Expression, function and regulation of the heat shock protein 70 (hsp70) gene during normal zebrafish (Danio rerio) embryogenesis

Heat shock proteins (Hsps) and heat shock transcription factors (HSFs) are critical to the ability of cells to cope with disturbances in their environment, and the function of Hsps and HSFs during the stress response has been well characterized. However, both classes of molecules are known to be expressed constitutively under non-stress conditions, and only recently have studies begun to address the specific consequences of inhibiting Hsp or HSF expression. Interestingly, these studies have demonstrated that both Hsps and HSFs are important players in eukaryotic development. Accordingly, this thesis focused on the expression, function, and regulation of the normally stress-inducible hsp70 gene during the normal development of the zebrafish ( Danio rerio).;The role of HSFs in normal zebrafish development, and whether these molecules are involved in regulating the expression of hsp70 in the zebrafish lens, has not been investigated prior to this thesis. Microinjecting MO targeted to the three zebrafish HSFs (HSF1, HSF2, and HSFX), demonstrated a requirement for HSF1 in normal zebrafish eye development. Specifically, hsf1-MO injected embryos phenocopy the lens and retinal defects observed with hsp70-MO injection. Furthermore, western blot analysis shows a marked decrease in Hsp70 in hsf1-MO injected embryos. Taken collectively, these data indicate that hsp70 is expressed during a brief window of lens fibre development, and that this expression is required for the proper development of the zebrafish lens. In addition, lens-specific hsp70 expression appears to be regulated by HSF1, as microinjection of hsf1-MO can phenocopy the defects observed in hsp70-MO injected embryos.;Whole mount in situ hybridization analysis was used to show that the heat-inducible zebrafish hsp70 gene is expressed during a distinct temporal window of embryonic lens formation at the normal growth temperature of 28°C. In addition, a 1.5 kb fragment of the zebrafish hsp70 gene promoter is sufficient to direct expression of a green fluorescent protein (GFP) reporter gene to the lens, suggesting that lens-specific expression of the hsp70 gene is regulated by promoter sequences and is expressed as part of the normal lens development program. To elucidate a possible role for the lens specific expression of hsp70 during normal embryonic development, morpholino modified antisense oligonucleotides (MO) directed against hsp70 mRNA (hsp70-MO) were microinjected into early zebrafish embryos to reduce the levels of Hsp70 during zebrafish development. hsp70-MO injected embryos exhibited a small eye phenotype relative to wildtype and control injected animals, with the phenotype discernable during the second day of development. Histological and immunological analysis revealed a small, underdeveloped lens and a disorganized retinal structure. Numerous TUNEL-positive nuclei also appeared in the lens of small eye embryos beyond 2 days of development, whereas they were no longer apparent in untreated embryos by 48 hours post-fertilization (hpf). Lenses transplanted from hsp70-MO injected embryos into wildtype hosts failed to recover and retained the immature morphology characteristic of the small eye phenotype, indicating that the phenotype is lens autonomous. These data suggest that the lens defect in hsp70-MO injected embryos is predominantly at the level of post-mitotic lens fibre differentiation, a result supported by the appearance of mature lens organization in these embryos at 5 days post fertilization (dpf), once MO degradation/dilution has occurred.