A cell atlas of the developing human outflow tract of the heart and its adult derivatives
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eLife Assessment
This study provides valuable insights into human valve development by integrating snRNA-seq and spatial transcriptomics to characterize cell populations and regulatory programs in the embryonic and fetal outflow tract. The methods, data, and analyses are solid overall, but with some weaknesses that can be strengthened. The findings will be of interest to those who work in the field of heart development and congenital heart disease.
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Abstract
The outflow tract (OFT) of the heart carries blood away from the heart into the great arteries. During embryogenesis, the OFT divides to form the aorta and pulmonary trunk, creating the double circulation present in mammals. Defects in this area account for one-third of all congenital heart disease cases. Here, we present comprehensive transcriptomic data on the developing OFT at two distinct timepoints (embryonic and fetal) and its adult derivatives, the aortic valves, and use spatial transcriptomics to define the distribution of cell populations. We uncover that distinctive embryonic signatures persist in adult cells and can be used as labels to retrospectively attribute relationships between cells separated by a large time scale. Single- cell regulatory network inference identifies GATA6, a transcription factor linked to common arterial trunk and bicuspid aortic valve, as a key regulator of valve precursor cells. Its downstream network reveals candidate drivers of human cardiac defects and illuminates the molecular mechanisms of both normal and pathological valve development. Our findings define the cellular and molecular signatures of the human OFT and its distinct cell lineages, which is critical for understanding congenital heart defects and developing cardiac tissue for regenerative medicine.
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eLife Assessment
This study provides valuable insights into human valve development by integrating snRNA-seq and spatial transcriptomics to characterize cell populations and regulatory programs in the embryonic and fetal outflow tract. The methods, data, and analyses are solid overall, but with some weaknesses that can be strengthened. The findings will be of interest to those who work in the field of heart development and congenital heart disease.
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Reviewer #1 (Public review):
Summary:
The study by Bobola et al reports single-nucleus expression analysis with some supporting spatial expression data of human embryonic and fetal cardiac outflow tracts compared to adult aortic valves. The transcription factor GATA6 is identified as a top regulator of one of the mesenchymal subpopulations, and potential interacting factors and downstream target genes are identified bioinformatically. Additional bioinformatic tools are used to describe cell lineage relationships and trajectories for developmental and adult cardiac cell types.
Strengths:
The studies of human tissue and extensive gene expression data will be valuable to the field.
Weaknesses:
(1) The expression data are largely confirmatory of previous studies in humans and mice. Thus, it is not clear what novel biological insights are …
Reviewer #1 (Public review):
Summary:
The study by Bobola et al reports single-nucleus expression analysis with some supporting spatial expression data of human embryonic and fetal cardiac outflow tracts compared to adult aortic valves. The transcription factor GATA6 is identified as a top regulator of one of the mesenchymal subpopulations, and potential interacting factors and downstream target genes are identified bioinformatically. Additional bioinformatic tools are used to describe cell lineage relationships and trajectories for developmental and adult cardiac cell types.
Strengths:
The studies of human tissue and extensive gene expression data will be valuable to the field.
Weaknesses:
(1) The expression data are largely confirmatory of previous studies in humans and mice. Thus, it is not clear what novel biological insights are being reported. While there is some novelty and impact in using human tissue, there are extensive existing publications and data sets in this area.
(2) Major conclusions regarding spatial localization, differential gene expression, or cell lineage relationships based on bioinformatic data are not validated in the context of intact tissues.
(3) The conclusions regarding lineage relationships are based on common gene expression in the current study and may not reflect cellular origins or lineage relationships that have previously been reported in genetic mouse models.
(4) An additional limitation is the exclusive examination of adult aortic valve leaflets that represent only a subset of outflow tract derivatives in the mature heart. The conclusion, as stated in the title regarding adult derivatives of the outflow tract, is not accurate based on the limited adult tissue evaluated, exclusive bioinformatic approach, and lack of experimental lineage analysis of cell origins.
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Reviewer #2 (Public review):
Summary:
The manuscript by Leshem et al. presents a transcriptomic analysis of the developing human outflow tract (OFT) at embryonic and fetal stages using snRNAseq and spatial transcriptomics. Additionally, the authors analyze transcriptomic data from the adult aortic valve to compare embryonic and adult cell populations, aiming to identify persistent embryonic transcriptional signatures in adult cells. A total of 15 clusters were identified from the embryonic and fetal OFT samples, including three mesenchymal and four endothelial clusters. Using SCENIC analysis on the embryonic snRNAseq data, the authors identified GATA6 as a key regulator of valve precursor cells. Spatial transcriptomic analysis of four fetal OFT sections further revealed the spatial distribution of mesenchymal nuclei, smooth muscle …
Reviewer #2 (Public review):
Summary:
The manuscript by Leshem et al. presents a transcriptomic analysis of the developing human outflow tract (OFT) at embryonic and fetal stages using snRNAseq and spatial transcriptomics. Additionally, the authors analyze transcriptomic data from the adult aortic valve to compare embryonic and adult cell populations, aiming to identify persistent embryonic transcriptional signatures in adult cells. A total of 15 clusters were identified from the embryonic and fetal OFT samples, including three mesenchymal and four endothelial clusters. Using SCENIC analysis on the embryonic snRNAseq data, the authors identified GATA6 as a key regulator of valve precursor cells. Spatial transcriptomic analysis of four fetal OFT sections further revealed the spatial distribution of mesenchymal nuclei, smooth muscle cells, and valvular interstitial cells. Trajectory analysis identified two distinct developmental origins of fetal mesenchymal cells: the neural crest and the second heart field. Finally, the authors used snRNAseq data from the adult aortic valve to propose that embryonic transcriptional signatures persist in a subset of adult cells.
Strengths:
(1) The study offers a rich and detailed dataset, combining snRNA-seq and spatial transcriptomics in human embryonic and fetal OFT, which are challenging to obtain.
(2) The use of SCENIC and trajectory analysis adds mechanistic insight into cell lineage and regulatory programs during valve development.
(3) This study confirms GATA6 as a key regulator of valve precursor cells.
(4) Comparison between embryonic/fetal and adult datasets represents a novel attempt to trace persistence of developmental transcriptional programs.
Weaknesses:
(1) A major limitation is the lack of experimental validation to support key conclusions, particularly the claim of persistent embryonic transcriptional signatures in adult cells.
(2) The manuscript would benefit from a clearer discussion of how these results advance beyond previous studies in human heart and valve development.
(3) The comparison between embryonic and adult data is interesting, but would be more convincing with additional evidence supporting the proposed persistence of embryonic transcriptional signatures in adult cells.
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Reviewer #3 (Public review):
Leshem et al have generated a transcriptional cell atlas of the human outflow tract at two developmental timepoints and its adult valvular derivatives. This carefully performed study provides a useful resource for the study of known genes implicated in outflow tract defects and potentially also for discovering new disease genes. The authors reveal neural crest and mesodermal contributions to different outflow tract components and show that GATA6, known to play a role in arterial valve development, controls a set of genes expressed in endocardium-derived cells during valve development. Interestingly, the results suggest lineage persistence of expression of certain genes through to the adult timepoint, a main new finding of this study.
The following points should be addressed to reinforce the conclusions and …
Reviewer #3 (Public review):
Leshem et al have generated a transcriptional cell atlas of the human outflow tract at two developmental timepoints and its adult valvular derivatives. This carefully performed study provides a useful resource for the study of known genes implicated in outflow tract defects and potentially also for discovering new disease genes. The authors reveal neural crest and mesodermal contributions to different outflow tract components and show that GATA6, known to play a role in arterial valve development, controls a set of genes expressed in endocardium-derived cells during valve development. Interestingly, the results suggest lineage persistence of expression of certain genes through to the adult timepoint, a main new finding of this study.
The following points should be addressed to reinforce the conclusions and emphasize the novel features of this study.
(1) It would be helpful to clarify how these new findings confirm or diverge from what is known from analysis of neural crest and mesodermal lineage contributions to different cell populations in the mouse heart. Did the authors identify any human-specific populations of cells, such as the LGR5 population reported by Sahara et al?
(2) The authors should clarify in the introduction and results that they consider the endocardium to be on the SHF trajectory as indicated in Figure S4C. Please add a reference for this point.
(3) The GATA6 results are interesting and support this experimental approach. The paper would be reinforced if the authors could provide any functional validation (in addition to their GATA6 genomic occupancy data) that the designated target genes are regulated by GATA6. This might involve looking at mutant mouse embryos or cultured cells. Do the authors consider that GATA6 may regulate the endocardial to mesenchymal transition during the early stages of valve development? Or the valve interstitial cell versus fibroblast fate choice?
(4) Do the new findings reveal whether human valves have a direct SHF to VIC trajectory (ie, without transiting through endocardium) as has been recently shown in the murine non-coronary valve leaflet? Relevant to this point, Figure 5E appears to show contributions to a single adult aortic valve leaflet - this should be explained, or corrected.
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Author response:
We thank the editors and reviewers for the time and effort they have invested in evaluating our manuscript. We appreciate the constructive feedback, which highlights both the strengths of the work and areas for improvement. We will carefully consider all comments and, in the coming months, revise the manuscript to incorporate additional data, address the concerns regarding limited referencing, and provide further clarification on the points raised.
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