Notochord and axial progenitor generation by timely BMP and NODAL inhibition during vertebrate trunk formation (human 3D)

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 trunk tissue formation, we developed a 3D in vitro assay using the human embryonic stem cell line H9 on low-bind 96 well plates. The protocol employs WNT(CHIR) + FGF together with timed BMP and NODAL inhibition (at 18h, 24h, 30h post-induction) for the first 3 days following four days with a RA precursor. Samples represent different days and inhibition delays. Cells of all conditions were dissociated and the transcriptome of single cells analysed using the 10X Genomics platform (Single Cell 3' v3.1).