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Ross F Collery, Terri L Young, Brian Link; Investigation of the role of ZNF644 in emmetropization and refractive error using zebrafish. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Multiple pedigree analyses have shown that mutations in human ZNF644 are associated with high-grade myopia. ZNF644 is widely expressed in many tissues throughout the body, including the eye. ZNF644 has recently been shown to regulate gene repression through its interaction and recruitment of the G9/GLP histone lysine-specific methyltransferase complex. It is thought that ZNF644, via its zinc finger domains, binds DNA at defined recognition sites and provides locus specificity for H3K9 methylation. To better understand the role of ZNF644 in myopia and emmetropia, we have inactivated zebrafish orthologs znf644 a and b. We also overexpressed wild-type and myopia-associated mutants of human ZNF644 in zebrafish to assess the effect on eye size and refractive state.
Zebrafish ZNF644 orthologs were inactivated using CRISPR/Cas9 methods. Transgenic zebrafish expressing human ZN644 were generated using Tol2 methods. Eye size and refractive state were measured using spectral-domain optical coherence tomography.
znf644a and b are expressed throughout the developing embryo, with enrichment within the nervous system and eye. Inactivation of znf644b in zebrafish increased eye size (n=8 eyes; p=0.0005, Mann-Whitney test). Human ZNF644-eGFP can be ubiquitously expressed in zebrafish without affecting survival. Consistent with its known role in gene regulation, overexpressed ZNF644 protein is enriched within the nucleus.
Inactivating ZNF644 orthologs in zebrafish provide insight into the function of the human gene and its effects on eye size and refractive state in wild-type and mutant contexts. In a complementary fashion, zebrafish engineered to overexpress human normal and disease-associated variants of ZNF644, may shed light on the precise links and mechanisms underlying ZNF644 alteration and myopia.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
A, B. in situ hybridization showing znf644a and znf644b mRNA expression at 3 dpf. C, D, D’. Human ZNF644-eGFP is enriched in the nucleus in zebrafish epithelial and muscle cells. E, F. inactivation of zebrafish znf644b causes enlargement of the eye axial length and lens diamter with respect to body length.
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