Notochord and axial progenitor generation by timely BMP and NODAL inhibition during vertebrate trunk formation (human). Notochord and axial progenitor generation by timely BMP and NODAL inhibition during vertebrate trunk formation (human)

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 tissues they generate, we developed an in vitro assay using the human embryonic stem cell line H9 growing on micropatterned laminin substrates. The control dataset employs WNT(CHIR) + FGF together with BMP and NODAL inhibition for 3 days. The two remaining datasets have a 24h and 48h delay in the addition of BMP and NODAL inhibitors whilst keeping the same day3 end-point. Cells of all conditions were dissociated and the transcriptome of single cells analysed using the 10X Genomics platform (Single Cell 3′ v3.1).

脊椎动物躯体的形成，由胚胎后部的祖细胞库逐步、协同地产生躯干组织所驱动。该过程的部分特征可通过基于多能干细胞（pluripotent stem cells, PSCs）的体外模型得以重现。然而，现有模型缺少脊椎动物躯干中正常存在的多种组织组分。最为突出的是，脊索（notochord）——脊索动物的标志性结构，亦是调控周围组织模式形成的中线信号来源——在当前的人类躯干形成模型中仍未出现。

为探究躯干组织的形成机制，我们对鸡胚实施了单细胞转录组分析（single-cell transcriptomic analysis）。该分析明确了分子层面上具有特异性的离散祖细胞群，确定了它们在胚胎中的空间定位，并关联了其信号通路活性。以此图谱为指导，我们阐明了分化中的人类多能干细胞内，组织类型的典型空间结构是如何形成的：该过程涉及LATS1/2介导的YAP活性抑制以促进WNT信号通路（WNT signalling），后者与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信号级联的适时抑制，可调控所产生的不同组织类型的比例，其中包括脊索细胞。我们利用这一机制构建了整合的人类脊索与神经组织形成的3D模型。本研究的数据不仅揭示了构成脊椎动物躯干的组织的形成机制，更为未来在类组织环境中开展模式形成研究铺平了道路。

总体实验设计：为探究轴向祖细胞库及其所产生的躯干组织的出现过程，我们建立了体外培养体系，使用在微图案化层粘连蛋白底物上培养的人类胚胎干细胞系H9。对照组数据集采用WNT（CHIR）+FGF联合BMP与NODAL抑制剂处理3天。其余两组数据集则在添加BMP与NODAL抑制剂时分别延迟24小时与48小时，保持相同的第3天作为实验终点。将所有实验组的细胞解离后，采用10X Genomics平台（Single Cell 3′ v3.1）对单细胞转录组进行分析。