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.     

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.     

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.     

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.     

Delta-sarcoglycan is necessary for early heart and muscle development in zebrafish

Delta-sarcoglycan, one member of the sarcoglycan complex, is a very conservative muscle-specific protein exclusively expressed in the skeletal and cardiac muscles of vertebrates. Mutations in sarcoglycans are known to be involved in limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy (DCM) in humans. To address the role of delta-sarcoglycan gene in zebrafish development, we have studied expression pattern of delta-sarcoglycan in zebrafish embryos and examined the role of delta-sarcoglycan in zebrafish embryonic development by morpholino. Strong expression of delta-sarcoglycan was observed in various muscles including those of the segment, heart, eye, jaw, pectoral fin, branchial arches, and swim bladder in zebrafish embryo. Delta-sarcoglycan was also expressed in midbrain and retina. Knockdown of delta-sarcoglycan resulted in severe abnormality in both the cardiac and skeletal muscles. Some severe ones displayed serious morphological abnormality such as hypoplastic head, linear heart, very weak heartbeats, and runtish trunk, all dead within 5 dpf. Whole-mount in situ hybridization analysis showed that adaxial cells and muscle pioneers were affected in delta-sarcoglycan knockdown embryos. In addition, absence of delta-sarcoglycan protein severely delayed the cardiac development and influenced the differentiation of cardiac muscle, and the cardiac left-right asymmetry was dramatically changed in morpholino-treated embryos. These data together suggest that delta-sarcoglycan plays an important role in early heart and muscle development.     

Heart C-protein is transiently expressed during skeletal muscle development in the embryo, but persists in cultured myogenic cells

The expression of cardiac and white skeletal C-protein isoforms was analyzed in developing chicken embryos and in primary skeletal muscle cell cultures by immunoblot and immunofluorescence staining using polyclonal antibodies specific for both of the two different proteins. In the embryo, cardiac C-protein was detected in the developing heart from very early stages through adulthood. In skeletal muscle, cardiac C-protein is shown to be transiently expressed between Days 3 and 15 during development. In contrast, the expression of white skeletal C-protein is gradual and progressive starting approximately from Day 15 on in development. In primary cell cultures of skeletal muscle, however, cardiac C-protein remained expressed throughout prolonged culture time, this in conjunction with white skeletal C-protein. Thus the down regulation of cardiac C-protein and the transition from cardiac C-protein to adult skeletal (white) C-protein which was observed during skeletal muscle development in vivo, does not seem to go to completion in the in vitro system.     

Thermotolerance and HSP70 expression in the Mediterranean fruit fly Ceratitis capitata

The relationship between Hsp70 expression and thermotolerance has been well documented in Drosophila melanogaster. However, there is limited information on this relationship in other insect species. In this report we describe the Hsp70-thermotolerance relationship in one of the major fruit fly pests, Ceratitis capitata (medfly). Hsp70 expression and thermotolerance were assayed at a range of temperatures in several stages of medfly development. The most thermotolerant stage was found to be the late larval stage (100% survival at 41 °C) followed by adult flies and late embryos (100% survival at 39 °C). These three stages showed a positive relationship between Hsp70 expression and thermotolerance. Mid-larval and mid-embryonic stages were found less thermotolerant and the Hsp70-thermotolerance relationship was not evident. Early embryos did not express Hsp70 at any temperature and exhibited the lowest thermotolerance. The relationship between Hsp70 and inducible thermotolerance was also studied in late larvae. A pretreatment at 37-39 °C increased thermotolerance at higher temperatures by approximately 1 °C. In parallel, the pretreatment increased Hsp70 expression suggesting a close link between Hsp70 expression and inducible thermotolerance. The increased Hsp70 levels after pretreatment were found to be due to the increased levels of the hsp70 RNA.     

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.     

Heat shock proteins Hsp70-1 and Hsp70-3 Are necessary and sufficient to prevent arsenite-induced dysmorphology in mouse embryos.

Heat Shock Proteins (HSPs) represent a variety of protein families that are induced by stressors such as heat and toxicants, and the induction of HSPs in the organogenesis stage rodent embryo is well established. It has been proposed that thermotolerance and chemotolerance result from expression of the HSPs. However, whether these proteins function to prevent dysmorphogenesis and which family members serve this function are unknown. Therefore, we evaluated the specific ability of stress-inducible Hsp70-1 and Hsp70-3 to prevent arsenite-induced dysmorphology in the cultured mouse embryo using gain- and loss-of-function models. Loss of HSP function was accomplished by injecting antisense oligonucleotides directed against hsp70-1 and hsp 70-3 mRNAs into the amniotic cavity of cultured Day 9 mouse embryos. Suppression of hsp70-1 and hsp70-3 expression resulted in an up to six-fold increase in the incidence of arsenite-induced neural tube defects. Gain of HSP function was accomplished by microinjecting a transgene with a constitutive promotor driving expression of the hsp70-1 coding region, and resulted in a decreased incidence of arsenite-induced neural tube defects. These results indicate that Hsp70-1 and Hsp70-3 are both necessary and sufficient for preventing arsenite-induced dysmorphology in early-somite staged mouse embryos. Mol. Reprod. Dev. 59:285-293, 2001.     

Extreme thermotolerance and behavioral induction of 70-kDa heat shock proteins and their encoding genes in honey bees

Foraging honey bees frequently leave the hive to gather pollen and nectar for the colony. This period of their lives is marked by periodic extremes of body temperature, metabolic expenditure, and flight muscle activity. Following ecologically relevant episodes of hyperthermia between 33°C and 50°C, heat shock protein 70 (Hsp70) expression and hsp70/hsc70-4 activity in brains of nonflying laboratory-held bees increased by only two to three times baseline at temperatures 46–50°C. Induction was undetectable in thoracic–flight muscles. Yet, thorax hsp70 mRNA (but not hsc70-4 mRNA) levels were up to ten times higher in flight-capable hive bees and foraging bees compared to 1-day-old, flight-incapable bees, while brain hsp70/hsc70-4 mRNA levels were low and varied little among behavioral groups. These data suggest honey bee tissues, especially flight muscles, are extremely thermotolerant. Furthermore, Hsp70 expression in the thoraces of flight-capable bees is probably flight-induced by oxidative and mechanical damage to flight muscle proteins rather than temperature.     

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions.     

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole‐mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker‐mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2‐kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5‐kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8‐kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters. Dev. Genet. 25:158–167, 1999. © 1999 Wiley‐Liss, Inc.     

Hsp27 associates with the titin filament system in heat-shocked zebrafish cardiomyocytes

Injury to muscle tissue plays a central role in various cardiovascular pathologies. Overexpression of the small heat shock protein Hsp27 protects muscle cells against thermal, oxidative and ischemic stress. However, underlying mechanisms of this protection have not been resolved. A distinctive feature of muscle cells is the stress-induced association of Hsp27 with the sarcomere. The association of Hsp27 with the cytoskeleton, in both muscle and non-muscle cells, is thought to represent interaction with Z-line components or filamentous actin. Here, we examined the association of Hsp27 with myofibrils in adult zebrafish myocardium subjected to hyperthermia and mechanical stretching. Consistent with previously published results, Hsp27 in resting length myofibrils localized to narrowly defined regions, or bands, which colocalized with Z-line markers. However, analysis of stretched myofibrils revealed that the association of Hsp27 with myofibrils was independent of desmin, alpha-actinin, myosin, and filamentous actin. Instead, Hsp27 maintained a consistent relationship with a marker for the titin A/I border over various sarcomeric lengths. Finally, extraction of actin filaments revealed that Hsp27 binds to a component of the remaining sarcomere. Together, these novel data support a mechanism of Hsp27 function where interactions with the titin filament system protect myofibrils from stress-induced degradation.