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.
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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 Wadsworth, S. Wallace, and M. Zhen for reagents, as well as M. Katz for sharing unpublished information.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.
The authors declare no competing financial interests.
Integrated supplementary information
(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.
(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.
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.
(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.
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 Figures 1–8 and Supplementary Tables 1–5 (PDF 4503 kb)
New mutant alleles generated in this study (XLSX 29 kb)
Genomic information of the new mutant alleles generated in this study.
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.
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.
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.
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.
Movie proceeds from posterior to anterior. Bottom right is ventral, top right is left. (MOV 6212 kb)
Movie proceeds from posterior to anterior. Bottom right is ventral, top right is left. (MOV 4196 kb)
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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
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