The common ancestor of all vertebrates had a highly sophisticated nervous system, but questions remain about the evolution of vertebrate neural cell types. The amphioxus, a chordate that diverged before the origin of vertebrates, can inform vertebrate evolution. Here we develop and analyse a single-cell RNA-sequencing dataset from seven amphioxus embryo stages to understand chordate cell type evolution and to study vertebrate neural cell type origins. We identified many new amphioxus cell types, including homologues to the vertebrate hypothalamus and neurohypophysis, rooting the evolutionary origin of these structures. On the basis of ancestor–descendant reconstruction of cell trajectories of the amphioxus and other species, we inferred expression dynamics of transcription factor genes throughout embryogenesis and identified three ancient developmental routes forming chordate neurons. We characterized cell specification at the mechanistic level and generated mutant lines to examine the function of five key transcription factors involved in neural specification. Our results show three developmental origins for the vertebrate nervous system: an anterior FoxQ2-dependent mechanism that is deeply conserved in invertebrates, a less-conserved route leading to more posterior neurons in the vertebrate spinal cord and a mechanism for specifying neuromesoderm progenitors that is restricted to chordates. The evolution of neuromesoderm progenitors may have led to a dramatic shift in posterior neural and mesodermal cell fate decisions and the body elongation process in a stem chordate.

所有脊椎动物的共同祖先都拥有高度复杂的神经系统，但关于脊椎动物神经细胞类型的进化仍存在疑问。文昌鱼是一种在脊椎动物起源之前就分化的脊索动物，可以为脊椎动物的进化提供信息。在这里，我们开发并分析了文昌鱼七个胚胎阶段的单细胞 RNA 测序数据集，以了解脊索动物细胞类型的进化并研究脊椎动物神经细胞类型的起源。我们发现了许多新的文昌鱼细胞类型，包括脊椎动物下丘脑和神经垂体的同源物，奠定了这些结构的进化起源。在对文昌鱼和其他物种的细胞轨迹进行祖先-后代重建的基础上，我们推断了整个胚胎发生过程中转录因子基因的表达动态，并确定了形成脊索动物神经元的三种古老的发育途径。我们在机制水平上表征了细胞规范，并生成了突变株系来检查参与神经规范的五个关键转录因子的功能。我们的结果显示了脊椎动物神经系统的三个发育起源：在无脊椎动物中高度保守的前部FoxQ2依赖性机制、导致脊椎动物脊髓中更多后部神经元的不太保守的途径以及限制指定神经中胚层祖细胞的机制到脊索动物。神经中胚层祖细胞的进化可能导致后神经和中胚层细胞命运决定以及干脊索动物身体伸长过程的巨大转变。