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Smriti Mishra, Sanjay Mishra, Abigail Poff, Alexandra W. Fuller, Shu-Yu Wu, Hassane S Mchaourab; Phenotypic characterization of Hsp27 Knock-out of Danio rerio. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3038.
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© ARVO (1962-2015); The Authors (2016-present)
Small heat shock proteins (sHSPs) including α-crystallins and Hsp27 play important role in cellular proteostasis. Hsp27 along with αB crystallin is a stress inducible and ubiquitously expressed sHSP. Hsp27 has detectable levels in all regions of the lens, retina and cornea besides robust expressions in extraocular tissues including heart and brain. To understand the role of Hsp27, we have generated Hsp27 knock-out (KO) mutants of zebrafish using CRISPR-Cas9 system that disrupted the Hsp27 (hspb1) gene. We have also cloned zebrafish Hsp27 and investigated its chaperone activity in vitro. Zebrafish (Danio rerio) is a useful model system to understand the role of sHSP because it expresses all homologs of human sHSP, including Hsp27.
Zebrafish larvae, grown at normal temperature (28°C) for 4 day post fertilization (dpf), were subjected to heat stress of different intensity and duration by incubating in water bath and were examined after recovering for 24 hours at normal permissive temperature (28°C). We have recently established that the glucocorticoid receptor signaling (GR) interacts with the proteostatic network in the zebrafish heart. To understand the role of Hsp27 in the heart proteostasis, we challenged Hsp27 knock-out larvae with glucocorticoid signaling stress using an established dexamethasone treatment regimen.
Hsp27 KO homozygous embryos had lower survivability compared to WT at adulthood although the mutation is not lethal. The homozygous adults were morphologically indistinguishable from the WT siblings. While the embryos of the heterozygous in-cross were born at the Mendelian ratio; three months post fertilization the homozygotes survived at significantly lower than the Mendelian ratio. Disruption of Hsp27 sensitized zebrafish embryos to heat stress and a significantly higher number of KO embryos did not survive compared to the WT. On treatment with dexamethasone, Hsp27 KO developed heart edema phenotype similar to those observed in αB-crystallins KO, but no lens phenotype was observed in Hsp27 KO.
Although preliminary, our results suggest that Hsp27 is important for stress resistance for zebrafish embryos. The knock-out zebrafish can be used as a valuable animal model to study the role of Hsp27 in proteostasis.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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