Glia initiate brain assembly through noncanonical Chimaerin–Furin axon guidance in C. elegans

Abstract

Brain assembly is hypothesized to begin when pioneer axons extend over non-neuronal cells, forming tracts guiding follower axons. Yet pioneer-neuron identities, their guidance substrates, and their interactions are not well understood. Here, using time-lapse embryonic imaging, genetics, protein-interaction, and functional studies, we uncover the early events of C. elegans brain assembly. We demonstrate that C. elegans glia are key for assembly initiation, guiding pioneer and follower axons using distinct signals. Pioneer sublateral neurons, with unique growth properties, anatomy, and innervation, cooperate with glia to mediate follower-axon guidance. We further identify a Chimaerin (CHIN-1)– Furin (KPC-1) double-mutant that severely disrupts assembly. CHIN-1 and KPC-1 function noncanonically, in glia and pioneer neurons, for guidance-cue trafficking. We exploit this bottleneck to define roles for glial Netrin and Semaphorin in pioneer- and follower-axon guidance, respectively, and for glial and pioneer-neuron Flamingo (CELSR) in follower-axon navigation. Taken together, our studies reveal previously undescribed glial roles in pioneer-axon guidance, suggesting conserved principles of brain assembly.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Hierarchical assembly of the embryonic NR.
Figure 2: CEPsh glia and SubL axons functionally pioneer the NR.
Figure 3: NR axon entry is disrupted in kpc-1;chin-1 mutants.
Figure 4: KPC-1 and CHIN-1 act in NR pioneers at the onset of NR assembly.
Figure 5: Glia direct pioneer- and follower-axon guidance using distinct signaling pathways.
Figure 6: KPC-1 and CHIN-1 control guidance-cue trafficking.

References

  1. 1

    Easter, S.S. Jr., Ross, L.S. & Frankfurter, A. Initial tract formation in the mouse brain. J. Neurosci. 13, 285–299 (1993).

    PubMed  PubMed Central  Google Scholar 

  2. 2

    Chédotal, A. & Richards, L.J. Wiring the brain: the biology of neuronal guidance. Cold Spring Harb. Perspect. Biol. 2, a001917 (2010).

    PubMed  PubMed Central  Google Scholar 

  3. 3

    Jacobs, J.R., Goodman, C.S. & Ii, C.N.S. Embryonic development of axon pathways in the Drosophila CNS. II. Behavior of pioneer growth cones. J. Neurosci. 9, 2412–2422 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Hidalgo, A., Urban, J. & Brand, A.H. Targeted ablation of glia disrupts axon tract formation in the Drosophila CNS. Development 121, 3703–3712 (1995).

    CAS  PubMed  Google Scholar 

  5. 5

    Hidalgo, A. & Booth, G.E. Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS. Development 127, 393–402 (2000).

    CAS  PubMed  Google Scholar 

  6. 6

    Whitington, P.M., Quilkey, C. & Sink, H. Necessity and redundancy of guidepost cells in the embryonic Drosophila CNS. Int. J. Dev. Neurosci. 22, 157–163 (2004).

    CAS  PubMed  Google Scholar 

  7. 7

    Takizawa, K. & Hotta, Y. Pathfinding analysis in a glia-less gcm mutant in Drosophila. Dev. Genes Evol. 211, 30–36 (2001).

    CAS  PubMed  Google Scholar 

  8. 8

    Placzek, M. & Briscoe, J. The floor plate: multiple cells, multiple signals. Nat. Rev. Neurosci. 6, 230–240 (2005).

    CAS  PubMed  Google Scholar 

  9. 9

    Minocha, S. et al. Nkx2.1-derived astrocytes and neurons together with Slit2 are indispensable for anterior commissure formation. Nat. Commun. 6, 6887 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Kolodkin, A.L. & Tessier-Lavigne, M. Mechanisms and molecules of neuronal wiring: a primer. Cold Spring Harb. Perspect. Biol. 3, a001727 (2011).

    PubMed  PubMed Central  Google Scholar 

  11. 11

    Iwasato, T. et al. Rac-GAP α-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling. Cell 130, 742–753 (2007).

    CAS  PubMed  Google Scholar 

  12. 12

    Jaworski, A. et al. Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2. Science 350, 961–965 (2015).

    CAS  PubMed  Google Scholar 

  13. 13

    Jorgensen, E.M. & Mango, S.E. The art and design of genetic screens: caenorhabditis elegans. Nat. Rev. Genet. 3, 356–369 (2002).

    CAS  PubMed  Google Scholar 

  14. 14

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Oikonomou, G. & Shaham, S. The glia of Caenorhabditis elegans. Glia 59, 1253–1263 (2011).

    PubMed  Google Scholar 

  16. 16

    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).

    CAS  Google Scholar 

  17. 17

    MacNeil, L.T., Hardy, W.R., Pawson, T., Wrana, J.L. & Culotti, J.G. UNC-129 regulates the balance between UNC-40 dependent and independent UNC-5 signaling pathways. Nat. Neurosci. 12, 150–155 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Zallen, J.A., Kirch, S.A. & Bargmann, C.I. Genes required for axon pathfinding and extension in the C. elegans nerve ring. Development 126, 3679–3692 (1999).

    CAS  PubMed  Google Scholar 

  19. 19

    Hedgecock, E.M., Culotti, J.G. & Hall, D.H. The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 4, 61–85 (1990).

    CAS  PubMed  Google Scholar 

  20. 20

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    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).

    CAS  PubMed  Google Scholar 

  22. 22

    Durbin, R.M. Studies on the development and organisation of the nervous system of Caenorhabditis elegans. PhD dissertation, Cambridge Univ. (1987).

  23. 23

    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).

    CAS  PubMed  Google Scholar 

  24. 24

    Troemel, E.R., Sagasti, A. & Bargmann, C.I. Lateral signaling mediated by axon contact and calcium entry regulates asymmetric odorant receptor expression in C. elegans. Cell 99, 387–398 (1999).

    CAS  PubMed  Google Scholar 

  25. 25

    Schroeder, N.E. et al. Dauer-specific dendrite arborization in C. elegans is regulated by KPC-1/Furin. Curr. Biol. 23, 1527–1535 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Thacker, C. & Rose, A.M. A look at the Caenorhabditis elegans Kex2/Subtilisin-like proprotein convertase family. BioEssays 22, 545–553 (2000).

    CAS  PubMed  Google Scholar 

  27. 27

    Mason, C. & Erskine, L. Growth cone form, behavior, and interactions in vivo: retinal axon pathfinding as a model. J. Neurobiol. 44, 260–270 (2000).

    CAS  PubMed  Google Scholar 

  28. 28

    Kumfer, K.T. et al. CGEF-1 and CHIN-1 regulate CDC-42 activity during asymmetric division in the Caenorhabditis elegans embryo. Mol. Biol. Cell 21, 266–277 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Roy, P.J., Zheng, H., Warren, C.E. & Culotti, J.G. mab-20 encodes Semaphorin-2a and is required to prevent ectopic cell contacts during epidermal morphogenesis in Caenorhabditis elegans. Development 127, 755–767 (2000).

    CAS  PubMed  Google Scholar 

  30. 30

    Steimel, A. et al. The Flamingo ortholog FMI-1 controls pioneer-dependent navigation of follower axons in C. elegans. Development 137, 3663–3673 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Organisti, C., Hein, I., Grunwald Kadow, I.C. & Suzuki, T. Flamingo, a seven-pass transmembrane cadherin, cooperates with Netrin/Frazzled in Drosophila midline guidance. Genes Cells 20, 50–67. http://dx.doi.org/10.1111/gtc.12202 (2015).

    CAS  PubMed  Google Scholar 

  32. 32

    Feng, J. et al. Celsr3 and Fzd3 organize a pioneer neuron scaffold to steer growing thalamocortical axons. Cereb. Cortex 26, 3323–3334 (2016).

    PubMed  PubMed Central  Google Scholar 

  33. 33

    Ferrario, J.E. et al. Axon guidance in the developing ocular motor system and Duane retraction syndrome depends on Semaphorin signaling via alpha2-chimaerin. Proc. Natl. Acad. Sci. USA 109, 14669–14674 (2012).

    CAS  PubMed  Google Scholar 

  34. 34

    Pan, X., Eathiraj, S., Munson, M. & Lambright, D.G. TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature 442, 303–306 (2006).

    CAS  PubMed  Google Scholar 

  35. 35

    Chen, W., Lim, H.H. & Lim, L. The CDC42 homologue from Caenorhabditis elegans. Complementation of yeast mutation. J. Biol. Chem. 268, 13280–13285 (1993).

    CAS  PubMed  Google Scholar 

  36. 36

    Lipschutz, J.H. & Mostov, K.E. Exocytosis: the many masters of the exocyst. Curr. Biol. 12, R212–R214 (2002).

    CAS  PubMed  Google Scholar 

  37. 37

    Hosaka, M. et al. Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway. J. Biol. Chem. 266, 12127–12130 (1991).

    CAS  PubMed  Google Scholar 

  38. 38

    Adams, R.H., Lohrum, M., Klostermann, A., Betz, H. & Püschel, A.W. The chemorepulsive activity of secreted semaphorins is regulated by furin-dependent proteolytic processing. EMBO J. 16, 6077–6086 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Sadeqzadeh, E. et al. Furin processing dictates ectodomain shedding of human FAT1 cadherin. Exp. Cell Res. 323, 41–55 (2014).

    CAS  PubMed  Google Scholar 

  40. 40

    Hung, W.L., Wang, Y., Chitturi, J. & Zhen, M. A Caenorhabditis elegans developmental decision requires insulin signaling-mediated neuron-intestine communication. Development 141, 1767–1779 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Tassew, N.G., Charish, J., Seidah, N.G. & Monnier, P.P. SKI-1 and Furin generate multiple RGMa fragments that regulate axonal growth. Dev. Cell 22, 391–402 (2012).

    CAS  PubMed  Google Scholar 

  42. 42

    Riccomagno, M.M. et al. The RacGAP β2-Chimaerin selectively mediates axonal pruning in the hippocampus. Cell 149, 1594–1606 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Miyake, N. et al. Human CHN1 mutations hyperactivate alpha2-chimaerin and cause Duane's retraction syndrome. Science 321, 839–843 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Barry, D.S., Pakan, J.M.P. & McDermott, K.W. Radial glial cells: key organisers in CNS development. Int. J. Biochem. Cell Biol. 46, 76–79 (2014).

    CAS  PubMed  Google Scholar 

  45. 45

    Rakic, P. Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electronmicroscopic study in Macacusrhesus. J. Comp. Neurol. 141, 283–312 (1971).

    CAS  PubMed  Google Scholar 

  46. 46

    Kuwajima, T. et al. Optic chiasm presentation of Semaphorin6D in the context of Plexin-A1 and Nr-CAM promotes retinal axon midline crossing. Neuron 74, 676–690 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Métin, C., Deléglise, D., Serafini, T., Kennedy, T.E. & Tessier-Lavigne, M. A role for netrin-1 in the guidance of cortical efferents. Development 124, 5063–5074 (1997).

    PubMed  Google Scholar 

  48. 48

    Dominici, C. et al. Floor-plate-derived netrin-1 is dispensable for commissural axon guidance. Nature 545, 350–354 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Varadarajan, S.G. et al. Netrin1 produced by neural progenitors, not floor plate cells, is required for axon guidance in the spinal cord. Neuron 94, 790–799 e3 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Voigt, T. Development of glial cells in the cerebral wall of ferrets: direct tracing of their transformation from radial glia into astrocytes. J. Comp. Neurol. 289, 74–88 (1989).

    CAS  PubMed  Google Scholar 

  51. 51

    Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Stiernagle, T. Maintenance of C. elegans. In C. elegans (ed. Hope, I.A.) 51–67 (Oxford, 1999).

  53. 53

    Mello, C.C., Kramer, J.M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Sulston, J.E. & Horvitz, H.R. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol. 56, 110–156 (1977).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Hedgecock, E.M., Culotti, J.G., Thomson, J.N. & Perkins, L.A. Axonal guidance mutants of Caenorhabditis elegans identified by filling sensory neurons with fluorescein dyes. Dev. Biol. 111, 158–170 (1985).

    CAS  PubMed  Google Scholar 

  56. 56

    Dickinson, D.J., Ward, J.D., Reiner, D.J. & Goldstein, B. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat. Methods 10, 1028–1034 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Ward, J.D. Rapid and precise engineering of the Caenorhabditis elegans genome with lethal mutation co-conversion and inactivation of NHEJ repair. Genetics 199, 363–377 (2015).

    PubMed  PubMed Central  Google Scholar 

  58. 58

    Duckert, P., Brunak, S. & Blom, N. Prediction of proprotein convertase cleavage sites. Protein Eng. Des. Sel. 17, 107–112 (2004).

    CAS  PubMed  Google Scholar 

  59. 59

    Tavernarakis, N., Wang, S.L., Dorovkov, M., Ryazanov, A. & Driscoll, M. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat. Genet. 24, 180–183 (2000).

    CAS  PubMed  Google Scholar 

  60. 60

    Kamath, R.S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Heiman, M.G. & Shaham, S. DEX-1 and DYF-7 establish sensory dendrite length by anchoring dendritic tips during cell migration. Cell 137, 344–355 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    Yochem, J. & Herman, R.K. Investigating C. elegans development through mosaic analysis. Development 130, 4761–4768 (2003).

    CAS  PubMed  Google Scholar 

  63. 63

    Conradt, B. & Horvitz, H.R. The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell 93, 519–529 (1998).

    CAS  PubMed  Google Scholar 

  64. 64

    Singhal, A. & Shaham, S. Infrared laser-induced gene expression for tracking development and function of single C. elegans embryonic neurons. Nat Commun 8, 1–13 (2017).

    Google Scholar 

  65. 65

    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).

    CAS  PubMed  Google Scholar 

  66. 66

    Bargmann, C.I., Hartwieg, E. & Horvitz, H.R. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell 74, 515–527 (1993).

    CAS  PubMed  Google Scholar 

  67. 67

    Seligman, A.M., Wasserkrug, H.L. & Hanker, J.S. A new staining method (OTO) for enhancing contrast of lipid--containing membranes and droplets in osmium tetroxide--fixed tissue with osmiophilic thiocarbohydrazide(TCH). J. Cell Biol. 30, 424–432 (1966).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68

    Lowe, D.G. Object recognition from local scale-invariant features. Proc. Seventh IEEE Int. Conf. Comput. Vis. 2, 1150–1157 (1999).

    Google Scholar 

  69. 69

    Scheffzek, K. et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333–338 (1997).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Bargmann, V. Bertrand, L. Cochella, L. Chen, J. Culotti, O. Hobert, H. Hutter, L. Kutscher, J. Malin, G. Oikonomou, N. Pujol, P. Sengupta, B. Tursun, WG. Wadsworth, S. Wallace, and M. Zhen for reagents, as well as M. Katz for sharing unpublished information. Some strains were provided by the CGC, funded by NIH (P40 OD010440). We thank the Rockefeller University Bio-Imaging and Electron Microscopy Resource Centers for technical help, W.J. Rice at the Simons Electron Microscopy Center (NYSBC) for help with FIB-SEM imaging, and C. Bargmann and the Shaham lab for insights. G.R. was supported by a Shelby White and Leon Levy Foundation fellowship. This work was supported in part by NIH grants NS064273 and NS073121 to S.S.

Author information

Affiliations

Authors

Contributions

G.R. performed all experiments except the electron microscopy studies, which were performed by Y.L. C.L. and A.S. assisted with generation of plasmids, strains and yeast-two-hybrid screens. S.S. supervised the project. G.R and S.S. wrote the manuscript.

Corresponding authors

Correspondence to Georgia Rapti or Shai Shaham.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Timeline and electron micrographs of C. elegans embryonic stages

(a-f) DIC images of embryonic stages. Hatching occurs at 880 minutes post-fertilization (20°C).(g,h) Electron micrographs of NR region of embryos of comma (g) or 1.5-fold stages (h). (g)The region outlined in the blue box corresponds to the region magnified in Fig. 1p. Ventral, bottom. In schematics gray rectangles indicate section plane of electron micrographs. In inset of panels g, h the a,p,v,d indicate: anterior, posterior, ventral, dorsal respectively. Scale bars: 2μm.

Supplementary Figure 2 Growth and nerve-ring entry of later nerve ring components

(a,e,h,k,m,p,r) Head regions of embryos as indicated by the dotted boxes in schematics. Embryos express (b-d) Pceh-17::GFP labeling SubL (SIA, SIB) neurons and Pttx-3::mCherry labeling SMDD neurons; (f-g) Pflp-10::GFP labeling BAG and AUA neurons,; (i,j,l) Pttx-1::GFP, labeling AFD neurons (pseudocolored blue) or Plsy-6::GFP, labeling ASE neurons; (n) Pflp-8::GFP labeling the ADA neuron; (o,q) Pser-2::GFP labeling BDU neurons; (s-u) Pttx-3::mCherry labeling SMDD (and thus the sublateral commissure bundle, with which SMDD has earlier fasciculated) and Phlh-1::myristoylated-GFP labeling muscle cell membranes. NR: nerve ring. Red dotted outline marks the nerve ring path. White arrows: axons, yellow arrowheads: muscle arms growing near the nerve ring. Scale bar 10μm.

Supplementary Figure 3 Neuron and glia soma and processes in kpc-1;chin-1 mutants

Animals of L3 stage expressing Pnpr-11::RFP to label the PVQ neuron (a-d) or Prab-3::RFP labeling all neurons (e-h). Black dotted line: lateral midline. Animals of L2 stage expressing Pmir-228::GFP (i,j) labeling all glia or Phlh-17:: myristoylated-GFP (k,l) labeling CEPsh glia. NR: nerve ring, VNC: ventral nerve cord, arrows: axons in NR, open arrowheads: axons in ventral nerve cord (VNC), full arrowheads: peripheral motoneuron commissures, black asterisks: intestine autofluorescence, white asterisk: vulva, doted white outline: pharyngeal bulbs. A: anterior, P: posterior, V: ventral, D: dorsal. Scale bar: 10μm.

Supplementary Figure 4 Gene and protein structures and expression patterns of KPC-1 and CHIN-1

(a,b) Protein domains and mutant lesions of kpc-1 (a) and chin-1 (b) loci. Amino acid conservation of Arginine motif of Chimaerins indicated in (b). (c,d) Protein expression patterns for KPC-1 and CHIN-1 using the genomic DNA fragments Pkpc-1::kpc-1::SL2::mCherry and Pchin-1::GFP. Dark dotted line: embryo outline. Embryonic stages as indicated. A: anterior, P: posterior, V: ventral, D: dorsal.

Supplementary Figure 5 AIY and SMDD axon growth in wild-type, kpc-1(gk8);chin-1(ns399), kpc-1(gk8) and chin-1(ns399) embryos

(a,b) Slopes of axon growth of AIY (a) or SMDD (b) axons presented in Fig. 4i,j, respectively. Slopes calculated for each embryo as μm/stage, for the following embryonic stage transitions: bean to comma, comma to 1.5-fold and bean to 1.5-fold stage. Dot: slope of individual axon in one stage transition, bar: average of slopes of all axons of given genotype in one stage transition. Numbers above bars, exact p values by t- test (GraphPad). ns: non significant (when P value>0.05). Number of degrees of freedom equals the number of pairs minus 1. Number of animals analyzed: (a) n=7 for WT, n=6 for chin-1; kpc-1 mutant, (b) n=8 for WT, n=7 for chin-1; kpc-1 mutant. (a) t ratios for comparisons of axon-growth slopes for stage transitions bean-comma, comma-1,5fold, and bean-1.5fold are (a) 2.7, 3.1, 3.3 respectively for AIY growth and (b) 0.1, 3.8, 2.8 respectively for SMDD growth. (c,d) Axon length (μm) of neurons AIY (c) or SMDD (d) in wild-type (WT) and single kpc-1(gk8) or chin-1(ns399) mutant embryos. Square bars: individual axon measurement in given embryonic stage. Line follows individual axon. Number of animals analyzed: (c) n=6 for WT, n=9 and n=10 for chin-1(ns399) and kpc-1(gk8) single mutants respectively, (d) n=7 for WT, n=4 for each of chin-1(ns399) and kpc-1(gk8) single mutants. (a-b) Numbers above bars are exact p values using Fisher’s exact test. ns: non significant.

Supplementary Figure 6 Protein structures, positions of mutant lesions and predicted furin-recognition motifs for FMI-1/Flamingo, MAB-20/Semaphorin, and UNC-6/Netrin

Isoforms, protein structures, motifs, and alleles of FMI-1/Flamingo/CELSR (a), of MAB-20/Semaphorin and human Sema6C (b) and UNC-6/Netrin and human Netrin1 proteins (c). Position of allele mutations and furin motifs are indicated by triangles and lines, respectively. Domain identity is indicated in box. fmi-1 alleles: rh308 and ns701, ns717, ns742 (this study, see Supplemental Information). mab-20 alleles: ev57429, and ns789 (this study, CRISPR-generated allele, see Supplemental experimental procedures). unc-6(ev400) allele21 EGF: Epithelial Growth Factor domain, GPS: GPCR proteolytic site, PSI: Plexin Semaphorin Integrin domain. Definition of the predicted protein domains can be found in the online tools of protein domain prediction: NCBI conserved domain and EMBL-SMART protein. (d) Protein domains legend.

Supplementary Figure 7 FMI-1-GFP signal localization

FMI-1-GFP ectopic signal is represented as relative intensities of regions, quantified as described in online Methods. Regions of interest I, II, III of cell bodies refer to the regions of blue boxes outlined in Figure 5a-d. Number of animals analyzed appears in the graph (n). Mean ± Error bars: SEM. Numbers above bars are exact p values by t-test (GraphPad). ns: non significant (when P value>0.05).

Supplementary Figure 8 Model for nerve ring assembly.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1–5 (PDF 4503 kb)

Life Sciences Reporting Summary (PDF 158 kb)

Supplementary Table 6

New mutant alleles generated in this study (XLSX 29 kb)

Genomic information of the new mutant alleles generated in this study.

Supplementary Table 7

Unstable extra-chromosomal transgenes used (XLSX 30 kb)

Information of the unstable extra-chromosomal transgenes generated in this study. Information on allele number, DNA injected and relevant background strain is provided.

Supplementary Table 8

Stably integrated transgenes used (XLSX 12 kb)

Information of the stably integrated transgenes used in this study. Information on allele number and relevant citations of published transgenes is provided.

Supplementary Table 9

List of plasmids used (XLSX 13 kb)

Information about the plasmids used in this study, as well as citations when applicable, is provided. DNA sequences of pGR plasmids (generated in this study) are available upon request.

Supplementary Table 10

Expression patterns of reporters used (XLSX 12 kb)

Information on expression patterns of transgene reporters used in this study is provided. Relevant citations are also provided when applicable.

Head region of wild-type L1 animal reconstructed using FIB-SEM.

Movie proceeds from posterior to anterior. Bottom right is ventral, top right is left. (MOV 6212 kb)

Head region of kpc-1(gk8); chin-1(ns399) L1 animal reconstructed using FIB-SEM

Movie proceeds from posterior to anterior. Bottom right is ventral, top right is left. (MOV 4196 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rapti, G., Li, C., Shan, A. et al. Glia initiate brain assembly through noncanonical Chimaerin–Furin axon guidance in C. elegans. Nat Neurosci 20, 1350–1360 (2017). https://doi.org/10.1038/nn.4630

Download citation

Further reading

Search

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