TY - JOUR
T1 - Forward genetic analysis using OCT screening identifies Sfxn3 mutations leading to progressive outer retinal degeneration in mice
AU - Chen, Bo
AU - Aredo, Bogale
AU - Ding, Yi
AU - Zhong, Xin
AU - Zhu, Yuanfei
AU - Zhao, Cynthia X.
AU - Kumar, Ashwani
AU - Xing, Chao
AU - Gautron, Laurent
AU - Lyon, Stephen
AU - Russell, Jamie
AU - Li, Xiaohong
AU - Tang, Miao
AU - Anderton, Priscilla
AU - Ludwig, Sara
AU - Moresco, Eva Marie Y.
AU - Beutler, Bruce
AU - Ufret-Vincenty, Rafael L.
N1 - Publisher Copyright:
© 2020 National Academy of Sciences. All rights reserved.
PY - 2020/6/9
Y1 - 2020/6/9
N2 - Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N-ethyl-N-nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3−/− mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3−/− mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function.
AB - Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N-ethyl-N-nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3−/− mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3−/− mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function.
KW - CRISPR
KW - ENU mutagenesis
KW - Forward genetics
KW - Retinal degeneration
KW - Sfxn3
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U2 - 10.1073/pnas.1921224117
DO - 10.1073/pnas.1921224117
M3 - Article
C2 - 32457148
AN - SCOPUS:85086257998
SN - 0027-8424
VL - 117
SP - 12931
EP - 12942
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 23
ER -