water-soluble fraction. Lower panel: western blot of the lysosomal fraction obtained by subcellular fractionation with antibodies against cathepsin L, aldolase, and HSC70. The red arrow indicates proteolytic products found in the burnt tuna muscle. A: Internal portion in the white muscle. B: Lateral portion in the white muscle. C: Dorsal portion of the white muscle. D: Dark muscle.

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Finally, the autophagic mechanism has been induced in the muscle tissues of bluefin tuna, producing abnormal softened meat referred to as "burnt tuna meat" (Yamashita 2010), and in chum salmon during the spawning migration (Yamashita and Konagaya 1991). In both cases, the muscle showed high cathepsin L activity, and the lysosomal fraction of the muscle contained HSP70/HSC70, aldolase, and cathepsin L. This finding suggests the induced activation of CMA during and/or after the capture of fish in vivo. Therefore, CMA may be important for understanding fish physiology as well as controlling fish meat quality under a variety of environmental circumstances.

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Acknowledgments

This work was supported in part by grants from the Fisheries Research Agency, the Japan Society of the Promotion of Science, the Ministry of Agriculture, Forestry and Fisheries, and the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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List of Figures

Fig. 1. Expression of HSP70 in the heat-shocked zebrafish embryo. Embryos maintained at 28.5°C were transferred to 37°C for 1 h. The embryos were fixed with 10% formalin in PBS, and HSP70 was stained with a specific monoclonal antibody (Embiotech Laboratories, Tokyo, Japan) vs. heat-inducible HSP70 raised against the C-terminal region of zebrafish HSP70-a by detection of signals with BM Blue POD substrate (Roche Japan, Tokyo, Japan). (A) Whole mount immunostaining showed HSP70 expressed in tissue specific manners. (B) Western blotting.

Fig. 2. Model of molecular chaperone functions of the HSP70 family. HSP70 and HSC70 function as intracellular chaperones for other proteins, regulating protein-protein interactions, such as protein folding, establishment of protein disassembly and rearrangement, prevention of protein aggregation, transport of target proteins into the intracellular compartments, and translocation of proteins across membranes (Welch and Feramisco 1985). MtHSP70 and GRP78 are localized to the mitochondria and the endoplasmic reticulum, respectively (Yamashita et al. 2004).

Fig. 3. Amino acid sequences of deduced proteins encoded in the heat-inducible hsp70 genes identified in the zebrafish genome. Comparisons of the predicted amino acid sequences. Residues identical to the amino acid in the zebrafish HSP70-a sequence are indicated by dots. Amino acids that are present in zebrafish HSP70-a, but not in other HSP70s, are marked by dashes. The zebrafish cDNA sequence for HSP70-a has been deposited in the DDBJ database with the accession number AB062116. The genes encoding HSP70-b and HSP70-ctg9500 are present on zebrafish chromosome 16 and 8, respectively (Yamashita et al. 2004).

Fig. 4. Molecular phylogenic tree of the HSP70 family members. Amino acid sequences of the vertebrate HSP70s were compared by the neighbor-joining method with the CLUSTAL W program (version 1.83). Bootstrap confidence values for the sequence groupings are indicated in the tree (n = 1000). The scale indicates the evolutionary distance of one amino acid substitution per site. Sequence database accession numbers in GenBank or genomic contig scaffold numbers in the zebrafish and Takifugu Ensenbl Genome Servers are indicated in parentheses.

Fig. 5. Gene structure of the hsp70 genes in zebrafish and human genomes. (A) The zebrafish genome contains three copies of the hsp70 genes, and HSP70-a was the only heat-inducible member of the HSP70 family. The gene encoding HSP70-b is tandemly linked to the gene encoding HSP70-a on the zebrafish chromosome 16, and the gene encoding HSP70-ctg9500 is present on zebrafish chromosome 8 (Yamashita et al. 2004). Arrows indicate the heat-inducibility of these genes measured by RT-PCR (Yabu et al. unpublished). (B) The human genome contains five copies of the hsp70 and its related genes; hsp70-1, hsp70-2, and hsp70B' are heat-inducible, whereas Hsp70HOM and Hst70 are testis-specific.

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Fig. 6. The nucleotide sequence of the 5'-upstream region of the zebrafish hsp70-a gene. The transciption start site was determined by primer extension. The nucleotide sequences in exons are underlined. The zebrafish hsp70 gene contains only one intron in the 5'-noncoding region. The translation start ATG codon is in italics. The heat-inducible expression of the zebrafish hsp70 gene is suggested to be regulated by a dual promoter system, i.e., a heat-shock promoter containing HSE upstream of the transcription start site and another constitutive promoter in the first intron (Yabu et al. unpublished).

Fig. 7. Comparisons of the predicted amino acid sequence of zebrafish HSF with those of human HSF1, mouse HSF1, chicken HSF1, and rainbow trout HSF1a proteins. Residues identical to the amino acid in the zebrafish HSF (accession number AB062117) are indicated by dots. Amino acids that are present in zebrafish HSF, but not in other HSFs, are marked by dashes. The regions boxed with a solid line or broken line indicate the predicted DNA binding domains and hydrophobic heptad repeats, respectively. The potent heat-activation domain boxed in red is suggested to regulate temperature ranges for HSF activation and conformational changes.

Fig. 8. Northern blot analysis of hsp70 and hsf mRNAs from zebrafish embryos. Total RNA was isolated from embryos incubated at 28.5°C and heat-shocked embryos that had undergone a 1-h temperature shift to 37°C, and then subjected to Northern analysis. (A) hsp70 mRNA, (B) hsf mRNA. The hsf gene was expressed as a primary transcript of 6 kb and a mature form of 2 kb after the gastrula stage, which coincided with the expression pattern of the hsf gene. Embryonic staging was according to Kimmel et al. (1995).

Fig. 9. Heat-inducible expression of the hsp70 gene detected by in situ hybridization. The fish hsp70 gene showed stress-inducible gene expression. In zebrafish embryos, hsp70 mRNA was expressed at the segmentation stage after the gastrula period under heat-shock conditions. During the cleavage and blastula periods, no apparent expression of hsp70 mRNA was seen, even in the heat-shocked 3-h embryos (B). However, heat-shock induction of hsp70 mRNA was found in the embryos 6 h after fertilization (D). A temperature shift from 28.5°C to 37°C for 1 h induced hsp70 mRNA expression throughout the entire embryo during the gastrula and segmentation periods (B), (D). Conversely, control embryos cultured at 28.5°C showed no apparent expression of hsp70 mRNA in any of the stages tested in this study (A), (C).

Fig. 10. Heat-inducible expression of the hsp70 gene detected by in situ hybridization. Most notably, strong expression of the hsp70 gene was observed in the brain, notochord, and yolk sac of heat-shocked 1- to 2-day-old embryos. In the case of the 2-day-old heat-shocked embryos, hsp70 mRNA expression was elevated in the forebrain, hindbrain, notochord (nt), otic vesicle (ov), yolk sac (yc), and skeletal muscle (m) in the middle part of the body. Thus, the hsp70 gene was expressed in stage- and tissue-specific manners.

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Fig. 11. Expression of hsf mRNA by in situ hybridization. The hsf gene was expressed after the gastrula period. The hsf gene is induced by zygotic expression during early development, similar to most other housekeeping genes. hsf mRNA was not observed in embryos within 3 h of fertilization (A) as shown by Northern blot analysis (see Fig. 8). After the gastrula period, hsf mRNA was observed throughout the developmental stages of zebrafish embryos (B)–(D). In 2-day-old embryos, HSF mRNA was highly expressed in the brain, notochord (nt), otic vesicle (ov), and yolk sac (yo) (D). Thus, the spatiotemporal expression of zebrafish hsf mRNA accounts for the tissue-specific heat-inducibility of HSP70 under stress conditions.

Fig. 12. Transactivation activities of zebrafish HSF. (A) Gene expression induced by overexpression of HSF. The cRNA encoding zebrafish HSF (HSF, lane 2) and the HSF mutants, HSF-Del1 (Del1, lane 3) and HSF-Del2 (Del2, lane 4), was introduced into zebrafish embryos at the one-cell stage. The embryos were then incubated at 28.5°C for 12 h. The control embryos were maintained at 28.5°C (control, lane 1), and the heat-shocked embryos were incubated at 37°C from the 10 h post-fertilization (hpf) to the 12 hpf (heat shock, lane 5). In the 12-h embryos, the mRNA expression levels of hsp70-a, hsp47, hsc70-1, and hsf were examined by RT-PCR. (B) The cRNA encoding zebrafish HSF or that of the truncated mutants, HSF-Del1 (Del1) and HSF-Del2 (Del2), was microinjected into zebrafish embryos at the one-cell stage, along with the hsp70-GFP gene construct. The induction of GFP expression under the control of the hsp70 promoter was observed in 12-h embryos by fluorescence microscopy.

Fig. 13. The model of divergence of the HSP70 family members during vertebrate evolution. The hsp70 genes may have diverged during vertebrate evolution several times. The duplication of the hsp70 genes may have occurred during fish evolution (open circle) or mammalian evolution (close circle).

Fig. 14. Expression of the β-galactosidase gene under the control of the hsp70 promoter. Each 72-h-stage transgenic zebrafish of the F1 generation was fixed and stained with X-gal (Yamashita 1999). The control transgenic fish (control) was cultured at 28.5°C for 72 h after fertilization. The DNA construct of the hsp70 promoter-lacZ gene was microinjected into the cytoplasm of the one-cell-stage embryo. The injected embryos were cultured for 5 days in sterilized tap water in an incubator at 28.5°C, and juvenile fish were then cultivated to adult fish. The transgenic F1 generation was treated with 100 μM sodium arsenite for the last 8 h (As) or heat-shocked in warm water at 37°C for the last 6 h (HS). The tissue-specific expression of the transgene was induced in the lens and yolk extension by sodium arsenite.

Fig. 15. Dose response of the β-galactosidase gene expression under the control of the hsp70 promoter to sodium arsenite. The transgenic embryos showing β-galactosidase activity in the eyes were counted, and 50% of the fish positive for β-galactosidase gene expression could be determined at 5 μM sodium arsenite (filled triangles), whereas lethality (50% lethal dose, LD50) following a 24-h arsenite treatment was observed at 560 μM (filled circles). This finding indicates that transgene expression is a more sensitive marker than lethality by approximately 100-fold.

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Fig. 16. Transgenic zebrafish expressing green fluorescent protein (GFP) under the control of the heat-shock promoter. Heat shock (A) and γ-irradiation (B) induced the expression of the hsp70-GFP transgene in some surface cells in the head and yolk sac in the living transgenic embryos in comparison with no apparent GFP expression in the control embryo (C). Photo shows fluorescent view of the transgenic embryos at 36 hpf. Panel D indicates the gene construct of the GFP gene under the control of the rainbow trout heat shock promoter (Yamashita 1999).

Fig. 17. Activation of HSF. (A) Model of HSF activation under stress conditions as described in Yamashita et al. (2003). (B) The HSF constructs lacking a heat-activation domain used to obtain stress-resistant transgenic lines. The nucleotide sequences of the gene constructs have been deposited to the DDBJ/EMBL/GenBank DNA Database (AB062118-AB062120).

Fig. 18. Transgene expression in zebrafish. Induced expression of HSP70/HSC70 was assayed in the embryos overexpressing HSF mutants Del1 and Del2. Transgenic embryos (lanes 1, 2: Del1 individual; lanes 3, 4: Del2 individual; lane 5: wild-type embryo) at 48 hpf were used for Western blotting with anti-His-Tag (Roche) and anti-bovine HSC70 (Sigma-Aldrich) antibodies.

Fig. 19. Survival of transgenic embryos following UV irradiation. The embryos were maintained at 28.5°C after UV-irradiation at 60 mJ/cm2 and 12 hpf. Survival rate was assayed for wild-type zebrafish (open circle) and transgenic strains with Del1 mutant (filled triangle) or Del2 mutant (filled circle). Each value represents the mean of three independent experiments. Error bars represent one standard deviation (n = 30).

Fig. 20. Induction of Smad2 phosphorylation in the transgenic zebrafish embryos at 9 hpf by whole-mount staining with antibodies vs. phosphorylated Smad2 and HSP70. The embryos overexpressing platyfish HSP70 under the control of the HSC70 promoter (Yamashita and Hojo 2004) were fixed with 10% formalin in PBS, and phosphorylated Smad2 and HSP70/HSC70 were stained with anti-phosphorylated Smad2 rabbit monoclonal antibody (Cell Signaling) and anti-bovine HSC70 (Sigma-Aldrich) by detection of signals with BM Blue POD substrate (Roche Japan).

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Fig. 21. Model for the formation and activation of Activin/Nodal/TGF-β and BMP receptors by HSP70/HSC70. HSP70/HSC70 binds directly to the type II receptor kinases of the receptors and facilitates Nodal signaling.

Fig. 22. Induction of autophagy. Two pathways, i.e., CMA and macroautophagy, are characterized in fish cells (Yamashita 2010; Yabu et al. unpubilished). The selective proteolysis of cytosolic protein substrates by CMA produces amino acids as an energy source under the heat-shocked conditions mediated by the heat-inducible members of the HSP70 family. In addition, macroautophagy that forms autophagic vacuoles (i.e., autophagosomes) is responsible for the survival of starved cells by intracellular protein bulk degradation.

Fig. 23. Induction of CMA in the muscles of bluefin tuna. The burnt tuna meat showed induction of CMA and extensive proteolysis (Yamashita 2010). Three individuals with normal, burnt, and severely (heavy) burnt muscles were used for the assay. Upper panel: SDS-PAGE on the water-soluble fraction. Lower panel: western blot of the lysosomal fraction obtained by subcellular fractionation with antibodies against cathepsin L, aldolase, and HSC70. The red arrow indicates proteolytic products found in the burnt tuna muscle. A: Internal portion in the white muscle. B: Lateral portion in the white muscle. C: Dorsal portion of the white muscle. D: Dark muscle.

Table 1. Zebrafish genes neighboring the HSP70 genes.

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