DEVELOPMENTAL BIOLOGY 117,456-487 (1986) Mutant Sensory Cilia in the Nematode Caenorhabditis elegans LIZABETH A. PERKINS,*,’ EDWARD M . HEDGECOCK,-/-’ J. NICHOL THOMSON,? AND JOSEPH G. CULOTTI* *Department of Biochemistry, Molecular and Cellular Siology a.nd Department of Neurobiology and Physiology, Northwest emz University, Evanston, Illinois 60201, and tDivision of Cell Biology, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England Received August 6, 1985; accepted in revised form March 31, 1986 Eight class es of chemosensory neurons in C. elegana fill with fluorescein when living anima ls are placed in a dye solution. Fluorescein enters the neurons through their exposed sensory cilia. Mutations in 14 genes prevent dye uptake and disrupt chemosensory behaviors. Each of these genes affects the ultrastructure of the chemosensory cilia or their accessory cells. In each case, the cilia are shorter or less exposed than normal, suggesting that dye contact is the principal factor under selection. Ten genes affect many or all of the sensory cilia in the head. The daf-19 (ti6) mutation eliminates all cilia, leaving only occasio nal centrioles in the dendrites. The cilia in the-13 (el805), osm-1 (p808), osm-5 (p813), and osm-6 (~811) mutants have normal transition zones and severely shortened axonemes. Doublet-microtubules, attached to the membrane by Y links, assemble ectopically proximal to the cilia in these mutants. The amphid cilia in the-11 (el810) are irregular in diameter and contain dark ground material in the middle of the axonemes. Certain mech anocilia are also affected. The amphid cilia in the-10 (e1809) apparently degenerate, leaving de ndrites with bulb- shaped endings filled with d ark ground material. The mech anocilia lack striated rootlets. Cilia defects have also been found in the-2, the- 3, and daf-10 mutants. The osm-3 (~802) mutation specifica lly eliminates the distal segment of the amphid cilia. Mutations in three genes affect sensillar support cells. The the-12 (e1812) mutation eliminates matrix material normally secreted by the amphid sheath cell. The the-14 (e1960) mutation disrupts the joining of the amphid sheath and socket ce lls to form the receptor channel. A similar defect has been observed in daf-6 mutants. Four add itional genes affect specific classes of ciliated sensory neurons. The met-l and met-8 (e398) mutations disrupt the fascicu lation of the amphid cilia. The cat-6 (e1861) mutation disrupts the tubular bodies of the CEP mechanocilia. A cryophilic thermotaxis mutant, ttx- 1 (p7’67), lacks fingers on the AFD dendrite, suggesting this neuron is thermosensory. D 1986 Academic Press, Inc INTRODUCTION Cilia and flagella are ubiquitous eukaryotic organelles that have been adapted for two seemingly unrelated functions, sensory transduction and cell motility. In the unicellular eukar yotes, Chlamydomonas and Parame- cium, for example, they are used for swimming. Simi- larly , flagella propel the sperm of man y animals and lower plants. Arrays of motile cilia line various epithelia, including the respiratory tracts, the oviducts, and the ventricles of the brain, where they propel fluid or par- ticles along the surface. Senso ry cilia are found in the rod and cone cells of the ey e, the hair cells of the ear, and the olfactory re- ceptor neurons. In nematodes, cilia are found only in the nervous system where they are sensory receptors spe- cialized for diverse modalities (Ward et al., 1975; Ware et al., 1975). Of the 118 clas ses of neurons in Caenorha- biditis elegant hermaphrodites, 24 clas ses hav e cilia (White et al, 1986). ’ Current address: Department of Developmental Genetics and Anatomy, Case Western Reserve Uni versity, Cleveland, Ohio 44106. * Current address: Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, N.J. 07110. The common plan of both motile and sen sory cilia i s a membrane-bound cylinde r of nine doublet microtu- bules that extend from a centriole. Ma ny cili a have ad- ditional structures that adapt them to specific tasks. As they are biochemically complex structures and, in many cases, present in limited numbers, genetic studie s have been helpful in understanding the assembly and function of cili a (Afzelius, 1981). In Chlamydommas and Para- mecium, genes coding for ciliary proteins hav e been identified by selecting for mutants with abnormal swi mming (Luck, 1984; Kung et ab, 1975). In humans, genetic disorders of ciliary motili ty produce a syndrome of male infertility and respiratory dis tress (Afzelius, 1976). In C. elegans, several collections of mutants hav e been obtained by select ing for altered sensory behavior (Du- senbery et al., 1975; Hedgeco ck and Ru ssell, 1975; Lewis and Hodgkin, 1977;Culotti and Russe ll, 1978;Chalfie and Sulston, 1981; Riddle et al., 1981; Hodgkin, 1983; and Trent et ab, 1983). While some of these mutations affe ct the sensory organs themselves (Lewis and Hodgkin, 1977; Albert et ab, 1981; Chalfie and Sulston, 1981; R. War e, D. Dusenbery, D. Clark, M. Sz alay , and R. Russ ell, per- sonal communication), others presumably disrupt be- havior at steps downstream of t ransduction. 0012-1606/86 $3.00 Press, Inc. All rights of reproduction in any for m reserved. 456
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Mutant Sensory Cilia in the Nematode Caenorhabditis elegans
LIZABETH A. PERKINS,*,’ EDWARD M. HEDGECOCK,-/-’ J. NICHOL THOMSON,? AND JOSEPH G. CULOTTI*
*Department of Biochemistry, Molecular and Cellular Siology a.nd Department of Neurobiology and Physiology, Northwestemz University,
Evanston, Illinois 60201, and tDivision of Cell Biology, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England
Received August 6, 1985; accepted in revised form March 31, 1986
Eight class es of chemosensory neurons in C. elegana fill with fluorescein when living anima ls are placed in a dye
solution. Fluorescein enters the neurons through their exposed sensory cilia. Mutations in 14 genes prevent dye uptake
and disrupt chemosensory behaviors. Each of these genes affects the ultrastructure of the chemosensory cilia or their
accessory cells. In each case, the cilia are shorter or less exposed than normal, suggesting that dye contact is the
principal factor under selection. Ten genes affect many or all of the sensory cilia in the head. The daf-19 (ti6) mutation
eliminates all cilia, leaving only occasio nal centrioles in the dendrites. The cilia in the-13 (el805), osm-1 (p808), osm-5
(p813), and osm-6 (~811) mutants have normal transition zones and severely shortened axonemes. Doublet-microtubules,
attached to the membrane by Y links, assemble ectopically proximal to the cilia in these mutants. The amphid cilia in
the-11 (el810) are irregular in diameter and contain dark ground material in the middle of the axonemes. Certainmech anocilia are also affected. The amphid cilia in the-10 (e1809) apparently degenerate, leaving de ndrites with bulb-
shaped endings filled with dark ground material. The mech anocilia lack striated rootlets. Cilia defects have also been
found in the-2, the-3, and daf-10 mutants. The osm-3 (~802) mutation specifica lly eliminates the distal segment of the
amphid cilia. Mutations in three genes affect sensillar support cells. The the-12 (e1812) mutation eliminates matrix
material normally secreted by the amphid sheath cell. The the-14 (e1960) mutation disrupts the joining of the amphid
sheath and socket ce lls to form the receptor channel. A similar defect has been observed in daf-6 mutants. Four add itional
genes affect specific classes of ciliated sensory neurons. The met-l and met-8 (e398) mutations disrupt the fascicu lation
of the amphid cilia. The cat-6 (e1861) mutation disrupts the tubular bodies of the CEP mechanocilia. A cryophilic
thermotaxis mutant, ttx-1 (p7’67), lacks fingers on the AFD dendrite, suggesting this neuron is thermosensory. D 1986
Academic Press, Inc
INTRODUCTION
Cilia and flagella are ubiquitous eukaryotic organelles
that have been adapted for two seemingly unrelated
functions, sensory transduction and cell motility. In the
unicellular eukaryotes, Chlamydomonas and Parame-
cium, for example, they are used for swimming. Simi-
larly, flagella propel the sperm of many animals and
lower plants. Arrays of motile cilia line various epithelia,
including the respiratory tracts, the oviducts, and the
ventricles of the brain, where they propel fluid or par-
ticles along the surface.
Sensory cilia are found in the rod and cone cells of
the eye, the hair cells of the ear, and the olfactory re-
ceptor neurons. In nematodes, cilia are found only in the
nervous system where they are sensory receptors spe-cialized for diverse modalities (Ward et al., 1975; Ware
et al., 1975). Of the 118 classes of neurons in Caenorha-
biditis elegant hermaphrodites, 24 classes have cilia
(White et al, 1986).
’ Current address: Department of Developmental Genetics and
Anatomy, Case Western Reserve University, Cleveland, Ohio 44106.
* Current address: Department of Cell Biology, Roche Institute of
Molecular Biology, Nutley, N.J. 07110.
The common plan of both motile and sensory cilia is
a membrane-bound cylinder of nine doublet microtu-
bules that extend from a centriole. Many cilia have ad-ditional structures that adapt them to specific tasks. As
they are biochemically complex structures and, in many
cases, present in limited numbers, genetic studies have
been helpful in understanding the assembly and function
of cilia (Afzelius, 1981). In Chlamydommas and Para-
mecium, genes coding for ciliary proteins have been
identified by selecting for mutants with abnormal
swimming (Luck, 1984; Kung et ab, 1975). In humans,
genetic disorders of ciliary motility produce a syndrome
of male infertility and respiratory distress (Afzelius,
1976).
In C. elegans, several collections of mutants have been
obtained by selecting for altered sensory behavior (Du-senbery et al., 1975; Hedgecock and Russell, 1975; Lewis
and Hodgkin, 1977; Culotti and Russell, 1978; Chalfie andSulston, 1981; Riddle et al., 1981; Hodgkin, 1983; and
Trent et ab, 1983). While some of these mutations affect
the sensory organs themselves (Lewis and Hodgkin, 1977;
Albert et ab, 1981; Chalfie and Sulston, 1981; R. Ware,
D. Dusenbery, D. Clark, M. Szalay, and R. Russell, per-
FIG. 1. Anterior sens illa in wild-type hermaphrodite. Section 4.0 nrn from tip of head. The fascic les of amphid channel cilia (AMPHID),
positioned laterally, have just entered the socket channels. The wings of the AWC c ilia (arrows) are spread vertically in the amphid sheath
cell. Six pairs of inner labial dendrites (IL1 and IL2) invaginate the inner labial sheath cells. A large striated rootlet is visible in each IL1
dendrite. Dorsally and ventrally, the four CEP and four OLO cilia are sectioned through their middle segments. The squares of microtubules
in the OLQ cilia are oriented with corners circumferential and radial. The two OLL dendrites, positioned laterally, are sectioned through theirjunctions with sheath cells. The cilia of the BAG and FLP neurons are also visible. The left FLP cilium and the right BAG cilium are sectioned
through their transition zones. Scale bar is 1.0 pm.
channel lumen (Wright, 1980). The matrix material of terial, though separating the cilia in the posterior chan-
the amphid sheath cells, and a similar material in the nel, gradually thins until the membranes of the channel
other sensilla, is not well preserved in animals fixed with cili a are in direct apposi tion in the anterior sheath and
OsOl alone. In consequence, several published reports the socket channel (Fig. 3). The pattern of fasciculat ion
erroneously describe an empty space around the cil ia or of the channel cil ia is invariant in wild-type animals
empty vesicles in the sheath cytoplasm. The matrix ma- (Ward et al., 1975; Ware et al., 1975).
FIG. 3. Amphid socket and sheath channe ls in wild-type. (a) Section through amphid socket c ell about 3.0 pm from the tip of the head. The
distal segments of the ten channel cilia are present. The cilia contain both large (13 protofilament) and sma ll (11 protofilament) diameter
microtubules. These are the A fibers of the nine doublet microtubules and the inner singlet microtubules, respectively (Chalfie and Thoms on,
1982). The socket channel is lined by cuticle (black arrow). The self-junction (JN) and an associate d scaffold of intermediate filaments (FS)
are also visible. (b) Section 2.5 pm posterior to (a) through the amphid sheath cell showing the middle segmen ts of the channel cilia. The B
subfibers of the doublets are complete. A variable number of inner singlet microtubules are also present. Traces of matrix (M) surround and
separate the cilia at this level and more posteriorly. The channel is lined by a dark material (white arrow) and the surrounding cytoplasm is
filled by a scaffold of longitudinal microtubules and intermediate filaments (FS). A rare circumferential filament is seen in the plane of section
(small black arrows). Part of the belt junction (JN) between the sheath (sh) and socket (so) cells is also visible. The dark linin g and filament
scaffold are interrupted where the AWB cilium se parates from the main fascic le and invaginates the sheath cell (arrowhead). Scale bar is
0.5 pm.
title. Most of the structural components of the amphid
sensilla described above are also found, reduced in size,
in these sensilla.
The tip of the head has six symmetrically arranged
lips (2 dorsal, 2 ventral, and 2 lateral). An inner labial
sensillum is found on the apex of each lip. These sensilla
each contain two ciliated dendrites (IL1 and IL2) (Fig.
1). The dorsal and ventral lips also contain a cephalic
and an outer labial quadrant sensillum. The cephalic
sensilla have a single dendrite (CEP) in hermaphrodites
and an additional dendrite (CEM) in males. The outer
labial quadrant sensilla have a single dendrite (OLQ).The lateral lips contain, in addition to an inner labialand an amphid sensillum, an outer labial lateral sensil-
lum. The outer labial lateral sensilla have a single den-
drite (OLL).
After passing through the socket channels, the ILl,
CEP, OLQ, and OLL cilia end embedded in the subcuticle
and are believed to be mechanosensory. In contrast, thetips of the IL2 and CEM cilia completely penetrate the
cuticle and are believed to be chemosensory.
Finally, two classes of ciliated dendrites (BAG and
FLP) found in the lateral lips are not surrounded by
support cells (Fig. 1). Their cilia end somewhat behind
the cuticle in bag and flap-shaped sheets, respectively,
that envelop short projections from the inner labial
socket cells.
Ultrastructure of Amphid Cilia
The dendrites of amphid channel neurons ASE, ASG,
ASH, ASI, ASJ, and ASK each end with a single cilium
about 7.5 pm long in adults (Ward et al., 1975; Ware et
al., 1975). The dendrites of channel neurons ADF andADL are similar but each ends in a pair of cilia (Figs.
2,3). Three segments can be distinguished in these cilia.
The proximal segment, which corresponds to the tran-
sition zone of the motile flagella in Chlamydomonas(Ringo, 1967), is a constriction at the base of the cilium
about 0.27 pm in diameter and up to 1.0 pm in length.It comprises nine doublet-microtubules joined to the
membrane by Y-shaped links (Gilula and Satir, 1972)
and drawn inward by attachments to a central cylinder
FIG. 5. CEP and OLQ cilia in wild-type. (a) Section through the distal segments of CEP (white arrow) and OLQ (black arrow) cilia in wild
type. The CEP cilium is filled with microtubules interspersed with an amorphous dark tubule-associated material (TAM). The outermost
microtubules appear to have fine attachments to the membrane. The OLQ cilium conta ins four doublet microtubules joined together into a
square by thick cross-bridges. The corners of the square point radially and circumferentially. Inside the square, fine radial arms connect the
doublets to a sma ll hub. Sma ll lumps of dark material flank the circumferential doublets. This tubule-associated material (TAM) may also be
attached to the membrane. (b) Section 0.15 pm posterior to (a) showing the end of the cuticle-ass ociated nubbin (CN) of the CEP cilium . The
OLQ cilium has a similar nubbin about 1 +rn more anterior. (c) Section 0.6 pm posterior to (a). The supernumerary microtubules and the dark
tubule-associated material of the CEP cilium are reduced. The tubule-associated material of the OLQ cilium is no longer present. (d) Section
2.0 pm posterior to (a) through the middle segment of the CEP cilium. No supernumerary microtubules or tubule-associated material are
present. Nine doublet microtubules are present in the OLQ cilium, four of which are joined by cross-bridges. The A and B subfibers of most
of the microtubules appear filled . The A subfibers of three doublet microtubules in the square appear empty. (e) Section 2.1 pm posterior to
(a) through the transition zone of the OLQ cilium . A ll nine doublet microtubules are attached to the membrane by Y-shaped links. Matrix (M)surrounds the cilium . (f) Section 3.3 Frn posterior to (a) through the transition zone of the CEP cilium . The doublet microtubules are attached
to the membrane by Y-shaped links. In contrast to the OLQ cilium, the A and B subfibers of the CEP cilium appear empty. Matrix (M)
surrounds the cilium. A large striated ciliary rootlet (SR) is present in the OLQ dendrite. (g) Section 3.8 pm posterior to (a) through the
transitional fibers of the CEP cilium . (h) Section 4.5 pm posterior to (a) through neuron/sheath junctions (JN). The CEP dendrite has no
prominent rootlet. Scale bar is 0.5 pm.
actually embedded in the amorphous root and extend to of neurofilaments extends to the base of the cilia. Finally,
the base of the cili a. The amorphous root is reduced or numerous coated pits and vesicles are found in al l the
absent in the AFD dendrites. In those cells, a fascicle amphid dendrites just proximal to the cil ia (Fig. 4d).
FIG. 7. IL1 and IL2 cilia in wild-type. (a) Section through the dark membrane-attached disc (D) at the tip of the IL1 cilium . The sma ll IL2
cilium (black arrow) continues anterior to a sma ll opening in the cuticle. The IL1 disc is positioned in the cuticle in such a way as to be
compressed by head-on collisio ns of the animal. (b) Section through the transition zone of an IL1 neuron (white arrow). A striated ciliary
rootlet (SR) extends into the center of the cilium . The dendrite of an IL2 neuron (black arrow) shares the sensillum . (c) Section 0.15 micron
posterior to (b) showing transitional fibers (arrowheads) in the IL1 cilium . Note the increase in diameter of the cilium at this point. Matrix-
filled vesicles (M) in the sheath cytoplasm. (d) Section 0.3 pm posterior to (b) showing the striated rootlet (SR). (e) Section 0.8 pm posteriorto (b) showing the ILl/sheath junction (JN). The striated rootlet (SR) continues for about 9 pm. Scale bars are 0.5 Frn.
than nine doublets in the shaft and no well-formed
transition zone.
Mechanism of Dye Filling
When l iving C. elegans are placed in solutions of 5-
fluorescein isothiocyanate (FITC), six pairs of neurons
in the head and two pairs in the tail f ill with dye (Fig.
8a). Their cel l bodies and processes become visible within
5 min and reach a maximum brightness within about 2hr when stained in 0.1 mg/m l FITC. Dye fil ling proceeds
equally well at 0’ as at 20’. Once fil led with 5-fluorescein
isothiocyanate, the neurons remain brightly stained for
many hours in the absence of dye. Staining with fluo-
rescein, in contrast, reverses completely in the course
of an hour. Presumably, 5-fluorescein isothiocyanate, but
not free fluorescein, can combine with amino groups
within the cell and become either immobile or imper-
meant to cel l membranes. In support of this, 5-fluores-
cein isothiocyanate, when coupled to bovine serum al-
bumin, cannot enter the neurons from the outside.
We tested a variety of other fluorescent dyes and none,
except certain fluorescein derivatives, accumulate in the
amphid and phasmid neurons. The fluorescein deriva-
tives that stain the neurons are weak acids and exist as
both neutral and anionic forms within the physiological
range of pH values. In their uncharged forms, favored
by lower pH, they can probably diffuse across cel l mem-
branes.
The FITC-filled neurons in the head and tail were
identif ied as amphid channel neurons (ADF, ASH, ASI,
ASJ, ASK, and ADL) and phasmid channel neurons
(PHA and PHB), respectively (Hedgecock et ah, 1985).
These cells stain in larvae of al l stages and in adults.
To learn whether fluorescein enters these neurons
through their exposed sensory cilia, we killed the phas-
mid support cells in newly hatched larvae using a laser
microbeam (Sulston and White, 1980). These animals
were tested as adults for dye uptake into the phasmid
neurons. Kil ling the socket cell (2 animals), which pre-
sumably disconnects the sheath and cil ia from the cu-
ticle, or the sheath cell (1 animal) abolished filli ng of
the ipsilateral neurons without affecting the neurons ofthe contralateral phasmid sensillum. Control ablations
of neighboring cells did not affect dye uptake.
The amphid channel neurons ASE and ASG, the IL2
neurons, and the various male-specific chemosensory
neurons do not appear to fi ll with fluorescein dyes. Thus
access of the sensory dendrites to the dye is apparently
necessary but not sufficient to ensure fill ing . Apparently
a physiological property, shared by some but not all sen-
sory neurons, is also required for fil ling. A simple sug-
gestion is that for dye to fill the entire neuron, the rate
of dye entry through the sensory receptor must be
greater than the rate of dye leakage into the body cavity
from the sensory process. The rate of entry is contro lled
by the geometry, and possibly, membrane properties of
the exposed dendrites. The rate of leakage from the pro-
cesses might depend on membrane potential or intra-
cellular pH.
Identi&ation of Behavioral Mutants with Impaired
FITC Uptake
Mutants with sensory defects have been isolated by
selections involving chemotaxis toward Na+ or Cl- ions
FIG. 21. Amphid channel cilia in osm-3 (~802) mutant. (a) Section through amphid socket ce ll. The channel (star) is empty of cilia. (b) Section
2.0 pm posterior to (a) at the junction between the sheath and socket c ells (JN). Only four c ilia extend this far in the channel. The center of
the channel is occupied by matrix (M). (c, d) Section s 2.7 and 3.0 pm posterior to (a) through the amphid sheath cell. All ten channel cilia are
present in (d).
ment is not absolute as hybrid sensilla can form when interactions in add ition to the neuron/sheath interac-normal partners are removed (Sulston et aZ., 1983). tions observed in al l sensilla. The 12 dendrites normally
Invagination may be an early step in neuron/sheath invaginate the sheath cell roughly in register. The chan-
interaction. In daf-19 mutants, where cil ia are appar- nel cil ia form a tight fascicle in the sheath ce ll which
ently not formed, the sensory dendrites still invaginate extends into the socket channel. Curiously, the arrange-
their sheath cells and form normal bel t junctions. ment of cil ia within this fascicle is invariant in wild-
The amphid dendrites show specific neuron/neuron type animals (Ward et al., 1975; Ware et al., 1975). It is
FIG. 22. Matrix in amphid sheath cells of wild-type and OWL-J mutant. (a) Section through the amphid sheath cell in wild-type showing a
few matrix-filled vesicles (M) fusing with the channel lumen. (b) Comparable section through mm-3 (~802) showing an abnormal accumu lation
of large matrix-filled vesicles throughout the sheath cell cytoplasm. Scale bar is 0.5 pm.
FIG. 23. Amphid sheath channel in wild type and the-12 mutant. (a) Wild-type amphid sheath cell showing matrix-filled vesicles (MV) fusing
with channel. The ten cilia in the channel are also surrounded by matrix. Fingers of the AFD neuron are shown by arrows. (b) Comparablesection from &e-I& (e1812) mutant. The matrix vesicles (MV) appear pale or empty. The channel appears devoid of matrix and the channel
cilia are abnormally dark. The extracellular space between the sheath ce ll and the AFD fingers (arrows) is abnormally pale. Abnormal vesicles
(arrowheads) are found between the layers of the cuticle. Scale bar is 0.5 pm.
unknown whether the ciliary pattern is inherited from
the more complex pattern of the papillary nerves.
In met-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 met-1 mutants (Lewis
and Hodgkin, 1977; Chalf ie and Sulston, 1981). Conceiv-
ably the met-1 and met-8 genes specify adhesive mole-
cules that determine pairwise affinit ies of the amphid
dendrites or their cilia. In addition, the met-1 and mec-
8 mutations disrupt the function of certain nonciliated
mechanosensory neurons (Chalfie and S&ton, 1981). The
met-1 mutations were shown to prevent the normal at-
tachment 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 me-8 mutants form separate channelsthat 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 met-8 mutants with displaced cilia,
the matrix material still deposits along them. It is also
deposited around the ectopic cilia-like projections found
in the-13, osm-1, and osm-5 dendrites.
In mutants with short or absent cilia, matrix material
accumulates in large vesicles in the anterior sheath cy-
toplasm. Abnormal accumulations of large matrix ves-
icles have also been reported in the amphid sheath cells
of the-2, the-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 am-
phid openings. This would explain why it accumulates
in the the-14, daf-6, and met-8 mutants that have ap-
parently normal cilia but obstructed channels.
The the-12 mutation appears to disrupt the synthesis
or secretion of matrix by the sheath cells. Interestingly,
empty vesicles still form at the lamellae, transport for-
ward, and fuse with the channel lumen. Presumably the
abnormal darkening of the channel cilia in the the-12
mutants is a degenerative change resulting from the loss
of matrix normally surrounding the cilia. These mutantsalso 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 en-
velop. 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-
FIG. 25. Amphid cilia in the-1.4 (e1960) mutant. (a) Section through the amphid socket cel l (so). The cuticle-lined channel (star) ends blindly
without connecting to the channel of the sheath cell. The self-junction (JN) of the socket ce ll is still formed. The main fascic le of channel cilia
(C) is deflected laterally in the sheath cell and ends blindly in a large de posit of matrix (M) surrounded by a thin sheet of sheath cell cytoplasm.
Two cilia (C) separate from the main fa scicle, exit the sheath cell, and invaginate the cytoplasm of the socket cell. (b) Section 0.45 Mm posterior
to (a) through junction (JN) of the sheath (sh) and socket (so) cells. Scale bar is 0.5 pm.
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 the-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 embryosor LI 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. Alterna-
tively, it may affect the entire cilium, perhaps being as-
sociated 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 com-
prising fewer than nine doublets (Ward et al, 1975; Ware
et al., 1975). Interestingly, in the the-13, osm-1, osm-5,
and osm-6 mutants with truncated cilia, the transition
zones of the IL2 cilia and the various mechanocilia areactually longer and better formed, in the sense of show-ing 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 dis-
sociate or rearrange, leaving fewer microtubules and no
osm-1, osm-5, and osm-6, are also required for mating
(Table 1). These mutations likely prevent mating by dis-
rupting male-specific sensilla in the tai l. Most of these
mutants show occasional fluorescein uptake into ray
neurons, indicating that the ray sensilla are abnormal.
The mating defect in the-10 (e1809) may be the conse-
quence of missing striated rootlets normally found in
dendrites of the ray, hook, and postcloacal sensilla
(Sulston et ah, 1980).
As expected, the various neurons of the amphid and
phasmid sensilla are probably not important for male
mating behavior as the-12, daf-6, osm-3, and ttx-1 mutants
FIG. 30. Schem atic longitudinal section through CEP cilium in cat- all mate efficiently (Table 1). Similarly, the efficient
6 (elSS1). Rod-shaped aggregates of tubule-associated material (TAM) mating of cat-6 males implies that the ADE, CEP, andand supernumerary microtubules assem ble along the entire cilium PDE neurons are not involved.and below it. They also extend into the cuticle-attached nubbin (CN)
which i s larger than normal and often penetrates the cuticle. Scale
bar is 1.0 pm.Possible Thermosenwry Role of Amphid Finger
Neuron (AFD)
The AFD dendrites are unique among sensory recep-
induced to form dauer larva by introducing second mu- tors in C. elegans in having numerous fingers that in-
tations which favor dauer format ion (Albert et ah, 1981; vaginate the surrounding sheath cell . These fingers,
Riddle et al., 1981). In these genet ic backgrounds, the which are topo logically proximal to the AFD cilia, do
daf-6 and daf-10 mutations inhib it exit from the dauer not depend on the cilia for formation since mutants with
stage perhaps by prevent ing detect ion of a food signal. reduced axonemes (the-13, osm-1, osm-5, and osm-6) orParadoxically, the daf-19 mutants with no sensory cilia no cilia (daf-19) have normal fingers.
form dauer larva constitutively in the absence of crowd- R. Ware has suggested, based on his unpublished ob-ing or starvation (D. Riddle, personal communication). servations on ttx-l(p767) mutants, that the AFD neurons
This suggests that mutations affecting the sensory cilia may be thermosensory. As confirmed here, the AFD fin-
FIG. 29. Amphid sens illum in met-8 (eS98). (a, b) Sections through the socket cell (so) showing disarrayed intermediate filaments (FS) of the
scaffold associate d with a self-junction (JN). The cuticle-lined channel has failed to extend th is far posteriorly. A few isolated cilia (C) are
visible in the sheath cell (sh). (c) Section 1.3 pm posterior to (b) showing sheath/socket junction (JN). The cilium and fingers of the AFD
dendrite are visible as is an isolated channel cilium (C). (d) Section 1.6 pm posterior to (b) showing four isolated channel cilia (C) and the
distal end of a fascic le of three cilia. The fasciele is surrounded by the matrix material, dark lining (black arrows), and filamentous scaffold
that surround the channel cilia in wild-type. (e-g) S ections 6.5, 6.7, and 6.9 pm posterior to (b). Three cilia form a fascic le (white arrow). The
cilium of another neuron (1) makes a complete U-turn and extends posteriorly into the sheath cell. The paired cilia of another neuron (2),
probably AWB, are orthogonal, rather than parallel, at their bases . Scale bar is 0.5 Wm.
Hodgkin (1977), for example, had normal or nearly nor-
mal AFD dendrites.
It may be possible to confirm a role for the AFD neu-
rons 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).
Photosensory Behavior
Burr (1985) has reported that C. elegans responds to
light by reversing, and consequently changing the di-
rection of movement, more frequently than in the dark.
This is a nonoriented response but, in principle, could
be used to keep animals away from ligh ted areas
(Fraenkel and Gunn, 1961). The light appears to act di-
rectly and not by radiant heating.
In nematodes with true phototaxis, the oce lli comprise
a pair of amphid dendrites plus nearby pigment spots
in the pharynx which provide shadowing (see Burr, 1985).Although G! elegans lacks obvious photopigments or
shadowing pigments, the AWC neurons are plausible
candidates for photoreceptors as their c ilia have ex-
tremely large membrane areas. Tests of mutants such
as daf-19 may help ascertain whether the photoresponse
in C. elegans is mediated by cilia ted sensory neurons.
Evolution of Sensory Cilia
Motile cilia , found in unicellular eukaryotes, lower
plants, and animals, are believed to be ancient organ-
elles. The sensory cil ia of animals probably arose by later
modification of motile cilia. In nematodes, the motile
functions of cilia have apparently been lost. Their sper-
matozoa are nonflage llated and move by extending con-
tractile pseudopodia (Ward et al., 1982), and there are
no cilia ted epi thelia. In contrast, the sensory functions
of cil ia are high ly elaborated. Wright (1983) has sug-
gested, that since there is no selective pressure to main-
tain ciliary structures used strictly for mot ility , nema-
tode cilia may be simpler than in other animals. For
example, the dynein and nexin arms, rad ial 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 theyare believed to have two functions, one of which is es-
sential. First, they are the templates for the ninefold
structure of the axonemes. The nine doublet microtu-
bules 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 cili um to
the cytoskeleton. This coupling is essential for trans-
mitting 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 transi-
tional fibers themselves could be the residue of the cen-triole. 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). Final ly, in view of these departures
from what are usually regarded as universal character-
istics 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).
Our many colleague s who generously provided strains are mentioned
under Materials and Methods. We thank R. Ware for sharing his un-
published observations on the sens illa of chemosensory and thermo-
sensory mutants; J. Weis s for illustrations; and E. Aamodt, P. Albert,
D. Albertson, A. Burr, M. Chalfie, D. Dusenbery, L. Gremke, D. Hall,
R. Herman, J. Hodgkin, C. Kenyon, B. Menco, D. Riddle, R. Russ ell, S.
Siddiqui, J. Sulston, S. Ward, J. White, and K. Wright for ideas and
discu ssions . In sadnes s, we acknowledge the assistan ce and kindness
of Kay Buck who died unexpectedly during the course of this work.
Part of this research was supported by a Bas il O’Connor starter grant
from the March of Dimes Foundation and by NIH Grants NS16510
and NS20258 to J.C. E.H. was recipient of postdoctoral fellowships
from the Muscular Dystrophy Asso ciation of America and the NIH.
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