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A zebrafish model of foxe3 deficiency demonstrates lens and eye defects with dysregulation of key genes involved in cataract formation in humans

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Abstract

The Forkhead box E3 (FOXE3) gene encodes a transcription factor with a forkhead/winged helix domain that is critical for development of the lens and anterior segment of the eye. Monoallelic and biallelic deleterious sequence variants in FOXE3 cause aphakia, cataracts, sclerocornea and microphthalmia in humans. We used clustered regularly interspaced short palindromic repeats/Cas9 injections to target the foxe3 transcript in zebrafish in order to create an experimental model of loss of function for this gene. Larvae that were homozygous for an indel variant, c.296_300delTGCAG, predicting p.(Val99Alafs*2), demonstrated severe eye defects, including small or absent lenses and microphthalmia. The lenses of the homozygous foxe3 indel mutants showed more intense staining with zl-1 antibody compared to control lenses, consistent with increased lens fiber cell differentiation. Whole genome transcriptome analysis (RNA-Seq) on RNA isolated from wildtype larvae and larvae with eye defects that were putative homozygotes for the foxe3 indel variant found significant dysregulation of genes expressed in the lens and eye whose orthologues are associated with cataracts in human patients, including cryba2a, cryba1l1, mipa and hsf4. Comparative analysis of this RNA-seq data with iSyTE data identified several lens-enriched genes to be down-regulated in foxe3 indel mutants. We also noted upregulation of lgsn and crygmxl2 and downregulation of fmodb and cx43.4, genes that are expressed in the zebrafish lens, but that are not yet associated with an eye phenotype in humans. These findings demonstrate that this new zebrafish foxe3 mutant model is highly relevant to the study of the gene regulatory networks conserved in vertebrate lens and eye development.

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Acknowledgements

Library preparation and QCs for sequencing was conducted by Yanxia Hao, Jim McGuire and Natasha Carli, PhD, at the Gladstone Genomics Core.

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Authors and Affiliations

  1. Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA

    M. Krall, S. Htun & A. M. Slavotinek

  2. Department of Biological Sciences, University of Delaware, Newark, DE, USA

    D. Anand & S. A. Lachke

  3. Department of Biochemistry and Biophysics, Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA

    D. Hart

  4. Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE, USA

    S. A. Lachke

  5. Department of Pediatrics, University of California San Francisco, Room RH384C, 1550 4th St, San Francisco, CA, 94143-2711, USA

    A. M. Slavotinek

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  1. M. Krall
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439_2018_1884_MOESM7_ESM.tif

Fig. S1. Chromatograms demonstrating indel variants in F1 fish after CRISPR/Cas9 injections targeting foxe3 (TIF 734 KB)

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Krall, M., Htun, S., Anand, D. et al. A zebrafish model of foxe3 deficiency demonstrates lens and eye defects with dysregulation of key genes involved in cataract formation in humans. Hum Genet 137, 315–328 (2018). https://doi.org/10.1007/s00439-018-1884-1

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