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Brian S Clark, Genevieve Stein-O'Brien, Fatemeh Rajaii, Gabrielle H Cannon, Fion Shiau, Erik Aranda-Michel, Elana J Fertig, Loyal A Goff, Seth Blackshaw; Mouse retinal development at single-cell resolution. Invest. Ophthalmol. Vis. Sci. 2018;59(9):587.
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© ARVO (1962-2015); The Authors (2016-present)
Work on retinal development over the past decades has begun to elucidate the transcriptional networks required for cell type specification; however, few efforts have focused on identifying the mechanisms by which an individual retinal progenitor cell (RPC) is selected at a given point in developmental time to differentiate as a specific retinal neuron or Müller glia. Additionally, our knowledge of the transcriptional changes within RPCs that govern the ability of RPCs to gain and/or lose the ability to generate a specific cell type over developmental time (competence model) remains limited. We seek to identify the genes and transcriptional networks that facilitate temporally-regulated cell fate specification.
Single cell RNA-sequencing (scRNA-seq) was used to assess the transcriptional profile of individual mouse cells from across retinal development. A modified Smart-Seq2 protocol assessed transcriptional profiles of isolated RPCs from Chx10::GFP+ retinas at E14, E18, and P2. The 10x Genomics Chromium Single Cell system was used to assess transcriptional profiles of single cells at 10 time-points (E11-P14). scRNA-seq bioinformatics analyses were performed using Monocle.
We have profiled >800 RPCs using the Smart-Seq2 protocol and >140,000 cells using the 10x Genomics system. Using bioinformatic analyses we are identifying the genes and transcriptional networks that correlate with RPC competence. Additionally, we are identifying the genes that demarcate retinal cell types. Furthermore, pseudotime analyses elicit the temporal ordering of gene usage that marks and/or govern the 1) selection of a RPC to exit the cell-cycle; 2) specification of cell identity; and 3) maturation of specified cell type. The function of identified novel candidate genes in these processes is currently being tested through gain and loss-of-function studies.
Using scRNA-seq, we are gathering insight into the mechanisms of cell fate specification in the retina. Our studies have readily identified transcriptional differences amongst RPCs across development, consistent with changes in RPC competence, and novel genes and transcriptional networks that are expressed as cells exit the cell-cycle and differentiate as mature retinal cells. This study provides a wealth of novel candidate genes that can be used to mark specific populations of cells within the developing retina and facilitate the temporal regulation of cell fate specification.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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