The Chick Caudolateral Epiblast Acts as a Permissive Niche for Generating Neuromesodermal Progenitor Behaviours

Abstract

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, which 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 behaviour 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 behaviour in 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. Together this suggests that in vitro-derived NMp-like cells maintain an ability to contribute to multiple germ layers that is also present within pluripotent ES cells, rather than acquiring a neuromesodermal competent state through differentiation.

An Epiblast Stem Cell derived multipotent progenitor population for axial extension

“…Our observations are in agreement with recent descriptions of the development of the tail bud (Wymeersch et al, 2019) and suggest that such a multipotent population might be an obligatory intermediate for the emergence of the NMPs in vitro. ESC-based protocols yield similar populations that can be differentiated into mesodermal and neural progenitors but lack several features characteristic of NMPs, in particular their ability to self-renew and to contribute significantly to axial extension (Baillie-Johnson et al, 2018;Gouti et al, 2014;Turner et al, 2014). Furthermore, as we have shown here, these populations represent highly heterogeneous populations with a low representation of NMPs.…”

“…However, as pluripotent stem cells can also contribute to spinal cord and somitic mesoderm upon transplantation (Baillie-Johnson et al, 2018), we have focussed our assessment of NMP behaviour on the length of time that the transplanted cells remain within the NMP niche and continually generate these progenitor populations. This is, in turn, reflected by the length of labelled cells distributed along the anteroposterior axis (Baillie-Johnson et al, 2018).…”

“…In our experiments, we transplanted cells from the EpiSC, Epi-NMP and Epi-meso conditions into a small region caudal and lateral to the node of the developing chick embryo. This region corresponds to a region that has been shown to contribute to spinal cord and paraxial mesoderm progenitors by fate mapping (Baillie-Johnson et al, 2018) and is outlined by dotted boxes in Fig. 6 (for details see Fig.…”

“…Labelled tissues were grafted into the region of the chick caudal lateral epiblast as described in Fig. S7 and (Baillie-Johnson et al, 2018;Gouti et al, 2014), using an eyebrow knife tool. Embryo cultures were imaged at single time points and as time-lapses for 15-18 h after grafting.…”

Understanding axial progenitor biology in vivo and in vitro

“…Resulting PSC-derived cell populations expressed caudal markers (Edri et al, 2019b;Frith et al, 2018;Gouti et al, 2017Gouti et al, , 2014Verrier et al, 2018), exhibited the ability to generate neural and mesodermal cell types in vitro (Frith et al, 2018;Gouti et al, 2014;Turner et al, 2014) and/or contributed to both the neural tube and paraxial mesoderm after engraftment into host chick or mouse embryos (Baillie-Johnson et al, 2018;Edri et al, 2019a;Gouti et al, 2014) (Table 3). Embryo grafting in these cases has provided a useful assay for the developmental potential of in vitro-derived axial progenitors, although the early somite mouse embryo appears to offer a more stringent host environment for distinguishing between NM bipotency versus pluripotency compared with their late gastrula chick counterparts (Baillie-Johnson et al, 2018;Gouti et al, 2014;Huang et al, 2012;Tsakiridis et al, 2014). NMP-like cells have also been reported to arise in a regionalised manner, in 3D self-organising aggregates of PSCs following a short timed pulse of CHIR (Beccari et al, 2018;Faustino Martins et al, 2020;Libby et al, 2020 preprint;Turner et al, 2014;van den Brink et al, 2020;Veenvliet et al, 2020).…”