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Single-cell multimodal analyses reveal epigenomic and transcriptomic basis for birth defects in maternal diabetes

Nature Cardiovascular Research volume 2, pages 1190–1203 (2023)Cite this article

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Abstract

Maternal diabetes mellitus is among the most frequent environmental contributors to congenital birth defects, including heart defects and craniofacial anomalies, yet the cell types affected and mechanisms of disruption are largely unknown. Here, using multimodal single-cell analyses, we show that maternal diabetes affects the epigenomic landscape of specific subsets of cardiac and craniofacial progenitors during embryogenesis. A previously unrecognized cardiac progenitor subpopulation expressing the homeodomain-containing protein ALX3 showed prominent chromatin accessibility changes and acquired a more posterior identity. Similarly, a subpopulation of neural crest-derived cells in the second pharyngeal arch, which contributes to craniofacial structures, displayed abnormalities in the epigenetic landscape and axial patterning defects. Chromatin accessibility changes in both populations were associated with increased retinoic acid signaling, known to establish anterior–posterior identity. This work highlights how an environmental insult can have highly selective epigenomic consequences on discrete cell types leading to developmental patterning defects.

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Fig. 1: Integrated analysis of scRNA-seq and scATAC-seq reveals highly selective chromatin accessibility alterations in PGDM.
Fig. 2: Maternal diabetes disrupts the epigenomic landscape of craniofacial neural crest cells in the second pharyngeal arch.
Fig. 3: Maternal diabetes epigenetically alters Alx3-positive AHF cells, which contribute to the aortic valve and atrial wall.
Fig. 4: Maternal diabetes epigenetically alters Alx3-positive AHF cells with disruption of A–P patterning.
Fig. 5: Disruption of RA signaling associated with pharyngeal arch and Alx3pos AHF A–P patterning defect.

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Data availability

Data are available in the main text and the supplementary materials. All the sequencing data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number GSE198905. Source data are provided with this paper.

Code availability

All codes are available on GitHub (https://github.com/SrivastavaLab-Gladstone/Nishino_DM_2022).

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Acknowledgements

We thank members of the Srivastava laboratory for discussion and feedback; B. Taylor from Gladstone Institutes for editorial and graphics assistance; G. Maki from Gladstone Institutes for graphics assistance; and K. Claiborn from Gladstone Institutes for editorial review. We acknowledge the Center for Advanced Technology (CAT) for sequencing; the Gladstone Histology and Light Microscopy Core for their technical support; and the Gladstone Animal Facility for support with mouse colonies. Figures 1a and 5g, Extended Data Fig. 1a and Supplementary Fig. 1a were created with BioRender.com. National Institutes of Health/NHLBI grant P01 HL146366, R01 HL057181, R01 HL015100, R01 HL127240, Roddenberry Foundation, L.K. Whittier Foundation, Dario and Irina Sattui, Younger Family Fund, and Additional Ventures to D.S. The Japan Society for the Promotion of Science Overseas Research Fellowship to T.N. Additional Ventures to S.S.R. American Heart Association Postdoctoral Fellowship (#899270) to B.J.v.S. National Institutes of Health grant K08 HL157700, Sarnoff Cardiovascular Research Foundation, Frank A. Campini Foundation and Michael Antonov Charitable Foundation to A. Padmanabhan.

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Authors and Affiliations

  1. Gladstone Institutes, San Francisco, CA, USA

    Tomohiro Nishino, Sanjeev S. Ranade, Angelo Pelonero, Benjamin J. van Soldt, Lin Ye, Michael Alexanian, Frances Koback, Yu Huang, Langley Grace Wallace, Nandhini Sadagopan, Adrienne Lam, Lyandysha V. Zholudeva, Feiya Li, Arun Padmanabhan, Reuben Thomas, Joke G. van Bemmel, Casey A. Gifford, Mauro W. Costa & Deepak Srivastava

  2. Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, CA, USA

    Tomohiro Nishino, Sanjeev S. Ranade, Angelo Pelonero, Benjamin J. van Soldt, Lin Ye, Michael Alexanian, Frances Koback, Yu Huang, Langley Grace Wallace, Nandhini Sadagopan, Adrienne Lam, Lyandysha V. Zholudeva, Feiya Li, Arun Padmanabhan, Joke G. van Bemmel, Casey A. Gifford, Mauro W. Costa & Deepak Srivastava

  3. Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA

    Michael Alexanian & Deepak Srivastava

  4. Division of Cardiology, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA

    Nandhini Sadagopan & Arun Padmanabhan

  5. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Arun Padmanabhan

  6. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA

    Deepak Srivastava

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  1. Tomohiro Nishino
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Contributions

T.N. and D.S. conceived and directed the study. T.N. and Y.H. performed animal work. T.N., L.G.W. and S.S.R. collected heart tissues and isolated single cells for subsequent scRNA-seq and scATAC-seq. T.N., S.S.R., A. Pelonero, B.J.v.S. and F.K. analyzed scRNA-seq and scATAT-seq and developed computational methods. T.N., B.J.v.S., L.V.Z. and F.L. performed RNA in situ hybridization and subsequent tissue clearing and imaging. T.N., L.Y., N.S., A.L., A. Padmanabhan and M.W.C designed, performed and analyzed luciferase assay and mouse lineage trace experiment. T.N., S.S.R., M.A., A. Pelonero, J.G.v.B, C.A.G., M.W.C. and D.S. interpreted the data. R.T. reviewed statistical methods. T.N., M.W.C. and D.S. wrote the manuscript with contributions of M.A.

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Correspondence to Deepak Srivastava.

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Competing interests

D.S. is a scientific co-founder, shareholder and director of Tenaya Therapeutics. The remaining authors declare no competing interests.

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Nature Cardiovascular Research thanks Professor Hiroki Kurihara, and the other, anonymous, reviewer for their contribution to the peer review of this work. Primary Handling Editor: Vesna Todorovic, in collaboration with the Nature Cardiovascular Research team.

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Extended data

Extended Data Fig. 1 Histological and micro-CT validation of the maternal diabetes model.

(A) The design of the in vivo maternal diabetic model experiment. After administration of either VEH or STZ, females in the STZ group with confirmed diabetes were mated with normoglycemic males, and heart samples at embryonic day 18.5 (E18.5) or postnatal day 0 (P0) were collected for histological examination. (B) Representative micro-CT images of the heart phenotypes detected in the diabetic model. The prevalence of each malformation is shown in Supplementary Table 1. The scale bar represents 500 µm.

Extended Data Fig. 2 Single cell multimodal analysis of cardio-pharyngeal region in maternal diabetes.

(A) Representative image of E10.5 embryo with detailed micro-dissected region used for scRNA/scATAC-seq experiment. Scale bar represents 1 mm. (B) scRNA-seq (left) or scATAC-seq (right) UMAP presentation colored by conditions. Different colors overlayed delineate cell type cluster annotations. (C) Expression patterns of representative cell type specific marker genes plotted on UMAP space shown in Fig. 1b. (D) Heatmap of marker gene scores per cluster of scATAC-seq. (E) Heatmap of Jaccard indices calculated between scRNA-seq and scATAC-seq after integration. Values range from 0 to 1 (higher value represents closer annotation matching between the two modalities). (F) Genomic distribution of all the called peaks color coded by the genomic location as shown. Total called peaks = 492,330. (G) Distribution of all called peaks based on the distance from transcription start sites.

Extended Data Fig. 3 Maternal diabetes dysregulates epigenomic landscape of neural crest cells in pharyngeal arches 4 and 6.

(A) Population distribution by sub-cell-type normalized to total number of cells per sample in neural crest cell subset data of scRNA-seq. Numbers inside the barplot represent the percentage of cell types of the total cell number. Statistics performed by permutation test in scRNA-seq data, comparing STZ vs. VEH, for NC-prog, FDR < 0.001, Log2FD = 1.88; for SMC-prog, FDR < 0.001, Log2FD = −0.33. (B) Heatmap of Jaccard indices calculated between neural crest cell scRNA-seq and scATAC-seq cell annotations after integration. Values range from 0 to 1 (the higher value represents closer annotation matching between those two modalities). (C) MA plot of DARs in PA3/4/6 population between VEH and STZ. Red dots represent the more accessible (open) (FDR < = 0.05 & Log2FC > = 1) and blue dots represent less accessible (closed) DARs in STZ (FDR < = 0.05 & Log2FC < = −1). (D) Enriched TF binding motifs in more accessible (left) or less accessible (right) DARs in STZ vs. VEH within the PA3/4/6 population. (E) scATAC-seq UMAP representation of neural crest cell C20 subset population colored by clusters (PA3 – dark red; PA4/6 – dark blue). (F) Heatmap of Gene Scores (GS) of curated marker genes based on scRNA-seq data for PA3 and PA4/6 neural crest. Scale indicates z-scored GS values. (G) MA plot of DARs between VEH and STZ in PA3 population (left) and PA4/6 population (right). Red dots represent the more accessible (open) (FDR < = 0.05 & Log2FC > = 1) and blue dots represent less accessible (closed) DARs in STZ (FDR < = 0.05 & Log2FC < = −1). (H) Enriched TF binding motifs in more accessible (left) and less accessible (right) DARs in STZ in PA4/6 population. NC-prog, neural crest cell progenitors; PA2, pharyngeal arch 2; PA3, pharyngeal arch 3; PA4/6, pharyngeal arch 4/6; SMC, smooth muscle cells; SMC-prog, smooth muscle cell progenitors.

Source data

Extended Data Fig. 4 Maternal diabetes dysregulates epigenomic and transcriptional landscape associated with cell differentiation and patterning in pharyngeal arch 2 neural crest.

(A) Violin plot of Tfap2a expression levels in cluster 0 of UMAP in Fig. 2i across 3 VEH and 3 STZ embryos (Wilcoxon Rank Sum test). (B) Expression of Nr2f1 mRNA on UMAP space for PA2 neural crest cells (VEH – left top; STZ – left bottom). Scale bar indicates z-scored expression values. Violin plot of Nr2f1 expression levels in cluster 0 of UMAP in Fig. 2i (right) (Wilcoxon Rank Sum test). (C) Expression of indicated genes on UMAP space for PA2 neural crest cells. Scale bar indicates z-scored expression values. (D) Violin plots of Dlx5 (left) and Dlx6 (right) expression levels in cluster 0 of UMAP in Fig. 2i (Wilcoxon Rank Sum test). (E) Enriched GO terms in detected DARs in PA2 population using GREAT analysis. (F) Enriched GO terms in detected DARs in PA3/4/6 population using GREAT analysis.

Extended Data Fig. 5 Identification of distinct subsets of AHF progenitors.

(A) scRNA-seq UMAP representation of mesodermal population (‘Meso/CPP’, ‘Cardiomyocyte’, or ‘Epicardium’ in Fig. 1b) colored by conditions (VEH – blue; STZ – light red). (B) Heatmap of Jaccard indices between mesoderm cell scRNA-seq and scATAC-seq annotations after integration. Values range from 0 to 1 (the higher value represents closer annotation matching between those two modalities). CM_V, ventricular cardiomyocyte; CM_AVC, atrioventricular canal cardiomyocyte; CM_A, atrial cardiomyocyte; CM_SV, sinus venosus cardiomyocyte; CM_OFT, outflow tract cardiomyocyte; pSHF1/2, posterior second heart field 1/2; EndoMT, endothelial mesenchymal transition; EpiC, Epicardium; AHF1/2, anterior heart field 1/2; PharyngealMeso, pharyngeal mesoderm; ParaxialMeso1/2, paraxial mesoderm 1/2; BrM, branchiomeric muscle. (C) Expression pattern of Hand2 (left) and Rgs5 (right) on UMAP space. AHF1 and 2 are circled in red. Scale bar indicates z-scored expression values.

Extended Data Fig. 6 Alx3Pos cells are a distinct subset of the AHF population.

(A) Representative images from RNA in situ hybridization for Armh4 (green) and Alx3 (red) in an E10.5 embryo from VEH treated female. The scale bar represents 500 µm. (B) Representative images from whole mount RNA in situ hybridization of E10.5 embryos using light sheet microscopy. Armh4 (green) and Alx3 (red) expression is shown from the dorsal view (D – left) and the right oblique view (O – right). A white bracket (left) highlights the anterior part of Alx3Pos cells. A white dotted oval (right) highlights the Alx3Pos cell streak on left side of the embryo from outflow tract (OFT) towards the posterolateral region. Still images were extracted from Supplementary video 2. Scale bar represents 100 µm. PA2, pharyngeal arch 2; BW, body wall. (C) The distribution of Alx3 positive cells by scRNA-seq between E7.75 and E9.25. (D) scRNA-seq UMAP of cardiac progenitor cells at E9.25 from the same data as (C) color coded by cell type annotation. AHF, anterior heart field; BM progenitors, branchiomeric muscle progenitors; pSHF, posterior second heart field. (E) Expression of Alx3 on the same UMAP as (D). (F) Heatmap of differentially expressed genes (DEGs) between Alx3Neg AHF and Alx3Pos AHF at E9.25. All detected DEGs that attained adjusted p-val < 0.05 and Log2FC > 0.25 are shown. Top GO terms enriched in upregulated or downregulated DEGs are shown with representative genes composing each GO (Fisher’s exact test, corrected for multiple testing using the Benjamini-Hochberg method). Scale bar indicates z-scored expression values. (G) Heatmap presentation of DEGs between AHF1 and AHF2 at E10.5 using only VEH cells in the scRNA-seq data. All detected DEGs that attained adjusted p-val < 0.05 and Log2FC > 0.25 are shown. Top GO terms enriched in upregulated or downregulated DEGs are shown with representative genes composing each GO (Fisher’s exact test, corrected for multiple testing using the Benjamini-Hochberg method). Scale bar indicates z-scored expression values. (H) Venn diagram representing the intersect between DEGs shown in (F-G).

Extended Data Fig. 7 PGDM disrupts anterior-posterior patterning in AHF2.

(A) Enriched GO terms in VEH vs. STZ DARs in AHF2 population using GREAT analysis. (B) Heatmap of marker genes of each of three subclusters found in Alx3Pos AHF2. These marker genes were detected using only VEH-treated Alx3Pos AHF2 cells (left). All marker genes that attained an adjusted p-val < 0.05 and Log2FC > 0.25 are shown. Scale bar indicates z-scored expression values. Top GO terms enriched in marker genes for each sub cluster with statistical information and representative maker genes to corresponding GO term are shown (Fisher’s exact test, corrected for multiple testing using the Benjamini-Hochberg method) (right). (C) Genome browser plots for Hoxb1 locus. The top two rows represent the chromatin accessibility in VEH and in STZ within AHF2. The third track from the top shows the genomic location of the DAR with more accessibility in STZ (red rectangles, highlighted by yellow box). The second track from the bottom represent the links between peaks and gene (‘Peak2GeneLinks’), calculated by ArchR. Darker lines represent stronger links. The bottom track shows the gene location and transcriptional direction (red – positive strand; blue – negative strand).

Extended Data Fig. 8 Enhanced retinoic acid signaling in pharyngeal arch 2 and AHF2 in response to hyperglycemia.

(A) Box plots of the distribution of ChromVAR deviation score for RAR and RXR transcription factor motifs for each cluster in the neural crest cell population. PA2 neural crest cells are highlighted in red. The X-axis shows the distribution of the Z-score. (STZ – red; VEH – blue) (n = 6 biological samples. n = 3 VEH replicates and n = 3 STZ replicates). (B) Box plots of the distribution of ChromVAR deviation score for RAR and RXR transcription factor motifs for each cluster in the mesoderm population. AHF2 cells are highlighted in red. The X-axis shows the distribution of the Z-score. (STZ – red; VEH – blue) (n = 6 biological samples. n = 3 VEH replicates and n = 3 STZ replicates). In the box plots, the central line indicates the median, box bounds represent the 25th and 75th percentiles, whiskers extend to values within 1.5 times the interquartile range, and outliers lie beyond this range.

Extended Data Fig. 9 Disrupted retinoic acid signaling is associated with dysregulation of gene regulatory networks in pharyngeal arch 2 and AHF2.

(A) Dot plot demonstrating the distribution of the WGCNA modules per cluster. X axis shows the Z-score differences of WGCNA module score per cluster between STZ and VEH and Y axis shows the statistical significance of the differences. Modules that are not statistically significant are shown in blue, and those that are statistically significant are shown in green or pink. Red label highlights selected module used for subsequent analysis. (B) Module scores for a gene module detected in the WGCNA analysis that showed statistically significant variation between VEH and STZ only in PA2. Linear mixed effects models with mouse id as the random effect was used to test the significance of the mean difference in the module score between VEH and STZ (n = 6 biological samples. n = 3 VEH replicates and n = 3 STZ replicates) (linear mixed-effects model with Benjamini-Hochberg multiple-testing correction). (C) Map of functional protein-protein interactions (PPI) of genes composing the module described in (B), depicted using STRING. Genes composing a core of the PPI network and being downstream of Tfap2 are highlighted in red and bold. (D) Dot plot demonstrating the distribution of the WGCNA modules per cluster. X axis shows the Z-score differences of WGCNA module score per cluster between STZ and VEH and Y axis shows the statistical significance of the differences. Modules that are not statistically significant are shown in blue, and those that are statistically significant are shown in green or pink. Red label highlights selected module used for subsequent analysis. (E) Module scores for a cardiac gene regulatory module detected in the WGCNA analysis that showed statistically significant variation between VEH and STZ only in AHF2. The same statistical test as (B) was used (n = 6 biological samples. n = 3 VEH replicates and n = 3 STZ replicates) (linear mixed-effects model with Benjamini-Hochberg multiple-testing correction). (F) Map of PPI of genes composing the module described in (E), depicted using STRING. Genes composing a core of the PPI network and being critical cardiac TFs or signaling genes are highlighted in red and bold. NC-prog, neural crest cell progenitors; PA2, pharyngeal arch 2; PA3, pharyngeal arch 3; PA4/6, pharyngeal arch 4/6; SMCs, smooth muscle cells; SMC-Prog, smooth muscle cell progenitors; pSHF1/2, posterior second heart field 1/2; AHF1/2, anterior heart field 1/2; ParaxialMeso1/2, paraxial mesoderm 1/2. In the box plots, the central line indicates the median, box bounds represent the 25th and 75th percentiles, whiskers extend to values within 1.5 times the interquartile range, and outliers lie beyond this range.

Extended Data Fig. 10 Anterior extension of retinoic acid signaling activity in STZ in vivo (complementary to Fig. 5e, f).

(A) X-gal staining of RARE-LacZ mouse fetuses at E10.5 from VEH and STZ groups (N = 3 each). Second heart field area and outflow tract area are circled with black lines. Highlighted area in magnified panels to the right show LacZ positive areas detected by threshold analysis as described in methods. The scale bar represents 1 mm. (B) Measurements of area of second heart field and outflow tract circled with black line in (A) (N = 3 each). (C) Percentage of LacZ positive area with in the second heart field area and outflow tract area circled with black line in (A) (N = 3 each).

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Supplementary Information

Supplementary Fig. 1, Tables 1–5 and Videos 1–5 and source data for the supplementary figure.

Reporting Summary

Supplementary Tables

Supplementary Table 1. Cardiac phenotypes at E18.5 from VEH- or STZ-treated females by micro-CT. This table presents the number and types of cardiac phenotypes detected by micro-CT, including atrial septal defect (ASD), patent foramen ovale (PFO), ventricular septal defect (VSD), atrioventricular septal defect (AVSD) and OFT anomalies, in E18.5 embryos from VEH- or STZ-treated females after cesarean section. Representative micro-CT images are shown in Extended Data Fig. 1b and Supplementary Video 1a,b. There were significant differences in the presence of cardiac developmental abnormalities between the VEH and STZ groups by two-sided Fisher’s exact test (P = 0.0004). Supplementary Table 2. Statistical results for the population changes in scRNA-seq and scATAC-seq data for Fig. 1g, left (a), for Fig. 1g, right (b), and for Fig. 4b (c). Supplementary Table 3. Statistical results for the ChromVAR analysis. This table presents the statistical results from two-sided Wilcoxon rank-sum test with Benjamini–Hochberg multiple-testing correction to determine the differences between the bias-corrected deviations for a TF motif between VEH and STZ conditions per each cell type displayed in Extended Data Fig. 9a,b. Gray highlighted cell types show the statistical differences. Supplementary Table 4. Primer lists. Primers for cloning the candidate distal regulatory regions and deletions discussed in Fig. 5a–d and Extended Data Fig. 7a,b. Supplementary Table 5. Guide RNA and HDR template sequence used for Alx3–Cre target allele generation.

Supplementary Video 1a

3D micro-CT images of E18.5 embryonic hearts from VEH and STZ conditions. a, 3D micro-CT images of the E18.5 embryonic heart from VEH that is shown in Extended Data Fig. 1b. Pulmonary artery (light green), aorta (light red), right ventricle chamber (dark green) and left ventricle chamber (dark green) are highlighted. b, 3D micro-CT images of the E18.5 embryonic heart from STZ that is shown in Extended Data Fig. 1b. Pulmonary artery (light green), aorta (light red), right ventricle chamber (dark green), left ventricle chamber (dark green) and conotruncal ventricular septal defect (purple) are highlighted.

Supplementary Video 1b

3D micro-CT images of E18.5 embryonic hearts from VEH and STZ conditions. a, 3D micro-CT images of the E18.5 embryonic heart from VEH that is shown in Extended Data Fig. 1b. Pulmonary artery (light green), aorta (light red), right ventricle chamber (dark green) and left ventricle chamber (dark green) are highlighted. b, 3D micro-CT images of the E18.5 embryonic heart from STZ that is shown in Extended Data Fig. 1b. Pulmonary artery (light green), aorta (light red), right ventricle chamber (dark green), left ventricle chamber (dark green) and conotruncal ventricular septal defect (purple) are highlighted.

Supplementary Video 2

Distribution of Alx3-positive cells in Mef2c–AHF–Cre:Ai6 fetus at E10.5. Serial coronal optical sections from ventral to dorsal of whole-mount RNA in situ hybridization. Alx3 (red), ZsGreen (green) and DAPI (blue) are shown. Scale bar, 100 µm.

Supplementary Video 3

The spatial relationship between Alx3-positive cells and Armh4-positive cells. The whole-mount RNA in situ hybridization for Alx3 (red), Armh4 (green) with DAPI (blue). Scale bar, 300 µm.

Supplementary Video 4

The distribution of Alx3–Cre:Ai6 lineage-traced cells in neonatal hearts. Serial coronal optical sections from dorsal to ventral of the Alx3Cre/+:Ai6 mouse neonatal heart shown in Fig. 3g.

Supplementary Video 5a

RARE activity is enhanced in the second heart field at E10.5 in maternal diabetes. a, The whole-mount RNA in situ hybridization for Alx3 (green), LacZ (red) with DAPI (blue) in an E10.5 RARE–LacZ fetus from VEH-treated female. Scale bar, 300 µm. b, The whole-mount RNA in situ hybridization for Alx3 (green), LacZ (red) with DAPI (blue) in an E10.5 RARE–LacZ fetus from STZ-treated female. Scale bar, 300 µm.

Supplementary Video 5b

RARE activity is enhanced in the second heart field at E10.5 in maternal diabetes. a, The whole-mount RNA in situ hybridization for Alx3 (green), LacZ (red) with DAPI (blue) in an E10.5 RARE–LacZ fetus from VEH-treated female. Scale bar, 300 µm. b, The whole-mount RNA in situ hybridization for Alx3 (green), LacZ (red) with DAPI (blue) in an E10.5 RARE–LacZ fetus from STZ-treated female. Scale bar, 300 µm.

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Source Data Extended Data Fig. 3

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Source Data Extended Data Fig. 10

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Nishino, T., Ranade, S.S., Pelonero, A. et al. Single-cell multimodal analyses reveal epigenomic and transcriptomic basis for birth defects in maternal diabetes. Nat Cardiovasc Res 2, 1190–1203 (2023). https://doi.org/10.1038/s44161-023-00367-y

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