Transcriptomes of mouse wildtype and Hoxa2-/- pharyngeal arches 1 and 2 at E9.0, E9.5, E10.0, and E10.5.. Transcriptomes of mouse wildtype and Hoxa2-/- pharyngeal arches 1 and 2 at E9.0, E9.5, E10.0, and 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 specification of axial identity in neural crest cells (NCCs), we collected pharyngeal arches (PAs) 1 and 2 from mouse embryos at four time points during NCC migration and the beginning of differentiation (E9.0, E9.5, E10.0, and E10.5). We collected these tissues from both wildtype embryos and embryos homozygous for a mutation abrogating Hoxa2 expression. By performing comparative analysis of these tissues, we set out to understand the molecular underpinnings of NCC axial identity, as well as the mechanistic role of Hoxa2 in establishing PA2 identity. Overall design: We examine 3-5 biological replicates of PA1 and PA2 collected from a single embryo for each genotype at each timepoint.

同源框基因（Hox genes）在调控迁移至发育中小鼠胚胎咽弓（pharyngeal arches）的神经嵴细胞（neural crest cells, NCCs）的轴向特性指定过程中发挥关键作用。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）的神经嵴细胞所形成的结构。本研究采用批量及单细胞RNA测序（bulk and single-cell RNAseq）技术，阐明驱动该表型逆转至基础状态的分子机制。通过比较野生型（wildtype）与纯合Hoxa2敲除（Hoxa2-/-）小鼠胚胎在神经嵴细胞迁移及分化阶段的第一、第二咽弓转录组，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"。这种表型与分子状态的分离，对我们理解神经嵴细胞生物学具有重要意义。本研究还鉴定出HOXA2可能借以赋予PA2轴向特性的潜在靶标。这些靶标在咽弓内的异质性表达及其对HOXA2缺失的响应提示，神经嵴细胞的不同亚群可能以与其最终命运相关的不同方式响应HOXA2的活性。为解析神经嵴细胞的轴向特性指定机制，我们在神经嵴细胞迁移及分化起始阶段（E9.0、E9.5、E10.0及E10.5）的四个时间点，从小鼠胚胎中收集了第一、第二咽弓组织。上述组织分别来源于野生型胚胎以及携带破坏Hoxa2表达突变的纯合突变胚胎。通过对这些组织开展比较分析，本研究旨在阐明神经嵴细胞轴向特性的分子基础，以及Hoxa2在建立PA2轴向特性中的机制性作用。整体实验设计：我们为每个时间点、每种基因型的单胚胎来源的PA1与PA2样本设置3-5个生物学重复。