Intrinsic and extrinsic cues time somite progenitor contribution to the vertebrate primary body axis

  1. Lara Busby
  2. Guillermo Serrano Nájera
  3. Benjamin John Steventon

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Abstract

During embryonic development, the timing of events at the cellular level must be coordinated across multiple length scales to ensure the formation of a well-proportioned body plan. This is clear during somitogenesis, where progenitors must be allocated to the axis over time whilst maintaining a progenitor population for continued elaboration of the body plan. However, the relative importance of intrinsic and extrinsic signals in timing progenitor addition at the single-cell level is not yet understood. Heterochronic grafts from older to younger embryos have suggested a level of intrinsic timing whereby later staged cells contribute to more posterior portions of the axis. To determine the precise step at which cells are delayed, we performed single-cell transcriptomic analysis on heterochronic grafts of somite progenitors in the chicken embryo. This revealed a previously undescribed cell state within which heterochronic grafted cells are stalled. The delayed exit of older cells from this state correlates with expression of posterior Hox genes. Using grafting and explant culture, we find that both Hox gene expression and the migratory capabilities of progenitor populations are intrinsically regulated at the population level. However, by grafting varied sizes of tissue, we find that small heterochronic grafts disperse more readily and contribute to more anterior portions of the body axis while still maintaining Hox gene expression. This enhanced dispersion is not replicated in explant culture, suggesting that it is a consequence of interaction between host and donor tissue and thus extrinsic to the donor tissue. Therefore, we demonstrate that the timing of cell dispersion and resulting axis contribution is impacted by a combination of both intrinsic and extrinsic cues.

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  1. Version published to 10.7554/elife.90499 on eLife
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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript entitled "A population instrinsic timer controls Hox gene expression and cell dispersion during progenitor addition to the body axis", Busby and colleagues investigate the topic of "cell type identity" in the context of body axis elongation in chick embryos. To this end, they performed heterochronic grafts from HH8 stage embryos to HH4 stage embryos and compared these to HH4 homochronic grafts. They found that HH8 grafts ingressed but were then delayed at a stage they termed cell dispersion. By scRNAseq this new cell state was characterized further. While HH8 cells adjusted their expression pattern to their surroundings, Hox gene expression was maintained as in the host developmental stage. Hox gene expression and collinearity of expression changes were also maintained when HH4 cells were grafted into HH8 embryos or cells were cultured ex vivo. Finally, the authors found differences in migration properties between HH4 and HH8 cells, when cultured ex vivo, with HH4 cells migrating faster than HH8.

    This constitutes an elegant work to describe the existence of a "cell-intrinsic timer" that regulates cell identity and progressive body axis extension. Experiments and analysis have been performed adequately and conclusions have been drawn appropriately.

    There are a rather minor comments I would suggest for further analysis, discussion and potentially experiments to further support this paper:

    • A major finding is that grafted cells keep their Hox expression pattern, independent of whether it is from HH4 to HH8 or vice versa. Moreover, grafted HH8 cells pause at the cell dispersion stage and do not mix, unless grafted in very low cell populations. The authors conclude that Hox gene expression seems to be cell intrinsically regulated. However, for pausing of cells after ingression, I wonder if it is rather the difference to the neighbors than a cell-intrinsic effect that prevents the cells from dispersing. One possibility is that differences in adhesion could account for this, since sorting of cell populations based on differential expression of adhesion molecules has been observed in various model systems. This possibility is excluded here, since adhesion-related genes were not differentially expressed in their expression data. However, I would not exclude this possibility at this stage for the following reasons: 1. The authors detect different migration speeds for HH4 and HH8 cell clusters with HH4 cells migrating faster. Differential migration rate could indeed hint at differential adhesion and mechanical properties of the cells. 2. Hox genes have been shown to be upstream of and modulate adhesion molecules, which might be an interesting link. 3. So far, the authors have only analyzed expression of adhesion molecules at mRNA levels. However, the functional components are the adhesion proteins themselves. It might therefore be useful to stain embryos for some "obvious" candidate adhesion molecules, such as cadherins. If no further experiments are performed, then this should at least be discussed.
    • The authors describe a new, intermediate stage, namely cell dispersion, in which HH8 MSP pause when grafted into HH4 embryos. They perform scRNAseq and GO term analyses to analyze these cells in more detail. The also perform gene set enrichment analysis. However, I am still wondering about the exact identity of these cells. What are they? What markers do they express? Do they upregulate certain signalling pathways? Etc. I would for instance be interested if there are differences in FGF or Wnt levels/ activities. It would be useful if the authors could analyze their scRNAseq data further in this regard.
    • At several points in the manuscript, expression levels and patterns of HH4 and HH8 grafts are compared to each other. It does not become clear what the differences and similarities to the non-grafted cells of the same clusters are. Does grafting itself change the expression patterns?
    • The authors found differences in cell cycle stages of HH4 and HH8 grafts. A more detailed discussion of this aspect would be useful rather than just excluding any cell cycle-related genes from the comparisons. Why could there be this difference? What effect could this have? Etc.

    Optional: Other experiments that could increase the relevance of the work:

    • As discussed by the authors, they specifically compare HH4 stage to HH8, which represents primitive streak stage and 4-somite stage, respectively. It would therefore be interesting to perform grafts from HH8 to later stages, such as HH10, or vice versa, when the process of somitogenesis is more similar. This could reveal if their findings are specific for pre to post node development or more general. However, this might be outside of the scope of this study.

    Significance

    General assessment: This study provides a systematic analysis of the interaction of embryonic cell clusters from different developmental stages. To this end, "classical" developmental biology techniques, i.e. grafting (complicated techniques that probably less and less people can perform nowadays), is combined with more modern ways of analysis, i.e. scRNAseq. This allows the authors to dissect the differential behaviour of hetero- and homochonic grafts. In the longer term this data can provide the basis for further in-depth mechanistic analyses, some of which could be added here already. The involvement of Hox genes in the control of developmental time is interesting and should be placed into context of our current knowledge. Here, Hox genes are rather used as readout of developmental time rather than active players.

    Advance: How developmental time is maintained during embryonic development is a long-standing question in the field. This study provides conceptual advance in this question by describing a cell-intrinsic timer.

    Audience: This study is relevant for developmental biologists in general, since it describes how developmental time can be kept by a cell-intrinsic timer, at least in early stages of somite formation in chick embryos.

    My expertise: developmental biology, somitogenesis

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, the authors investigate the importance of intrinsic and extrinsic factors in the timing of progenitor addition to the elongating primary body axis. During development, progenitor populations have to combine their self-renewal with the gradual contribution to the full length of the body axis. The mechanisms underlying the population dynamics that ensure the formation of a proportioned body plan remain poorly understood. By combining heterochronic (HH8 to HH4) and homochronic grafting (HH4 to HH4) of somitic progenitors with next generation sequencing and imaging, the authors observe that the older HH8 tissue shows intrinsic delays in migration and does not disperse within the surrounding mesodermal tissue after ingression through the primitive streak. This behavior correlates with intrinsic and tissue-specific differences in the expression of Hox genes but not with differences in the expression of cell adhesion/migration genes.

    Overall, this study provides new data exploring how progenitors control their contribution to the body axis. By combining classic embryology techniques with single-cell sequencing, the authors describe novel cell states that might help understand the progenitor population dynamics. There are however a number of further analyses and experiments that should be performed to support the main claims of the manuscript.

    Major comments:

    1. The authors claim that grafted HH8 cells are paused after the ingression stage and before the dispersion stage. The grafted cells ingress through the primitive streak and then remain as a distinct cluster of cells that does not disperse throughout the mesoderm. This is in contrast with other observations where overexpression of late hox genes delays the cells at the point of ingression. The authors should better demonstrate that their grafts are actually ingressing and then stopping once in the mesoderm compartment. Figure 3B' shows grafted HH8 cells (GFP positive) present in the mesoderm (ME) compartment 3 h after grafting. It is surprising that a cluster of cells can ingress through the primitive streak in a short period of time and then remain paused. It would be helpful to have the equivalent figure right after grafting to assess the differences in the location of the HH8 GFP+ cells and potentially observe them while ingressing.
    2. The authors describe a novel transcription state, namely clusters 6, 12 and 8 in Figure 2B, populated by HH8 cells 3 h after grafting. It is surprising that the UMAP looks very different between 0 h and 3 h in the HH8-HH4 grafts (Figure 2E and F). The authors should clarify where the HH4 (GFP negative) cells are present in Figure F, I. In the current figures, it looks as if both HH8 and HH4 cells changed completely their transcription profile in only 3 h and populated the central clusters (6, 12, 8). The authors claim that these central clusters are present in normal development and that cells rapidly transit through them. However, it is not clear whether this state happens before or after HH4. For example, the cells may be moving from right to left in UMAP_1 according to time (HH4 in the right, HH8 in the left and a central transient cluster). This would mean that in Figure 2F HH8 grafted cells are regressing to an earlier development state and not a new one. Including RNA velocity analysis could help clarify how the cells are changing their expression profiles.
    3. Related to the previous point, the striking changes between 0 h and 3 h in the HH8-HH4 grafts (Figure 2E and F) may suggest an effect of the grafting procedure on the transcription profile of the cells. The authors should demonstrate that the grafting of cells does not have a huge impact on the transcriptome and that these changes are specific to the previously undescribed delayed state of HH8 cells. For this, they should include scRNA data of HH4-HH4 3 h. If grafting does not have a significant effect on the transcriptome, they should see GFP positive and negative cells in HH4-HH4 3 h remain intermixed.
    4. The authors observe that when doing smaller heterochronic grafts, cells can disperse throughout the mesoderm. Nevertheless, the Hox gene expression does not change depending on the size of the grafts. This is in sharp contrast with their observations and claims done for big heterochronic grafts. The result is interesting as it demonstrates that the expression of Hox genes, but not dispersion, is cell intrinsic. However, the uncoupling of hox gene expression and cell dispersion requires further investigation. The authors should repeat the heterochronic grafting of Figure 1 using smaller grafts and check the contribution of grafted cells to the somites. If cells can readily disperse without delay, they might be able to contribute to all somites as observed with homochronic grafts. Similarly, the authors should repeat the explant spreading assay using smaller HH8 grafts and quantify whether differences in the migratory dynamics are observed. The authors already discuss the possibility that other factors apart from Hox expression might affect dispersion. Nevertheless, they should assess the importance of graft size in their experimental system. If smaller grafts maintain the expression profile but have a different capacity to contribute to the body axis, the initial observations might have been influenced by extrinsic factors of the graft (size, cell-to-cell contact, ECM...) and not by cell intrinsic properties (gene expression). This would change the conclusion of the work.

    Minor comments:

    1. It would be informative to have a better time-resolved description of the heterochronic graft behavior in Figure 1. For homochronic grafts, several timepoints are provided allowing the visualization of cells travelling through the body axis. For heterochronic grafts, by contrast, only an early and final timepoint are provided.
    2. In Figure 4, the authors show that explants of HH4 and HH8 embryos have different migratory dynamics, with HH4 cells migrating faster than HH8. In Figure 4E, HH8 explants seem not to change their area for about 15 h and then start spreading. This indicates that there is a great delay in migration compared to HH4 explants. However, once they start spreading, it seems that the area starts to increase exponentially in a similar manner to what is observed for HH4 at earlier time points. It would be interesting to monitor the HH8 for a longer time to see the behaviors at later time points. HH8 explants may be just delayed, and once they start fully spreading, the speed may not be so different from the one of HH4 explants.
    3. The authors conclude in Supp. Table 2 that HH8 and HH4 do not have different expressions of adhesion-related genes upon grafting. This observation is very important to understand the potential mechanism behind the different dispersion behaviors, and thus it should be included in the main figure.

    Significance

    The authors combine classic embryology with single-cell RNA-seq and imaging techniques to explore progenitor population dynamics during addition to the body axis. They conclude that the delayed contribution of older cells to axis formation correlates with the intrinsic expression of posterior hox genes. While the idea of intrinsic regulation of hox genes during axial specification is not conceptually new, the authors use modern techniques to describe with finer detail the progenitor population states. For this reason, this manuscript will be of interest to researchers in the development field who want to better understand the hox control and influence during axial elongation.

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    Referee #1

    Evidence, reproducibility and clarity

    This manuscript describes the differential behavior of the epiblast region of chicken embryos containing the progenitor cells for the medial half of the somites (MSP) at HH4 (building the first 4-5 somites) and HH8 (building more caudal somites). Their approach combines grafting experiments with imaging, single cell and whole mount expression analyses of the grafts. The basic experiment involves the comparison of HH4 to HH4 homochronic grafts with HH8 to HH4 heterochronic grafts. They show that homochronic grafts undergo a dispersion stage after ingression through the primitive streak before they contribute to somites. The same region of HH8 embryos, when grafted into the MSP region of HH4 embryos, however, fail to undergo this dispersion and do not contribute to the first 4-5 somites. They also show that Hox gene expression follows the patterns observed in the grafted tissue, failing to acquire the expression profiles of the receiving host. The authors conclude that the MSP cells contain an internal timer involved in the regulation of their changing behavior as development proceeds.

    The general findings reported in this manuscript are novel and can provide insights to further our understanding of the differences between formation of the first 4-5 somites and more caudal somites. However, I think that ADDITIONAL EXPERIMENTS are important to properly evaluate the data and the conclusions of this manuscript.

    1. A control HH8 to HH8 homochronic graft to check the behavior of the grafted cells: do they disperse in their natural environment after ingression through the primitive streak or they are also paused as a distinctive cell cluster?
    2. The reverse heterochronic grafting experiment, namely HH4 cells into HH8. Do HH4 cells maintain their dispersal behavior at the ectopic position, or they behave differently?

    While the authors assessed the intrinsic properties of HH4 and HH8 tissue by incubating it on fibronectin, this experiment does not properly reproduce the environment of the embryonic region receiving the graft, which might be different at HH4 and HH8. The experiments I am suggesting take this variable into consideration and will therefore help assessing the possible involvement of the host tissue in the behavior of the grafts.

    In addition to those experiments, a MORE EXTENSIVE ANALYSES of the already reported experiments could also improve the manuscript.

    1. When the cells staying in the MSP region after homochronic HH4 grafts reach later stages (e.g. approaching HH8), do they keep dispersing as at earlier stages after ingression through the primitive streak or they remain as a distinct cluster? And does Hox gene expression within those grafts follow the same activation profile observed in the host cells as development proceeds?
    2. In the experiment reported on fig. 5I, HH4 MSP grafted into HH8 embryos fail to activate Hox genes like Hoxa2, even after 6 hours of incubation. When these grafted embryos develop even further (for the period of time required for a HH4 embryo to reach the HH8 stage), do they activate Hoxa2 or Hoxa3 or they remain negative for these genes?
    3. The differential GO terms between HH4 and HH8 tissue in cluster 6 include chromatin organization, DNA methylation and C5-methylation of cytosine. This suggests that epigenetic changes might be involved in the behavioral differences between the MSP of the two stages, which can affect many different processes involved in cell activity, including the activation of Hox genes.

    SOME COMMENTS ON DATA INTERPRETATION.

    1. It is clear that Hox gene expression in the grafts matches the profile of the donor tissue, indicating the existence of a Hox "timer". However, in my opinion, the authors place too much emphasis on the possible meaning of these observations in what concerns the differential behavior of the grafted cells. If they want to focus on Hox genes they should include some experiment testing their involvement in cell dispersal, either by misexpression or downregulation of specific genes (although there is plenty of information arguing against this possibility, maybe with the exception of that of Iimura and Pourquie, 2006; in this regard, the authors' own data already indicate that dispersion is independent of Hox gene expression).
    2. The authors disregard the involvement of differential patterns of cell adhesion molecules as the origin of the differential behavior of HH4 and HH8 grafts in the HH4 context. However, in their data on supplementary fig. 2A there are several genes differentially expressed between the HH8 and HH4 cells (e.g. Ptk7, Spon1 or Nfasc) that could indeed play a role in the differential interaction between cells from the two embryonic stages. It might be interesting to perform HCR experiments with some of these factors to see if they are differentially expressed at the two embryonic stages. Also, although it might be somewhat far reaching, if differential expression is observed by HCR, it might be interesting to experimentally manipulate expression of the relevant gene (s) (misexpression or down-regulation, depending on the stage) to evaluate its/their potential functional relevance.

    Minor points

    1. The authors write that the HH8 specific clusters are 0, 4 and 6. However, I think that it is #7 and not #6 the one belonging to this group. I guess that this is typo, but becomes confusing, as a large part of the analysis of the single cell data is centered on cluster #6.
    2. In the introduction the authors state that the first 4-5 somites do not develop ganglia, citing Lim et al 1987. I think that the way this is written is imprecise, as it sort of implies that more caudal somites develop ganglia (which would mean that the dorsal root ganglia are somite derivatives). However, somites at any level do not develop ganglia; the anterior half of their sclerotomes are permissive to migration of the neural crest that will eventually build the ganglia, something that seems not to happen in the more anterior somites.

    A side note

    Different alternative transcripts have been reported at least for Ptk7 and Nfasc. This might be relevant considering that another of the prominent differential GO terms identified in supplementary fig 2C is related to RNA splicing. Would different alternative transcripts for some of these genes be specifically associated with the cells from one of the embryonic stages?

    Significance

    It has been known for many decades that the first 4-5 somites of amniotes are different to the rest of the somites in several ways, from the structures they generate to the way they are generated or the gene regulatory networks controlling their morphogenesis. Much is known about how the posterior somites are generated and the mechanisms of their differentiation. Conversely, relatively little is known about the same processes in the most anterior somites. The work described in this manuscript shows that the progenitor cells from the epiblast that will contribute to the 4-5 first somites already behave different than those generating more caudal somites. Also, they show that progenitors generating more caudal somites are unable to contribute to the rostral somites. These two sets of observations show that the differences in the rostral and caudal somites are already present in their progenitors and that those features are quite stable within the cells, at least when they are kept as a group.

    So far, the single cell analyses shown in this manuscript failed to provide clear hints to explain the different behavior of the two sets of progenitors. However, they represent an important resource to further explore this important biological question. The authors focus on Hox genes as potential regulators of the differential behavior of the HH4 and HH8 MSPs, I guess that prompted by the report by Iimura and Pourquie (2006) indicating the involvement of Hox genes in the migratory properties of the somite progenitors. However, there is plenty of information, mostly genetic studies in mice, indicating that Hox genes might have very little influence in the differential behavior of rostral and caudal somites. In this regard, expression does not mean causation. I think that this manuscript is interesting, most particularly for developmental biologists involved in understanding the mechanisms governing the basic layout of the vertebrate body plan.

    Research in my laboratory also explores this type of biological questions, although using more genetic approaches and in a different model system, namely the mouse. I therefore consider myself in a position that allows a knowledgeable evaluation of this manuscript.

  6. Version published to 10.1101/2023.05.10.540133v1 on bioRxiv