June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Single cell transcriptome analysis of periocular mesenchyme during anterior segment development
Author Affiliations & Notes
  • Oliver Voecking
    Biology, University of Kentucky, Lexington, Kentucky, United States
  • Jeramiah J. Smith
    Biology, University of Kentucky, Lexington, Kentucky, United States
  • Jakub Famulski
    Biology, University of Kentucky, Lexington, Kentucky, United States
  • Footnotes
    Commercial Relationships   Oliver Voecking, None; Jeramiah Smith, None; Jakub Famulski, None
  • Footnotes
    Support  Knights Templar Eye Foundation Career Starter Grant
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 2610. doi:
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      Oliver Voecking, Jeramiah J. Smith, Jakub Famulski; Single cell transcriptome analysis of periocular mesenchyme during anterior segment development. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2610.

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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 of the eye. Despite the importance for the development of a healthy eye, knowledge about this cell group is limited. Particularly, only very few genetic markers and their respective roles are known. The purpose of this study is to identify formerly unknown markers of POM cells, to further understand their role in eye development and the genetic interactions required for AS formation. To do so, we employed newly available scRNA analysis over the course of AS development.

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 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 studied via in situ hybridization and gene function via Morpholino and Alt R CRISPR induced knockouts.

Results : We collected a total of more than 40,000 Foxc1b+ and Lmx1b+ cells from eyes only, including one biological replicate for each individual time point. Transcriptome analyses showed that these cells were organized in re-occurring clusters during zebrafish anterior segment development. These clusters are partly localized to different structures within the eye, including the cornea, iridocorneal angle, retina and retinal pigment epithelium. Additionally, we found several new markers with specific expression within these spatially limited areas with apparent importance for general eye development, as proven by genetic knockout. These genes include hgd, si:ch211-251b21.1, slc22a7a, ppil1, stmn1a, seta, hmgb2a and b.

Conclusions : Our results provide a base for a single cell transcriptome atlas for anterior segment development in zebrafish. Not only do they reveal previously unknown and uncharacterized genetic markers, but they also give a first insight into potential genetic interactions necessary anterior segment development. This new knowledge might further enable clinicians to increase their genetic screening for anterior segment related diseases and treatment of ocular diseases such as glaucoma.

This is a 2021 ARVO Annual Meeting abstract.

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