June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Single cell transcriptome and gene knockout analysis reveals novel candidate genes with impact on anterior segment development
Author Affiliations & Notes
  • Oliver Voecking
    Biology, University of Kentucky College of Arts and Sciences, Lexington, Kentucky, United States
  • Jeramiah J. Smith
    Biology, University of Kentucky College of Arts and Sciences, Lexington, Kentucky, United States
  • Jakub K. Famulski
    Biology, University of Kentucky College of Arts and Sciences, Lexington, Kentucky, United States
  • Footnotes
    Commercial Relationships   Oliver Voecking None; Jeramiah Smith None; Jakub Famulski None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1173 – A0027. doi:
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      Oliver Voecking, Jeramiah J. Smith, Jakub K. Famulski; Single cell transcriptome and gene knockout analysis reveals novel candidate genes with impact on anterior segment development. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1173 – A0027.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Periocular mesenchyme (POM) is a subgroup of neural crest cells, responsible for forming anterior structures, including the anterior segment (AS) of the eye. Despite the importance for the development of a healthy eye, molecular knowledge about this cell group is limited. The purpose of this study is to characterize genes involved in regulation of POM cells as they form the zebrafish AS. We employed scRNA analyses over the course of AS development to identify potential candidates and used CRISPR/Cas9 knockouts to analyze resulting phenotypes.

Methods : Larval eyes of transgenic zebrafish Tg(Foxc1b:GFP) and Tg(Lmx1b:GFP) were collected every 24 hours between 48hpf and 144hpf. GFP+ cells were isolated via FACS cell sorting and processed with the 10x genomics chromium single cell transcriptome kit. The subsequently resulting Illumina sequencing single cell transcriptomes were processed with the Cell Ranger pipeline. Analysis was done with the Cell Loupe Browser 5.0 and Monocle3. Gene expression was confirmed via in situ hybridization and gene function via Alt-R-CRISPR induced knockouts.

Results : Over 40,000 Foxc1b+ and Lmx1b+ cells from eyes only were collected. Transcriptome analyses showed that these cells were organized in re-occurring clusters during zebrafish anterior segment development. We identified and tracked clusters representing the iridocorneal angle (ICA) and the cornea. Our analysis revealed several genes associated within these clusters, including hgd, si:ch211-251b21.1, nusap1 and apoda1 with the cornea and ano9a, mdka, clu and zgc158463 with the ICA. Initial gene knockouts and drug treatments showed a crucial role in eye development for hgd and si:ch211-251b21.1. Specifically, the drug Nitisionone induced hgd inhibition, resulting in cloudy lenses and an overall maldeveloped eye morphology.

Conclusions : Our results provide the first single cell transcriptome atlas for AS development in zebrafish. Our efforts have identified several new genes previously not associated with AS development. Particularly, we have discovered that hgd and si:ch211-251b21.1 play roles in AS development. Ultimately, this type of approach is likely to advance potential for genetic screening of anterior segment related diseases and/or treatment of ocular diseases such as glaucoma.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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