Live-imaging reveals Coordinated Cell Migration and Cardiac Fate Determination during Mammalian Gastrulation
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Review Commons)
Abstract
Heart development involves the specification of cardiac progenitors at distinct stages and locations. Using live-imaging of mouse embryos between gastrulation and heart tube formation, we tracked individual mesodermal cells and reconstructed their lineage trees for up to five cell divisions. We found independent unipotent progenitors emerging at specific times, contributing exclusively to either left ventricle/atrioventricular canal (LV/AVC) or atrial myocytes. LV/AVC progenitors differentiated early to form the cardiac crescent, while atrial progenitors later generated the heart tube’s inflow tract during morphogenesis. We also identified short-lived bipotent progenitors with broad potential, illustrating early developmental plasticity. Sister cells from bipotent progenitors displayed greater dispersion and more diverse migratory trajectories within the anterior mesoderm than those from unipotent progenitors. Bipotent progenitors contributing to extraembryonic mesoderm (ExEm) exhibited the fastest and most dispersed migrations, whereas those giving rise to endocardial, LV/AVC, and pericardial cells showed a more gradual divergence, with late-stage behavioural shifts: endocardial cells increased in speed, while pericardial cells slowed relative to LV/AVC cells. Together the data reveal the regulation of individual cell directionality and cardiac fate allocation within the seemingly unorganised migratory pattern of mesoderm cells.
Article activity feed
-
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1
Evidence, reproducibility and clarity
The manuscript describes the tracking of individual mesoderm cells through live imaging. Through a combination of reporters including a novel cardiomyocyte reporter and a combined nuclear GFP-inducible Cre reporter under the dependance of the Brachyury promoter, the authors label mesoderm cells at different stages of gastrulation then perform long term (>30h) live imaging of late gastrulation embryo up to the cardiac crescent and heart tube stages. They use elaborate analysis tools as well as manual tracking to reconstruct cells' trajectory, lineage trees, and various behavioral traits.
The study is …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1
Evidence, reproducibility and clarity
The manuscript describes the tracking of individual mesoderm cells through live imaging. Through a combination of reporters including a novel cardiomyocyte reporter and a combined nuclear GFP-inducible Cre reporter under the dependance of the Brachyury promoter, the authors label mesoderm cells at different stages of gastrulation then perform long term (>30h) live imaging of late gastrulation embryo up to the cardiac crescent and heart tube stages. They use elaborate analysis tools as well as manual tracking to reconstruct cells' trajectory, lineage trees, and various behavioral traits.
The study is well designed. Experiments are technically challenging, well executed, and carefully analysed.
Methods are clear and complete so that experiments should be faithfully reproduced provided availability of an appropriate microscope.
The description of the results of the live imaging experiments is not easy to read and understand, but I believe this is inherent to the complexity of the results themselves and due to the high diversity of behaviors observed. Similarly the figures are extremely dense ans some graphs would benefit from a more didactic legend.
I realize the difficulty of being more concise due to the large amount of information and its diversity. If possible, I would suggest integrating tables within the results section that may help shorten the text, and may be easier to grasp.
We will add tables describing the numbers of uni-fated and multi-potent mothers, cell speeds, and dispersion. We will also split the figures to reduce the amount of information in each figure; and improve the legends by providing more detailed explanations.
The interpretation of the results is fair and in line with previous studies, which are adequately cited.
A discussion on the reasons why a large proportion of cells could not determined as uni or multipotent might be useful. Instinctively I would imagine that a majority of those are multipotent and therefore garder to track, so if the authors do not agree with this interpretation it may be useful to detail technical reasons why those cells cannot be fully interpreted.
We have discussed further reasons why a large proportion of cells could not be classified as uni-fated or multipotent. Indeed, while our analysis revealed a predominance of uni-fated progenitors (n=98, generating 728 descendants) over bifated/trifated progenitors (n=18, generating 302 descendants), a significant number of mother cells (n=111) produced progeny whose fates could not be determined. This is due to multiple factors, as explained below.
First, we were unable to fully track a large proportion of cells that generate short tracks. This limitation hindered our ability to determine their final fates. One key reason for these shorter tracks was the occasional high density of labeling, which, coupled with the spatiotemporal resolution of our imaging setup (0.347 x 2 µm z-stacks acquired every 2 minutes), was insufficient to consistently and unambiguously curate some cell tracks. We agree with the reviewer that the difficulty in tracking was probably exacerbated by the high dispersion of cells during the earliest stages, which is particularly high for multipotent mother cells. To avoid introducing erroneous lineage assumptions, we opted to stop tracking under such conditions.
Another contributing factor is related to cells migrating to deeper regions of the heart tube. Over extended timeframes, these cells often relocated towards the more dorsal regions of the forming heart tube, where they became dimmer due to their position along the z-axis. Consequently, many daughter cells did not meet the GFP intensity threshold required to classify them as myocytes and were thus labeled as mesodermal (line 194 and see Fig. 7C for an example). Additionally, some cells could not be tracked for prolonged periods, especially as they moved dorsally during the transformation of the cardiac crescent into the heart tube. A limitation of light-sheet imaging is its reduced capacity to capture high-quality images in deeper tissues due to light scattering. Addressing this limitation and improving imaging depth will be critical in future studies.
We also acknowledge the graded expression pattern of cTnnT2-GFP in the forming heart tube, with early and higher levels in LV/AVC myocytes and later, lower levels in inflow myocytes. To maintain consistency, we refrained from using different thresholds to account for these regional intensity differences. While this choice could have led to false negatives (e.g., inflow cells not meeting the GFP threshold), we believe this approach minimises the risk of false positives. Any daughter cells failing to meet the threshold were conservatively classified as mesodermal (meso GFP-), even though they may have been myocyte progenitors.
Additionally, some cells contributing to the inflow/atria regions may not have passed the GFP threshold during the imaging period but could have done so at later developmental stages. These cells were also classified as mesodermal, as their myocyte progenitor status could not be determined. This conservative approach prioritises accuracy over overestimation. We have included all these explanations in the main text and Materials and Methods.
Significance
Strengths: novel transgenic tools, powerful imaging technique, thorough quantified nalysis. Limitations: the development of embryos after E7.75-E8 is never completely normal ex vivo, particularly when there is no rotation. This is visible in the pictures of the embryos post culture (ballooned yolk sac, unattached allantois). It is probably not a limitation regarding cardiac development but may influence other mesoderm lineages notably ExE. Advance: It is a unique study dur to the labelling strategy, the length of imaging, and thereby the faithful tracking of cell lineages across several rounds of division. The information provided corroborates what previous hypothesis in the field based on less direct assessment, and is here very strong and unbiased. The research is of great interest for developmental biologists (including but not limited to the heart field), cell biologists (notably those working on stem cells and organoids as it provides a ground truth), microscopy and image analysis experts.
Reviewer #2
Evidence, reproducibility and clarity
The authors perform an elegant "tour de force" lineage relationships during mouse heart development. They perform long-term live imaging and single-cell tracking in mouse embryos from early gastrulation to stages of heart tube formation. They then track the progeny of individual cells and reconstruct the lineage tree of tracked cells. They analyze how their migratory paths of cells correlate with cell fate in the heart. Altogether, the manuscript presents a highly detailed live-imaging lineage tracing study of a subset of cells in the cardiac crescent in mouse. This presents a nice contribution to the literature, but would be improved by the suggestions below.
Major comments:
- Can the authors be sure they can track all of the derivatives of labeled cells? They are claiming to be able to follow complete lineages, but I worry if they may lose progeny in their tracking or incorrectly conclude that cells are lineally related. wonder how you could show how accurate it really is. Perhaps if the authors could include a movie where they trace what they claim as an entire lineage of a single cell and show this with the mother and daughter cells labelled throughout the movie, that would at least provide an example for readers to make their own decisions about how reliable the lineage tracing is. Would it be feasible to include an interactive movie where the reader can move the embryo around in 3D at each time point?
We have not tracked all the derivatives of labeled cells, as explained in our response to Reviewer 1. A number of mother cells (n=111) produced progeny whose fates could not be determined. Each cell track (up to 1,000 time points) required manual curation and verification, as even a single linkage error would compromise conclusions. When a track could not be unambiguously determined, we stopped tracking those cells. We have acknowledged this limitation in the manuscript.
We also agree with the reviewer that it is important to show the tracks, and we will therefore include supplementary movies displaying all the cells tracks. Furthermore, we are submitting all our datasets to the Image Data Resource (IDR) (https://idr.openmicroscopy.org/). Our datasets have been accepted, and the IDR team is currently assessing our track data, cell annotations, and metadata. This will enable users to download the data and fully assess them interactively in 3D using MaMuT or Mastodon (https://mastodon.readthedocs.io/en/latest/index.html) for cell tracking, as well as to generate their own tracking data. The availability of our data through this resource will significantly enhance its value to the community.
The authors describe the lineage labeled cells as unipotent, bipotent, etc. But they cannot really say anything about developmental potential as they are only looking at normal fate which is less that their potential. Without manipulation of the cells through transplantation etc., the use of the term 'potential' or 'potent' is not appropriate except when they find cells that are multipotent. Rather than calling cells unipotent, I would suggest using the phrase 'assume a single fate'.
We have replaced all instances of unipotent with uni-fated.
Lines 112-115, the authors state that variability in embryonic stages likely explains differences in labelling. Are there any morphological characteristics across the embryos that support this variability in stages? For example, any characteristics that suggest that the n=3 embryos are slightly older, and the n=7 embryos are slightly younger (line 111)?
We thank the reviewer for this excellent suggestion. Unfortunately, as the embryos were collected at different times, it is not possible to directly compare embryos from different litters. To address this, we would need to repeat the lineage tracing experiments by collecting embryos at fixed time points. This approach would allow us to compare variability in developmental stages at the time of collection while accounting for differences in labeling. Our live analysis shows that the early and late mesoderm contribute to the cardiac crescent and heart tube inflows, respectively, supporting our interpretation of the lineage tracing results.
Paragraph beginning on line 116: Please clarify how cells were counted, from the wholemount/across sections?
We counted the tdTomato+ cells across sections in wholemount embryos using the Cell Counter plugin in Fiji. We added this information to the Methods section.
- Line 165: Authors state that in the absence of tamoxifen, tdTomato-positive cells were identified in one embryo. Please state here the total number of embryos out of which this one embryo was counted.
Done.
- Line 190: 'Figure 2-Supplementary Figure 3A-F' doesn't exist. Do they mean Fig.3 supplementary 3A-F?
Yes, thank you, we corrected. Fig.3 supplementary 3A-F is now Fig.4 supplementary 3A-F.
Figure 1F-G: For cross sections in 'G' please show the level they were taken from in 'F'.
The cross-section shown in panel G (now Figure 2B) was not taken from the same embryo depicted in panel F (now Figure 2C). We apologize for the confusion and have clarified this point in the text.
Figure 4I: There is a large disparity in cell dispersion across movies. Please comment on why this could be. Is there a difference in stage/morphology etc..
Movies 1 and 2 depict embryos cultured at earlier stages, while Movies 3 to 5 show embryos cultured at later stages. The later the embryonic stage at the start of culture, the less dispersed along the anterior-posterior (AP) and dorsal-ventral (DV) axes of the heart tube the clones were. This is consistent with the idea that cell dispersion was more prominent during the earliest phases of migration taking place in the earlier embryos, consistent with the results from Dominguez et al. 2022. We will add a graph comparing the stages at which the cells were tracked (based on the alignment of the movies shown in Figure 5B) to cell dispersion to illustrate this point and have clarified in the manuscript.
Figure 4K-L: The arrowhead color is too similar to the cell fluorescence color, making the visualization a little confusing. Changing the color of the arrowheads may be helpful. This is also true for some of the other figures (red arrowheads).
We have changed all the red arrows to white arrows.
Significance
This is a well-done study that will be useful to developmental biologists as well as cardiologists. The experiments seem very well done and beautifully executed. With the proposed modifications, it will make a very nice contribution to the literature.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
In their manuscript, Abukar et al. investigate the origins and migratory behaviors of cardiac progenitor cells, in mice, from gastrulation to early heart tube formation. They use sophisticated live imaging to tracks individual mesodermal cells, reconstructing their lineage and fate over several generations. The findings reveal distinct unipotent progenitors that contribute exclusively to specific cardiac regions, such as the left ventricle/atrioventricular canal (LV/AVC) or atrial cardiomyocytes. LV/AVC progenitors differentiate early, forming the cardiac crescent, while atrial progenitors differentiate later, contributing to the venous poles of the heart tube. Additionally, the study identifies multipotent mesodermal progenitors contributing to various mesodermal cell types, including the endocardium, pericardium and extraembryonic tissues.
Major comments:
- Important conclusions of the manuscript rely on the expression of a reporter line (cTnnt2-2a-eGFP) as well as on the position of tdTomoto+ cells in relation to the reporter. We feel that markers of non-myocardial lineages should have been used to better characterize these populations. We acknowledge the technical challenge of live imaging, which may not allow labeling of all lineages. We believe that a better description of the final stages of investigation with markers of endocardium, pericardium, extra-embryonic mesoderm together with the eGFP of the reporter will strengthen the conclusions drawn on the multipotency of the progenitors. If not addressed, some claims may appear more speculative and would benefit from being toned down.
We agree that the use of additional specific reporters and endogenous marker gene expression data would provide further insights and have now acknowledge this point in the Discussion. For example, the extra-embryonic mesoderm is situated in the extra-embryonic space, and additional markers would help identify which cell types within the ExEm compartment were traced. Similarly, many cells were classified as meso but could not be defined further in the absence of suitable markers in our live imaging experiments.
However, we stand by our assertion that the spatial distribution of progenitors in the heart tube regions, as observed in our live-imaging data-particularly within the somatic and inner endocardial layers surrounding the cTnnT-2a-GFP+ myocardial layer-provides the most compelling evidence.
Gene expression is not always a perfect proxy for assigning cell fates without carefully documented spatial context, as transcription factors (TFs) are often expressed in multiple cell types. For example, Hand1 is expressed in the pericardium, ExEm, and left ventricle myocardium, while Nr2f2 is expressed throughout the posterior mesoderm and not exclusively in myocytes (as shown in Fig. 1H). Similarly, Tal1 is expressed in hemogenic endothelial/blood progenitors located in the ExEm and endocardial lineages.
Therefore, we stand by our cell annotations. This approach, based on cell location, aligns with well-established lineage mapping studies that have long demonstrated the predictive power of spatial and morphological information in early development. For instance, Wei et al. (2000) successfully predicted early segregation between myocardial and endocardial lineages solely based on cell location within these layers of the heart tube. Decades-old research has provided clear evidence that the pericardial (somatic), myocardial (splanchnic), and endocardial layers are distinguishable in E7.5 mouse embryos (see DeRuiter et al., 1992, PMID: 1567022, Figure 2A-F). In fact, cell types were often defined through morphological observation long before gene expression techniques became available. Such approaches remain relevant for elucidating cell fates, particularly in early embryogenesis, when spatial information plays a crucial role in defining progenitors.
- Similarly, since all the results of the manuscript derive from five movies of five independent embryos, it would be important to provide a more detailed description (for example, in a table) of the experimental setup. This could include the timing of tamoxifen induction (+7h or +21h?), the stage of dissection (based on anatomical landmarks rather than dissection stage - see atlas of gastrulation), the duration of the movies, and the stage at the final time point. Providing this information would greatly enhance the ability to robustly compare each movie and ensure reproducibility. Of note, the methods section could benefit from additional clarity. For example, in line 594, the embryo from Movie1 is described as being dissected in the morning, while the next sentence states it was dissected in the afternoon, similar to the embryo in Movie5. To avoid confusion and ensure greater rigor, describing the developmental stage of the embryos rather than the time of dissection would be more precise and biologically meaningful.
We thank the reviewer for this suggestion. While we have already temporally aligned our movies based on the timing of the first LV/AVC progenitors and atrial progenitors passing the threshold to be considered as myocytes (Fig. 5B), we will provide additional staging of the embryos based on morphological landmarks at T0. This will include the extent of the nGFP+ primitive streak and the normalized intensity of the nGFP signal. Additionally, the duration of the movies and the timing of tamoxifen induction will be indicated in the table, as suggested by the reviewer. We removed the statement on the dissection in the morning and afternoon since it was clumsy.
- This manuscript focuses primarily on LV/AVC progenitors and likely a subpopulation of atrial cardiomyocytes, leaving other cardiac progenitor populations unaddressed. While it is understandable that the study focuses on specific populations, the authors should further discuss the limitations of their approach and explain why not all cardiac progenitors were targeted. A discussion of how these limitations might impact the broader interpretation of their findings would also be valuable.
We agree with the reviewer that our analysis focuses mainly on the LV/AVC and atrial progenitors and have now mentioned these limitations in our Discussion. However, the HCN4+ inflow structures of the heart tube we are analysing likely contribute to most (if not all) of the atria later in development, rather than constituting a subpopulation. Published lineage tracing of HCN4+ cells using a tamoxifen inducible system suggests that these cells contribute to most of E19.5 atria (Fig. 2b in Später et al., 2013), raising the question of the extent of the contribution from an additional HCN4- population to the atria. However, we agree that this question warrants further investigation.
Regarding the progenitors contributing to the RV and OFT, we agree with the reviewer that our analysis does not fully address these progenitors. While we did analyse a subset of distal mesodermal cells contributing to the pharyngeal mesoderm (labeled in red in Fig.), the absence of a live marker prevented us from determining whether these cells localized in this part of the embryo were part of the cardiopharyngeal mesoderm. Consequently, we labeled these cells as meso GFP- in our results.
We suspect that mesodermal cells contributing to the pharyngeal mesoderm may arise earlier than atrial progenitors and are currently investigating their origin using a new Tbx1-2a-tdTomato reporter line (Figure 1). However, as these findings are still preliminary and require further work, which is beyond the scope of this manuscript, we prefer not to include these data at this stage.
More broadly, we fully agree with the reviewer that the inclusion of additional markers in future studies will provide a more comprehensive understanding of cardiac development, and we are excited to pursue this work in the coming years.
- Since a recent preprint (Sendra et al.), already cited in the manuscript, used complementary approaches to investigate endothelial/endocardial cell fate during gastrulation, we feel that a more in-depth discussion is warranted. In particular, how the results presented here align with the early segregation between endocardial and myocardial lineages observed by Sendra et al. could be clarified. Additionally, it is unclear how these findings correlate with Foxa2 lineage tracing. Addressing these points could further strengthen the contextualization and impact of the manuscript.
We agree with the reviewer and have highlighted in our Discussion how our findings align with the Sendra et al. study. Specifically, our observation of short-lived multipotent progenitors supports the hypothesis that mesodermal lineages, including endocardial lineage, are rapidly established during gastrulation. Our observation of rare endo-myo bipotent progenitors is consistent with these findings and aligns with clonal analyses by Devine et al., which identified a shared mesodermal progenitor between these two lineages (Figure 1J in Devine et al., 2014).
However, we believe that the scATAC-seq evidence for an earlier lineage bias specifically toward the endocardial lineage warrants further investigation. In our opinion, it remains unclear whether the nuclei analyzed in their study represent prospective endocardium equivalent to the cells we observed in the live-imaging experiments. Notably, both Nfatc1 and Notch1 exhibit broader expression patterns beyond the endocardium, including in yolk sac endothelial cells and the allantois (see J Cell Biol (2022) 221 (6): e202108093, and doi.org/10.1002/dvdy.21246). Thus, it is plausible that the first mesodermal lineage decision observed in the Sendra et al. scATAC-seq analysis corresponds to the establishment of ExEm hemato/endothelial cells, which are the first mesoderm to ingress in the primitive streak at E6.5 (Development (1999) 126 (21): 4691-4701). Moreover, the scATAC-seq analysis does not demonstrate that the cells analysed are irreversibly excluded from a myocardial fate at these early stages. Instead, their data likely reflect chromatin reconfiguration within a subset of posterior epiblast cells in response to signaling.
We have clarified our mention of Foxa2 lineage tracing. In a previous manuscript (Ivanovitch et al. 2021), we identified a Foxa2+/T+ primitive streak (PS) region that contributes to the LV myocardium but not to the endocardial lineage at the midstreak stage, further supporting the finding that a population of uni-LV/AVC-fated progenitors exists.
Minor comments:
- For all figures, annotations, axes and/or schematics would greatly help readers outside the field to locate the regions of interest within the embryo.
We have added axes on all our figures and added annotated.
- Interesting questions that could be easily addressed and added in the manuscript: are mother cells T-nGFP positives? If so, do they have different levels of GFP expression? From the different movies, is there a hot spot of cell division? What is the frequency of progenitors that adopt a sustained interaction with their sister cells?
We thank the reviewer for these great suggestions. We will analyse the nGFP signals in mother cells and test whether those that are nGFP+ exhibit different levels of GFP expression. We are particularly interested on this question since we hypothesised in our previous manuscript (Ivanovitch et al., 2021, Figure 1J-K and S4 Fig) that LV progenitors express lower levels of T/Bra and, consequently, lower levels of nGFP expression compared to Atria progenitors. Furthermore, we will analyse the frequency of progenitors that adopt sustained interactions with their sister cells.
We also explored the reviewer's suggestion to analyse whether there is a hotspot of cell division. However, we found this analysis to be complex and will require spatial and temporal registration of the embryos. We feel this falls outside the scope of the present manuscript. That said, we fully agree with the reviewer that this is an intriguing question.
Reviewer #3 (Significance (Required)):
The manuscript presents a technically original study, offering one of the first prospective clonal analyses of cardiac progenitors during mouse gastrulation. While previous studies have addressed the fate of cardiac progenitors using retrospective clonal analysis or lineage tracing (e.g., Meilhac et al., 2004; Devine et al., 2014; Lescroart et al., 2014; Bardot et al., 2017; Ivanovitch et al., 2021; Tyser et al., 2021; Zhang et al., 2021), this work provides new insights into the temporal and spatial dynamics of cardiac progenitor migration and fate allocation. Notably, the study's investigation of the pericardium-a rarely studied cardiac mesodermal fate-adds significant novelty.
However, a limitation of the study is its focus on a relatively small region of the heart, primarily the left ventricle, atrioventricular canal, and atrium, which may not fully represent the broader diversity of cardiac progenitor behaviors across other regions of the developing heart. Additionally, the lack of markers for non-myocardial cell lineages leaves open questions regarding the full spectrum of progenitor fates. These aspects could be addressed in future studies to provide a more comprehensive understanding of cardiac development.
A complementary preprint by the Torres group (Sendra et al., 2024) combines retrospective and prospective clonal analyses and highlights the multipotency of early mesodermal progenitors, particularly those contributing to non-cardiac fates. While both studies reveal the plasticity of early mesoderm, this manuscript by Abukar et al. focuses specifically on cardiac progenitors, offering unique insights into their behaviors and fate decisions.
The study is poised to have a broad impact on the fields of cardiac development and early mouse development. The tools and concepts developed here could also find applications in broader developmental biology studies. This review is written with expertise in cardiac development. I do not have sufficient expertise to evaluate computational modeling within the manuscript.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
In their manuscript, Abukar et al. investigate the origins and migratory behaviors of cardiac progenitor cells, in mice, from gastrulation to early heart tube formation. They use sophisticated live imaging to tracks individual mesodermal cells, reconstructing their lineage and fate over several generations. The findings reveal distinct unipotent progenitors that contribute exclusively to specific cardiac regions, such as the left ventricle/atrioventricular canal (LV/AVC) or atrial cardiomyocytes. LV/AVC progenitors differentiate early, forming the cardiac crescent, while atrial progenitors differentiate later, contributing to the …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
In their manuscript, Abukar et al. investigate the origins and migratory behaviors of cardiac progenitor cells, in mice, from gastrulation to early heart tube formation. They use sophisticated live imaging to tracks individual mesodermal cells, reconstructing their lineage and fate over several generations. The findings reveal distinct unipotent progenitors that contribute exclusively to specific cardiac regions, such as the left ventricle/atrioventricular canal (LV/AVC) or atrial cardiomyocytes. LV/AVC progenitors differentiate early, forming the cardiac crescent, while atrial progenitors differentiate later, contributing to the venous poles of the heart tube. Additionally, the study identifies multipotent mesodermal progenitors contributing to various mesodermal cell types, including the endocardium, pericardium and extraembryonic tissues.
Major comments:
- Important conclusions of the manuscript rely on the expression of a reporter line (cTnnt2-2a-eGFP) as well as on the position of tdTomoto+ cells in relation to the reporter. We feel that markers of non-myocardial lineages should have been used to better characterize these populations. We acknowledge the technical challenge of live imaging, which may not allow labeling of all lineages. We believe that a better description of the final stages of investigation with markers of endocardium, pericardium, extra-embryonic mesoderm together with the eGFP of the reporter will strengthen the conclusions drawn on the multipotency of the progenitors. If not addressed, some claims may appear more speculative and would benefit from being toned down.
- Similarly, since all the results of the manuscript derive from five movies of five independent embryos, it would be important to provide a more detailed description (for example, in a table) of the experimental setup. This could include the timing of tamoxifen induction (+7h or +21h?), the stage of dissection (based on anatomical landmarks rather than dissection stage - see atlas of gastrulation), the duration of the movies, and the stage at the final time point. Providing this information would greatly enhance the ability to robustly compare each movie and ensure reproducibility. Of note, the methods section could benefit from additional clarity. For example, in line 594, the embryo from Movie1 is described as being dissected in the morning, while the next sentence states it was dissected in the afternoon, similar to the embryo in Movie5. To avoid confusion and ensure greater rigor, describing the developmental stage of the embryos rather than the time of dissection would be more precise and biologically meaningful.
- This manuscript focuses primarily on LV/AVC progenitors and likely a subpopulation of atrial cardiomyocytes, leaving other cardiac progenitor populations unaddressed. While it is understandable that the study focuses on specific populations, the authors should further discuss the limitations of their approach and explain why not all cardiac progenitors were targeted. A discussion of how these limitations might impact the broader interpretation of their findings would also be valuable.
- Since a recent preprint (Sendra et al.), already cited in the manuscript, used complementary approaches to investigate endothelial/endocardial cell fate during gastrulation, we feel that a more in-depth discussion is warranted. In particular, how the results presented here align with the early segregation between endocardial and myocardial lineages observed by Sendra et al. could be clarified. Additionally, it is unclear how these findings correlate with Foxa2 lineage tracing. Addressing these points could further strengthen the contextualization and impact of the manuscript.
Minor comments:
- For all figures, annotations, axes and/or schematics would greatly help readers outside the field to locate the regions of interest within the embryo.
- Interesting questions that could be easily addressed and added in the manuscript: are mother cells T-nGFP positives? If so, do they have different levels of GFP expression? From the different movies, is there a hot spot of cell division? What is the frequency of progenitors that adopt a sustained interaction with their sister cells?
Significance
The manuscript presents a technically original study, offering one of the first prospective clonal analyses of cardiac progenitors during mouse gastrulation. While previous studies have addressed the fate of cardiac progenitors using retrospective clonal analysis or lineage tracing (e.g., Meilhac et al., 2004; Devine et al., 2014; Lescroart et al., 2014; Bardot et al., 2017; Ivanovitch et al., 2021; Tyser et al., 2021; Zhang et al., 2021), this work provides new insights into the temporal and spatial dynamics of cardiac progenitor migration and fate allocation. Notably, the study's investigation of the pericardium-a rarely studied cardiac mesodermal fate-adds significant novelty.
However, a limitation of the study is its focus on a relatively small region of the heart, primarily the left ventricle, atrioventricular canal, and atrium, which may not fully represent the broader diversity of cardiac progenitor behaviors across other regions of the developing heart. Additionally, the lack of markers for non-myocardial cell lineages leaves open questions regarding the full spectrum of progenitor fates. These aspects could be addressed in future studies to provide a more comprehensive understanding of cardiac development.
A complementary preprint by the Torres group (Sendra et al., 2024) combines retrospective and prospective clonal analyses and highlights the multipotency of early mesodermal progenitors, particularly those contributing to non-cardiac fates. While both studies reveal the plasticity of early mesoderm, this manuscript by Abukar et al. focuses specifically on cardiac progenitors, offering unique insights into their behaviors and fate decisions.
The study is poised to have a broad impact on the fields of cardiac development and early mouse development. The tools and concepts developed here could also find applications in broader developmental biology studies. This review is written with expertise in cardiac development. I do not have sufficient expertise to evaluate computational modeling within the manuscript.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
The authors perform an elegant "tour de force" lineage relationships during mouse heart development. They perform long-term live imaging and single-cell tracking in mouse embryos from early gastrulation to stages of heart tube formation. They then track the progeny of individual cells and reconstruct the lineage tree of tracked cells. They analyze how their migratory paths of cells correlate with cell fate in the heart. Altogether, the manuscript presents a highly detailed live-imaging lineage tracing study of a subset of cells in the cardiac crescent in mouse. This presents a nice contribution to the literature, but would be …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
The authors perform an elegant "tour de force" lineage relationships during mouse heart development. They perform long-term live imaging and single-cell tracking in mouse embryos from early gastrulation to stages of heart tube formation. They then track the progeny of individual cells and reconstruct the lineage tree of tracked cells. They analyze how their migratory paths of cells correlate with cell fate in the heart. Altogether, the manuscript presents a highly detailed live-imaging lineage tracing study of a subset of cells in the cardiac crescent in mouse. This presents a nice contribution to the literature, but would be improved by the suggestions below.
Major comments:
- Can the authors be sure they can track all of the derivatives of labeled cells? They are claiming to be able to follow complete lineages, but I worry if they may lose progeny in their tracking or incorrectly conclude that cells are lineally related. wonder how you could show how accurate it really is. Perhaps if the authors could include a movie where they trace what they claim as an entire lineage of a single cell and show this with the mother and daughter cells labelled throughout the movie, that would at least provide an example for readers to make their own decisions about how reliable the lineage tracing is. Would it be feasible to include an interactive movie where the reader can move the embryo around in 3D at each time point?
- The authors describe the lineage labeled cells as unipotent, bipotent, etc. But they cannot really say anything about developmental potential as they are only looking at normal fate which is less that their potential. Without manipulation of the cells through transplantation etc., the use of the term 'potential' or 'potent' is not appropriate except when they find cells that are multipotent. Rather than calling cells unipotent, I would suggest using the phrase 'assume a single fate'.
- Lines 112-115, the authors state that variability in embryonic stages likely explains differences in labelling. Are there any morphological characteristics across the embryos that support this variability in stages? For example, any characteristics that suggest that the n=3 embryos are slightly older, and the n=7 embryos are slightly younger (line 111)?
- Paragraph beginning on line 116: Please clarify how cells were counted, from the wholemount/across sections?
- Line 165: Authors state that in the absence of tamoxifen, tdTomato-positive cells were identified in one embryo. Please state here the total number of embryos out of which this one embryo was counted.
- Line 190: 'Figure 2-Supplementary Figure 3A-F' doesn't exist. Do they mean Fig.3 supplementary 3A-F?
- Figure 1F-G: For cross sections in 'G' please show the level they were taken from in 'F'.
- Figure 4I: There is a large disparity in cell dispersion across movies. Please comment on why this could be. Is there a difference in stage/morphology etc..
- Figure 4K-L: The arrowhead color is too similar to the cell fluorescence color, making the visualization a little confusing. Changing the color of the arrowheads may be helpful. This is also true for some of the other figures (red arrowheads).
Significance
This is a well-done study that will be useful to developmental biologists as well as cardiologists. The experiments seem very well done and beautifully executed. With the proposed modifications, it will make a very nice contribution to the literature.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The manuscript describes the tracking of individual mesoderm cells through live imaging. Through a combination of reporters including a novel cardiomyocyte reporter and a combined nuclear GFP-inducible Cre reporter under the dependance of the Brachyury promoter, the authors label mesoderm cells at different stages of gastrulation then perform long term (>30h) live imaging of late gastrulation embryo up to the cardiac crescent and heart tube stages. They use elaborate analysis tools as well as manual tracking to reconstruct cells' trajectory, lineage trees, and various behavioral traits.
The study is well designed. Experiments are …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The manuscript describes the tracking of individual mesoderm cells through live imaging. Through a combination of reporters including a novel cardiomyocyte reporter and a combined nuclear GFP-inducible Cre reporter under the dependance of the Brachyury promoter, the authors label mesoderm cells at different stages of gastrulation then perform long term (>30h) live imaging of late gastrulation embryo up to the cardiac crescent and heart tube stages. They use elaborate analysis tools as well as manual tracking to reconstruct cells' trajectory, lineage trees, and various behavioral traits.
The study is well designed. Experiments are technically challenging, well executed, and carefully analysed.
Methods are clear and complete so that experiments should be faithfully reproduced provided availability of an appropriate microscope.
The description of the results of the live imaging experiments is not easy to read and understand, but I believe this is inherent to the complexity of the results themselves and due to the high diversity of behaviors observed. Similarly the figures are extremely dense ans some graphs would benefit from a more didactic legend.
I realize the difficulty of being more concise due to the large amount of information and its diversity. If possible, I would suggest integrating tables within the results section that may help shorten the text, and may be easier to grasp.
The interpretation of the results is fair and in line with previous studies, which are adequately cited.
A discussion on the reasons why a large proportion of cells could not determined as uni or multipotent might be useful. Instinctively I would imagine that a majority of those are multipotent and therefore garder to track, so if the authors do not agree with this interpretation it may be useful to detail technical reasons why those cells cannot be fully interpreted.
Significance
Strengths: novel transgenic tools, powerful imaging technique, thorough quantified nalysis.
Limitations: the development of embryos after E7.75-E8 is never completely normal ex vivo, particularly when there is no rotation. This is visible in the pictures of the embryos post culture (ballooned yolk sac, unattached allantois). It is probably not a limitation regarding cardiac development but may influence other mesoderm lineages notably ExE.
Advance: It is a unique study dur to the labelling strategy, the length of imaging, and thereby the faithful tracking of cell lineages across several rounds of division. The information provided corroborates what previous hypothesis in the field based on less direct assessment, and is here very strong and unbiased. The research is of great interest for developmental biologists (including but not limited to the heart field), cell biologists (notably those working on stem cells and organoids as it provides a ground truth), microscopy and image analysis experts.
-
-