The alpha-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the alpha-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with null mutations for each of the three alpha-crystallin genes (cryaa, cryaba and cryabb). The absence of normal protein was confirmed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of alpha A-crystallin produced a variety of lens defects with varying severity in larval lenses at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of either alpha Ba- or full-length alpha Bb-crystallin produced no substantial lens defects. Mutation of each alpha-crystallin gene did not alter the expression levels of the remaining two, suggesting a lack of genetic compensation. These data confirm a developmental role for alphaA-crystallin in lens development, but the range of phenotype severity suggests that its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels in lens epithelial and fiber cells. Future experiments can explore the molecular consequences of cryaa mutation and causes of lens defects in this null mutant, as well as the roles of other genes in lens development and function.
Zebrafish have been a valuable model for investigating lens crystallin function. Zebrafish and human lenses are made of similar proteins, and the mechanisms underlying their development are conserved. The production of large numbers of transparent embryos and the ease with which zebrafish can be genetically manipulated make them a good tool for examining crystallin function. The presence of two alpha B-crystallin paralog genes in zebrafish provides an opportunity to dissect the roles of this multifunctional single mammalian protein. The accessibility of early-stage embryos and fast development times make zebrafish ideal for studying the roles of alpha-crystallins in early lens development. These studies in zebrafish are more convenient and less costly than in mammalian systems such as mice.
Various genetic knockout and translation blocking techniques have been used to investigate the impacts of alpha-crystallin loss on zebrafish lens development. These experiments showed that loss of any of the three alpha-crystallin genes produced lens defects in 3- and 4-day-old fish. This result differed from work in mice, which showed that while the loss of alpha A-crystallin led to reduced lens size and early cataract development, the loss of alpha B-crystallin did not produce a lens phenotype, although heart defects were found. Furthermore, our recent analysis of zebrafish gene expression at single-cell resolution indicated that neither of the two alpha B-crystallin genes were expressed in the lens at substantial levels through five days of development. Therefore, there seem to be conflicting reports on what role, if any, alpha B-crystallins play in early lens development.
In the present study, we examined adult lenses in newly generated knockout lines for all three zebrafish alpha-crystallin proteins. A spectral library for adult zebrafish lens was produced from a data-dependent acquisition analysis of wildtype lens digests, and subsequent parallel reaction monitoring runs targeting 3 peptides from each alpha A, alpha Ba, and alpha Bb proteins were analyzed to confirm the absence of the three proteins in the respective knock out animals.
A detailed protocol outlining the sample preparation of zebrafish lenses to detect the presence of alpha-crystallins was previously published (Posner et al., Peer J, Nov27:5:e4093, 2017). Briefly, 50 µg portions of protein derived from either one or two dissected lenses were digested overnight with trypsin in the presence of ProteaseMax™ detergent as recommended by the manufacturer (ProMega, Madison, WI, USA). One microgram of each tryptic digest was then injected at 10 µl/min onto a Symmetry C18 peptide trap (Waters Corporation, Milford, MA, USA) for 0-5 min using 98% water, 2% acetonitrile (ACN), and 0.1% formic acid mobile phase and then switched onto a 75 µm x 250 mm NanoAcquity BEH 130 C18 column (Waters Corporation) using mobile phases water (A) and ACN (B), each containing 0.1% formic acid. Peptides were separated using 7.5-30% ACN over 5-35 min, 30-98% from 35-36 min, 98% ACN from 36-41 min, 98-2% ACN from 41-42 min, and 2% ACN from 41-60 min at a 300 nL/min flow rate. Eluent was analyzed using a Q-Exactive HF mass spectrometer using electrospray ionization with a Nano Flex Ion Spray Source fitted with a 20 µm stainless steel nanobore emitter spray tip and 1.0 kV source voltage (Thermo Fisher Scientific, San Jose, CA, USA). Xcalibur version 4.0 was used to control the system. Prior to the targeted parallel reaction monitoring data collection, a digest from an adult WT lens was analyzed by data-dependent acquisition (DDA) to produce a zebrafish spectral library to design a targeted parallel reaction monitoring (PRM) method to measure the abundance of αA, αBa, and αBb- crystallins in wild type and knockout fish. Precursor mass spectra were acquired over m/z 375 to 1,400 m/z at 120,000 resolution, automatic gain control (AGC) target 3 x 106, maximum ion time (MIT) of 50 ms, profile mode, and lock mass correction using m/z = 445.12002 and 391.28429 polysiloxane ions. MS2 scans were acquired from 200-2000 m/z, intensity threshold of 5 x 10E4, automatic gain control (AGC) 1 x 10E5, maximum ion time (MIT) 100 ms, 30,000 resolution, profile mode, normalized collision energy (NCE) 30, isolation window of 1.2 m/z, loop count 10, and exclusion of +1 and unassigned charges. MS2 results were searched using Sequest within Protein Discoverer 1.4 (Thermo Fisher Scientific) using a tryptic search, 10 ppm precursor mass tolerance, 0.1 Da fragment ion tolerance, dynamic modification for oxidation of methionine, and a static modification of cysteines for carbamidomethyl alkylation. A uniprot filtered proteome 000000437 zebrafish protein database containing 43,607 entries downloaded on 5/17/2022 was used. Control of peptide false discovery used a reversed sequence strategy and Percolator software to calculate q scores. Skyline (v.220.127.116.11) was then used to create a Biblio Spec spectral library containing 564 peptide entries using a cutoff score of 0.95. These peptides were associated with the above uniprot zebrafish database to match to 434 proteins and peptides matching αA-, αBa-, and αBb-crystallins were added to the target list. The raw file used to create this spectral library was then imported into Skyline and 3 peptides from each protein were selected that had both strong precursor intensities and complete fragment ion series. This precursor list of was exported to create a PRM method within the instrument control software with identical chromatographic conditions as used in the DDA run. Precursor scans from 400-1000 m/z at 120,000 resolution were used with AGC 1 x 10E6, MIT 200 ms, profile mode, and mass lock mass correction as above. PRM scans used a loop count of 10, 15,000 resolution, AGC target 2 x 10E5, MIT 100 ms, NCE 26, and isolation window 2.0 m/z.