Single-cell analysis of cardiogenesis reveals basis for organ-level developmental defects

Abstract

Organogenesis involves integration of diverse cell types; dysregulation of cell-type-specific gene networks results in birth defects, which affect 5% of live births. Congenital heart defects are the most common malformations, and result from disruption of discrete subsets of cardiac progenitor cells1, but the transcriptional changes in individual progenitors that lead to organ-level defects remain unknown. Here we used single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular subpopulations has catastrophic consequences. A network-based computational method for single-cell RNA-sequencing analysis that predicts lineage-specifying transcription factors2,3 identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite the failure of right ventricular formation in Hand2-null mice4. Temporal single-cell-transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium was specified but failed to properly differentiate and migrate. Loss of Hand2 also led to dysregulation of retinoic acid signalling and disruption of anterior–posterior patterning of cardiac progenitors. This work reveals transcriptional determinants that specify fate and differentiation in individual cardiac progenitor cells, and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework for investigating congenital heart defects.

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Fig. 1: Single-cell RNA sequencing reveals heterogeneity of cardiogenic regions during early embryonic development.
Fig. 2: Analysis of CPC populations reveals early specification dynamics of myocardial subtypes.
Fig. 3: Transcriptional dysregulation in Hand2-null embryos reveals ectopic retinoic acid signalling with posteriorization of AHF derivatives.
Fig. 4: Loss of Hand2 disrupts OFT myocardial cell specification and RV myocardial cell differentiation and migration.

Data availability

All source data, including sequencing reads and single-cell expression matrices have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number GSE126128. Data underlying each figure are available as Source Data, in the Supplementary Information and on the UCSC cell browser at https://mouse-cardiac.cells.ucsc.edu. Users can use the cell browser to explore the data, view expression of genes of interest in each UMAP plot and download datasets for custom analysis.

Code availability

All analyses were performed using standard protocols with previously described R packages6,7. The R scripts are available upon request.

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Acknowledgements

We thank B. Bruneau and members of the Srivastava laboratory for helpful discussion and feedback and C. Trapnell for guidance on single-cell RNA-sequencing analysis. We acknowledge the Gladstone Histology and Light Microscopy Core, the Gladstone Genomics Core and the Gladstone Bioinformatics Core for their technical expertise and the Gladstone Animal Facility for support with mouse colony maintenance. We thank T. Marsh, J. Debnath, H. Yin, F. Chanut, B. Taylor, T. Roberts and G. Maki for their assistance with imaging, literature review, editing and graphics. We thank M. Haeussler and M. Speir for formatting and hosting the processed datasets on the UCSC cell browser. T.Y.d.S. was supported by the UCSF Chancellor’s Fellowship, Genentech Foundation Fellowship, Discovery Fellows Program and Phi Beta Kappa Graduate Scholarship. C.A.G. is a HHMI fellow of the Damon Runyon Cancer Research Foundation (DRG-2206-14). S.S.R. is a Winslow Fellow. D.S. is supported by the National Heart Lung and Blood Institute (R01 HL057181, P01 HL089707, UM1HL098179, UM1HL128761), the California Institute for Regenerative Medicine (DISC2-09098), the Roddenberry Foundation, the L. K. Whittier Foundation, and the Younger Family Fund. S.O. is supported by an FNR CORE grant (C15/BM/10397420) and S.R. is supported by the University of Luxembourg IRP Grant (R-AGR-3227-11). This work was also supported by NIH/NCRR grant C06 RR018928 to the Gladstone Institutes.

Author information

T.Y.d.S., C.A.G. and D.S. conceived the study, interpreted the data and wrote the manuscript. T.Y.d.S. prepared chromium libraries, performed in situ hybridization experiments and analysed data with Seurat and Monocle. T.Y.d.S. and S.S.R. dissected and processed embryos for single-cell library preparation and in situ hybridization experiments and conducted whole-mount and section imaging. T.Y.d.S. and A.S. performed genotyping of mice. S.O. and S.R. performed, and A.d.S. conducted the computational modelling for cell-fate determinant predictions. Y.H. and H.T.S. identified pregnant female mice by echocardiography.

Correspondence to Casey A. Gifford or Deepak Srivastava.

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Competing interests

D.S. is a co-founder and member of the board of directors of Tenaya Therapeutics and has equity in Tenaya Therapeutics. The other authors declare no competing interests.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Genes associated with congenital heart defects are enriched in specific cardiac populations.

a, b, UMAP plot of all captured cell populations coloured by cluster (a) and embryonic stage of collection (b). c, UMAP feature plot showing expression of marker genes used to identify and remove endoderm (Epcam), ectoderm (Sox2) and blood (Hbb-y) cell populations. Statistics for differential gene expression tests were applied to n = 36,777 cells. d, UMAP plot of all mesodermal and neural crest populations captured at E7.75, E8.25 and E9.25 coloured by cluster identity as in Fig. 1d. e, Expression of Flt4 and Upp1 in endocardium or endothelium population. f, Expression of Rrad, Ank3 and Prkaa2 in subpopulations of the myocardium. Statistics for differential gene expression tests were applied to n = 21,366 cells. All genes represented in c, e, f, have a Bonferroni correction adjusted P < 1 × 10−4 (two-sided Wilcoxon rank-sum test). Source data

Extended Data Fig. 2 Focused analyses of cardiac populations.

Schema of progressive subdivisions of broadly clustered cell populations from Fig. 1d that are discussed in the manuscript.

Extended Data Fig. 3 Spatial validation of marker gene expression by in situ hybridization.

a, Ventral view of Tdgf1 and Tnnt2 expression in the cardiac crescent (CC), right lateral views of Wnt5a and Bmp4 in the OFT, Mab21l2 and Shox2 in the SV, and Hoxa1 and Hoxb1 in the pSHF by in situ hybridization at the indicated embryonic stages, which informed assignment of population identities in Extended Data Figs. 4c, 5a. b, Expression of Tbx1 in the SHF and pharyngeal arches (PA) and of novel unannotated gene 3632451O06Rik in the OFT at E8.25 and E9.25. Scale bars indicate 200 μm unless otherwise noted. c, Expression of Rgs5 and Isl1 in the SHF, Hand2 in the SHF, OFT and RV, and Tbx18 in the proepicardial organ (PEO) of E9.25 embryos. d, In situ hybridization of mRNA expression of Isl1, Fgf8 and Hoxb1 at E8.25 and Nr2f2 at E9.25 in right lateral histologic sections. Scale bars, 50 μm. n = 2 independent embryos per gene for all panels.

Extended Data Fig. 4 Heterogeneity in endocardium or endothelium and multipotent progenitor populations.

a, UMAP plot of reclustered endocardium or endothelium population coloured by cluster and embryonic stage of collection. b, Violin plot of markers indicating distinct subpopulations of endocardium or endothelial cells. Summary statistics reported in violin plots: the centre white line represents median gene expression and the central black rectangle spans the first quartile to the third quartile of the data distribution. The whiskers above or below the box indicate values that are 1.5× the interquartile range above the third quartile or below the first quartile. Statistics for differential gene expression tests were applied to n = 2,199 cells. c, UMAP plot of reclustered multipotent progenitor populations coloured by cluster and embryonic stage of collection. d, Heat map showing curated list of marker genes that identify pSHF, AHF and branchiomeric muscle progenitors. Scale indicates z-scored expression values. Statistics for differential gene expression tests were applied to n = 5,376 cells. HE, haematoendothelial progenitors; EndMT, endothelial–mesenchymal transition cells. All genes represented in b, d, have a Bonferroni correction adjusted P < 1 × 10−4 (two-sided Wilcoxon rank-sum test). Source data

Extended Data Fig. 5 Focused analyses of myocardial populations and spatial validation of RV markers.

a, UMAP plot of reclustered ‘myocardium’ population coloured by cluster and embryonic stage of collection. b, Heat map of highly and uniquely expressed genes in myocardial subpopulations. Scale indicates z-scored expression values. Statistics for differential gene expression tests were applied to n = 6,474 cells. c, UMAP plot of reclustered ventricle populations coloured by cluster and embryonic stage of collection. d, Heat map showing curated list of genes that identify LV, RV and early RV cells. Scale indicates z-scored expression values. Statistics for differential gene expression tests were applied to n = 1,976 cells. All genes represented in b, d, have a Bonferroni correction adjusted P < 1 × 10−4 (two-sided Wilcoxon rank-sum test). e, mRNA expression of LV marker Hand1 (green) and Pln (red) in frontal view of the E9.5 heart showing enrichment of Pln in the RV region by whole-mount in situ hybridization. n = 2 independent embryos per gene. Scale bar, 200 μm. f, Breeding scheme for lineage-tracing Cck expressing cells. g, mRNA expression of endogenous Cck and TdTomato driven by Cck-cre transgene at E9.25 in right oblique view of the heart. n = 2 independent embryos per gene. Scale bars, 200 μm. h, Expression of TdTomato in whole-mount and sectioned P1 heart from Ai14 × Cck-cre lineage-traced mice showing location of progeny of Cck-expressing cells. Left panels show bright-field view (top) or TdTomato (bottom) of whole-mount P1 heart; right panels show sections of TdTomato and DAPI (top) or TdTomato alone (bottom) in P1 heart section. n = 2 independent embryos. Scale bars, 100 μm. A, atria; V, ventricle. Source data

Extended Data Fig. 6 Pseudotemporal ordering of myocardium populations.

ad, Pseudotime trajectory of myocardium populations coloured by pseudotime value (a), cluster identity (b), embryonic stage of collection (c) and cell state (d). Pseudotime trajectory analysis was applied to n = 6,474 cells. e, Percentage of cells in each state that were captured at E7.75, E8.25 or E9.25. f, Violin plots showing expression of Nppa and Fgf8 in state e and state f from pseudotime trajectory in d. Statistics for differential gene expression tests were applied to n = 455 cells from each state. Bonferroni correction adjusted P < 1 × 10−4 (two-sided Wilcoxon rank-sum test). Summary statistics reported in violin plots: the centre white line represents median gene expression and the central black rectangle spans the first quartile to the third quartile of the data distribution. The whiskers above or below the box indicate values that are 1.5× the interquartile range above the third quartile or below the first quartile. Source data

Extended Data Fig. 7 Endoderm populations adjacent to the cardiac crescent.

a, UMAP plot of endoderm populations captured at E7.75 coloured by cluster. b, Dot plot highlighting expression patterns of known and novel endodermal secreted factors, Fgf8, Bmp4, Bmp2 and Wnt5a. The size of the dot indicates the percentage of cells expressing that gene within a cluster (% exp) and the colour indicates the average expression level of that gene within a cluster. c, Expression heat map of the top-ten marker genes of each endodermal population and secreted factors from b. Scale indicates z-scored expression values. Statistics for differential gene expression tests were applied to n = 915 cells. All genes represented have a Bonferroni correction adjusted P < 1 × 10−4 (two-sided Wilcoxon rank-sum test). Source data

Extended Data Fig. 8 Transcriptional perturbation in Hand2-null embryos.

a, Heat map of marker genes of populations from Fig. 3a. Statistics for differential gene expression tests were applied to n = 13,185 cells. b, c, Heat map of differentially expressed genes between wild-type and Hand2-null OFT (b) and RV (c) cells captured at E7.75 and E8.25. Statistics were applied to n = 253 OFT cells per genotype at E7.75, n = 276 OFT cells per genotype at E8.25, n = 132 RV cells per genotype at E7.75, and n = 331 RV cells per genotype at E8.25. Scale indicates z-scored expression values. d, Quantification of fluorescence signal for indicated genes in f and Fig. 3h–j. n = 3 independent embryos per genotype. The mean ± s.e.m. is indicated. Two-tailed t-test. *P < 0.05 and **P < 0.01. e, Violin plots showing expression of Smyd1 and Sema3c in wild-type and Hand2-null E8.25 AHF cells. f, Expression of Hoxb1 in wild-type and Hand2-null embryos at E9.25 by whole-mount in situ hybridization in right lateral view. Arrowheads indicate expanded anterior Hoxb1 expression in Hand2-null embryos. Scale bars, 500 μm. n = 3 independent experiments with similar results. g, Proportion of wild-type and Hand2-null cells from each population captured at E7.75. n = 5 wild-type embryos and n = 3 Hand2-null embryos. The mean ± s.e.m. is indicated. Two-tailed t-test. *P < 0.05 and **P < 0.01. h, Violin plots showing expression of Nppa and Nppb in E8.25 wild-type and Hand2-null RV cells. All genes in ac, e, h have a Bonferroni correction adjusted P < 1 × 10−4 (two-sided Wilcoxon rank-sum test). Violin plot summary statistics: centre white line represents median gene expression and central black rectangle spans the first to third quartile of the data distribution. The whiskers indicate values that are 1.5× the interquartile range above the third quartile or below the first quartile. Source data

Extended Data Fig. 9 RV cells are present in Hand2-null embryos at E9.25.

a, Violin plots showing expression of Nppa and Nppb in RV states 1 and 2 from Fig. 4c. Statistics for differential gene expression tests were applied to n = 251 cells from each state. b, c, UMAP plot of subset of cardiac populations captured at E9.25 coloured by cluster (b) and genotype (c). d, Curated list of highly and uniquely enriched genes in cardiac populations at E9.25. Scale indicates z-scored expression values. e, UMAP feature plot showing expression domains of Irx4, Cited1 and Cck, indicating presence of LV and RV at E9.25. Statistics for differential gene expression tests for d and e were applied to n = 5,211 cells. f, Violin plots of genes differentially expressed in wild-type versus Hand2-null AHF cells captured at E9.25. Isl1 is shown to indicate equivalent expression, and thus progenitor identity, in wild-type and Hand2-null cells. ns, not significant. All other genes represented in a, df, have a Bonferroni correction adjusted P < 1 × 10−4 (two-sided Wilcoxon rank-sum test). Violin plot summary statistics: centre white line represents median gene expression and the central black rectangle spans the first quartile to the third quartile of the data distribution. The whiskers indicate values that are 1.5× the interquartile range above the third quartile or below the first quartile. Source data

Extended Data Fig. 10 RV cell migration is impaired in Hand2-null embryos.

a, b, Whole-mount in situ hybridization for Irx4 and Cck in right lateral view (a) and transverse sections (b) at E9.25, indicating presence of RV cells in Hand2 mutants (arrowheads). n = 2 independent experiments with similar results. Scale bars, 200 μm. c, Quantification of Sema3c fluorescence signal in Fig. 4f. n = 3 replicate embryos per genotype. The mean ± s.e.m. indicated. Two-tailed t-test: *P < 0.05. d, e, Violin plots of Wnt5a and Tbx2 expression in wild-type and Hand2-null RV cells at E7.75 and E8.25, respectively (d), and Hand1 and Hand2 expression in LV and OFT cells at E9.25 (e). All genes in d and e have a Bonferroni correction adjusted P < 1 × 10−4 (two-sided Wilcoxon rank-sum test). Violin plot summary statistics: centre white line represents median gene expression and central black rectangle spans the first to third quartile of the data distribution. Whiskers indicate values that are 1.5× the interquartile range above the third quartile or below the first quartile. f, In situ hybridization for Hand1 in wild-type and Hand2-null embryos at E9.25 in frontal view. n = 3 independent experiments with similar results. g, Quantification of Hand1 fluorescent signal in the OFT. n = 3 replicated embryos per genotype. The mean ± s.e.m. is indicated. Two-tailed t-test. *P < 0.05. h, GO biological process terms of differentially expressed genes in wild-type and Hand2-null AHF (n = 406 cells per genotype), OFT (n = 362 cells per genotype) or RV (n = 227 cells per genotype) cells at E9.25, as determined with DAVID v.6.8. Significant functional enrichment was statistically determined using a modified Fisher’s exact test (EASE score) followed by Benjamini–Hochberg correction for multiple comparisons, with 0.01 as a P-value cut-off. Source data

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1Summary of single-cell RNA-sequencing data. This table summarizes features of analyzed cells at each embryonic stage for each biological replicate of wild type and Hand2-null embryos (Table 1a). This table also contains information about cells captured from wild type and Hand2-null embryos at each time point for each population discussed in the manuscript (Table 1b).

Supplementary Table

Supplementary Table 2Wild type cardiogenesis analysis. This table lists differentially expressed genes used to identify and distinguish populations discussed in Fig. 1, Fig. 2, and Extended Data Fig. 4, 5, and 7. Several genes have been reported to be cell type specific in the heart at the embryonic stages under study, and these reports are referenced herein. This table also lists differential gene expression analyses for the myocardium pseudotime analysis (Extended Data Fig. 6d, f), Pitx2 high vs low expressing cells, and the cell fate prediction method. Differentially expressed genes between populations were statistically determined using the Wilcoxon rank sum test (two-sided) for single-cell gene expression with Bonferroni correction and an adjusted p-value cut-off < 1x10-4. The exact number of cells (n) to which statistics for differential gene expression tests were applied is indicated in each sheet or sheet tab.

Supplementary Table

Supplementary Table 3Hand2-null cardiogenesis analysis. This table lists differentially expressed genes between wild type and Hand2-null populations, by embryonic stage, as well as marker gene lists that were used to assign identities to populations described in Fig. 3, Extended Data Fig. 8, and Extended Data Fig. 9. Differentially expressed genes were statistically determined using the Wilcoxon rank sum test (two-sided) for single-cell gene expression with Bonferroni correction and an adjusted p-value cut-off < 1x10-4. The exact number of cells (n) per genotype to which statistics for differential gene expression tests were applied is indicated in each sheet or sheet tab.

Video 1: Expression of Sema3c and Tbx2 in wild type embryos at E8.25.

3D reconstructed lightsheet videos of replicate wild type embryos indicating expression of Sema3c (red) and Tbx2 (green) by in situ hybridization. n = 2 independent experiments with similar results.

Video 2: Expression of Sema3c and Tbx2 in wild type embryos at E8.25.

3D reconstructed lightsheet videos of replicate wild type embryos indicating expression of Sema3c (red) and Tbx2 (green) by in situ hybridization. n = 2 independent experiments with similar results.

Video 3: Expression of Sema3c and Tbx2 in Hand2-null embryos at E8.25.

3D reconstructed lightsheet videos of replicate Hand2-null embryos indicating expression of Sema3c (red) and Tbx2 (green) by in situ hybridization. n = 2 independent experiments with similar results.

Video 4: Expression of Sema3c and Tbx2 in Hand2-null embryos at E8.25.

3D reconstructed lightsheet videos of replicate Hand2-null embryos indicating expression of Sema3c (red) and Tbx2 (green) by in situ hybridization. n = 2 independent experiments with similar results.

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