Forrester-Gauntlett Blaise, Peters Linda, Oback Björn
Animal Biotech, AgResearch, Hamilton, New Zealand.
School of Science, University of Waikato, Hamilton, New Zealand.
Front Cell Dev Biol. 2023 Sep 7;11:1112069. doi: 10.3389/fcell.2023.1112069. eCollection 2023.
Mutations in the transcription factor gene grainyhead-like 2 () are associated with progressive non-syndromic sensorineural deafness autosomal dominant type 28 () in humans. Since complete loss of is lethal in mouse embryos, we studied its role during inner ear pathology and hearing loss . To this end, we generated different homozygous deletions to knockout in mouse embryonic stem cells ( ESCs), including some mimicking naturally occurring truncations in the dimerisation domain related to human . Under naïve culture conditions, cells in suspension were more heterogenous in size and larger than wild-type controls. Adherent cells were also larger, with a less uniform shape, flattened, less circular morphology, forming loose monolayer colonies with poorly defined edges. These changes correlated with lower expression of epithelial cadherin but no changes in tight junction markers () or other isoforms (). Clonogenicity from single cells, proliferation rates of cell populations and proliferation markers were reduced in ESCs. We next induced stepwise directed differentiation of ESCs along an otic pathway, giving rise to three-dimensional inner ear-like organoids (IELOs). Quantitative morphometry revealed that cells initially formed larger IELOs with a less compacted structure, more eccentric shape and increased surface area. These morphological changes persisted for up to one week. They were partially rescued by forced cell aggregation and fully restored by stably overexpressing exogenous in ESCs, indicating that Grhl2 alters cell-cell interactions. On day 8, aggregates were transferred into minimal maturation medium to allow self-guided organogenesis for another two weeks. During this period, cells and wild-type controls developed similarly, expressing neural, neuronal and sensory hair cell markers, while maintaining their initial differences in size and shape. In summary, Grhl2 is required for morphological maintenance of ESCs and orderly formation of IELOs, consistent with an essential role in organising epithelial integrity during inner ear development. Our findings validate quantitative morphometry as a useful, non-invasive screening method for molecular phenotyping of candidate mutations during organoid development.
转录因子基因颗粒头样蛋白2（Grhl2）的突变与人类常染色体显性遗传28型进行性非综合征性感音神经性耳聋（DFNA28）相关。由于Grhl2的完全缺失在小鼠胚胎中是致死性的，我们研究了其在内耳病理和听力损失中的作用。为此，我们在小鼠胚胎干细胞（mESCs）中产生了不同的纯合缺失以敲除Grhl2，包括一些模拟与人类相关的二聚化结构域中自然发生的截短突变。在原始培养条件下，悬浮的Grhl2缺失细胞在大小上更具异质性且比野生型对照更大。贴壁的Grhl2缺失细胞也更大，形状更不均匀，扁平，圆形形态减少，形成边缘不清晰的松散单层集落。这些变化与上皮钙黏蛋白E-cadherin的表达降低相关，但紧密连接标记物（ZO-1）或其他Grhl2亚型没有变化。Grhl2缺失的mESCs中单细胞的克隆形成能力、细胞群体的增殖率和增殖标记物均降低。接下来，我们沿着耳途径诱导Grhl2缺失的mESCs进行逐步定向分化，产生三维内耳样类器官（IELOs）。定量形态学分析显示，Grhl2缺失细胞最初形成的IELOs更大，结构更疏松，形状更偏心且表面积增加。这些形态变化持续长达一周。通过强制细胞聚集可部分挽救这些变化，通过在Grhl2缺失的mESCs中稳定过表达外源性Grhl2可完全恢复，这表明Grhl2改变了细胞间相互作用。在第8天，将聚集体转移到最小成熟培养基中，以允许自我引导的器官发生再过两周。在此期间，Grhl2缺失细胞和野生型对照的发育相似，表达神经、神经元和感觉毛细胞标记物，同时保持其在大小和形状上的初始差异。总之，Grhl2是mESCs形态维持和IELOs有序形成所必需的，这与它在内耳发育过程中组织上皮完整性的重要作用一致。我们的研究结果验证了定量形态学分析作为一种有用的、非侵入性的筛选方法，可用于类器官发育过程中候选突变的分子表型分析。
