Single cell profiling of CRISPR/Cas9-induced OTX2 deficient retinas reveals fate switch from restricted progenitors
Single cell profiling of CRISPR/Cas9-induced OTX2 deficient retinas reveals fate switch from restricted progenitors
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CRISPR/Cas9诱导的OTX2缺陷视网膜的单细胞分析揭示了受限祖细胞的命运转变
DOI：
10.1101/538710
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Mark M. Emerson
中科院分区：
文献类型：
--
作者：
Miruna G. Ghinia Tegla;Diego F. Buenaventura;Diana Y Kim;C. Thakurdin;Kevin C. Gonzalez;Mark M. Emerson
像许多发育系统一样，脊椎动物眼睛的发育取决于在多个不同过程中反复使用的基因。OTX2转录因子是这样一种基因，其需要眼形成、光感受器形成和视网膜色素上皮特化等。最近的证据表明，OTX2也在具有有限命运选择的视网膜祖细胞亚群中表达。然而，考虑到OTX2的多种作用和常规条件敲除策略的局限性，该表达的功能意义尚不清楚。在这里，我们使用CRISPR/Cas9基因编辑来产生OTX2的突变，识别出与人类患者中观察到的表型相似的表型。此外，我们使用单细胞RNA测序来确定通过CRISPR/Cas9对源自表达OTX2的视网膜祖细胞的细胞群体进行OTX2基因编辑的功能后果。我们不仅证实了OTX2是光感受器产生所必需的，而且也是维持细胞增殖潜力和抑制特定视网膜命运形成所必需的。这些包括视网膜神经节和水平细胞的亚型，通常与这些祖细胞类型相关，表明在这种情况下，OTX2的功能是抑制姐妹细胞的命运选择。观察到参与这些细胞形成的关键转录因子的上调，表明OTX2是这些替代命运的基因调控网络的关键节点的上游。
Development of the vertebrate eye, like many developmental systems, depends on genes that are used iteratively in multiple distinct processes. The OTX2 transcription factor is one such gene, with a requirement for eye formation, photoreceptor formation, and retinal pigment epithelium specification, among others. Recent evidence has suggested that OTX2 is also expressed in subsets of retinal progenitor cells with restricted fate choices. However, given the multiple roles for OTX2 and limitations of conventional conditional knockout strategies, the functional significance of this expression is unknown. Here we use CRISPR/Cas9 gene editing to produce mutations of OTX2, identifying similar phenotypes to those observed in human patients. In addition, we use single cell RNA sequencing to determine the functional consequences of OTX2 gene editing by CRISPR/Cas9 on the population of cells derived from OTX2-expressing retinal progenitor cells. We not only confirm that OTX2 is required for the generation of photoreceptors, but also for maintaining the proliferative potential of cells and suppressing the formation of specific retinal fates. These include subtypes of retinal ganglion and horizontal cells normally associated with these progenitor types, suggesting that in this context OTX2 functions to repress sister cell fate choices. Upregulation of key transcription factors involved in the formation of these cells was observed suggesting that OTX2 is upstream of critical nodes of gene regulatory networks of these alternative fates.
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影响因子：
4.6
作者：
Wei Liu;Suvarna L. Khare;Xuelian Liang;Maureen A. Peters;Xiaoying Liu;C. Cepko;M. Xiang
通讯作者：
Wei Liu;Suvarna L. Khare;Xuelian Liang;Maureen A. Peters;Xiaoying Liu;C. Cepko;M. Xiang
影响因子：
56.9
作者：
Bunz, F;Dutriaux, A;Vogelstein, B
通讯作者：
Vogelstein, B
影响因子：
4.6
作者：
Schick, Estie;McCaffery, Sean D.;Emerson, Mark M.
通讯作者：
Emerson, Mark M.
影响因子：
11.8
作者：
Emerson, Mark M.;Surzenko, Natalia;Goetz, Jillian J.;Trimarchi, Jeffrey;Cepko, Constance L.
通讯作者：
Cepko, Constance L.
影响因子：
2.7
作者：
Gandhi S;Piacentino ML;Vieceli FM;Bronner ME
通讯作者：
Bronner ME