Notochord and axial progenitor generation by timely BMP and NODAL inhibition during vertebrate trunk formation

The formation of the vertebrate body is driven by the progressive and coordinated production of trunk tissues from pools of progenitors located in the posterior of the embryo. Aspects of this process are recapitulated by in vitro models based on pluripotent stem cells (PSCs). However, these models lack several tissue components normally found in the vertebrate trunk. Most strikingly, the notochord, a hallmark of chordates and the source of midline signals that pattern surrounding tissues, is absent from current models of human trunk formation. To investigate how trunk tissue is formed, we performed single-cell transcriptomic analysis of chick embryos. This delineated molecularly discrete progenitor populations, which we spatially locate in the embryo and relate to signalling activity. Guided by this map, we determined how a stereotypical spatial organization of tissue types arises in differentiating human PSCs. This involved LATS1/2 mediated repression of YAP activity facilitating WNT signalling, that, together with FGF mediated ERK1/2 activation, induces the transcription factor Bra/TBXT. In addition, timely inhibition of a WNT-induced NODAL and BMP signalling cascade regulates the proportions of different tissue types produced, including notochordal cells. We exploit this to develop an integrated 3D model of human notochord and neural tissue formation. Together the data provide insight into the mechanisms responsible for the formation of the tissues that comprise the vertebrate trunk and pave the way for future studies of patterning in a tissue-like environment. Overall design: To investigate the emergence of axial progenitor pools and the trunk tissue they generate, we took advantage of precise staging of chick embryos to select four closely timed stages, approximately 5-6h apart, with somite (S) numbers: 4S, 7S, 10S and 13S (HH8-11, Hamburger Hamilton Stages). Tissue caudal to the third somite pair was dissected and the transcriptome of single cells analysed using the 10X Genomics platform.

脊椎动物体的形成，由胚胎后部的祖细胞库逐步、协同地产生躯干组织的过程所驱动。基于多能干细胞（PSCs）的体外模型可重现该过程的部分特征。然而，此类体外模型缺少脊椎动物躯干中天然存在的多种组织组分。其中最为显著的缺陷是：作为脊索动物（chordates）的标志性特征，同时作为调控周围组织模式形成的中线信号来源的脊索（notochord），在当前已有的人类躯干形成体外模型中均未出现。

为探究躯干组织的形成机制，我们对鸡胚开展了单细胞转录组分析。该分析鉴定出了具有明确分子特征的不同祖细胞群，并明确了这些细胞群在胚胎内的空间定位及其与信号通路活性的关联。依托该细胞空间定位图谱，我们明确了分化中的人类多能干细胞如何形成具有典型特征的组织类型空间排布。该过程涉及LATS1/2介导的YAP活性抑制，该抑制可增强WNT信号通路活性；WNT信号与FGF介导的ERK1/2激活共同诱导转录因子Bra/TBXT的表达。此外，对WNT诱导的NODAL与BMP信号级联反应的适时抑制，可调控包括脊索细胞在内的不同组织类型的生成比例。

基于上述研究发现，我们构建了可模拟人类脊索与神经组织形成的整合式三维模型。本研究数据为解析构成脊椎动物躯干的组织的形成机制提供了新的认知，并为未来在类组织环境中开展组织模式形成相关研究铺平了道路。

实验整体设计：为探究轴祖细胞库及其所产生的躯干组织的形成过程，我们借助精准分期的鸡胚，选取了4个时间间隔约5-6小时的紧密相邻的发育阶段，对应体节（somite）数分别为4S、7S、10S和13S（HH8-11期，即Hamburger Hamilton分期系统）。我们分离了第三对体节尾侧的组织，并利用10X Genomics平台对其单细胞转录组进行了分析。