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. 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).