Single-cell transcriptomics of mouse wildtype and Hoxa2-/- pharyngeal arches 1 and 2 at E10.5.

The Hox genes play a key role in specifying the axial identity of neural crest cells (NCCs) migrating into the pharyngeal arches of the developing mouse embryo. In the absence of Hoxa2, NCC derivatives of the second pharyngeal arch (PA2) duplicate those formed by NCCs of the first arch (PA1). In this study, we use bulk and single-cell RNAseq to establish the molecular mechanisms driving this phenotypic reversion to a ground state. Comparing the transcriptomes of PA1 and PA2 in wildtype and Hoxa2-/- embryos during NCC migration and differentiation, we find that Hoxa2-/- PA2 does not revert to a molecular "ground state" corresponding to the NCC derivatives. This separation of phenotypic and molecular states has significant implications for our understanding of NCC biology. We also identify putative targets through which HOXA2 likely acts to impart PA2 identity. The heterogenous expression of these targets within the PAs and their responses to the absence of HOXA2 suggest that subsets of NCCs may respond to HOXA2 activity in distinct manners related to their ultimate fate. To understand the heterogeneity of neural crest cells (NCCs) and other tissues of pharyngeal arches (PAs) 1 and 2 in the mouse embryo at E10.5, during NCC differentiation, we collected PA1 and PA2 from wildtype embryos and Hoxa2-/- embryos. We used the 10x Chromium single-cell sequencing system to compare the transcriptional profiles between PA1 and PA2 as well as wildtype and Hoxa2-/- PAs. We manually dissected PA1 and PA2 from six wildtype embryos and five Hoxa2-/- embryos, pooling according to PA and genotype, then produced a single-cell suspension for each and sequenced using 10x Chromium machine.Hox基因在调控迁移至发育中小鼠胚胎咽弓（pharyngeal arches, PAs）的神经嵴细胞（neural crest cells, NCCs）的轴向身份确立过程中发挥核心作用。当缺失Hoxa2时，第二咽弓（PA2）的神经嵴细胞衍生物会复刻第一咽弓（PA1）神经嵴细胞所形成的细胞结构与类型。本研究利用批量RNA测序（bulk RNAseq）与单细胞RNA测序技术，阐明驱动这一表型逆转至分子基础状态的具体机制。通过对比野生型与Hoxa2基因敲除（Hoxa2-/-）胚胎在神经嵴细胞迁移及分化阶段第一、第二咽弓的转录组特征，我们发现Hoxa2-/-的PA2并未逆转至对应神经嵴细胞衍生物的分子"基础状态"。这种表型状态与分子状态的分离，为我们理解神经嵴细胞生物学提供了重要的研究启示。我们还鉴定出HOXA2可能借以赋予PA2身份特异性的潜在靶基因。这些靶基因在咽弓中的异质性表达，以及它们在HOXA2缺失后的响应模式，提示神经嵴细胞亚群可能以与其最终命运相关的不同方式响应HOXA2的调控活性。为解析小鼠胚胎E10.5阶段（神经嵴细胞分化期）第一、第二咽弓的神经嵴细胞及其他组织的异质性，我们从6个野生型胚胎与5个Hoxa2基因敲除（Hoxa2-/-）胚胎中手动解剖得到PA1与PA2，并按照咽弓类型与基因型进行样本混合；随后为各样本制备单细胞悬液，利用10x Chromium单细胞测序系统完成转录组测序。