Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest– and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.
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I.A. was supported by the Paradifference Foundation, the Swedish Cancer Society, the Bertil Hallsten Research Foundation, a Knut and Alice Wallenberg Foundation project grant, an ERACoSysMed 4D-Healing grant, the Swedish Research Council, ERC Consolidator (‘STEMMING FROM NERVE’, 647844), ERC Synergy (‘KILL OR DIFFERENTIATE’, 856529, ERC-2019-SyG), an Austrian Science Fund (FWF) grant and EMBO Young Investigator grants. O.G. was supported by the Russian Science Foundation (grant N19-14-00260 for regulatory element analysis). M.V. was supported by the Ministry of Science and Higher Education of the Russian Federation (agreement no. 075-15-2020-784). L.F. was supported by the Austrian Science Fund (DOC 33-B27). M.E.K. was supported by the Novo Nordisk Foundation (Postdoctoral Fellowship in Endocrinology and Metabolism at International Elite Environments, NNF17OC0026874) and Stiftelsen Riksbankens Jubileumsfond (Erik Rönnbergs fond stipend). P. Kogner was funded by the Swedish Research Council, the Swedish Childhood Cancer Fund, the Swedish Cancer Society and the Swedish Foundation for Strategic Research. T.K.O., N.B. and P. Kogner were financially supported by the Knut and Alice Wallenberg Foundation as part of the National Bioinformatics Infrastructure Sweden at SciLifeLab. N.A. was supported by the Russian Science Foundation (grant no. 16-15-10273 for immunohistochemical validation of bioinformatic predictions). A.V.A. was supported by the Russian Science Foundation (grant no. 19-15-00241 for bioinformatic analysis of the mouse dataset) and an ERACoSysMed 4D-Healing grant. T.B. was supported by the Lise Meitner Programme. A.E. was supported by StratNeuro SRP Postdoctoral Research 2020–2021 (C333740002), E.R.A. and B.S. were supported by the Swedish Research Council, Karolinska Institutet (KI Foundations, Career Development grant, PhD student KID funding and SFO StratNeuro funding, the Center of Innovative Medicine), the Ollie and Elof Ericssons Foundation, the Tornspiran Foundation, the Jeanssons Foundation, a Sven and Ebba-Christina Hagbergs prize and research grant, a Knut and Alice Wallenberg project grant, the Fredrik and Ingrid Thurings Foundation, Lars Hiertas Minne, the Childhood Cancer Foundation (Barncancerfonden), the Åhlen Foundation, the Åke Wibergs Foundation, the Tore Nilssons Foundation and a Swedish Foundations starting grant. E.S. acknowledges the Knut and Alice Wallenberg Foundation and the Erling Person Foundation for generous support. M.R. was supported by a DFG research fellowship (RA 2889/1-1). J.F. was supported by the Swedish Foundation for Strategic Research. P.V.K. and S.M. were supported by the NIH (1R01HL131768 from NHLBI) and the NSF (CAREER 1452964). We thank the KI Developmental Tissue Bank for providing prenatal human tissue. We thank O. Kharchenko for help with illustrations. We also thank the Karolinska Eukaryotic Single Cell Genomics Facility (M. Erickson and K. Wallenborg) at SciLifeLab, Sweden for assistance with sequencing single cells. The 2h3 antibody (P12839) developed by T.M. Jessell and J. Dodd was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the Department of Biology, University of Iowa, Iowa City, IA 52242.
P.V.K. serves on scientific advisory boards for Celsius Therapeutics Inc. and Biomage Inc. The other authors declare no conflict of interests.
Peer review information Nature Genetics thanks Guoji Guo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, UMAP embedding of 99,553 cells from 11 individual human fetuses (including the 74,401 cells shown on Fig. 1a and additional samples collected with varying procedure see Methods). b, Numbers of detected genes per cell (upper panel) and numbers of UMIs per cell (lower panel) in each sample. Probability of cell doublets (Scrublet score) per sample. Color legend is the same for all plots. c, Dot plot with key genes for the clusters in (a). d, Probability of cell doublets (Scrublet score) plotted on the general UMAP embedding. e, Numbers of UMIs per cell plotted on a general UMAP embedding. f, Numbers of detected genes per cell plotted on a general UMAP embedding. g, Key genes for cell clusters plotted on a general UMAP embedding h, UMAP embedding of cells from each individual human embryo sample. Note: embryo 2 from week 9 and embryo 2 from week 2 have two biopsies combined (see Methods).
Extended Data Fig. 2 Quality controls and examples of cells in transition between SCPs and sympathoadrenal fates.
a, Probability of cell doublets (Scrublet score) per sample and sympathoadrenal clustering. b, Fate transitions shown per time point. c, Immunohistochemistry against SOX10 (marker of SCPs), HAND2 (marker of developing sympathoblasts and chromaffin cells), ISL1/2 (marker of sympathoblasts) on the cross-section of adrenal gland at week 6 of human development. Scale bar is 10 µm.
Extended Data Fig. 3 Comparative histological analysis of the developing adrenal glands from human and mouse.
a, The distribution of chromaffin cells as shown by TH immunostaining in the human adrenal gland. Scale bar is 200 µm. Note the diffuse nature of chromaffin cell localization as opposed to the compact medulla in mouse adrenal glands (d). b, c, The ISL1/2high/HUC/D+/THlow intra-adrenal ganglia-like structures identified at all investigated stages of the human development are proliferative (according to MKI67 immunoreactivity) and can be discriminated from ISL1/2low/HUC/D−/THhigh chromaffin cells. Scale bar is 200 µm for the main panel and 20 µm for the insets. Note that chromaffin cells are associated with 2H3+ (Neurofilament+) axons. CART+ extrinsic innervation is found next to HUC/D+ intra-adrenal ganglia-like structures and is not associated with chromaffin cell and sympathetic cells somas at this stage. Later on CART+ expression segregates to chromaffin cell population (for staining see Extended Data Fig. 5). Hematoxilin/Eosin staining (H&E) as well as immunohistochemistry against TH, CHGA and VIP of postnatal human adrenal glands, shows intra-adrenal sympathetic neurons in adult human adrenal glands. RNA scope in situ hybridization against CHGA, STMN2, VIP on postnatal human adrenal glands, showing clusters of intra-adrenal sympathetic neurons. Scale bar is 100 µm for the main panel and 20 µm for the insets. d, Analysis of the developing mouse adrenal gland showing HUC/D+ or ISL1/2high cells within the medulla at E18.5 developmental stage. Note CART+/HUC/D+ and ISL1high cells in the sympathetic ganglion outside of the gland. Scale bar is 100 µm for the main panel and 20 µm for the insets. AM: adrenal medulla, ChCs: chromaffin cells.
Extended Data Fig. 4 Comparison of human intra-adrenal, extra-adrenal sympathoblasts and chromaffin cells.
a, UMAP embedding resulting from the re-clustering of sympathoblasts from 4 embryonic stages (see Methods). b, Dot plot of key genes expressed in proliferating and non-proliferating sympathoblasts. c, UMAP embedding with highlighted positions of intra-adrenal and extra-adrenal sympathoblasts. Please note the uniform mixing of both populations. d, UMAP embedding with defined samples contribution. e, Key genes of sympathetic lineage. f, Immunohistochemistry against TH, NPY, VMAT2, NF200, PRPH, HUC/D and RNA in situ hybridization against EYA4, STMN2 on cross-sections of week 8, 9, 11 and 14 human adrenal gland showing that intra- and extra-adrenal sympathoblasts are comprised of the same populations across different developmental stages. Note the EYA4 negative area inside the ganglion at week 14 showing the mature sympathoblasts. g, UMAP embedding showing the positions of SCPs, proliferating sympathoblasts, sympathoblasts, chromaffin cells with corresponding markers. h, Immunohistochemistry against CART, TH, HUC/D and RNA in situ hybridization against PENK on cross-sections of week 14 human adrenal gland showing differences in the marker expression discriminating sympathoblasts and chromaffin cells. Scale bar is 20 µm, scale bar on insets is 10 µm.
a, UMAP of human sympathoadrenal cells with expression of PRPH (marker of sympathoblasts), PENK (marker of chromaffin cells) and CARTPT showing the specific expression in the chromaffin cells cluster. b, Immunohistochemistry against CART, TH and HUC/D on cross-sections of week 11 human adrenal gland showing external ganglion, internal ganglia-like structure and chromaffin cells. c, UMAP of mouse sympathoadrenal cells with expression of Prph (marker of sympathoblasts), Chga (marker of chromaffin cells), and Cartpt. Note that Cartpt shows the specific expression in sympathoblasts cluster. Scale bar is 20 µm, scale bar on insets is 10 µm. d, Immunohistochemistry against NF200, TH, HUCD, and CART on the cross-sections of E12.5, E13.5, E16.5 mouse adrenal glands showing sympathetic chain ganglia (SG), suprarenal ganglia (SRG) and adrenal medullae (AM). Scale bar is 20 µm for E12.5 and E13.5. Scale bar is 100 µm for E16.5.
a–c, Trajectories corresponding to Transition 1 and Transition 2 with heat maps show genes with non-linear patterns of mixing.
Extended Data Fig. 7 Stability of transitions between SCPs and sympathoadrenal cell types after cell cycle genes removal.
UMAP embedding of SCPs and sympathoadrenal populations without cell cycle-associated genes. Note that the exact same cells occupy the same mapped transitions in clustering before and after extensive removal of cell cycle-associated genes (see Methods).
Extended Data Fig. 8 Integrated analysis of sympatho-adrenal lineage sequenced by different methods across mouse and human species.
a, Probability of cell doublets (Scrublet score) plotted on the UMAP embedding of mouse SCPs and sympathoadrenal cells. b, UMAP embedding of mouse SCPs and sympathoadrenal cells without cell cycle genes. c, tSNE embedding of 356 cells sequenced by Smart-seq2 from the mouse adrenal gland with associated sympathetic ganglia at E13.5. The color-code reflects the identified cell types and shows gene expression aspects of sympathoblasts and chromaffin cell markers (right). d, Joint analysis of sympathoadrenal lineage from mouse embryos at E13.5 as sequenced by 10x Chromium and Smart-seq2 platforms (upper). Early expression of chromaffin cells program in the transient cells (bottom). e, Expression of the Bridge cell marker genes according to the annotation in Smart-seq2 dataset from Furlan et al. Science 2017 in the joint embedding of Smart-seq2 (upper) and 10x Chromium (middle) embedding as integrated by Conos. The lower panels show the Bridge cell markers in the original embedding of mouse scRNA-seq generated by 10x. f, Integrated analysis of sympathoadrenal lineage from human and mouse embryos both sequenced by 10x platform. g, number of clones that are present in different combinations of cell types: SCPs, chromaffin cells and sympathoblasts. h, Illustration of potential differentiation models (Model 1 and 3), with the corresponding likelihood tests. Pχ2 – P value of the likelihood ratio test comparing Model 3 and Model 1 (one-tailed chi-squared probability; H0: Model 3 is not more likely than Model 1). n = 322 independent clones were analyzed. Model 3 that includes the possibility of a direct transition from SCP to sympathoblasts does not show any improvement as compared to Model 1.
Extended Data Fig. 9 Fate convergence of the neural crest cell-derived sympathoblasts and sympathoblasts originating from SCP-dependent pathways in mouse and human.
a, Scheme depicting convergence of sympathoblasts generated from the neural crest cells (NCC, early pathway) with sympathoblasts generated by alternative late pathways in mouse and human on the UMAP embedding. The indicated transitions from NCCs are based on published results and are not captured in the present datasets due to the early onset. b, Ternary plot of extra-medullary and intra-medullary cells from week 12 of human adrenal gland (sequenced separately) indicate convergence of differentiated sympathoadrenal cells independently of their origin.
Extended Data Fig. 10 Correlation of gene expression signature of selected mesoderm-derived populations with survival probabilities of neuroblastoma patients and bootstrap validation for the survival analysis.
a, Correlation of gene expression signature of selected mesoderm-derived populations with survival probabilities of patients with different MYCN-amplified (88 samples) and non-MYCN-amplified (353 samples) neuroblastoma subtypes. P-values of the two-sided log-rank test between Kaplan–Meier curves for the top and the bottom quarter of tumors sorted according to a gene expression signature. Non-MYCN-amplified tumors: immune cells P = 0.83, liver P = 0.5, erythroid cells P = 0.1, intermediate mesoderm P = 0.077, endotelial cells P = 0.24, Melanocytes P<0.0001. MYCN-amplified tumors: immune cells P = 0.41, liver P = 0.95, erythroid cells P = 0.42, intermediate mesoderm P = 0.72, endotelial cells P = 0.066, Melanocytes P = 0.047. b, Bootstrap validation for the survival analysis of cluster-specific gene expression signatures in non-MYCN-amplified and MYCN-amplified neuroblastoma subtypes. For a set of marker genes, we took 100 random samples of the same size with replacement (see Methods). Time is measured in days.
Supplementary Note, Methods and Figs. 1–5
Sheet 1, individual sample contribution to cell clusters on the general UMAP embedding. Sheet 2, individual sample contribution to cell clusters on the sympathoadrenal UMAP subembedding.
Gene expression patterns in chromaffin cells and sympathoblasts in mice and humans.
Single-cell clones in the mouse sympathoadrenal domain.
Survival analysis for the expression of individual marker genes in selected sympathoadrenal clusters in non-MYCN-amplified (353 samples) or MYCN-amplified (88 samples) neuroblastoma subtypes. P values are shown for two-sided log-rank tests between Kaplan–Meier curves for the top and the bottom quarters of tumors sorted according to gene expression.
Table of adrenal cortex disease-associated genes expressed during development.
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Kameneva, P., Artemov, A.V., Kastriti, M.E. et al. Single-cell transcriptomics of human embryos identifies multiple sympathoblast lineages with potential implications for neuroblastoma origin. Nat Genet 53, 694–706 (2021). https://doi.org/10.1038/s41588-021-00818-x
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