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:

Structural and developmental principles of neuropil assembly in C. elegans

Nature volume 591, pages 99–104 (2021)Cite this article

Subjects

Abstract

Neuropil is a fundamental form of tissue organization within the brain1, in which densely packed neurons synaptically interconnect into precise circuit architecture2,3. However, the structural and developmental principles that govern this nanoscale precision remain largely unknown4,5. Here we use an iterative data coarse-graining algorithm termed ‘diffusion condensation’6 to identify nested circuit structures within the Caenorhabditis elegans neuropil, which is known as the nerve ring. We show that the nerve ring neuropil is largely organized into four strata that are composed of related behavioural circuits. The stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create neural structures that cage the strata within the nerve ring. We use high resolution light-sheet microscopy7,8 coupled with lineage-tracing and cell-tracking algorithms9,10 to resolve the developmental sequence and reveal principles of cell position, migration and outgrowth that guide stratified neuropil organization. Our results uncover conserved structural design principles that underlie the architecture and function of the nerve ring neuropil, and reveal a temporal progression of outgrowth—based on pioneer neurons—that guides the hierarchical development of the layered neuropil. Our findings provide a systematic blueprint for using structural and developmental approaches to understand neuropil organization within the brain.

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: Computational detection of a hierarchical tree of neurite organization in the C. elegans neuropil.
Fig. 2: Neuropil architecture reflects functional segregation of sensory and motor outputs.
Fig. 3: Developmental processes guide layered neuropil assembly.
Fig. 4: Temporal progression of outgrowth guided by pioneer neurons results in inside-out neuropil development.

Similar content being viewed by others

Data availability

The datasets generated during and/or analysed during the study are available from the corresponding author upon request. To facilitate exploration of the placement of neurites in the C-PHATE diagrams, we have generated a 3D interactive version of the C-PHATE plots. Plots can be downloaded, and neurite condensation and position can be examined. These 3D interactive versions enable identification of any neuron within the C-PHATE plot and provide the iteration number and total neurons found within any cluster. See Supplementary Discussion 2 for instructions on how to access the data. Source data are provided with this paper.

Code availability

Electron micrograph segmentation adjacency analysis code is available in ref. 18. Diffusion condensation analysis code6 is available at https://github.com/agonopol/worm_brain. C-PHATE visualization code is available to download at http://dccphate.wormguides.org/CPHATE_pythonCode.zip.

References

  1. Maynard, D. M. Organization of neuropil. Am. Zool. 2, 79–96 (1962).

    Article  Google Scholar 

  2. Schürmann, F. W. Fine structure of synaptic sites and circuits in mushroom bodies of insect brains. Arthropod Struct. Dev. 45, 399–421 (2016).

    Article  PubMed  Google Scholar 

  3. White, J. G., Southgate, E., Thomson, J. N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. B 314, 1–340 (1986).

    Article  CAS  ADS  Google Scholar 

  4. Soiza-Reilly, M. & Commons, K. G. Unraveling the architecture of the dorsal raphe synaptic neuropil using high-resolution neuroanatomy. Front. Neural Circuits 8, 105 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Zheng, Z. et al. A complete electron microscopy volume of the brain of adult Drosophila melanogaster. Cell 174, 730–743.e22 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Brugnone, N. et al. Coarse graining of data via inhomogeneous diffusion condensation. In 2019 IEEE International Conference on Big Data 2624–2633 (IEEE, 2019).

  7. Kumar, A. et al. Dual-view plane illumination microscopy for rapid and spatially isotropic imaging. Nat. Protoc. 9, 2555–2573 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wu, Y. et al. Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nat. Biotechnol. 31, 1032–1038 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bao, Z. et al. Automated cell lineage tracing in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 103, 2707–2712 (2006).

    Article  CAS  PubMed  ADS  Google Scholar 

  10. Boyle, T. J., Bao, Z., Murray, J. I., Araya, C. L. & Waterston, R. H. AceTree: a tool for visual analysis of Caenorhabditis elegans embryogenesis. BMC Bioinformatics 7, 275 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Sulston, J. E., Schierenberg, E., White, J. G. & Thomson, J. N. The embryonic-cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64–119 (1983).

    Article  CAS  PubMed  Google Scholar 

  12. Azulay, A., Itskovits, E. & Zaslaver, A. The C. elegans connectome consists of homogenous circuits with defined functional roles. PLoS Comput. Biol. 12, e1005021 (2016).

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  13. Chatterjee, N. & Sinha, S. Understanding the mind of a worm: hierarchical network structure underlying nervous system function in C. elegans. Prog. Brain Res. 168, 145–153 (2008).

    Article  PubMed  ADS  Google Scholar 

  14. Cook, S. J. et al. Whole-animal connectomes of both Caenorhabditis elegans sexes. Nature 571, 63–71 (2019).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  15. Towlson, E. K., Vertes, P. E., Ahnert, S. E., Schafer, W. R. & Bullmore, E. T. The rich club of the C. elegans neuronal connectome. J. Neurosci. 33, 6380–6387 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Varshney, L. R., Chen, B. L., Paniagua, E., Hall, D. H. & Chklovskii, D. B. Structural properties of the Caenorhabditis elegans neuronal network. PLoS Comput. Biol. 7, e1001066 (2011).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  17. Yan, G. et al. Network control principles predict neuron function in the Caenorhabditis elegans connectome. Nature 550, 519–523 (2017).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  18. Brittin, C. A., Cook, S. J., Hall, D. H., Emmons, S. W. & Cohen, N. Volumetric reconstruction of main Caenorhabditis elegans neuropil at two different time points. Preprint at https://doi.org/10.1101/485771 (2018).

  19. Brittin, C. A., Cook, S. J., Hall, D. H., Emmons, S. W. & Cohen, N. A multi-scale brain map derived from whole-brain volumetric reconstructions. Nature https://doi.org/10.1038/s41586-021-03284-x (2021).

  20. Sabrin, K. M., Wei, Y., van den Heuvel, M. P. & Dovrolis, C. The hourglass organization of the Caenorhabditis elegans connectome. PLoS Comput. Biol. 16, e1007526 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kennerdell, J. R., Fetter, R. D. & Bargmann, C. I. Wnt-Ror signaling to SIA and SIB neurons directs anterior axon guidance and nerve ring placement in C. elegans. Development 136, 3801–3810 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rapti, G., Li, C., Shan, A., Lu, Y. & Shaham, S. Glia initiate brain assembly through noncanonical Chimaerin–Furin axon guidance in C. elegans. Nat. Neurosci. 20, 1350–1360 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wadsworth, W. G., Bhatt, H. & Hedgecock, E. M. Neuroglia and pioneer neurons express UNC-6 to provide global and local netrin cues for guiding migrations in C. elegans. Neuron 16, 35–46 (1996).

    Article  CAS  PubMed  Google Scholar 

  24. Yoshimura, S., Murray, J. I., Lu, Y., Waterston, R. H. & Shaham, S. mls-2 and vab-3 control glia development, hlh-17/Olig expression and glia-dependent neurite extension in C. elegans. Development 135, 2263–2275 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Blondel, V. D., Guillaume, J. L., Lambiotte, R. & Lefebvre, E. Fast unfolding of communities in large networks. J. Stat. Mech. Theory Exp. P10008, (2008).

  26. Dasgupta, A., Hopcroft, J., Kannan, R. & Mitra, P. Spectral clustering by recursive partitioning. Lect. Notes Comput. Sci. 4168, 256–267 (2006).

    Article  MathSciNet  MATH  Google Scholar 

  27. Kaufman, L. & Rousseeuw, P. J. Finding Groups in Data: An Introduction to Cluster Analysis (Wiley, 2005).

  28. Moon, K. R. et al. Visualizing structure and transitions in high-dimensional biological data. Nat. Biotechnol. 37, 1482–1492 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bargmann, C. I. Chemosensation in C. elegans. In WormBook (ed. The C. elegans Research Community) https://doi.org/10.1895/wormbook.1.123.1 (2006).

  30. Goodman, M. B. Mechanosensation. In WormBook (ed. The C. elegans Research Community) https://doi.org/10.1895/wormbook.1.62.1 (2006).

  31. Goodman, M. B. & Sengupta, P. How Caenorhabditis elegans senses mechanical stress, temperature, and other physical stimuli. Genetics 212, 25–51 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Newman, M. E. Modularity and community structure in networks. Proc. Natl Acad. Sci. USA 103, 8577–8582 (2006).

    Article  CAS  PubMed  ADS  Google Scholar 

  33. Millard, S. S. & Pecot, M. Y. Strategies for assembling columns and layers in the Drosophila visual system. Neural Dev. 13, 11 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Sanes, J. R. & Zipursky, S. L. Design principles of insect and vertebrate visual systems. Neuron 66, 15–36 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Baier, H. Synaptic laminae in the visual system: molecular mechanisms forming layers of perception. Annu. Rev. Cell Dev. Biol. 29, 385–416 (2013).

    Article  CAS  PubMed  Google Scholar 

  36. Ware, R. W., Clark, D., Crossland, K. & Russell, R. L. Nerve ring of nematode Caenorhabditis elegans: sensory input and motor output. J. Comp. Neurol. 162, 71–110 (1975).

    Article  Google Scholar 

  37. Ward, S., Thomson, N., White, J. G. & Brenner, S. Electron microscopical reconstruction of the anterior sensory anatomy of the nematode Caenorhabditis elegans. J. Comp. Neurol. 160, 313–337 (1975).

    Article  CAS  PubMed  Google Scholar 

  38. Riddle, D. L., Blumenthal, T., Meyer, B. J. & Priess, J. R. (eds) C. elegans II 2nd edn (Cold Spring Harbor Lab. Press, 1997).

  39. White, J. Clues to basis of exploratory behaviour of the C. elegans snout from head somatotropy. Phil. Trans. R. Soc. Lond. B 373, (2018).

  40. Groh, J. M. Making Space: How The Brain Knows Where Things Are (Harvard Univ. Press, 2014).

  41. Kaas, J. H. Topographic maps are fundamental to sensory processing. Brain Res. Bull. 44, 107–112 (1997).

    Article  CAS  PubMed  Google Scholar 

  42. Sasakura, H. & Mori, I. Behavioral plasticity, learning, and memory in C. elegans. Curr. Opin. Neurobiol. 23, 92–99 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Chalfie, M. et al. The neural circuit for touch sensitivity in Caenorhabditis elegans. J. Neurosci. 5, 956–964 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Gray, J. M., Hill, J. J. & Bargmann, C. I. A circuit for navigation in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 102, 3184–3191 (2005).

    Article  CAS  PubMed  ADS  Google Scholar 

  45. Lockery, S. R. Neuroscience: A social hub for worms. Nature 458, 1124–1125 (2009).

    Article  CAS  PubMed  ADS  Google Scholar 

  46. Macosko, E. Z. et al. A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans. Nature 458, 1171–1175 (2009).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  47. White, J. G., Southgate, E., Thomson, J. N. & Brenner, S. Factors that determine connectivity in the nervous system of Caenorhabditis elegans. Cold Spring Harb. Symp. Quant. Biol. 48, 633–640 (1983).

    Article  PubMed  Google Scholar 

  48. Wakabayashi, T., Kitagawa, I. & Shingai, R. Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103–111 (2004).

    Article  PubMed  Google Scholar 

  49. Duncan, L. H. et al. Isotropic light-sheet microscopy and automated cell lineage analyses to catalogue Caenorhabditis elegans embryogenesis with subcellular resolution. J. Vis. Exp. (2019).

  50. Guo, M. et al. Rapid image deconvolution and multiview fusion for optical microscopy. Nat. Biotechnol. (2020).

  51. Santella, A. et al. WormGUIDES: an interactive single cell developmental atlas and tool for collaborative multidimensional data exploration. BMC Bioinformatics 16, 189 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Ayala, R., Shu, T. & Tsai, L. H. Trekking across the brain: the journey of neuronal migration. Cell 128, 29–43 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Chelur, D. S. & Chalfie, M. Targeted cell killing by reconstituted caspases. Proc. Natl Acad. Sci. USA 104, 2283–2288 (2007).

    Article  CAS  PubMed  ADS  Google Scholar 

  54. Brandes, U. et al. On modularity clustering. IEEE Trans. Knowl. Data Eng. 20, 172–188 (2008).

    Article  Google Scholar 

  55. Wicks, S. R. & Rankin, C. H. Integration of mechanosensory stimuli in Caenorhabditis elegans. J. Neurosci. 15, 2434–2444 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Walthall, W. W. & Chalfie, M. Cell–cell interactions in the guidance of late-developing neurons in Caenorhabditis elegans. Science 239, 643–645 (1988).

    Article  CAS  PubMed  ADS  Google Scholar 

  57. Kindt, K. S. et al. Caenorhabditis elegans TRPA-1 functions in mechanosensation. Nat. Neurosci. 10, 568–577 (2007).

    Article  CAS  PubMed  Google Scholar 

  58. Insley, P. & Shaham, S. Automated C. elegans embryo alignments reveal brain neuropil position invariance despite lax cell body placement. PLoS ONE 13, e0194861 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Shah, P. K. et al. An in toto approach to dissecting cellular interactions in complex tissues. Dev. Cell 43, 530–540.e4 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Lu, N., Yu, X., He, X. & Zhou, Z. Detecting apoptotic cells and monitoring their clearance in the nematode Caenorhabditis elegans. Methods Mol. Biol. 559, 357–370 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank S. Emmons, S. Cook and C. Brittin for sharing the segmented electron microscopy datasets and adjacency analysis code, and for comments on this work; O. Hobert for sharing the ceh-48p promoter and for advice; Z. Zhou for sharing a 2xFYVE-containing plasmid; J. White for transferring the electron microscopy data archive from MCR/LMB to the laboratory of D. Hall for long-term curation and display on the WormImage website (website supported by NIH grant no. OD010943 to D.H.); D. Hall for use of the young adult scanning electron microscopy images, for advice, and for help with electron microscopy images; R. Sommer for use of an electron microscopy image; H. Eden for comments on the manuscript; and members of the D.A.C-R. laboratory for comments during manuscript preparation. We thank the Caenorhabditis Genetic Center (funded by National Institutes of Health (NIH) Office of Research Infrastructure Programs P40 OD010440) for C. elegans strains; the Research Center for Minority Institutions program; the Marine Biological Laboratories (MBL); and the Instituto de Neurobiología de la Universidad de Puerto Rico for providing meeting and brainstorming platforms. H.S. and D.A.C-R. acknowledge the Whitman and Fellows program at MBL for providing funding and space for discussions valuable to this work. H.S. and D.A.C-R. are MBL Fellows. Research in the D.A.C.-R., W.A.M. and Z.B. laboratories was supported by NIH grant no. R24-OD016474. M.W.M. was supported by NIH grant no. F32-NS098616. Research in the H.S. laboratory was further supported by the intramural research program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), NIH. Research in the Z.B. laboratory was further supported by an NIH center grant to MSKCC (P30CA008748). Research in the D.A.C.-R. laboratory was further supported by NIH grant nos R01NS076558 and DP1NS111778 and by an HHMI Scholar Award.

Author information

Author notes

  1. These authors jointly supervised this work: Smita Krishnaswamy, Zhirong Bao, Hari Shroff, William A. Mohler, Daniel A. Colón-Ramos

Authors and Affiliations

  1. Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA

    Mark W. Moyle, Leighton H. Duncan, Titas Sengupta, Lin Shao & Daniel A. Colón-Ramos

  2. Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA

    Kristopher M. Barnes, Anthony Santella & Zhirong Bao

  3. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

    Manik Kuchroo, Alex Gonopolskiy & Smita Krishnaswamy

  4. Laboratory of High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, USA

    Min Guo, Ryan Christensen, Yicong Wu & Hari Shroff

  5. Marine Biological Laboratory, Woods Hole, MA, USA

    Abhishek Kumar, Hari Shroff & Daniel A. Colón-Ramos

  6. Department of Mathematics and Statistics, Utah State University, Logan, UT, USA

    Kevin R. Moon

  7. Department of Mathematics and Statistics, Université de Montréal, Montreal, Quebec, Canada

    Guy Wolf

  8. Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT, USA

    William A. Mohler

  9. Instituto de Neurobiología, Recinto de Ciencias Médicas, Universidad de Puerto Rico, San Juan, Puerto Rico

    Daniel A. Colón-Ramos

Authors
  1. Mark W. Moyle
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kristopher M. Barnes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Manik Kuchroo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alex Gonopolskiy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Leighton H. Duncan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Titas Sengupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lin Shao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Min Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Anthony Santella
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ryan Christensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Abhishek Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yicong Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Kevin R. Moon
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Guy Wolf
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Smita Krishnaswamy
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Zhirong Bao
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Hari Shroff
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. William A. Mohler
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Daniel A. Colón-Ramos
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.W.M., K.M.B., M.K., A.G., L.H.D., A.S., K.R.M., G.W., S.K., Z.B., H.S., W.A.M. and D.A.C.-R. designed experiments; M.W.M., K.M.B. and L.H.D. performed biological experiments; M.W.M., K.M.B., M.K., A.G., A.S., G.W. and W.A.M. performed computational experiments; M.W.M., L.H.D. and T.S. generated reagents; M.W.M., T.S., L.S., M.G., A.S., A.K., Y.W. and W.A.M. built instrumentation and analysis software; M.W.M., K.M.B., L.H.D., A.S., R.C., Z.B. and W.A.M. contributed lineaging data and expertise; M.W.M., W.A.M. and D.A.C.-R. prepared the manuscript with assistance from all authors; S.K., Z.B., H.S., W.A.M. and D.A.C.-R. supervised the research; and D.A.C.-R. directed the research.

Corresponding author

Correspondence to Daniel A. Colón-Ramos.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks John White and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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

Extended data figures and tables

Extended Data Fig. 1 Diffusion condensation analyses of the nerve ring neuropil.

a, b, Quantification of network modularity32,54 for diffusion condensation (DC) analysis of L4 (a) and adult (b) animals. DC iteratively groups neurons on the basis of quantitative similarities of each neuron’s contact profile, unveiling data relationships at varying scales of granularity (Fig. 1b). We calculated network modularity for each iteration. The highest modularity score was for the iteration with four clusters (in the L4 stage animal) and 6 clusters (in the adult animal). Comparisons revealed that for the adult animal, there were four large clusters similar to the L4 stage animal, and two smaller ones (Supplementary Methods; Extended Data Fig. 2a–d). Some of these neurons from the two smaller clusters in the adult also clustered in the L4 animal, but in an earlier iteration (Extended Data Fig. 2m, n). The iterations with the highest modularity scores were then used for subsequent strata analyses in the manuscript (Supplementary Methods), but we emphasize that other iterations reveal other valuable data relationships at varying scale of granularity, including cell–cell and circuit– circuit interactions (Extended Data Fig. 3a, b). c, Volumetric reconstruction of the L4 stage C. elegans neuropil (from EM serial sections3) with the 4 strata and unassigned neurons individually coloured. Above, schematic of worm head with nerve ring neuropil (dashed box). Location of EM sections displayed in d–g shown below (arrows and corresponding section numbering). Scale bar, 5 μm. d–g, Segmented serial section electron micrographs3, neurons coloured as in c. Original EM slices 41, 92, 152, 206 shown in d–g, respectively. Electron microscopy images used with permission from D. Hall. Scale bar (2.5 μm, in d) applies to d–g. h, Listing of neuronal classes in the 4 strata, and the ‘unassigned’ group. The number to the right of each neuron represents total neurons for each class, for all 181 neurons in the nerve ring neuropil.

Extended Data Fig. 2 Analyses of the L4 and adult contact adjacency matrices, DC outputs, and C-PHATE plots.

a, b, Heat map of scaled neuron distances within the DC nested hierarchy outputs for the L4 (a) and adult (b) animal. Values were calculated by measuring all distances between neurons from one cluster to another cluster and then averaged (Supplementary Methods). This was done for the clusters at the iteration with the highest modularity score (see Fig. 1b and Extended Data Fig. 1a, b). The two smaller clusters from the adult were excluded. Colouring scale displayed to the right of the heat map. Darker colours indicate smaller average distance. Note how C3 is closer to C4 and C1 is closer to C2 in distance. c, d, As a, b, except scaled distance values were calculated by measuring the distances between every neuron within the DC output. Two additional clusters in the adult are coloured grey. Note varying scales of granularity in neuronal relationships that constitute the major clusters, and, similar to the C-PHATE plots, circuits such as thermotaxis and body mechanosensation can be found in the more granular areas (Extended Data Fig. 3a, b). e, Histogram of the distribution of contact profile differences for each neuron between the L4 and adult contact adjacency matrices. In brief, we calculated an index of difference (Supplementary Methods section (in Supplementary Information) entitled ‘Contact adjacency data analysis’) for each neuron, comparing the contact profiles of the adult and L4 animals. We also generated 2,004 random adjacency matrices to simulate distributions of random contact profile differences. We determined that the contact profiles of 49/178 neurons (28%) were significantly more similar than random (P < 0.05), whereas the contact profiles of 96/178 neurons (54%) were significantly more different (P < 0.05; by unpaired two-tailed Student’s t-test between L4 vs. Adult and null distribution; degrees of freedom (df) = 178,532, no adjustments were made for multiple comparisons; Supplementary Methods). Therefore, there are a substantial portion of neurons that have statistically different contact profiles between L4 and adult. Additionally, we calculated the Jaccard distance and found that the average neuron’s contact partner list was 37.6% different between the L4 and adult animal, consistent with previous analysis18. As further discussed in Supplementary Discussion 3, we think that the differences between the contact profiles in the connectomes could be due to a large number of small, varying contacts. In the graph, a higher index number reflects a neuron with a higher amount of contact differences between L4 and adult. Red bars represent the differences between L4 and adult (178 neurons in total). Grey bars are the null difference distribution. Asterisks denote the location of single neurons that are hard to see on the graph. f, Same as in e, except the histogram represents distribution of differences in neuronal distances within the DC hierarchical outputs (Supplementary Methods section ‘Diffusion Condensation Data Analysis’). Notably, we found that, unlike the contact adjacency data, the DC output clustering location was significantly similar in 127/178 neurons (71%) (P < 0.05), whereas only 9/178 neurons (5%) were significantly different than random (P < 0.05; by unpaired two-tailed Student’s t-test between L4 vs. Adult and null distribution; df = 881,276, no adjustments were made for multiple comparisons; Supplementary Methods). These analyses, in the context of the differences seen for the contact profiles (e), indicate that the DC algorithm identifies meaningful relationships within the neuropil’s tangled and varying contact profiles. g–i, C-PHATE plots of DC analyses preformed on adjacency matrices calculated using different contact thresholds for the L4 animal. Data presented in Fig. 1b was calculated with the 45 nm threshold. 45 nm is approximately 10 pixels in the EM datasets. DC correctly defines clusters even for the different thresholds. j–l, Same as in g–i, except for the adult animal. m, n, C-PHATE plots of the L4 and adult animal. The adult animal has 2 additional clusters at the highest modularity score. These clusters (shown in grey) are composed primarily of neurons that interact across multiple strata. Additionally, neurons from these smaller clusters in the adult also co-clustered in the L4 animal, but at an earlier iteration. Brackets indicate location of the cluster in the L4 and adult that is composed of similar neurons. The neurons within the cluster are listed between the two plots. o, p, C-PHATE plot of DC analyses preformed on the chemical synaptic connectomics data from the L4 animal (https://wormwiring.org). The C1 cluster, and part of the C3 cluster, are similar between the connectomic and contact DC analysis outputs. However, C2/C3/C4 are mixed in the connectomics DC output. p is a rotation of o, to show the partially retained C3 blue cluster, as seen at the top of the graph. Notably, we also found that the neuropil pioneers (Fig. 3i–k) that cluster in C2 in the contact DC analysis are now scattered among the different mixed clusters in the connectome DC analysis. The pioneering neurons SIA and SIB make almost no presynaptic contacts and very few postsynaptic contacts. This dispersion of the pioneers among different clusters in the connectome DC analysis highlights the value of using contact analysis to uncover the structural architecture of the neuropil, especially for neurons that are synaptically sparse. q–s, C-PHATE plots for the L4 animal (q), adult animal (r), and the adult animal where the neurons VB01 (and also HSNL and PVNR) were omitted from the adjacency matrices used to calculate the DC/C-PHATE plot (s). These are the only 3 neurons that are exclusively found in the adult because they haven’t grown into the neuropil at the L4 stage. We observed that AVAR (C-PHATE plot location annotated with arrows) is assigned to Cluster 3 in the L4 animal and to Cluster 5 in the adult animal. AVAR makes extensive contacts with VB01 in the adult animal that are absent in L4 animal. We eliminated the VB01 neuron profile and contacts from the adult dataset and, consistent with our analyses, observed that AVAR now similarly clustered, as in the L4 dataset, to Cluster 3 in the adult dataset lacking VB01. This demonstrates that developmental differences affecting the contact profile of neurons leads to differences in the DC outputs between the L4 and adult animals. AVAR does not contact the other two neurons that were omitted in these analyses (HSNL and PVNR), suggesting that omission of VB01 is causative for the change in AVAR clustering. Graphs or plots in a, c, g–i, m, o–q are coloured according to the 4 clusters identified in iteration 23 of the L4 animal as in Fig. 1b using the 45 nm threshold: C1, red; C2, purple; C3, blue; C4, green. Graphs or plots in b, d, j–l, n, r, s are coloured according to the 6 clusters identified in iteration 22 of the adult animal using the 45 nm threshold: C1, red; C2, purple; C3, blue; C4, green; C5/6, grey.

Source data

Extended Data Fig. 3 Examination of behavioural circuits in the DC/C-PHATE analyses.

a, C-PHATE plot of DC analyses for a larval stage 4 (L4) animal, with known behavioural circuit locations highlighted. b, Enlargement of inset from a displaying the condensation of a group of neurons corresponding to the mechanosensation circuit30,43,55,56. Neurons with their names in filled blue boxes are members of the anterior body mechanosensation circuit, neurons with their names in outlined blue boxes (BDUL and BDUR) have been proposed to guide the formation of the circuit during development56. c, Model of functional segregation of information streams within the neuropil. Papillary sensory information is processed in S1 and innervates head muscles to control head movement. Amphid sensory information is processed in S3/S4 and links to body muscles (via command interneurons) and neck muscles (via motor neurons in S1/S2) to control body locomotion29,38,42,57. Interneurons cross strata to functionally link these modular circuits (Extended Data Fig. 5). Individual neuron classes and muscle outputs are coloured according to the strata they belong to (for muscles, according to the strata the innervating neurons belong to). d, Schematic of C. elegans head highlighting area in e, f as dashed rectangle and line, respectively. e, Representation of S1 sensory organs, projected over a scanning EM of the C. elegans mouth. Numbers highlight the sixfold symmetry of the papillary organs. Image produced by and used with permission of D. Hall. Mouth sensilla coloured according to their strata assignment. Scale bar, 1 μm. Same image as Fig. 2b. f, Topographical map of the S1 shallow head circuit. The S1 motor neurons, which have a ‘fourfold-plus-two’ symmetric pattern39, make neuromuscular junctions (NMJs), projecting their symmetry onto the fourfold symmetrical muscle quadrants that control head movement (black)39 (compare to e above). Numbers represent the fourfold-plus-two symmetry of the S1 neurons in (e). In brief, for example, for IL1 neurons, the dorsal and ventral IL1 neuron pairs (in our schematic, 1, 2 and 4, 5) connect to the dorsal and ventral muscle octants, while the lateral IL1 neurons connect to the two lateral muscle octants (in our schematic, 6 and 3 represent the two lateral IL1 neurons)39. g, h, Axial view of the ‘rich-club’ AIB left (AIBL) interneuron15,20 (Fig. 2f) with distribution of the presynaptic (g) and postsynaptic (h) sites coloured according to the strata of the corresponding AIBL synaptic partner. The vertical dashed line indicates the division between proximal and distal regions of the neurite. Similar distribution was seen for AIB right (AIBR, not shown). i, C-PHATE plot of DC analysis for the larval stage 4 (L4) animal. AIBL/R are coloured yellow to highlight their location within the plot. j, Volumetric reconstruction of the unassigned (yellow), ‘rich-club’ AIBL interneurons15,20 depicting the regions of AIB that were partitioned in the EM data. The proximal region is in blue (AIBL1), the lateral region in yellow and the distal region in purple (AIBL2). The proximal region of AIB borders S3/S4, and the distal region borders S2/S3 (Fig. 2f–i). k, C-PHATE plot of DC analysis for the larval stage 4 (L4) animal after AIBs had been partitioned in the EM dataset. AIB1s and AIB2s are yellow and the AIB lateral regions seen in j are grey. After partitioning the proximal regions (AIB1s) remain within C4, while the distal regions (AIB2s) now cluster with C2, demonstrating that DC/C-PHATE analysis clusters neurons on the basis of their neighbouring contact profiles. Plots in a, b, i–k are coloured according to the 4 clusters identified in iteration 23 of the L4 animal as in Fig. 1b: C1, red; C2, purple; C3, blue; C4, green.

Extended Data Fig. 4 S1 structure precisely encases S2, S3 and S4 of the neuropil.

a, Illustration of the IL1L neuron based on data presented in ref. 3 (additional information available at https://wormatlas.org). Schematic of worm head and neuropil (dashed box) above. In the schematic below, the IL1 neuron is in red, with dendrite (left pointing arrow), soma (circle) and axon (right pointing loop, arrow). The pharynx (shaded grey) is shown for reference. b, Cartoon of IL1L loop in the context of the nerve ring neuropil (sensory dendrite not shown). c, Volumetric reconstruction of IL1L from L4 animal EMs. d, Overlay of IL1L volumetric reconstruction and synapses (grey spheres) highlighting the position of the synaptic endplate (after the neuron loops around the neuropil). e, Volumetric reconstruction of the L4 neuropil with individual neurons from the 4 strata coloured as follows: S1, red; S2, purple; S3, blue; S4, green). Scale bar, 5 μm. f, g, Schematic of the structure formed by the S1 looping structures, from a lateral (f) and axial (g) view with neuropil strata. h, Schematic of the S1 loops (lateral view) without the strata (as Fig. 1d). i, j, Volumetric reconstruction of dorsal right loop and S2 (i) and S3/S4 (j) with individual neurons that fall outside of the looping structure rule (yellow arrows). Although 90% of S2, 84% of S3 and 100% of S4 neurons are contained within the indicated loops, a minority of neurons belonging to S2 and S3 strata are not encased by the loops corresponding to the specific strata. We include the names of these neurons below the volumetric reconstructions; Supplementary Video 5. k, List of all neurons and their positions in the dorsal right loop structures, coloured according to the 4 strata (a complete listing of all neurons, and their positions within the sixfold symmetric honeycomb structure, can be found in Supplementary Table 1, and movie projections of the honeycomb-like structure in the context of the strata in Supplementary Videos 4, 5).

Extended Data Fig. 5 Neurons unassigned to the 4 strata anatomically contact multiple strata, and a subset belong to the highly interconnected ‘rich-club’ neurons.

a–d, Volumetric reconstructions of the unassigned AVE interneuron (yellow) in the context of nerve ring strata, with AIB (grey). Arrows indicate the two segments of AVE that border strata. AVE has a similar morphology to AIB (Fig. 2f) but is anteriorly displaced by one stratum: AVE borders S2/S3 (b, c), shifts along the A–P axis, and then borders S1/S2 (c, d). Lines in a, b indicates AVE shift along the A–P axis to shift strata; Supplementary Video 7. e, Analysis of the total number of neurons within each stratum, and in the unassigned group. f, Classification of ‘unassigned’ neurons. g, Stratum location of the rich club neurons15,20. Coloured box depicts strata assignment. These 14 neurons functionally consist of two groups: eight command interneurons (which modulate the backward and forward locomotion; AVA, AVB, AVD, PVC) and six nerve ring interneurons (AVE, AIBR, RIBL, RIAR, DVA). The six command interneurons that are not part of the unassigned group have neurites that remain within S2. The two command interneurons that are part of our unassigned group (AVA) border S2 and S3, and contain a large protrusion that crosses S3/S4 (Extended Data Fig. 5m). Of the six ‘rich-club’ interneurons, four of them were identified in our DC analyses as neurons that cross strata. One that was not identified in our analysis is RIAR, but it is a neuron that also crosses between S1 and S4. As such, our study extends the understanding of the rich-club interneurons in the context of the nerve ring, particularly the subgroup of rich-club interneurons that are not part of the command interneurons. h–u, Volumetric reconstructions of all unassigned neurons highlighting their strata interactions. h, AWA borders S3/S4. i, RIG borders S2/S3. j, RIR borders S3/S4. k, RIS borders S1/S2. l, AIZ shifts perpendicularly from S3 to the S2/S3 border, highlighted with arrow. m, AVA borders S2/S3 and protrudes into S3 and S4. n, PVR borders S1/S3 and protrudes into S1. o, RIB forms a cage-like structure around S2. p, RIM borders S2/S3 and protrudes into S3. q, RMG protrudes into S1/S2/S3. r, SIBV’s main neurite is in S2, but it sends a second neurite into S1. s, SDQ borders S2/S3. t, URX interacts with S1 and S4. u, FLP has sparse segmentation data in the nerve ring. Images are rotated relative to each other, and transparency settings vary between images, for clarity in display of their position within the nerve ring. Neurons are arranged according to f.

Extended Data Fig. 6 Analysis of the 4 strata in the context of the lineage tree.

Lineage tree for C. elegans (0–428 mpf). The terminal branch of each neuron is coloured according to its stratum assignment and marked with a similarly coloured sphere. The lower panel is part of the lineage tree above. Upon detailed examination of all 181 neuropil neurons in the context of the lineage tree, although we observe clusters of neurons, we could not systematically correlate those clusters (representative of terminal lineage positions) with stratum assignment. This image was generated using WormGUIDES51. For access to a fully interactive lineage tree, see Supplementary Discussion 1.

Extended Data Fig. 7 Early stereotypical segregation of neuronal somas correlates with neuropil strata architecture.

a–e, Time series of neuronal soma positions within the embryo (generated using WormGUIDES51). We analysed soma positions from around 0 to 430 mpf; during this interval the embryo proceeds through gastrulation, into the early stages of elongation, and the majority of the terminal neuronal cell divisions are completed. We found that soma segregation occurs between 330–420 mpf. S2 pioneer neuron, SIAD, is shown in white for reference of nerve ring position. White arrowheads in c, d highlight the growing tips of the pioneer SIAD. The white arrowhead in e highlights the dorsal midline (meeting point for the bilateral SIADs). S1 somas are anteriorly segregated before pioneer neurite outgrowth (Supplementary Video 8). f–h, 3D depth trajectories displaying the movement of cells that will extend their neurites to S2 (f), S3 (g) or S4 (h) (movement represented from neuronal cell birth to 420 mpf). S2 movement has a ventral bias. S3 movement is principally along the outermost embryonic edge, and S4 clusters into 2 bilaterally symmetric groups. i–k, 3D depth trajectories of S1-cell movements between neuronal cell birth and 420 mpf for 3 different lineaged embryos. The embryo in i is the same dataset as the embryo shown in a–h) and in Fig. 3b–d. The migration trajectories for all 3 embryos is stereotypical. The dashed box highlights the area shown in i’–k’. Scale bar (10 μm) applies to i–k. i’–k’, Neuronal soma positions for cells from the four strata just before twitching onset. Same embryos as in i–k. Similar to the migration trajectories (i–k) the positions of neuronal somas from each stratum are stereotypical across individuals. Scale bar (10 μm) applies to i’–k’. l, Quantifications of average 3D distances from selected neuronal somas to the ALA neuron soma over a 100 min interval up to the start of twitching (430 mpf). ALA was used as reference because its position was reported to be reproducible between individual embryos58. The migration paths for neurons from the 4 strata are stereotypical across animals, and we found that the average neuron’s 3D distance from ALA varies by a standard deviation of less than a micrometre (~0.73 μm). m, Quantification of average 3D distances from selected neuronal somas to the ALA neuron soma just before the onset of twitching (430 mpf). Neuron somas in all panels are coloured according to strata assignment (S1, red; S2, purple; S3, blue; S4, green). For l–m, means are from 3 embryos (same embryos as in i–k’). All error bars are mean ± s.e.m.

Source data

Extended Data Fig. 8 S2 pioneering neurons outgrowth initiate development of the stratified neuropil and are required for nerve ring neuropil development.

a, Schematic of C. elegans embryo depicting region displayed in b–f, red box. b–f, Time-lapse of the outgrowth dynamics of the neuropil (labelled with ubiquitous nhr-2p::membrane-tethered::gfp) and schematic. Arrowheads indicate first extensions entering the future neuropil. Deconvolved diSPIM maximum intensity projections are shown (n = 8 embryos; Supplementary Video 9). g, Schematic of C. elegans embryo depicting region displayed in h–j, red box. h–j, Comma stage embryo (400 mpf) co-labelled with ubiquitous (nhr-2p::membrane-tethered::gfp) and pioneer neuron (lim-4p::mCherry) markers. Membrane and pioneer expression co-localize in SIAD, SIBV, and SMDD confirming the lineaging analysis (Fig. 3e–h). Single z-slice acquired with a single diSPIM arm shown (n = 12 embryos). The arrow indicates co-labelling of the two markers. k, Schematic of C. elegans embryo depicting the region displayed in l–n, o–q, red box. l–n, Time-lapse of the outgrowth dynamics of pioneer neurons (labelled with lim-4p::membrane-tethered::gfp). The same image series was used in Fig. 3i, j and are displayed here for comparison with the next panels. Deconvolved diSPIM maximum intensity projections are shown (n = 16 embryos). o–q, Time-lapse of the outgrowth dynamics of the neuropil (labelled with rab-3p::membrane-tethered::gfp). Deconvolved diSPIM maximum intensity projections are shown (n = 3 embryos). Neuronal outgrowth into the neuropil occurs simultaneously for pioneers (l–n) and the first pan-neuronal outgrowth events detected (o–q), suggesting the S2 pioneers are the first to enter the developing neuropil (Supplementary Video 11). r, Analysis of timing for arrival to the dorsal midline in a strain co-labelled with rab-3p::membrane-tethered::gfp, and lim-4p::mCherry. Points connected with a line correspond to data points from the same embryo (n = 10 embryos). ns, not significant by paired two-tailed Student’s t-test. s, Schematic of caspase ablation strategy for pioneer neurons. The left panel depicts split-caspase induced cell ablation, as described in ref. 53. The right panel depicts pioneer-specific split-caspase ablation assay in embryos. The lim-4p promoter was used to drive caspase expression in the SIA, SIB, SMD, RIV and SAAV neurons, ablating them before neurite outgrowth. F1 is first generation after mating. t, u, Time-lapse of the outgrowth dynamics of the pioneering neurons in control (t) and pioneer ablated (u) embryos (labelled with lim-4p::mCherry). Pixel intensities are different for t and u owing to a significant decrease in signal in the ablated animals. Deconvolved diSPIM maximum intensity projections are shown (Ctrl, n = 20 embryos; Ablation, n = 10 embryos). Previous studies showed that laser-ablations of subsets of these pioneer neurons were wild-type59, suggesting the existence of functional redundancy in guiding nerve ring development. v, Quantification of the percentage of embryos forming a full neuropil ring in control and pioneer-ablation embryos. n = number of embryos scored. ****P < 0.0001 by two-sided Fisher’s exact test between control and ablation. w, Time-lapse of the dynamics of 2xFYVE on ablated pioneering neuron somas. 2xFYVE is a marker of cell death and appears around cell corpses as described60. To see cell corpses of pioneer neurons, embryos were labelled with ced-1p::2xFYVE::gfp(S65C/Q80R) (to image cell corpses) and lim-4p::mCherry (to image pioneer neurons). Single Z-plane from diSPIM dataset shown (ctrl, n = 7 embryos; ablation, n = 6 embryos). x, Quantification of 2xFYVE encasing ablated pioneer somas. ***P = 0.0006 by two-sided Fisher’s exact test between control and ablation. n = number of embryos scored. y, Volumetric reconstruction of the developing neuropil for control and pioneer ablated embryos. Volumes were acquired from diSPIM images analysed with 3D Object Counter (FIJI-ImageJ2; Supplementary Methods). Green arrowheads emphasize aberrant neuropil phenotypes in ablation animals (gaps in the neuropil and decreased widths). z, Analysis of pixel intensity within the neuropil volume of control and pioneer ablated embryos. Each dot represents the summation of all pixels within a neuropil volume for 1 embryo (Ctrl, n = 8; ablation, n = 7), quantified using 3D Object Counter (FIJI-ImageJ2; Supplementary Methods). **P = 0.0032 by two-tailed Student’s t-test between control and ablation. Error bars are mean ± s.e.m. Timing for all panels is mpf. Scale bar, 10 μm (b–e, h–j, l–n, o–q, t, u); 3 μm (w).

Source data

Extended Data Fig. 9 S2 pioneer neurons are required for the development of neurons from all four strata, and the unassigned neurons.

a, Schematic of embryo, highlighting area in b, c with a red rectangle. Promoters used are shown below schematic in italics. b, c, Time-lapse of outgrowth dynamics of stratum 3 neuron ASH in control (b) and pioneer-ablated (c) embryos, with schematic (right). Deconvolved diSPIM maximum intensity projections shown (Ctrl, n = 5 neurons; ablation, n = 6 neurons; Supplementary Video 14). d, e, Quantifications of ASH axon (d) or dendrite (e) outgrowth for control and ablated animals. Axons (which are in the nerve ring) are affected by nerve ring pioneer neuron ablations, whereas dendrites (which are not in the nerve ring) are not affected. n = number of neurons quantified. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired two-tailed Student’s t-test between control and ablation at each time point (see Supplementary Methods for exact P values). Time points without annotation are not significant. Error bars are mean ± s.e.m. f–i, As for a–d, but for unassigned interneuron AIB (Ctrl, n = 10 neurons; ablation, n = 5 neurons; Supplementary Video 15). j–n, As for a–e, but for S4 neuron BAG (Ctrl, n = 15 neurons; ablation, n = 6 neurons;). o, Quantification of the percentage of BAG neurons with defective morphologies at 444 mpf for control and pioneer-ablated animals. BAG neurons are delayed in early outgrowth, but eventually find their terminal locations, suggesting guidance of this neuron relies on redundant mechanisms. n = number of embryos. ns indicates not significant by two-sided Fisher’s exact test between control and ablation. p, As for d but for S2 neuron AVL shown in (Fig. 4d, e). q, Schematic of C. elegans head highlighting area in r, s as red rectangle. r, s, Larval stage 1 (L1) images of S1 neuron OLL in control and pioneer-ablated animals, with schematic (below). L1 images were taken because there were no available promoters to image OLL in embryos. Spinning disk confocal maximum intensity projections shown (Ctrl, n = 8 animals; ablation, n = 20 animals). t–v, As for q–s but for S3 neuron AIY (Ctrl, n = 10 animals; ablation, n = 16 animals). w–y, As for q–s but for unassigned neuron AIB (Ctrl, n = 6 animals; ablation, n = 9 animals). The AIB outgrowth defect in embryogenesis (h) persists to L1 (y). z, Quantification of the percentage of AIB neurons with defective morphologies in L1 animals for control and pioneer-ablated animals. n = number of animals scored. ***P = 0.0002 by two-sided Fisher’s exact test between control and ablation. For cell-specific labelling of neurons, see Supplementary Methods and Supplementary Tables 2, 3. Scale bar, (b, c, g, h, k, l, r, s, u, v, x, y) 10 μm; and timing for all panels is mpf. Neurons are coloured according to which strata they belong to (S1, red; S2, purple; S3, blue; S4, green; unassigned, yellow).

Source data

Extended Data Fig. 10 A temporal progression of outgrowth, beginning with the S2 pioneers, results in the inside-out development of the nerve ring.

a, Schematic of embryo, highlighting area in b–u with a red rectangle. b–e, Time-lapse of the outgrowth dynamics of S3 neuron ASH in control animal. For ASH cell-specific labelling, see Supplementary Methods. b’–e’, As b–e but includes S2 pioneer neurons (labelled with lim-4p::mCherry). Yellow arrowheads mark ASH axonal outgrowth in the context of the pioneers. White arrowheads mark dorsal midline. ASH outgrowth into the neuropil occurs after the pioneers have grown into the nerve ring. Deconvolved diSPIM maximum intensity projections shown (n = 4 embryos). f–j, Time-lapse of the outgrowth dynamics of unassigned neuron AIB in control animal. For AIB cell-specific labelling, see Supplementary Methods. f’–j’, As f–j, but includes S2 pioneer neurons (labelled with lim-4p::mCherry). Blue arrowheads mark AIB axonal outgrowth in the context of the pioneers. White arrowheads mark the dorsal midline. AIB enters the neuropil after the pioneers have reached the dorsal midline, and as ASH reaches the dorsal midline (compare e’ to h’). Data collected in this way b–j were used for indicated neurons in Fig. 4j. Deconvolved diSPIM maximum intensity projections shown (n = 6 embryos). k–r, Time-lapse of the outgrowth dynamics for S2 pioneer SAAV, and S4 neuron AWC. In k, red arrowheads mark outgrowth of SAAV. In l–r, red arrowheads mark the dorsal midline and yellow arrowheads mark the outgrowth of AWC. The S4 neuron AWC pauses for around 20 min near the SAAV soma before growing into the nerve ring. Deconvolved diSPIM maximum intensity projections shown. n = 7 embryos. The ceh-37p promoter expresses strongly in SAAV and AWC, but weakly in ADF, AFD, AWB. k’–r’, As k–r, but one side of the bilateral AWC neurons have been pseudocoloured green and the remaining image pseudocoloured red to highlight the outgrowth of the S4 neurons (n = 7 embryos; Supplementary Methods). l’’, Schematic of l’ depicting the growing SAAV (red) and AWC (green) neuron. s, Quantification of AWC pausing duration. Each dot represents an embryo (n = 7). AWC pauses at the SAAV cell body for around 20 min before entering the neuropil. Error bars are mean ± s.e.m. (see Supplementary Methods for quantification). t, u, Time-lapse of the outgrowth dynamics for S1 sensory neurons. Red arrowheads mark sensory endings. The yellow arrowhead marks outgrowth of a looping neuron. The dashed line in t corresponds to the position of the pioneering neurons (seen in Fig. 3i, j). The outgrowth of looping structures starts after 420 min, that is, after the pioneer neurons have grown out (compare to Fig. 3i, j). Image in u taken in a threefold embryo, which moved (therefore position of cell bodies is different between t and u). Deconvolved diSPIM maximum intensity projections shown. (n = 9 embryos; compare to Fig. 4j). u’, As u, but one S1 sensory neuron has been pseudocoloured red and the remaining image pseudocoloured green to highlight the looping outgrowth of the S1 neuron (Supplementary Methods). u”, Schematic of u’ depicting the growing loop of the S1 sensory neuron. Together with the temporal dynamics of outgrowth and the ablation studies, our findings support an inside-out model in which the strata are assembled through timed entry into the nerve ring, starting with a core unit of the pioneering bundle, proceeding to central S2, then to the peripherally located neurons in S1 (anterior) and S4 (posterior), followed by outgrowth of neurons which link the strata, such as the S1 looping neurons or the neurons that cross strata (like AIB). Scale bar, 10 μm (b–e’, f–j’, k–r’, t–u’), and timing for all panels is mpf. The promoter used to drive expression is shown in italics in b, f, k, t.

Source data

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3 and Supplementary Discussions 1-3. Supplementary Table 1: Analysis of neurons found within each loop of the S1 honeycomb-like structure. Supplementary Table 2: C. elegans strains used in this study. Supplementary Table 3: DNA plasmids used in this study. Supplementary Discussion 1: Protocol to access WormGUIDES desktop application. Supplementary Discussion 2: Protocol to access immersive 3D C-PHATE plots. Supplementary Discussion 3: Systematic quantitative examination of the differences in contact profiles and DC outputs between the L4 and adult datasets.

Reporting Summary

Supplementary Methods.

Supplementary Video 1

. C-PHATE plot reveals hierarchical nested clustering over the DC iterations for L4 C. elegans. 3D rendering of C-PHATE plot of DC analyses for a larval stage 4 (L4) animal. Individual neurons are located at edges of the graph and condense as they move centrally. Plot colored according to 4 clusters at iteration 23 (C1-Red, C2-Purple, C3-Blue, C4-Green).

Supplementary Video 2

. C-PHATE plot reveals hierarchical nested clustering over the DC iterations for adult C. elegans. 3D rendering of C-PHATE plot of DC analysis for an adult stage animal. Individual neurons are located at edges of the graph, with condensation iterations towards the center. Plot colored according to 6 clusters at iteration 22 (C1-Red, C2-Purple, C3-Blue, C4-Green, C5/6 Grey).

Supplementary Video 3

. The nerve ring neuropil is structurally organized into 4 strata. Volumetric reconstruction of the L4 C. elegans neuropil (from EM serial sections3) with neurons from the 4 strata highlighted (S1-Red, S2-Purple, S3-Blue, S4-Green).

Supplementary Video 4

. S1 neurons form a honeycomb-like structure encasing S2, S3, and S4. Volumetric reconstruction of the L4 animal honeycomb-like structure formed by the 32 neurons in S1. During first part of the movie, bilaterally symmetric neuron pairs are highlighted in a range of colors to differentiate them. In the second part, the color scheme changes, with the entire structure shown in red (due to their placement in S1) in the context of the remaining strata (S2-Purple, S3-Blue, S4-Green).

Supplementary Video 5

. The S1 honeycomb-like structure precisely loops at the boundaries of the indicated strata. Volumetric reconstruction of a subset of dorsal right loop neurons forming the honeycomb structure around the strata (Neurons: URADR, URYDR, IL1DR, and IL2DR, in red), in the context of S2 (purple), and then S3/S4 (blue/green).

Supplementary Video 6

. Unassigned interneuron AIB contacts multiple strata. Volumetric reconstruction of the unassigned (yellow) ‘rich-club’ AIB interneurons15,20 in the context of nerve ring strata. AIB borders S3/S4, shifts along the A-P axis, and then borders S2/S3.

Supplementary Video 7

. Unassigned interneuron AVE contacts multiple strata. Volumetric reconstructions of the unassigned (yellow) ‘rich-club’ AVE interneuron15,20 in the context of nerve ring strata and AIB (orange). AVE borders S2/S3, shifts along the A-P axis, and then borders S1/S2.

Supplementary Video 8

. Early segregation of neuronal somas followed by S2 pioneering neuron outgrowth correlate to development of the stratified neuropil. WormGUIDES atlas representation of the embryonic neuronal soma positions and S2 pioneer SIAD outgrowth between 330-427 mpf. Records of the positions of every cell at every minute during embryonic development for three embryos were used to generate a 4D representation of all cellular positions as described51. Somas colored to show their eventual neurite strata assignment (S1-Red, S2-Purple, S3-Blue, S4-Green), and pioneer SIAD neurons in white to depict the developing neuropil. S1 is anteriorly segregated prior to pioneer neurite outgrowth.

Supplementary Video 9

. Embryonic neuropil development begins between 390-400 mpf. Time-lapse of the outgrowth dynamics of the neuropil (labeled with ubiquitous nhr-2p::membrane-tethered::GFP). Neuropil visible as “omega” structure in the embryo head (anterior of the embryo is towards the top) and is highlighted with a white line midway through the movie. Images are deconvolved diSPIM maximum intensity projections. Time-lapse images in Extended Data Fig. 8b-e were generated from this dataset. Scale bar applies to entire movie, and timing is mpf.

Supplementary Video 10

. S2 pioneering neurons grow together and enter the presumptive neuropil between 390-400 mpf. Time-lapse of the outgrowth dynamics of pioneer neurons (labeled with lim-4p::membrane-tethered::GFP), followed by a 3D rotation of the last timepoint to highlight the early ring structure formed by the S2 pioneers. Images are deconvolved diSPIM maximum intensity projections. Time-lapse images in Fig. 3i-j are from this dataset. Scale bar applies to entire movie, and timing is mpf.

Supplementary Video 11

. Embryonic neuropil development begins between 390-400 mpf. Time-lapse of the outgrowth dynamics of the nerve ring (labeled with rab-3p::membrane-tethered::GFP), followed by a 3D rotation of the last timepoint to highlight the neuropil, which is the bright ring structure in the anterior part of the embryo (top). Embryo twitches during later development. Neuropil intensity increases in overtime, probably due to an increase in the number of neurons entering the neuropil. Images are deconvolved diSPIM maximum intensity projections. Time-lapse images in Extended Data Fig. 8o-q are from this dataset. Scale bar applies to entire movie, and timing is mpf.

Supplementary Video 12

. Embryonic neuropil development begins between 390-400 mpf. Time-lapse of the outgrowth dynamics of the nerve ring (labeled with ceh-48p::membrane-tethered::GFP), followed by a 3D rotation of the last timepoint to highlight the neuropil, which is the bright ring structure found near the top of the embryo. Embryo starts twitching during later development. Note that the neuropil increases in intensity overtime, probably due to an increase in the number of neurons entering the neuropil through time. Images are deconvolved diSPIM maximum intensity projections. Scale bar applies to entire movie, and timing is mpf.

Supplementary Video 13

. Outgrowth dynamics of the S2 neuron AVL. Time-lapse of the outgrowth dynamics of the AVL neuron (labeled with lim-6p::GFP), followed by a 3D rotation of the last timepoint to highlight AVL outgrowth into the neuropil. Embryo starts twitching during later development. Images are deconvolved diSPIM maximum intensity projections. Time-lapse image in Fig. 4d is from this same dataset. Scale bar applies to entire movie, and timing is mpf.

Supplementary Video 14

. Outgrowth dynamics of the S3 neuron ASH. Time-lapse of the outgrowth dynamics of the ASH neuron (labeled with unc-42p::ZF1::membrane-tethered::GFP + lim-4p::ZIF-1; see Supplementary Methods) followed by a 3D rotation of the last timepoint to highlight ASH outgrowth into the neuropil. ASH is the brightest neuron seen in the movie. Embryo starts twitching during later development. Images are deconvolved diSPIM maximum intensity projections. Time-lapse images in Extended Data Fig. 9b are from this dataset. Scale bar applies to entire movie, and timing is mpf.

Supplementary Video 15

. Outgrowth dynamics of the unassigned neuron AIB. Time-lapse of the outgrowth dynamics of the AIB neuron (labeled with unc-42p::ZF1::membrane-tethered::GFP + lim-4p::ZIF-1; see Supplementary Methods) followed by a 3D rotation of the last timepoint to highlight AIB outgrowth into the neuropil. AIB is the brightest neuron seen in the later time points. Images are deconvolved diSPIM maximum intensity projections. Time-lapse images in Extended Data Fig. 9g are from this same dataset. Scale bar applies to entire movie, and timing is mpf.

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Moyle, M.W., Barnes, K.M., Kuchroo, M. et al. Structural and developmental principles of neuropil assembly in C. elegans. Nature 591, 99–104 (2021). https://doi.org/10.1038/s41586-020-03169-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-020-03169-5

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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