The amniote primitive streak (PS), equivalent of the amphibian blastopore, forms at the midline of the embryo during gastrulation. It marks the location where epithelial cells of the epiblast undergo epithelium to mesenchyme conversion and ingress to form the mesoderm and endoderm. At the end of gastrulation, the PS has reached its maximum length and begins to regress, laying in its wake the tissues that will form the embryonic body. When regression is complete, the remnant of the PS morphs into a poorly organized mass of cells located at the posterior end of the embryo, the tail bud, which generates the posterior-most regions of the body (Stern, 2004). Fate mapping studies in avian and mouse embryos have shown that at the beginning of PS regression the mesodermal trunk progenitors are found in the epiblast along the PS (Kinder et al., 1999; Psychoyos and Stern, 1996; Schoenwolf et al., 1992; Smith et al., 1994; Spratt and Condon, 1947; Tam and Beddington, 1987; Wilson and Beddington, 1996). These studies demonstrated that the anteroposterior distribution of mesodermal progenitors in the PS epiblast reflects their future medio-lateral fate. Cells within the node generate the notochord, and the most anterior PS cells produce the paraxial mesoderm, while the territories of the intermediate, lateral plate and extraembryonic mesoderm lie in progressively more posterior regions of the PS. The recent discovery of a new population of axis progenitor stem cells, the neuromesodermal progenitors (NMPs) in mouse, has led to reconsider the PS role in amniote body formation (Tzouanacou et al., 2009). These bipotent stem cells generating large clones containing both neural and mesodermal derivatives along the forming body axis were identified by a retrospective strategy, which did not allow to precisely localize them in the embryo. Intriguingly, direct reconstruction of the epiblast cell lineage from high-resolution light-sheet imaging of developing mouse embryos did not identify bipotential NMP cells in the PS region but only cells fated to one or the other lineage (McDole et al., 2018). Similar retrospective tracking experiments based on time-lapse movies of fluorescent cells in transgenic chicken embryos expressing GFP have led to identify a small population of epiblast cells that gives rise to descendants lying in neural and mesodermal territories (Wood et al., 2019). In the chicken and mouse embryo, extensive fate mappings of the PS region have been performed, but they did not reveal the existence of NMPs giving rise to paraxial mesoderm and neural tube (Brown and Storey, 2000; Iimura et al., 2007; Psychoyos and Stern, 1996; Schoenwolf et al., 1992; Selleck and Stern, 1991; Tam and Beddington, 1987; Wilson and Beddington, 1996; Wilson et al., 2009). Grafts of small territories of the epiblast adjacent to the anterior PS in avian or mouse embryos can give rise to both neural and mesodermal derivatives, suggesting that this territory could contain NMPs (Garcia-Martinez et al., 1993; Iimura et al., 2007; Wymeersch et al., 2016). In mouse, cells of this region (caudal lateral epiblast and node streak border) coexpress the neural marker Sox2 and the mesodermal marker T/Brachyury and were proposed to include the NMP population (Wymeersch et al., 2016). However, these grafting experiments do not allow to distinguish between a population of bipotential cells and a mixture of precursors committed to each lineage. To our knowledge, the only direct evidence for epiblast precursors able to give rise to both neural and mesodermal lineages in amniotes comes from single-cell injections of horseradish peroxidase in the mouse epiblast followed by embryo culture (Forlani et al., 2003). In this work, six clones with descendants in both lineages have been obtained from injections between the late streak and head fold stage. However, this study was performed before identification of NMPs by retrospective cloning and the bifated clones are not discussed in the article. Thus, the localization and fate of NMPs in amniotes remain incompletely understood.

Here, we performed direct lineage analysis of cells in the anterior PS epiblast of chicken embryos using two different approaches: labeling cells with a barcoded retroviral library and a Brainbow-derived strategy (Loulier et al., 2014). We show that single cells of the SOX2/T-positive region of the anterior PS are NMPs that can contribute to both neural tube and paraxial mesoderm and self-renew during formation of more posterior regions of the body. As most published fate mappings have been analyzed only after short term and did not study formation of the posterior regions of the embryo, cells of the anterior epiblast were considered monopotent and their bipotentiality went undetected. Thus, our findings reconcile the existence of bipotential NMPs demonstrated in mouse with the large body of amniote fate mappings described in classical developmental biology literature. We also performed single-cell RNA-sequencing (scRNAseq) analyses of the region encompassing the anterior PS epiblast and tail bud during axis elongation. This led to the identification of a cluster exhibiting two developmental trajectories leading to neural and mesodermal fates as expected for NMPs. We identified a similar cluster from published scRNAseq datasets from posterior tissues of equivalent stages of developing mouse embryos. In mouse and chicken embryos, these NMP populations maintained a conserved transcriptional identity segregating from their neural and mesodermal descendants as a single-cell cluster. During formation of the posterior body, the NMP population nevertheless underwent maturation characterized by expression of posterior Hox genes and loss of the epithelial phenotype. Moreover, using in toto imaging, we show that a posterior-to-anterior gradient of cell convergence speed toward the PS coupled to ingression results in the progressive exhaustion of the PS from its posterior end. This leads to the successive disappearance of the territories of the extraembryonic, lateral plate, intermediate mesoderm, and non-NMP paraxial mesoderm precursors (PMPs). Increased proliferation combined with limited ingression of cells ensures the self-renewal and expansion of the territory of clonally related bipotential NMPs during body formation, which constitutes the last remnant of the PS to contribute to the tail bud. Thus, our work provides a direct demonstration of the existence of NMP cells in amniotes and a mechanistic understanding for their sustained contribution to axis elongation.

NMPs are a population of stem cells that generate most of the posterior spinal cord, vertebrae, and skeletal muscles. Surprisingly, this cell population was only recently discovered using retrospective clonal analysis in mouse (Tzouanacou et al., 2009). The identification of such an important population of stem cells came much as a surprise because none of the many classical fate mapping studies of the epiblast and PS of the chicken or mouse embryo performed so far ever reported the identification of such bipotent precursors (Brown and Storey, 2000; Iimura et al., 2007; Psychoyos and Stern, 1996; Schoenwolf et al., 1992; Selleck and Stern, 1991; Tam and Beddington, 1987; Wilson and Beddington, 1996; Wilson et al., 2009). While the retrospective technique employed to first identify mouse NMPs unambiguously identified these bipotent stem cells, it could however not locate them in the embryo (Tzouanacou et al., 2009). Thus, direct identification of the amniote NMPs and their location in the embryo is still lacking. Here, we used lineage tracing and scRNAseq to identify and characterize NMPs as a population of stem cells located in the SOX2/T-expressing region of the anterior PS epiblast in the chicken embryo. We show that cells expressing the mesodermal marker T and the neural marker SOX2 are first found in the epiblast adjacent to the anterior PS and Hensen’s node region at the beginning of PS regression. This domain appears at a similar stage of mouse development and occupies a position similar to the SOX2/T domain of the node-streak border and the caudal lateral epiblast proposed to contain NMPs in mouse embryos (Wymeersch et al., 2016). In both mouse and chicken embryos, this domain contains cells fated to give rise to both neural and mesodermal derivatives, suggesting that they are functionally equivalent (Garcia-Martinez et al., 1993; Wymeersch et al., 2016). However, so far the bipotentiality of these cells has not been established at the single-cell level. We performed lineage tracing using a barcoded retroviral library and Brainbow-derived MAGIC markers (Loulier et al., 2014) to show that single cells of the SOX2/T region are bipotential and can contribute both to the neural tube and paraxial mesoderm along the trunk axis in chicken embryos. We further demonstrate clonal continuity and transcriptional homogeneity between early NMPs of the PS and late ones in the tail bud.

The significant contribution of cells of the SOX2/T territory of the anterior PS to both neural and mesodermal lineages has been missed in previous fate mapping studies of this region in chicken embryos (Brown and Storey, 2000; Fernández-Garre et al., 2002; Henrique et al., 1997; Iimura et al., 2007; Psychoyos and Stern, 1996; Schoenwolf et al., 1992; Selleck and Stern, 1991). Compared to these studies, we analyzed our lineage-tracing experiments at significantly later stages after in ovo labeling (stage 17–20HH instead of stage 10–14HH). Thus, while these fate maps indicate that cells of the anterior PS epiblast initially produce either neural or mesoderm descendants, a trend also observed in mouse clones (Tzouanacou et al., 2009), NMPs can give rise to both lineages but mostly later, in more posterior regions of the body. These observations are consistent with recent grafts of the epiblast territory in 6-somite chicken embryos showing that the territory first produces neural and then both mesodermal and neural derivatives (Kawachi et al., 2020). Direct identification of bipotential cells with a neural and mesodermal fate has been reported in zebrafish where they segregate during gastrulation and contribute to the most posterior part of the axis (Attardi et al., 2018). In zebrafish, however, only monopotent cells were found in the tail bud (Attardi et al., 2018; Kanki and Ho, 1997), while this is not the case in chicken (this report) and mouse (Tzouanacou et al., 2009).

scRNAseq analysis of the posterior embryonic region during PS to tail bud transition in chicken and mouse revealed a distinct NMP cluster lying between a cluster of PSM cells and one of neural cells. Using two different trajectory inference analysis methods (diffusion pseudotime and WOT), we identified two developmental trajectories leading from NMPs to these neural and PSM clusters, consistent with the bipotentiality of these cells. While the mouse NMP cluster shows expression of the NMP markers T, Sox2, and Nkx1.2, only T was identified in the chicken NMP cluster. SOX2 and NKX1.2 (SAX1) expression was neither found in the NMP cluster nor in neural tissue, suggesting a problem with annotation in the chicken genome. Alternatively, this might reflect a difference in sequencing depth as the chicken dataset was obtained using the inDrops pipeline, whereas most of the mouse data was obtained with the 10X platform. From our analysis, we identified a signature of genes enriched in the NMP clusters, including a majority of effectors and targets of wnt signaling, which is known to play a key role in the differentiation of this lineage (Henrique et al., 2015). Most of these signature genes are expressed in all chicken and mouse NMP clusters. Using WOT trajectory analysis, we identified cells within the NMP cluster as ancestors of the paraxial mesoderm and neural clusters in the mouse and chicken embryo. We were not able to find any bias for the NMP contribution toward the neural or mesodermal fate. This result aligns with our lineage-tracing data, which does not show any preferential enrichment toward a specific fate in our analysis timeframe. Interestingly, we identified genes associated to the NMP trajectory, including previously undescribed ones such as RARG or MNX1 in both the mouse and chicken datasets. We also identified expected transcription factors that are predictive of the fate differentiation toward the neural (PAX6) and PSM lineages (MSGN1). It will be interesting to investigate how these genes function during differentiation of these lineages. Using diffusion pseudotime and WOT analyses, we also identified a developmental trajectory within the NMP cluster, suggesting that NMP cells undergo maturation during axis formation. This is supported by the identification of clusters of early and late NMPs characterized by expression of specific genes in both chicken and mouse. As reported for mouse embryos (Wymeersch et al., 2019), late NMPs are characterized by the expression of more posterior Hox genes in both chicken and mouse. We also observed that genes associated to the epithelial state such as EpCAM, CDH1, or GJA1 are downregulated while genes associated to the mesenchymal state are upregulated at tail bud stages in both species. This is consistent with observations in mouse where tail bud progenitors were shown to undergo incomplete EMT during later stages of axial elongation (Dias et al., 2020).

As reported for mouse embryos (Wymeersch et al., 2016; Wymeersch et al., 2019), we observe an increase in SOX2/T cell numbers during axis elongation, indicating that these cells can self-renew while giving rise to a progeny in the paraxial mesoderm and in the neural tube. Labeling experiments in mouse and chicken embryos have identified a population of epiblast cells in the region of the anterior PS and Hensen’s node, which behave as stem cells, giving rise to descendants in the paraxial mesoderm while being able to self-renew (Cambray and Wilson, 2002; Cambray and Wilson, 2007; Iimura et al., 2007; McGrew et al., 2008; Nicolas et al., 1996; Selleck and Stern, 1991; Wilson et al., 2009). These cells were proposed to contribute mostly to medial somites while lateral somitic cells are derived from more posterior areas of the PS (Iimura et al., 2007; Selleck and Stern, 1991). The anterior SOX2/T territory encompasses Hensen’s node and the epiblast adjacent to the anterior PS and approximately corresponds to the territory containing the stem cells fated to give rise to medial somites. The epiblast territory immediately posterior to the SOX2/T territory does not express SOX2 and exhibits many cells positive for the paraxial mesoderm-specific marker MSGN1 (this study). This territory likely corresponds to the prospective territory of lateral somites, which does not show a stem cell behavior, giving rise to descendants spanning only ~5–7 segments (Iimura et al., 2007; Psychoyos and Stern, 1996). Our data suggest that this territory becomes largely exhausted at the end of PS regression, resulting in posterior somites to derive mostly from the NMPs (Figure 8D). This is consistent with observations in chicken and mouse demonstrating that the selective contribution of different PS territories to medial or lateral somitic territories only applies to anterior somites (Cambray and Wilson, 2007; Psychoyos and Stern, 1996).

We observed a posterior to anterior gradient of convergence in the epiblast associated to a parallel gradient of ingression in the PS. A parallel posterior to anterior gradient of cell motility of ingressed mesodermal cells has been documented along the regressing PS (Zamir et al., 2006). These graded movements in the mesoderm are largely controlled by a gradient of Fgf8 acting as a repellent on newly ingressed mesodermal cells at this stage (Yang et al., 2002). This cellular dynamics suggests that the movement away from the posterior PS could act as a sink for the epiblastic territories generating these progenitors. These cell movements in the epiblast and the ingressing mesoderm could explain the progressive exhaustion of the PS from its posterior end first described for the extraembryonic territory (Spratt, 1947). Importantly, our data show that the precursor territories along the PS do not disappear at the same rate. We observe a sequential posterior to anterior exhaustion of the territories of the extraembryonic mesoderm, the lateral plate, and the SOX2-negative PMPs (Figure 8D). Combined to an increased proliferation in the NMP region, this could explain why the SOX2/T territory eventually remains as the major remnant of the PS in the tail bud after PS regression. Thus, while most of the PS behave as a transit zone for committed progenitors as proposed by Pasteels, 1937, its anterior-most region behaves more like a blastema, containing multipotential cells as predicted by Wetzel (Romanoff, 1960; Wetzel, 1929). The fact that the tail bud contains multipotent NMPs in addition to monopotent territories such as the precursors of the notochord or the hindgut could explain the long-standing controversy on whether the tail bud functions as a blastema or as a mosaic of committed precursors (Catala et al., 1995; Davis and Kirschner, 2000; Gont et al., 1993; Holmdahl, 1925).

Here, we show that NMPs can contribute to a single type of derivative (neural or mesodermal) for several segments followed by another type in the next segments, as reported in mouse embryos (Tzouanacou et al., 2009). This argues for a striking plasticity of NMPs and rules out simple models of asymmetric divisions for the generation of neural and mesodermal descendants. Furthermore, as reported in mouse, we also observed bifated clones that do not contribute to the tail bud (Tzouanacou et al., 2009). Plasticity of the NMPs is supported by heterotopic grafts of the NMP epiblast territory into territories fated to become neural or mesodermal, which resulted in the donor cells to adopt the fate of their new territory in chicken or mouse embryos (Garcia-Martinez et al., 1997; McGrew et al., 2008; Wymeersch et al., 2016). Our results also show that the transcriptional identity of the NMP population remains largely stable throughout axis formation. This suggests that the NMP population behaves as a unique stem cell population that remains uncommitted toward either lineage, with its descendants acquiring their identity only after entering the territory of the paraxial mesoderm or the neural tube. Such plasticity is supported by the observation that SOX2/T-positive NMP-like cells generated in vitro can be induced to differentiate to either neural or mesodermal cells by changing the culture conditions (Diaz-Cuadros et al., 2020; Edri et al., 2019a; Edri et al., 2019b; Gouti et al., 2017; Gouti et al., 2014; Henrique et al., 2015; Turner et al., 2014). The difficulty to observe NMPs in vivo contrasts with the ease with which NMP-like cells can be obtained in vitro from mouse or human pluripotent stem cells. NMPs have been generated in 2D cultures by activation of Wnt signaling at the epiblast-like stage (Diaz-Cuadros et al., 2020; Edri et al., 2019a; Edri et al., 2019b; Gouti et al., 2017; Gouti et al., 2014; Henrique et al., 2015; Turner et al., 2014). Analysis of 3D cultures such as gastruloids induced in vitro from mouse ES cells also shows that most of the cells forming these structures belong to the neural tube and paraxial mesoderm lineage, suggesting that they are largely derived from an initial NMP population (Beccari et al., 2018; Faustino Martins et al., 2020; van den Brink et al., 2020). NMP-like cells generated in vitro express both SOX2 and T and single-cell RNA-sequencing demonstrated that they form a very homogeneous population with characteristics similar to the endogenous SOX2/T cells (Diaz-Cuadros et al., 2020; Gouti et al., 2017). Our comparison of chicken, mouse, and human NMP cells identified by scRNAseq points to an significant level of conservation of the transcriptional signature of this population. Overall, our work answers an important conundrum on the existence and fate of the NMPs in vivo, an elusive stem cell population in amniotes.

Sequencing data have been deposited in GEO under accession codes GSE161905. All the raw data to generate the graphs are given in a txt file.

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Author details

1. Charlene Guillot

   1. Department of Pathology, Brigham and Women’s Hospital, Boston, United States
   2. Department of Genetics, Harvard Medical School, Boston, United States
   3. Harvard Stem Cell Institute, Boston, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   charlene_guillot@hms.harvard.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9214-7509

2. Yannis Djeffal

   1. Department of Pathology, Brigham and Women’s Hospital, Boston, United States
   2. Department of Genetics, Harvard Medical School, Boston, United States
   3. Harvard Stem Cell Institute, Boston, United States

   Contribution

   Formal analysis, YD performed the trajectory analysis in silico with CG and OP

   Competing interests

   No competing interests declared

3. Arthur Michaut

   1. Department of Pathology, Brigham and Women’s Hospital, Boston, United States
   2. Department of Genetics, Harvard Medical School, Boston, United States
   3. Harvard Stem Cell Institute, Boston, United States

   Contribution

   Software, AM wrote the code to analyze the cell trajectories and their speed

   Competing interests

   No competing interests declared

4. Brian Rabe

   1. Department of Genetics, Harvard Medical School, Boston, United States
   2. Howard Hughes Medical Institute, Boston, United States

   Contribution

   Resources, BR constructed the viral library and prepared the viral solutions

   Competing interests

   No competing interests declared

5. Olivier Pourquié

   1. Department of Pathology, Brigham and Women’s Hospital, Boston, United States
   2. Department of Genetics, Harvard Medical School, Boston, United States
   3. Harvard Stem Cell Institute, Boston, United States

   Contribution

   Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   pourquie@genetics.med.harvard.edu

   Competing interests

   No competing interests declared

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