Single-cell transcriptomics reveals the cellular identity of a novel progenitor population crucial for murine neural tube closure

Neural tube closure in vertebrates is achieved through a highly dynamic and coordinated series of morphogenic events involving neural plate, surface ectoderm, and neural plate border. Failure of this process in the caudal region causes spina bifida. Grainyhead-like 3 (GRHL3) is an indispensable transcription factor for neural tube closure as constitutive inactivation of which leads to fully penetrant spina bifida. Here, through single-cell transcriptomics we show that at E8.5, the time-point preceding mouse neural tube closure, the co-expression of Grhl3, Tfap2a, and Tfap2c defines a previously unrecognised progenitor population of surface ectoderm. Specific deletion of Grhl3 expression using Tfap2a-Cre recapitulate the spina bifida observed in Grhl3-null animals. Moreover, conditional inactivation of Tfap2c expression in Grhl3-expressing neural plate border cells also causes mild spina bifida. These findings clearly indicate that Grhl3-expressing neural plate border cells cohort is required for the early-stage neurulation. E8.5 wild-type and Grhl3-null embryos were harvested at 3 p.m. on the eighth day post-fertilisation. The caudal half of five wild-type and three Grhl3-null embryos was separated to generate single cell suspension. Cells from five wild-type embryos and three Grhl3-null embryos were then pooled. Two wild-type and two Grhl3-null single cell-RNA sequencing libraries were constructed for analysis.脊椎动物神经管闭合通过一系列高度动态且协同有序的形态发生事件实现，这些事件涉及神经板、表面外胚层与神经板边界。若该过程在尾部区域发生障碍，会引发脊柱裂（spina bifida）。Grainyhead样蛋白3（Grainyhead-like 3, GRHL3）是神经管闭合不可或缺的转录因子，对其进行组成型失活会导致完全外显的脊柱裂表型。本研究通过单细胞转录组学（single-cell transcriptomics）分析发现，在小鼠神经管闭合前的时间节点E8.5时，Grhl3、Tfap2a与Tfap2c的共表达可定义一类此前未被识别的表面外胚层祖细胞群。利用Tfap2a-Cre特异性敲除Grhl3的表达，能够重现Grhl3全基因敲除（Grhl3-null）动物中观察到的脊柱裂表型。此外，在表达Grhl3的神经板边界细胞中条件性失活Tfap2c的表达，同样会引发轻度脊柱裂。上述发现明确证实，表达Grhl3的神经板边界细胞群是早期神经胚形成过程所必需的。我们于受精后第8日下午3点收集了E8.5的野生型（wild-type）与Grhl3全基因敲除小鼠胚胎，分离5枚野生型胚胎与3枚Grhl3全基因敲除胚胎的尾部半侧组织以制备单细胞悬液，将来自5枚野生型胚胎与3枚Grhl3全基因敲除胚胎的细胞进行混合，随后构建2个野生型与2个Grhl3全基因敲除的单细胞RNA测序（single cell-RNA sequencing）文库用于后续分析。