Glia promote local synaptogenesis through UNC-6 (netrin) signaling in C. elegans

Abstract

Neural circuits are assembled through the coordinated innervation of pre- and postsynaptic partners. We show that connectivity between two interneurons, AIY and RIA, in Caenorhabditis elegans is orchestrated by a pair of glial cells that express UNC-6 (netrin). In the postsynaptic neuron RIA, the netrin receptor UNC-40 (DCC, deleted in colorectal cancer) plays a conventional guidance role, directing outgrowth of the RIA process ventrally toward the glia. In the presynaptic neuron AIY, UNC-40 (DCC) plays an unexpected and previously uncharacterized role: It cell-autonomously promotes assembly of presynaptic terminals in the immediate vicinity of the glial cell endfeet. These results indicate that netrin can be used both for guidance and local synaptogenesis and suggest that glial cells can function as guideposts during the assembly of neural circuits in vivo.

Fig. 1

AIY and RIA innervate at a specific spatial coordinate in the nerve ring. (A) Schematic diagram of the morphology of the presynaptic AIY and postsynaptic RIA neurons in the nerve ring (brackets). Modified image adapted from http://www.wormatlas.org with permission. (B) Representative adult animal expressing cytoplasmic fluorophores specifically in AIY (red) and RIA (green). In all images, the dashed box (zone 2 in text) delineates a spatial coordinate in the wild-type nerve ring where the RIA and the AIY interneurons converge and synapse onto each other. (C to H) Confocal micrographs and corresponding diagrams demonstrating the colocalization of AIY presynaptic vesicles [CFP::RAB-3 and pseudocolored red in (C)] with postsynaptic RIA glutamate receptors [GLR-1::YFP and pseudocolored green in (E)] in zone 2 (dashed box). Double labels are shown in (G). Schematic diagrams [(D), (F), and (H)] show the relation between AIY presynaptic terminals and the RIA postsynaptic region, which was highly reproducible across animals (n > 500). Scale bars, 5 µm.

Fig. 2

UNC-40 (DCC) is required for correct presynaptic patterning in AIY. (A to J) Distribution of presynaptic sites of AIY in wild-type [(A) to (E)] or unc-40 [(F) to (J)] animals. Confocal micrographs demonstrate the colocalization and patterning of AIY presynaptic vesicles [mCherry::RAB-3 pseudocolored red in (A) and (F)] and active zones [ELKS-1/ERC::YFP pseudocolored green in (B) and (G)] in a representative wild-type [(A) to (C)] or unc-40 [(F) to (H)] animal, evident in the double-label merge images [(C) and (H)] and represented in the diagram of presynaptic site distribution [(D) and (I)]. Three-dimension line scan profiles of the fluorescence intensity (arbitrary units, AU) distribution in (A) and (F). (K) Quantification comparing the relative distribution of synaptic vesicle fluorescence in wild-type (blue; n = 20 neurons) versus unc-40 animals (red; n = 31 neurons). Error bars represent standard error, and the asterisk represents statistical significance (P < 0.001). (L and M) unc-40 (e271) animals expressing an unstable transgene containing an unc-40 rescuing construct, and cytoplasmic cell-specific markers in AIY and RIA (L) or RIB/RIM (M) were scored for retention of the transgene and rescue of the AIY presynaptic phenotype. ***P < 0.001, **P < 0.01, and *P < 0.05 between indicated groups. Scale bars, 5 µm.

Fig. 3

Ventral cephalic sheath cells control UNC-40 (DCC) enrichment in presynaptic regions by secreting UNC-6 (netrin). (A and B) Localization of UNC-40::GFP in AIY of a representative wild-type (A) or unc-6 (B) animal. Note UNC-40:GFP enrichment in zones 2 and 3 in wild-type animals [(A) dashed box], as compared to diffuse localization in unc-6 animals (B). (C) Quantification of the enrichment of UNC-40::GFP in the presynaptic regions of wild-type versus unc-6 animals. Two different transgenic lines (lines 1 or 2), both expressing UNC-40::GFP, were scored. (D) Projection of confocal micrographs obtained from a representative animal simultaneously expressing hlh-17::mCherry (to label ventral cephalic sheath cells and pseudocolored blue), ttx-3::cfp::rab-3 (to label presynapses in AIY and pseudocolored red), and glr-3::glr-1::yfp (to label the glutamate receptors in RIA and pseudocolored green). (E) Volume rendering of (D) showing the relative positioning of the glial-like ventral cephalic sheath cells with respect to the region of innervation between AIY and RIA (29). (F) Diagram of (D) and (E). The asterisk marks the posterior extension of the sheath cell. (G) Volume rendering of the ventral sheath cell in (D). Note the groove in the boxed region where AIY and RIA are ensheathed by the ventral cephalic sheath cells and innervate each other. (H and I) Single confocal plane of (D), with presynaptic vesicles in AIY [labeled with cyan fluorescent protein (CFP)::RAB-3 and pseudocolored red in (H)], glutamate receptors in RIA [labeled with GLR-1:YFP and pseudocolored green in (I)] and a triple label including expression of the mCherry cytoplasmic fluorophore in the sheath cells [pseudocolored blue in (J)]. This anatomical relation was extremely stereotyped across animals (n > 500). Scale bars, 5 µm.

Fig. 4

Repositioning of the sheath cells affects RIA axon guidance and AIY presynapses. (A) Diagram showing the distended positioning of the ventral cephalic sheath cell in unc-34 animals. The asterisk marks the normal posterior boundary of a wild-type sheath cell (see Fig. 3F for comparison). In unc-34, animals sheath cells abnormally distend posteriorly in 47.9% of animals (n = 94 animals). (B to D) Confocal micrographs obtained from a representative unc-34 animal simultaneously expressing ttx-3::cfp::rab-3 [to label presynapses in AIY and pseudocolored red in (B) and (D)], glr-3::glr-1::yfp [to label the glutamate receptors in RIA and pseudocolored green in (C) and (D)], and hlh-17::mCherry [to label the ventral cephalic sheath cells and pseudocolored blue in (D)]. Note abnormal posterior extension of the sheath cell endfeet, corresponding ectopic AIY presynapses in zone 1, and distended RIA axon in the area now covered by the distended sheath cell (compare with Fig. 3D). (E to G) Confocal micrographs obtained from a representative unc-34 animal simultaneously expressing ttx-3:: mCherry::rab-3 [to label presynapses in AIY and pseudocolored red in (E) and (G)] and ttx-3::unc-40::gfp [to label the UNC-40 (DCC) receptors in AIY and pseudocolored green in (F) and (G)]. Scale bars, 5 µm.