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. Overall design: 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.