Expression of hsp90α and hsp90β during Xenopus laevis embryonic development

Background: Members of the eukaryotic Hsp90 family function as important molecular chaperones in the assembly, folding and activation of cellular signaling in development. Two hsp90 genes, hsp90α and hsp90β, have been identified in fish and homeothermic vertebrates but not in poikilothermic vertebrates. In the present study, the expression of hsp90α and hsp90β genes in Xenopus laevis, which is phylogenetically positioned between zebrafish and mammals, has been addressed. Methods: Partial Xenopus hsp90α and hsp90β cDNA were identified and isolated using RT-PCR, and a full-length Xenopus hsp90β cDNA was isolated from an embryonic cDNA library. Northern-blot analysis was used to study the expression of hsp90α and hsp90β genes in total RNA of the embryos and in situ hybridization was used to compare the expression of these genes with that of hsp70 and MyoD genes in Xenopus embryogenesis. Results: Northern-blot analysis revealed that the hsp90β gene was strongly expressed constitutively at all stages of embryogenesis, but weakly induced following the heat shock. In contrast, the hsp90α gene was weakly expressed in embryos at control temperature, but strongly up-regulated following heat shock. In situ hybridization results showed that hsp90α gene was observed predominantly in cells of the developing somite. Microscopic sections showed that hsp90α and MyoD mRNA are expressed in similar regions in somite and this pattern was distinct from that of hsp70 and hsp90β. Conclusion: These data support the hypothesis that the presence of hsp90α and hsp90β genes is conserved among vertebrates, and these genes are differentially regulated in a tissue, stress, and development stage-specific manner

Different interactions of Hsp90Hsp90 and Hsp90Hsp90 with different substrates

Background: The Hsp90 chaperone complex functions in assembly, folding and activation of numerous substrates. The two vertebrate homologues encoded by hsp90 and hsp90 genes are differentially expressed in embryonic and adult tissues and during stress, however, it is not known if they possess identical functional activities in chaperone complexes. This question was addressed by examining potential differences between the Hsp90 isoforms with respect to both co-chaperone and substrate interactions. Materials and Methods: Epitope-tagged proteins were expressed in mammalian cells or Xenopus oocytes and subjected to immunoprecipitation with an array of co-chaperones. Results: Both isoforms were shown to participate equally in multi-chaperone complexes and no significant difference in co-chaperone distribution was observed. The substrates Raf-1, HSF1, Cdc37 and Mek interacted with both Hsp90 and Hsp90, and the relative patterns of these interactions were not affected by heat shock. The substrates kinases c-Src, CKIIB, A-raf, and Erk interacted with both isoforms, however, significantly more Hsp90 was recovered after heat shock. Conclusion: The results demonstrate that the Hsp90 and Hsp90 exhibit similar interactions with co-chaperones, but significantly different behaviors with respect to substrate interactions under stress conditions

Heat-inducible expression of a reporter gene detected by transient assay in Zebrafish

Heat-inducibility of two reporter constructs expressing lacZ gene under the control of mouse and Xenopus hsp70 promoters was tested in zebrafish (Danio rerio) embryos using a transient expression system. Cells expressing β-galactosidase were stained blue by histochemical staining and their average number per embryo was used as an indicator of the expression level of the reporter gene. Both constructs were heat-inducible in the embryonic tissues and showed similar heat dependence (increasing expression levels from 35–36°C up to 39°C with an apparent decrease at 40°C), resembling that of the zebrafish hsp70 genes. However, their induction kinetics were different, which might be due to differences in their 5′ UTRs. Spatial expression patterns of the two hsp/lacZ constructs and an endogenous hsp70 gene were mostly similar on the RNA level. These results indicate that our approach is applicable for in vivo analysis of the heat-shock response and that exogenous heat-shock promoters may be useful for inducible expression of transgenes in fish