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Single-cell transcriptomics of human embryos identifies multiple sympathoblast lineages with potential implications for neuroblastoma origin

Abstract

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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Fig. 1: Heterogeneity of the human sympathoadrenal region across several developmental stages.
Fig. 2: Heterogeneity and transitions in the human sympathoadrenal system.
Fig. 3: Human-specific transition from sympathoblasts to chromaffin cells.
Fig. 4: Immature chromaffin cells give rise to sympathoblasts in a mouse embryo.
Fig. 5: Intra-adrenal sympathoblasts originate from Plp1+ SCPs specified under the control of Ret.
Fig. 6: Transcriptional signatures of neuroblastoma cells match with transcriptional signatures of fetal human cells.
Fig. 7: Heterogeneity of the developing human adrenal cortex, kidney and mesenchyme, and their ability to shape the microenvironment in neuroblastoma.

Data availability

Sequenced reads and h5 files with gene counts in individual cells were deposited at the GEO database under accession numbers GSE147821 (human datasets) and GSE166262 (mouse datasets).

Code availability

Source code is available at https://github.com/artem-artemov/adrenal. TREX source code is available under the MIT license at https://github.com/frisen-lab/TREX.

References

  1. 1.

    Del Valle, I. et al. A genomic atlas of human adrenal and gonad development. Wellcome Open Res. 2, 25 (2017).

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Nishikawa, M. et al. Role of the microenvironment of the embryonic aorta–gonad–mesonephros region in hematopoiesis. Ann. N. Y. Acad. Sci. 938, 109–116 (2001).

    CAS  PubMed  Google Scholar 

  3. 3.

    Ohneda, O. et al. Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta–gonad–mesonephros region-derived endothelium. Blood 92, 908–919 (1998).

    CAS  PubMed  Google Scholar 

  4. 4.

    Pietila, I. & Vainio, S. The embryonic aorta–gonad–mesonephros region as a generator of haematopoietic stem cells. APMIS 113, 804–812 (2005).

    PubMed  Google Scholar 

  5. 5.

    Huber, K., Kalcheim, C. & Unsicker, K. The development of the chromaffin cell lineage from the neural crest. Auton. Neurosci. 151, 10–16 (2009).

    CAS  PubMed  Google Scholar 

  6. 6.

    Saito, D., Takase, Y., Murai, H. & Takahashi, Y. The dorsal aorta initiates a molecular cascade that instructs sympatho-adrenal specification. Science 336, 1578–1581 (2012).

    CAS  PubMed  Google Scholar 

  7. 7.

    Furlan, A. et al. Multipotent peripheral glial cells generate neuroendocrine cells of the adrenal medulla. Science 357, eaal3753 (2017).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Lumb, R. et al. Neuropilins guide preganglionic sympathetic axons and chromaffin cell precursors to establish the adrenal medulla. Development 145, dev162552 (2018).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Huber, K., Janoueix-Lerosey, I., Kummer, W., Rohrer, H. & Tischler, A. S. The sympathetic nervous system: malignancy, disease, and novel functions. Cell Tissue Res. 372, 163–170 (2018).

    PubMed  Google Scholar 

  10. 10.

    Scriba, L. D. et al. Cancer stem cells in pheochromocytoma and paraganglioma. Front. Endocrinol. 11, 79 (2020).

    Google Scholar 

  11. 11.

    Lee, S. et al. Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: developmental culling and cancer. Cancer Cell 8, 155–167 (2005).

    PubMed  Google Scholar 

  12. 12.

    Stiller, C. A. & Parkin, D. M. International variations in the incidence of neuroblastoma. Int. J. Cancer 52, 538–543 (1992).

    CAS  PubMed  Google Scholar 

  13. 13.

    Johnsen, J. I., Kogner, P., Albihn, A. & Henriksson, M. A. Embryonal neural tumours and cell death. Apoptosis 14, 424–438 (2009).

    PubMed  Google Scholar 

  14. 14.

    Park, J. R., Eggert, A. & Caron, H. Neuroblastoma: biology, prognosis, and treatment. Hematol. Oncol. Clin. North Am. 24, 65–86 (2010).

    PubMed  Google Scholar 

  15. 15.

    Saxen, L. & Sariola, H. Early organogenesis of the kidney. Pediatr. Nephrol. 1, 385–392 (1987).

    CAS  PubMed  Google Scholar 

  16. 16.

    Kuure, S., Vuolteenaho, R. & Vainio, S. Kidney morphogenesis: cellular and molecular regulation. Mech. Dev. 92, 31–45 (2000).

    CAS  PubMed  Google Scholar 

  17. 17.

    Davidson, A. J. Mouse kidney development. In StemBook (Harvard Stem Cell Institute, 2008).

  18. 18.

    de Bruijn, M. F., Speck, N. A., Peeters, M. C. & Dzierzak, E. Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo. EMBO J. 19, 2465–2474 (2000).

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Muller, A. M., Medvinsky, A., Strouboulis, J., Grosveld, F. & Dzierzak, E. Development of hematopoietic stem cell activity in the mouse embryo. Immunity 1, 291–301 (1994).

    CAS  PubMed  Google Scholar 

  20. 20.

    Medvinsky, A. L., Samoylina, N. L., Muller, A. M. & Dzierzak, E. A. An early pre-liver intraembryonic source of CFU-S in the developing mouse. Nature 364, 64–67 (1993).

    CAS  PubMed  Google Scholar 

  21. 21.

    David, R. et al. The many faces of neuroblastoma. Radiographics 9, 859–882 (1989).

    CAS  PubMed  Google Scholar 

  22. 22.

    Unsicker, K. Fine structure and innervation of the avian adrenal gland. Z. Zellforsch. Mikrosk. Anat. 145, 389–416 (1973).

    CAS  PubMed  Google Scholar 

  23. 23.

    Unsicker, K., Krisch, B., Otten, U. & Thoenen, H. Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells: impairment by glucocorticoids. Proc. Natl Acad. Sci. USA 75, 3498–3502 (1978).

  24. 24.

    Schalling, M. et al. Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine. Proc. Natl Acad. Sci. USA 85, 8306–8310 (1988).

  25. 25.

    Cooper, M. J., Hutchins, G. M. & Israel, M. A. Histogenesis of the human adrenal medulla. An evaluation of the ontogeny of chromaffin and nonchromaffin lineages. Am. J. Pathol. 137, 605–615 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Dagerlind, Å., Pelto-Huikko, M., Diez, M. & Hokfelt, T. Adrenal medullary ganglion neurons project into the splanchnic nerve. Neuroscience 69, 1019–1023 (1995).

    CAS  PubMed  Google Scholar 

  27. 27.

    Katsetos, C. D. et al. Class III β-tubulin isotype (β III) in the adrenal medulla: I. Localization developing human adrenal medulla. Anat. Rec. 250, 335–343 (1998).

    CAS  PubMed  Google Scholar 

  28. 28.

    Stubbusch, J. et al. Synaptic protein and pan-neuronal gene expression and their regulation by Dicer-dependent mechanisms differ between neurons and neuroendocrine cells. Neural Dev. 8, 16 (2013).

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Moolenaar, W. H., Houben, A. J., Lee, S. J. & van Meeteren, L. A. Autotaxin in embryonic development. Biochim. Biophys. Acta 1831, 13–19 (2013).

    CAS  PubMed  Google Scholar 

  30. 30.

    Adameyko, I. et al. Sox2 and Mitf cross-regulatory interactions consolidate progenitor and melanocyte lineages in the cranial neural crest. Development 139, 397–410 (2012).

    PubMed  PubMed Central  Google Scholar 

  31. 31.

    Dyachuk, V. et al. Parasympathetic neurons originate from nerve-associated peripheral glial progenitors. Science 345, 82–87 (2014).

    CAS  PubMed  Google Scholar 

  32. 32.

    Chan, W. H. et al. Differences in CART expression and cell cycle behavior discriminate sympathetic neuroblast from chromaffin cell lineages in mouse sympathoadrenal cells. Dev. Neurobiol. 76, 137–149 (2016).

    CAS  PubMed  Google Scholar 

  33. 33.

    Olsen, T. K. et al. Malignant Schwann cell precursors mediate intratumoral plasticity in human neuroblastoma. Preprint at bioRxiv https://doi.org/10.1101/2020.05.04.077057 (2020).

  34. 34.

    Dong, R. et al. Single-cell characterization of malignant phenotypes and developmental trajectories of adrenal neuroblastoma. Cancer Cell 38, 716–733 (2020).

    CAS  PubMed  Google Scholar 

  35. 35.

    Boeva, V. et al. Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries. Nat. Genet. 49, 1408–1413 (2017).

    CAS  PubMed  Google Scholar 

  36. 36.

    van Groningen, T. et al. Neuroblastoma is composed of two super-enhancer-associated differentiation states. Nat. Genet. 49, 1261–1266 (2017).

    PubMed  Google Scholar 

  37. 37.

    Hochane, M. et al. Single-cell transcriptomics reveals gene expression dynamics of human fetal kidney development. PLoS Biol. 17, e3000152 (2019).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Gut, P. et al. Lack of an adrenal cortex in Sf1 mutant mice is compatible with the generation and differentiation of chromaffin cells. Development 132, 4611–4619 (2005).

  39. 39.

    Parlato, R. et al. Conditional inactivation of glucocorticoid receptor gene in dopamine-β-hydroxylase cells impairs chromaffin cell survival. Endocrinology 150, 1775–1781 (2009).

    CAS  PubMed  Google Scholar 

  40. 40.

    Nguyen, P. et al. Prenatal glucocorticoid exposure programs adrenal PNMT expression and adult hypertension. J. Endocrinol. 227, 117–127 (2015).

    Google Scholar 

  41. 41.

    Poli, G. et al. Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture. FASEB J. 33, 2263–2277 (2019).

    CAS  PubMed  Google Scholar 

  42. 42.

    Molenaar, W. M., Lee, V. M. Y. & Trojanowski, J. Q. Early fetal acquisition of the chromaffin and neuronal immunophenotype by human adrenal medullary cells. An immunohistological study using monoclonal antibodies to chromogranin A, synaptophysin, tyrosine hydroxylase, and neuronal cytoskeletal proteins. Exp. Neurol. 108, 1–9 (1990).

    CAS  PubMed  Google Scholar 

  43. 43.

    Farnsworth, D. R., Saunders, L. M. & Miller, A. C. A single-cell transcriptome atlas for zebrafish development. Dev. Biol. 459, 100–108 (2019).

  44. 44.

    Wagner, D. E. et al. Single-cell mapping of gene expression landscapes and lineage in the zebrafish embryo. Science 360, 981–987 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Briggs, J. A. et al. The dynamics of gene expression in vertebrate embryogenesis at single-cell resolution. Science 360, eaar5780 (2018).

    PubMed  PubMed Central  Google Scholar 

  46. 46.

    Pijuan-Sala, B. et al. A single-cell molecular map of mouse gastrulation and early organogenesis. Nature 566, 490–495 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Cao, J. et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature 566, 496–502 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Manzo, G. Similarities between embryo development and cancer process suggest new strategies for research and therapy of tumors: a new point of view. Front. Cell Dev. Biol. 7, 20 (2019).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Azzarelli, R., Simons, B. D. & Philpott, A. The developmental origin of brain tumours: a cellular and molecular framework. Development 145, dev162693 (2018).

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Rasmuson, A. et al. Tumor development, growth characteristics and spectrum of genetic aberrations in the TH-MYCN mouse model of neuroblastoma. PLoS ONE 7, e51297 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Ratner, N., Brodeur, G. M., Dale, R. C. & Schor, N. F. The ‘neuro’ of neuroblastoma: neuroblastoma as a neurodevelopmental disorder. Ann. Neurol. 80, 13–23 (2016).

    PubMed  PubMed Central  Google Scholar 

  52. 52.

    Papaioannou, G. & McHugh, K. Neuroblastoma in childhood: review and radiological findings. Cancer Imaging 5, 116–127 (2005).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Brisse, H. J. et al. Guidelines for imaging and staging of neuroblastic tumors: consensus report from the International Neuroblastoma Risk Group Project. Radiology 261, 243–257 (2011).

    PubMed  Google Scholar 

  54. 54.

    Kastriti, M. E. et al. Schwann cell precursors generate the majority of chromaffin cells in Zuckerkandl organ and some sympathetic neurons in paraganglia. Front. Mol. Neurosci. 12, 6 (2019).

  55. 55.

    Shimada, H. The International Neuroblastoma Pathology Classification. Pathologica 95, 240–241 (2003).

    CAS  PubMed  Google Scholar 

  56. 56.

    Nakazawa, A. et al. Correlation between the International Neuroblastoma Pathology Classification and genomic signature in neuroblastoma. Cancer Sci. 106, 766–771 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Beronja, S., Livshits, G., Williams, S. & Fuchs, E. Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nat. Med. 16, 821–827 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Zheng, G. X. et al. Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 8, 14049 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Wolock, S. L., Lopez, R. & Klein, A. M. Scrublet: computational identification of cell doublets in single-cell transcriptomic data. Cell Syst. 8, 281–291 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Barkas, N. et al. Joint analysis of heterogeneous single-cell RNA-seq dataset collections. Nat. Methods 16, 695–698 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Qiu, X. et al. Reversed graph embedding resolves complex single-cell trajectories. Nat. Methods 14, 979–982 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Csardi, G. & Nepusz, T. The igraph software package for complex network research. InterJournal Complex Systems, 1695 (2006).

  64. 64.

    La Manno, G. et al. RNA velocity of single cells. Nature 560, 494–498 (2018).

    PubMed  PubMed Central  Google Scholar 

  65. 65.

    Bergen, V., Lange, M., Peidli, S., Wolf, F. A. & Theis, F. J. Generalizing RNA velocity to transient cell states through dynamical modeling. Nat. Biotechnol. 38, 1408–1414 (2020).

    PubMed  Google Scholar 

  66. 66.

    Biddy, B. A. et al. Single-cell mapping of lineage and identity in direct reprogramming. Nature 564, 219–224 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Zhang, K., Peters, J., Janzing, D. & Schoelkopf, B. Kernel-based Conditional Independence test and application in causal discovery. Preprint at https://arxiv.org/abs/1202.3775v1 (2012).

  68. 68.

    Wang, F. et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 14, 22–29 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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.

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Authors

Contributions

I.A. and P.V.K. conceived and designed the main idea, drafted the manuscript and participated in data analysis. P. Kameneva and A.V.A. curated the data, designed and performed numerous experiments, analyzed the data and drafted the manuscript. T.K.O., J.O., N.B. and P. Kogner collected and sequenced neuroblastoma samples and provided expertise for the manuscript. M.E.K., T.B., M.R., A.E., N.A., A.S.T. and R.R.d.K. performed experiments and analyzed data. S.M., M.V., O.G. and L.F. participated in data analysis and drafted the manuscript. E.S. collected human fetal material and drafted the manuscript. K.F. and J.F. provided analysis expertise and drafted the manuscript. E.R.A. and B.S. contributed experimental expertise for in utero ultrasound-guided lentiviral library injection and clonal cell barcoding analysis and provided feedback on the manuscript.

Corresponding authors

Correspondence to Peter V. Kharchenko or Igor Adameyko.

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Competing interests

P.V.K. serves on scientific advisory boards for Celsius Therapeutics Inc. and Biomage Inc. The other authors declare no conflict of interests.

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Peer review information Nature Genetics thanks Guoji Guo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Technical quality controls.

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.

Extended Data Fig. 5 Expression of CARTPT in the sympathoadrenal domain in human and mouse.

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.

Extended Data Fig. 6 Transitions between SCPs and sympathoadrenal cell types.

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. Pχ2P 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 information

Supplementary Information

Supplementary Note, Methods and Figs. 1–5

Reporting Summary

Supplementary Table 1

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.

Supplementary Data 1

Gene expression patterns in chromaffin cells and sympathoblasts in mice and humans.

Supplementary Data 2

Single-cell clones in the mouse sympathoadrenal domain.

Supplementary Data 3

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.

Supplementary Data 4

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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