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.

Figure 1. Hierarchical assembly of the embryonic nerve ring

(a–b) The post-embryonic nerve ring is populated by neuronal commissures and enveloped by CEPsh glia. (a) Schematics of lateral view of post-embryonic nerve ring. (b) Imaging of post-embryonic nerve ring in L4 animals. Inset, cross-sectional view. Pttx-3::mCherry (AIY, red), Phlh-17::myristoylated-GFP (CEPsh glia). (c–n) Formation of the embryonic nerve ring starts at late bean stage, with early entry of sublateral commissure axons and CEPsh glia and later entry of other components. (c,d) Phis-72:: myristoylated-GFP (cell membranes). (c–k) Dotted line: embryo outline. D: dorsal, V: ventral, A: anterior, P: posterior. Arrow: axon, arrowhead: CEPsh glia. Scale bars: 10μm. (c-g,i-k,m,n) Pttx-3::mCherry (SMDD, red; AIY, pseudocolored orange in (I). (e–h) Pceh-17::GFP, sublateral neurons SIAV/D, SIBV/D. (j) Phlh-16::GFP (SIA/SIB, red; AWC, pseudocolored blue). (k–n) Pmir-228:: myristoylated-GFP (glia). (b–n) Expression patterns are described in Supplementary Methods and Supplementary Tables S8, S10. l–n: magnified view of boxed region in k. (o,p) A single pair of bilateral bundles are observed in electron micrographs of early comma stage embryos. Schematic (o) and magnification (p) of electron micrograph section. The magnified region corresponds to the region outlined in the blue box of Supplementary Fig. S1g. Red outline: axon bundle. Scale bar: 2μm. (q) Summary of imaging results. CEPsh glia and pioneer neurons enter NR path first, followed by amphid and AIY neurons.

Figure 2. CEPsh glia and SubL axons functionally pioneer the NR

Nerve-ring entry of AIY follower axons and fasciculation of sublateral commissure axons is abnormal in CEPsh-ablated animals. Ablation of neurons of the sublateral commissure results in abnormal AIY nerve-ring entry but spares CEPsh membrane growth. Scale bars: 10 μm. (a–d) Pttx-3::mCherry (AIY), Pmir-228::GFP (glia). (e–h) Pceh-24::GFP (SIA/SIB/SMD), Pptr-10::RFP (glia). Dotted white outline: 1st pharyngeal bulb. (i–p) Pceh-17::RFP (SIA/SIB), Pttx-3::GFP (AIY, green) or Phlh-17:: myristoylated-GFP (CEPsh glia, green). Reporter expression patterns are described in Supplementary Methods and Supplementary Tables S8, S10. Asterisk: NR axon gap.

Figure 3. NR axon entry is disrupted in kpc-1; chin-1 mutants

(a–h, m–o) Nerve-ring guidance of axons of different neuron subtypes, and in different commissures, is abnormal in kpc-1; chin-1 mutants. Asterisk, arrow, scale bars: 10μm. D: dorsal, V: ventral, A: anterior, P: posterior. (a,b) Pgcy-5::GFP. (c,d) Pttx-1::RFP. (e,f) Pttx-3::GFP. (b,d,f) kpc-1(ns623); chin-1(ns399). (g,h) Pnpr-11::RFP. (i–l) Nerve-ring structure of L1 animals is abnormal in kpc-1; chin-1 mutants compared to wild-type animals. FIBSEM images of WT (i,j) and mutant (k,l) NR region of age-matched L1 animals. Dotted red line: axons. (h,k,l,n) kpc-1(gk8); chin-1(ns399). (m,n,o) Fasciculation of sublateral commissure neurons is abnormal kpc-1; chin-1 L1 mutant animals compared to wild-type animals. Pceh-24::GFP. (a–h, m–n) Reporter expression patterns are described in Supplementary Methods and Supplementary Tables S8, S10. (o) Numbers inside bars: total animals scored per genotype, n=4 independent scoring experiments. Mean +/− Error bars: SEM. Numbers above bars of significance, p values from Fisher’s exact test. ns: non significant.

Figure 4. KPC-1 and CHIN-1 act in NR pioneers at the onset of NR assembly

(a–j) Extension of AIY axons and SubL commissure axons into the presumptive nerve-ring is delayed in kpc-1; chin-1 mutants compared to wild-type embryos. Histograms and scale bars, as in Fig. 3. (a–h) Head region (outlined in schematics) of bean or 1.5 fold embryos expressing Pttx-3::GFP (a-d) or Pceh-17::GFP (e-h). Arrows: SubL axons, arrowhead: AIY axon. Asterisk: growth cone. (i–j) Squares: individual axon measurement at given embryonic stage. Lines track individuals across stages. Number of animals analyzed: (i) n=7 for WT, n=6 for chin-1; kpc-1 mutants, (j) n=8 for WT, n=7 for chin-1; kpc-1 mutants. (k) CHIN-1 and KPC-1 expression is necessary prior to the embryonic comma stage for proper nerve-ring assembly. HS: Heat-Shock driving chin-1 or kpc-1 cDNA expression. (l,m) CHIN-1 and KPC-1, acting non-cell-autonomously from SubL neurons and glia, can rescue chin-1; kpc-1 mutant defects of follower axons. Furthermore, CHIN-1 and KPC-1 acting non-cell-autonomously from glia can rescue chin-1; kpc-1 mutant defects of pioneer SubL axons. Rescue of mutant defects by cDNA expression using indicated promoters (Expression patterns are described in Supplementary Methods and Supplementary Tables S8, S10). The following alleles are used unless otherwise indicated: kpc-1(gk8), chin-1(ns399). (k–m) Numbers inside bars: total animals scored per genotype, n=3 independent scoring experiments or number of transgenic lines in rescue experiments. Mean +/− Error bars: SEM. Numbers above bars of significance, p values from Fisher’s exact test. ns: non significant.

Figure 5. Glia direct pioneer and follower-axon guidance using distinct signaling pathways

(a, c) UNC-6/Netrin and MAB-20/Semaphorin regulate nerve-ring assembly. UNC-6 guides primarily pioneer SubL axons while MAB-20 specifically guides follower axons and both act from glia. (b,c) FMI-1/Flamingo/CELSR can act cooperatively from SubL commissure neurons and glia to drive nerve-ring assembly, by specifically guiding follower axons. (a–c) cDNA expression of MAB, 20, UNC-6 and FMI-1 is driven by Pmir-228 (glia), Pceh-17 or Pceh-24 (SubL neurons), or endogenous regulatory regions. Expression patterns are described in Supplementary Methods and Supplementary Tables S8, S10. Numbers inside bars: total animals scored per genotype, n=4 independent scoring experiments or number of transgenic lines in rescue experiments. Mean +/− Error bars: SEM. Numbers above bars, p values from Fisher’s exact test. ns: non significant.

Figure 6. KPC-1/CHIN-1 control guidance-cue trafficking

(a–e) FMI-1-GFP localizes to the NR bundle of 1.5-fold wild-type embryos but is detected abnormally outside the NR in kpc-1; chin-1 mutants. (a,c) 1.5-fold embryos expressing Pfmi-1::FMI-1-GFP and magnified view of their head regions (b,d). (b,d) Blue boxes outline regions of interest I (cell bodies, triangles), II (NR, arrows), and III (dendrites, arrowheads). Scale bar: 10 um. (e) FMI-1-GFP ectopic signal is quantified as described in Supplementary Methods (see also Supplementary Fig. S7). Number of animals analyzed appears in the graph (n). Numbers above bars of significance, p values from t-test test (GraphPad). ns: non-significant. t ratios for group comparisons of wild-type and kpc-1; chin-1, wild-type and kpc-1, wild-type and chin-1 are 4.95, 2.44, 2.95, respectively, for mean intensity ratio; and 2.55, 0.74 and 1.50, respectively, for max intensity ratio. Number of degrees of freedom equals the number of pairs minus 1. (f) Overexpression of UNC-6, MAB-20, FMI-1 but not UNC-129 can partially restore the AIY axon defects of kpc-1; chin-1 mutant animals. (g) chin-1(ns399) encodes a dominant-negative protein similar to CHIN-1 harboring an Arginine finger-mutation. (h) CHIN-1 specifically binds CDC-42 in yeast-two-hybrid assays. p53-DBD and HA-GAD: negative controls, CDC-42(Q61L)-DBD and CHIN-1(R270A)-GAD: positive interactors. His: histidine. (i) cdc-42 RNAi results in abnormal AIY axon nerve-ring entry. Mock RNAi: L4440 vertor. (j) MAB-20, UNC-6 and FMI-1 predicted furin motifs are important for AIY axon nerve-ring entry. (f,g; I,j) cDNA expression is driven by Pmir-228 (glia), Pceh-17 (SubL neurons), Pgly-18 (GLR cells) or endogenous regulatory regions (Online methods). (j) mab-20(ns789): CRISPR allele. (-Rn) or (-Kn): extrachromosomal array of unc-6 or fmi-1 cDNA with single amino-acid deletions perturbing predicted Furin-recognition motifs (Supplementary Fig. S6, Supplementary Table S5). Numbers inside bars: total animals scored per genotype, n=4 independent scoring experiments or number of transgenic lines in rescue experiments. Mean +/− Error bars: SEM. Numbers above bars, exact p values by Fisher’s exact test. ns: non-significant.