In classical descriptions of vertebrate development, the segregation of the three embryonic germ layers completes by the end of gastrulation. Body formation then proceeds in a head to tail fashion by progressive deposition of lineage-committed progenitors during regression of the primitive streak (PS) and tail bud (TB). The identification by retrospective clonal analysis of a population of neuromesodermal progenitors (NMPs) contributing to both musculoskeletal precursors  and spinal cord during axis formation challenged these notions. However, classical fate mapping studies of the PS region in amniotes have so far failed to provide direct evidence for such bipotential cells at the single-cell level. Here, using lineage tracing and single-cell RNA sequencing in the chicken embryo, we identify a resident cell population of the anterior PS epiblast, which contributes to neural and mesodermal lineages in trunk and tail. These cells initially behave as monopotent progenitors as classically described and only acquire a bipotential fate later, in more posterior regions. We show that NMPs exhibit a conserved transcriptomic signature during axis elongation but lose their epithelial characteristicsin the TB. Posterior to anterior gradients of convergence speed and ingression along the PS lead to asymmetric exhaustion of PS mesodermal precursor territories. Through limited ingression and increased proliferation, NMPs are maintained and amplified as a cell population which constitute the main progenitors in the TB. Together, our studies provide a novel understanding of the PS and TB contribution through the NMPs to the formation of the body of amniote embryos. 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 . Fate maHere, 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 . We show- HH . Heat maps showing the top differentially expressed genes used to identify the clusters are shown in To characterize the molecular identity of these NMPs, we performed scRNAseq of cells dissociated from a micro-dissected region encompassing the anterior PS in stage 5HH and in 6-somite embryos as well as the tail bud of 35-somite embryos . We usedSALL4 or FRZB. Another cluster contained cells co-expressing markers of paraxial mesoderm  and lateral plate , suggesting that they correspond to ingressed mesoderm contributing to the anterior-most somites as recently shown in mouse embryos  and the notochord , was also identified. The last cluster (yellow) expressing genes such as TBXT, CDH2, GJA1, and WNT8A is found in between neural and mesodermal clusters, suggesting that it could represent the NMP population  and LP  identities. We also identified clusters with neural , notochord (NOTO), and endoderm (SOX17) identities. A large cluster of cells connected to the neural and PSM clusters represents putative NMPs , blood (HBZ), notochord (CHRD), neural , and ectoderm . Cells with a PSM  and somitic identity  also formed separate clusters. As for the earlier stages, there was a putative NMP cluster lying between the neural and PSM clusters   with ourare NMPs . We nextare NMPs . The NMPare NMPs . Using pare NMPs . This isare NMPs , that isare NMPs , which care NMPs . A thirdare NMPs . This suare NMPs .Altogether, our data identify NMPs as a cell population conserved in chicken and mouse that exhibit a distinct identity from mesodermal and neural cells. These cells first become specified at the beginning of PS regression and remain as a population of bipotential precursors contributing both to the neural and mesodermal territories during formation of the posterior embryonic axis.We next used a trajectory inference technique based on optimal-transport (Waddington-OT [WOT]) to analyze quantitatively how NMP cells transition between the NMP, PSM, and neural states . This meWe also studied the transcription factor gene sets underlying the neural and mesodermal trajectories . The WOTEPCAM or CDH1 were significantly upregulated in the early cluster  and downregulated in the late ones  < 0.05) and mouse  datasets . We foun embryos  as well  embryos . We iden embryos . This li embryos . OverallLM) and anteroposterior (VAP) directions over time as a function of their position along the PS  revealed similar cell dynamics and trajectories for epiblast cells of the anterior PS region maintaining low lateral to medial and high anteroposterior speed , and U0 is the initial population, that is, 50 cells. To obtain a population of 550 cells, the model predicts n = 9 or 10 cell cycles , suggesting a cell cycle time around 4 hr. We manually tracked individual dividing cells and their daughter cells in the anterior PS region over 10 hr to determine the time between two divisions. We identified symmetric cell divisions where the two daughter cells remain in the epiblast after cell division, consistent with self-renewal of this population. The cell cycle time for such symmetric divisions is around 4.5 hr in agreement with the number of divisions predicted by the model , the PMP and lateral plate progenitors (LPPs), and LPP and extraembryonic mesoderm progenitor (EMP) , Video 7NMPs 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 . The ideThe 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 . CompareT, 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 , 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  lineage . Most of lineage , late NMongation .As reported for mouse embryos , we obseWe 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 . These gHere, we show that NMPs can contribute to a single type of derivative  for several segments followed by another type in the next segments, as reported in mouse embryos . This arAll animal experiments were performed in accordance to all relevant guidelines and regulations. The office for protection from Research Risks (OPRR) has interpreted ‘live vertebrate animal’ to apply to avians  only after hatching. All of the studies proposed in this project only concern early developmental stages (prior to 5 days of incubation); therefore, no IACUC-approved protocol is required. Fertilized chicken eggs were obtained from commercial sources. Fertilized eggs from transgenic chickens expressing cytoplasmic GFP ubiquitously  were obtFor whole-mount immunohistochemistry, stage 3–20HH chicken embryos were fixed in 4% paraformaldehyde  diluted in PBS 1X at 4°C overnight. The embryos were rinsed and permeabilized in PBS-0.1% Titon, three times 30 min, and incubated in blocking solution  prior to incubating with primary and secondary antibodies. Embryos were incubated in antibodies against T/Brachyury , SOX2 , E-cadherin (1/250 Abcam: ab76055), N-cadherin , fibronectin (1/50 DSHB: MT4S), laminin , phospho-histone 3 , MSGN1 (1/1000) , dilutedFor histological analysis, stage 5HH chicken embryos were fixed in 4% PFA. Embryos were then embedded in OCT compound and frozen in liquid nitrogen. Frozen sections (12 μm) were cut using a Leica Cryostat and incubated overnight at 4°C with the primary antibody diluted in blocking solution (same as above), and after washing in PBS-0.1% Triton, they were incubated overnight at 4°C with the secondary antibody conjugated with Alexa Fluor (Molecular probes) diluted in blocking solution. Sections were then washed in PBS-0.1% Triton before mounting in fluoromount-G (Thermo Fisher) and stored at 4°C overnight prior to imaging.Images were captured using a laser scanning confocal microscope with a 10× or 20× objective . To image the whole embryo, we used the tiling and stitching function of the microscope (5 by 2 matrix) and z-sectioning (5 μm). Later stages (from 17HH) were imaged in clearing solution using the scale A2 clearing protocol from pCAGG-H2B-Venus and pCAGG-H2B-RFP have been described in The nuclear red solution was prepared from the NucRed Live 647 ReadyProbes Reagent (Thermo Fisher) and diluted in PBS 1X as indicated by the manufacturer's experimental procedures. Sparse nuclear labeling of the dorsal epiblast was performed in ovo by injecting the nuclear red solution between the epiblast and the vitelline membrane at the PS level. The solution was left for 15 min to perform the long-term epiblast tracking and 30 min for monitoring ingression. The embryos were then dissected, rinsed in PBS, and mounted on paper filter for EC culture to perform live imaging from the dorsal side for the long-term epiblast tracking and from the ventral side for measuring cell ingression.Time-lapse imaging, ingression, and cell-cycle length measurements stage 5HH chicken embryos were cultured ventral side up on a microscope stage using a custom-built time-lapse station . We usedsinus rhomboidalis region during PS regression. The PSM progenitor domain is found posterior to the NMP territory in the anterior third of the PS approximately extending 670 μm posterior to the node. We observed that electroporation of the region located 750 μm posterior to the node is labeling LP cells  antibody  as described above. In the penultimate wash, Hoechst (1/1000) was added to label cell nuclei. The number of proliferative cells was measured in 250 by 250 μm squares at three different locations along the PS . Proliferative cells were identified by automated segmentation of pH3-positive cells using ImageJ via thresholding the images and using the analyze particle module. Hoechst-positive cells were also segmented using the same procedure and their number was used to normalize to the cell density in each square. The percentage of dividing cells was obtained by dividing the number of pH3 cells by the number of Hoechst-positive cells in each square and multiplying by 100. A minimum of five embryos was analyzed for each stage. We also performed similar measurements on live H2B:Cherry quail embryo at stage 5HH in the anterior (around the node) and posterior (LPP region). We used a high threshold, which identifies cells in mitosis , while all the other cells in the tissue are identified with a lower threshold.Chicken embryos from stage 4 to 20HH were harvested and fixed in 4% PFA at 4°C overnight. Embryos were immunostained in whole mount using T/Brachyury, SOX2, and Alexa Fluor 647 Phalloidin (1/500 Thermo Fisher: A22287) combinations and imaged by confocal laser microscopy . SOX2/T-double positive cells were segmented and counted using ImageJ software. To identify the double-positive cells, cells in the T and SOX2 channel were manually thresholded (around 1.3% of the histogram). We used the image calculator tool from ImageJ to generate a new image by performing the image operation T AND SOX2 and identify the double-positive cells. Cell were then counted automatically by the ‘Analyze Particle’ module on ImageJ (particle size < 500 pixel square). Embryos were grouped by stage, and data is shown in Escherichia coli. Following a 1 hr outgrowth in 12 ml SOC at 37°C with shaking, dilutions were plated on LB agarose plates with carbenicillin to determine complexity, and the remaining transformation was added to 500 ml of LB with carbenicillin and grown for 16 hr at 37°C. The liquid culture was split into four and plasmid was purified with the Qiagen Maxiprep Kit. A plate with 1/1 × 104 of the transformation grew approximately 1 × 103 colonies, indicating a total library complexity of 1 × 107.The plasmid library used to generate the barcoded retrovirus was based on the pQCXIX backbone. First, this backbone was digested with NotI and XhoI. A Gibson assembly reaction was performed using three PCR-generated fragments to insert the EGFP, IRES, and Cre recombinase, creating pQCGIC . These P8 infectious particles per milliliter.Replication-incompetent retrovirus was produced and concentrated as previously described . Briefly 1 μl of the virus preparation was diluted in 10 μl of PBS 1X and 0.1% Fast Green to form the virus mix. The virus mix was placed in ovo on top of the NMP region for 1 hr and washed with PBS 1X. The eggs where then sealed with tape and reincubated for 36 hr.TCTCTGTCTCGACAAGCCCAG, 1F-GATCATGCAAGCTGGTGGCTG. PCR primer 2: 2R-CTTACCTGCCCTTTATTGAGCCTG, 2F-CTGCTGGAAGATGGCGATGG.To retrieve the different barcodes, we manually harvested individual fluorescent cells from transverse sections of embryos fixed 36 hr post infection. GFP-positive cells were handpicked under a microscope (20× objective) using single-use needles (BD Precision). Each cell/needle was put in an individual PCR tube containing the buffer for the PCR1, and its localization was recorded. The tissue of origin of the cells was visually identified on the sections. The barcodes were retrieved using two consecutive nested PCR amplifications and sequenced using Sanger sequencing. PCR primer 1: 1R-Lineage tracing was performed by co-electroporating the NMP region at stage 5HH in ovo as described above with the following constructs: a self-excising Cre recombinase (se-Cre), the Nucbow construct, and the TolII transposase as described in To perform lineage analysis, we fixed the electroporated embryos at stage 17HH and 20HH, and imaged them in clearing solution ScaleA2 . The imaCells were manually segmented in the YFP and Cherry channel using ImageJ. Positions were assigned to the mesodermal and neural tube layers. Color retrieval was performed by measuring the intensity in the three channels, Cerulean, YFP, and Cherry so that the total of all the intensities was normalized to 1 and expressed in percentages similarly to Single-cell dissociation protocols were optimized to achieve >90% viability and minimize doublets before sample collection. To generate the samples, four embryos were harvested for each stage and cells were dissociated and captured on an inDrops  setup onhttps://github.com/indrops/indrops, (copy archived at swh:1:rev:2ad4669b72ea6ba794f962ed95b778560bdfde4a); Gallus gallus transcriptome built from the GRCg6a (GCA_000002315.5) genome assembly. Bowtie version 1.1.1 was used with parameter –e 200.Single-cell transcriptomes were barcoded using the inDrops pipeline using V3 sequencing adapters as previously reported . FollowiSingle-cell counts matrices were processed and analyzed using ScanPy (1.4.3) and custom Python scripts (Code Availability). Low-complexity cell barcodes, which can arise from droplets that lack a cell but contain background RNA, were filtered in two ways. First, inDrops data were initially filtered to only include transcript counts originating from abundantly sampled cell barcodes. This determination was performed by inspecting a weighted histogram of unique molecular identifier–gene pair counts for each cell barcode and manually thresholding to include the largest mode of the distribution. Second, low-complexity transcriptomes were filtered out by excluding cell barcodes associated with <400 expressed genes. Transcript unique molecular identifier counts for each biological sample were then reported as a transcript × cell table, adjusted by a total-count normalization, log-normalized, batch corrected (using bbknn module), and scaled to unit variance and zero mean. Note that for the WOT analysis the module ‘combat’ was also used for batch correction. Unless otherwise noted, each dataset was subset to the 1000 most highly variable genes, as determined by a bin-normalized overdispersion metric.k = 10 except 35-somite k = 15) nearest-neighbor graph using Euclidean distance and 50 PCA dimensions and visualized using UMAP representation. Clustering was performed using Leiden community detection algorithms.Processed single-cell data were projected into a 50-dimensional PCA subspace  are reported in t-test was then performed between these two sets of 100 expression measurements (FDR < 0.01). Scaled expression values for significant genes were then smoothened over a sliding window of 100 cells, ranked by peak expression and plotted as a heat map, shown in Pseudo-spatiotemporal orderings were constructed by randomly selecting a root cell from the NMP cluster and calculating the diffusion pseudotime distance of all remaining cells relative to the root. Trajectories were assembled for paths through specified clusters, with cells ordered by diffusion pseudotime values, as described in Cell state prediction using the chicken cell states was predicted using the LDA classifier trained on the mouse E9.5 embryos  after suWaddington-OT 1.0.7 conceptual framework  was usedhttp://www.gsea-msigdb.org), and only genes with p-values<0.05 were kept. Results are plotted in List of genes and log fold-change values for each clusters obtained by differential expression (Wilcoxon rank-sum) were used as input for computing overlap with the Hallmark Epithelial Mesenchymal Transition gene set collection using the GSEA tool  .Thank you for submitting your article "Dynamics of primitive streak regression controls the fate of neuro-mesodermal progenitor cells in the chicken embryo" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential Revisions1. Many of the concerns can be addressed by text changes and minor changes to the existing data presentation as detailed in the full reviews.2. The main problems are with the bioinformatic analysis, where both reviewers highlight issues where the analysis is too superficial and needs further work.3. Additional imaging quantification is required to support their findings.Reviewer #1:The paper entitled "Dynamics of primitive streak regression controls the fate of neuro-mesodermal progenitor cells in the chicken embryo" from Guillot and colleagues aims to characterize in the context of developing chick embryos the cellular dynamics of a population of stem cells, recently identified, called Neuro-Mesodermal Progenitors (NMPs). The authors provide several lines of evidence supporting their claims that overall look interesting, however, additional imaging quantification and more comprehensive analysis of their transcriptomic datasets are required to support their findings.Specifically, the authors generated an important scRNA-seq dataset of micro-dissect regions of the chick embryos across developmental time points from stage 5 to 35-somites. Despite the dataset represents a crucial part of the paper supporting the authors' findings of this NMP population, the analysis is not deep enough and does not report several controls and markers. In figure 2 the authors should show heatmap of the marker genes used for classification. Louvain markers genes that define each cluster should be showed in a supplementary not as an unreadable table, instead, a dotplot or heatmap would be better. It is unclear whether Louvain clustering has been used to label the data, did the authors find that each Louvain cluster correspond to a defined cell identity? The pseudotime analysis is incomplete and superficial. Diffusion pseudotime strongly depends on the root cell selection. RNA velocity analysis and/or another trajectory analysis are required to better show lineage trajectory. Figure 4 L-K, the pesudotime time is not progressively ordered and it would be advisable to perform an alternative analysis to visualize lineage trajectory. Furthermore, gene expression trends should be identified and further discussed over those trajectories. The supporting information about the classification analysis using the mouse dataset is incomplete.SOX2-T double staining. Panels A-B-C represent the first evidence of SOX2-T co-expression in stage 5 chick embryos. The merged image is not provided together with the single panels A-B and only appear in panel G of the same figure, this is confusing. Panel B has two scale bars. The authors tried to show SOX2-T co-expression by thresholding and masking SOX2-T signals in panel C and Suppl. Figure 1. The authors should clearly explain how this thresholding is obtained. Is this manually set? Is this set based on global or local signal intensity distribution? Otsu thresholding? In order to avoid artifacts due to thresholding, the authors should also display a scatter plot showing nuclear intensity values for SOX2-T. FACS analysis and quantitative assessments of the percentage of cells would further strengthen the authors' claim.– In figure 1 the authors show SOX2-T expression in quail embryos– The authors should validate – The authors should better discuss the mechanical forces that are implicated in epithelium to mesenchyme, and in the posterior gradient of proliferation that counteracts ingression in the anterior PS epiblast. Is this affecting/related to WNT or hyppo pathway?– Can the authors speculate about the conservation of this phenomena in the human context? What is the evidence that this conserved in human?– The authors speculate about in-vitro generated NMPs, did they try transplantation of in-vitro generate cells? Can they compare the transcriptomic signature of the human in-vitro generated NMP with the chick counterpart?– The tables in Suppl. Figure 4 should be a supplementary table.– In Suppl.Fig5D there is not scale bar to assess classification efficacy and material and methods do not describe how this analysis has been performed.– The authors should substantially revise scRNA-seq data.Reviewer #2 :Sox2 and T. However, these studies had not shown that individual cells in the Sox2/T positive region were dual-fated. The authors use three approaches: live imaging, and two prospective clonal labelling studies of the Sox2/T positive region to show, for the first time, that indeed this region harbours dual-fated cells. This is an important missing piece of the current volume of work on NMPs. Whilst the rationale for the experiments are clear, it might be better to present this work more in the context of previous studies. It seems a little harsh to dismiss population fate mapping studies as 'failing to identify' NMPs rather than complementing the clonal analysis by identifying regions potentially harbouring NMPs. In addition, there are interesting parallels with the clonal analysis carried out in mouse. For example, not all NM clones contribute to the tail bud; the observation in live imaging studies that NMPs first contribute to the neural tube and later to mesoderm is also similar to the observations from mouse retrospective clonal analysis; this could perhaps be discussed more fully.In this manuscript, the authors aimed firstly to prospectively identify cells with dual neural and mesodermal fate. Previous studies in mouse had determined that such dual-fated cells existed, and had narrowed down their location to subregions of the axial progenitor zone co-expressing Next, the authors carry out single cell RNA-seq analyses of three stages of chick axial elongation, and compare these with existing datasets in mouse, adding data from a later stage of mouse embryos. This aspect of the study has again confirmed and extended previous studies, showing both similarities and differences between chick and mouse NMPs. Trajectory analysis suggests progression of NMPs towards neural and mesodermal lineages in both chick and mouse. This is a valuable resource for the community. However once again it would be good to present this aspect of the work in the context of previous studies showing maturation of NMPs over time and an increased mesenchymal transcriptomic signature of NMPs at late compared to early timepoints.Finally, the authors carry out dynamic cell tracking and show that NMPs ingress later, and proliferate faster than more posteriorly-placed cells in the primitive streak, leading to exhaustion of the posterior populations and long-term retention of the NMP population. This part of the study is well-executed and contains important new information that advances our understanding of both NMPs and axial progenitors as a whole.Suggestions for strengthening the scienceSox2 and Msgn1 staining. Perhaps the thresholding strategy could be described in the supplementary data to support statements about Sox2 positive time of emergence and the location of Msgn1 positive/Sox2 negative axial levels. It would be good to cite the source of the fate mapping information locating NMP/PMP/LPP/EMP in Figure 1D- is it this study or another? The section at level 5 in Figure 1E is named LPP in 1E but corresponds to a region marked 'EMP' in Fig1D. Together these two factors make it hard for the reader to judge where the posterior limit of Sox2/T positive cells is, relative to Msgn1 and to regions of differential fate. Also, the text (p5) says there are 'low levels [of TBXT] in the node' but the expected high levels in the node/ emerging notochord are visible in Fig1E sections 1-2.1. The results presented in Figure 1 are a little difficult to interpret as there is some background fluorescence in 2. In Figure 2, the clustering shows two datasets, (which are presumably batches?) of cells for each stage in chick (st5 1 etc). However, the colour coding does not allow the reader to distinguish these. Do the batches occupy different positions in the UMAP plot? How was batch correction done?Sox2 from this dataset is not, in my opinion, especially troublesome as it is expressed at low levels. However, it means that the chick single cell transcriptomes cannot be independently verified as NMPs and distinguished from midline streak cells. Perhaps the mouse data can help here. Instead of using the chick as a starting point to define the NMP phenotype, if the authors can work back from cells dissected from the Sox2/T positive regions , as well as the NMP annotations in the mouse atlas data used here , how well do the chick cells correspond to the mouse ones? If a narrower definition of NMPs is used in chick, does this remove any of the variation seen between mouse and chick?3. The cells annotated as NMPs in chick show elevated expression of Hes5 (6-somite stage) and Dll1 (combined dataset) relative to the cells annotated as PSM or neural . Also, some of the NMP signature markers are expressed in a higher percentage of the NMP population in mouse compared to chick. This suggests that the cluster annotated as NMPs in chick also includes the midline primitive streak. The absence of 4. The mouse NMP trajectory analysis  shows two arrows. What do they represent? if just pseudotime, it looks more like a radiation in all directions from the centre. Interestingly this would suggest NMPs also progress (upwards on the Y axis) as NMPs in pseudotime. Is this effect seen in the chick NMPs?4. The annotations of chick versus mouse early and late clusters are confusing, In chick, there are two early clusters (coloured the same) annotated nmp-1 and nmp-2, but these do not correspond to the two earlier stages. What do they correspond to? Some discussion of the heterogeneity would be appropriate- or possibly a reanalysis of the NMP annotation in point 3 above may highlight that one of these clusters is less NMP-like. In the mouse, there is one early subcluster and two late ones. These are also annotated nmp-1 and nmp-2. A different nomenclature should be used to highlight that they are not the same as the chick ones. Unlike the chick clusters, these seem to correspond to different stages of development.Suggestions for improving the manuscript1. The statements that no previous studies have prospectively identified neuromesodermal-fated cells  should be modified. Forlani et al., Development 2003 10.1242/dev.00573 and Wood et al. BioRXiv https://doi.org/10.1101/622571 show dual-fated cells in mouse and chick. I think that it would be fairer in the context of previous studies to cite the population or oligoclonal studies of Iimura and Pourquié, (2006) 10.1073/pnas.0610997104, Cambray and Wilson, (2007) 10.1242/dev.02877 and Brown and Storey, (2000) 10.1016/s0960-9822(00)00601-1 as showing potential locations for cells of dual neuromesodermal fate.2. The words 'bipotent/monopotent' used throughout the manuscript is not appropriate for the studies here, which are demonstrating fate and not potency. I would suggest 'dual fate'.3. NMPs are presented here as 'stem cells' when the transcriptome analysis shows they mature with time. It would be good to define exactly in what sense the term is being used- it is valid since the manuscript describes an enduring progenitor producing neural and mesodermal tissue but should be used with care.4. p5. 'We identified 7 clones containing cells expressing the same barcodes' – could this text be modified to clarify, e.g. 'expressing unique (or clone-specific) barcodes'?5. p7 para 2 'the NMP lineage' should be changed to something not implying lineage, e.g. 'cluster'6. Figure S7 is entitled 'NMP early and late clusters are not due to different Hox genes expression'- since the authors show differential Hox gene expression, this statement should be moderated. The text describing this observation also suggests that the genes that are differentially expressed early and late in mouse and chick are different. However, many of the genes upregulated in the later chick stage are also upregulated in mouse, eg Wnt5a, Greb1, Fgf8, Figf18, Cyp26a1 . It would be good to comment on this.7. Can the authors consider whether 'persistence' of cell tracks is the optimal term? It could suggest persistence of movement rather than just their continued observation over time. Is 'longevity' a possible alternative? Essential Revisions1. Many of the concerns can be addressed by text changes and minor changes to the existing data presentation as detailed in the full reviews.2. The main problems are with the bioinformatic analysis, where both reviewers highlight issues where the analysis is too superficial and needs further work.3. Additional imaging quantification is required to support their findings.We thank the editor for the suggestions of improvement and have now thoroughly revised the text including new analyses as suggested. These are described below.Reviewer #1:The paper entitled "Dynamics of primitive streak regression controls the fate of neuro-mesodermal progenitor cells in the chicken embryo" from Guillot and colleagues aims to characterize in the context of developing chick embryos the cellular dynamics of a population of stem cells, recently identified, called Neuro-Mesodermal Progenitors (NMPs). The authors provide several lines of evidence supporting their claims that overall look interesting, however, additional imaging quantification and more comprehensive analysis of their transcriptomic datasets are required to support their findings.Specifically, the authors generated an important scRNA-seq dataset of micro-dissect regions of the chick embryos across developmental time points from stage 5 to 35-somites. Despite the dataset represents a crucial part of the paper supporting the authors' findings of this NMP population, the analysis is not deep enough and does not report several controls and markers.We agree with the reviewer and in the revised version, we now present a thoroughly revised analysis of the single cell RNAseq dataset as described below.In figure 2 the authors should show heatmap of the marker genes used for classification. Louvain markers genes that define each cluster should be showed in a supplementary not as an unreadable table, instead, a dotplot or heatmap would be better. It is unclear whether Louvain clustering has been used to label the data, did the authors find that each Louvain cluster correspond to a defined cell identity?We have now redone the entire analysis using Leiden clustering algorithm which performed better than Louvain at identifying tissue-specific clusters. As requested, we now present heatmaps showing the top 10 genes differentially expressed for each cluster in Figure 3 – Supplementary Figure 1 (for chicken) and in Figure 3 – Supplementary Figure 3 (for mouse). We have moved the gene tables originally presented in supplementary figures to a Supplementary File 2 which also includes dot plots showing the expression of genes used for identification of the clusters. We also discuss more in detail how the different clusters were identified at each developmental age in the revised text.The pseudotime analysis is incomplete and superficial. Diffusion pseudotime strongly depends on the root cell selection. RNA velocity analysis and/or another trajectory analysis are required to better show lineage trajectory. Figure 4 L-K, the pesudotime time is not progressively ordered and it would be advisable to perform an alternative analysis to visualize lineage trajectory. Furthermore, gene expression trends should be identified and further discussed over those trajectories. The supporting information about the classification analysis using the mouse dataset is incomplete.We now present a novel analysis of the developmental trajectories using the Waddington-OT pipeline developed by the Broad Institute . This analysis, which is based on optimal transport, shows that the cells we identify as NMPs are indeed the most likely ancestors of both the Neural and PSM cells in the datasets. By analyzing the transport maps, this allows us to show the self-renewal of the NMP cell population and its maturation during axis elongation. We also use this tool to identify transcription factors associated with these different fates. We have added novel figures  to the revised text to describe these new results.In addition, we have performed a Gene Set Enrichment Analysis of the early and late NMP clusters which shows an enrichment in genes associated to epithelium to mesenchyme transition in the late clusters in both species . We have also completed the supporting information on the mouse dataset.– In figure 1 the authors show SOX2-T double staining. Panels A-B-C represent the first evidence of SOX2-T co-expression in stage 5 chick embryos. The merged image is not provided together with the single panels A-B and only appear in panel G of the same figure, this is confusing.SOX2 and T expression in Figure 1C instead of showing only the double-positive cells as in the previous version. We also show a blow-up of the sections of the NMP and PMP regions in Figure E’-F’ and E’’-F’’ to better illustrate the difference between these two regions which both contribute to the paraxial mesoderm as shown by the MSGN1 antibody labeling in the ingressed mesoderm.In the revised version, we now show the merge of Panel B has two scale bars.This was corrected.The authors tried to show SOX2-T co-expression by thresholding and masking SOX2-T signals in panel C and Suppl. Figure 1. The authors should clearly explain how this thresholding is obtained. Is this manually set? Is this set based on global or local signal intensity distribution? Otsu thresholding?SOX2/T cells shown in Figure 1C and Figure 1 – Supplementary Figure 1G was done manually with image J. We have now replaced the subtracted image originally shown in Figure 1C by an image showing the merge between T and SOX2 staining which clearly identifies the double positive cells without thresholding. We have nevertheless left the subtracted image with the manual thresholding performed with image J in Figure 1 – Supplementary Figure 1G in the revised version. We also explained the thresholding in the legend of this figure and material and method section.Thresholding of the In order to avoid artifacts due to thresholding, the authors should also display a scatter plot showing nuclear intensity values for SOX2-T.Since all the thresholding was performed manually, we could verify that virtually all cells in the images  were indeed expressing both signals.FACS analysis and quantitative assessments of the percentage of cells would further strengthen the authors' claim.SOX2/T double positive cells is expected to be a few hundreds at the stages discussed. Thus, getting quantitative data on such a low number might be difficult. Moreover, since both markers are nuclear, this would require sorting after fixation and labeling of the cells which is much trickier than sorting for live cells labeled with a membrane marker. In addition, SOX2 and T are expressed in overlapping opposite antero-posterior gradients in epiblast cells with significant co-expression in the epiblast adjacent to the anterior PS. The positioning of the gates for FACS analysis would be somehow arbitrary as neither marker is truly specific for the NMP population.While this is an interesting suggestion, this experiment would be very challenging as the number of – The authors should validate SOX2-T expression in quail embryosSOX2 cells in the quail embryo and their distribution is similar to that we observe in the chicken embryo .Recent work from the Benazeraf laboratory has examined the distribution of T-– The authors should better discuss the mechanical forces that are implicated in epithelium to mesenchyme, and in the posterior gradient of proliferation that counteracts ingression in the anterior PS epiblast. Is this affecting/related to WNT or hyppo pathway?While this is indeed an interesting question, our paper is mostly focused on the lineage and fate of NMP cells in the chicken embryo. We have no experimental data addressing the mechanical properties of ingressing cells and we are not aware of such data available in the literature for the stages considered in our study. Thus, we feel this discussion is beyond the scope of the paper.– Can the authors speculate about the conservation of this phenomena in the human context? What is the evidence that this conserved in human?SOX2/T cells showing a similar identity to NMPs can be obtained from human pluripotent cells (ES/iPS) in vitro after Wnt activation. We have reanalyzed our human dataset from SOX2/T cells differentiated in vitro from iPS cells  and observed that the list of human SOX2/T cells signature genes was much more similar to that of chicken and mouse when looking at the genes differentially expressed with the undifferentiated iPS only rather than with all the clusters as performed in the original publication. This revised list of human NMP signature genes is shown in Supplementary File 4 together with the genes conserved with chicken and mouse NMPs. We find a significant degree of conservation among the signature NMP genes in the three species and discuss these comparisons in a new paragraph in the revised text.Several groups including ours have reported that – The authors speculate about in-vitro generated NMPs, did they try transplantation of in-vitro generate cells? Can they compare the transcriptomic signature of the human in-vitro generated NMP with the chick counterpart?We have not tried such transplantations of in vitro derived NMP cells and we believe this is beyond the scope of this work. Such work is however described in the following paper: “The Chick Caudolateral Epiblast Acts as a Permissive Niche for Generating Neuromesodermal Progenitor Behaviours Peter Baillie-Johnson, Octavian Voiculescu, Penny Hayward, and Benjamin Steventon”.As suggested and discussed above, we have now compared the transcriptomic signature of cells of the chicken and mouse NMP clusters with that of the human NMP-like cells. This is now shown in Supplementary File 4.– The tables in Suppl. Figure 4 should be a supplementary table.We have moved the gene tables shown in supplementary figures to the Supplementary File 2 in the revised MS.– In Suppl. Fig5D there is not scale bar to assess classification efficacy and material and methods do not describe how this analysis has been performed.We apologize for this mistake, and we have now added it to the figure. The details of the analysis is presented in the material and method section: “Machine-learning classification of cell states”. The classification of the different cellular states was performed using an LDA classifier as described in Diaz-Cuadros et al., in vitro characterization of the human segmentation clock. Nature 580, 113-118, doi:10.1038/s41586-019-1885-9 (2020), which we now cite in the material and methods section.– The authors should substantially revise scRNA-seq data.As described above, we now provide a much deeper analysis of our single cell RNAseq data in the revised MS. We improved our analysis by changing our clustering method to Leiden that allows better identification of cell identities in the data. All the figures were remade with Leiden clustering.We also provide an extended discussion of the datasets and the genes that were used to identify the various clusters in the revised text, as well as heat maps and dotplots of the genes used to identify the clusters in Figure 3 – Supplementary Figure 1 (chicken) and 6 (mouse) and in Supplementary File 2. In Figure 3F-M, we now show Diffmap plots to better illustrate the differentiation trajectories of the NMP cells.We also performed a new analysis of the data using the Waddington-OT pipeline . Unlike pseudotime, this analysis does not impose to select a root for the trajectories and considers the actual developmental time. This analysis shows that the NMP cells are the most probable common ancestors to the PSM and Neural cells in both chicken and mouse datasets. It also identifies a developmental trajectory in the NMP lineage which corresponds to their maturation during axis elongation. Using transport matrices, we could also quantify the contribution of NMPs to PSM, NT and NMPs and found equivalent contributions to these three lineages. This analysis therefore strongly supports the self-renewing properties of these cells. The WOT analysis also identified a number of interesting transcription factors associated with the NMP, PSM and neural fates. Expression patterns of a selection of transcription factors enriched in the NMPs is presented in Figure 4 – Supplementary Figure 1 and the extensive lists are provided in Supplementary File 3. We now also provide the expression levels of some genes involved in the NMP differentiation toward the mesodermal and neural cellular states in the new Figure 4 and in Figure 4 – Supplementary Figure 1. The results of the WOT analysis are described in a new paragraph in the revised text and shown as a new Figure 4.We have also performed a Gene set enrichment analysis (GSEA) to compare the NMP early and late clusters identified using Leiden algorithm. This revealed an enrichment in genes associated to epithelium to mesenchyme transition in late vs early NMP clusters in both species. This analysis is now shown in Figure 5.These new analyses led to new figures 3, 4 and 5 and Supplementary Figures 4, 5, 6, 7, 8 and 9 as well as new Supplementary Files 2, 3, 4 and 5.Reviewer #2:In this manuscript, the authors aimed firstly to prospectively identify cells with dual neural and mesodermal fate. Previous studies in mouse had determined that such dual-fated cells existed, and had narrowed down their location to subregions of the axial progenitor zone co-expressing Sox2 and T. However, these studies had not shown that individual cells in the Sox2/T positive region were dual-fated. The authors use three approaches: live imaging, and two prospective clonal labelling studies of the Sox2/T positive region to show, for the first time, that indeed this region harbours dual-fated cells. This is an important missing piece of the current volume of work on NMPs. Whilst the rationale for the experiments are clear, it might be better to present this work more in the context of previous studies. It seems a little harsh to dismiss population fate mapping studies as 'failing to identify' NMPs rather than complementing the clonal analysis by identifying regions potentially harbouring NMPs. In addition, there are interesting parallels with the clonal analysis carried out in mouse. For example, not all NM clones contribute to the tail bud; the observation in live imaging studies that NMPs first contribute to the neural tube and later to mesoderm is also similar to the observations from mouse retrospective clonal analysis; this could perhaps be discussed more fully.We agree and have revised the text along the lines suggested by the reviewer.Next, the authors carry out single cell RNA-seq analyses of three stages of chick axial elongation, and compare these with existing datasets in mouse, adding data from a later stage of mouse embryos. This aspect of the study has again confirmed and extended previous studies, showing both similarities and differences between chick and mouse NMPs. Trajectory analysis suggests progression of NMPs towards neural and mesodermal lineages in both chick and mouse. This is a valuable resource for the community. However once again it would be good to present this aspect of the work in the context of previous studies showing maturation of NMPs over time and an increased mesenchymal transcriptomic signature of NMPs at late compared to early timepoints.We agree and have now extended our analysis and compared the identified transcriptomic signature of the NMP clusters of the chicken and mouse datasets to a set of published NMP transcriptomic signatures from mouse  and human (in vitro derived) NMPs . We also compare the signature of the early and late clusters identified in our analysis to early and late NMP signatures described by Gouti et al., (2017) and E8.0 NMPs from Dias et al., 2020. We also separately compare the signature of the early and late NMPs to the genes up and down regulated in small dissected regions of the NMP from the Node streak border  and the Chordo neural hinge  in mouse from Wymeersch et al., 2019. We added a paragraph describing these comparisons in the revised text and present these data in Supplementary Files 4 and 5.Finally, the authors carry out dynamic cell tracking and show that NMPs ingress later, and proliferate faster than more posteriorly-placed cells in the primitive streak, leading to exhaustion of the posterior populations and long-term retention of the NMP population. This part of the study is well-executed and contains important new information that advances our understanding of both NMPs and axial progenitors as a whole.Suggestions for strengthening the science1. The results presented in Figure 1 are a little difficult to interpret as there is some background fluorescence in Sox2 and Msgn1 staining. Perhaps the thresholding strategy could be described in the supplementary data to support statements about Sox2 positive time of emergence and the location of Msgn1 positive/Sox2 negative axial levels.+/sox2+ and T+/SOX2- and their corresponding MSGN1 regions as Figure 1 E’-F’ and E’’-F’’. Without thresholding, the images shown clearly illustrates the difference between the two paraxial mesoderm-producing regions which both exhibit MSGN1 positive nuclei in the mesodermal layer while SOX2 is only seen in the epiblast of the anterior  and not in the posterior  regions. This shows that the paraxial mesoderm progenitor domain can be subdivided in 2 progenitor domains both producing paraxial mesodermal cells (MSGN1+).We now provide higher magnification images of the TIt would be good to cite the source of the fate mapping information locating NMP/PMP/LPP/EMP in Figure 1D- is it this study or another?The fate mapping used was from Psychoyos and Stern,  who generated a very detailed fate map of the PS of the chicken embryo encompassing the stages described here. We have added this information to the revised text.The section at level 5 in Figure 1E is named LPP in 1E but corresponds to a region marked 'EMP' in Fig1D. Together these two factors make it hard for the reader to judge where the posterior limit of Sox2/T positive cells is, relative to Msgn1 and to regions of differential fate.We apologize for the oversight. This study is focused on the fate of paraxial mesoderm and we made no attempt to distinguish between the lateral plate and extraembryonic mesoderm territories of the posterior primitive streak. We have now relabeled the posterior primitive streak territory as LPP/EMP on Figure 1E. This corresponds to the territory devoid of MSGN1 expression.Also, as a minor point, the text (p5) says there are 'low levels [of TBXT] in the node' but the expected high levels in the node/ emerging notochord are visible in Fig1E sections 1-2.It is indeed right that the node shows high TBXT expression but the level of TBXT in the epiblast around the Node is low as seen in our quantifications and transverse sections in Fig1E sections 1-2. We meant 'low levels [of TBXT] in epiblast around the node’ and have changed the text accordingly.2. In Figure 2, the clustering shows two datasets, (which are presumably batches?) of cells for each stage in chick (st5 1 etc). However, the colour coding does not allow the reader to distinguish these. Do the batches occupy different positions in the UMAP plot? How was batch correction done?The 2 datasets are technical replicates. We agree that the color coding is not properly showing the heterogeneity of the data and we now provide a Figure 3F and in Figure 3 – Supplementary Figure 1 with a new set of colors. As seen in the new figures, the 2 datasets do not occupy different parts of the UMAP. In most cases, we used the module Bbknn for batch correction, but for the WOT analysis, we used the Combat module. We have added this information to the M&Ms section.3. The cells annotated as NMPs in chick show elevated expression of Hes5 (6-somite stage) and Dll1 (combined dataset) relative to the cells annotated as PSM or neural . Also, some of the NMP signature markers are expressed in a higher percentage of the NMP population in mouse compared to chick. This suggests that the cluster annotated as NMPs in chick also includes the midline primitive streak. The absence of Sox2 from this dataset is not, in my opinion, especially troublesome as it is expressed at low levels. However, it means that the chick single cell transcriptomes cannot be independently verified as NMPs and distinguished from midline streak cells.SOX2 double positive cells are also present in the anterior primitive streak (in the groove) at stage 5HH . This suggests that the distribution of NMPs might be slightly different in chicken compared to mouse embryos. This is supported by the observation that we cannot identify any PS specific cluster, although we cannot rule out that this due to the limited resolution of the dataset.This is correct, as in our immunostaining we see that T-Perhaps the mouse data can help here. Instead of using the chick as a starting point to define the NMP phenotype, if the authors can work back from cells dissected from the Sox2/T positive regions , as well as the NMP annotations in the mouse atlas data used here , how well do the chick cells correspond to the mouse ones? If a narrower definition of NMPs is used in chick, does this remove any of the variation seen between mouse and chick?Sox2 or Nkx1.2 in our chicken dataset. This is also why it is difficult to identify the chicken NMPs based on the mouse NMP signature genes. However, the most stringent definition of NMPs is not based on expression of marker genes  but is based on their ability to give rise to both neural tube and PSM. This is the criterion we use to define NMPs in this analysis. Our new WOT analysis strongly argues that in both chicken and mouse, cells of the identified NMP clusters are ancestors to the PSM and Neural Tube.We appreciate the suggestion which in principle could be a good way to present the data. The problem we are facing here, which we did not sufficiently highlight in the previous version of the paper, is that chicken cells were sequenced with Indrops, which provides limited depth, whereas mouse cells were sequenced with SMARTseq  or 10x  which allows for more sequencing depth. This is probably the main reason why we do not identify eLife 2020; Guibentif et al., Dev Cell, 2021, Diaz-Cuadros et al., Nature 2020. This analysis shows significant conservation of genes between these different lists. While only 38 genes are conserved in 4 or 5 of the 6 datasets compared. The most striking characteristic of this list is the strong enrichment in genes of the Wnt pathway, consistent with the requirement for Wnt signaling for NMP differentiation in vivo and in vitro. We also find an enrichment of genes involved in glycolysis in line with recent papers describing higher glycolytic activity required for Wnt signaling in the tail bud . This new analysis is detailed in a new paragraph in the revised text and described in Supplementary File 4 and 5.We now present a comparison between the NMP signature we identified in chicken and mouse embryos and lists of genes identified as NMP signatures in mouse embryos and human NMP-like cells differentiated in vitro in the following papers: Gouti et al., Dev Cell 2017; Wymeersch et al., 2019; Dias et al., 4. The mouse NMP trajectory analysis  shows two arrows. What do they represent? if just pseudotime, it looks more like a radiation in all directions from the centre. Interestingly this would suggest NMPs also progress (upwards on the Y axis) as NMPs in pseudotime. Is this effect seen in the chick NMPs?As suggested, we now present diffusion maps plots in Figure 3 that better show the pseudotime trajectories toward the neural and mesodermal fates in chicken and mouse cells. Indeed, NMP also exhibit changes in pseudotime that parallel developmental time in both species although this is more subtle in chick than in mouse probably because of sequencing depth. We also performed a new analysis of the mouse and chicken datasets using the Waddington OT pipeline . This analysis shows that the NMPs are the most probable ancestors of the PSM and neural cells. It also identifies a trajectory in the NMP cluster (confirming the trajectories observed in pseudotime) correlating with the age of the samples, suggesting maturation of these cells. We now describe these new analyses in detail and have added a new figure 4 to present this data.4. The annotations of chick versus mouse early and late clusters are confusing, In chick, there are two early clusters (coloured the same) annotated nmp-1 and nmp-2, but these do not correspond to the two earlier stages. What do they correspond to? Some discussion of the heterogeneity would be appropriate- or possibly a reanalysis of the NMP annotation in point 3 above may highlight that one of these clusters is less NMP-like. In the mouse, there is one early subcluster and two late ones. These are also annotated nmp-1 and nmp-2. A different nomenclature should be used to highlight that they are not the same as the chick ones. Unlike the chick clusters, these seem to correspond to different stages of development.The analysis of the early and late NMP clusters has been revisited using Leiden clustering . We now only find one early and late clusters in both species. We perform a GSEA analysis using the gene set “Epithelium to mesenchyme transition” which shows enrichment in the late clusters in both species. We also compared these early and late clusters to other similar datasets published in the literature . This part has been modified in the revised text and a new figure 5, Supplementary File 4 and Figure 5 – supplementary Figure 1 have been generated with the new data.Suggestions for improving the manuscript1. The statements that no previous studies have prospectively identified neuromesodermal-fated cells  should be modified. Forlani et al., Development 2003 10.1242/dev.00573 and Wood et al., BioRXiv https://doi.org/10.1101/622571 show dual-fated cells in mouse and chick.We are well aware of the Forlani paper, where they perform single cell injections of HRP in epiblast cells. However, this study was intended for a different purpose (comparing the anterior boundaries of the clones with the timing of Hox gene activation). It is true that they show a few bipotent clones in their Figure 7 but this work was done before the identification of the NMPs in 2009 and in no place in the text do they refer to bi-potential precursors. We nevertheless introduced and discussed this reference in the text.With respect to the Wood preprint, I don’t think they demonstrate the existence of dual-fated cells. Their data arguing for the dual fate is shown in Figure 5a-c which shows cell fate tracked for only 15 hours (ie up to the 5-somite stage approximately). Their designation of the neuro-mesodermal region is quite arbitrary. The main argument in support of a dual fate of these cells is that their descendants span neural and mesodermal territories after 15 hours when backtracked from time lapse movies . However, at these stages, these two territories are not clearly separated into neural tube and paraxial mesoderm and thus the designation of the final fate is not supported by any anatomical or molecular landmark. This study is extremely preliminary and lacking critical details to evaluate the findings. I don’t think it would be fair to consider that this work  first identified dual-fated cells in the chicken embryo. We nevertheless discuss this reference in the revised version of the manuscript.I think that it would be fairer in the context of previous studies to cite the population or oligoclonal studies of Iimura and Pourquié, (2006) 10.1073/pnas.0610997104, Cambray and Wilson, (2007) 10.1242/dev.02877 and Brown and Storey, (2000) 10.1016/s0960-9822(00)00601-1 as showing potential locations for cells of dual neuromesodermal fate.We were already discussing these studies in the previous version of this paper but have rewritten this part. We were not trying to diminish the merit of these oligoclonal studies, which are very elegant and compelling. Nevertheless, we believe it is fair to say that due to their design, these studies cannot prospectively identify bipotential cells, only territories.2. The words 'bipotent/monopotent' used throughout the manuscript is not appropriate for the studies here, which are demonstrating fate and not potency. I would suggest 'dual fate'.We respectfully disagree about using dual fated to describe NMP cells as dual fated means that the two fates can be observed. Thus, we believe using this term would only be appropriate for the description of a subset of the cells identified by the lineage analysis (and we do use it in some places) as in these experiments we can visualize the fate of the cells. However, in many cases, only one of the fates is realized even though the precursors (the NMPs) appear to be bipotent at the population level. For the scRNAseq analysis, the cells of the NMP cluster cannot be called dual-fated as the analysis does not reveal the actual fate of the cells. We believe the cells identified as NMP using this approach should be called bipotent.3. NMPs are presented here as 'stem cells' when the transcriptome analysis shows they mature with time. It would be good to define exactly in what sense the term is being used- it is valid since the manuscript describes an enduring progenitor producing neural and mesodermal tissue but should be used with care.We define NMPs as stem cells because they can give rise to differentiating progeny and self-renew. We now add further evidence for a self-renewing behavior with the WOT analysis transport maps which show that NMP contribute significantly to the NMP population itself over time. This is now shown in the revised Figure 4C-D. The fact that NMPs can also mature is a situation frequently observed for other stem cell types . We have however removed stem cell from the title of the revised paper.4. p5. 'We identified 7 clones containing cells expressing the same barcodes' – could this text be modified to clarify, e.g. 'expressing unique (or clone-specific) barcodes'?This has been modified accordingly.5. p7 para 2 'the NMP lineage' should be changed to something not implying lineage, e.g. 'cluster'We agree and have removed NMP lineage from the revised text and replaced it by the appropriate clusters.6. Figure S7 is entitled 'NMP early and late clusters are not due to different Hox genes expression'- since the authors show differential Hox gene expression, this statement should be moderated. The text describing this observation also suggests that the genes that are differentially expressed early and late in mouse and chick are different. However, many of the genes upregulated in the later chick stage are also upregulated in mouse, eg Wnt5a, Greb1, Fgf8, Figf18, Cyp26a1 . It would be good to comment on this.We agree and have changed the title of what is now Figure 5 – supplementary Figure 1 with “Characterization of the early and late NMP clusters”. We have also performed a series of comparisons between the differentially expressed genes from the early and late NMP clusters identified in our chicken and mouse datasets and with the datasets from Gouti et al., 2017, Wymeersch et al., 2019, Dias et al., 2020 and Dias-Cuadros et al., 2020. We do find similarities between the gene signatures identified in our mouse and chicken early and late NMPs clusters and those identified in these studies and the results are now shown in the Supplementary File 4.7. Can the authors consider whether 'persistence' of cell tracks is the optimal term? It could suggest persistence of movement rather than just their continued observation over time. Is 'longevity' a possible alternative?We have made the corresponding change.


The detection of organophosphates, a wide class of pesticides, in water-solution has a huge impact in environmental monitoring. Acoustic transducers are used to design passive wireless sensors for the direct detection of pesticides in water-solution by using tailored polymers as sensitive layers. We demonstrate by combining analytical chemistry tools that organophosphate molecules strongly alter polymer layers widely used in acoustic sensors in the presence of water. This chemical degradation can limit the use of these polymers in detection of organophosphates in water-solution. Monitoring the increasing number of pollutants in the water table is an ongoing concern for water treatment systems and water resource managers . PesticiIn this work, we monitor the acoustic response of SAW devices functionalized with PECH, PIB and PBMA thin films exposed to chlorpyrifos water-solution in environmental conditions. Then, we assess the instability of the measurements by considering analytical characterization of the thin films prior and after exposure to chlorpyrifos using Fourier-Transform InfraRed Spectroscopy (FT-IR) and Atomic Force Microscopy (AFM). We demonstrate the irreversible degradation of the thin polymer films exposed to chlorpyrifos in water-solution. This instability of sensitive layers seems to be a strong limitation to the popularization of this kind of acoustic sensors in environmental challenge and water resource management in practical deployment scenarios.Surface Acoustic Wave (SAW) reflective delay lines were manufactured by depositing 200 nm thick aluminum interdigitated electrodes acting as transducer IDT in  and mirrThe four areas are selected with two areas patterned with the same sensing polymer to cancel the impact of the probing electronic unit to sensor range ; a third area patterned with a polymer insensitive to the compound to be detected for temperature compensation; and a fourth area used here to assess reproducibility, but which could be used to pattern a second sensing polymer to improve selectivity by inducing a different response to the same compound.11 transfer function on a 20 MHz bandwidth—a 20% relative bandwidth selected as greater than the sensor transfer function width determined by the electromechanical coupling coefficient—in order to post-process the spectral domain response to reach the time domain response by inverse Fourier-transform. Echoes are identified as local maxima of the magnitude, and the phase is recorded at the associated time delay found, by design of the reflective SAW delay line, between 0.8 and 2 microseconds (Thin polymer films are spin-coated to reach thicknesses between 500 and 1200 nm aimed at confining acoustic energy in a Love mode guided in the polymer (see below). The IDT is wired to an Agilent E5071B network analyzer for probing the sensor acoustic response once every 30 s by recording the Soseconds .−1 and a nominal tip radius of 2 nm.All commercially available starting materials and solvents were purchased from SigmaAldrich, Fisher and used without further purification. All polymer deposits were performed in a Class-100 clean-room on the Karl Suss RC-8 CT-62 spin-coater. All surface activations were performed on PVA/Tepla Giga BATCH 360 M. The thickness of polymer layers was performed with a stylus profilometer Dektak xt (C9). All layers of polymers were characterized by Fourier-transform Infrared-Spectroscopy (FT-IR) by using a spectrum two FT-IR spectrometer from Perkin Elmer. AFM measurements were performed with a Bruker Icon AFM connected to a Nanoscope V controller. The AFM was employed in peak-force tapping mode using Scanasyst-Air-HPI Bruker probes of nominal stiffness of 0.4 N m2, 300 W, 10 min). A volume of 250 µL of polymer solution was then deposited on clean surface by using a plastic pipette. PBMA solution was spread at a speed of 2500 rpm with an acceleration of 800 rpm/sec for 30 s. The polymer layers covering contact pads were removed with a cotton-bud dipped in acetone. Then, an annealing at 100 °C for 2 min give a clear, translucent, and homogenous polymer film with a thickness of 980 nm.PBMA (90 mg) was dissolved in toluene (1000 mg) and stirred for 30 min at room temperature. The chip sensor surface  was activated by plasma . A volume of 250 µL of polymer solution was then deposited on clean surface by using a plastic pipette. PBMA solution was spread at a speed of 2500 rpm with an acceleration of 800 rpm/sec for 30 s. Then, an annealing at 100 °C for 2 min give a clear, translucent, and homogenous polymer film with a thickness of 200 nm. We used a thinner silicon substrate and a thinner layer of polymer for FT-IR analysis than for AFM or SAW devices in order to decrease the light absorption.PBMA (42 mg) was dissolved in toluene (1000 mg) and stirred for 30 min at room temperature. Silicon wafers  were cut into 1.5 cm × 2 cm pieces. Then, they were cleaned in piranha solution for 2 min and rinsed with clean water. Silicon surface was activated by plasma . The thickness of resulting polymer layers was 500 nm.Dektak Thickness measurements were collected by using this set of parameters, with range of 6.5 µm, hills and valleys as profile, 0.5 mg for stylus force, and a resolution close to 0.1 µm/pt.Thin scratches were applied to the corner and to the center of the PBMA-coated pieces.An amount of 40 mg of chlorpyrifos was dissolved into 40 mL of acetonitrile. The resulting solution was then diluted in 960 mL of pure water.The sensor was designed for wireless sensing of an analyte in liquid phase: such conditions were simulated in a controlled laboratory environment by flowing solutions in an open-well liquid cell with fluids continuously injected and sucked by a peristaltic pump (Peripro-4HS from WPI) running at 5 mL/min. In order to obtain a homogenous aqueous phase, we used a water solution containing 4% of acetonitrile which strongly promotes the dissolution of chlorpyrifos. This solution is named ‘blank’ in the following parts. All measurements were simultaneously recorded on three parallel sensors for reproducibility assessment, and each cycle of blank-analyte-blank was repeated to assess the stability of the polymer layer. Phase differences between second and first echo on one hand, and between third and first echo on the other hand, aim at cancelling correlated noise such as fluctuations in the level of liquid in the flow cell. Each cycle was completed when an asymptotic behavior was reached, with kinetics lasting multiple hours as shown in We observe that the phase variations, decreasing being usually associated with mass loading when absorbing analyte molecules in the polymer matrix and increasing being associated with layer stiffening or desorption, are not consistent with injection steps of the pesticide or blank solutions. Indeed, the phase starts dropping upon exposure to the pesticide prior to rising again after 10 h exposure a. On theMagnitude of the insertion losses of echo 2 is shown in The same evolution for phase and amplitude is observed for PECH and PIB polymers see .The lack of reproducibility on the quantitative evolution of the phase from one cycle to another leads to the investigation on the stability of the various polymers upon exposure to chlorpyrifos using analytical chemistry techniques.In order to understand the variation and the non-reproducible phase variation of SAW devices, we investigated the morphology of the sensitive polymer layers by AFM. The three polymers were deposited by spin-coating onto a silicon substrate as a model of atomically flat surface. We performed three different AFM mappings on (i) the raw polymer layer after deposition by spin-coating onto silicon substrate; (ii) this polymer layer after an immersion into a water-acetonitrile (96:4) of chloropyrifos (40 mg/L) during 12 h; and finally (iii) the latter polymer layer after an immersion in water-acetonitrile (96:4) during 12 h. AFM was used to acquire images in the PeakForce Tapping mode of Bruker Icon. All images were recorded under ambient  conditions with 512 points × 512 lines.The layers obtained by spin-coating a solution of PBMA, PECH or PIB onto a silicon substrate are flat with a few nanometers peak to peak roughness and homogenous, as shown by AFM images depicted in In the case of PBMA layer, the roughness is below 2 nm for a length of 30 microns  and after rinsing with water-acetonitrile solution (yellow line), the three bands are still observed, and some new bands centered at 1576, 1542, 1436, 1412, 1023 and 846 cm−1, respectively, are clearly identified (The spectrum of PBMA layer (blue line in entified b.−1 and at 1022 and 967 cm−1 for PIB and PECH respectively, after immersion to chlorpyrifos water solution and rinsing with a water-acetonitrile solution (In the case of PIB and PECH layers, similar behaviors are observed. New extra-bands are observed in FT-IR spectra, centered at 1164 and 1025 cmsolution  in ESI.The phase and amplitude variations of the acoustic sensors upon exposure to a water-acetonitrile solution of chlorpyrifos are not reproductible See . AFM expIn this case, no hole is observed. The layer before and after exposure to the buffered solution exhibit a similar roughness, on the order of a few nanometers as opposed to the holes penetrating through the polymer layer in the absence of PBS .+ in water (+, due to the hydrolysis of chlorpyrifos, is enhanced at this interface, which leads to the enhancement of acidic degradation of the PBMA layer as observed in the AFM images. In addition, pH can be very different at solid-liquid interface from the solution as it has been reported in the case of polymer–water interface [Consequently, we suggest that the degradation of polymer layers could be attributed to local variation of pH due to the presence of chlorpyrifos. Indeed, in aqueous media, chlorpyrifos is hydrolyzed which leads to the formation of 3,4,6-trichloro-2-pyridinol and O,O-diethyl hydrogen thiophosphate. These two molecules are weak acids, which can lead to the formation of Hin water . As the nterface . Figure In the context of developing surface acoustic wave transducers acting as passive, wireless cooperative targets for direct detection water table pollutant sensors, we have identified degradation processes when exposing PBMA, PIB and PECH to chlorpyrifos pesticide dissolved in water with concentration determined by the addition of acetonitrile. The degradation is explained following atomic force microscopy and Fourier-transform InfraRed characterization, and is the cause of unstable sensor response over timespans of several hours of exposure of the sensing area to the pesticide. Our work highlights the important role of deep investigations to determine the stability of polymers in their environmental conditions before they are used as a sensitive layer: the results presented here can be generalized to organophosphate direct detection by thin polymer coated sensors whose detection mechanism is more complex than surface absorption of molecule leading to mass loading as found in the literature. The development of new inert polymers for direct detection water table pollutant sensors is currently investigated.


However, so far CTCs have been barely used for studying their chemosensitivity due to the lack of technology to obtain wholly intact and viable CTCs.The prognosis for small cell lung cancer (SCLC) patients receiving later-line treatment is very poor and there is still no standard treatments after the second-line setting. Analyzing the susceptibility of chemotherapeutic drugs with circulating tumor cells (CTCs) cultured in vitro obtained from ClearCell FX® System was tested and the therapy response was evaluated.Based on a retrospective study of the therapeutic response of 99 patients with unresectable SCLC, the CTC count in 14 SCLC patients was detected before and after chemotherapy to evaluate its role as a potential marker of response. Furthermore, the drug susceptibility of CTCs cultured All of the 99 patients received the first-line chemotherapy and the objective response rate (ORR) was 74.7%. A total of 36 patients received the second-line therapy and the average duration was 2.6 months, and only 11 cases out of them received the third-line therapy but no one responded. The change of CTC counts was identified to be correlated with therapy response. However all the five SCLC patients who were administered with the drugs according to the drug susceptibility test of CTCs for two cycles underwent progression of disease.in vitro may not benefit the improvement of therapeutic regimen of SCLC patients.The results showed that the responses of chemotherapy are very poor in later lines and the drug susceptibility test using CTCs primary cultured  According to the statistics, there would be 2.2 million new lung cancer cases in 2020, 15-20% of which were SCLC . The reaTopotecan is the only Grade I recommended chemotherapeutic drug in second-line therapy for recurrent SCLC patients. There is no standard therapy for those patients eventually progressed on second-line chemotherapy. Palliative care/best supportive care (BSC) or other systemic chemotherapy can be the alternative option based on patient’s performance status. Some new drugs, such as immunotherapy agents (Nivolumab and Pembrolizumab) and the multi-targeting tyrosine kinase inhibitor Anlotinib, have been approved by Chinese Food and Drug Administration (CFDA) and are already available to physicians in China, but their survival benefits are very limited and most patients cannot afford these drugs which are not included in the coverage of medical insurance reimbursement.CTCs are tumor cells that shed from primary and metastatic sites and circulate in the peripheral blood and can be detected by many advanced technologies. Hou et al. reported that CTCs were present in 85% of SCLC patients with the abundance of 1,589 ± 5,565 cells/7.5 mL blood .in vitro may provide some useful information for optimizing the therapeutic regimen and prolong survival time of SCLC patients. So far CTC has been barely studied for its chemosensitivity due to the lack of technology for obtaining wholly intact and viable CTCs. In this study, the ClearCell FX System was used to get intact and viable CTCs and then the CTCs were primary cultured in vitro and employed for investigations of their drug sensitivity profiles.As there is no standard care and the prognosis and outcomes of SCLC patients are very poor in later lines of therapy, the precision treatment holds great promise for cancer patients. With the potential to address challenges associated with drug susceptibility and the variability among the patients, analyzing the susceptibility of chemotherapeutic drugs with primary cultured CTCs We retrospectively reviewed the medical records of patients with unresectable SCLC treated at Blinded for peer review between January 2014 and December 2019. All patients displayed measurable disease by the Response Evaluation Criteria in Solid Tumors  and an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of less than or equal to 2. The information were collected including age, gender, laboratory results, diagnoses, stage, anatomic sites of involvement, sites of metastases, treatment plan, specific therapy, other medications such as supportive care agents, and performance status. The therapy response was evaluated after the two cycles of chemotherapy and every two subsequent cycles after the first evaluation until the disease progressed, and the results were recorded as complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) according to RECIST 1.1 criteria.These studies were prospective single-institution clinical studies conducted at the Blinded for peer review. Patients aged ≥18 years with histologically or cytologically confirmed unresectable SCLC were enrolled. The therapy response was evaluated after the two cycles of chemotherapy and every two subsequent months after the first evaluation until the disease progressed, and was recorded as CR, PR,SD, and PD according to RECIST 1.1 criteria. The ORR was defined as the sum of CR plus PR. The disease control rate (DCR) was defined as the sum of CR plus PR plus SD. The treatment response was evaluated by CT scan two months after the initiation of chemotherapy and then every two months. Our study was approved by The Ethics Committee of Blinded for peer review . All patients provided written informed consent prior to enrollment in the study.In this study, patients were administrated with the first-line carboplatin plus etoposide chemotherapy and some of them were screened for CTC counts test using folate receptor targeted PCR by GENO Biology in China, within one week before and after two cycles of chemotherapy.® circulating lung cancer cell kit on ABI StepOne™ system (Life technologies). Two and half microliters of the prepared samples were added into a 25ml PCR reaction system following the manufacturer’s instruction manual. The PCR reaction conditions were as follows: denaturation at 95°C for 2 min, annealing at 40°C for 30 s, extension at 72°C for 30 s, then cooling at 8°C for 5 min; 40 cycles of denaturation at 95°C for 10 s, annealing at 35°C for 30 s, and extension at 72°Cfor 10 s. A serial of standards containing oligonucleotides  were used for CTC quantification. All patients’ samples were tested in duplicates with 6 standards and 3 quality controls. Following the manufacturer’s protocol, the mean intraassay variance (the maximum difference between duplicates) should be < 0.5 threshold cycle for the standards and quality controls, and < 1 threshold cycle for tested samples  was firstly used to lyse the anticoagulant whole blood samples for 15min on ice. Then 200ml anti-CD45 coated magnetic beads were used to treat the cells for 30 min to deplete leucocytes. After that, CTCs were incubated with 10ml labeling buffer (folate-linked oligonucleotide) for 40min at room temperature. The cells were then washed 3 times with 1ml wash buffer at 500g. Finally, the cells were treated with 120ml stripping buffer to remove the ligand-oligonucleotide conjugates. The supernatant were collected by centrifugation and neutralized by 24ml neutralization buffer for further PCR analysis. Real time quantitative polymerase chain reaction was performed using the CytoploRare samples .In this study, the patients resistant to at least the first-line chemotherapy (etoposide plus cisplatin or carboplatin) were enrolled. CTCs collection and the drug susceptibility tests were done by Polaris Biology in China. About 6 chemotherapeutic agents per patient were tested based on their previous medication histories if the numbers of CTCs collected were enough. Then these patients were treated with the highly sensitive chemotherapeutic drugs according to the test results.® tubes  and processed within 24h, respectively. Next, red blood cell (RBC) lysis buffer was used to treat the prepared whole blood and the nucleated cell fraction was recovered. The nucleated cells were suspended in the custom ClearCell resuspension buffer, and loaded on the ClearCell FX® System. A new CTChip was loaded on the machine and the automated protocol was run. Within an hour, the enriched CTCs were collected in a 15ml centrifuge tube in suspension format and seamlessly integrated into downstream assays. After the enrichment, the system ran a cleaning cycle to avoid cross-contamination between samples. Because of the fast metabolic rate, cancer cells can rapidly absorb glucose, which has become the basic detection principle of PET-CT. So, PET-CT was used to identify and confirmed the CTCs  patients received Irinotecan followed by platinum-based chemotherapy. For all the second-line patients, the median PFS was 2.6 months, with only 2.8% surviving for more than a year. Within the third-line setting, 11 (11.1%) patients received chemotherapy and the others received BSC. Among actively treated patients, taxane-based single-agent regimens were the most commonly used regimens followed by Irinotecan. Only 1 patient was treated with immunotherapy across all lines of therapies. The average duration of chemotherapy was 2.4 months in the third-line setting and no one responded to chemotherapy Table 2.p<0.001.To evaluate the change of CTC counts as a potential marker for monitoring therapy response, fourteen patients with SCLC in first-line setting were screened for CTCs counts test within one week before and after two cycles of chemotherapy, and then the patients were grouped into responsive, stable, and progressive disease based on therapeutic efficacy according to RECIST criteria 1.1. The results showed that the patients with responsive diseases had reduced CTC counts with a median decrease of 6.96 CTCs, and the patients with stable diseases had a median decrease of 3.34 CTCs, whereas the patients with progressive diseases got a median increase of 13.05 CTCs after chemotherapy Table 3,in vitro, ten never used chemotherapeutic agents were tested for their sensitivities in five patients with SCLC after their second-line chemotherapy. The test reports are shown in in vitro may not have distinct effect on clinical efficacy of patients with SCLC.To evaluate the clinical value of this drug susceptibility platform using CTCs primary cultured in vitro provides an opportunity to study patterns of drug susceptibility that is unique to an individual tumor although this technique is not well developed in patients with SCLC now.In our study there are 99 patients with unresectable SCLC received platinum-based doublet chemotherapy in the first-line setting and the ORR was 74.7%. Due to the inaccessibility of topotecan and irinotecan was not used in first-line therapy, the patients with SCLC in our hospital received the treatment of irinotecan in second-line setting. Among them, 36 patients received Irinotecan treatment in the second-line setting, but the median PFS was only 2.6 months, and only 2.8% surviving for more than one year. In the third-line setting, 11 patients received chemotherapy, but no one responded. However, in the patients receiving BSC, more than half were classified as treatment-eligible to receive active treatments. So this study hopes to find some drugs sensitive to patients. Finally, we have shown that the culture of CTCs CTCs circulating in the peripheral blood, with their role as a “tumor liquid biopsy”, provide convenient access to all disease sites. It is conceivable that detecting and analyzing CTCs will provide insightful information in assessing the disease status and monitoring the response of anticancer drugs. However, identifying CTCs in patient blood samples is technically challenging due to the extremely low abundance of CTCs among a large number of hematologic cells. The size of circulating tumor cells (~15-20 um) is significantly different from that of red blood cells (~8 um) and white blood cells (~8-15 um). In addition, CTC has higher nuclear-cytoplasmic ratio and irregular cell morphology. These characteristics make CTCs different from other cells in fluid characteristics and flow rate . Researcin vitro. The ClearCell® FX System, a label-free microfluidics technology that utilizes Dean Flow Fractionation principle in a spiral microfluidics system to separate the larger CTCs from smaller blood cells, driven by the CTChip® FR biochip, is one of the world’s first automated cell retrieval systems that can enrich wholly intact and viable CTCs from blood in a relatively short time. The automated system performs a single-step CTCs isolation and retrieval and collects the enriched CTCs in suspension format, achieving extremely high recovery rates. CTCs with high activity and no damage can be naturally separated from other cells in the sample based on the difference of flow velocity , in which the blood sample is depleted of leukocytes by immunomagnetically targeting both the common leukocyte antigen CD45 and the granulocyte marker CD15 . The othvelocity . After tin vitro may have little effect on the clinical efficacy of SCLC, it still needs to be validated in larger studies. The other possible reason for this result may be that SCLC is a highly aggressive malignant disease and resistant to all the old drugs in our tests or that the tumor microenvironment has some influences on the drug efficacy. Krohn et al. found that many tumors acquired drug resistance and their neuroendocrine differentiation was lost during epithelial-mesenchymal transition (EMT) of SCLC cells, indicating that drug resistance is one characteristic of EMT . The patients/participants provided their written informed consent to participate in this study.JL, YJ, ZC and CS: substantial contributions to the conception or design, acquisition, analysis, or interpretation of data, critical revision for important intellectual content, final approval of the version to be published. DZ and LM: coordinate and to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work. All authors contributed to the article and approved the submitted version.Science and Technology Commission of Shanghai Municipality ; National Natural Science Foundation of China (81207106); Shanghai Pulmonary Hospital (fkgg1807).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.