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Development of the Sensilla
The assembly of an individual sensillum requires interactions between at least four cell types. One or more neurons invaginate and form junctions with the sheath cell, the sheath cell forms junctions with the socket cell, and the socket cell forms junctions with adjacent epidermal cells. Presumably a specific cell-cell adhesion is required before any permanent junction can form. In the embryo, many sensilla are assembled concurrently in a small region of the head. Thus each cell must adhere to its correct partners despite possible competition from nearby cells of similar type. This specificity of attachment is not absolute as hybrid sensilla can form when normal partners are removed (Sulston et al., 1983).
Invagination may be an early step in neuron/sheath interaction. In daf-19 mutants, where cilia are apparently not formed, the sensory dendrites still invaginate their sheath cells and form normal belt junctions.
The amphid dendrites show specific neuron/neuron interactions in addition to the neuron/sheath interactions observed in all sensilla. The 12 dendrites normally invaginate the sheath cell roughly in register. The channel cilia form a tight fascicle in the sheath cell which extends into the socket channel. Curiously, the arrangement of cilia within this fascicle is invariant in wild-type animals (Ward et al, 1975; Ware et al, 1975). It is unknown whether the ciliary pattern is inherited from the more complex pattern of the papillary nerves.
In mec-8 mutants, the amphid dendrites invaginate the sheath cell at irregular levels and their cilia do not fasciculate fully. A similar, if milder, defect in amphid fasciculation has been observed in mec-1 mutants (Lewis and Hodgkin, 1977; Chalfie and Sulston, 1981). Conceivably the mec-1 and mec-8 genes specify adhesive molecules that determine pairwise affinities of the amphid dendrites or their cilia. In addition, the mec-1 and mec-8 mutations disrupt the function of certain nonciliated mechanosensory neurons (Chalfie and Sulston, 1981). The mec-1 mutations were shown to prevent the normal attachment of these neurons to the hypodermis.
The lining and scaffold of the amphid sheath channel assemble around the fascicle of cilia. The sheath channel forms correctly in mutants with truncated or missing cilia suggesting that the dendrites, and not exclusively their cilia, can induce these structures. Small fascicles or isolated cilia in mec-8 mutants form separate channels that can accrete a scaffold and dark lining resembling the normal sheath channel.
The sheath matrix material appears to be synthesized at the lamellae, transported forward in membrane bound vesicles, and secreted from these vesicles into the sheath channel near the base of the cilia (Wright, 1980). The cilia themselves appear to induce the deposition of the matrix material. In mec-8 mutants with displaced cilia, the matrix material still deposits along them. It is also deposited around the ectopic cilia-like projections found in che-13, osm-1, and osm-5 dendrites.
In mutants with short or absent cilia, matrix material accumulates in large vesicles in the anterior sheath cytoplasm. Abnormal accumulations of large matrix vesicles have also been reported in the amphid sheath cells of che-2, che-3, and daf-6 mutants (Lewis and Hodgkin, 1977; Albert et al, 1981). It may be that matrix material is normally discharged from the cilia through the amphid openings. This would explain why it accumulates in the che-14, daf-6, and mec-8 mutants that have apparently normal cilia but obstructed channels.
The che-12 mutation appears to disrupt the synthesis or secretion of matrix by the sheath cells. Interestingly, empty vesicles still form at the lamellae, transport forward, and fuse with the channel lumen. Presumably the abnormal darkening of the channel cilia in the che-12 mutants is a degenerative change resulting from the loss of matrix normally surrounding the cilia. These mutants also have a defect in cuticle secretion by the epidermis.
The socket channel has a rather different origin than the sheath channel (Wright, 1980). The socket cells can wrap around and form junctions with themselves, thus creating a channel, even when there are no cilia to envelop. The scaffold that assembles around the channel cilia in the sheath cell may be important in joining the sheath and socket channels. In the absence of a well defined sheath channel, the socket channel sometimes ends in a blind pocket in mec-8 mutants. The sheath channel appears nearly normal in che-14 mutants but the join with the socket channel is defective. Conceivably the primary defect is in the sheath or socket scaffolds. The published description of daf-6 (e1377) mutants suggests they, too, may be defective in the joining of the sheath and socket channels (Albert et al, 1981). Consistent with this idea, Herman (1984) has shown that the genetic focus of the daf-6 phenotype is probably the sheath or the socket cell, or possibly both, but not the neurons (Table 2).
Assembly of Sensory Cilia
All classes of sensory cilia are absent in daf-19 mutants. Vestigial centrioles, without membrane attachments, were found in a few dendrites. No membrane-linked microtubules assemble ectopically in these mutants, suggesting (see below) that the wild type daf-19 product directly affects the peripheral doublets or their Y links. A mutation disrupting doublet-microtubules has been described in Chlamydomonas (Goodenough and St. Clair, 1975).
The che-13, osm-1, osm-5, and osm-6 mutations shorten the axonemes of all classes of cilia. Microtubules, attached to the membrane by Y links, assemble ectopically in these mutants. The number and lengths of these ectopic microtubules vary by neuron type and roughly parallel the normal lengths of cilia in these cells. We suggest these ectopic microtubules are misassembled components of the axoneme. Thus the peripheral doublets and Y links can apparently self-assemble and the wild-type products of these four genes are needed to ensure they assemble only on the ciliary template. Interestingly, the transition zones are fairly normal in these mutants. Also, the OLQ axonemes are probably less affected than other classes. It may be that additional structures, such as the apical ring in the transition zone and the filled microtubules or crossbridges in the OLQ axonemes, increase the stability of these segments even in the absence of normal che-13, osm-1, osm-5, and osm-6 products. Consistent with the idea that these genes encode ciliary components, Herman (1984) has shown that the wild-type osm-1 gene must be expressed in the neurons themselves for normal cilia as judged by FITC uptake.
All classes of sensory cilia in che-2 and che-3 mutants have been previously shown to have normal transition zones and truncated axonemes (Lewis and Hodgkin, 1977; Albert et al, 1981). It was reported that microtubules assemble ectopically below the transition zones in both of these mutants. Whereas the truncated amphid cilia in che-2, che-13, osm-1, osm-5, and osm-6 mutants are normal in diameter, the amphid cilia in che-3 mutants have enlarged, bulb-shaped endings filled with dark ground material (Lewis and Hodgkin, 1977).
The amphid channel cilia of che-11 and daf-10 mutants are irregular in contour, variably enlarged in diameter, and may contain dark ground material in the center of the axoneme (Albert et al, 1981). The che-11 channel cilia are nearly normal in length whereas the daf-10 cilia are described as foreshortened (Albert et al, 1981). Both mutants also affect the CEP cilia and, at least for che-11, probably other cilia. Perhaps related, dark ground material has been observed in the center of the axonemes in the bronchial epithelium of a human subject with immotile cilia (Afzelius, 1976).
The amphid cilia are usually absent in che-10 mutants. Instead, the dendrites have large, bulb-shaped endings filled with dark ground material. Occasional dendrites have well-formed cilia, suggesting the amphid defect is degenerative rather than developmental. The mechano-cilia appear normal but lack striated rootlets. It may be interesting to examine the amphid dendrites in embryos or L1 larvae of this strain.
The osm-3 mutation specifically eliminates the distal segments of the amphid channel cilia, leaving the middle segment and the transition zone unaffected. The distal segment differs from the middle segment in that the B subfibers of the peripheral doublets, and the membrane-links, are absent. The osm-3 product may be a protein specific to the distal segments of these cilia. Alternatively, it may affect the entire cilium, perhaps being associated with the A subfibers, but the distal segment is most vulnerable to its absence.
Dissociation of the IL2 Cilia
In wild-type adults, the IL2 neurons, and possibly some mechanosensory neurons, have incomplete cilia comprising fewer than nine doublets (Ward et al, 1975; Ware et al., 1975). Interestingly, in the che-13, osm-1, osm-5, and osm-6 mutants with truncated cilia, the transition zones of the IL2 cilia and the various mechanocilia are actually longer and better formed, in the sense of showing nine Y-linked doublet microtubules drawn together in a ring, than in wild type. We speculate that when they form all classes of cilia have complete transition zones, but certain classes, particularly the IL2 cilia, later dissociate or rearrange, leaving fewer microtubules and no recognizable nine-fold organization. Rearrangement might be expected if, for example, the neurons undersynthesize ciliary proteins during dendrite growth. Mutants which destabilize the axoneme might actually leave more material available for maintaining the transition zone than in the wild type. It may be interesting to examine the IL2 cilia in embryos or L1 larvae.
Striated rootlets are frequently associated with the basal bodies of both sensory and motile cilia but their function is unknown. Salisbury and Floyd (1978) have shown that the rootlets of certain flagellate alga are contractile and that contraction is induced by calcium. A calcium-binding phosphoprotein of 20,000 MW is the principal component of the contractile rootlets from Tetraselmis striata (Salisbury et al, 1984). Striated rootlets have also been purified from several other sources (see Salisbury et al, 1984). In each case, only one or two proteins account for most of the protein in the purified rootlets. However, the molecular weights of these proteins vary widely and it remains to be seen how they are related.
The daf-19 mutation eliminates cilium formation, but striated
rootlets still assemble in the appropriate dendrites. Interestingly, certain
sensory neurons in C. elegans males contain striated rootlets but no
associated cilia (Sulston et al., 1980). The rootlets are attached to dark plates,
resembling hemidesmosomes, at the dendritic tips. These observations imply that
rootlet and cilium formation can occur independently and an unknown mechanism
ensures that the distal end of the rootlet attaches to the base of the cilium.
A similar conclusion has been reached using basal body defective and rootlet
defective mutants of Chlamydomonas (Goodenough and St. Clair, 1975; Wright
et al., 1983). Interestingly, the ectopic membrane-linked microtubules in the
che-13, osm-1, osm-5, and osm-6 mutants can recruit small rootlets.
Presumably, the same affinity exists between the rootlets and microtubules of
The che-10 mutation eliminates striated rootlets from the mechanosensory cilia of the head. The wild type che-10 product may be a rootlet component. The amphid cilia, which lack striated rootlets in the wild-type, are badly degenerated in this strain. A possible explanation, that the strain harbors two mutations, one responsible for the rootlet defect and one for the amphid defect, can be resolved by isolating a second, independent che-10 mutant and examining its rootlets.
Mechanosensory Specializations and Modalities
The distal tips of the IL1 cilia each contain a dark membrane-attached disc. These discs are positioned in the cuticle at the base of the papillae in such a way as to be compressed by head-on contacts of the animal. The mutants with truncated or missing IL1 cilia show that the discs can assemble normally in the absence of cilia but they require the cilia to position them at the tip of the dendrite.
The supernumerary microtubules and associated dark material of the CEP cilia closely resemble the tubular bodies found in proved mechanocilia of insects (Thurm et al, 1983). The dark material, itself amorphous, appears to be molded into rods by the associated microtubules. A similar dark material is found at the tips of the OLL cilia where it forms a ball. The difference in shape may reflect the comparative paucity of microtubules in the OLL cilia. In mutants with truncated or missing cilia, the supernumerary microtubules and dark tubule-associated material in the CEP neurons assemble ectopi-cally along the dendrite in both rod- and ball-shaped aggregates. This shows that these specializations can self-assemble but the cilia are needed to position them at the distal tip of the dendrite. In the cat-6 mutants, both the CEP cilia and their specializations appear normal but the association between them is specifically disrupted.
Amorphous dark material is also present in the OLQ cilia as small lumps that flank the circumferential corners of the joined square of microtubules. These lumps may also be connected to the membrane. In the wild-type, the corners of the square always point radially and circumferentially. There is no obvious structure joining the OLQ axoneme to the support cells or the cuticle that might provide orientation. A simple suggestion is that the square and associated dark material are aligned passively by repeated deformation of the cuticle as might occur during stimulation. Interestingly, the OLQ squares are sometimes misoriented and the dark lumps are fragmented and mispositioned in che-14 mutants which have abnormally thick subcuticle adjacent to the cilia. Perhaps relevant, the cilia of the respiratory epithelia, which normally are oriented to beat in a common direction, are randomly oriented, as judged by their basal feet, in subjects with immotile cilia (Afzelius, 1981). Here, it is thought that cilia form in random orientations and become oriented through a mechanism involving active beating.
As the CEP, OLL, and OLQ cilia are situated somewhat posterior to the IL1 cilia, they probably detect radial, rather than axial, displacements. The geometry of the OLQ cilia suggests they have substantial directional discrimination. The adjacent CEP cilia may be lower threshold, isotropic detectors.
The dark, cuticle-embedded nubbins of the CEP, OLL, and OLQ dendrites presumably provide mechanical anchorage of the dendrite to the cuticle. They are not a ciliary specialization as such since they persist in mutants with truncated cilia and, at least for the CEP dendrites, in the daf-19 mutants without cilia. In the cat-6 mutants with enlarged CEP nubbins or the che-14 mutants with abnormally thin cuticle, the nubbins can completely penetrate the cuticle and expose the dendrite to the medium.
A similar cuticle-embedded nubbin occurs in males at the distal tips of the CEM cilia. Here, it penetrates the cuticle and is believed to provide access of the CEM dendrite to the chemical environment. As there is no cuticular opening in the cephalic sensilla of hermaphrodites which lack the CEM neurons, the openings in males must be created by the CEM dendrites and not the cephalic socket cells. In contrast, the raised cuticle and pore of the inner labial sensilla appear to be formed by the innerlabial socket cell. They persist in mutants with truncated or missing IL1 and IL2 cilia.
C. elegans has at least five distinct chemosensory behaviors. First, it is attracted or repelled by a variety of small molecules at low concentrations (10 -3 M or below) (Ward, 1973; Dusenbery, 1974, 1975, 1976a, 1980a,c). Second, it is repelled by very high concentrations of various chemically unrelated solutes, including NaCl and fructose (Ward, 1973; Culotti and Russell, 1978). Third, when starved under crowded conditions, young larvae may differentiate into dauer larvae, a non-feeding, arrested stage, adapted for long-term survival and dispersal (Cassada and Russell, 1975). Crowding is sensed by the accumulation of a fatty-acid-like pheromone made constitutively by all animals (Golden and Riddle, 1982, 1984a,b). Fourth, chemical cues influence egg laying (Horvitz et al, 1982; Golden and Riddle, 1982; Trent et al, 1983). Fifth, males are attracted to hermaphrodites by an unknown attractant (H. Horvitz and J. Sulston, personal communication). The cephalic companions (CEM), a class of chemosensory neurons found only in males, may be detectors for an hermaphrodite pheromone.
The amphid and phasmid sensilla have long been suspected of mediating many of these chemosensory behaviors (Wright, 1980). Each class of neurons in these sensilla has distinct synaptic outputs, suggesting their cilia may detect different chemicals (Hall and Russell, 1986; White et al, 1986). The clearest evidence that the ten classes of channel cilia are required for chemotaxis, osmotic avoidance, and dauer larvae formation comes from the mutants osm-3, (p802), and daf-6 (el377). The ultrastructural defects in the heads of these mutants are confined to the amphid channel cilia and amphid sheath cell, respectively (Albert et al, 1981). These mutants fail to form dauer larva in response to pheromone, to avoid concentrated NaCl or fructose, or to chemotax toward dilute NaCl (Culotti and Russell, 1978; Albert et al, 1981).
Interestingly, osm-3 (p802) mutants are still responsive to some chemicals including pyridine, C02 and H+ (Dusenbery, 1980b). This may reflect some residual responsiveness of the shortened channel cilia. Alternatively, the amphid wing neurons or the IL2 neurons may be the principal detectors for these chemical species. Significantly, the osm-1 (p808) mutation, which affects the assembly of all classes of cilia, abolishes even these responses (Dusenbery, 1980b). Hence, all known chemical attractants, repellants, and pheromones are apparently sensed through ciliated receptors in C. elegans.
Competing levels of food and crowding pheromome are believed to regulate both entry into and exit from the dauer larval stage. Mutants with abnormal amphid cilia are generally incapable of forming dauer larva in response to crowding and starvation (Tables 1 and 2) or direct application of pheromome (Golden and Riddle, 1984a). Two of these mutants, daf-6 and daf-10, have been induced to form dauer larva by introducing second mutations which favor dauer formation (Albert et al, 1981; Riddle et al, 1981). In these genetic backgrounds, the daf-6 and daf-10 mutations inhibit exit from the dauer stage perhaps by preventing detection of a food signal. Paradoxically, the daf-19 mutants with no sensory cilia form dauer larva constitutively in the absence of crowding or starvation (D. Riddle, personal communication). This suggests that mutations affecting the sensory cilia can shift decisions to enter or exit the dauer stage in either direction.
Horvitz et al. (1982) have reported that che-3, daf-10, and osm-3 mutants lack biochemically detectable levels of octopamine, a presumptive neurotransmitter found in the wild-type. The common defect of these three mutants is a disruption of the amphid and phasmid cilia. This suggests that these neurons either make octopamine or regulate the neurons which do.
Mating by C. elegans males is a complex behavior involving ten classes of male-specific sensory neurons (Ward et al, 1975; Sulston et al, 1980; Hodgkin, 1983). Ten genes that affect mechanosensory receptors in the head, che-2, che-3, che-10, che-11, che-13, daf-10, daf-19, osm-1, osm-5, and osm-6, are also required for mating (Table 1). These mutations likely prevent mating by disrupting male-specific sensilla in the tail. Most of these mutants show occasional fluorescein uptake into ray neurons, indicating that the ray sensilla are abnormal. The mating defect in che-10 (e1809) may be the consequence of missing striated rootlets normally found in dendrites of the ray, hook, and postcloacal sensilla (Sulston et al, 1980).
As expected, the various neurons of the amphid and phasmid sensilla are probably not important for male mating behavior as che-12, daf-6, osm-3, and ttx-1 mutants all mate efficiently (Table 1). Similarly, the efficient mating of cat-6 males implies that the ADE, CEP, and PDE neurons are not involved.
Possible Thermosensory Role of Amphid Finger Neuron (AFD)
The AFD dendrites are unique among sensory receptors in C. elegans in having numerous fingers that invaginate the surrounding sheath cell. These fingers, which are topologically proximal to the AFD cilia, do not depend on the cilia for formation since mutants with reduced axonemes (che-13, osm-1, osm-5, and osm-6) or no cilia (daf-19) have normal fingers.
R. Ware has suggested, based on his unpublished observations on ttx-1 (p767) mutants, that the AFD neurons may be thermosensory. As confirmed here, the AFD fingers are entirely missing and the AFD cilia are longer than normal in ttx-1 mutants. The other sensory receptors in the head appear ultrastructurally normal.
When placed in a thermal gradient, wild-type animals move toward the temperature at which they were previously raised (Hedgecock and Russell, 1975). The ttx-1 thermotaxis mutants seek the cold regardless of their thermal history. These mutants are also hyper-responsive to dauer-inducing pheromone (Golden and Riddle, 1984a,b). Elevated temperatures are known to lower the pheromone threshold for dauer-larva formation in wild-type animals. This suggests that both the cryophilia and heightened pheromone sensitivity of the ttx-1 mutants may reflect a common sensory defect in which the animal perceives a higher temperature than actual. Other sensory behaviors, including chemotaxis and mating, are normal in this strain (Hedgecock and Russell, 1975; Dusenbery and Barr, 1980).
A reduction in the number of fingers on the AFD dendrites has also been reported for the che-1 mutants, e1034 and a74 (formerly DD74) (Lewis and Hodgkin, 1977; R. Ware, D. Clark, M. Salzay, and R. Russell, personal communication). No thermotaxis defects were detected in population assays of che-1 mutants (Hedgecock and Russell, 1975). However, the finger abnormality in che-1 mutants is variable and comparatively mild. About half of the che-1 (e1034) animals examined by Lewis and Hodgkin (1977), for example, had normal or nearly normal AFD dendrites.
It may be possible to confirm a role for the AFD neurons in thermal behavior by killing these cells with a laser microbeam (Sulston and White, 1980) and testing the animals in individual thermotaxis assays (Hedgecock and Russell, 1975).
Burr (1985) has reported that C. elegans responds to light by reversing, and consequently changing the direction of movement, more frequently than in the dark. This is a nonoriented response but, in principle, could be used to keep animals away from lighted areas (Fraenkel and Gunn, 1961). The light appears to act directly and not by radiant heating.
In nematodes with true phototaxis, the ocelli comprise a pair of amphid dendrites plus nearby pigment spots in the pharynx which provide shadowing (see Burr, 1985). Although C. elegans lacks obvious photopigments or shadowing pigments, the AWC neurons are plausible candidates for photoreceptors as their cilia have extremely large membrane areas. Tests of mutants such as daf-19 may help ascertain whether the photoresponse in C. elegans is mediated by ciliated sensory neurons.
Evolution of Sensory Cilia
Motile cilia, found in unicellular eukaryotes, lower plants, and animals, are believed to be ancient organelles. The sensory cilia of animals probably arose by later modification of motile cilia. In nematodes, the motile functions of cilia have apparently been lost. Their spermatozoa are nonflagellated and move by extending contractile pseudopodia (Ward et al, 1982), and there are no ciliated epithelia. In contrast, the sensory functions of cilia are highly elaborated. Wright (1983) has suggested, that since there is no selective pressure to maintain ciliary structures used strictly for motility, nematode cilia may be simpler than in other animals. For example, the dynein and nexin arms, radial spokes, and central pair of singlet microtubules that generate the sliding force in motile axonemes and control the flexion are all apparently absent in nematode cilia.
The absence of basal bodies seems a paradox as they are believed to have two functions, one of which is essential. First, they are the templates for the ninefold structure of the axonemes. The nine doublet microtubules of the axoneme are a direct extension of the A and B subfibers of the nine triplet-microtubules in the basal body. Second, basal bodies are attachment points for cytoplasmic microtubules which anchor the cilium to the cytoskeleton. This coupling is essential for transmitting force from a beating cilium into cell motion. It may also be useful for holding cilia erect from the cell surface.
In nematodes, the mechanical role of the basal body is probably not needed. The template role would be filled if the centriole is present only transiently to initiate the cilium and then disappears. Alternatively, the transitional fibers themselves could be the residue of the centriole. Importantly, nematode centrioles are composed of singlet microtubules, plus some attachments that may be vestiges of B and C subfibers, rather than triplets (D. Albertson, A. Crowther, and J. N. Thomson, personal communication). Finally, in view of these departures from what are usually regarded as universal characteristics of centrioles and cilia, it is worth mentioning that microtubules themselves may be unusual in nematodes. Cytoplasmic microtubules in nerve processes contain only 11 protofilaments rather than the more usual 13 protofilaments (Chalfie and Thomson, 1982).
Adapted by Yusuf KARABEY for WORMATLAS, 2003