GRHL2 coordinates regeneration of a polarized mucociliary epithelium from basal stem cells.
GRHL2 coordinates regeneration of a polarized mucociliary epithelium from basal stem cells.
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DOI：
10.1083/jcb.201506014
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发表时间：
2015-11-09
期刊：
影响因子：
--
通讯作者：
Hogan BL
中科院分区：
文献类型：
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作者：
Gao X;Bali AS;Randell SH;Hogan BL
Crispr/Cas9介导的转录因子GRHL 2或其预测的下游靶点ZNF 750和SMAGP在原代人支气管上皮基底细胞中的突变导致纤毛发生和/或屏障功能的缺陷。肺的假复层气道上皮由基底干/祖细胞维持的极化纤毛和分泌细胞组成。一个重要的问题是谱系选择和分化是如何与顶基极性和上皮形态发生协调的。我们以前的研究表明，一个关键的整合作用的转录因子Grainyhead样2（Grhl 2）。在这项研究中，我们提出了进一步的证据，该模型使用条件基因删除过程中的气道上皮细胞和克隆类器官培养的再生。我们还在体外将CRISPR/Cas9基因组编辑用于原代人基底细胞分化成类器官和粘膜纤毛上皮。Grhl 2的缺失抑制类器官形态发生和纤毛细胞的分化，并降低具有不同Grhl 2调节位点的notch和纤毛发生基因（Mcidas、Rfx 2和Myb）的表达。其他推定的靶基因的基因组编辑揭示了锌指转录因子Znf 750和小膜粘附糖蛋白在促进纤毛发生和屏障功能中的作用，作为Grhl 2协调调控的基因网络的一部分。
Crispr/Cas9-mediated mutation of the transcription factor GRHL2 or either of its predicted downstream targets ZNF750 and SMAGP in primary human bronchial epithelial basal cells leads to defects in ciliogenesis and/or barrier function. Pseudostratified airway epithelium of the lung is composed of polarized ciliated and secretory cells maintained by basal stem/progenitor cells. An important question is how lineage choice and differentiation are coordinated with apical–basal polarity and epithelial morphogenesis. Our previous studies indicated a key integrative role for the transcription factor Grainyhead-like 2 (Grhl2). In this study, we present further evidence for this model using conditional gene deletion during the regeneration of airway epithelium and clonal organoid culture. We also use CRISPR/Cas9 genome editing in primary human basal cells differentiating into organoids and mucociliary epithelium in vitro. Loss of Grhl2 inhibits organoid morphogenesis and the differentiation of ciliated cells and reduces the expression of both notch and ciliogenesis genes (Mcidas, Rfx2, and Myb) with distinct Grhl2 regulatory sites. The genome editing of other putative target genes reveals roles for zinc finger transcription factor Znf750 and small membrane adhesion glycoprotein in promoting ciliogenesis and barrier function as part of a network of genes coordinately regulated by Grhl2.