Expression and function of heat shock factors in zebrafish (Danio rerio)

Abstract

Heat shock proteins (hsp) and heat shock transcription factors (HSF) have important roles in the development of the eye lens. Our lab previously demonstrated that knockdown of hsp70 gene expression using morpholino antisense technology (MO) resulted in a small lens phenotype in zebrafish (Danio rerio) embryos. A less severe phenotype was seen with knockdown of hsf1, suggesting other factors that regulate hsp70 are involved during lens formation. Both HSF1 and HSF4 are known to play a role in mammalian lens development. An expressed sequence tag encoding zebrafish HSF4, named hsf4a, has been identified and a second splice variant, hsf4b, has been predicted in the Ensembl database. The objectives of this thesis were to characterize the zebrafish HSF4 and compare its expression to other HSFs as well as investigate its role in lens development. Analysis of zebrafish HSF4 sequence was performed using standard in silico analytical software. The deduced amino acid sequence of HSF4a shares structural similarities with mammalian HSF4 including the lack of an HR-C domain. This domain is absent due to a C-terminal truncation within zebrafish HSF4a relative to the mammalian protein. HSF4b is identical to the HSF4a sequence with the exception of an additional 155 amino acids at the carboxyl end of the protein which contains an HR-C domain, unlike mammalian HSF4. Surprisingly, electrophoretic mobility shift assays (EMSA) demonstrated that the binding affinity of zebrafish HSF4 to discontinuous HSEs is more similar to HSF1 than to other HSF4 proteins. The amino acid sequence of zebrafish HSF4 DNA binding domain was also more similar to HSF1 than other HSF4 proteins. These results, along with a phylogenetic analysis of HSF proteins from eleven species, suggest that HSF1 was an evolutionary precursor of HSF4 and that functions of this protein may differ between zebrafish and mammals. The expression level for each of the three zebrafish HSFs was determined in adult tissues and in developing embryos by quantitative reverse transcription polymerase chain reaction (qPCR) analysis. Expression of both hsf4 transcripts was observed predominantly in the eye but only observed in developing embryonic tissue at 60 hours post fertilization or later. This, together with the lack of an observable phenotype following MO knockdown of hsf4, suggests that HSF4 likely has a role in later stages of lens development. Additionally, hsf1 and hsf2 expression were detected in all tissues and in all stages of development as well as being present as maternal transcripts in zebrafish eggs. The results presented in this thesis demonstrate that while zebrafish HSFs share some similarity with HSF proteins from other species, they also have structural characteristics and expression patterns unique to the zebrafish.