Spatial pattern of constitutive and heat shock-induced expression of the small heat shock protein gene family, Hsp30, in Xenopus laevis tailbud embryos

We employed whole-mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock-induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33 degrees C heatshock. The lowest temperature capable of inducing this pattern was 30 degrees C. Placement of embryos at 22 degrees C following a 1-h 33 degrees C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues.     

Spatial pattern of constitutive and heat shock‐induced expression of the small heat shock protein gene family,hsp30, in Xenopus laevis tailbud embryos

We employed whole‐mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock‐induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33°C heatshock. The lowest temperature capable of inducing this pattern was 30°C. Placement of embryos at 22°C following a 1‐h 33°C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues. Dev. Genet. 25:365–374, 1999. © 1999 Wiley‐Liss, Inc.     

Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos

Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33-35 degrees C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27-37 degrees C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 degrees C, with relatively greater levels at 30-35 degrees C and lower levels at 37 degrees C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27-35 degrees C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33-35 degrees C induced maximum relative levels of hsp 70 mRNA within 1-1.5 h, while at 30 and 27 degrees C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Acquisition of the heat-shock response and thermotolerance during early development of Xenopus laevis

The ability to synthesize a 68,000- to 70,000-Da protein (hsp) in heat-shocked early Xenopus laevis embryos is dependent on the stage of development. Whereas late blastula and later stage embryos synthesize hsp68-70 after heat shock, cleavage stages are incompetent with respect to hsp synthesis. In vitro translation experiments and RNA blot analyses demonstrate that enhanced synthesis of hsp68-70 is associated with an accumulation of hsp68-70 mRNA. Examination of the effect of heat shock on preexisting actin mRNA reveals that heat shock promotes a reduction in the levels of actin mRNA in cleavage embryos but has no discernible effect on actin mRNA levels in neurula embryos. Finally, the acquisition of the heat-shock response (i.e., synthesis of hsp68-70 and accumulation of hsp70 mRNA) during early Xenopus development is correlated with the acquisition of thermotolerance.     

Comparison of the effect of heat shock factor inhibitor, KNK437, on heat shock- and chemical stress-induced hsp30 gene expression in Xenopus laevis A6 cells

In this study, we compared the effect of KNK437 (N-formyl-3, 4-methylenedioxy-benzylidene-gamma-butyrolactam), a benzylidene lactam compound, on heat shock and chemical stressor-induced hsp30 gene expression in Xenopus laevis A6 kidney epithelial cells. Previously, KNK437 was shown to inhibit HSE-HSF1 binding activity and heat-induced hsp gene expression. In the present study, Northern and Western blot analysis revealed that pretreatment of A6 cells with KNK437 inhibited hsp30 mRNA and HSP30 and HSP70 protein accumulation induced by chemical stressors including sodium arsenite, cadmium chloride and herbimycin A. In A6 cells subjected to sodium arsenite, cadmium chloride, herbimycin A or a 33 degrees C heat shock treatment, immunocytochemistry and confocal microscopy revealed that HSP30 accumulated primarily in the cytoplasm. However, incubation of A6 cells at 35 degrees C resulted in enhanced HSP30 accumulation in the nucleus. Pre-treatment with 100 microM KNK437 completely inhibited HSP30 accumulation in A6 cells heat shocked at 33 or 35 degrees C as well as cells treated with 10 microM sodium arsenite, 100 microM cadmium chloride or 1 microg/mL herbimycin A. These results show that KNK437 is effective at inhibiting both heat shock- and chemical stress-induced hsp gene expression in amphibian cells.     

Examination of KNK437- and quercetin-mediated inhibition of heat shock-induced heat shock protein gene expression in Xenopus laevis cultured cells

We examined the effect of quercetin (3,3',4',5,7-pentahydroxyflavon) and KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolactam), a benzylidene lactam compound, on heat-induced heat shock protein (hsp) gene expression in Xenopus laevis A6 kidney epithelial cells. In previous studies, both quercetin and KNK437 inhibited heat shock factor activity resulting in a repression of hsp mRNA and protein accumulation in human cultured cells. In this first study of the effect of these hsp gene expression inhibitors in a non-mammalian cell line, we report that both quercetin and KNK437 reduced the heat shock-induced accumulation of hsp30, hsp47 and hsp70 mRNA in X. laevis cultured cells. However, these inhibitors had no effect on the relative level of a non-heat shock protein mRNA, ef1alpha, in either control or heat shocked cells. Western blot and immunocytochemical analyses revealed that quercetin partially inhibited HSP30 protein accumulation. In contrast, HSP30 protein was not detectable in KNK437-treated cells. Finally, treatment of A6 cells with KNK437 inhibited the heat shock-induced acquisition of thermotolerance, as determined by preservation of actin filaments and cellular morphology using immunocytochemistry and laser scanning confocal microscopy.     

Role of glutathione and hsp 70 in the acquisition of thermotolerance in postimplantation rat embryos

Studies were initiated to determine the extent to which reduced glutathione (GSH) may be involved in the capacity of cultured rat embryos to develop heat-induced tolerance to the deleterious effects of exposure to high temperatures (heat shock). Investigations of the modulation of dysmorphogenic responses of embryos to heat shock (43 degrees C, 30 min) as well as to the expression of the hsp70 gene and subsequent formation of hsps indicated that the acquisition of thermotolerance by rat embryos could be significantly influenced by the inhibition of GSH synthesis. Treatment of conceptuses with L-buthionine-S,R-sulfoximine (BSO) reduced intracellular GSH concentrations and compromised the capacity of embryos to mount a thermotolerance response as assessed by alterations in indices of growth and development. Embryonic thermotolerance elicited by preexposure to 42 degrees C for 30 min was accompanied by increases in GSH to levels greater than those measured in control embryos at 37 degrees C just prior to the subsequent 43 degrees C heat exposure. Expression of hsp70 mRNA was detectable soon after elevation of the temperature to 42 degrees C and reached its highest level of accumulation 1.5 hr after the 43 degrees C heat shock. BSO treatment had little if any effect on hsp70 message levels or on the synthesis of hsp70. The fact that BSO-treatment attenuated the thermotolerance response but did not produce a decrease in hsp70 RNA or the synthesis of hsp70 suggests that hsp70 alone is not sufficient to confer thermotolerance upon cultured rat embryos.     

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.     

Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.     

Expression analysis of heat shock genes in the skin, spleen and blood of common carp (Cyprinus carpio) after cadmium exposure and hypothermia

Heat shock proteins are chaperones that play a pivotal role in controling multiple regulatory pathways such as stress defense, hormone signaling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, the expression patterns of four well-known heat shock genes (hsp70, hsc70-1, hsc70-2 and hsp90α) were characterized in the skin, spleen and blood cells of the common carp, under unstressed conditions and after Cd2+ treatment or hypothermia. The examined genes were expressed in a tissue-specific manner: hsc70-2 was expressed constitutively, and was at best only slightly inducible; hsp90α exhibited a high basic expression in all three tissues, whereas hsc70-1 did so only in the blood cells, the expression of hsp70 proved to be below the level of detection in unstressed fish. Cold shock induced the expression of hsp genes in the spleen (hsp90α) and blood cells (hsp70, hsc70-1 and hsp90α), while Cd2+ treatment has no effect on the expression pattern. The highest inducibilities were detected in the skin: for hsp70 an induction of at least 20-fold after cadmium exposure, for hsc70-1 of at least 30-fold and for hsp90α of 3-fold after hypothermia.