Formation of a second lens in the zebrafish occhiolino/collagen4a5 mutant.

Abstract

Purpose: Characterization of the mechanism for the visual defects in the zebrafish occhiolino (occ) mutant.

Methods: In an N-ethyl-N-nitrosourea (ENU) screen for eye phenotypes occ was identified as having a small pupil and lens. Because of visual deficits, occ mutants behave as blind fish and have difficulty feeding beyond 5 days post fertilization (dpf). Tissue sections of occ and wild-type (WT) embryos were compared using H&E staining. Live occ and WT embryos were mounted in soft agar and lens development was compared by multiphoton imaging over the first 5dpf. Impairment of visual function was evaluated using the optokinetic response (OKR). Retinal function was evaluated using electroretinograms (ERG). Whole genome sequencing and SNP-mapping was utilized to identify the causative mutation.

Results: In H&E stained sections, disruption of the original lens and formation of a second lens in occ mutants was observed by 5dpf. Live embryo imaging with multiphoton microscopy starting at 1dpf determined that occ mutants and WT embryos developed similarly to approximately 3dpf. After 3dpf, a second lens cell mass separated from the surface ectoderm and formed anterior to the original lens in occ. Retinal structure was similar in occ mutants and WT embryos. OKR screening confirmed visual defects in occ embryos. The number of saccades per 20 sec decreased in occ to less than one-half of WT embryos. ERG wave amplitudes and timing were nearly identical in occ and WT embryos confirming normal photoreceptor function. The formation of the second lens leads to defective optics without an effect on retinal structure or function. Whole genome sequencing and SNP-mapping identified a T>G mutation in the intron 41 splice-donor site of the collagen4a5 (col4a5) gene. This mutation resulted in the inclusion of intron 41 and a premature stop codon. Complementation testing with two other col4a5 mutant alleles (col4a5^uta8 and col4a5^sa1609) resulted in embryos with lens defects, supporting the identification of col4a5 as the causative mutation in occ.

Conclusions: A mutation in the splice-donor site of the collagen4a5 gene leads to development of a second lens mass at 3 dpf in the zebrafish mutant occhiolino, leading to altered optical function with normal retinal ERGs. Marked thinning of the human lens capsule is associated with collagen4a5 mutation. Zebrafish occ is a model for the importance of lens capsule in the development of visual function.

Keywords: 497 development • 604 mutations • 519 extracellular matrix