Single cell analysis of lymphatic endothelial cell fate specification and differentiation during zebrafish development

  1. Lin Grimm
  2. Elizabeth Mason
  3. Oliver Yu
  4. Stefanie Dudczig
  5. Virginia Panara
  6. Tyrone Chen
  7. Neil I. Bower
  8. Scott Paterson
  9. Kazuhide Okuda
  10. Maria Rondon Galeano
  11. Sakurako Kobayashi
  12. Anne Senabouth
  13. Anne K. Lagendijk
  14. Joseph Powell
  15. Kelly A. Smith
  16. Katarzyna Koltowska
  17. Benjamin M. Hogan

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Abstract

During development, the lymphatic vasculature forms as a second, new vascular network derived from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is the first step in this process. Specification, differentiation and maintenance of LEC fate are all driven by the transcription factor Prox1, yet downstream mechanisms remain to be elucidated. We present a single cell transcriptomic atlas of lymphangiogenesis in zebrafish revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a VEC-LEC fate reversion during differentiation. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new transcriptional targets and regulatory regions of the genome downstream of Prox1 in LEC maintenance, as well as showing that Prox1 specifies LEC fate primarily by limiting blood vascular and hematopoietic fate. This extensive single cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.

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    Reply to the reviewers

    We thank the reviewers for their thoughtful comments and suggestions and were pleased to note the quality of work and the findings were well received. Both reviewers commented that the datasets and findings represent a valuable resource for the field, and that this is a valuable resource paper. The only major concern related to conceptual advance and we provide a clear plan below that we believe will thoroughly address this issue.

    Below, we provide point-by-point responses to each of the reviewer’s comments. These are presented to improve the conceptual advance in section 1 and address all other issues raised in other minor comments in section 2.

    SPECIFIC ISSUES:

    Section 1: Conceptual A____dvance

    One main concern raised by both of the reviewers was that the main biological findings did not represent a major new conceptual advance, which is encapsulated by the comments below:

    Reviewer 1:

    “Major comments:

    The conclusions of the manuscript are convincing. The robust data generated is inherently valuable and is of great interest of the field. However, my impression is that the authors did not utilize the power of their studies. The main message - Prox1 is a key regulator in promoting and maintaining lymphatic cell fate - is well accepted and has been intensively studied. Therefore, the main findings presented in the current manuscript are not conceptually-advancing.

    Additional studies focusing on the function of some of the identified hit genes, such as cdh6, slc7a7, fabp11a in lymphatics - either in fish or in vitro - would significantly improve the novelty of the article. Zebrafish is an ideal experimental model that enable a relatively easy and quick way to address these questions. However, considering the time and expense of those experiments, in vitro studies would be also well appreciated instead of fish.”

    Reviewer 2:

    “While the work presented in this manuscript could be an interesting resource for the researchers in the field, it does not provide significant conceptual advances in the field.”

    “CROSS-CONSULTATION COMMENTS

    I agree with the excellent technical and statistical comments of Rev. 2. Overall, we are in agreement regarding the strength of the datasets as a resource for the field, but with limited conceptual novelty.”

    We appreciate both reviewers’ feedback and take this concern seriously. We believe that the paper would be improved by utilizing the unique and extensive single cell resource to develop a deeper new understanding of the molecular control of lymphatic development. We also believe that the novel and new biology already presented in the paper can be better highlighted by re-writing the paper in key places.

    In revision, we will therefore provide two major improvements to address these comments on conceptual advance.

    Firstly, we will re-write relevant sections of the paper to provide an improved focus on the new biology uncovered and less of a technical focus on the resource generated. Specifically, the key new biological insights our thorough analysis already made in the manuscript that will be better highlighted include:

    1. We define the precise timing of differentiation of lymphatics in vivo. Specified LECs in the cardinal vein are not significantly differentiated from their venous neighbours, rather they become transcriptionally distinct between 40 and 72 hours post fertilisation (hpf), well after the sprouting events by which they emerge from the vein. This was not previously shown in any study.
    2. We show in definitive Prox1 null maternal and zygotic double mutant zebrafish for the first time that the sprouting of LECs from the cardinal vein occurs independently of Prox1 function, separating the initial sprouting and fating events. This is different from earlier findings in mouse (Yang, Garcia-Verdugo et al. 2012) and offers a unique new understanding.
    3. We define the transcriptional program controlled by Prox1 during the maintenance of LEC fate in vivo at a whole transcriptome level. This has never actually been done before in any published literature. This reveals that Prox1 simultaneously up-regulates lymphatic marker genes and down-regulates blood vascular marker genes (which was known for a small number of markers). However, it also demonstrates the surprising finding that none of the change in fate from blood vascular to lymphatic vascular is regulated independently of Prox1 function. This shows that Prox1 is not “a” master regulator but “the only” master regulator of this fate decision in a definitive manner for the first time.
    4. In contrast to how Prox1 maintains lymphatic fate, we also provide a challenging and important analysis of Prox1 function at the earliest stages of lymphatic specification and transdifferentiation from blood vascular to lymphatic vasculature. For the first time in any published literature, we show that the role of Prox1 in vivo in this fate decision is primarily to negatively regulate blood vascular and hematopoietic cell fate and not to positively regulate a lymphatic specification gene network. Importantly, this suggests that lymphatic fate transition begins by blocking what may be a default blood vascular cell fate. This was not previously shown in vivo.
    5. Finally, at a molecular level we have demonstrated that Prox1 regulates chromatin accessibility across the genome. Specifically, mutants show a unique and unexpected chromatin signature, whereby chromatin is opened up at many key lymphatic developmental genes but these genes are not transcribed. This discordance in chromatin state and gene transcription appears to be consistent with ectopic activity of early blood and blood vascular transcription factors. This unique finding indicates that Prox1 function negatively regulates blood and blood vascular transcriptional control across diverse enhancers and regulatory elements, and that Prox1 function determines normal chromatin state changes to regulate cell fate. We believe that we did not do a good job of highlighting these important biological observations for the reviewers. Our revision will better emphasise the biological meaning in our data, rather than emphasising the technical aspects of the work.

    Secondly, we agree with the reviewers that extracting a new biological finding or understanding from the data will improve the impact of this study. A longstanding question in the field of lymphangiogenesis has been what precise role Notch signalling plays in cell fate decisions and in vessel network growth. The literature is very murky on the role of Notch signalling in the specification of lymphatics. For example:

    1. Human in vitro work (Kang, Yoo et al. 2010) showed that increased Notch pathway function repressed expression of key transcription factors Prox1 and CoupTFII and the subsequent induction of LEC fate, but this was not confirmed in vivo.
    2. In mice, Murtomaki et al (2013) reported that Notch signalling negatively regulates VEC to LEC transition via suppression of Prox1 expression at the earliest stages of specification of LECs from the cardinal veins (Murtomaki, Uh et al. 2013).
    3. This work contradicts the rather definitive observation that endothelial deletion of the core Notch effector Rbpj (Tie2:Cre, Rbpj-f/f) has no effect on the expression of Prox1 in the cardinal veins (Srinivasan, Geng et al. 2010).
    4. In zebrafish, it was found that lymphatics don’t form in the absence of Notch signalling (Geudens, Herpers et al. 2010), but in this study we found no evidence of active Notch signalling during LEC specification and sprouting. This was recently explained with the demonstration that it was arterial Notch signalling responsible for the abnormal wiring of zebrafish vessels and loss of lymphatics (Geudens, Coxam et al. 2019). These studies suggest that the role of Notch signalling in zebrafish is not autonomous to the developing veins and lymphatics. Thus, it is currently very unclear if Notch signalling plays a specific role in developing lymphatics or if so, when and how it controls lymphangiogenesis in a cell autonomous manner.

    Upon re-evaluating our data, we examined all of the known Notch ligands, receptors and target genes with single cell resolution. To our surprise we found that:

    • jag2b expression is a specific marker of the fate-shifted LECs in Zprox1a-/- mutants at 4dpf, switching on when LEC fate is not maintained by Prox1.
    • *notch1a and notch1b *are the key lymphatic expressed receptors for the pathway in zebrafish.
    • The main downstream target expressed was *her6, *which was expressed in a specific manner in vasculature in the maturing LECs.
    • Strikingly, there was little to no expression of these key pathway components at specification of LEC fate stages, but rather the Notch pathway is active at later stages when LECs differentiate and grow in the embryo. This prompted us to examine a unique notch1b mutant that we have in the lab. We found that this mutant has clear defects in lymphangiogenesis that impact later stages of development but do not impact early specification.

    For our revision, we therefore plan to include one additional large Figure of data. This figure will build of a deeper analysis of Notch signalling in our single cell RNA and ATAC sequencing resource and use our new mutant strain to definitively demonstrate the importance of and timing of Notch signalling in the development of lymphatic vessels. We believe that this will clear up the mystery of when and how Notch controls lymphangiogenesis and will add important new conceptual advance to the paper.

    Section 2: Other minor comments

    Reviewers’ Comments:

    Reviewer #1

    An article in 2017 presented abundant expression of fabp11a in zebrafish and suggested its function in brain vessel integrity (PMID: 28443032). In the current manuscript however, the authors did not find fabp11a expression in the head vasculature. Did the authors not detect expression of fabp11a in brain blood vessel endothelial cells at the investigated stages of the zebrafish development? In this case, how would they discuss this seeming contradiction?

    We thank the reviewer for pointing out this study. In the paper from Zhang et al. (2017), the authors showed blood vessel expression of fabp11a at earlier stages than we have examined in our images here. In particular, the expression in blood vessels in the head was shown at 1.5, 2 and 3 dpf. We have examined our transgenic line only at 5 dpf. At 5 dpf we do see expression in the trunk veins, which is consistent with the Zhang paper, but we have not looked at the cranial blood vessels at early stages.

    In our revision, we will image earlier stage brain blood vessels using our new transgenic line to address this issue and provide additional confidence in our findings.

    Minor comments:

    In Figure 1a, authors show LEC sprouts in the trunk region at 40 hpf. At 3 dpf however, these LECs sprouts are not shown, but parachordal LECs only. Do these LEC sprouts disappear by 3 dpf? Cartoons on later timepoints suggest that LEC sprouts shown at 40 hpf remain in their location and make connection with parachordal LECs, but the panel in its current form is misleading.

    We thank the reviewer for this feedback. We will correct this figure to better indicate these key stages and we will include a full reference at this point of the article to our previous review article Hogan and Schulte-Merker (2017) in which we describe this process in detail and in full (Hogan and Schulte-Merker 2017).

    Although I appreciate that the authors were consistent with the colour coding in the graphs, some combinations should be revised. Although the light blue/dark blue colour combination works well in other places, in Figure 4a, it is hard to distinguish those colours. Use of a higher contrast colour combination would be better.

    We will correct this by using high-contrast colour combinations as requested.

    In Figure 1b, similar colours are used for different purposes. Orange in the upper panel shows 40 hpf cluster, while a very similar colour is used for the VEC_preLEC cluster in the lower panels. Although I recognize the overlay between these clusters, a different colour coding would be more accurate. Maybe, clusters from the upper panel (Stage) should be show individually, just like genes in panel c, to help the reader identifying those clusters at different timepoints.

    We will correct this by selecting different colours for VEC_preLEC cluster and cells collected at the 40hpf time point.

    Reviewer #2:

    Specific Comments:

    In general, the authors need to be more precise and cautious in interpreting the RNA velocity analyses. For instance, in Fig 1b, there are two potential regions which could reflect VEC to LEC transition (the one which is connected to LEC sub-cluster and the other which is located in between LEC and VEC/preLEC sub-clusters.) Which trajectory are the authors referring to? In addition, in Fig 3c, the authors claim that RNA velocity analyses showed that the cells within the mutant cluster, however, since cells located within the edge of the clusters tend to have similar trajectory (for instance, cells in the right edge within the LEC_S1 sub-cluster and those in the top left edge within the LEC_S2 sub-cluster), it is difficult to assess whether the trajectory the authors indicated in the mutant sub-cluster is biologically meaningful and relevant. Finally, in Fig 7a, further analyses are needed to support the authors claim which is solely based on RNA velocity analyses.

    We thank the reviewer for this feedback and will ensure the size of arrows on our Velocity analysis are increased, to facilitate interpretation of the data. Further to this we will include a second trajectory analysis (Street, Risso et al. 2018) in Extended data figures 1, 2 and 7 that we expect will validate our observations made in Figures 1b, 3c and 7a respectively.

    In Fig. 1b, it is not clear whether arterial and venous ECs were excluded from the analyses, if so, the authors need to state how these cell types were identified and excluded. In addition, it would be helpful if the authors show the actual number of cells in each sub-cluster, so the readers could estimate the prevalence of each sub-cluster.

    We agree that this information can be more explicitly described, and will include the number of cells per cluster in the legend of Figure 1b, and all single cell RNA-seq UMAPs that define sub-populations. Furthermore, we will include an extra column in Extended Data Table 1a detailing the number of cells per cluster, expand Extended Figure 1 to describe step-wise sub setting of data. We will do this for all 3 single cell datasets. This information will also be written into the Results and Methods.

    In Fig 2a, the authors claim that the level of gene expression is different between head and trunk region using cropped fluorescence microscopy images. It would be more convincing if the authors show both head and trunk regions in a single image.

    We will address this by using images taken of the entire fish including both head and trunk.

    In Fig. 1c, could the authors include an UMAP image showing the expression level of prox1b? It would be helpful for the readers to compare the expressivity of prox1b over time.

    We will amend Figure 1c by replacing UMAP images of hexa with prox1b (prox3).

    In Fig. 1d, the authors need to explain why the expression of LEC markers diminish at 5dpf.

    We thank the reviewer for pointing this out. The 4 dpf single cell RNA-seq libraries are larger than the other libraries included in our developmental time course. While the normalisation (Stuart, Butler et al. 2019) and integration (He, Brazovskaja et al. 2020) approaches have partially corrected this, we believe the higher expression at 4 dpf can be attributed to library size rather than biology. In our revision we will include an analysis that applies down-sampling to larger libraries, that we believe will reduce the contribution of library size to gene expression patterns reported in the developmental time-course.

    In Fig. 3a, it would be helpful if the authors show arterial ECs as well, so the readers could assess the characteristics of mutant clusters in a more general context.

    We thank the reviewer for this feedback. This information is detailed in Extended Data Figure 2a, which shows UMAPS for all cells in the Zprox1a-/- mutant scRNA-seq dataset. We will expand this figure and include a separate panel with additional UMAP images and dot plots of all endothelial cell types including AEC (arterial endothelial cells), and believe that this will allow readers to better appreciate how different sub-populations of ECs relate to each other.

    In the current Figure 3 we focus exclusively on evaluating the LEC, VEC and mutant sub-populations, allowing the reader to hone in on our key points.

    In Fig. 3a and 3b, the authors state that Zprox1a null cells generate a peculiar VEC cluster (mutant cluster). Does prox1a influence the transcriptomic profile of VECs as well?

    We thank the reviewer for this important question. We will expand the Extended Data Table 2 to include differential expression analyses between Zprox1a-/- mutant and WT AEC (arterial endothelial cells), and VEC (venous endothelial cells). We will include a dot plot in Extended Data Figure 2 that includes cluster specific markers of the mutant cluster with Zprox1a-/- mutant and WT AEC and VEC phenotypes. This demonstrates that the changes in Prox1 mutants are restricted to the cells that normally express Prox1 (i.e. LECs).

    It is not clear how the normalization was done in Fig. 3d.

    We will include this information in the Results section text more clearly upon revision.

    In Fig. 3f, the number of the genes do not match with the extended data table 2b (1034 vs 1107, and 294 vs 326).

    We thank the reviewer for picking up these errors. Figure 3f includes all genes that are considered differentially expressed (Wilcoxin Rank Sum adjusted p value

    In Fig. 3i and 3k, the authors show the quantification of cdh5/kdrl intensity within the thoracic duct. It would be helpful if the authors could correlate the location of the area used for quantification (whether the quantification represents LEC cluster or mutant cluster).

    We thank the reviewer for this suggestion and will add a clear box displaying where measurements were made. We will also amend the text for clarity.

    Can the authors specify the unique characteristics of mutant clusters such as the presence of specific markers?

    We thank the reviewer for this suggestion, and will amend the text for clarity. We will include a dot plot of top cluster specific markers for all clusters (including the mutant specific cluster) in Extended Data Figure 2.

    In Fig. 4g, how prevalent is prox1a/b binding sites and what is the P value?

    This is a great question from the reviewer. The Prox1-motif has been problematic but we have now developed robust approaches to identify predicted Prox1-motifs in our snATAC identified peaks. We have now performed a Prox1 motif analysis and will update Figure 4g to include these results. We will include a quantitative comparison of the frequency of Prox1 motifs in LEC, VEC and AEC specific peaks identified in our ATAC analyses.

    In Fig. 5a and 5b, the authors assume that the mutant cluster in scRNA-seq data and the mutant cluster in snATAC-seq data are the same population. Is there any validation done?

    We thank the reviewer for pointing this out. We will clarify in text that we believe that these are the same cell population for two reasons:

    1. They are the only populations in both the scRNA-seq and snATAC-seq data composed almost entirely of Zprox1a-/- mutant cells.
    2. Furthermore, all other endothelial cell phenotypes (eg. AEC, VEC, LEC, muLEC, Endocardium) are accounted for in both datasets. At the transcriptional level (in our scRNA-seq) the mutant specific cluster co-expresses LEC and VEC markers, suggesting it is a hybrid cell type that sits between LEC and VEC phenotypes. However, at key lymphatic genes chromatin accessibility and gene expression (comparing snATAC and scRNA-seq) become discordant in the mutant specific clusters, which gives us confidence that we are observing a fate shift due to loss of Prox1 in this specific type of cell. This also suggests that Prox1 is required for concordant chromatin accessibility and gene expression.

    In Fig. 5c, figure legend and the extended data table 4a did not match. In Fig 5c, the figure legend says the cut off was set by Wilcoxon Rank Sum, FDRWe thank the reviewer for picking up these errors. As in Figure 3f, Figure 5c includes peaks that are considered differentially accessible (Wilcoxin Rank Sum FDR In Fig. 7d and 7e, it is not clear how the clustering was performed. Based on the image shown in the Fig. 7d/e, three sub-clusters do not seem to clearly separate from one another. It would be helpful if the authors clearly state what was the criteria used for the clustering.

    We thank the reviewer for this suggestion. The reason that the clusters sit close together is because these cell types are not yet differentiated from each other. This can be appreciated by looking at clustering of all endothelial cells in 7a. In response to this comment we will no longer show subsetted and re-clustered data in 7d (we will move this to Extended Data), instead will display 7d and 7e using the same UMAP used in 7a with other endothelial cells (AEC, VEC, Endocardium) coloured light grey. We will also expand our description of clustering in the Results and Methods.

    Overall, the dot plots should be replaced with the violin plots to better reflect potential heterogeneity within sub-clusters.

    We agree that for key points, violin plots could be helpful. We will include violin plots in Extended Data Figures for key data points that include the following: Figures 1c, 3b and 7e. This will ensure that readers have a clear appreciation for heterogeneity within sub-clusters for all key markers that define phenotype in each dataset.

    __Other comments from reviewers: __

    Reviewer 1:

    Significance:

    The manuscript uses state of the art approaches to characterize Prox1-dependent transcriptional and chromatin accessibility changes that define LEC fate and lymphatic sprouting in zebrafish models.

    The key role of Prox1 in LEC differentiation and maintenance of lymphatic cell fate and lymphatic development is well known based on previous findings. Strength of the current manuscript is the massive dataset generated, which opens the opportunity to identify downstream players of Prox1 in regulating lymphatic fate and expansion. The authors, however, did not utilize this opportunity for elucidating novel conceptual findings about lymphatic endothelial fate, development or function.

    The presented results will be of interest for experts in vascular biology, lymphatic biology, developmental biology and genetics. The generated data may be further used in studies investigating the function of the hit genes highlighted in this manuscript in lymphatic vessels.

    Reviewer 2:

    Grimm and colleagues analysed developmental lymphangiogenesis in zebrafish embryos using single cell transcriptomics. They identified a number of novel targets of Prox1a, the master regulator for the LEC fate. In addition, the authors have identified a novel mutant-specific sub-cluster in Zprox1a mutant embryos, reiterating the importance of prox1a in the specification and differentiation of LECs.

    Significance:

    While the work presented in this manuscript could be an interesting resource for the researchers in the field, it does not provide significant conceptual advances in the field. Moreover, there are some technical issues that needs to be resolved prior to the publication of the manuscript.

    We thank the reviewers for their positive response and feedback.

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    Referee #2

    Evidence, reproducibility and clarity

    Grimm and colleagues analyzed developmental lymphangiogenesis in zebrafish embryos using single cell transcriptomics. They identified a number of novel targets of Prox1a, the master regulator for the LEC fate. In addition, the authors have identified a novel mutant specific subclusters in Zprox1a mutant embryos, reiterating the importance of prox1a in the specification and differentiation of LECs.

    Specific Comments:

    1. In general, the authors need to be more precise and cautious in interpreting the RNA velocity analyses. For instance, in Fig 1b, there are two potential regions which could reflect VEC to LEC transition (the one which is connected to LEC subcluster and the other which is located in between LEC and VEC/preLEC subclsuters.) Which trajectory are the authors referring to? In addition, in Fig 3c, the authors claim that RNA velocity analyses showed that the cells within the mutant cluster, however, since cells located within the edge of the clusters tend to have similar trajectory (for instance, cells in the right edge within the LEC_S1 subcluster and those in the top left edge within the LEC_S2 subcluster), it is difficult to assess whether the trajectory the authors indicated in the mutant subcluster is biologically meaningful and relevant. Finally, in Fig 7a, further analyses are needed to support the authors claim which is solely based on RNA velocity analyses.
    2. In Fig. 1b, it is not clear whether arterial and venous ECs were excluded from the analyses, if so, the authors need to state how these cell types were identified and excluded. In addition, it would be helpful if the authors show the actual number of cells in each subcluster, so the readers could estimate the prevalence of each subcluster.
    3. In Fig 2a, the authors claims that the level of gene expression is different between head and trunk region using cropped fluorescence microscopy images. It would be more convincing if the authors show both head and trunk regions in a single image.
    4. In Fig. 1c, could the authors include an UMAP image showing the expression level of prox1b? It would be helpful for the readers to compare the expressivity of prox1b over time.
    5. In Fig. 1d, the authors need to explain why the expression of LEC markers diminish at 5dpf.
    6. In Fig. 3a, it would be helpful if the authors show arterial ECs as well, so the readers could assess the characteristics of mutant clusters in a more general context.
    7. In Fig. 3a and 3b, the authors state that Zprox1a null cells generate a peculiar VEC cluster (mutant cluster). Does prox1a influence the transcriptomic profile of VECs as well?
    8. It is not clear how the normalization was done in Fig. 3d.
    9. In Fig. 3f, the number of the genes do not match with the extended data table 2b (1034 vs1107, and 294 vs 326).
    10. In Fig. 3i and 3k, the authors show the quantification of cdh5/kdrl intensity within the thoracic duct. It would be helpful if the authors could correlate the location of the area used for quantification (whether the quantification represents LEC cluster or mutant cluster).
    11. Can the authors specify the unique characteristics of mutant clusters such as the presence of specific markers?
    12. In Fig. 4g, how prevalent is prox1a/b binding sites and what is the P value?
    13. In Fig. 5a and 5b, the authors assume that the mutant cluster in scRNA-seq data and the mutant cluster in snATAC-seq data are the same population. Is there any validation done?
    14. In Fig. 5c, figure legend and the extended data table 4a did not match. In Fig 5c, the figure legend says the cut off was set by Wilcoxon Rank Sum, FDR<0.05. However, in the extended data table 4a, different cut off was used. Similarly, figure legend for Fig. 5e needs to be revised as well.
    15. In Fig. 7d and 7e, it is not clear how the clustering was performed. Based on the image shown in the Fig. 7d/e, three subclusters do not seem to clearly separate from one another. It would be helpful if the authors clearly state what was the criteria used for the clustering.
    16. Overall, the dot plots should be replaced with the violin plots to better reflect potential heterogeneity within subclusters.

    Significance

    While the work presented in this manuscript could be an interesting resource for the researchers in the field, it does not provide significant conceptual advances in the field. Moreover, there are some technical issues that needs to be resolved prior to the publication of the manuscript.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors provide a comprehensive transcriptomic and chromatin accessibility atlas of embryonic lymphangiogenesis in fish using state-of-the-art sc-RNAseq and single cell ATAC sequencing approaches. Furthermore, they present data to prove that Prox1 is a key factor of maintaining LEC identity and promoting lymphatic vascular fate and lymphatic sprouting. Using novel reporter models, they further analyzed the spatial expression pattern of numerous hit proteins in the presence or absence of Prox1 genes.

    The manuscript is well written, clear and reproducible based on the given information.

    Major comments:

    The conclusions of the manuscript are convincing. The robust data generated is inherently valuable and is of great interest of the field. However, my impression is that the authors did not utilize the power of their studies. The main message - Prox1 is a key regulator in promoting and maintaining lymphatic cell fate - is well accepted and has been intensively studied. Therefore, the main findings presented in the current manuscript are not conceptually-advancing.

    Additional studies focusing on the function of some of the identified hit genes, such as cdh6, slc7a7, fabp11a in lymphatics - either in fish or in vitro - would significantly improve the novelty of the article. Zebrafish is an ideal experimental model that enable a relatively easy and quick way to address these questions. However, considering the time and expense of those experiments, in vitro studies would be also well appreciated instead of fish.

    An article in 2017 presented abundant expression of fabp11a in zebrafish and suggested its function in brain vessel integrity (PMID: 28443032). In the current manuscript however, the authors did not find fabp11a expression in the head vasculature. Did the authors not detect expression of fabp11a in brain blood vessel endothelial cells at the investigated stages of the zebrafish development? In this case, how would they discuss this seeming contradiction?

    Minor comments:

    In Figure 1a, authors show LEC sprouts in the trunk region at 40 hpf. At 3 dpf however, these LECs sprouts are not shown, but parachordial LECs only. Do these LEC sprouts disappear by 3 dpf? Cartoons on later timepoints suggest that LEC sprouts shown at 40 hpf remain in their location and make connection with parachordial LECs, but the panel in its current form is misleading.

    Although I appreciate that the authors were consistent with the color coding in the graphs, some combinations should be revised. Although the light blue/dark blue color combination works well in other places, in Figure 4a, it is hard to distinguish those colors. Use of a higher contrast color combination would be better.

    In Figure 1b, similar colors are used for different purposes. Orange in the upper panel shows 40 hpf cluster, while a very similar color is used for the VEC_preLEC cluster in the lower panels. Although I recognize the overlay between these clusters, a different color coding would be more accurate. Maybe, clusters from the upper panel (Stage) should be show individually, just like genes in panel c, to help the reader identifying those clusters at different timepoints.

    Referees cross-commenting

    I agree with the excellent technical and statistical comments of Rev. 2. Overall, we are in agreement regarding the strength of the datasets as a resource for the field, but with limited conceptual novelty.

    Significance

    The manuscript uses state of the art approaches to characterize Prox1-dependent transcriptional and chromatin accessibility changes that define LEC fate and lymphatic sprouting in zebrafish models.

    The key role of Prox1 in LEC differentiation and maintenance of lymphatic cell fate and lymphatic development is well known based on previous findings. Strength of the current manuscript is the massive dataset generated, which opens the opportunity to identify downstream players of Prox1 in regulating lymphatic fate and expansion. The authors, however, did not utilize this opportunity for elucidating novel conceptual findings about lymphatic endothelial fate, development or function.

    The presented results will be of interest for experts in vascular biology, lymphatic biology, developmental biology and genetics. The generated data may be further used in studies investigating the function of the hit genes highlighted in this manuscript in lymphatic vessels.

  4. Version published to 10.1101/2022.02.10.479999v2 on bioRxiv
  5. Version published to 10.1101/2022.02.10.479999v1 on bioRxiv