Heat-inducible and constitutive expression of the 90 kD heat shock protein gene, Hsp90, during zebrafish embryogenesis
Sass, Jennifer Beth
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The objective of my thesis work is to examine the in vivo transcript accumulation of the 90 kD heat shock protein, 'hsp90α' during zebrafish embryogenesis in order to identify stage- and tissue-dependent localization. Northern blot analysis indicated that 'hsp90β' mRNA was visible at high levels constitutively, and only mildly up-regulated following heat shock, whereas, 'hsp90α' mRNA was undetectable at control temperatures (28.5°C), but was dramatically upregulated following heat shock (1 hr at 37°C). Whole mount in situ hybridization with gene specific riboprobes demonstrated that constitutive accumulation of ' hsp90β' mRNA was evident in a wide range of tissues throughout the embryo, whereas 'hsp90α' mRNA was localized within putative myogenic tissue of the zebrafish trunk and pectoral fin bud during myogenesis. Observation of tissue sections confirmed high levels of ' hsp90α' mRNA within cells of the presegmental and segmental plate mesoderm, and not within the axial structures (neural tube and notochord). Similar tissue- and stage-specific accumulation of 'hsp90α' mRNA was also seen in mid-somitogenic chicken embryos. Following exposure of zebrafish and chicken embryos to heat shock ' hsp90α' was dramatically upregulated throughout all embryonic tissues, demonstrating that heat-induced expression of 'hsp90α ' is regulated independent of constitutive expression, and that the expression of the two 'hsp90' genes are regulated separately during vertebrate embryogenesis. I examined the altered pattern of 'hsp90α' transcript accumulation within three zebrafish mutant embryo types, 'spadetail, no tail', and 'floating head', which exhibit aberrant myogenesis. In all cases 'hsp90α' mRNA was localized within cells of the paraxial mesoderm which are fated to form striated trunk muscle (identified by expression of 'myoD'), suggesting that its upregulation within myogenic tissue is regulated as part of myogenesis. The work presented in this dissertation demonstrates for the first time the independent regulation of 'hsp90α' and 'hsp90β ' gene expression at the mRNA level during embryogenesis. Further, the highly restricted pattern of 'hsp90α' mRNA localization during myogenesis, in contrast to its ubiquitous high level accumulation following heat shock, suggests that the protein may perform a specific function during skeletal myogenesis in addition to a more general protective function during periods of environmental stress.