July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Single-cell transcriptomic analysis of human and murine NRL-null retinas
Author Affiliations & Notes
  • Alyssa Kallman
    Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Elizabeth E Capowski
    Waisman Center, University of Wisconsin - Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin - Madison, Madison, Wisconsin, United States
  • Aniruddha M. Kaushik
    Department of Mechanical Engineering, Johns Hopkins University, Baltimore, Maryland, United States
  • Melissa Liu
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Baranda S. Hansen
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Liben Chen
    Department of Mechanical Engineering, Johns Hopkins University, Baltimore, Maryland, United States
  • Jie Cheng
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Karl Wahlin
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
    Shiley Eye Institute, University of California, San Diego, La Jolla, California, United States
  • Ming-Wen Hu
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Loyal A Goff
    Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
    Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Jiang Qian
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Cynthia Berlinicke
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Jeff (Tza-Huei) Wang
    Department of Mechanical Engineering, Johns Hopkins University, Baltimore, Maryland, United States
    Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, United States
  • David M Gamm
    Waisman Center, University of Wisconsin - Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin - Madison, Madison, Wisconsin, United States
  • Donald J Zack
    Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
    Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Alyssa Kallman, None; Elizabeth Capowski, None; Aniruddha Kaushik, None; Melissa Liu, None; Baranda Hansen, None; Liben Chen, None; Jie Cheng, None; Karl Wahlin, None; Ming-Wen Hu, None; Loyal Goff, None; Jiang Qian, None; Cynthia Berlinicke, None; Jeff (Tza-Huei) Wang, None; David Gamm, None; Donald Zack, None
  • Footnotes
    Support  NIH, Thome Foundation, Beckman Foundation, Maryland Stem Cell Research Fund, Foundation Fighting Blindness, Research to Prevent Blindness, Bright Focus Foundation, and Guerrieri Family Foundation
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3105. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Alyssa Kallman, Elizabeth E Capowski, Aniruddha M. Kaushik, Melissa Liu, Baranda S. Hansen, Liben Chen, Jie Cheng, Karl Wahlin, Ming-Wen Hu, Loyal A Goff, Jiang Qian, Cynthia Berlinicke, Jeff (Tza-Huei) Wang, David M Gamm, Donald J Zack; Single-cell transcriptomic analysis of human and murine NRL-null retinas. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3105.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Retinal degenerative diseases (RD) remain largely untreatable, partly due to limited understanding of the complex gene-expression patterns directing photoreceptor (PR) development. By performing single-cell transcriptomic analysis of human in vitro and mouse in vivo models of retinal development in the presence and absence of Neural Retina-specific Leucine zipper protein (NRL), a transcription factor vital to rod PR development and degeneration, we are working to systematically define the gene networks involved in PR differentiation. Pathways that have been previously unappreciated as being important for PR differentiation and maintenance could potentially be therapeutic targets for RD.

Methods : We used two microfluidic-based single-cell RNA capturing platforms, Dropseq and 10X Genomics, to obtain transcriptomic data for retinal cells. Human induced pluripotent stem cell control and NRL-null lines were differentiated into retinal organoids and samples were collected at time points for single cell analysis. We performed parallel analyses in wild type (WT) and Nrl-null mice at postnatal days 7, 11, 18 and 25. We analyzed the transcriptomic data by principle component analysis and t-distributed stochastic neighbor embedding (t-SNE) to cluster cells by variably expressed genes. Using marker genes to determine cluster identity, we compared PR populations across WT and NRL-null datasets.

Results : WT human stem cell-derived retinal organoids yielded characteristic retinal cell populations, including rod and cone PRs. The NRL-null models produced only cone PR populations, with S-cones as the dominant subtype. Consistent with the human datasets, Nrl-null mouse retinas contained only cone PRs with S-opsin more highly expressed than M-opsin. Comparative analysis of the murine and human cells is providing additional insights into retinal cell type-specific expression patterns.

Conclusions : Consistent with the function of NRL in directing PRs toward a rod fate, human and murine retinas lacking NRL led to development of cone-dominant PR populations. Further analysis comparing the nature of PRs which develop with and without NRL will better define the regulatory networks involved in PR differentiation.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

t-SNE representation of 5,514 cells from human NRL-null retinal organoids demonstrating absence of rod PRs.

t-SNE representation of 5,514 cells from human NRL-null retinal organoids demonstrating absence of rod PRs.

 

t-SNE representation of 3,504 cells from human control retinal organoids demonstrating presence of rod PRs.

t-SNE representation of 3,504 cells from human control retinal organoids demonstrating presence of rod PRs.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×