Differential expression of glutamate receptor subunits in the nervous system of Caenorhabditis elegans and their regulation by the homeodomain protein UNC-42

Abstract

In almost all nervous systems, rapid excitatory synaptic communication is mediated by a diversity of ionotropic glutamate receptors. In Caenorhabditis elegans, 10 putative ionotropic glutamate receptor subunits have been identified, a surprising number for an organism with only 302 neurons. Sequence analysis of the predicted proteins identified two NMDA and eight non-NMDA receptor subunits. Here we describe the complete distribution of these subunits in the nervous system of C. elegans. Receptor subunits were found almost exclusively in interneurons and motor neurons, but no expression was detected in muscle cells. Interestingly, some neurons expressed only a single subunit, suggesting that these may form functional homomeric channels. Conversely, interneurons of the locomotory control circuit (AVA, AVB, AVD, AVE, and PVC) coexpressed up to six subunits, suggesting that these subunits interact to generate a diversity of heteromeric glutamate receptor channels that regulate various aspects of worm movement. We also show that expression of these subunits in this circuit is differentially regulated by the homeodomain protein UNC-42 and that UNC-42 is also required for axonal pathfinding of neurons in the circuit. In wild-type worms, the axons of AVA, AVD, and AVE lie in the ventral cord, whereas in unc-42 mutants, the axons are anteriorly, laterally, or dorsally displaced, and the mutant worms have sensory and locomotory defects.

Fig. 1.

Partial sequence alignments of predicted C. elegans ionotropic glutamate receptors. Sequence alignment of a highly conserved region of ∼400 amino acids of GLR-1 and the additional nine putative C. elegans glutamate receptor subunits is shown. This region includes the four hydrophobic domains (TMI–TMIV, solid underline) and two ligand-binding domains (S1, S2, dashed underline witharrows) that are similar in sequence to bacterial amino acid-binding proteins (Nakanishi et al., 1990; Stern-Bach et al., 1994). The polypeptide sequences were derived from partial cDNAs amplified from mixed-stage first-strand cDNA and are numbered on theleft beginning with the first amino acid included in the alignment. Identical or similar residues are shaded in blackor gray, respectively. Ce, C. elegans.

Fig. 2.

Comparison of C. elegans,Drosophila, and rat ionotropic glutamate receptor subunits. Sequence alignment of C. elegans,Drosophila, and rat ionotropic glutamate receptor subunits beginning immediately at TMI and terminating at TMIII is shown. C. elegans subunits have signature features found in both vertebrate and invertebrate subunits. These include predicted hydrophobic domains (solid underline), a conserved glutamine residue in TMII (filled circle; a target of RNA editing in a subset of vertebrate non-NMDA subunits), and a sequence of nine amino acids in TMIII (dashed line) found in almost all known ionotropic glutamate receptor subunits. This region also contains a conserved alanine residue (filled triangle) that when mutated to threonine causes the ion channel to be constitutively open (Zuo et al., 1997; Zheng et al., 1999). Amino acids are numbered on the left beginning with the first residue in TMI. Identical or similar residues are shaded inblack or gray, respectively. Dro,Drosophila.

Fig. 3.

Phylogenetic tree of the amino acid sequences for glutamate receptor subunits. Unrooted phylogenetic tree of the amino acid sequences for the 10 identified C. elegans putative glutamate receptor subunits and selected rat,Drosophila, and Arabidopsis subunits (see Materials and Methods) is shown. The predicted C. elegans polypeptides are based on the cloned partial cDNAs (Fig. 1). The schematic shows clusters of relationships between the amino acid alignments of the glutamate receptor subtypes. The total length of the horizontal lines between different receptors is proportional to the difference between sequences; distance along the vertical axis has no significance. Five hundred bootstrap replicates were performed, and the bootstrap values are indicated by dots on the supporting branches.No dot indicates a value <50%. At,A. thaliana.

Fig. 4.

Glutamate receptor–GFP fusion proteins. Schematic representation of the genomic fusions to GFP for each of the glutamate receptor subunits. Boxes represent exons, andhorizontal lines between boxes represent introns. Black and gray boxes represent genomic sequence that was either included or omitted from the GFP fusion construct, respectively. The underlined regionsrepresent intron and exon boundaries that have been confirmed by cDNA sequence that was used to predict the amino acid sequence shown in Figures 1 and 2. The amount of upstream regulatory sequence included in each fusion is indicated as is the site of GFP fusion (inverted triangle).

Fig. 5.

Four non-NMDA and two NMDA ionotropic glutamate receptor subunits are expressed in the command interneurons of the locomotory control circuit. Confocal micrographs of transgenic worms expressing GFP fusions to glr-1, glr-2,glr-4, glr-5, nmr-1, andnmr-2 are shown. Only the anterior head and posterior tail regions of the worm are shown (two left columns). A schematic reconstruction of the neuronal cell bodies in the head and tail of the worm are also shown [two right columns; adapted from White et al. (1986)]. Cells filled in grayand black represent neurons that express the respective subunit. Each of these GFP fusions is expressed in at least one of the five pairs of command interneurons, AVA, AVB, AVD, AVE, and PVC (black cells). Neurons that express at least one of the six receptor subunits have been labeled in the bottom panel. The command interneurons of the locomotory control circuit are filled in black. Scale bars, 5 μm.

Fig. 6.

Two ionotropic glutamate receptor subunits are exclusively coexpressed in the thermoregulatory interneuron RIA (arrows). A, B, Confocal micrographs (anterior region only) of transgenic worms expressingglr-3:: GFP (A) andglr-6:: GFP (B) are shown. C, Expression of both fusion proteins is detected in a single neuron, the thermoregulatory interneuron RIA (arrow). Scale bars, 5 μm.

Fig. 7.

Two ionotropic glutamate receptors are coexpressed in the pharyngeal nervous system. A, Confocal image of a transgenic worm expressing the glr-7:: GFPtransgene in the pharyngeal nervous system (head region only).B, Diagram of the pharynx and the neuronal cell bodies that express glr-7:: GFP. C, D, Confocal images of a transgenic worm expressingglr-8:: GFP in the pharyngeal nervous system (C, head region only) and in a single neuron in the tail (arrow; D). E, F, Neuronal cell bodies that express glr-8:: GFP. Neurons filled in black coexpressglr-7:: GFP andglr-8:: GFP. Neurons filled ingray only express glr-8:: GFP in either the pharyngeal nervous system (E; URB is the only neuron of this subset that is not considered part of the pharyngeal nervous system) or the tail (F). Scale bars, 5 μm.

Fig. 8.

Expression of ionotropic glutamate receptor subunits can be detected during embryogenesis. A–C, Image of a twofold stage embryo that expressedglr-5:: GFP (arrowheads). Expression of this fusion protein was detected the earliest of all of the GFP fusion transgenes. D–F, Image of a threefold stage embryo that expressed glr-1:: GFP. Most of the GFP fusions were first detected at this stage. G, Time course of embryogenesis (minutes) indicating the approximate time of the earliest detected expression of each of the receptor subunits.glr-3:: GFP andglr-6:: GFP expression was not detected during embryogenesis. A, D, Nomarski. B, E, FITC. C, F, Overlay. Scale bars, 5 μm.

Fig. 9.

UNC-42 is required for expression of GLR-1, GLR-4, and GLR-5. A–C, Confocal images ofunc-42(e270) transgenic worms that expressedglr-1:: GFP (A),glr-4:: GFP (B), andglr-5:: GFP (C).D–F, Diagram of neuronal cell bodies that expressglr-1 (D), glr-4(E), and glr-5(F). Cell bodies filled in grayand black represent those that expressed the respective subunits in wild-type worms. Cell bodies filled in grayrepresent those that no longer express the subunits inunc-42 mutants. unc-42 is required forglr-1 expression in the command interneurons AVA, AVD, and AVE and in the RMD motor neurons (Baran et al., 1999). It is also required for glr-4 and glr-5 expression in these same cells. Scale bars, 5 μm.

Fig. 10.

unc-42 disrupts the axon outgrowth of AVA, AVD, and AVE. A–D, Confocal images of transgenic unc-42(e419) mutants (A, B) and unc-42(e270) mutants (C, D) that expressed the nmr-1:: GFP transgene. Abnormal processes can be observed extending anterior to the nerve ring (arrows) or along dorsal or lateral paths (arrowheads). E, Confocal image of a transgenic unc-42(e270) mutant expressing bothnmr-1:: GFP and a wild-typeunc-42 genomic clone. Axon outgrowth is indistinguishable from that of wild-type worms. F, Wild-type worm that expressed nmr-1:: GFP. All images show the head region only, with anterior on the left and more posterior regions on the right of each panel. See Figure 5 for the identity of neurons that expressnmr-1:: GFP. G, Processes of the ventral cord in an nmr-1:: GFP; unc-42(e270)mutant. Only four processes were visible in the ventral cord (three of these indicated by arrows), whereas nine are expected in a wild-type worm. Migration of a subset of the processes terminated prematurely in the ventral cord (arrowhead). H, I, Schematic diagram of the locomotory control circuit in wild-type (H) and unc-42mutants (I). The circuits have been divided into forward (black) and backward (gray) components. Gap junctions and chemical synapses between sensory neurons (triangles), command interneurons (hexagons), and motor neurons (circles) are represented by lines andarrows, respectively. In wild-type worms (H) the DA motor neurons that direct backward movement receive synaptic input from the backward command interneurons (AVA, AVD, and AVE). In unc-42mutants (I) these connections are disrupted, and the worms have difficulty moving backward. Note that both the forward and backward command interneurons receive synaptic input from sensory neurons (AVM and ASH) that normally drive backward movement in response to anterior tactile stimulation. Scale bars, 5 μm.