Viterbi Family Department of Ophthalmology, Shiley Eye Institute, University of California San Diego, La Jolla, CA 92093, USA.
Department of Bioengineering, University of California San Diego, La Jolla, CA 92093, USA.
Cells. 2022 Oct 28;11(21):3412. doi: 10.3390/cells11213412.
Retinogenesis involves the specification of retinal cell types during early vertebrate development. While model organisms have been critical for determining the role of dynamic chromatin and cell-type specific transcriptional networks during this process, an enhanced understanding of the developing human retina has been more elusive due to the requirement for human fetal tissue. Pluripotent stem cell (PSC) derived retinal organoids offer an experimentally accessible solution for investigating the developing human retina. To investigate cellular and molecular changes in developing early retinal organoids, we developed SIX6-GFP and VSX2-tdTomato (or VSX2-h2b-mRuby3) dual fluorescent reporters. When differentiated as 3D organoids these expressed GFP at day 15 and tdTomato (or mRuby3) at day 25, respectively. This enabled us to explore transcriptional and chromatin related changes using RNA-seq and ATAC-seq from pluripotency through early retina specification. Pathway analysis of developing organoids revealed a stepwise loss of pluripotency, while optic vesicle and retina pathways became progressively more prevalent. Correlating gene transcription with chromatin accessibility in early eye field development showed that retinal cells underwent a clear change in chromatin landscape, as well as gene expression profiles. While each dataset alone provided valuable information, considering both in parallel provided an informative glimpse into the molecular nature eye development.
视网膜发生涉及早期脊椎动物发育过程中视网膜细胞类型的特化。虽然模式生物对于确定动态染色质和细胞类型特异性转录网络在这个过程中的作用至关重要，但由于需要人胎儿组织，对发育中的人类视网膜的理解更加难以捉摸。多能干细胞（PSC）衍生的视网膜类器官为研究发育中的人类视网膜提供了一种实验上可接近的解决方案。为了研究早期视网膜类器官中的细胞和分子变化，我们开发了 SIX6-GFP 和 VSX2-tdTomato（或 VSX2-h2b-mRuby3）双荧光报告基因。当分化为 3D 类器官时，它们分别在第 15 天表达 GFP 和第 25 天表达 tdTomato（或 mRuby3）。这使我们能够使用 RNA-seq 和 ATAC-seq 从多能性到早期视网膜特化来探索转录和染色质相关的变化。发育中类器官的途径分析显示出多能性的逐步丧失，而视囊和视网膜途径变得越来越普遍。将早期眼区发育中的基因转录与染色质可及性相关联表明，视网膜细胞经历了染色质景观以及基因表达谱的明显变化。虽然每个数据集本身都提供了有价值的信息，但同时考虑两个数据集可以更深入地了解眼睛发育的分子本质。
