Research Article: Why does the zebrafish cloche mutant develop lens cataract?

Date Published: March 12, 2019

Publisher: Public Library of Science

Author(s): Mason Posner, Matthew S. McDonald, Kelly L. Murray, Andor J. Kiss, Usha P. Andley.


The zebrafish has become a valuable model for examining ocular lens development, physiology and disease. The zebrafish cloche mutant, first described for its loss of hematopoiesis, also shows reduced eye and lens size, interruption in lens cell differentiation and a cataract likely caused by abnormal protein aggregation. To facilitate the use of the cloche mutant for studies on cataract development and prevention we characterized variation in the lens phenotype, quantified changes in gene expression by qRT-PCR and RNA-Seq and compared the ability of two promoters to drive expression of introduced proteins into the cloche lens. We found that the severity of cloche embryo lens cataract varied, while the decrease in lens diameter and retention of nuclei in differentiating lens fiber cells was constant. We found very low expression of both αB-crystallin genes (cryaba and cryabb) at 4 days post fertilization (dpf) by both qRT-PCR and RNA-Seq in cloche, cloche sibling and wildtype embryos and no significant difference in αA-crystallin (cryaa) expression. RNA-Seq analysis of 4 dpf embryos identified transcripts from 25,281 genes, with 1,329 showing statistically significantly different expression between cloche and wildtype samples. Downregulation of eight lens β- and γM-crystallin genes and 22 retinal related genes may reflect a general reduction in eye development and growth. Six stress response genes were upregulated. We did not find misregulation of any known components of lens development gene regulatory networks. These results suggest that the cloche lens cataract is not caused by loss of αA-crystallin or changes to lens gene regulatory networks. Instead, we propose that the cataract results from general physiological stress related to loss of hematopoiesis. Our finding that the zebrafish αA-crystallin promoter drove strong GFP expression in the cloche lens demonstrates its use as a tool for examining the effects of introduced proteins on lens crystallin aggregation and cataract prevention.

Partial Text

The ocular lens provides an excellent model system for examining tissue development and physiology due to its transparency, accessibility and the presence of only two cell types. Cataract, the development of opacities that interfere with the transmittance of light to the retina, continues to be the leading cause of human blindness worldwide [1]. A better understanding of the mechanisms leading to lens cataract could foster the development of preventative strategies. In recent years the zebrafish has become a powerful model for examining eye lens biology [2,3]. Not only do their transparent, external embryos facilitate experiments with the lens, but it is also relatively easy to express introduced proteins and explore their impact on lens function [4,5]. Multiple studies have shown that lens development and protein content are well conserved between zebrafish and mammals, making zebrafish studies translatable to our understanding of human lens disease [5–9].

Homozygous cloche embryos were recognizable by the presence of cardiac edema (Fig 1A compared to Fig 1B), an abnormally shaped heart (Fig 1C and 1D), bent trunks, reduced eye size compared to non-phenotypic cloche siblings, and the lack of circulating red blood cells. Some of these features, such as cardiac edema and bent trunks, are typical of many abnormal embryos phenotypes. However, the loss of red blood cells and reduced eye size was diagnostic for the mutation. We confirmed that our cloche fish heterozygote breeding population contained the clochem39 allele by PCR amplifying the identified region of mutation using primers from [14] (Fig 1E).

Our work and that from others has identified at least three features of the cloche lens phenotype. The lens is smaller than normal, shows arrested denucleation of fiber cells and contains a noticeable central irregularity that previous studies have shown scatters light [15,32]. Data in this present study show that lens size and retention of fiber cells are similar in all cloche lenses, while the central irregularity can vary in severity. Goishi et al. [15] showed that cloche lenses include aggregated γ-crystallins, which likely contributes to the visual roughness seen by DIC microscopy. The variability in appearance of this lens irregularity suggests that it is stochastic. Whatever physiological and/or molecular mechanisms lead to reduced eye size and fiber cell denucleation arrest do not similarly dictate cataract formation, but may make the lens more prone to protein aggregation.




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