June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
The impact of ectopic expression of the Kruppel-like Factors on mammalian retinal development
Author Affiliations & Notes
  • Patrick Leavey
    Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Clayton Santiago
    Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Lizhi Jiang
    Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Seth Blackshaw
    Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Patrick Leavey, None; Clayton Santiago, None; Lizhi Jiang, None; Seth Blackshaw, None
  • Footnotes
    Support  Funding support to SB include NIH R01EY020560-08
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 1692. doi:
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      Patrick Leavey, Clayton Santiago, Lizhi Jiang, Seth Blackshaw; The impact of ectopic expression of the Kruppel-like Factors on mammalian retinal development. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1692.

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

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Abstract

Purpose : The seven neuronal cell types of the retina arise in a well defined sequence of temporal windows from retinal progenitor cells (RPCs). Over the course of neurogenesis, RPCs express different transcription factors predicted to control gene regulatory networks guiding cell fate decisions and pluripotency capacity. The Kruppel-like factor (KLF) family is enriched within the late stage RPC population. We hypothesize that the KLF family members dictate cell cycle exit and cell lineage within the developing retina, and maintain this capacity outside their developmental expression window.

Methods : All procedures with animals were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. CD-1 wildtype E14 and P0 (representing early and late stages of neurogenesis) retinas were harvested and electroporated with pCAGIG overexpression plasmids encoding just the IRES-GFP cassette (control) and KLF3/6/9/13. Retinal explants were cultured for two days and subsequently dissociated into a single cell suspension. GFP positive cells were obtained by flow sorting and prepared for single cell RNA sequencing (scRNA-Seq). Cell suspensions for control and KLF3/6/9/13 electroporated E14 and P0 retinas were process on the 10x Genomics Chromium Single Cell System to assess the KLF family’s impact on the transcriptional profile at a single cell level after two days of overexpression. Downstream analysis was performed using the Seurat package in R.

Results : Clusters of retinal neurogenic cells and retinal neurons were identified by the expression of marker genes for each timepoint for the control and KLF3/6/9/13 scRNA-seq profiled cells. In addition, when comparing the gene expression patterns within the RPC and neurogenic precursors between the control and KLF samples, changes were observed in genes dictating cell cycle and neurogenesis.

Conclusions : Determining which transcription factors control the transition from early and late-stage RPCs as well as the temporal patterning dictating cell fate and divisions is paramount to understanding how certain cell types arise within the mammalian retina. By establishing the KLF family as a group of transcription factors regulating retinal neurogenesis, this work can potentially inform the design of stem cell-based therapies for retinal dystrophies.

This is a 2021 ARVO Annual Meeting abstract.

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