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.

1. 1. Guillot C
   2. Pourquie O

   (2019) NCBI Gene Expression Omnibus

   ID GSE161905. Single cell sequencing of posterior axis development in Gallus gallus.

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161905

1. 1. Pijuan-Sala

   (2017) ArrayExpress

   ID E-MTAB-6967. Timecourse single-cell RNAseq of whole mouse embryos harvested between days 6.5 and 8.5 of development.

   https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6967/
2. 1. Diaz-Cuadros

   (2020) NCBI Gene Expression Omnibus

   ID GSE114186. In vitro characterization of the human segmentation clock.

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE114186

1. 1. Attardi A
   2. Fulton T
   3. Florescu M
   4. Shah G
   5. Muresan L
   6. Lenz MO
   7. Lancaster C
   8. Huisken J
   9. van Oudenaarden A
   10. Steventon B

   (2018) Neuromesodermal progenitors are a conserved source of spinal cord with divergent growth dynamics

   Development 145:dev166728.

   https://doi.org/10.1242/dev.166728

   • PubMed
   • Google Scholar
2. 1. Beccari L
   2. Moris N
   3. Girgin M
   4. Turner DA
   5. Baillie-Johnson P
   6. Cossy AC
   7. Lutolf MP
   8. Duboule D
   9. Arias AM

   (2018) Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids

   Nature 562:272–276.

   https://doi.org/10.1038/s41586-018-0578-0

   • PubMed
   • Google Scholar
3. 1. Beier KT
   2. Samson ME
   3. Matsuda T
   4. Cepko CL

   (2011) Conditional expression of the TVA receptor allows clonal analysis of descendents from Cre-expressing progenitor cells

   Developmental Biology 353:309–320.

   https://doi.org/10.1016/j.ydbio.2011.03.004

   • PubMed
   • Google Scholar
4. 1. Bénazéraf B
   2. Francois P
   3. Baker RE
   4. Denans N
   5. Little CD
   6. Pourquié O

   (2010) A random cell motility gradient downstream of FGF controls elongation of an amniote embryo

   Nature 466:248–252.

   https://doi.org/10.1038/nature09151

   • PubMed
   • Google Scholar
5. 1. Bénazéraf B
   2. Beaupeux M
   3. Tchernookov M
   4. Wallingford A
   5. Salisbury T
   6. Shirtz A
   7. Shirtz A
   8. Huss D
   9. Pourquié O
   10. François P
   11. Lansford R

   (2017) Multi-scale quantification of tissue behavior during amniote embryo Axis elongation

   Development 144:4462–4472.

   https://doi.org/10.1242/dev.150557

   • PubMed
   • Google Scholar
6. 1. Brown JM
   2. Storey KG

   (2000) A region of the vertebrate neural plate in which neighbouring cells can adopt neural or epidermal fates

   Current Biology 10:869–872.

   https://doi.org/10.1016/S0960-9822(00)00601-1

   • PubMed
   • Google Scholar
7. 1. Bulusu V
   2. Prior N
   3. Snaebjornsson MT
   4. Kuehne A
   5. Sonnen KF
   6. Kress J
   7. Stein F
   8. Schultz C
   9. Sauer U
   10. Aulehla A

   (2017) Spatiotemporal analysis of a glycolytic activity gradient linked to mouse embryo mesoderm development

   Developmental Cell 40:331–341.

   https://doi.org/10.1016/j.devcel.2017.01.015

   • Google Scholar
8. 1. Cambray N
   2. Wilson V

   (2002) Axial progenitors with extensive potency are localised to the mouse chordoneural hinge

   Development 129:4855–4866.

   https://doi.org/10.1242/dev.129.20.4855

   • PubMed
   • Google Scholar
9. 1. Cambray N
   2. Wilson V

   (2007) Two distinct sources for a population of maturing axial progenitors

   Development 134:2829–2840.

   https://doi.org/10.1242/dev.02877

   • PubMed
   • Google Scholar
10. 1. Catala M
    2. Teillet M-A
    3. Le Douarin NM

    (1995) Organization and development of the tail bud analyzed with the quail-chick Chimaera system

    Mechanisms of Development 51:51–65.

    https://doi.org/10.1016/0925-4773(95)00350-A

    • Google Scholar
11. 1. Cepko C
    2. Pear W

    (2001) Retrovirus infection of cells in vitro and in vivo

    Current Protocols in Molecular Biology 9:14.

    https://doi.org/10.1002/0471142727.mb0914s36

    • PubMed
    • Google Scholar
12. 1. Chapman SC
    2. Collignon J
    3. Schoenwolf GC
    4. Lumsden A

    (2001) Improved method for chick whole-embryo culture using a filter paper carrier

    Developmental Dynamics 220:284–289.

    https://doi.org/10.1002/1097-0177(20010301)220:3<284::AID-DVDY1102>3.0.CO;2-5

    • PubMed
    • Google Scholar
13. 1. Davis RL
    2. Kirschner MW

    (2000) The fate of cells in the tailbud of Xenopus laevis

    Development 127:255–267.

    https://doi.org/10.1242/dev.127.2.255

    • PubMed
    • Google Scholar
14. 1. Denans N
    2. Iimura T
    3. Pourquié O

    (2015) Hox genes control vertebrate body elongation by collinear wnt repression

    eLife 4:e04379.

    https://doi.org/10.7554/eLife.04379

    • Google Scholar
15. 1. Dias A
    2. Lozovska A
    3. Wymeersch FJ
    4. Nóvoa A
    5. Binagui-Casas A
    6. Sobral D
    7. Martins GG
    8. Wilson V
    9. Mallo M

    (2020) A Tgfbr1/Snai1-dependent developmental module at the core of vertebrate axial elongation

    eLife 9:e56615.

    https://doi.org/10.7554/eLife.56615

    • PubMed
    • Google Scholar
16. 1. Diaz-Cuadros M
    2. Wagner DE
    3. Budjan C
    4. Hubaud A
    5. Tarazona OA
    6. Donelly S
    7. Michaut A
    8. Al Tanoury Z
    9. Yoshioka-Kobayashi K
    10. Niino Y
    11. Kageyama R
    12. Miyawaki A
    13. Touboul J
    14. Pourquié O

    (2020) In vitro characterization of the human segmentation clock

    Nature 580:113–118.

    https://doi.org/10.1038/s41586-019-1885-9

    • PubMed
    • Google Scholar
17. 1. Edri S
    2. Hayward P
    3. Baillie-Johnson P
    4. Steventon BJ
    5. Martinez Arias A

    (2019a) An epiblast stem cell-derived multipotent progenitor population for axial extension

    Development 146:dev168187.

    https://doi.org/10.1242/dev.168187

    • PubMed
    • Google Scholar
18. 1. Edri S
    2. Hayward P
    3. Jawaid W
    4. Martinez Arias A

    (2019b) Neuro-mesodermal progenitors (NMPs): a comparative study between pluripotent stem cells and embryo-derived populations

    Development 146:dev180190.

    https://doi.org/10.1242/dev.180190

    • PubMed
    • Google Scholar
19. 1. Faustino Martins J-M
    2. Fischer C
    3. Urzi A
    4. Vidal R
    5. Kunz S
    6. Ruffault P-L
    7. Kabuss L
    8. Hube I
    9. Gazzerro E
    10. Birchmeier C
    11. Spuler S
    12. Sauer S
    13. Gouti M

    (2020) Self-Organizing 3D human trunk neuromuscular organoids

    Cell Stem Cell 26:172–186.

    https://doi.org/10.1016/j.stem.2019.12.007

    • Google Scholar
20. 1. Fernández-Garre P
    2. Rodríguez-Gallardo L
    3. Gallego-Díaz V
    4. Alvarez IS
    5. Puelles L

    (2002) Fate map of the chicken neural plate at stage 4

    Development 129:2807–2822.

    https://doi.org/10.1242/dev.129.12.2807

    • PubMed
    • Google Scholar
21. 1. Forlani S
    2. Lawson KA
    3. Deschamps J

    (2003) Acquisition of hox codes during gastrulation and axial elongation in the mouse embryo

    Development 130:3807–3819.

    https://doi.org/10.1242/dev.00573

    • Google Scholar
22. 1. Garcia-Martinez V
    2. Alvarez IS
    3. Schoenwolf GC

    (1993) Locations of the ectodermal and nonectodermal subdivisions of the epiblast at stages 3 and 4 of avian gastrulation and neurulation

    Journal of Experimental Zoology 267:431–446.

    https://doi.org/10.1002/jez.1402670409

    • PubMed
    • Google Scholar
23. 1. Garcia-Martinez V
    2. Darnell DK
    3. Lopez-Sanchez C
    4. Sosic D
    5. Olson EN
    6. Schoenwolf GC

    (1997) State of commitment of prospective neural plate and prospective mesoderm in late gastrula/early neurula stages of avian embryos

    Developmental Biology 181:102–115.

    https://doi.org/10.1006/dbio.1996.8439

    • PubMed
    • Google Scholar
24. 1. Gibson DG
    2. Young L
    3. Chuang RY
    4. Venter JC
    5. Hutchison CA
    6. Smith HO

    (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases

    Nature Methods 6:343–345.

    https://doi.org/10.1038/nmeth.1318

    • PubMed
    • Google Scholar
25. 1. Gont LK
    2. Steinbeisser H
    3. Blumberg B
    4. de Robertis EM

    (1993) Tail formation as a continuation of gastrulation: the multiple cell populations of the Xenopus tailbud derive from the late blastopore lip

    Development 119:991–1004.

    https://doi.org/10.1242/dev.119.4.991

    • PubMed
    • Google Scholar
26. 1. Gouti M
    2. Tsakiridis A
    3. Wymeersch FJ
    4. Huang Y
    5. Kleinjung J
    6. Wilson V
    7. Briscoe J

    (2014) In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity

    PLOS Biology 12:e1001937.

    https://doi.org/10.1371/journal.pbio.1001937

    • PubMed
    • Google Scholar
27. 1. Gouti M
    2. Delile J
    3. Stamataki D
    4. Wymeersch FJ
    5. Huang Y
    6. Kleinjung J
    7. Wilson V
    8. Briscoe J

    (2017) A gene regulatory network balances neural and mesoderm specification during vertebrate trunk development

    Developmental Cell 41:243–261.

    https://doi.org/10.1016/j.devcel.2017.04.002

    • Google Scholar
28. 1. Guibentif C
    2. Griffiths JA
    3. Imaz-Rosshandler I
    4. Ghazanfar S
    5. Nichols J
    6. Wilson V
    7. Göttgens B
    8. Marioni JC

    (2021) Diverse routes toward early somites in the mouse embryo

    Developmental Cell 56:141–153.

    https://doi.org/10.1016/j.devcel.2020.11.013

    • Google Scholar
29. 1. Hama H
    2. Kurokawa H
    3. Kawano H
    4. Ando R
    5. Shimogori T
    6. Noda H
    7. Fukami K
    8. Sakaue-Sawano A
    9. Miyawaki A

    (2011) Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain

    Nature Neuroscience 14:1481–1488.

    https://doi.org/10.1038/nn.2928

    • PubMed
    • Google Scholar
30. 1. Hamburger V
    2. Hamilton HL

    (1992) A series of normal stages in the development of the chick embryo. 1951

    Developmental Dynamics 195:231–272.

    https://doi.org/10.1002/aja.1001950404

    • PubMed
    • Google Scholar
31. 1. Harwell CC
    2. Fuentealba LC
    3. Gonzalez-Cerrillo A
    4. Parker PR
    5. Gertz CC
    6. Mazzola E
    7. Garcia MT
    8. Alvarez-Buylla A
    9. Cepko CL
    10. Kriegstein AR

    (2015) Wide dispersion and diversity of clonally related inhibitory interneurons

    Neuron 87:999–1007.

    https://doi.org/10.1016/j.neuron.2015.07.030

    • PubMed
    • Google Scholar
32. 1. Henrique D
    2. Tyler D
    3. Kintner C
    4. Heath JK
    5. Lewis JH
    6. Ish-Horowicz D
    7. Storey KG

    (1997) cash4, a novel achaete-scute homolog induced by Hensen's node during generation of the posterior nervous system

    Genes & Development 11:603–615.

    https://doi.org/10.1101/gad.11.5.603

    • PubMed
    • Google Scholar
33. 1. Henrique D
    2. Abranches E
    3. Verrier L
    4. Storey KG

    (2015) Neuromesodermal progenitors and the making of the spinal cord

    Development 142:2864–2875.

    https://doi.org/10.1242/dev.119768

    • PubMed
    • Google Scholar
34. 1. Holmdahl DE

    (1925)

    Experimentelle untersuchungen über die lage der grenze zwischen primärer und sekundärer körperentwicklung beim huhn

    Anatomischer Anzeiger 59:393–396.

    • Google Scholar
35. 1. Hunter JD

    (2007) Matplotlib: a 2D graphics environment

    Computing in Science & Engineering 9:90–95.

    https://doi.org/10.1109/MCSE.2007.55

    • Google Scholar
36. 1. Iimura T
    2. Yang X
    3. Weijer CJ
    4. Pourquié O

    (2007) Dual mode of paraxial mesoderm formation during chick gastrulation

    PNAS 104:2744–2749.

    https://doi.org/10.1073/pnas.0610997104

    • PubMed
    • Google Scholar
37. 1. Iimura T
    2. Pourquié O

    (2008) Manipulation and electroporation of the avian segmental plate and somites in vitro

    Methods in Cell Biology 87:257–270.

    https://doi.org/10.1016/S0091-679X(08)00213-6

    • PubMed
    • Google Scholar
38. 1. Kanki JP
    2. Ho RK

    (1997) The development of the posterior body in zebrafish

    Development 124:881–893.

    https://doi.org/10.1242/dev.124.4.881

    • PubMed
    • Google Scholar
39. 1. Kawachi T
    2. Shimokita E
    3. Kudo R
    4. Tadokoro R
    5. Takahashi Y

    (2020) Neural-fated self-renewing cells regulated by Sox2 during secondary neurulation in chicken tail bud

    Developmental Biology 461:160–171.

    https://doi.org/10.1016/j.ydbio.2020.02.007

    • PubMed
    • Google Scholar
40. 1. Kinder SJ
    2. Tsang TE
    3. Quinlan GA
    4. Hadjantonakis AK
    5. Nagy A
    6. Tam PP

    (1999) The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior Axis during gastrulation of the mouse embryo

    Development 126:4691–4701.

    https://doi.org/10.1242/dev.126.21.4691

    • PubMed
    • Google Scholar
41. 1. Klein AM
    2. Mazutis L
    3. Akartuna I
    4. Tallapragada N
    5. Veres A
    6. Li V
    7. Peshkin L
    8. Weitz DA
    9. Kirschner MW

    (2015) Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells

    Cell 161:1187–1201.

    https://doi.org/10.1016/j.cell.2015.04.044

    • PubMed
    • Google Scholar
42. 1. Landau NR
    2. Littman DR

    (1992) Packaging system for rapid production of murine leukemia virus vectors with variable tropism

    Journal of Virology 66:5110–5113.

    https://doi.org/10.1128/jvi.66.8.5110-5113.1992

    • PubMed
    • Google Scholar
43. 1. Le Douarin NM
    2. Teillet MA
    3. Catala M

    (1998)

    Neurulation in amniote vertebrates: a novel view deduced from the use of quail-chick chimeras

    The International Journal of Developmental Biology 42:909–916.

    • PubMed
    • Google Scholar
44. 1. Liberzon A
    2. Subramanian A
    3. Pinchback R
    4. Thorvaldsdóttir H
    5. Tamayo P
    6. Mesirov JP

    (2011) Molecular signatures database (MSigDB) 3.0

    Bioinformatics 27:1739–1740.

    https://doi.org/10.1093/bioinformatics/btr260

    • PubMed
    • Google Scholar
45. 1. Loulier K
    2. Barry R
    3. Mahou P
    4. Le Franc Y
    5. Supatto W
    6. Matho KS
    7. Ieng S
    8. Fouquet S
    9. Dupin E
    10. Benosman R
    11. Chédotal A
    12. Beaurepaire E
    13. Morin X
    14. Livet J

    (2014) Multiplex cell and lineage tracking with combinatorial labels

    Neuron 81:505–520.

    https://doi.org/10.1016/j.neuron.2013.12.016

    • PubMed
    • Google Scholar
46. 1. McDole K
    2. Guignard L
    3. Amat F
    4. Berger A
    5. Malandain G
    6. Royer LA
    7. Turaga SC
    8. Branson K
    9. Keller PJ

    (2018) In toto imaging and reconstruction of Post-Implantation mouse development at the Single-Cell level

    Cell 175:859–876.

    https://doi.org/10.1016/j.cell.2018.09.031

    • Google Scholar
47. 1. McGrew MJ
    2. Sherman A
    3. Ellard FM
    4. Lillico SG
    5. Gilhooley HJ
    6. Kingsman AJ
    7. Mitrophanous KA
    8. Sang H

    (2004) Efficient production of germline transgenic chickens using lentiviral vectors

    EMBO Reports 5:728–733.

    https://doi.org/10.1038/sj.embor.7400171

    • PubMed
    • Google Scholar
48. 1. McGrew MJ
    2. Sherman A
    3. Lillico SG
    4. Ellard FM
    5. Radcliffe PA
    6. Gilhooley HJ
    7. Mitrophanous KA
    8. Cambray N
    9. Wilson V
    10. Sang H

    (2008) Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior hox identity

    Development 135:2289–2299.

    https://doi.org/10.1242/dev.022020

    • PubMed
    • Google Scholar
49. 1. Moreau C
    2. Caldarelli P
    3. Rocancourt D
    4. Roussel J
    5. Denans N
    6. Pourquie O
    7. Gros J

    (2019) Timed collinear activation of hox genes during gastrulation controls the avian forelimb position

    Current Biology 29:35–50.

    https://doi.org/10.1016/j.cub.2018.11.009

    • Google Scholar
50. 1. Nicolas JF
    2. Mathis L
    3. Bonnerot C
    4. Saurin W

    (1996) Evidence in the mouse for self-renewing stem cells in the formation of a segmented longitudinal structure, the myotome

    Development 122:2933–2946.

    https://doi.org/10.1242/dev.122.9.2933

    • PubMed
    • Google Scholar
51. 1. Oginuma M
    2. Moncuquet P
    3. Xiong F
    4. Karoly E
    5. Chal J
    6. Guevorkian K
    7. Pourquié O

    (2017) A gradient of glycolytic activity coordinates FGF and wnt signaling during elongation of the body Axis in amniote embryos

    Developmental Cell 40:342–353.

    https://doi.org/10.1016/j.devcel.2017.02.001

    • Google Scholar
52. 1. Olivera-Martinez I
    2. Harada H
    3. Halley PA
    4. Storey KG

    (2012) Loss of FGF-Dependent mesoderm identity and rise of endogenous retinoid signalling determine cessation of body Axis elongation

    PLOS Biology 10:e1001415.

    https://doi.org/10.1371/journal.pbio.1001415

    • Google Scholar
53. 1. Ory DS
    2. Neugeboren BA
    3. Mulligan RC

    (1996) A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes

    PNAS 93:11400–11406.

    https://doi.org/10.1073/pnas.93.21.11400

    • PubMed
    • Google Scholar
54. 1. Pasteels J

    (1937)

    Etudes sur la gastrulation des vertébrés méroblastiques III Oiseaux IV Conclusions générales

    ArchBiol 48:381–488.

    • Google Scholar
55. 1. Pijuan-Sala B
    2. Griffiths JA
    3. Guibentif C
    4. Hiscock TW
    5. Jawaid W
    6. Calero-Nieto FJ
    7. Mulas C
    8. Ibarra-Soria X
    9. Tyser RCV
    10. Ho DLL
    11. Reik W
    12. Srinivas S
    13. Simons BD
    14. Nichols J
    15. Marioni JC
    16. Göttgens B

    (2019) A single-cell molecular map of mouse gastrulation and early organogenesis

    Nature 566:490–495.

    https://doi.org/10.1038/s41586-019-0933-9

    • PubMed
    • Google Scholar
56. Software

    1. Pourquie Lab

    (2021) Guillot_2021, version swh:1:rev:3d80f422bc5a675c08546cbb10ab79211e430b1e

    Software Heritage.

    https://archive.softwareheritage.org/swh:1:rev:3d80f422bc5a675c08546cbb10ab79211e430b1e
57. 1. Psychoyos D
    2. Stern CD

    (1996) Fates and migratory routes of primitive streak cells in the chick embryo

    Development 122:1523–1534.

    https://doi.org/10.1242/dev.122.5.1523

    • PubMed
    • Google Scholar
58. Book

    1. Romanoff A

    (1960)

    The Avian Embryo: Structural and Functional Development

    The Macmillan Company.

    • Google Scholar
59. 1. Sbalzarini IF
    2. Koumoutsakos P

    (2005) Feature point tracking and trajectory analysis for video imaging in cell biology

    Journal of Structural Biology 151:182–195.

    https://doi.org/10.1016/j.jsb.2005.06.002

    • PubMed
    • Google Scholar
60. 1. Schiebinger G
    2. Shu J
    3. Tabaka M
    4. Cleary B
    5. Subramanian V
    6. Solomon A
    7. Gould J
    8. Liu S
    9. Lin S
    10. Berube P
    11. Lee L
    12. Chen J
    13. Brumbaugh J
    14. Rigollet P
    15. Hochedlinger K
    16. Jaenisch R
    17. Regev A
    18. Lander ES

    (2019) Optimal-Transport analysis of Single-Cell gene expression identifies developmental trajectories in reprogramming

    Cell 176:928–943.

    https://doi.org/10.1016/j.cell.2019.01.006

    • Google Scholar
61. 1. Schoenwolf GC
    2. Garcia-Martinez V
    3. Dias MS

    (1992) Mesoderm movement and fate during avian gastrulation and neurulation

    Developmental Dynamics 193:235–248.

    https://doi.org/10.1002/aja.1001930304

    • PubMed
    • Google Scholar
62. 1. scikit-image contributors
    2. van der Walt S
    3. Schönberger JL
    4. Nunez-Iglesias J
    5. Boulogne F
    6. Warner JD
    7. Yager N
    8. Gouillart E
    9. Yu T

    (2014) scikit-image: image processing in Python

    PeerJ 2:e453.

    https://doi.org/10.7717/peerj.453

    • PubMed
    • Google Scholar
63. 1. Selleck MA
    2. Stern CD

    (1991) Fate mapping and cell lineage analysis of Hensen's node in the chick embryo

    Development 112:615–626.

    https://doi.org/10.1242/dev.112.2.615

    • PubMed
    • Google Scholar
64. 1. Smith JL
    2. Gesteland KM
    3. Schoenwolf GC

    (1994) Prospective fate map of the mouse primitive streak at 7.5 days of gestation

    Developmental Dynamics 201:279–289.

    https://doi.org/10.1002/aja.1002010310

    • PubMed
    • Google Scholar
65. 1. Spratt NT

    (1947) Regression and shortening of the primitive streak in the explanted chick blastoderm

    Journal of Experimental Zoology 104:69–100.

    https://doi.org/10.1002/jez.1401040105

    • PubMed
    • Google Scholar
66. 1. Spratt NT
    2. Condon L

    (1947)

    Localization of prospective chordo and somite mesoderm during regression of the primitive streak in the chick blastoderm

    The Anatomical Record 99:653.

    • Google Scholar
67. 1. Stern CD

    (1979) A re-examination of mitotic activity in the early chick embryo

    Anatomy and Embryology 156:319–329.

    https://doi.org/10.1007/BF00299630

    • Google Scholar
68. Book

    1. Stern CD

    (2004)

    Gastrulation in the chick

    In: Stern C. D, editors. Gastrulation: From Cells to Embryos. Cold Spring Harbor Laboratory Press. pp. 219–232.

    • Google Scholar
69. 1. Subramanian V
    2. Meyer BI
    3. Gruss P

    (1995) Disruption of the murine homeobox gene Cdx1 affects axial skeletal identities by altering the mesodermal expression domains of hox genes

    Cell 83:641–653.

    https://doi.org/10.1016/0092-8674(95)90104-3

    • PubMed
    • Google Scholar
70. 1. Subramanian A
    2. Tamayo P
    3. Mootha VK
    4. Mukherjee S
    5. Ebert BL
    6. Gillette MA
    7. Paulovich A
    8. Pomeroy SL
    9. Golub TR
    10. Lander ES
    11. Mesirov JP

    (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles

    PNAS 102:15545–15550.

    https://doi.org/10.1073/pnas.0506580102

    • PubMed
    • Google Scholar
71. 1. Tam PP
    2. Beddington RS

    (1987) The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis

    Development 99:109–126.

    https://doi.org/10.1242/dev.99.1.109

    • PubMed
    • Google Scholar
72. 1. Turner DA
    2. Hayward PC
    3. Baillie-Johnson P
    4. Rué P
    5. Broome R
    6. Faunes F
    7. Martinez Arias A

    (2014) Wnt/β-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor in ensembles of mouse embryonic stem cells

    Development 141:4243–4253.

    https://doi.org/10.1242/dev.112979

    • PubMed
    • Google Scholar
73. 1. Tzouanacou E
    2. Wegener A
    3. Wymeersch FJ
    4. Wilson V
    5. Nicolas JF

    (2009) Redefining the progression of lineage segregations during mammalian embryogenesis by clonal analysis

    Developmental Cell 17:365–376.

    https://doi.org/10.1016/j.devcel.2009.08.002

    • PubMed
    • Google Scholar
74. 1. van den Brink SC
    2. Alemany A
    3. van Batenburg V
    4. Moris N
    5. Blotenburg M
    6. Vivié J
    7. Baillie-Johnson P
    8. Nichols J
    9. Sonnen KF
    10. Martinez Arias A
    11. van Oudenaarden A

    (2020) Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids

    Nature 582:405–409.

    https://doi.org/10.1038/s41586-020-2024-3

    • PubMed
    • Google Scholar
75. Software

    1. Veres A

    (2021) Project YAML file, version swh:1:rev:2ad4669b72ea6ba794f962ed95b778560bdfde4a

    Software Heritage.

    https://archive.softwareheritage.org/swh:1:rev:2ad4669b72ea6ba794f962ed95b778560bdfde4a
76. 1. Wetzel R

    (1929) Untersuchungen am hühnchen die entwicklung des keims während der ersten beiden bruttage

    Wilhelm Roux' Archiv Für Entwicklungsmechanik Der Organismen 119:188–321.

    https://doi.org/10.1007/BF02111186

    • Google Scholar
77. 1. Wilson V
    2. Olivera-Martinez I
    3. Storey KG

    (2009) Stem cells, signals and vertebrate body Axis extension

    Development 136:1591–1604.

    https://doi.org/10.1242/dev.021246

    • PubMed
    • Google Scholar
78. 1. Wilson V
    2. Beddington RS

    (1996) Cell fate and morphogenetic movement in the late mouse primitive streak

    Mechanisms of Development 55:79–89.

    https://doi.org/10.1016/0925-4773(95)00493-9

    • PubMed
    • Google Scholar
79. Preprint

    1. Wood TR
    2. Kyrsting A
    3. Stegmaier J
    4. Kucinski I
    5. Kaminski CF
    6. Mikut R
    7. Voiculescu O

    (2019) Neuromesodermal progenitors separate the axial stem zones while producing few single- and dual-fated descendants

    bioRxiv.

    https://doi.org/10.1101/622571

    • Google Scholar
80. 1. Wymeersch FJ
    2. Huang Y
    3. Blin G
    4. Cambray N
    5. Wilkie R
    6. Wong FC
    7. Wilson V

    (2016) Position-dependent plasticity of distinct progenitor types in the primitive streak

    eLife 5:e10042.

    https://doi.org/10.7554/eLife.10042

    • PubMed
    • Google Scholar
81. 1. Wymeersch FJ
    2. Skylaki S
    3. Huang Y
    4. Watson JA
    5. Economou C
    6. Marek-Johnston C
    7. Tomlinson SR
    8. Wilson V

    (2019) Transcriptionally dynamic progenitor populations organised around a stable niche drive axial patterning

    Development 146:dev168161.

    https://doi.org/10.1242/dev.168161

    • PubMed
    • Google Scholar
82. 1. Yang X
    2. Dormann D
    3. Münsterberg AE
    4. Weijer CJ

    (2002) Cell movement patterns during gastrulation in the chick are controlled by positive and negative chemotaxis mediated by FGF4 and FGF8

    Developmental Cell 3:425–437.

    https://doi.org/10.1016/S1534-5807(02)00256-3

    • PubMed
    • Google Scholar
83. 1. Zamir EA
    2. Czirók A
    3. Cui C
    4. Little CD
    5. Rongish BJ

    (2006) Mesodermal cell displacements during avian gastrulation are due to both individual cell-autonomous and convective tissue movements

    PNAS 103:19806–19811.

    https://doi.org/10.1073/pnas.0606100103

    • PubMed
    • Google Scholar

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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