CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids.
CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids.
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DOI：
10.3389/fcell.2021.764725
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发表时间：
2021
影响因子：
5.5
通讯作者：
Zack DJ
中科院分区：
文献类型：
--
作者：
Wahlin KJ;Cheng J;Jurlina SL;Jones MK;Dash NR;Ogata A;Kibria N;Ray S;Eldred KC;Kim C;Heng JS;Phillips J;Johnston RJ Jr;Gamm DM;Berlinicke C;Zack DJ
人类多能干细胞（PSC）是研究人眼发育和疾病的强大工具。当在 3D 环境中生长时，它们可以自组装成层状组织的视网膜；然而，各个类器官的大小、形状和成分存在差异。驱动视网膜发生的微环境和关键生长因子的时间尚不清楚。为了探索早期视网膜发育，我们开发了一种 SIX6-GFP 报告基因，能够系统优化促进视泡形成的条件。我们证明早期低氧生长条件增强了 SIX6 的表达并促进了眼睛的形成。 SIX6 的表达通过连续抑制 Wnt 和激活 sonic hedgehog 信号而进一步增强。 SIX6 + 视神经泡显示出与视网膜特征一致的 RNA 表达谱；然而，腹侧间脑标记也存在。为了证明视神经泡导致真正的“视网膜样”结构，我们生成了 SIX6-GFP/POU4F2-tdTomato 双报告系，分别标记整个发育中的视网膜和视网膜神经节细胞。通过分别收获 SIX6 + /POU4F2- 和 SIX6- 类器官来鉴定其他大脑区域，包括下丘脑和中脑-后脑 (MBHB) 区域。使用 RNAseq 研究转录谱，我们证明 SIX6-GFP 和 POU4F2-tdTomato 报告基因为人类视网膜、下丘脑和中脑/后脑类器官的发育提供了可靠的读数。
Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood. To explore early retinal development, we developed a SIX6-GFP reporter that enabled the systematic optimization of conditions that promote optic vesicle formation. We demonstrated that early hypoxic growth conditions enhanced SIX6 expression and promoted eye formation. SIX6 expression was further enhanced by sequential inhibition of Wnt and activation of sonic hedgehog signaling. SIX6 + optic vesicles showed RNA expression profiles that were consistent with a retinal identity; however, ventral diencephalic markers were also present. To demonstrate that optic vesicles lead to bona fide “retina-like” structures we generated a SIX6-GFP/POU4F2-tdTomato dual reporter line that labeled the entire developing retina and retinal ganglion cells, respectively. Additional brain regions, including the hypothalamus and midbrain-hindbrain (MBHB) territories were identified by harvesting SIX6 + /POU4F2- and SIX6- organoids, respectively. Using RNAseq to study transcriptional profiles we demonstrated that SIX6-GFP and POU4F2-tdTomato reporters provided a reliable readout for developing human retina, hypothalamus, and midbrain/hindbrain organoids.
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影响因子：
3
作者：
Bardou P;Mariette J;Escudié F;Djemiel C;Klopp C
通讯作者：
Klopp C
影响因子：
5.2
作者：
Capowski, Elizabeth E.;Wright, Lynda S.;Liang, Kun;Phillips, M. Joseph;Wallace, Kyle;Petelinsek, Anna;Hagstrom, Anna;Pinilla, Isabel;Borys, Katarzyna;Lien, Jessica;Min, Jee Hong;Keles, Sunduz;Thomson, James A.;Gamm, David M.
通讯作者：
Gamm, David M.
DOI：
10.1002/cne.23952
发表时间：
2016-08-01
期刊：
The Journal of comparative neurology
影响因子：
--
作者：
Byun H;Kwon S;Ahn HJ;Liu H;Forrest D;Demb JB;Kim IJ
通讯作者：
Kim IJ
影响因子：
3.7
作者：
de Jonge HJ;Fehrmann RS;de Bont ES;Hofstra RM;Gerbens F;Kamps WA;de Vries EG;van der Zee AG;te Meerman GJ;ter Elst A
通讯作者：
ter Elst A
影响因子：
9.2
作者：
EKKER, SC;UNGAR, AR;BEACHY, PA
通讯作者：
BEACHY, PA