Suppression of heat shock transcription factor HSF1 in zebrafish causes heat‐induced apoptosis

Abstract

The heat shock transcription factor HSF1 regulates the stress-inducible expression of heat shock proteins (HSPs) and other molecular chaperones that are involved in higher order protein structure and assembly. HSPs protect organisms against environmental challenges. Targeted disruption of the murine Hsf1 gene results in massive heat-induced apoptosis in cells deficient in HSF1. To explore whether zebrafish HSF1 has overlapping function with mammalian HSF1, we inhibited zebrafish HSF1 expression during development using morpholino oligonucleotides specific for the HSF1 sequence. The zebrafish HSF1 morpholinos were injected at the one-to eight-cell-stage embryos of a transgenic zebrafish carrying an Hsp70-promoter-GFP (Hsp70-EGFP) reporter construct. The injected embryos were heated and analyzed for GFP expression and the presence of apoptotic cells 24 h after fertilization. Our data indicate that the morpholino-injected embryos exhibit a severe reduction in expression of the heat-induced HSP70-GFP reporter gene. Furthermore, there was an enhancement of apoptotic response in these transgenic zebrafish embryos at elevated temperatures. Thus, the function of HSF1 is conserved in zebrafish and higher vertebrates.Diverse organisms from bacteria to humans respond to environmental challenges that cause protein damage by synthesizing HSPs. HSPs protect cells and tissues against such challenges by assisting in the repair of protein damage, thereby protecting cells from self-destruction or apoptosis (Li and Mivechi, 1986;Mivechi et al., 1994;Mosser et al., 1997;Morimoto, 1998; L. Huang, N. F. Mivechi, and D. Moskophidis, unpublished data). The inducible regulation of HSP expression is achieved by heat shock transcription factors (HSFs). In lower organisms such as yeast and Drosophila, control of the heat shock response is accomplished by a single Hsf gene (Wu, 1995). In higher eukaryotes, however, there are multiple Hsf genes that respond to diverse environmental insults, suggesting that individual HSF proteins may be responsible for different biological functions (Morimoto, 1998). In zebrafish, at least two Hsf genes are expressed to encode multiple isoforms of each protein. cDNAs for HSF1a and HSF1b have been recently reported (Rabergh, 2000) and an EST clone for a second HSF family member with high homology with HSF2 has been deposited in the database. In this report, we have isolated and analyzed a unique isoform of HSF1 (designated thereafter as HSF1c) in zebrafish (Fig. 1A, B). In situ hybridization indicated that HSF1c was widely expressed throughout early development (Fig. 1C). Both the zebrafish HSF1a and HSF1c isoforms are capable of binding to the heat shock element (HSE), and their DNA binding abilities are enhanced if the in vitro translated HSF1a or HSF1c is incubated at elevated temperature (data not shown). HSF1 proteins in different species are highly conserved in the DNA binding domain and oligomerization domains. HSF1 exists as an inactive monomer under physiological conditions and acquires DNA binding ...

Differential gene expression associated with euryhalinity in sea bream (Sparus sarba)

“…Expression of hsf1 was found to increase only in gills of seawater-and hypersaline-adapted sea bream, an effect that followed the same profile as hsp70. It is likely that transcription of hsp70 is regulated via hsf1, since recent studies using fish and mammalian models have provided conclusive evidence for a regulatory link between these two genes (42,55,60). An elevated hsf1 transcription would be a critical determining factor for maintaining sufficient hsp70, and as such the combined upregulation of both of these genes could be one reason as to why gills of euryhaline fish have the capability to maintain cellular functions, even under severe stress such as hypersaline exposure.…”

CHIP Suppresses Polyglutamine Aggregation and ToxicityIn VitroandIn Vivo

“…Although these features are also readily recapitulated in other model systems, zebrafish embryos offer certain advantages for modeling polyQ disease: they develop rapidly and externally, can be produced quickly in large numbers, and are transparent, permitting direct analysis of organs, tissues, and fluorescently tagged proteins. In addition, pathways and components of protein QC have been identified that are conserved from zebrafish to humans, including an HSF1-dependent stress response and a zebrafish ortholog of human CHIP (Wang et al, 2001;Strausberg et al, 2002). Antisense morpholinos can efficiently knock down expression of zebrafish genes, raising the possibility of studying candidate suppressor and enhancer genes (Nasevicius and Ekker, 2000).…”

Critical role of Brg1 member of the SWI/SNF chromatin remodeling complex during neurogenesis and neural crest induction in zebrafish

“…The anti-sense morpholino oligonucleotides were dissolved in 1ϫ Danieau's buffer in a stock concentration of 20 ng/nl (Nasevicius and Ekker, 2000). For morpholino microinjection, one-to two-cellstage zebrafish embryos were injected with either Brg1-MO or control-MO (5Ј-CCTCTTACCTCAGTTACAATTT-ATA-3Ј) (5 or 10 ng) (Wang et al, 2001). At different stages post-morpholino injections, embryos were fixed in 4% paraformaldehyde (PFA) and gene expression was assayed by in situ hybridization.…”