Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions [scRNA-seq]. Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions [scRNA-seq]

Neural crest cells migrate throughout the embryo, but how cells move in a directed and collective manner has remained unclear. Here, we perform the first single-cell transcriptome analysis of cranial neural crest cell migration at three progressive stages in chick and identify and establish hierarchical relationships between cell position and time-specific transcriptional signatures. We determine a novel transcriptional signature of the most invasive neural crest Trailblazer cells that is consistent during migration and enriched for approximately 900 genes. Knockdown of several Trailblazer genes shows significant but modest changes to total distance migrated. However, in vivo expression analysis by RNAscope and immunohistochemistry reveals some salt and pepper patterns that include strong individual Trailblazer gene expression in cells within other subregions of the migratory stream. These data provide new insights into the molecular diversity and dynamics within a neural crest cell migratory stream that underlie complex directed and collective cell behaviors. Overall design: 501 single cells analyzed from various parts of the migrating neural crest during various timepoints in development. 96 cells from HH11 Trailblazers, 50 cells from HH13 Trailblazers, 50 cells from HH13 Leaders, 60 cells from HH13 Trailers, 50 each from HH15 Trailblazers, Leaders, and Trailers. For definitions, see the extract protocol. Neural crest from a neural tube placed in culture were also analyzed.

神经嵴细胞（neural crest cells）可在胚胎内广泛迁移，但细胞如何以定向且集体的方式完成运动，这一科学问题迄今尚未阐明。本研究首次针对鸡胚中三个发育进程阶段的颅神经嵴细胞（cranial neural crest cells）迁移开展单细胞转录组分析（single-cell transcriptome analysis），明确并建立了细胞位置与时间特异性转录特征之间的层级关系。本研究鉴定出侵袭能力最强的神经嵴先驱细胞（Trailblazer cells）的全新转录特征，该特征在迁移过程中保持稳定，且富集了约900个基因。对数个先驱细胞相关基因进行敲低（knockdown）实验后发现，细胞总迁移距离出现显著但轻微的改变。不过，通过RNAscope原位杂交技术与免疫组化（immunohistochemistry）开展的体内表达分析，揭示出部分椒盐样分布模式：在迁移流的其他亚区域内，部分细胞中可检测到高强度的先驱细胞特征基因表达。本研究数据为解析调控复杂定向集体细胞运动的神经嵴细胞迁移流内部分子多样性与动态变化提供了全新视角。
实验设计：共分析501个取自发育不同时间点、迁移中神经嵴不同区域的单细胞。其中包括96个HH11时期的先驱细胞，50个HH13时期的先驱细胞、50个HH13时期的领头细胞（Leaders）、60个HH13时期的尾随细胞（Trailers），以及HH15时期的先驱细胞、领头细胞与尾随细胞各50个。相关术语定义详见提取实验方案。本研究同时分析了体外培养神经管来源的神经嵴细胞。