1. Author Response:

    Reviewer #1:

    This manuscript shows cell to cell variability in the relative levels of Sox2 and Brachyury (Bra) expression by individual cells within the region of the epiblast containing axial progenitors (the progenitor zone, PZ). Accordingly, some cells express high Bra and low Sox2 levels, others high Sox2 and low Bra and a third group expressing equivalent levels of both transcription factors. They then show that by experimentally promoting high Sox2 expression cells enter neural tube (NT) fates, whereas high Bra brings cells in the progenitor zone to enter the presomitic mesoderm (PSM). The authors then complement these experiments with evaluation of cell movements within the PZ, NT and PSM to show that cells in the NT are much less motile than those in the PZ and PSM. These data led the authors to propose a fundamental role for Sox2/Bra heterogeneity to maintain a pool of resident progenitors and that it is the high cell motility promoted by high Bra levels what pushes cells to join the PSM, whereas high Sox2 levels inhibit cell movement forcing cells to take NT fates. To validate their hypothesis, the authors generated a mathematical model to show that those expression and motility characteristics can indeed lead to axial extension generating NT and PSM derivatives in the proper positions, while keeping a PZ at the posterior end.

    Some specific comments on the manuscript are specified below.

    1. Although the description of cells within the PZ containing different Sox2 and Bra expression ratios is more explicit and quantitative in the present manuscript, this has already been previously reported by different methods including immunofluorescence (e.g., Wymeersch et al, 2016). Similarly, that breaking the Sox2/Bra balance towards high Sox2 or Bra is an essential step to bring the progenitors towards NT or PSM fates has also been previously shown in different ways. These observations are, therefore, not totally new. The novel contribution of this paper is the authors' interpretation that "heterogeneity among a population of progenitor cells is fundamental to maintain a pool of resident progenitors". In this work, however, this conclusion is only supported by their mathematical simulation, as the experiments described in this manuscript are not aimed at homogenizing Sox2/Bra expression levels in the progenitor cells (meaning keeping the double positive feature) but, instead, forcing the progenitors to express Sox2 or Bra alone, which permits evaluation of differentiation routes rather than how to maintain the resident progenitor pool. Interestingly, their alternative mathematical model in which the relative Sox2/Bra levels follow an anterior-posterior gradient (which is actually a feature observed in the embryo) was also successful in producing an extending embryo. This model was not favored by the authors (but see my comment below). According to this model, the progenitor zone could be maintained by a cell pool containing equivalent Sox2/Bra levels; when this balance is broken cells eventually enter NT or PSM routes. Therefore, while expression heterogeneity can be observed in the PZ, I am not sure that the work shown in this manuscript is conclusive enough to claim an essential role of such heterogeneity to maintain the progenitor pool.

    We acknowledge that regional heterogeneity of Sox2 and Bra has been described in the PZ and we made sure that we cite the bibliography including Wymeersch et al, 2016 and Kawachi,2020. Although these papers described different levels of Sox2 and Bra in the PZ, they did not clearly reported and quantified the fact that direct neighboring cells have very different levels of Sox2 and Bra, therefore we believe that our description of a “random-like” pattern of heterogeneity constitutes a real novelty. In the same lines, we are aware of the several studies independently showing that gain or loss of-function of Sox2 or Bra can act on the progenitor decision to join either the NT or the PSM (these references are cited l.70, l.72). However, we believe that our study is the first to test systematically both overexpression and downregulation of Sox2 and Bra on progenitor distribution in the same biological system and to link Sox2/Bra functions to cellular motility.

    Testing the requirements of spatial cell-to-cell heterogeneity to maintain a pool of progenitors is experimentally challenging and even if we were able to homogenize Sox2 and Bra expression, we would have to do it in all progenitors, which is not, so far, technically possible using bird embryo as a model system. We are well aware of these limitations and have toned down claims on the essential role of heterogeneity to maintain progenitor pool. In particular, we have changed the abstract (we removed the last sentence stating that heterogeneity is fundamental to maintain a pool of resident progenitors), as well as the end of the introduction (we removed “while progenitors expressing intermediate/equivalent levels of the two proteins tend to remain resident”). We have pondered our model in the discussion in saying by cell with comparable levels of Sox2 and Bra “could” remain resident (L.370)

    To better apprehend the role of cell-to-cell spatial heterogeneity, we have developed a new mathematical model (Figure 5) which integrates both gradient and random heterogeneity in Sox2/Bra values within the PZ and thus fits better to our biological results. In the new version of the manuscript, we compared this model with a model in which the PZ is fully gradient-like and second one in which it is completely random. These comparisons allow us to describe better what properties random and patterned heterogeneities could bring to the system (Figure 6).

    1. The other main novelty of this manuscript is the idea that differences in cell motility derived from their Sox2 or Bra contents are a major force driving the generation of NT and PSM from the progenitors in the PZ. While there are clear differences between cell motility in the NT and the other two regions, the differences between what is observed in the PSM and PZ is not that high (actually, from the data presented it is not clear that such differences actually exist). However, independently of motility differences, there is no experimental evidence demonstrating that the essential driver of the cell fate choices is motility itself. Differences in cell motility could be just one of the results of more fundamental (and causal) changes in cell characteristics triggered by Sox2 or Bra activity. Indeed, NT and PSM cells are different in many different ways, including adhesion properties, which are normally a major determinant of tissue morphogenesis. Cell motility could, therefore, be one of the factors but it is not clear that it plays the essential role proposed by the authors. (see also next comment).

    Cell motility distributions in the PZ are slightly different from that of the PSM since slower cells were found in the PZ. We agree with the reviewer that this difference might be difficult to see because the average motilities between the two tissues are very similar (Figure 3 and Figure 3-figure Supplement 1). To reveal this difference more clearly we have used a reporter gene for Sox2 and analyze progenitor motility by time lapse imaging. We have specifically tracked GFP positive cells (reporter gene for Sox2) in the PZ and compared them to cells which are not expressing GFP. The result is that Sox2 high progenitors are globally slower than other progenitors clearly revealing heterogeneity in cell movements within the PZ and its relation to Sox2 expression (L.225-232, Figure 3-figure Supplement 1B, video 2).

    We agree that there is no experimental evidence that motility itself is the driver of the cell fate choices. To test if the effect on cell motility is taking place downstream of differentiation events, we have analyzed the expression of markers for mesodermal and neural fate (Msgn1 and Pax6) 7hrs after overexpression of Sox2 and Bra. While Sox2 or Bra overexpression triggers changes on cell motility in this short time window, we did not observe any changes in Msgn1 and Pax6 expression (L.267-274, Figure 4-figure Supplement 2) arguing that the effect on motility is an early consequence of the Bra and Sox2 misexpression. Nevertheless, we are aware that this is not a strict demonstration that the effect on fate are coming from the differential motility only. We have therefore toned down our arguments and changed the title of the manuscript (“....guides destiny by controlling their motility “ has been replaced by “...guides motility and destiny”) .

    The effects on cell motility we observe could be a consequence of Sox2 and Bra effect on adhesion as suggested by the reviewer, this is an interesting possibility that we cannot and don’t want to rule out. The effect on cell adhesion is taken into account in our model and we discuss this hypothesis in the new version of the manuscript (L. 456-459). Identifying the mechanisms underlying the effects of Sox2 and Bra on cell motility is an extremely interesting project we want to pursue but we consider that this aspect goes beyond the scope of the current manuscript.

    1. The authors developed a mathematical model to confirm their hypothesis that Sox2/Bra expression diversity combined with different motility of cells with high, low or intermediate relative levels of Sox2 and Bra expression are the key to guarantee proper axial elongation from the PZ. I am, however, not sure that the model, the way it was designed, actually proves their point. In particular, because it introduces an additional variable that might actually be the essential parameter for the success of the mathematical model: physical boundaries between NT and PSM cells, meaning that cells with high Sox2 or high Bra are unable to mix. As I commented above, this variable reflects a key biological property of the two tissues involved, one epithelial and the other mesenchymal in nature, which might be more relevant that the motility of the cells themselves (e.g. by different cell adhesion properties). How would a model that does not include such physical barriers work? Conversely, how would a model work in which only physical barriers are applied, using similar starting conditions: a prefigured central neural tube (Sox2 high), flanked at both sides by PSM (Brachyury high) and with the PZ (variable Sox2/Bra levels) just posterior to the neural tube?

    We agree that adhesion and non-mixing properties are essential to our models. Because it was not clear in the previous version, we have explained them in more details in the new version of the manuscript (l.295-300 and Appendix 1). To assess their roles, we have made two new simulations one without the regulation of non-mixing /adhesion properties and one without motility control by Sox2/Bra. Both simulations show strong defects in morphogenesis arguing that motility on its own is a key component of the system and that the non-mixing and adhesion properties are also important but not sufficient to drive morphogenesis (Figure 5F). Having the same non-mixing/adhesion and motility properties downstream of Sox2 and Bra in all our models allows us to isolate the phenomena we wish to study: the role of the distribution of cell -to cell heterogeneity in the PZ (Figure 6).

    1. The authors generate two mathematical models, differing in whether they start with a random distribution of Sox2 and Bra expression throughout the PZ or with prefigured opposing Sox2 and Bra expression gradients, somehow resembling the image observed in the embryo. The two models generated structures resembling the elongating embryo, although with small differences in the extension process and the extension rate. After analyzing the behavior of those models, they concluded that the random model fits better with the expectations from the in vivo characteristics in the embryo. I am however not sure that I agree with the authors' interpretation. First, because the gradient model includes a natural characteristic observed in the embryo, which the random model does not. Second, because one of the deciding characteristics, namely the slower extension rate observed in the gradient model, does not necessarily make it worse than the random model, as it is not possible to properly determine which extension rate actually resembles more accurately axial extension in the embryo. Third, because the observation that in the gradient model the PZ undergoes fewer transient deformations and self-corrective behaviour is in my view an argument to favor, instead of to disfavor the gradient model, both because the final result is at least as good as the one obtained with the random model and it is actually not clear that in the embryo the PZ undergoes such clearly visible deformations and self-corrections during axial extension. In addition, the gradient model generates a "pure" PZ (just yellow cells) in the posterior end of the structure, while in the random model the PZ contains some islands of NT cells, which is not what is observed in the embryo. According to the last features, the gradient model seems better than the random model.

    To answer the reviewer’s concern about similarity to the embryo, we have developed a new model that is clearly closer to the biological system because it integrates both the gradient and the random ratio distributions (new Figure 5). Interestingly, by comparing it to the two extreme models (random and gradient), we found that this more “natural” model combines the stability and fluidity brought by the gradient model and the random model, respectively. As pointed out by the reviewer, we found that graded distribution brings more stability to the system with a “purest” PZ. At the opposite, random distribution allows more tissue fluidity and cell rearrangements as well as tissue shape conservation (Figure 6). We want to thank the reviewer for his or her input; we think that the new model and the comparison with the two extreme cases allowed us to reveal more clearly properties that are specific to the two types of spatial distributions and therefore to point out what general morphogenetic properties could emerge from random- like heterogeneity in the embryo.

    Reviewer #2:

    In this manuscript, Romanos et al show firstly that there is extensive cell-to-cell heterogeneity in the relative levels of Sox2 and Bra in the region containing progenitors for neural and paraxial mesoderm, gradually resolving towards high Bra/low Sox2 in the mesoderm or high Sox2/low Bra in emerging neurectoderm. They then show that overexpression of Sox2/morpholino-based inhibition of Bra or vice versa lead cells to favour neurectoderm or mesoderm respectively. Next they show that cells expressing high Bra are more motile than those expressing Sox2, and show using mathematical modelling that these behaviours can explain many aspects of the eventual segregation of Sox2-high neurectoderm and Bra-high mesoderm.

    This interesting and well-presented work leads to the elegant and novel hypothesis that random cell motility induced by Bra and inhibited by Sox2 are sufficient to explain the segregation of NMps towards mesoderm and neurectoderm respectively. The work will be of broad interest to developmental and mathematical biologists interested in the cell biological basis of self-organising cell behaviours. Nevertheless there are some concerns to address in order to solidify the claims in the manuscript.

    1. The section where Sox2 and Bra levels are manipulated (line 152 onwards) is somewhat under-analysed. Results are presented as supporting a model where the two proteins mutually repress each other and lead to segregation of neural (high Sox2) and mesodermal (high Bra) cells. However the data presented does not unequivocally support the claims in the manuscript and would require further clarification.

    In the new version of our manuscript, we give more details on the analysis of Sox2 Bra levels manipulations. In particular, we provide data showing the tissue localization of manipulated cells on transverse sections (L. 192, Figure 2-figure supplement 3). We have also studied the effects of Sox2 and Bra ovexpression on cell fate maturation in the PZ and provide some evidence that progenitors do not yet express differentiation markers as they acquire specific motile properties in response to Sox2 or Bra overexpression (L. 267-273, Figure 4-figure supplement 1). According to our results and to the literature, we revised the text by removing mentions to Sox2 and Bra mutual repression (L 171, L 386, L389).

    1. The mathematical model may be an oversimplification of the role of these two genes in organising a balanced production of neurectoderm and mesoderm.

    In the new version of our manuscript, we have made significant efforts to better explain how non- mixing properties are taken into consideration in our models and thus, hopefully, to avoid an impression of oversimplification. We would like to point out that simulations performed to evaluate the impact of non-mixing properties on the elongation process, indicate that adhesion and non- mixing properties alone cannot account for the morphogenetic events we modelled (new Figure 5F), thus reinforcing the view that regulation of cell motility is a key element in the system. Furthermore, we have designed a new mathematical model, which is closer to the biological system because it integrates both graded and random distribution of Sox2/ Bra values (as observed in vivo) (new Figure 5). As explained above in response to reviewer 1, comparison of this model with our previous models, based on either graded or random distribution of the Sox2/ Bra values, points out the importance of random like cell-to-cell heterogeneity in this morphogenetic process.

    Reviewer #3:

    The manuscript by Romanos and colleagues examines how Sox2 and Brachyury control the behavior and cell fate of neuro-mesodermal progenitors (NMPs) in avian embryos. Using immunohistochemistry, the authors showed that the cells residing in the progenitor zone (PZ) display high variability in Sox2/Bra expression. Manipulation on the levels of the two transcription factors affected NMPs' choice to stay or exit the PZ and their future tissue contributions. This motivated the authors to employ an agent-based computational model and additional functional experiments to explore the importance of Sox2/Bra for cellular motility. The results led the authors to propose that (i) heterogeneity in Sox2/Bra ratio is important for the spatial organization of the PZ and its derivatives and that (ii) Sox2/Bra determine the fate of progenitor cells by controlling cellular movements.

    This is a technically sound report that combines single-cell analysis, in vivo functional experiments, and mathematical modeling to explore the link between cell motility and cell identity. While the model proposed by the authors is intriguing, I found that the study should provide evidence placing Sox2/Bra as primary regulators of cell motility in the context of the PZ. Given the extensively-studied role of these transcription factors in NMPs, it is challenging to decouple cellular behavior from cellular identity during tissue formation. The study would benefit from further demonstration that cell fate commitment is regulated by - and not a regulator of - cell migration of NMPs.

    We have now tested the effect of Sox2 and Bra overexpression on cell identity. We show that, 7 hrs after electroporation (a time at which we observe an effect on cell movement), no modification of the expression of neural (Pax6) and mesodermal (Msgn1) maturating markers. These data thus indicate that the effect on cell motility happens without a major acceleration of the maturation program (Figure 4 figure supplement 2). However, as mentioned in response to Reviewer 1, these experiments are correlative and do not demonstrate that the effect of Sox2 and Bra on neural and mesodermal differentiation programs are going only thought cell motility, therefore we have accordingly toned down our arguments in the new version of our manuscript.

    Strengths and Weaknesses:

    • The idea that heterogeneity in cellular behaviors within a progenitor field may act as a driver of morphogenesis is interesting and nicely supported by the agent-based model.

    We want to thank the reviewer for this comment. We believe that in the new version of the manuscript we go even further by developing a new model (Figure 5) which is closer to reality and by testing the influence of random versus gradient Sox2/Bra distribution on morphogenesis (Figure 6)

    • One of the premises of the model (Fig 4) is that Sox2/Bra ratio determines how much cells move, but this is not clear from the in vivo experiments and seems speculative. A clear demonstration of correlation between Sox2/Bra ratio and cellular motility is necessary for proper support of the model.

    The role of the Sox2 to Bra ratio on PZ cell motility is demonstrated in Figure 4. In the new version of the manuscript, these results are presented before the modelling section, we hope that it would help clarifying any doubt the reader can have on the fact that we do demonstrate clearly a role of Sox2 and Bra in controlling PZ cell motility in vivo.

    • The authors found that manipulation in the levels of the TFs results in changes in NMP motility, but it is not clear if this the cause or a consequence of commitment to a neural or mesodermal fate. Could Bra-High cell moving more because they have been specified to a mesodermal fate? Conversely, Sox2-High cells might migrate less since they get incorporated into the neural tube. Establishing the timing of cell fate commitment is necessary to resolve this issue

    We agree with the reviewer that it is an interesting issue; we have checked for expression of specification markers 7hrs after electroporation of Sox2 and Bra expression vectors, a time point at which electroporated cells did not yet leaved the PZ but have already changed their motility. In these conditions, overexpression of Sox2 and Bra had no discernable effect on expression of the neural marker Pax6 and on the PSM marker Msgn1, respectively (Figure 4 figure supplement 2).

    • The study's impact and novelty depend on the demonstration that the primary function of Sox2/Bra in NMPs is to drive cell movement. This is not sufficiently explored in the study, and there are no proposed mechanisms for how Sox2/Bra modulate cellular behavior.

    We do have shown that Sox2 and Bra act on progenitor motility in vivo (Figure 4). As a mechanism, we propose that Sox2 and Bra could act directly on motility or indirectly by regulating differential adhesion. Cell adhesion control by Sox2/Bra is part of our modeling assumptions and is therefore a hypothesis that will be the subject of future investigations in the lab. This hypothesis is part of the discussion in the new version of the manuscript (L.457).

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  2. Evaluation Summary:

    This study is of potential interest to biologists interested in developmental patterning and the link between cellular identity and behavior. The authors perform experiments in the progenitor zone of avian embryos to propose that heterogeneity of cellular behaviors may drive morphogenesis and underlie cell fate choices. The work is nicely done but might need some additional experimental validation of the proposed hypothesis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    This manuscript shows cell to cell variability in the relative levels of Sox2 and Brachyury (Bra) expression by individual cells within the region of the epiblast containing axial progenitors (the progenitor zone, PZ). Accordingly, some cells express high Bra and low Sox2 levels, others high Sox2 and low Bra and a third group expressing equivalent levels of both transcription factors. They then show that by experimentally promoting high Sox2 expression cells enter neural tube (NT) fates, whereas high Bra brings cells in the progenitor zone to enter the presomitic mesoderm (PSM). The authors then complement these experiments with evaluation of cell movements within the PZ, NT and PSM to show that cells in the NT are much less motile than those in the PZ and PSM. These data led the authors to propose a fundamental role for Sox2/Bra heterogeneity to maintain a pool of resident progenitors and that it is the high cell motility promoted by high Bra levels what pushes cells to join the PSM, whereas high Sox2 levels inhibit cell movement forcing cells to take NT fates. To validate their hypothesis, the authors generated a mathematical model to show that those expression and motility characteristics can indeed lead to axial extension generating NT and PSM derivatives in the proper positions, while keeping a PZ at the posterior end.

    Some specific comments on the manuscript are specified below.

    1. Although the description of cells within the PZ containing different Sox2 and Bra expression ratios is more explicit and quantitative in the present manuscript, this has already been previously reported by different methods including immunofluorescence (e.g., Wymeersch et al, 2016). Similarly, that breaking the Sox2/Bra balance towards high Sox2 or Bra is an essential step to bring the progenitors towards NT or PSM fates has also been previously shown in different ways. These observations are, therefore, not totally new. The novel contribution of this paper is the authors' interpretation that "heterogeneity among a population of progenitor cells is fundamental to maintain a pool of resident progenitors". In this work, however, this conclusion is only supported by their mathematical simulation, as the experiments described in this manuscript are not aimed at homogenizing Sox2/Bra expression levels in the progenitor cells (meaning keeping the double positive feature) but, instead, forcing the progenitors to express Sox2 or Bra alone, which permits evaluation of differentiation routes rather than how to maintain the resident progenitor pool. Interestingly, their alternative mathematical model in which the relative Sox2/Bra levels follow an anterior-posterior gradient (which is actually a feature observed in the embryo) was also successful in producing an extending embryo. This model was not favored by the authors (but see my comment below). According to this model, the progenitor zone could be maintained by a cell pool containing equivalent Sox2/Bra levels; when this balance is broken cells eventually enter NT or PSM routes. Therefore, while expression heterogeneity can be observed in the PZ, I am not sure that the work shown in this manuscript is conclusive enough to claim an essential role of such heterogeneity to maintain the progenitor pool.

    2. The other main novelty of this manuscript is the idea that differences in cell motility derived from their Sox2 or Bra contents are a major force driving the generation of NT and PSM from the progenitors in the PZ. While there are clear differences between cell motility in the NT and the other two regions, the differences between what is observed in the PSM and PZ is not that high (actually, from the data presented it is not clear that such differences actually exist). However, independently of motility differences, there is no experimental evidence demonstrating that the essential driver of the cell fate choices is motility itself. Differences in cell motility could be just one of the results of more fundamental (and causal) changes in cell characteristics triggered by Sox2 or Bra activity. Indeed, NT and PSM cells are different in many different ways, including adhesion properties, which are normally a major determinant of tissue morphogenesis. Cell motility could, therefore, be one of the factors but it is not clear that it plays the essential role proposed by the authors. (see also next comment).

    3. The authors developed a mathematical model to confirm their hypothesis that Sox2/Bra expression diversity combined with different motility of cells with high, low or intermediate relative levels of Sox2 and Bra expression are the key to guarantee proper axial elongation from the PZ. I am, however, not sure that the model, the way it was designed, actually proves their point. In particular, because it introduces an additional variable that might actually be the essential parameter for the success of the mathematical model: physical boundaries between NT and PSM cells, meaning that cells with high Sox2 or high Bra are unable to mix. As I commented above, this variable reflects a key biological property of the two tissues involved, one epithelial and the other mesenchymal in nature, which might be more relevant that the motility of the cells themselves (e.g. by different cell adhesion properties). How would a model that does not include such physical barriers work? Conversely, how would a model work in which only physical barriers are applied, using similar starting conditions: a prefigured central neural tube (Sox2 high), flanked at both sides by PSM (Brachyury high) and with the PZ (variable Sox2/Bra levels) just posterior to the neural tube?

    4. The authors generate two mathematical models, differing in whether they start with a random distribution of Sox2 and Bra expression throughout the PZ or with prefigured opposing Sox2 and Bra expression gradients, somehow resembling the image observed in the embryo. The two models generated structures resembling the elongating embryo, although with small differences in the extension process and the extension rate. After analyzing the behavior of those models, they concluded that the random model fits better with the expectations from the in vivo characteristics in the embryo. I am however not sure that I agree with the authors' interpretation. First, because the gradient model includes a natural characteristic observed in the embryo, which the random model does not. Second, because one of the deciding characteristics, namely the slower extension rate observed in the gradient model, does not necessarily make it worse than the random model, as it is not possible to properly determine which extension rate actually resembles more accurately axial extension in the embryo. Third, because the observation that in the gradient model the PZ undergoes fewer transient deformations and self-corrective behaviour is in my view an argument to favor, instead of to disfavor the gradient model, both because the final result is at least as good as the one obtained with the random model and it is actually not clear that in the embryo the PZ undergoes such clearly visible deformations and self-corrections during axial extension. In addition, the gradient model generates a "pure" PZ (just yellow cells) in the posterior end of the structure, while in the random model the PZ contains some islands of NT cells, which is not what is observed in the embryo. According to the last features, the gradient model seems better than the random model.

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  4. Reviewer #2 (Public Review):

    In this manuscript, Romanos et al show firstly that there is extensive cell-to-cell heterogeneity in the relative levels of Sox2 and Bra in the region containing progenitors for neural and paraxial mesoderm, gradually resolving towards high Bra/low Sox2 in the mesoderm or high Sox2/low Bra in emerging neurectoderm. They then show that overexpression of Sox2/morpholino-based inhibition of Bra or vice versa lead cells to favour neurectoderm or mesoderm respectively. Next they show that cells expressing high Bra are more motile than those expressing Sox2, and show using mathematical modelling that these behaviours can explain many aspects of the eventual segregation of Sox2-high neurectoderm and Bra-high mesoderm.

    This interesting and well-presented work leads to the elegant and novel hypothesis that random cell motility induced by Bra and inhibited by Sox2 are sufficient to explain the segregation of NMps towards mesoderm and neurectoderm respectively. The work will be of broad interest to developmental and mathematical biologists interested in the cell biological basis of self-organising cell behaviours. Nevertheless there are some concerns to address in order to solidify the claims in the manuscript.

    1. The section where Sox2 and Bra levels are manipulated (line 152 onwards) is somewhat under-analysed. Results are presented as supporting a model where the two proteins mutually repress each other and lead to segregation of neural (high Sox2) and mesodermal (high Bra) cells. However the data presented does not unequivocally support the claims in the manuscript and would require further clarification.

    2. The mathematical model may be an oversimplification of the role of these two genes in organising a balanced production of neurectoderm and mesoderm.

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  5. Reviewer #3 (Public Review):

    The manuscript by Romanos and colleagues examines how Sox2 and Brachyury control the behavior and cell fate of neuro-mesodermal progenitors (NMPs) in avian embryos. Using immunohistochemistry, the authors showed that the cells residing in the progenitor zone (PZ) display high variability in Sox2/Bra expression. Manipulation on the levels of the two transcription factors affected NMPs' choice to stay or exit the PZ and their future tissue contributions. This motivated the authors to employ an agent-based computational model and additional functional experiments to explore the importance of Sox2/Bra for cellular motility. The results led the authors to propose that (i) heterogeneity in Sox2/Bra ratio is important for the spatial organization of the PZ and its derivatives and that (ii) Sox2/Bra determine the fate of progenitor cells by controlling cellular movements.

    This is a technically sound report that combines single-cell analysis, in vivo functional experiments, and mathematical modeling to explore the link between cell motility and cell identity. While the model proposed by the authors is intriguing, I found that the study should provide evidence placing Sox2/Bra as primary regulators of cell motility in the context of the PZ. Given the extensively-studied role of these transcription factors in NMPs, it is challenging to decouple cellular behavior from cellular identity during tissue formation. The study would benefit from further demonstration that cell fate commitment is regulated by - and not a regulator of - cell migration of NMPs.

    Strengths and Weaknesses:

    - The idea that heterogeneity in cellular behaviors within a progenitor field may act as a driver of morphogenesis is interesting and nicely supported by the agent-based model.

    - One of the premises of the model (Fig 4) is that Sox2/Bra ratio determines how much cells move, but this is not clear from the in vivo experiments and seems speculative. A clear demonstration of correlation between Sox2/Bra ratio and cellular motility is necessary for proper support of the model.

    - The authors found that manipulation in the levels of the TFs results in changes in NMP motility, but it is not clear if this the cause or a consequence of commitment to a neural or mesodermal fate. Could Bra-High cell moving more because they have been specified to a mesodermal fate? Conversely, Sox2-High cells might migrate less since they get incorporated into the neural tube. Establishing the timing of cell fate commitment is necessary to resolve this issue

    - The study's impact and novelty depend on the demonstration that the primary function of Sox2/Bra in NMPs is to drive cell movement. This is not sufficiently explored in the study, and there are no proposed mechanisms for how Sox2/Bra modulate cellular behavior.

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