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During vertebrate embryogenesis, the body axis elongates posteriorly through a self renewing population of precursor cells in the tail bud. The Caudal Lateral Epiblast (CLE) of the mammalian embryo harbours a stem cell/progenitor zone of bipotent progenitors that contribute to both the elongating spinal cord and the paraxial mesoderm of the embryo. These progenitors, called the Neural Mesodermal Progenitors (NMPs), are characterized by the coexpression of Sox2 and T. A number of in vitro studies have been able to produce NMP-like (NMP-l) cells from Pluripotent Stem Cells (PSCs). However, in contrast with embryonic NMPs these in vitro derived ones do not self renew. Here we use different protocols for NMP-l cells and find that, in addition to NMPs, all produce cells with the potential to differentiate into Lateral Plate and Intermediate Mesoderm precursors. We show that Epiblast Stem Cells (EpiSCs) are a better starting source to produce NMPs and show that a specific differentiation protocol yields cells with the potential to self renew in vitro and to make large contributions in xenotransplant assay to axial elongation in both neural and mesodermal tissue. These cells are derived from a population that resembles the Caudal Epiblast (CE) of the embryo at the time of the appearance of the node and we show that a balance between Nodal and BMP signalling is important to define this population. Our study suggests that at the end of gastrulation and associated with the node, a multipotential progenitor population emerges that will give rise to the spinal cord and the mesodermal populations posterior to the brain.

Neuromesodermal progenitors (NMps) are a population of bipotent progenitors that maintain competence to generate both spinal cord and paraxial mesoderm throughout the elongation of the posterior body axis. Recent studies have generated populations of NMp-like cells in culture and have been shown to differentiate to both neural and mesodermal cell fates when transplanted into either mouse or chick embryos. Here, we aim to compare the potential of mouse embryonic stem (ES) cell-derived progenitor populations to generate NMp behavior against both undifferentiated and differentiated populations. We define NMp behaviour as the ability of cells to i) contribute to a significant proportion of the anterior-posterior body axis, ii) enter into both posterior neural and somitic compartments and, iii) retain a proportion of the progenitor population within the posterior growth zone. We compare previously identified ES cell-derived NMp-like populations to undifferentiated mouse ES cells and find that they all display similar potentials to generate NMp behaviourin vivo. To assess whether this competence is lost upon further differentiation, we generated anterior and posterior embryonic cell types through the generation of 3D gastruloids and show that NMp competence is lost within the anterior (Brachyury negative) portion of the gastruloid. Taken together, this demonstrates that the chick caudo-lateral epiblast is a highly permissive environment for testing NMp competence and is therefore not suitable as a positive test of neuromesodermal progenitor identity(ie. specification). However, it does act as an appropriate system to test for the loss of NMp potential, and therefore offers insight as a functional test for the regulation of NMp competencein vivo.

Generation of asymmetry within the early embryo is a critical step in the establishment of the three body axes, providing a reference for the patterning of the organism. To study the establishment of asymmetry and the development of the anteroposterior axis (AP) in culture, we utilised our ‘Gastruloid’ model system. ‘Gastruloids’, highly reproducible embryonic organoids formed from aggregates of mouse embryonic stem cells, display symmetry-breaking, polarised gene expression and axial development, mirroring the processes on a time-scale similar to that of the mouse embyro. UsingGastruloidsformed from mouse ESCs containing reporters for Wnt, FGF and Nodal signalling, we were able to quantitatively assess the contribution of these signalling pathways to the establishment of asymmetry through single time-point and live-cell fluorescence microscopy.During the first 24-48h of culture, interactions between the Wnt/β-Catenin and Nodal/TGF/βsignalling pathways promote the initial symmetry-breaking event, manifested through polarisedBrachyury(T/Bra) expression. Neither BMP nor FGF signalling is required for the establishment of asymmetry, however Wnt signalling is essential for the amplification and stability of the initial patterning event. Additionally, low, endogenous levels of FGF (24-48h) has a role in the amplification of the established pattern at later time-points.Our results confirm thatGastruloidsbehave like epiblast cells in the embryo, leading us to translate the processes and signalling involved in pattern formation ofGastruloidsin culture to the development of the embryo, firmly establishingGastruloidsas a highly reproducible, robust model system for studying cell fate decisions and early pattern formation in culture.

Dissociated mouse embryonic stem (ES) cells were cultured to form aggregates in small volumes of basal medium in U-bottomed, non tissue-culture-treated 96-well plates and subsequently maintained in suspension culture. After growth for 48 hours, the aggregates are competent to respond to ubiquitous experimental signals which result in their symmetry-breaking and generation of defined polarised structures by 96 hours. It is envisaged that this system can be applied both to the study of early developmental events and more broadly to the processes of self-organisation and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo but unobtainable from conventional adherent culture.