Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D

Nature Methods volume 18, pages 1542–1551 (2021)Cite this article

Subjects

Abstract

Transneuronal viruses are powerful tools for tracing neuronal circuits or delivering genes to specific neurons in the brain. While there are multiple retrograde viruses, few anterograde viruses are available. Further, available anterograde viruses often have limitations such as retrograde transport, high neuronal toxicity or weak signals. We developed an anterograde viral system based on a live attenuated vaccine for yellow fever—YFV-17D. Replication- or packaging-deficient mutants of YFV-17D can be reconstituted in the brain, leading to efficient synapse-specific and anterograde-only transneuronal spreading, which can be controlled to achieve either monosynaptic or polysynaptic tracing. Moreover, inducible transient replication of YFV-17D mutant is sufficient to induce permanent transneuronal genetic modifications without causing neuronal toxicity. The engineered YFV-17D systems can be used to express fluorescent markers, sensors or effectors in downstream neurons, thus providing versatile tools for mapping and functionally controlling neuronal circuits.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Controlled anterograde transneuronal spreading of YFV-17D.
Fig. 2: Anterograde-only tracing by inducible replication of YFV∆NS1.
Fig. 3: Dual fluorescence tracing of parallel circuits.
Fig. 4: Transneuronal genetic control by YFV∆NS1-Cre.
Fig. 5: Mapping monosynaptic projectomes with YFV∆CME.
Fig. 6: Monosynaptic transneuronal genetic control with YFV∆CMENS1.

Similar content being viewed by others

Data availability

The original data in this study include images of brain sections, calcium imaging data of freely moving mice and sequences of plasmids. The sequences of the plasmids have been deposited to GenBank as follows: pAAV-DIO-NS1 (MZ708030), pAAV-Syn-NS1-p2A-NLSdTomato (MZ695807), pAAV-rtTA (MZ708018), pAAV-SynaptoTAG2 (MZ708019), pAAV-TRE-C-prM-E-NS1 (MZ708020), pAAV-TRE-NS1-dTomato (MZ708021), pAAV-TRE-NS1NF (MZ708022), pAAV-tTA (MZ708023), pFUW-C-prM-E-NS1 (MZ708024), pYFVdelCME-mVenus (MZ708025), pYFVdeltaCMENS1-Cre (MZ708026), pYFVdeltaNS1-Cre (MZ708027), pYFVdeltaNS1-mCherry (MZ708028) and pYFVdeltaNS1-mVenus (MZ708029). The plasmids have been deposited to Addgene as follows: pAAV-DIO-NS1 (plasmid 175273), pAAV-Syn-NS1-p2A-NLSdTomato (plasmid 175276), pAAV-rtTA (plasmid 175274), pAAV-SynaptoTAG2 (plasmid 175275), pAAV-TRE-C-prM-E-NS1 (plasmid 175277), pAAV-TRE-NS1-dTomato (plasmid 175278), pAAV-TRE-NS1NF (plasmid 175279), pAAV-tTA (plasmid 175280) and pFUW-C-prM-E-NS1 (plasmid 175281). The other plasmids or reagents can be requested from the corresponding authors. The raw imaging data are in large files (total >100 GB) and can be requested from the corresponding authors. Source data are provided with this paper.

References

  1. Zeng, H. & Sanes, J. R. Neuronal cell-type classification: challenges, opportunities and the path forward. Nat. Rev. Neurosci. 18, 530–546 (2017).

    Article  CAS  PubMed  Google Scholar 

  2. Callaway, E. M. Transneuronal circuit tracing with neurotropic viruses. Curr. Opin. Neurobiol. 18, 617–623 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Ekstrand, M. I., Enquist, L. W. & Pomeranz, L. E. The alpha-herpesviruses: molecular pathfinders in nervous system circuits. Trends Mol. Med. 14, 134–140 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Miyamichi, K. et al. Cortical representations of olfactory input by trans-synaptic tracing. Nature 472, 191–196 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Oyibo, H. K., Znamenskiy, P., Oviedo, H. V., Enquist, L. W. & Zador, A. M. Long-term Cre-mediated retrograde tagging of neurons using a novel recombinant pseudorabies virus. Front Neuroanat. 8, 86 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Wickersham, I. R. et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53, 639–647 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Osakada, F. et al. New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron 71, 617–631 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Enquist, L. W. Exploiting circuit-specific spread of pseudorabies virus in the central nervous system: insights to pathogenesis and circuit tracers. J. Infect. Dis. 186, S209–S214 (2002).

    Article  PubMed  Google Scholar 

  9. Wojaczynski, G. J., Engel, E. A., Steren, K. E., Enquist, L. W. & Patrick Card, J. The neuroinvasive profiles of H129 (herpes simplex virus type 1) recombinants with putative anterograde-only transneuronal spread properties. Brain Struct. Funct. 220, 1395–1420 (2015).

    Article  CAS  PubMed  Google Scholar 

  10. Zemanick, M. C., Strick, P. L. & Dix, R. D. Direction of transneuronal transport of herpes simplex virus 1 in the primate motor system is strain-dependent. Proc. Natl Acad. Sci. USA 88, 8048–8051 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lo, L. & Anderson, D. J. A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons. Neuron 72, 938–950 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zeng, W. B. et al. Anterograde monosynaptic transneuronal tracers derived from herpes simplex virus 1 strain H129. Mol. Neurodegener. 12, 38 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Dix, R. D., McKendall, R. R. & Baringer, J. R. Comparative neurovirulence of herpes simplex virus type 1 strains after peripheral or intracerebral inoculation of BALB/c mice. Infect. Immun. 40, 103–112 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Itzhaki, R. F. Corroboration of a major role for herpes simplex virus type 1 in Alzheimer’s disease. Front Aging Neurosci. 10, 324 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Beier, K. T. et al. Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors. Proc. Natl Acad. Sci. USA 108, 15414–15419 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zingg, B. et al. AAV-mediated anterograde transsynaptic tagging: mapping corticocollicular input-defined neural pathways for defense behaviors. Neuron 93, 33–47 (2017).

    Article  CAS  PubMed  Google Scholar 

  17. Castle, M. J., Gershenson, Z. T., Giles, A. R., Holzbaur, E. L. & Wolfe, J. H. Adeno-associated virus serotypes 1, 8, and 9 share conserved mechanisms for anterograde and retrograde axonal transport. Hum. Gene Ther. 25, 705–720 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Burger, C. et al. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol. Ther. 10, 302–317 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Mukhopadhyay, S., Kuhn, R. J. & Rossmann, M. G. A structural perspective of the flavivirus life cycle. Nat. Rev. Microbiol. 3, 13–22 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Yi, Z. et al. Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator. PLoS Pathog. 7, e1001255 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Voorn, P., Vanderschuren, L. J., Groenewegen, H. J., Robbins, T. W. & Pennartz, C. M. Putting a spin on the dorsal-ventral divide of the striatum. Trends Neurosci. 27, 468–474 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Bienkowski, M. S. et al. Integration of gene expression and brain-wide connectivity reveals the multiscale organization of mouse hippocampal networks. Nat. Neurosci. 21, 1628–1643 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Tervo, D. G. et al. A designer AAV variant permits efficient retrograde access to projection neurons. Neuron 92, 372–382 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lindenbach, B. D. & Rice, C. M. Trans-complementation of yellow fever virus NS1 reveals a role in early RNA replication. J. Virol. 71, 9608–9617 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Dana, H. et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat. Methods 16, 649–657 (2019).

    Article  CAS  PubMed  Google Scholar 

  26. Boudkkazi, S. et al. Release-dependent variations in synaptic latency: a putative code for short- and long-term synaptic dynamics. Neuron 56, 1048–1060 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Barbera, G. et al. Spatially compact neural clusters in the dorsal striatum encode locomotion relevant information. Neuron 92, 202–213 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Xu, W. & Sudhof, T. C. A neural circuit for memory specificity and generalization. Science 339, 1290–1295 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. van Strien, N. M., Cappaert, N. L. & Witter, M. P. The anatomy of memory: an interactive overview of the parahippocampal-hippocampal network. Nat. Rev. Neurosci. 10, 272–282 (2009).

    Article  PubMed  Google Scholar 

  30. Reardon, T. R. et al. Rabies virus CVS-N2c(DeltaG) strain enhances retrograde synaptic transfer and neuronal viability. Neuron 89, 711–724 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Chatterjee, S. et al. Nontoxic, double-deletion-mutant rabies viral vectors for retrograde targeting of projection neurons. Nat. Neurosci. 21, 638–646 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ciabatti, E., Gonzalez-Rueda, A., Mariotti, L., Morgese, F. & Tripodi, M. Life-long genetic and functional access to neural circuits using self-inactivating rabies virus. Cell 170, 382–392 e314 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Libbrecht, S., Van den Haute, C., Malinouskaya, L., Gijsbers, R. & Baekelandt, V. Evaluation of WGA-Cre-dependent topological transgene expression in the rodent brain. Brain Struct. Funct. 222, 717–733 (2017).

    Article  CAS  PubMed  Google Scholar 

  35. Inagaki, H. K. et al. Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing. Cell 148, 583–595 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Jagadish, S., Barnea, G., Clandinin, T. R. & Axel, R. Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace. Neuron 83, 630–644 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Huang, T. H. et al. Tracing neuronal circuits in transgenic animals by transneuronal control of transcription (TRACT). eLife 6, e32027 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Barrett, A. D. T. Yellow fever live attenuated vaccine: a very successful live attenuated vaccine but still we have problems controlling the disease. Vaccine 35, 5951–5955 (2017).

    Article  PubMed  Google Scholar 

  39. Zolotukhin, S. et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6, 973–985 (1999).

    Article  CAS  PubMed  Google Scholar 

  40. Pnevmatikakis, E. A. & Giovannucci, A. NoRMCorre: an online algorithm for piecewise rigid motion correction of calcium imaging data. J. Neurosci. Methods 291, 83–94 (2017).

    Article  CAS  PubMed  Google Scholar 

  41. Zhou, P. et al. Efficient and accurate extraction of in vivo calcium signals from microendoscopic video data. eLife 7, e28728 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank E. Kavalali for critical comments and suggestions. This study was supported by Klingenstein-Simons Fellowship Awards in Neuroscience (to W.X.) and grants NIH/NINDS (no. NS104828 to W.X.), NIH/NIMH (no. MH099153 to W.X.) and NIH/NIAID (no. AI117922 to J.W.S.). UT BRAIN seed grants (no. 365231) and a Texas Institute for Brain Injury and Repair pilot grant provided funds to initiate this study. We thank D. Ramirez, J. Meeks and the Whole Brain Microscopy Facility at UT Southwestern and S. Yamazaki (Neuroscience Microscopy Facility at UT Southwestern) for help with imaging.

Author information

Author notes

  1. These authors contributed equally: Elizabeth Li, Jun Guo, So Jung Oh.

Authors and Affiliations

  1. Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Elizabeth Li, Jun Guo, So Jung Oh, Yi Luo, Heankel Cantu Oliveros, Wenqin Du, Rachel Arano, Yerim Kim, Yuh-Tarng Chen, Ying Li, Todd Roberts & Wei Xu

  2. Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Jennifer Eitson & John W. Schoggins

  3. Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA

    Da-Ting Lin

Authors
  1. Elizabeth Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jun Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. So Jung Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yi Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heankel Cantu Oliveros
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wenqin Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rachel Arano
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yerim Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yuh-Tarng Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jennifer Eitson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Da-Ting Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ying Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Todd Roberts
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. John W. Schoggins
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Wei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.X. initiated, designed and oversaw this study. W.X., J.W.S., T.R. and J.G. designed the experiments. E.L., J.G., S.J.O., Y.L., H.C.O., W.D., R.A., Y.K., Y.-T.C., J.E., D.-T.L., Y.L., T.R., J.W.S. and W.X. participated in conducting the experiments, analyzing the results and writing the paper.

Corresponding authors

Correspondence to John W. Schoggins or Wei Xu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Methods thanks Esteban Engel, Liqun Luo and Jennifer Treweek for their contribution to the peer review of this work. Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Extended data

Extended Data Fig. 1 Anterograde transport of YFV-17D in the PFC-striatum-SN pathway.

(a) Schematics showing the PFC-striatum-SN pathway. (b) Expression of mVenus after injection of YFV-mVenus into the PFC. Brains were collected 3, 6 or 9 days after injection. All images are tile scans of brain sections. The blue color is counterstaining with DAPI. (c) Quantification of images in b: Density of mVenus-positive neurons in each brain region on day 3, 6, and 9. The bars are mean±SEM, n=10-12 sections from 3 mice for each brain region.

Source data

Extended Data Fig. 2 Transneuronal spreading of YFV-17D in hippocampal pathways.

(a)The dentate gyrus (DG)-CA3-CA1-subiculum (SUB)-striatum pathway. (b) Expression of mVenus after injection of YFV-mVenus into the DG. Brains were collected 3, 6 or 10 days after injection. (c-e) YFV-mVenus spread from the CA1 to SUB and dorsal striatum along polysynaptic pathways. mVenus expression after YFV-mVenus injection into dorsal CA1. Brains were collected 6 (c), 9 (d) or 11 (e) days after injection. (e) Images of brain sections containing the striatum from a mouse 11 days after YFV-mVenus injection at dorsal CA1 that were immunostained with MOR, a marker for striosomes. The experiments were repeated 3 times with similar results. All images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Fig. 3 mVenus co-localizes with YFV viral proteins.

(a) Images of brain sections from mice infected with YFV-mVenus that were immunostained with antibody for YFV protein E. The green fluorescence was from native mVenus without immunostaining. The arrowheads indicate cells positive for E but not mVenus. (b) Quantification of mVenus-positive cells expressing E, and E-positive cells expressing mVenus. (c) Images of brain sections from mice infected with YFV-mVenus that were immunostained with antibody for NS1. The arrowheads indicate cells positive for NS1 but not mVenus. (d) Quantification of mVenus-positive cells expressing NS1, or NS1-positive cells expressing mVenus. n=14 brain sections from 3 mice for ‘E’ and n=14 brain sections from 4 mice for ‘NS1’. All images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Source data

Extended Data Fig. 4 Preferential infection of neurons by YFV-mVenus.

Expression of the neuronal marker NeuN in brain sections from mice infected with YFV-mVenus. The green fluorescence was from native mVenus without immunostaining. The arrowheads indicate a non-neuronal cell expressing mVenus. n=22 brain sections from 3 mice. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Source data

Extended Data Fig. 5 Immunostaining of NG2 on YFV-mVenus-infected brain sections.

(a) NG2 (Neuron-glial antigen 2) expression in brain sections from mice infected with YFV-mVenus. NG2 is a marker of NG2 cells (also referred to as oligodendrocyte precursor cells). The green fluorescence was from native mVenus without immunostaining. (b) High-resolution images of the areas indicated by arrowheads in a, showing a NG2-positive cell next to a mVenus-positive cell. (n=25 brain sections from 3 mice). The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Fig. 6 Immunostaining of Olig2 on YFV-mVenus-infected sections.

(a) Olig2 (a marker of oligodendrocytes) expression in brain sections from mice infected with YFV-mVenus. The green fluorescence was from native mVenus without immunostaining. (b) High-resolution images of the cropped areas in a. CC: corpus callosum. n=26 brain sections from 3 mice. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Fig. 7 Broad tropism of YFV-17D.

(a) Expression of mVenus in multiple brain regions after injection of YFV-mVenus into the PFC and fixation of the brains 15 days later. (b) Expression of mVenus in a cerebellar Purkinje cell. The experiments were repeated 6 times with similar results. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Fig. 8 Delayed retrograde transport of YFV∆NS1-mVenus.

Expression of mVenus and tdTomato after injection of AAVDJ-Syn-NS1 into the PFC and the striatum, and injection of YFV∆NS1-mVenus into the striatum. The brains were fixed 6 (a) or 12 (b) days after YFV∆NS1-mVenus injection. The experiments were repeated 3 times with similar results. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Fig. 9 Efficiency of YFV∆NS1-Cre without post-synaptic NS1.

(a) The experimental design was the same as in Fig. 4b–d except that no AAVs for NS1 expression were injected into the striatum. We fixed the brains 15 days after YFV∆NS1-Cre injections. (b) jGCaMP7f-positive neurons in the striatum. The image is a tile scan of the brain section. The blue color is counterstaining with DAPI. (c) The density of labeled cells in mice with or without NS1 in the striatum (n=15 sections from 4 mice for ‘with NS1’ and n=14 sections from 4 mice ‘without NS1’, *** P=0.000000026, Mann–Whitney test, two tailed). The data of the group ‘with NS1’ were also shown in Fig. 4d.

Source data

Extended Data Fig. 10 YFV∆CME-mVenus tracing from pontine nuclei to cerebellum.

(a) Image of a brain section after injection of AAV-tTA, AAV-TRE-C-prM-E-NS1 and YFV∆CME-mVenus into pontine nucleus and adjacent reticular tegmental nucleus. (b) mVenus-positive cells in the granular layer of the cerebellar cortex. The sections were counterstained with a marker for Purkinje cells, PCP4 (red), and DAPI (Blue). The experiments were repeated 4 times with similar results. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Supplementary information

Supplementary Information

Supplementary Figs. 1–17, Table 1 and step-by-step protocol.

Reporting Summary

Supplementary Video 1

Calcium activities of traced striatal neurons in a mouse undergoing open-field test 42 days after YFV∆NS1-Cre injection. Video is played at 5× speed.

Supplementary Video 2

Calcium activities of traced striatal neurons in a mouse undergoing open-field test 49 days after YFV∆NS1-Cre injection. Video is played at 5× speed.

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, E., Guo, J., Oh, S.J. et al. Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D. Nat Methods 18, 1542–1551 (2021). https://doi.org/10.1038/s41592-021-01319-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-021-01319-9

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing