A cell atlas of the chick retina based on single cell transcriptomics
- Harvard University, United States
Abstract
Retinal structure and function have been studied in many vertebrate orders, but molecular characterization has been largely confined to mammals. We used single-cell RNA sequencing (scRNA-seq) to generate a cell atlas of the chick retina. We identified 136 cell types plus 14 positional or developmental intermediates distributed among the six classes conserved across vertebrates – photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and glial cells. To assess morphology of molecularly defined types, we adapted a method for CRISPR-based integration of reporters into selectively expressed genes. For Müller glia, we found that transcriptionally distinct cells were regionally localized along the anterior-posterior, dorsal-ventral and central-peripheral retinal axes. We also identified immature photoreceptor, horizontal cell and oligodendrocyte types that persist into late embryonic stages. Finally, we analyzed relationships among chick, mouse and primate retinal cell classes and types. Our results provide a foundation for anatomical, physiological, evolutionary, and developmental studies of the avian visual system.
Data availability
Sequencing data have been deposited in GEO under accession GSE159107. Data can be visualized at the Broad Institute Single Cell Portal using the link: https://singlecell.broadinstitute.org/single_cell/study/SCP1159.
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human retinaNCBI Gene Expression Omnibus, GSE148077.
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mouse rodsNCBI Gene Expression Omnibus, GSE63472.
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mouse bipolarNCBI Gene Expression Omnibus, GSE81904.
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mouse amacrinesNCBI Gene Expression Omnibus, GSE149715.
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macaque retinaNCBI Gene Expression Omnibus, GSE118852.
Article and author information
Author details
Funding
National Eye Institute (RO1EY022073)
- Masahito Yamagata
- Wenjun Yan
- Joshua R Sanes
National Institute of Neurological Disorders and Stroke (NS029269)
- Masahito Yamagata
- Wenjun Yan
- Joshua R Sanes
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#24-10) of Harvard University.
Copyright
© 2021, Yamagata et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A cell atlas of the chick retina based on single cell transcriptomics
eLife 10:e63907.
https://doi.org/10.7554/eLife.63907
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We present near-atomic-resolution cryoEM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9 Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2–2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.
