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. 2015 Sep:138:104-13.
doi: 10.1016/j.exer.2015.07.001. Epub 2015 Jul 4.

A conserved role of αA-crystallin in the development of the zebrafish embryonic lens

Ping Zou  1 , Shu-Yu Wu  1 , Hanane A Koteiche  1 , Sanjay Mishra  1 , Daniel S Levic  2 , Ela Knapik  2 , Wenbiao Chen  1 , Hassane S Mchaourab  3
Affiliations

A conserved role of αA-crystallin in the development of the zebrafish embryonic lens

Ping Zou et al. Exp Eye Res. 2015 Sep.

Abstract

αA- and αB-crystallins are small heat shock proteins that bind thermodynamically destabilized proteins thereby inhibiting their aggregation. Highly expressed in the mammalian lens, the α-crystallins have been postulated to play a critical role in the maintenance of lens optical properties by sequestering age-damaged proteins prone to aggregation as well as through a multitude of roles in lens epithelial cells. Here, we have examined the role of α-crystallins in the development of the vertebrate zebrafish lens. For this purpose, we have carried out morpholino-mediated knockdown of αA-, αBa- and αBb-crystallin and characterized the gross morphology of the lens. We observed lens abnormalities, including increased reflectance intensity, as a consequence of the interference with expression of these proteins. These abnormalities were less frequent in transgenic zebrafish embryos expressing rat αA-crystallin suggesting a specific role of α-crystallins in embryonic lens development. To extend and confirm these findings, we generated an αA-crystallin knockout zebrafish line. A more consistent and severe lens phenotype was evident in maternal/zygotic αA-crystallin mutants compared to those observed by morpholino knockdown. The penetrance of the lens phenotype was reduced by transgenic expression of rat αA-crystallin and its severity was attenuated by maternal αA-crystallin expression. These findings demonstrate that the role of α-crystallins in lens development is conserved from mammals to zebrafish and set the stage for using the embryonic lens as a model system to test mechanistic aspects of α-crystallin chaperone activity and to develop strategies to fine-tune protein-protein interactions in aging and cataracts.

Keywords: Alpha-crystallin; Cataract; Chaperone; Lens development; Maternal transcript; Morpholino; Small heat shock protein; TALEN; Zebrafish.

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Figures

Figure 1
Figure 1. Suppression of cloche lens phenotypes by expression of Rno.Cryaa transgene
Cloche mutants showed apparent lens defects (A) and transgenic expression of Tg(cryaa:Rno.Cryaa) rescued, at least partially, the lens defects in cloche mutants (B, E). Zoomed-in images (C, D) of the lenses in cloche−/− and cloche−/−; Tg(cryaa:Rno.Cryaa), denoted by white-dotted boxes (A, B). Embryos of lens-specific Rno.Cryaa transgene displayed Cerulean marker in the heart (F; Tg(cryaa:Rno.Cryaa,myl7:Cerulean), white arrow)
Figure 2
Figure 2. Transgenic expression of rat Cryaa rescues lens phenotypes induced by α-crystallin morpholino knockdown in 4dpf embryos
DIC images from wild-type (WT) lens (A), MO-cryaa lens (B), MO-cryaba lens (C) and MO-cryabb lens (D) at 4dpf demonstrating the severe lens phenotype observed upon α-crystallin knockdown. The H&E staining on histological sections from WT lens (E) and MO-cryaa lens (F). The slightly immature retina in the MO-cryaa sample might be due to non-specific effects of MOs. Western blot (G) against zebrafish Cryaa protein suggested that MO-cryaa interfered the translation of cryaa gene. RT-PCR analysis (H) of cryaba and cryabb showed that zebrafish 3B-cystallins were expressed relatively early (starting from 24hpf). Significant numbers of MO-cryabb knockdown embryos (10ng) showed heart edema and tail abnormality (I). Statistic analyses of Rno.Cryaa-rescue effect on MO-cryaa, MO-cryaba and MO-cryabb (J).
Figure 3
Figure 3. Cryaa mutants show lens defects at 4dpf
Compared to WT siblings (A), cryaa−/− embryos (B) showed no morphological or anatomical difference at 4dpf. High-magnification representative images from Cryaa−/− lens (D–F) at 4dpf revealed abnormal features, different from WT lens (C). (I) Reflectance analysis at 4dpf showed that the reflectance intensity was higher (t-test; p=0.0009) in cryaa−/− lens (H) than in WT lens (G). Lens diameters were measured and no difference was observed between cryaa−/− and WT embryos (J).
Figure 4
Figure 4. The percentage of cryaa mutant embryos with lens abnormalities depends on the genotype of the parents
Progenies from the cryaa+/− and cryaa−/− incross showed lens defects, which were graded into three categories based on the severity of the abnormalities: WT-like, minor defects and major defects, in cryaa−/− embryos at 4dpf. In cryaa−/− embryos from cryaa+/− incross, 51% showed lens defects and 24% of which were major defects (left column). When compared to the cryaa−/− embryos from cryaa+/− incross, the resulting cryaa−/− embryos from cryaa−/− incross showed significantly higher percentage of embryos with lens defects (p=0.02), as well as ones with major defects, even though both were the same genotype (right column).
Figure 5
Figure 5. Maternal expression of zebrafish cryaa was detectable in early stage embryos
Four sets of crossing pairs: WT female x WT male, WT female x cryaa+/− male, cryaa+/− female x cryaa−/− male, cryaa−/− female x cryaa−/− male. Twenty embryos from each cross were pooled together for RT-PCR at 0hpf, 1hpf, 3hpf, 6hpf, and 48hpf.
Figure 6
Figure 6. Maternal expression of cryaa plays a role in embryonic lens development
A significantly higher proportion of cryaa−/− embryos showed major lens defects produced from the homozygote females (~69%) when compared to those from heterozygote females (~19%), which were crossed to heterozygote and homozygote males, respectively.
Figure 7
Figure 7. Transgenic expression of exogenous Cryaa suppresses the lens defects of cryaa+/− embryos
Adult cryaa−/− zebrafish was crossed with Tg(cryaa:Rno.Cryaa) zebrafish line. The lens of resulting embryos (cryaa+/−) were examined and classified into three categories as previously mentioned. The proportion of cryaa+/− embryos showing lens defects was significantly lower with the presence of Rno.Cryaa transgene.
Figure 8
Figure 8. The influence of genetic modifier(s) on lens phenotype severity
The embryos from a pair of cryaa+/− adults that presented major embryonic lens defects exhibited a higher fraction of lens defects (right column) than those from a randomly selected pair with the same genotype, cryaa+/− (left column).
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