Special Reviews

Neurobiology of the Caenorhabditis elegans Genome

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Science  11 Dec 1998:
Vol. 282, Issue 5396, pp. 2028-2033
DOI: 10.1126/science.282.5396.2028

Abstract

Neurotransmitter receptors, neurotransmitter synthesis and release pathways, and heterotrimeric GTP–binding protein (G protein)–coupled second messenger pathways are highly conserved between Caenorhabditis elegans and mammals, but gap junctions and chemosensory receptors have independent origins in vertebrates and nematodes. Most ion channels are similar to vertebrate channels but there are no predicted voltage-activated sodium channels. The C. elegans genome encodes at least 80 potassium channels, 90 neurotransmitter-gated ion channels, 50 peptide receptors, and up to 1000 orphan receptors that may be chemoreceptors. For many gene families, C. elegans has both conventional members and divergent outliers with weak homology to known genes; these outliers may provide insights into previously unknown functions of conserved protein families.

The nervous system of C. elegans has an unprecedented set of tools that are available for its analysis: a complete cell lineage that reveals the developmental origin of every neuron, a wiring diagram from serial section electron micrographs that describes all the synapses between neurons, and now the genome sequence with all the genes required to build the animal (1, 2) (Table 1). The nervous system contains about one-third of all the somatic cells in C. elegans and probably dominates a comparable portion of the genes, but at this point only a handful of those genes are understood.

Table 1

Genes that affect nervous system function (see text for references). PLC, phospholipase C; PDE, phosphodiesterase; ChAT, choline acetyltransferase; AChE, acetylcholinesterase; VChAT, vesicular ChAT.

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Comparison of the predicted C. elegans genes with molecules in the vertebrate nervous system reveals many parallels and a few striking differences (3). Conserved gene systems include the neurotransmitter biosynthetic enzymes, synaptic release mechanisms, and neurotransmitter receptors, including both ligand-gated ion channels and G protein–coupled receptors. Most ion channels and second messenger pathways are also highly conserved. The gene for the voltage-activated sodium channel is absent, although voltage-gated potassium and calcium channels abound (4).Caenorhabditis elegans also lacks the rhodopsin molecules that are widely used for vertebrate and invertebrate vision. In two cases, gap junctions and olfactory receptors, the nematode and vertebrate gene families are unrelated in sequence but have similar properties.

Caenorhabditis elegans often has one recognizable member of a gene family where vertebrates have three or four very similar genes, consistent with the general model that the mammalian genome experienced two large-scale genome duplications early in chordate evolution (5). A few C. elegans gene families are represented by dozens or even hundreds of family members; these families are often quite large in vertebrates as well. The multigene families provide insight into the aspects of neuronal function that define different types of neurons or distinct functions within one neuron. The C. elegans genome highlights the special importance of diversity in sensory recognition (olfactory receptors), neuronal excitability (potassium channels), and information transfer and cell-cell recognition at the synapse (neurotransmitter receptors and gap junctions).

Regulation of Excitability

There is no voltage-activated sodium channel in the C. elegans genome, which is consistent with the absence of such an activity in electrophysiological recordings of Ascaris motor neurons (4, 6). Jellyfish are more primitive invertebrates than nematodes, and they do possess voltage-activated sodium channels, which suggests that the ability to generate a sodium-based action potential was lost during nematode evolution (7). Caenorhabditis elegans neurons are small and have high membrane resistance, and they are able to propagate signals efficiently without the large-scale amplification provided by the sodium channel (8, 9).

On the other hand, voltage-activated calcium channels and potassium currents have been observed in recordings from C. elegansneurons and muscles (9, 10). Voltage-activated calcium channels depolarize cells and allow calcium entry for synaptic exocytosis and muscle contraction. They consist of one α1 subunit that defines the intrinsic properties of the channel and very different accessory α2/δ, β, and sometimes γ subunits that modify channel kinetics and density. Caenorhabditis elegans has five predicted α1 subunits, one related to α1D/L-type channels (EGL-19), one related to α1A/α1B/N/P/Q-type channels (UNC-2), one related to α1G/T-type channels, and two outliers that are most similar to one another and to a channel in the yeastSchizosaccharomyces pombe. This is one of the cases in whichC. elegans encodes both conventional genes and outliers that are the most divergent known members of the gene family. There are also predicted α2 and β subunits and a potential ryanodine receptor that could release calcium from intracellular stores (unc-68)(11, 12).

Mutations are known for two α1 subunits, egl-19 (also known as pat-5 or eat-12) and unc-2.egl-19 is likely to encode the muscle voltage-activated calcium channel: null mutants in egl-19 are lethal and appear to lack almost all muscle contraction (10).unc-2 is required for normal locomotion and acts in C. elegans neurons, where it might have a role at the synapse (13).

In contrast to the small number of calcium channel genes, there are many diverse potassium channel genes in the C. elegansgenome (14). Potassium entry drives a cell to its resting potential, and potassium channels shape the electrical response of an excitable cell, determining its susceptibility to depolarization, its resting potential, and its recovery from depolarization.

There are three major classes of potassium channels: the inward rectifier channels, with two transmembrane domains (2 TM), the two pore/TWIK channels, with four transmembrane domains (4 TM), and the voltage-regulated potassium channels, with six transmembrane domains (6 TM) (15). For the well-characterized 2 TM and 6 TM proteins, a single channel is formed by four monomers encoded by one or more similar genes. Additional dissimilar β subunits can modify channel properties. The 6 TM channels can be regulated by voltage; in addition, the intracellular domains of all channels define their regulation by second messengers, their inactivation, and their multimerization.Caenorhabditis elegans has about 80 predicted potassium channels including members of 2 TM, 4 TM, and 6 TM families; within the 6 TM family there are members of five different subclasses—the voltage-gated Shaker-type potassium channels (Shak/shab/shaw/shal), the calcium-activated Slo potassium channels, the calcium-regulated SK potassium channels, the eag/erg channels with cyclic nucleotide binding domains, and the KQT channels. One of the Slo channels and all the inward rectifiers are potentially interesting outliers compared with the vertebrate genes. The 4 TM TWIK channels are the most striking group of outliers. These channels represent the largest worm family and include about 50 different genes. There are also many mammalian TWIK homologs in expressed sequence tag (EST) databases, but they have not been extensively studied. Some of the TWIK channels have unusual pore domains, so they may not all encode potassium channels (16).

One Shaw-like gene is mutated in egl-36 mutants (17, 18), and its properties hint at the reason for potassium channel diversity. The egl-36 mutation is a gain of function (gf) that allows potassium entry at inappropriate membrane potentials, resulting in constitutive inhibition of the egg-laying muscles that express the gene; its loss-of-function phenotype is subtle. The channel is expressed in only a few neurons and the egg-laying muscles, which suggests that its restricted function is due to restricted expression. Similarly, unpublished gf mutations in five additional potassium channel genes result in severe phenotypes that are much more striking than loss of function of the same genes. The simplest explanation for this result is that gf mutations can silence a cell entirely, whereas loss-of-function mutations affect the same cell slightly. The relatively mild loss-of-function phenotypes are probably due to a combination of redundancy of potassium channels within a given cell and restricted expression of each channel in small groups of cells.

Why are there so few calcium channels and so many potassium channels in both nematodes and vertebrates? Perhaps calcium channels provide a function that is regulated in similar ways in all cells, a common language of excitability. The potassium channels may provide the modulatory functions that make excitable cells different from one another.

Sensory Channels and Other Functions

The functions of some channels are best understood because of their roles in sensory transduction. The degenerin family of potential mechanosensory channels was identified by genetic analysis of touch sensation (19); this family also includes vertebrate epithelial sodium channels. These proteins have two predicted membrane-spanning domains and a predicted pore domain, together with large extracellular domains that could interact with extracellular signals. Two C. elegans degenerin genes required for touch sensation, mec-4 and mec-10, can mutate to give dominant hyperactive channels that cause the touch cells to swell, degenerate, and die (thus degenerins) (19, 20). Three other degenerin genes might function as stretch receptors, a function analogous to touch sensation (21–23). Like the potassium channels, this family of channels appears to have a subtle or regulatory role in most cells that express it rather than an essential role.

The genetic analysis of touch sensation also identifiedmec-2, which is related to the erythrocyte band 7.2b integral membrane protein stomatin (24). There are eight other predicted stomatin-related genes, including unc-24,which is required for normal locomotion (25).

Cyclic nucleotide–gated channels are a prominent feature of vertebrate vision and olfaction, and two of the six C. elegans cyclic nucleotide–gated channel genes are essential for some forms of chemosensation and thermosensation(tax-2 and tax-4), consistent with a function in sensory transduction (26, 27). Other forms of chemosensation depend on the trp-like channel osm-9, whose closest vertebrate homolog is the capsaicin receptor expressed on pain-sensing nociceptive sensory neurons (28, 29). The capsaicin receptor is directly gated by noxious heat, whereas thetrp channel is known from Drosophilaphototransduction to be regulated by phospholipase C.Caenorhabditis elegans has 11 trp/osm-9-like genes, including 3 genes in a novel branch of the gene family. It also has six predicted chloride channels.

Neurotransmitters and Exocytosis

Like other animals, C. elegans uses the small molecule neurotransmitters acetylcholine, dopamine, serotonin, γ-aminobutyric acid (GABA), excitatory amino acids (glutamate), and the invertebrate catecholamine octopamine. At the neuromuscular junction, excitatory motor neurons release acetylcholine and inhibitory motor neurons release GABA (30, 31). The pharynx has specialized muscles that are also excited by acetylcholine but are inhibited by glutamate (32–34).

Caenorhabditis elegans and vertebrates appear to use similar molecules for neurotransmitter synthesis, packaging into synaptic vesicles, and reuptake or destruction. Mutations in genes for acetylcholine function are known: C. elegans has the biosynthetic enzyme choline acetyltransferase (cha-1) (35), a vesicle transporter (unc-17)(36), and cholinesterases that are highly similar to the vertebrate genes (37, 38) (indeed, unc-17 was the first cloned vesicular acetylcholine transporter). GABA in C. elegans is presumably synthesized from glutamate by the enzyme glutamic acid decarboxylase (GAD) and packaged into vesicles by a novel transporter encoded by the unc-47 gene (39). Vertebrate homologs of unc-47 can package either GABA or glycine into synaptic vesicles (39). The genome includes several genes that are related to unc-47, and perhaps these molecules encode vesicular transporters for other small molecules.

The enzymes required for dopamine, serotonin, and octopamine synthesis (tyrosine hydroxylase, dopamine β-hydroxylase, aromatic acid decarboxylase, tryptophan hydroxylase) and vesicular transport (VMAT, the monoamine transporter) are present in the C. elegansgenome. Candidate molecules to clear transmitters include monoamine oxidase, a putative cocaine-sensitive dopamine transporter, and a putative prozac-sensitive serotonin transporter. Glutamate reuptake from the synapse is mediated by the EAAT transporters, which may be encoded by six C. elegans genes.

In addition to small-molecule transmitters, C. elegans uses neuropeptides as transmitters. One widespread class of nematode neuropeptides has the sequence FMRF-amide (Phe-Met-Arg-Phe-amide) or closely related sequences at the COOH-terminus (40, 41). There are at least 15 genes in C. elegans that encode at least 56 potential FMRF-amide-like peptides (42). As is observed in other species, multiple peptides are likely to be produced from each peptide precursor gene by cleavage and processing. Mutations in one FMRF-amide gene, flp-1, cause defective locomotion and chemosensation (43). In addition to the FMRF-amide genes, there are at least 15 other genes that could encode 58 neuropeptides; most are nematode specific but some are related to snail peptides such as buccalin and pedal peptide. Caenorhabditis elegans does not have highly conserved homologs of the mammalian neuropeptide endorphins, enkephalins, dynorphins, substance P, substance K, and neuropeptide Y. These peptides are small and may be difficult to recognize after evolutionary divergence; the C. elegans genome does have many receptors that are related to the mammalian receptors for these peptides.

Many of the genes involved in neurotransmitter release have been identified because of their defects in cholinergic neurotransmission at the neuromuscular junction (44–48). The essential components of exocytosis are highly conserved between mammals and nematodes, with each important gene (syntaxin, synaptobrevin,unc-18/nsec-1, synaptotagmin, rab-3) represented by a highly conserved C. elegans homolog (45,49–53). Most of the mutants in this pathway have severe uncoordinated phenotypes that are lethal or almost lethal, but a few like synaptotagmin and rab-3 have relatively mild defects. The genome also encodes outlier proteins related to the exocytosis genes. Some of these genes probably have nonneuronal functions (54), but others might act in neurons. Their expression patterns should indicate whether they act in different types of neurons or whether they activate different classes of vesicles (for example, classic neurotransmitters versus neuropeptides).

Ligand-Gated Ion Channels Act in Neurons and at the Neuromuscular Junction

The ligand-gated ion channels are neurotransmitter receptors that open rapidly and desensitize rapidly, which makes them ideal for short-term signaling. Each channel is made up of four or five similar subunits that can be encoded by one to five different genes.Caenorhabditis elegans has about 100 genes for ligand-gated ion channels, including excitatory receptors for acetylcholine and glutamate and inhibitory receptors for GABA and glutamate.

At the nematode and vertebrate neuromuscular junction, excitatory motor neurons release acetylcholine, which drives muscle contraction (30, 46). In the nematode Ascaris, motor neurons also use acetylcholine to communicate with each other (55). There are about 40 predicted acetylcholine receptor subunits inC. elegans that fall into the ligand-gated (nicotinic) class. Three subunits of a levamisole-sensitive muscle acetylcholine receptor are encoded by the unc-29, unc-38, andlev-1 genes (56). The roles of the other >35 genes are mostly unknown. There are other muscle acetylcholine receptors (33, 46). Dominant alleles in one neuronal acetylcholine receptor (deg-3) are associated with cell-autonomous sensory neuron degeneration, but this receptor has no obvious loss-of-function phenotype (57).

Excitatory glutamate receptors are unrelated in sequence to the acetylcholine receptors, although they share a similar multimeric subunit structure. Caenorhabditis elegans has six glutamate receptors of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate class, for which mutations have been described in one: glr-1mutants have defects in their locomotor response to mechanosensory stimuli (58, 59). There are also two potential glutamate receptors in the N-methyl-d-aspartate (NMDA) receptor class, one type 1 and one type 2 subunit (NMDA receptors are typically type 1/type 2 heteromeric channels), and two unusual outlier genes related to glutamate receptors.

Caenorhabditis elegans has over 30 different genes that could encode inhibitory GABA-A, glycine, and glutamate receptors, which are distantly related to the excitatory nicotinic acetylcholine receptors and very closely related to one another. There is a major GABA receptor on nematode body muscle, but again the diversity of the gene family suggests other functions that are not known. Clear orthologs of glycine receptors were not found, and it is not known whether these will be present in C. elegans. The glutamate-gated chloride channels are the major molecular target for a class of nematocide drugs, the ivermectins, and they do not have known vertebrate homologs (60). Mutations in these channels have been identified in screens for high-level ivermectin resistance; these mutants also have defects in pharyngeal function due to a loss of glutamate-mediated inhibition (61).

Several C. elegans ligand-gated ion channel genes are outliers that might encode previously unidentified receptor types. For example, the invertebrate histamine-gated chloride channel, which has not been cloned, might be one of these receptors.

The number of ligand-gated ion channel genes is unexpectedly large. One possible explanation for this diversity is that these channels may be localized to particular synapses or regions of the cell. In mammals, a peptide-binding motif called the PDZ domain binds to the COOH-terminus of many channels, including the glutamate-gated ion channels, and clusters them at synaptic regions. PDZ proteins also organize proteins in nonneuronal cells; the C. elegans lin-2, lin-7, and lin-10 genes are required for localization of the epidermal growth factor receptor protein LET-23 in vulval precursor cells (62). PDZ-binding motifs are present in manyC. elegans receptors and channels, including the glutamate receptor GLR-1, and the PDZ protein LIN-10 is involved in GLR-1 localization (63). There are at least 30 PDZ domain–containing proteins in C. elegans, which suggests that there are many different types of receptor clusters.

G Protein–Coupled Receptors

G protein–coupled receptors typically generate slower and longer-lasting changes in neuronal excitability than ligand-gated ion channels. G protein–coupled receptors have seven transmembrane domains, and they are associated with a cytoplasmic heterotrimeric G protein consisting of a guanosine triphosphate (GTP)– or guanosine diphosphate–binding α subunit and β and γ subunits. Upon ligand binding, the receptor causes GTP exchange and dissociation of the Gα subunit from Gβγ; both Gα and Gβγ subunits can interact with effectors in the cell.

Remarkably, about 5% of all C. elegans genes encode G protein–coupled receptors. The receptors fall into two groups: those with clear relationships to receptors identified in other animals (about 100 genes), and “orphan” worm-specific receptors (about 1000 genes). Most of the worm-specific receptors are thought to encode chemoreceptors. Most of the other genes are probably neurotransmitter receptors.

Both vertebrates and invertebrates have several families of conserved G protein–coupled receptors. Class A, the rhodopsin-like receptor family, has different subclasses with different ligands. The class A amine receptors interact with small molecule neurotransmitters such as acetylcholine, catecholamines, dopamine, and serotonin. The amine receptors are quite similar, so the ligands for the approximately 18 C. elegans genes in this family are uncertain. Based on sequence similarity there might be acetylcholine receptors, tyramine/octopamine receptors, serotonin receptors, and dopamine receptors. One of the predicted serotonin receptors has been shown to interact with serotonergic agonists and antagonists when expressed in mammalian cells (64).

Class A peptide receptors have peptide ligands. The 50 or more C. elegans genes in this group are less closely related to their vertebrate cousins than the amine receptors. Moreover, there are no clear C. elegans homologs of the vertebrate neuropeptides (see above). Nonetheless, it is possible to identify subgroups of theC. elegans receptors that have similarity to subgroups of vertebrate peptide receptors such as tachykinin receptors, neurotensin receptors, and neuropeptide Y receptors. No authentic rhodopsin is present.

The C. elegans–specific subfamily of about 80 orphan class A receptors is uncharacterized, but it might encode chemoreceptors like the other large orphan families of G protein–coupled receptors (see below) (65).

The class B or secretin receptor family of peptide receptors is completely unrelated to the class A family by sequence. This gene family has at least four members in C. elegans. By analogy to similar receptors in other animals, the C. elegansreceptors may bind peptide hormones and perhaps have endocrine roles. The G protein–coupled metabotropic glutamate receptors are different in sequence from either class A or class B receptors.Caenorhabditis elegans has four genes that are similar to the glutamate receptors and related extracellular calcium receptors. The GABA-B receptor is distantly related to these receptors, and there are three C. elegans genes in the GABA-B subfamily. Each of the receptor families mentioned above includes at least one unusualC. elegans outlier gene.

Of the approximately 80 receptor genes described above, mutations have been described only for npr-1, a class A receptor most closely related to neuropeptide Y receptors. npr-1 mutant animals associate with one another in groups, and they have subtle alterations in feeding behavior (66).

Chemosensory neurons make up about 10% of the C. elegansnervous system, and it is likely that many of the 1000 orphan G protein–coupled receptors act in chemosensation. A single olfactory receptor, odr-10, has been identified through mutation; it is predicted to be a G protein–coupled receptor in a novel sequence family (67, 68) The C. elegansgenome contains over 600 genes that are odr-10–like as well as several additional families of orphan G protein–coupled chemoreceptors (69). Many but not all of these genes are expressed in chemosensory neurons (69), and about one-third have stop codons and frameshifts. These 1000 genes might encode 500 chemoreceptors, 200 genes with functions in other cells, and 300 pseudogenes.

The chemosensory receptors are often found in clusters that contain as many as 10 or 15 closely related genes. Clusters of two or three related genes are sometimes seen for other C. elegans genes, but the clustering of chemoreceptors is extreme. Clustering provides an opportunity for gene addition and loss by unequal sister chromatid exchange, perhaps explaining why these receptors evolve rapidly—a partial sequence from the nematode Caenorhabditis briggsaeshows that as many as 20% of the genes may be present in only one of the two species (68). The nematode genes are not closely related to the three mammalian families of olfactory receptors, but the mammalian genes also accumulate in clusters.

Another proposed family of chemoreceptors is encoded by 26 predicted transmembrane receptor-type guanylyl cyclases (70). Some mammalian receptor-type guanylyl cyclases respond directly to peptide ligands, and the retinal receptor-type cyclases act as a constitutive source of guanosine 3′,5′-monophosphate (cGMP) for signaling by the G protein–coupled receptor rhodopsin. Many of the C. elegans cyclases are expressed in small subsets of chemosensory neurons, and they could subserve either an odorant-regulated or a constitutive role in those neurons (70). The one gene for which a mutation is known, daf-11, is required for responses to many odorants, but others could be more specific (71). One cyclase is expressed in the thermosensory AFD neurons, so it could function in thermosensation rather than chemosensation.

Caenorhabditis elegans encodes 20 different Gα subunits. These include one Go-, Gq-, and Gs-like protein, as well as 17 GPA proteins that fall loosely within the Gi group. Go and Gq proteins are expressed in neurons and affect locomotion and egg laying (72–74). The Gs-like protein is expressed in neurons and its loss of function phenotype is lethal at the early larval stage (75). Three Gi-like proteins act in olfaction and pheromone sensation (76, 77).

There are two Gβ subunits. One Gβ mutant is lethal, with neuronal defects that are reminiscent of Gq protein loss of function (78). In addition, Gβ orients cell divisions in the early embryo. Twelve potential RGS (regulators of G protein signaling) proteins are present, and mutations in one are found in theegl-10 mutant, which has defective egg laying (93).

The best characterized targets of G proteins are adenylyl cyclase (Gs and Gi), phospholipase Cβ (Gqand Gβγ), potassium channels (Gi and Gβγ), calcium channels (Go), and cGMP phosphodiesterase (Gi). The G protein–regulated second messengers cAMP, inositol trisphosphate, diacylglycerol, and cGMP can indirectly modulate many enzymes within the cell. C. elegans has homologs of all these major classes of G protein targets. Mutations in one adenylyl cyclase have been identified, but their phenotype is not visibly abnormal (79).

Gap Junctions

The C. elegans nervous system has about 600 highly reproducible neuronal gap junctions, or electrical synapses, in addition to the 5000 predicted chemical synapses (2). Neuronal gap junctions are important in locomotion and touch-withdrawal behaviors (80), and gap junctions also synchronize muscle cell contractions (81). However, C. elegans does not have any close relatives of the mammalian connexin gap junction gene family. Instead, its genome has over 20 innexin genes that are defined by similarity to the Drosophila Passover and l(1)ogre genes and the C. elegans unc-7 and eat-5genes. Mutations in innexin genes can lead to a loss of electrical coupling between cells, and expression of a Drosophilainnexin protein in paired Xenopus oocytes leads to electrical coupling between the two oocytes (82). There are no innexins in the existing vertebrate genomic sequence or EST sequences, which suggests that the gap junctions of invertebrates and vertebrates have independent and convergent origins. A detailed alignment of 24 predicted C. elegans innexin proteins has been published (83). The innexins are predicted to have four membrane-spanning domains, like the vertebrate connexins, with variable intracellular NH2- and COOH-terminal domains that might have regulatory functions.

Mutations in the innexin eat-5 gene affect pharyngeal muscle coupling (81), whereas mutations in unc-7 andunc-9 affect locomotion (83, 84).unc-7 and unc-9 alter neuronal gap junctions observed by electron microscopy, but these effects are subtle, which suggests that neither gene is essential for morphological gap junctions.

If each of the innexins can form a working gap junction only with a cell expressing certain other innexins, it is possible that the expression pattern of the 24 predicted innexin subunits specifies the pattern of gap junctions between cells. It is likely that some of the innexins function outside the nervous system. Innexin expression has been observed in the two-cell embryo, and many embryonic cells have gap junctions (81).

Does C. elegans Learn?

Behavioral plasticity in C. elegans has been described for thermotactic, chemotactic, and mechanosensory behaviors, but the genes and molecules responsible are mostly unknown (85–88). In other animals, protein kinases that respond to second messengers like calcium (CAM kinases), adenosine 3′,5′-monophosphate (cAMP) (protein kinase A), and cGMP (protein kinase G) are implicated in synaptic plasticity and learning, and each of these kinases is represented by one or two genes in the C. elegans genome. Because there are over 300 predicted protein kinases, it is likely that others also function in the nervous system. cAMP signaling pathways are important for learning in Drosophila, Aplysia, and mammals— in behavioral paradigms as well as in models of synaptic plasticity (89, 90). In particular, the transcription factor CREB (cAMP response element–binding protein) plays a role in long-term memories that depend on altered gene expression. C. elegans has one predicted CREB gene.

Synaptic plasticity in other animals can also be influenced by the NMDA-type glutamate receptors, which appear to be present in C. elegans, and nitric oxide synthase and neurotrophins, which are not present in a recognizable form. Further genetic analysis of these pathways would benefit from better assays for learning and assays for synaptic plasticity, which have not yet been developed in C. elegans.

Conclusions

The C. elegans genome reveals many potential targets for investigation. Caenorhabditis elegans homologs of highly conserved neuronal genes and human disease genes are open to standard methods for isolating mutations and characterizing gene networks by enhancer and suppressor analysis.

The nematode-specific systems are potential targets for nematode control. This principle has already worked for the ivermectin-sensitive glutamate-gated chloride channel and might also be useful for the innexin gap junctions and the chemosensory receptors.

Within a gene family, it is often true that C. eleganshas genes that are more divergent than any of the known vertebrate members of the gene family. These outlier family members deserve special attention: in some cases, the highly divergent vertebrate genes probably exist but have not been found, although in other cases theC. elegans genes may be nematode-specific genes. Searching for functions of the outlier nematode sequences could be a fruitful way of exploring new functions in multigene families. Indeed, the mammalian T-type calcium channel was identified starting from the sequence of a divergent nematode channel that later proved to be T-type–like (91). Intriguing outliers include calcium channel subunits, potassium channels, ligand-gated ion channels, trp-like channels, GABA transporters, and neuropeptide receptors.

Although C. elegans is intensively studied, there are no known mutations in most of the receptor and channel genes. This absence is especially striking in the large gene families like the G protein–coupled receptors and the potassium channels. By contrast, numerous mutations are known in the small gene families such as calcium channel subunits and synaptic vesicle proteins. In the short term, analyzing the new genes will be facilitated by describing their patterns of expression, including subcellular localization. In the long term, the genome sequence points to the need for better behavioral and electrophysiological assays in C. elegans neurobiology. Most mutant screens conducted in the past have required a substantial defect in neuromuscular function such as uncoordinated movement. These screens revealed genes with widespread function and even weak mutations in lethal genes, but they overlooked most genes with subtle, modulatory, or cell-specific functions. The limiting steps for understanding gene function are defining the function of each neuron (still unknown for many C. elegans neurons) and devising better assays for neuronal function in vivo. In the end, instead of transcending neurobiology, the genome leads back to it.

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