Hsp27 is persistently expressed in zebrafish skeletal and cardiac muscle tissues but dispensable for their morphogenesis

Constitutive expression of Hsp27 has been demonstrated in vertebrate embryos, especially in developing skeletal and cardiac muscle. Results of several previous studies have indicated that Hsp27 could play a role in the development of these tissues. For example, inhibition of Hsp27 expression has been reported to cause defective development of mammalian myoblasts in vitro and frog embryos in vivo. In contrast, transgenic mice lacking Hsp27 develop normally. Here, we examined the distribution of Hsp27 protein in developing and adult zebrafish and effects of suppressing Hsp27 expression using phosphorodiamidate morpholino oligonucleotides (PMO) on zebrafish development. Consistent with our previous analysis of hsp27 messenger RNA expression, we detected the protein Hsp27 in cardiac, smooth, and skeletal muscle of both embryonic and adult zebrafish. However, embryos lacking detectable Hsp27 after injection of antisense hsp27 PMO exhibited comparable heart beat rates to that of control embryos and cardiac morphology was indistinguishable in the presence or absence of Hsp27. Loss of Hsp27 also had no effect on the structure of the skeletal muscle myotomes in the developing embryo. Finally, embryos injected with antisense hsp27 and scrambled control PMO displayed equal motility. We conclude that Hsp27 is dispensable for zebrafish morphogenesis but could play a role in long-term maintenance of heart and muscle tissues. Tucker and Ustyugov contributed equally to this work.     

Development of a heat shock inducible gfp transgenic zebrafish line by using the zebrafish hsp27 promoter

In the present study, a zebrafish hsp27 promoter was isolated and used to develop heat shock inducible gfp transgenic zebrafish. The endogenous hsp27 mRNAs were constitutively expressed from 4 hpf and increased in several regions of brain, heart and somites in early embryogenesis until 24 hpf. Subsequently, the expression was reduced significantly but maintained in the heart and ears. Heat shock induced hsp27 mRNAs in the blastoderm from 6 hpf and later in somites, branchial arches and several regions of brain. Similarly in hsp27-gfp transgenic zebrafish, constitutive GFP expression was observed from 11 hpf. GFP expression was mainly in the skin cells and increased to the peak level at 7 dpf, followed by a reduction. The constitutive GFP expression in the heart was initiated from 50 hpf and maintained even in the adult fish. After heat shock, GFP expression was mainly induced in the muscle in addition to a mild increase in the skin and heart. The early stages of the embryos were more sensitive than late stages as the time required for induced GFP expression in the muscle is shorter. Thus, the hsp27-gfp transgenic line generally recapitulates the expression pattern and heat shock inducibility of endogenous hsp27 RNAs. We also tested the potential of using the hsp27-gfp transgenic zebrafish embryos for heavy metal induction and demonstrated the inducibility of GFP expression by arsenic; this pattern of induction was also supported by examination of endogenous hsp27 mRNA.     

Zebrafish Hsp70 is required for embryonic lens formation

Heat shock proteins (Hsps) were originally identified as proteins expressed after exposure of cells to environmental stress. Several Hsps were subsequently shown to play roles as molecular chaperones in normal intracellular protein folding and targeting events and to be expressed during discrete periods in the development of several embryonic tissues. However, only recently have studies begun to address the specific developmental consequences of inhibiting Hsp expression to determine whether these molecular chaperones are required for specific developmental events. We have previously shown that the heat-inducible zebrafish hsp70 gene is expressed during a distinct temporal window of embryonic lens formation at normal growth temperatures. In addition, a 1.5-kb fragment of the zebrafish hsp70 gene promoter is sufficient to direct expression of a gfp reporter gene to the lens, suggesting that the hsp70 gene is expressed as part of the normal lens development program. Here, we used microinjection of morpholino-modified antisense oligonucleotides (MOs) to reduce Hsp70 levels during zebrafish development and to show that Hsp70 is required for normal lens formation. Hsp70-MO-injected embryos exhibited a small-eye phenotype relative to wild-type and control-injected animals, with the phenotype discernable during the second day of development. Histological and immunological analysis revealed a small, underdeveloped lens. Numerous terminal deoxynucleotidyl transferase-mediated dUTP-fluoroscein nick-end labeling (TUNEL)-positive nuclei appeared in the lens of small-eye embryos after 48 hours postfertilization (hpf), whereas they were no longer apparent in untreated embryos by this age. Lenses transplanted from hsp70-MO-injected embryos into wild-type hosts failed to recover and retained the immature morphology characteristic of the small-eye phenotype, indicating that the lens phenotype is lens autonomous. Our data suggest that the lens defect in hsp70-MO-injected embryos is predominantly at the level of postmitotic lens fiber differentiation, a result supported by the appearance of mature lens organization in these embryos by 5 days postfertilization, once morpholino degradation or dilution has occurred.     

Examination of the expression of the heat shock protein gene, hsp110, in Xenopus laevis cultured cells and embryos

Eukaryotic organisms respond to various stresses with the synthesis of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is part of the HSP70/DnaK superfamily. In this study, we have examined, for the first time, the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. Sequence analysis revealed that the protein encoded by the hsp110 cDNA exhibited 74% identity with its counterparts in mammals and only 27-29% with members of the Xenopus HSP70 family. Hsp110 mRNA and/or protein was detected constitutively in A6 kidney epithelial cells and was inducible by heat shock, sodium arsenite, and cadmium chloride. However, treatment with ethanol or copper sulfate had no detectable effect on hsp110 mRNA levels. Similar results were obtained for hsp70 mRNA except that it was inducible with ethanol. In Xenopus embryos, hsp110 mRNA was present constitutively during development. Heat shock-inducible accumulation of hsp110 mRNA occurred only after the midblastula stage. Whole mount in situ hybridization analysis revealed that hsp110 mRNA accumulation in control and heat shocked embryos was enriched in selected tissues. These studies demonstrate that Xenopus hsp110 gene expression is constitutive and stress inducible in cultured cells and developmentally- and tissue specifically-regulated during early embryogenesis.     

Cloning, characterization, and heat stress-induced redistribution of a protein homologous to human hsp27 in the zebrafish Danio rerio

Hsp27 is a small heat shock protein (shsp) regulating stress tolerance and increasingly thought to play roles in tissue homeostasis and differentiation. The zebrafish Danio rerio is an important model for the study of developmental processes, but little is known regarding shsps in this animal. Here, we report the sequence, expression, regulation, and function of a zebrafish protein (zfHsp27) homologous to human Hsp27. zfHsp27 contains three conserved phosphorylatable serines and a cysteine important for regulation of apoptosis, but it lacks much of a C-terminal tail domain and shows low homology in two putative actin interacting domains that are features of mammalian Hsp27. zfHsp27 mRNA is most abundant in adult skeletal muscle and heart and is upregulated during early embryogenesis. zfHsp27 expressed in mammalian fibroblasts was phosphorylated in response to heat stress and anisomycin, and this phosphorylation was prevented by treatment with SB202190, an inhibitor of p38 MAPK. Expression of zfHsp27 and human Hsp27 in mammalian fibroblasts promoted a similar degree of tolerance to heat stress. zfHsp27 fusion proteins entered the nucleus and associated with the cytoskeleton of heat stressed cells in vitro and in zebrafish embryos. These results reveal conservation in regulation and function of mammalian and teleost Hsp27 proteins and define zebrafish as a new model for the study of Hsp27 function.     

Hsp70 expression and function during embryogenesis

This review focuses on the expression and function of 70-kDa heat shock proteins (Hsp70s) during mammalian embryogenesis, though many features of embryogenesis and the developmental expression of Hsp70s are conserved between mammals and other vertebrates. A variety of Hsp70s are expressed from the point of zygotic gene activation in cleavage-stage embryos, through blastulation, implantation, gastrulation, neurulation, organogenesis, and on throughout fetal maturation. The regulation and patterns of hsp70 gene expression and the known and putative Hsp70 protein functions vary from constitutive and metabolic housekeeping to stress-inducible and embryo-protective roles. Understanding the genetic regulation and molecular function of Hsp70s has been pursued by developmental biologists interested in the control of gene expression in early embryos as well as reproductive toxicologists and teratologists interested in how Hsp70s protect embryos from the adverse effects of environmental exposures. These efforts have also been joined by those interested in the chaperone functions of Hsp70s, and this confluence of effort has yielded many advances in our understanding of Hsp70s during critical phases of embryonic development and cellular differentiation.     

Accumulation of a specific subset of D. melanogaster heat shock mRNAs in normal development without heat shock

During normal development in D. melanogaster, messenger RNAs for three of the seven heat shock proteins (hsp83, hsp28 and hsp26) accumulate in adult ovaries and are abundant in embryos until blastoderm. The three mRNAs appear to originate in nurse cells and subsequently pass, during stages 10-11, into the oocyte. Little if any of the four other heat shock mRNAs is present in unshocked ovaries or embryos at any time examined. Pre-blastoderm embryos fail to accumulate these heat shock mRNAs even if subjected to heat shock. The accumulation in normal oogenesis of mRNAs for only three of the seven heat shock proteins indicates the existence of differential, possibly multiple controls of heat shock gene expression, and suggests that heat shock proteins hsp83, hsp28 and hsp26 function in the oocyte or early embryo.     

Inhibition of hsp70–1 and hsp70–3 expression disrupts preimplantation embryogenesis and heightens embryo sensitivity to arsenic

Mouse 70-kDa heat shock proteins Hsp70–1 and Hsp70–3 (Hsp70–1/3) are stress-inducible protein chaperones thought to protect embryonic cells and tissues from the effects of a wide range of environmental exposures. Hsp70–1/3 are expressed constitutively, and at times are stress-inducible during various stages of preimplantation embryogenesis. In order to elucidate the functions of constitutive and stress-inducible Hsp70 expression in mouse preimplantation embryos, the consequences of inhibiting expression with antisense oligonucleotides complementary to the mRNAs of hsp70–1 and hsp70–3 (AO70–1/3) were evaluated. Transfection of preimplantation embryos (four-cell stage) with 2.5 μM AO70–1/3 had no effect on in vitro blastocoel formation. However, transfection with 5 or 10 μM AO70–1/3 reduced in vitro blastocyst development to 30% and 0%, respectively (approximately 90% control embryos developed to blastocyst). Thus constitutive expression of Hsp70–1/3 appears significant to preimplantation embryogenesis. Limiting expression of Hsp70–1/3 with 5 μM AO70–1/3 also heightened embryo sensitivity to arsenic, resulting in less than 5% in vitro development to blastocyst in the presence of the subtoxic dose of 0.4 μM sodium arsenite. Whether the combined effect of AO70–1/3 and arsenic is due to blocking inducible expression of the Hsp70s, or due to further reducing the amount of constitutively expressed Hsp70s available to the embryo is not known at this time. However, these results clearly indicate that some minimal amount of Hsp70–1 and/or Hsp70–3 is required for preimplantation embryogenesis, and that increasing the demand for Hsp70s by arsenic exposure heightens this requirement. Mol. Reprod. Dev. 51:373–380, 1998. © 1998 Wiley-Liss, Inc.     

The heat-inducible zebrafish hsp70 gene is expressed during normal lens development under non-stress conditions

In the present study, we show that the stress-inducible hsp70 gene in zebrafish is strongly and specifically expressed during normal lens formation from 28 to 38 hours post-fertilization, and is subsequently downregulated by 2 days of age. Only weak constitutive hsp70 mRNA signal was sporadically observed in other embryonic tissues. Similarly, transgenic fish carrying a 1.5 kb fragment of the hsp70 promoter linked to eGFP exhibited fluorescence only in the lens. In contrast, both the endogenous hsp70 gene and the transgene were strongly expressed throughout the embryo following heat shock at the same developmental stages.     

Expression of the murine small heat shock proteins hsp 25 and alpha B crystallin in the absence of stress

Stress induces the synthesis of several large and small heat shock proteins (hsp's). Two related small hsp's, hsp25 and alpha B crystallin exist in mice. alpha B crystallin is an abundant protein in several tissues even in the absence of stress. Particularly high amounts accumulate in the eye lens. Here we show that hsp25 is likewise constitutively expressed in many normal adult tissues. In the absence of stress the protein is most abundant in the eye lens, heart, stomach, colon, lung, and bladder. The stress-independent expression pattern of the two small hsp's is distinct. In several tissues the amount of hsp25 exceeds that accumulating in NIH 3T3 fibroblasts in response to heat stress. hsp25, like alpha B crystallin, exists in a highly aggregated form in the eye lens. The expression of hsp25 and alpha B crystallin in normal tissues suggests an essential, but distinct function of the two related proteins under standard physiological conditions.     

Heat-induced expression of albumin during early stages of rat embryo development.

An examination of heat-induced expression of proteins in tissues from adult and embryonic liver in rats shows that albumin, which is constitutively expressed in adult liver and is not synthesized in embryos before 16 days of gestation, appears in liver cells at earlier stages of development upon heat shock. On the basis of available evidence for the expression of heat shock proteins at distinct stages of development and on the basis of our findings, it may be argued that there could be common molecular events taking place during development and as a result of heat shock. We suggest also that one of the consequences of heat shock could be an internal change of pH within the cell which, in turn, might trigger alterations in gene expression.     

Restricted expression of the zebrafish hsp90alpha gene in slow and fast muscle fiber lineages

Members of the heat shock protein 90 (Hsp90) family of molecular chaperones play important roles in allowing a select group of intracellular signaling molecules reach and maintain functionally active conformations. We have previously shown that hsp90alpha gene expression in early zebrafish embryos is restricted to a subgroup of paraxial-mesoderm derived somitic cells prior to muscle formation and that the gene is downregulated in mature trunk and tail muscle fibers. Here we have compared the expression of the hsp90alpha gene to muscle regulatory genes during development of slow and fast muscle fibers in normal embryos and in embryos carrying mutations which affect somitic muscle formation. We show that hsp90alpha is first expressed early during the development of slow somitic muscle progenitors shortly following myoD activation and at a point prior to or co-incident with the expression of other known muscle regulatory genes. Expression of hsp90alpha is also activated in the midline of flh mutants when these cells switch from a notochord to a muscle fate. Conversely, expression is not detectable in cells of the paraxial mesoderm lineage which fail to converge in spt mutants and which do not activate expression of other muscle specific marker genes. Finally, expression of hsp90alpha is downregulated in slow muscle fibers by 24 h of age but becomes detectable in the later developing fast fibers at this time. Thus, hsp90alpha is expressed in developing muscle progenitors during short temporal and spatial windows of both slow and fast fiber lineages in the zebrafish somite.     

Examination of the stress-induced expression of the collagen binding heat shock protein, hsp47, in Xenopus laevis cultured cells and embryos

HSP47 is an endoplasmic reticulum (ER)-resident molecular chaperone involved in collagen production. This study examined the stress-induced pattern of hsp47 gene expression in Xenopus cultured cells and embryos. Sequence analysis revealed that protein encoded by the hsp47 cDNA exhibited 70-77% identity with fish, avian and mammalian HSP47. In A6 kidney epithelial cells hsp47 mRNA and HSP47 were present constitutively and inducible by heat shock but not ER stressors including tunicamycin and A23187, both of which enhanced BiP mRNA. Furthermore A23187 treatment inhibited constitutive accumulation of hsp47 mRNA and retarded heat-induced accumulation of hsp47 and hsp70 mRNA. Interestingly, hsp47 gene expression but not hsp70 or BiP mRNA accumulation was enhanced by treatment with a procollagen-specific stressor, beta-aminopropionitrile. In Xenopus embryos hsp47 mRNA was present constitutively throughout development. In tailbud embryos hsp47 mRNA was enriched in tissues associated with collagen production including notochord, somites and head region. Heat shock-induced accumulation of hsp47 mRNA was enhanced primarily in embryonic tissues already exhibiting hsp47 mRNA accumulation. These studies suggest that the pattern of Xenopus hsp47 gene expression is similar to hsp70 in response to heat shock but also displays unique features including a response to a procollagen-specific stressor and preferential expression in collagen-containing tissues.