Fig 1.
Transgenic expression of murine Cryaa mutants induced lens defects in 4dpf zebrafish embryos.
(A) The lens-specific murine Cryaa transgenes displayed Cerulean marker in the heart. Upper Panel, DIC image. Lower panel, a white arrow marks the Cerulean marker in the heart. No overall morphology changes were observed in all transgenic carriers. (B) Representative DIC images of lenses from embryos expressing cataract-linked αA-crystallin mutants. The cataract phenotypes have been classified on the basis of the severity of defects. (i) WT, (ii, iii) minor defects (iv) major defects. (C) Embryos of lens-specific Rno.Cryaa transgenes (R49C and R116C mutations) displayed lens defects with various penetrance (scored at 4dpf). Removal of endogenous cryaa alleles exacerbated the lens defects. Error bars represented standard errors. Number of crosses is denoted in parentheses, n is the total number of screened embryos.
Fig 2.
Apoptotic cell death was not induced by transgenic expression of murine Cryaa mutants.
TUNEL staining (A), embryos of Tg(cryaa:Rno.Cryaa_R49C) and Tg(cryaa:Rno.Cryaa_R116C) did not show increase of apoptosis in the lens when compared to non-transgenic (WT) siblings (B). Center black lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Individual sample points are shown by red circle.
Fig 3.
Synergistic effects on embryonic lens defects were selectively observed in transgenic co-expression of the human CRYGD mutant and murine Cryaa mutants.
Compared to the siblings carrying single transgene, Tg(cryaa:Hsa.CRYGD_I4F), double transgenic embryos at 4dpf exhibited enhanced penetrance of lens defects when Tg(cryaa:Rno.Cryaa_R49C) was co-expressed, but not Tg(cryaa:Rno.Cryaa_R116C). Error bars represented standard errors. Number of crosses is denoted in parentheses.
Fig 4.
Differential modulation of γD-crystallin protein aggregation by transgenes of murine Cryaa mutants.
(A) Schematic of the experiment utilizing the Gal4/UAS targeted gene expression system. Double transgenic line, Tg[cryaa:Gal4]; Tg[UAS:GFP], were outcrossed to either αA-R49C, cryaa-/-;αA-R49C, αA-R116C (shown in Fig 4B) or αB-R120G (shown in Fig 5B) transgenic lines. Fertilized zygotes were injected with tol2 mRNA and UAS responder Tol2 constructs expressing fluorescently tagged human γD-crystallin (Hsa.CRYGD_I4F-mCherry). Embryos possessing lens fiber cells positive for mCherry mosaic expression were selected and imaged at 4dpf. (B) The percentage of embryos showing γD-crystallin_I4F-mCherry punctuates in the lens were significantly increased when Tg(cryaa:Rno.Cryaa_R49C) was co-expressed, but not with Tg(cryaa:Rno.Cryaa_R116C). Error bars represented standard deviations in αA-R49C crosses (left panel,) and standard errors in αA-R116C crosses (right panel). Number of crosses is denoted in parentheses.
Fig 5.
The effects on γD-crystallin protein aggregation by transgenes of murine αB-crystallin R120G mutant.
(A) Transgenic expression of mouse Cryab R120G mutant induced lens defects in 4dpf zebrafish embryos, and lens defects were significantly exacerbated in the background of αB-crystallin null (cryaba-/-; cryabb-/-). (B) The percentage of embryos showing mCherry-tagged γD-I4F punctates in the lens were not changed when co-expressed with Tg(cryaa:Mmu.Cryab_R120G). Error bars represented standard deviations in (A) and standard errors in (B). Number of crosses is denoted in parentheses.