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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, 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-
sonal communication), others presumably disrupt be-
havior at steps downstream of transduction.
0012-1606/86 $3.00
Copyright C 1986 by Academ ic Press, Inc.
All rights of reproduction in any for m reserved.
456
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PERKINS ET AL. Sensory Cilia, in Nema,todes 457
Recently we found that certain sensory neurons in C.
eZegans accumulate fluorescein when liv ing animals are
placed in a solution of this dye (Hedgecock et al., 1985).
In this paper, we show that these neurons are chemo-
sensory and that dye uptake occurs through their ex-
posed cilia. We have used this dye-filling technique to
identify a subset of behavioral mutants with primary
defects in sensory cil ia or their support cells. These mu-
tations both prevent dye uptake and disrupt sensory be-
haviors.
MATERIALS AND METHODS
Brenner (1974) describes cultur ing and genetic ma-
nipulation of Caenorhabditis elegant. Strains were kindly
provided by Martin Chalf ie, David Dusenbery, Jonathan
Hodgkin, Donald Riddle, Richard Russell, and the Cae-
norhabditis Genetics Center at the University of Mis-
souri, Columbia.
Chemosensory neurons were stained with fluorescein
isothiocyanate and examined by fluorescence microscopyas in Hedgecock et al. (1985). New mutants with abnor-
mal staining were induced with ethylmethanesulphonate
(Brenner, 1974). The cat-6 (elSS1) mutat ion was separated
from the strain CB246.
Animals were fixed for electron microscopy using glu-
taraldehyde and then osmium as in Sulston et al. (1983).
Usually, two or three individuals of each mutant strain
were embedded and sectioned together. About 200 con-
tiguous sections, 50 nm thick, were collected from the
tips of the heads. One animal, selected for good fixation
quality and orientation, was photographed approxi-
mately every third section at 5000X magnification to re-
construct the head sensilla. The other animals were ex-
amined directly in the microscope.
RESULTS
Description of the Amphid and Phasmid Sensilla
The amphids, a pair of lateral sensilla in the head,
are the principal chemosensory organs of nematodes
(Fig. 1). In C. elegaxs, each amphid comprises the ciliated
dendrites of 12 sensory neurons plus two support cells
called sheath and socket cells (Ward et al., 1975; Ware
et al., 1975; White et al., 1986). The sheath and socket
cells form a cylindrical channel to the outside (Fig. 2).Of the 12 amphid neurons, 8 (ASE, ADF, ASG, ASH,
ASI, ASJ, ASK, and ADL) are evidently chemosensory
in that their cilia extend into the channel of the socket
cell and are thereby exposed to the external medium.
The cilia of three additional neurons (AWA, AWB, and
AWC), called wing cells, also share the main lumen of
the sheath cell . The wing cil ia separate from the others,
and invaginate individually into the sheath cell, proxi-
mal to where the fascicle of channel cil ia enters the
socket cell . Final ly, the dendri te of a neuron (AFD),
called the finger cell, remains separate from the other
dendrites in the sheath cell. It has only a rudimentary
cilium but, proximal to the cilium, the dendritic mem-
brane expands into about fifty vi lli, called fingers, that
invaginate the sheath cel l (Fig. 2). These fingers are
about 0.15 pm in diameter and 2 pm long. No internal
microfilaments or microtubules have been seen in them
but they tend to be oriented anteriorly or posteriorly in
the sheath cell.
The phasmids, a pair of lateral sensilla in the tail, are
similar but smaller chemosensory organs (Sulston et al.
1980; Hall and Russell 1986; White et al., 1986). In newly
hatched larvae, each phasmid comprises two ciliated
dendrites (PHA and PHB), a sheath cell , and a socket
cell. The neurons resemble the amphid channel neurons
in that their cilia extend into a socket channel that is
open to the external medium.
Below the cili a, the sensory dendrites are joined to
the sheath cell by belt junctions (Fig. 2). These havebeen described both as tight junctions (Ward et al, 1975)
and as desmosomes (Ware et al., 1975) and may have
properties of both. Similar belt junctions encircle the
channels, joining the sheath and the socket cells to-
gether. Finally, belt junctions join the socket cells to the
surrounding hypodermis.
The channels of the amphid sheath and socket cells
appear to originate by different mechanisms (Wright,
1980). The sensory dendrites deeply invag inate and, ex-
cepting the AFD neuron, completely penetrate the
sheath cel l so that it is topo logically a solid torus with
11 holes. In contrast, the socket cell wraps around the
receptor channel and forms a typical intercellular belt
junct ion where i t meets with itself. Thus topologically
it has no hole.
The channel of the socket cell is lined with cutic le that
is continuous with the external cuticle (Fig. 3a). The
sheath cell channel is not lined with cuticle . Instead, the
anterior sheath channel, in the region where the cilia
draw together into a tight fascicle, has a characteristic
dark lining (Fig. 3b). More posterior, nearer the bases
of the cilia, the dark lining is interrupted by matrix-
fil led vesicles fusing with the lumen. The cytoplasm ad-
jacent to the anterior sheath channel contains longitu-
dinally aligned microtubules and intermediate filaments.These filaments may form a scaffold for the receptor
channel (Wright, 1980). A much thinner scaffold, joined
at its ends to the self-junct ion, wraps around the socket
channel (Fig. 3a).
In glutaraldehyde fixed animals, a dark matrix sur-
rounds the cil ia in the posterior sheath channel (Figure
4a). The matrix material appears to be synthesized at
lamellae posterior to the cil ia and transported forward
in membrane-bound vesicles which later fuse with the
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DEVELOPMENTAL BIOLOGY VOLUME 117, 1986
?ZEP
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).
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PERKINS ET AL. Sensory Cilia in Nematodes 459
e
i
FIG. 2. Schem atic longitudinal section through amphid sensillu m in wild-type. The amphid channel is formed from a soeket cell (so) and a
sheath cell (sh). The socket cell is joined by belt junctions to surrounding hypodermal cells (not shown). The socket channe l is lined with
cuticle that is continuous with the external cuticle. The anterior sheath channel has a dark, noncuticular lining surrounded by a filamentous
scaffold (FS). The sheath and socket cells are joined together by belt junctions encircling the channel. The space between the cilia in the
posterior sheath channel is filled with a dark matrix (M) that appears to be packaged into vesicles further posterior, transported forward, and
deposited around the cilia. The dendrites of three channel neurons and one wing neuron (AWA) are shown. The distal segment of the AWA
cilium leaves the fascic le of channel cilia to re-invaginate the sheath cell. The AFD dendrite remains separate from the fascic le of wing and
channel cilia. AlI of the dendrites form belt-shaped junctions with the sheath cell near their point of invagination. The inset shows enlarged
cross sections of a channel dendrite through the distal segme nt (a), the middle segment (b), the transition zone (c), the neuron/sheath junction
(d), and the main dendrite in the papillary nerve (e). Main scale bar is 1.0 pm.
Description of Other Head Sensilla (Ward et al., 1975; Ware et al., 1975). They resemble the
larger amphid sensilla in having two support cells, a
In addition to the amphids, four classes of cuticular sheath and a socket, that form channels around the cil-
sensilla (cephalic, inner labia l, outer labia l quadrant, iated portion of the dendrites. They differ from the am-
and outer labia l lateral) are found in the tip of the head phids in that the socket channels are not lined with cu-
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460 DEVELOPMENTA L BIOLOGY VOLUME 117, 1986
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
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PERKINS ET AL. Sensory Cilia in Nematodes
FIG. 4. Amphid channel cilia in wild-type. (a) Section through the middle segment of a channel cilium . Nine doublet microtubules are attached
to the membrane and seven smaller singlet microtubules occupy the center. Matrix (M) separates the several cilia in the sheath channel. (b)
Section 0.8 lrn posterior to (a) through the transition zone. The nine doublets are drawn together by the apical ring. The links are clearly Y
shaped at their attachment to the membrane. The seven singlets are attached to the inner face of the apical ring. (c) Section 1.6 pm posterior
to (a) through the transitional fibers (arrowheads) that join the ends of the doublets radially to the cell membrane. There is no basal body in
the center of the dendrite but only an amorphous root. (d) Section 2.5 pm posterior to (a). The dendrite is much larger in diameter than at the
cilium and is filled with coated p its and vesicles (CV). The amorphous root (AR) may contain neurofilaments. (e) Section 6.6 pm posterior to
(a) and proximal to the neuron/sheath junction. The amorphous root has gradually thinned to reveal a fascic le of ten neurofilaments (NF).
Scale bar is 0.5 pm.
(Fig. 4b). A variable number of singlet microtubules are
attached to the inner surface of the cylinder. The central
cylinder may correspond to the apical rings found in the
transition zones of cilia in some organisms. The inner
singlets in C. elegans differ from the central pair of mi-
crotubules found in motile cilia in that they originate
at the base of the transition zone rather than above it.
The inner singlets, like axonal microtubules in C. elegans,
have only 11 protofilaments whereas the A and B subfi-
bers of the peripheral doublets have 13 and 11 protofi-
laments, respectively (Chalfie and Thomson, 1982).
The middle segment differs from the transition zone
in lacking the central cylinder. The doublets, still linked
to the membrane, spread apart somewhat and the cilium
flares in diameter (Fig. 4a). The Y-shaped bases of the
membrane links are no longer apparent, perhaps relax-
ing against the membrane in the absence of inward ten-
sion on the doublets. The inner singlet microtubules
continue, unattached, in the center of the cilium. The
middle segment of the channel cilia corresponds to the
flagellar shaft in Chlamydomonas and continues for
about 4 pm.
The B subfibers of the doublet microtubules are grad-
ually lost near the end of the middle segment (Fig. 3b).The distal segment, about 2.5 pm long, contains only A
subfibers and inner singlet microtubules (Fig. 3a). The
membrane links are probably also lost. The distal seg-
ment, roughly the portion in the socket channel, may be
the transducing region of the cilium.
The amphid cilia, like sensory cilia in nematodes gen-
erally have no apparent basal bodies (Wright, 1980). The
cilia terminate proximally in connections from the pe-
ripheral doublets to the cell membrane (Fig. 4~; see also
Figs. 5g, 7~). These terminal connections may be equiv-
alent to the transitional fibers seen in other organisms
(Reese, 1965; Ringo, 1967). As they have complex sub-
structure, they conceivably also contain some residue of
the nematode centriole.
In some of the published descriptions of nematode
cilia, the proximal segment, identified in this paper as
the transition zone, has been incorrectly called a basal
body. To reduce confusion, we reserve the word basal
body for the modified centrioles found proximal to the
transition zone in more conventional cilia.
Unlike the channel cilia which are all cylindrical, each
of the amphid wing cilia (AWA, AWB, and AWC) has
a unique shape (Ward et al., 1975; Ware et al., 1975). The
AWC cilium spreads vertically into two enormous sheets,
resembling wings. These wings and the surrounding
sheath cell, fill much of the left and right hemisectors
at the tip of the animal (Fig. 1). The AWA and AWB
cilia are smaller than AWC and comparable in size to
the channel cilia. The distal segments of the AWA cilia
split into several small projections each containing one
or more of the original nine doublet microtubules (Fig.
2). The AWB dendrite, like ADF and ADL, ends in a
pair of cilia. The distal segments of the AWB cilia donot split like the AWA cilia but are somewhat flattened
and irregular.
None of the amphid dendrites contain striated ciliary
rootlets. Instead, an amorphous gray material extends
posteriorly from the centers of the channel and wing
cilia for about a micrometer (Fig. 4d). This material
gradually thins, revealing a fascicle of 3 to 12 neurofil-aments that continue at least several micrometers fur-
ther (Fig. 4e). It is likely that these neurofilaments are
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462 DEVELOPMENTAL BIOLOGY VOLUME 117, 1986
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).
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PERKINS ET AL. Sensory Cilia in Nematodes 463
Ultrastructure and Specialization of Mechanocilia
The transition zones of the various mechanocilia re-
semble those of the amphid cilia. In particular, central
structures, probably short cylinders, join the inner faces
of the doublets. In many of the mechanocilia, some pe-
ripheral doublets terminate just distal to the transition
zone. In the CEP and OLL cilia, for example, usuallyonly five membrane-linked doublets continue in the
middle segments (Figs. 5e,6). The distal segments of the
CEP and OLL cilia contain an amorphous dark material
and associated microtubules common to proved mech-
anocilia in insects (Ward et al., 1975; Ware et al., 1975;
Thurm et aZ., 1983). In the CEP cilia, the microtubules
are interspersed with the dark material and mold it into
irregular rods (Fig. 5a). In the OLL cilia, the dark ma-
4uticle-- --/
sh
FIG. 6. Schem atic of longitudinal section through the CEP sensillu m
in wild-type showing the receptor channel formed by the sheath (sh),
socket (so), and hypodermis. The distal segment, containing super-
numerary microtubules and dark tubule-associated material (TAM),
is embedded in the subcu ticle. A sma ll nubbin (CN) extends into the
cuticle near the base of the distal segment. Coated vesicles (circles)
are present in the CEP dendrite proximal to the cilium and distal to
the neuron/sheath junction. Scale bar is 1.0 pm.
terial is not interspersed with microtubules but forms
a large aggregate surrounded by a single layer of mi-
crotubules. In both the CEP and OLL cilia, the outermost
microtubules appear to have fine attachments to the
membrane. The microtubules in the distal segments are
all singlets and, at least a majority, are supernumerary
in that they do not derive from the nine-doublet micro-tubules of the axoneme nor are they central singlet mi-
crotubules arising at the apical ring as in the amphid
cilia. The supernumerary microtubules and the dark tu-
bule-associated material are confined to the region
embedded in the cuticle (Fig. 6).
The OLQ cilia are unique in two respects. First, the
A and B subfibers have filled cores giving the doublets
an exceptionally dark appearance. Second, exactly four
of the nine doublets extend through the cilium (Figs.
5a-e). These four doublets are not membrane linked but
are joined along their lengths by thick cross-bridges to
form a square. Fine radial arms join these doublets to
a small hub in the center of the square. The corners ofthe square always point radially or circumferentially in
the wild type. In the distal segment, embedded in the
subcuticle, one or two small aggregates of amorphous
dark material, resembling the tubule-associated mate-
rial of the CEP and OLL cilia, flank the doublet micro-
tubules at the circumferential corners, but not the radial
corners (Figs. 5a,b). This material may also be connected
to the membrane.
The tips of the IL1 cilia contain a disc of dark material
attached on both faces to the ciliary membrane (Fig.
7a). This dark material is positioned in the cuticle in
such a way as to be compressed by outward radial de-
flections of the papillary protrusions caused by head-on
contact with external objects.
The distal segments of the CEP, OLL, and OLQ cilia
are anchored in cuticle by a small dark nubbin (Ward
et ah, 1975; Ware et ah, 1975). In the CEP and OLQ neu-
rons the nubbin occurs at the base of the transducing
region (Figs. 5b, 6). The OLL cilia differ in that the nub-
bin is at the distal tip of the cilium and the supernu-
merary microtubules and tubule-associated material are
proximal to the nubbin.
Finally, three classes of sensory cilia (BAG, ILl, and
OLQ) in the hermaphrodite have large striated rootlets
(Ward et al., 1975; Ware et al., 1975). The rootlets extendinto the center of the transition zone (Figs. 7b,c).
Fewer than nine peripheral doublets have been re-
ported for some classes of cilia in C. elegant (Ward et
ah, 1975; Ware et ab, 1975). Using glutaraldehyde-fixed
adults, we consistently found nine doublets in the tran-
sition zone of the BAG, CEP, ILl, and OLQ cilia. Since
not all nine doublets extend into the shaft in some of
these classes, they could be overlooked in a coarse series.
All the IL2 cilia examined in wild-type adults have fewer
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464 DEVELOPMENTAL BIOLO GY VOLUME 117, 1986
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
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PERKINS ET AL. Sensory Cilia in Nematodes 465
FIG. 8. FITC uptake by amphid neurons in living animals, (a) Ventral
view of the wild type animal. Six cells on each side, not all resolved
in this focal plane, are filled with dye (Hedgecock et al, 1985). Processe s
from the sensory cilia (arrowheads) and processes to the nerve ring
neuropil (arrows) are also visible. (b) Ventral view of the-10 (e1809j
mutant. One cell on each side is brightly stained in this individual. A
second cell is faintly stained on the right side. (c) Ventral view of che-
10 (e1809) mutant. No cells are stained in this individual. The bright
central stripe i s fluorescence from dye bound to the sclerotized cuticle
lining the pharynx. Scale bar is 20 Frn.
(tax and the genes: Dusenbery et al., 1975; Lewis and
Hodgkin, 1977), thermotaxis (ttx genes: Hedgecock andRussell, 1975), male mating (Lewis and Hodgkin, 1977,
Hodgkin, 1983), avoidance of solutions of high osmotic
strength (osm genes: Culotti and Russell, 1978), dauer
larva formation (dufgenes: Riddle et al., 1981), coarsemechanical stimulation (met genes: Chalfie and Sulston,
1981), egg-laying (egl genes: Trent et al., 1983), and form-
aldehyde-induced fluorescence (FIF) to visualize cate-
cholamine (dopamine) containing mechanosensory neu-
rons (CEP, ADE, and PDE) (cut genes: Sulston et al.,
1975).
We examined alleles of all the published cat, the, daf,
met, osm, tax, and ttx genes for defects in FITC uptake
into chemosensory neurons, All of the cat, ttx, and met
mutants, with the exceptions of met-1 and met-8, were
essentially normal in dye filling. In contrast, all of the
osm mutants and some of the the, duf, and tax mutants
are defective in dye uptake, affecting both the amphidand phasmid neurons (Fig. 9, Table 1).
We tested whether any of these mutations, isolated
in different laboratories, fail to complement. Indeed, the
mutations the-3 (e1124), the-8 (e1253), and osm-2 (~801)
on linkage group I all fail to complement for FITC up-
take. Similarly, mutations daf-10 (e1387)and osm-4 (~821)
on linkage group IV represent a single gene. Finally, the
unmapped tax mutation, a83 (formerly RS3, Dusenbery
et al., 1975) is an allele of osm-1.
We also isolated nine new mutants with reduced dye
uptake. These fall in two of the known osm genes and five
new genes designated the-10 through the-14. Excluding
the met-1 and met-8 alleles, there are now 25 mutations,defining 14 complementation groups, which reduce or
eliminate FITC uptake by amphid and phasmid neurons
(Table 1, Fig. 9). A spectrum of behaviors was tested for
each mutant (Table 1).
Dye Filling of Mutant Mechanosenswy Neurons
Mechanosensory neurons do not normally fill with
FITC. In some chemosensory mutants, however, certain
mechanosensory neurons, including CEP, ADE, and PDE
neurons, occasionally stain brightly (Table 1). In many
of the mutants showing occasional staining of mechano-sensory neurons in hermaphrodites, occasional ray neu-
rons also stain in males (Table 1). We examined ray
staining in detail in osm-1 (~808) males. It appears that
neurons from each of the 18 ray sensilla are capable of
staining. Apparently only one neuron per sensillum can
fill with dye. We speculate that the stained cells are RnA
neurons, rather than RnB neurons, as the RnB dendrites
are externally exposed, yet nonstaining, in wild-type
males (Sulston et ah, 1980).
Mutants of two genes, cat-6 and the-14, show a much
higher frequency of dye filling by mechanosensory neu-
rons. In cat-6 mutants, the amphid and phasmid neurons
stain normally, but the CEP, ADE, and PDE neuronsalso stain brightly in many animals. The proportion of
these mechanosensory neurons staining is greatest just
after molts (Fig. 10). In the-14 mutants, the phasmid
neurons never stain and the amphid neurons frequentlyfail to stain (Table 1). The CEP, ADE, and PDE neurons
stain brightly in many animals as do additional, un-
identified sensory neurons in the head. As shown below,
the CEP dendrites, and presumably the other classes
that stain, have abnormal access o the external medium
in cat-6 and the-14 mutants.
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466 DEVELOPMENTAL BIOLO GY VOLUME 117, 1986
Linkage Group I. the-3 (e1124), the-13 (e1805), the-14 (e1960), dpy-5
(eSl), and uric-13 (e51).
2F distance s: dpy-5 (6/132) the-3
dpy-5 (12/184) the-13
3F distance s: dpy-5 (13/13) (WE-B &e-d)
dpy-5 (12/12) (uric-13 the-13)
dpy-5 (3/12) the-14 (9/12) uric-13
Linkage Group II. the-10 (e1809), dcbf-19 (m86), dpy-10 (el28), and
uric-4 (el20).
2F distance s: uric-$ (4/120) the-10
dpy-10 (O/50) daf-19
3F distances: (the-IO dpy-IO) (5/5) uric-4
dpy-10 (7/7) (uric-4 daf-19)
Linkage Group IV. dpy-13 (el84), daf-10 (p821), him-8 (e1489). and
osm-3 (~802)
3F distances : (osm-3 dpy-13) (1202) him-8
dpy-13(8/11) daf-10 (3111) him-8
Linkage Group V. cat-6 (e1861), the-11 (e1810), the-12 (e1812), dpy-
11 (e224), osm -6 (p811), sma-1 (e30), uric-42 (e270), a nd uric-76 (e911).
2F distances : dpy-11(3/60) cat-6
dpy-11 (g/120) the-11
dpy-11 (6/120) the-12
dpyll (7/120) osm-6
3F distance s: dpg-11 (11/11) (tine-42 cat-6)
dpy-11(4/4) (uric-42 the-11)
dpy-11 (9/9) (uric-42 the-12)dpy-11 (13/13) (uric-42 osm-6)
(the-11 sma-I) (3/3) uric-76
(the-12 sma-1) (3/3) uric-76
(osm-6 sma-1) (717) uric-76
Linkage Group X, the-2 (elO33), daf-6 (e1377), lmz-2 (e678), osm -I
(p808), os m-5 (p813), and uric-6 (e78).
2F distance s: lmz-2 (‘7/82) osm-5
uric-6 (55/174) the-2
3F distance s: (osm-5 ion-2) (20/20) uric-6
(the-2 km -Z) (919) uric-6
Mutants with Short Axonemes in al l Classes of Cilia
Mutations in three genes, the-13 (e1805), osm-1 (p808),
and osm-5 (p813), shorten the axonemes of all classes of
sensory cilia in the head. Singlet or doublet microtubules,
joined to the membrane by Y links, assemble below the
transition zones. The various distal specializations of
the mechanoc ilia also assemble ectopically in these mu-
tan ts.
The peripheral doublets of the amphid channel cilia
end within about 2 pm of the transition zone (Fig. 11).
The inner singlets do not extend beyond the apical ring.
The wing cil ia are similar ly affected. Interestingly, the
AWC cili um fails to spread into sheets and the sur-
rounding sheath cell is correspondingly reduced. The
AFD cili a, although fairly short in wild-type, are reduced
further and often tilted. The AFD fingers themselves
are unaffected in number or appearance.
Doublet microtubules, joined to the membrane by Y
links, assemble below the cil ia in these mutants. Thesedoublets are not continuous with the nine peripheral
doublets of the cilium (Fig. 12a). The ectopic doublets
do not generally cross the neuron/sheath junctions but
instead create a posterior projection within the sheath
cell (Fig. 12b). Like normal cilia, these projections are
topologica lly distal to the junctions. They strikingly
mimic the middle segment of a normal cilium (Fig. 12~).
They end blindly within the sheath cell and are usually
fil led with vesicles where they terminate (Fig. 12d). The
occasional doublets that cross the neuron/sheath junc-
tion, lose their membrane links below the junct ion.
As judged by the hooks on microtubules with part ial
B subfibers, the ectopic doublets have the opposite
clocksense to the nine ciliary doublets in adjacent sec-
tions. As the ectopic tubules project posteriorly and the
cil ia project anterior ly, both classes of doublets have
the same relative clocksense. In particular, the B subfi-
bers are counterclockwise of their respective A fibers
for a viewer looking from proximal to distal.
The amphid sheath channel in these mutants contains
more matrix than wild-type and much of the space nor-
mally occupied by cilia is filled with matrix instead. Ab-
FIG. 9. Genetic map. Map positions of genes affecting FITC uptake
are shown below the lines. Marker genes are shown above the lines.
The positions are based on the data of Lewis and Hodgkin (1977),
Culotti and Russ ell (1978), Riddle et al. (1981), Rand and Russ ell (1984).
R. Herman (1984), and new data, listed below, obtained using the dye-
uptake phenotypes of the mutants. Two-factor distance s, obtained by
scoring the DPY, UNC, or LON progeny of cis- linked heterozygotes,
are expressed as the number of recombinant chromosom es to total
chromosome s examined. No corrections are made for multiple events.
Three-factor gene orders and distance s are shown in the format of
the map database maintained by the Caenorhabditis Genetics Center
(see Swanson et aZ., 1984).
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PERKINS ET AL. Sensory Ci a in Nematodes
TABLE 1
BEHAVIORALMUTAN TSAFFECTING FITC UPTA KE
467
FITC uptake”.*
CE P Sensory behaviors’
PHA ADE
Gene Allele ADF ASH AS1 ASJ ASK ADL PHB PDE RAY OSM CTX DAF TTX
Wild-type
cat-6 (V) elX61
the-Z (X) e1033
the-9 (I) ellL4
e1253
elY79
p801
ChP10 (II) e 1809
the-11 (V) e1810
elXl5
chv-12 (V) e181S
chr-13 (I) elXO5
the-14 (I) 41.960”dqf-6 (X) elY77
duf-10 (IV) elY87
m 79
p&21
daf-19 (II) m86’
met-1 (V) elO66f
met-8 (I) 62398
II 74
osnr-1 (X) a83
e1803
pxox
p816
osm-3 (IV) e1806
elKl1p803
own-*5 (X) p813
osm-6 (V) @I1
3
3
0
0
0
0
0
2
2w
2w
3
0
2
0
278
2w
2w
0
3
2
2
0
0
0
0
0
00
0
0
3
3
0
0
0
0
0
0
0
0
3w
0
2
0
0
2w
0
0
2
2
2
0
0
0
0
0
00
0
0
3
3
0
0
0
0
0
0
0
0
3w
0
2
0
0
0
0
0
2
0
0
0
0
0
0
0
00
0
0
3
3
0
0
0
0
0
0
0
0
3w
0
2
0
0
0
0
0
2
0
0
0
0
0
0
0
00
0
0
3
3
0
0
0
0
0
0
0
0
3w
0
2
0
0
0
0
0
2
0
0
0
0
0
0
0
00
0
0
3
3
0
0
0
0
0
2
2w
2w
3w
0
2
0
2w
2w
2w
0
2
0
0
0
0
0
0
0
00
0
0
3
3
0
0
0
0
0
0
0
2w
3w
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
00
0
0
0
2
1
1
1
1
0
0
1
1
0
1
2
0
1
1
1
0
0
1
0
1
1
1
1
0
00
1
1
0
0
1
1
0
1
1
0
1
1
0
1
00
1
1
1
0
0
0
0
1
1
1
1
0
00
1
1
+
+
-
k-
+
+
+
-
--
-
+
+
f-
k
f
+
+
2
+
+-
-
+
+
+
-
-
-
4
*-
-
-
+
*
-
zk
-.
-
f
-
+
-
-
zk
+
*
+
+
-
-
-
t-.
-
+
+
+
++++
+
++
+
+
++
+++
+
t
+t
tt+t
+
t+
t
t
MEC MAT
+
t
t
tt
t+
t
t
t
t
t
t+
t
t+
t
-
-
tt
+
+
t
+t
+
+
4
4
0
0
0
3
2
0
0
2
3
0
3
4
0
0
1
0
2
2
3
0
0
2
1
3
43
1
1
” The following mutants were found to have normal FITC uptake: he-5 (e1073), the-6 (e1126), the-7 (ell%), daf-1 (e1287), daf-2 (e1370), dafi
3 (e1376), daf-4 (e1364), d af-5 (e1386), da &7 (e1372), daf-8 (e139 3), daf-9 (e1406), d af-11 (m47), daf-12 (mZO), daf-13 (m66), daf-14 (m77), daf-15
(&I), daf-16 (m26), daf17 (m27), da f-18 (e1375), an d daf-20 (m25). Heat-sensitive alleles were tested at nonpermissive temperature (25’). In
the-1 (e1034) mutants, an additional clas s of amphid neurons often stains.
*The frequency and intensity of staining of neurons is indicated qualitatively: 3, usually or always stains; 2, frequently stains; 1, occasion ally
stains; 0, rarely or never stain s. A suffix w indicates that the staining intensity is much weaker than in wild-type.
‘Avoidance of concentrated NaCl (osmotic, OSM) was tested with a population assay (Culotti and Russe ll, 1978). Attraction (chemotaxis,
CTX) was tested individually using dilute gradients of NaCl (Ward, 1973). Dauer larva formation (DAF) was tested on crowded, starved plates
using sodium dodecyl sulfate to kill nondauer larva (Cassada and Russe ll, 1975). The cuticle s of survivors were examined using Nomarski
optics to confirm the presence of dauer-specific alae. Ability to follow isotherms (thermotaxis, TTX ) was tested individually in radial temperature
gradients (Hedgecock and Russe ll, 1975). Touch sensitivity (mechanosensory, MEC) was tested with an eyebrow hair (Chalfie and Sulston,
1981). Males were obtained from him-5 (elQ90) double mutants and their mating ability (MAT) was tested by the procedure of Hodgkin (1983).
All behaviors, except mating, were scored either (-) no response, (+) intermediate response, or (t) essentially wild-type response. Male mating
ability was scored according to Hodgkin (1983): 4, very efficient mating (30-100% of wild-type efficiency); 3, efficient mating (lo-30% of wild-
type); 2, poor mating (l-10% of wild-type); 1, very poor mating (less than lYO of wild-type); and 0, no detected matings.
’ For each amphid sensillu m in &e-14 (e1960), either all six neurons stain or none stain. In addition to the CEP neurons, unidentified sensory
neurons with cell bodies anterior to the nerve ring frequently stain in the-I$ (e1960). In the OSM assay, about 10% of the the-14 (e1960) animals
failed to avoid concentrated NaCl.
e The daf-19 (m86hs) mutants form dauer larvae constitutively, particularly at high temperature (D. Riddle, personal communication). There
is no FITC staining at either permissive (15”, adults and dauers) or nonpermissive temperature. (25”, dauers only).
‘The phasm id neurons were examined in forty met-1 (e1066) mutants. Both neurons stained brightly in 58 sensilla , only one neuron stained
in 15 sens illa, and no neurons stained in 7 sens illa. In comparison, both phasmid neurons stained in 78 sens illa and no neurons stained in 2
sens illa in 40 wild-types.
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468 DEVELOPMENTAL BIOLOGY VOLUME 117, 1986
CEP
a t” : +L ElLl L2 t L3 t L4 t ADULT
(31 12)416)6) 141 1716)5)8131 (51 ~4113)(91 (81
t I I 1 I I I I I I I I I I I )Wh
0 16 25 34 45 50 70 a70
Age In hours
FIG. 10. FITC Uptake by CEP and PDE neurons in cat-6 (~~1861) mu-
tants. Anim als were stained with FITC for 2 hr and then examined
by fluorescence microscopy for uptake into CEP and PDE neurons and
by Nomarski microscopy to determine their approximate age. The av-
erage number of stained neurons per animal is shown as a function
of age. Arrows mark the four larval molts. The star indicates the time
of birth of the PDE neurons (Sulston and Horvitz, 19’77). Numbers in
parentheses indicate how many an imals in each age group were ex-
amined. Each anim al has a total of four CEP neurons and two PDE
neurons (White et al., 1986).
normal large matrix-filled vesicles accumulate in the
anterior cytoplasm of the sheath cell . Often these ves-
icles are part ially fused with the channel.
The CEP, ILl, IL2, OLL, and OLQ axonemes are
greatly reduced in length in the-13 (e1805), osm-1 (p808),and osm-5 (~813) mutants (Fig. 13). The dendrites them-
selves, however, continue and may reach the cutic le. In
particular, the CEP, OLL and OLQ dendrites form cu-
ticle-attached nubbins. Empty tunnels in the subcuticle
are found anterior to CEP and, less often, the OLQ den-
drites suggesting that these dendrites once extended
somewhat further but have retracted, usually to the
nubbin.
The transition zones of the CEP, ILl, IL2, OLL, and
OLQ cilia, although normal in structure, are frequently
mispositioned along the dendrite either anteriorly, to
the leve l of the socket channel or beyond (Fig. 14a), or
posteriorly, to the level of the neuron/sheath junction
(Fig. 16a) or even into the ectopic posterior projections.
As in the amphid cilia, membrane-linked microtubules
assemble ectopically behind the cilia. These microtubules
are generally fewer and shorter than in the amphid cil ia
and are more often singlets than doublets. Again, these
ectopic membrane-linked microtubules do not cross the
neuron/sheath junct ion but instead create a posterior
projection within the sheath cell .
The supernumerary microtubules and associated dark
material normally found in the distal segments of the
CEP and OLL cilia were present but positioned irregu-
larly along the dendrites, both distal and proximal to
the residual cilia. Large, ball-shaped aggregates of the
tubule-associated material were often found in the ec-
topic posterior projections of the CEP cil ia (Fig. 14b).
The joined square of doublets is formed in the OLQcilia but generally fails to extend past the sheath chan-
nel. In many cilia, the corners of the square do not point
radially and circumferent ially. In a few cases, five rather
than four doublets were joined by cross-bridges to make
an irregular pentagon with two central hubs (Fig. 15).
\ ,----cuticle
FIG. 11. Schematic of amphid sensil lum in mm-1 (~808). The amphid
cilia are extremely short. Doublet microtubules attached to the mem-
brane by Y links, assemble ectopically below the transition zone. These
membrane-linked doublets, like the normal cilia, are topologically distal
to the neuron sheath junction. They create cilia-like posterior projec-
tions that terminate in vesicle-filled swelling s. Abnormal large matrix-
fi l led vesicles accumulate in the sheath cell. Insets show cross sections
through the level of the neuron/sheath junction (a) and through the
ectopic posterior projection (b). Scale bar is 1.0 pm.
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PERKINS ET AL. Sensory Cilia in Nematodes 469
FIG. 12. Amphid cilia in osm-5 (p813) mutant. (a) Section through an ADF dendrite. The transitional fibers (TF) of one cilium are visible in
the upper left. The transition zone of the second c ilium is 0.5 pm distal to this sectio n in the upper right. In the lower part, ectopic doublet
microtubules are attached to the membrane by Y links (arrows). Matrix material (M) surrounds the dendrites. (b) Section 0.8 pm posterior to
(a) showing the main dendrite (star) leaving the sheath c ell. The ectopic doub lets (arrows) segregate into a posterior projection that, like a
normal cilium , is topologieally distal to the neuron/sheath junction (JN). (c) Section 1.2 pm posterior to (a). The ectopic pro jection (P) is
completely separated from the main dendrite (star). Except for the absence of inner singlet microtubules, the projection strikingly resembles
the middle segment of a normal cilium. (d) Section 3.0 pm posterior to (a). The ectopic doublets have terminated and the ectopic projection
(P) terminates in a vesicle-filled swelling within the sheath cell. The main dendrite (star) continues toward the neuron cell body. Scale bar is
0.5 pm .
The dark material that normally flanks the circumfer-
ential corners was fragmented and mispositioned.
The dark membrane-attached discs normally found
at the tips of the IL1 cilia were present but displaced
posteriorly in these mutants, often to the level of the
transition zone (Fig. 16a).
The striated ciliary rootlets of the ILl, OLQ, and BAG
neurons are normal in these mutants and attach prop-
erly to the transition zone. Interesting, the ectopic
membrane-attached microtubules found in these mu-
tants also recruit small rootlets (Figs. 16b, c).
In an unexpected contrast to wild-type, well-formed
transition zones comprising a tight ring of nine Y-linked
doublet microtubules were found in all classes of cilia,
including IL& in the-13, osm-1, and osm-5 mutants.
The osm-6 (~811) mutant has a similar, though perhaps
less severe, ultrastructural phenotype than the the-13,osm-1, and osm-5 mutants. The microtubules of the var-
ious classes of cilia extend further than in the other
mutants but ectopic membrane-attached microtubules
still assemble proximal to the cilia. The large wings of
the AWC cilia are reduced but not eliminated. The tran-
sition zones of the mechanocilia in osm-6 fp811), in con-
trast to the other three mutants, are positioned normally
along the dendrites. The dark discs in the IL1 dendrites
are also positioned normally at the tips but another
mechanosensory specialization, the supernumerary mi-
crotubules and dark tubule-associated material of the
CEP dendrites, assembles ectopically. Possibly signifi-
cant, the amphid sheath cytoplasm contains an excess
of small, unfused matrix-filled vesicles rather than the
large vesicles found in the other mutants. The osm-6
(~811) vesicles resemble the unfused matrix-filled vesi-
cles found in wild type except for their greater numbers.
daf-19 Mutants Lack All Classesof Cilia
The sensory dendrites in daf-19 (rn86] mutants entirely
lack cilia including the transition zones. Vestigial cen-
trioles, without membrane attachments, are found in a
few of the amphid dendrites (Fig. 1’7). No ectopic mem-
brane-linked microtubules are found in the amphid den-
drites. A few membrane-associated singlet microtubulesare found in the CEP, ILl, and OLQ cilia. The amphid
dendrites, and most of the mechanosensory dendrites,
terminate in club-shaped endings after invaginating, and
forming belt-shaped junctions with their respective
sheath cells. The CEP dendrites, though not the OLL
and OLQ dendrites, extend through their socket channels
to end in cuticle-attached nubbins. Supernumerary mi-
crotubules and associated dark material are present,
though mispositioned, in CEP and OLL dendrites. Sim-
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470 DEVELOPMENTAL BIOLOGY VOLUME 117, 1986
TAM-
FIG. 13. Schem atic longitudinal section through the CEP cilium in
osm-5 (~81.3) mutant. The cilium is truncated distal to the transition
zone. Normal rod-shaped and large, ball-shaped aggregates of tubule-
associate d material (TAM) and supernumerary microtubules assem ble
both distal and proximal to the cilium. The dendrite forms a normal
cuticle-attached nubbin (CN). An empty tunne l (star) in the subcu ticle
sugges ts that the distal dendrite has retracted. Scale bar is 1 pm.
ilarly, the disc-shaped accessories normally found at the
tips of the IL1 cilia are present in the mutant dendrites
immediately distal to the neuron/sheath junctions.
Striated rootlets are present in IL1 and OLQ dendrites,
some in their normal position and others in ectopic pos-terior projections of the dendrite distal to the neuron/
sheath junctions. The fingers of the AFD neuron are
normal. Abnormal large matrix-filled vesicles accumu-
late in the amphid sheath cell.
the-11 Cilia Contain Abnormal Ground Material
In contrast to the mutants mentioned above, the am-
phid wing and channel cilia in the-11 (el810) are nearly
normal in length and arrangement of microtubules.
However, these cilia contain abnormal dark ground ma-
terial interspersed among the microtubules of the axo-
neme (Fig. 18). Some of the cilia are slightly enlarged
in diameter and irregular in contour. The dendrites be-
low the cilia also contain dark ground material and few,
if any, membrane-attached microtubules. The AWC ciliafail to spread into wing-shaped sheets. Abnormal large
matrix-filled vesicles accumulate in the amphid sheath
cell. In one sensillum examined, many of the interme-
diate filaments in the sheath scaffold are oriented cir-
cumferentially rather than longitudinally.
The CEP cilia in the-11 (el810) mutants are reduced
in length and largely resemble the cilia in the-13, osm-
1, osmd, and osm-6. Dark material and associated mi-
crotubules assemble in both rod- and ball-shaped ag-
gregates along the dendrites and in ectopic posterior
projections. The transition zones are often displaced.
Empty tunnels are present in the subcuticle distal to
the cuticle-attached nubbin. In contrast to the other four
mutants, the posterior projections are filled with dark
ground material and numerous vesicles.
The ILl, IL2, OLL, and OLQ axonemes are nearly nor-
mal in length and the transition zones are positioned
correctly in the-11 (el810). The joined squares in the OLQ
cilia are oriented normally but the flanking dark ma-
terial is fragmented and mispositioned. The distal seg-
ments of some OLQ cilia have unattached singlet mi-
crotubules in addition to the joined square. The dark
discs of the IL1 cilia and the amorphous dark material
in the OLL cilia were positioned normally. A fe w mem-
brane-attached singlet microtubules were found belowthe IL1 cilia.
the-10 Mutants Lack Amphid Cilia and
Striated Rootlets
Most of the amphid wing and channel dendrites in
the-10 (e1809) mutants have no recognizable transition
zones or axonemes. These dendrites generally have en-
larged bulb-shaped endings filled with dark ground ma-
terial (Fig. 19b). However, usually one or two dendrites
per sensillum have well-formed cilia with normal tran-
sition zones and nearly full-length axonemes (Fig. 19a).
The wing-shaped sheets of the AWC cilia are present.The AFD cilia are absent or tilted but the fingers are
normal. Abnormal large matrix-filled vesicles accumu-
late in the sheath cell.
The striated rootlets normally found at the base of
the cilia in the IL1 (Fig. 20), OLQ, and BAG neurons are
entirely missing in the mutant the-10 (el809). The distal
specializations of these cilia, and the other mech-
anosensory cilia of the head, are normal.
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PERKINS ET AL. Sensory Cilia in Nematodes 471
FIG. 14. CEP cilia in osw-1 (~808). (a) Section through the transition zone of a CEP cilium. The axoneme is abnormally short and the transition
zone is displace d forward to the level of the sheath/socket junction (JN). Excess d endritic membrane is drawn aside from the cilium (white
arrow). (b) Section 2.7 Frn posterior to (a). The main CEP dendrite (star) has passed out of the sheath cell. An ectopic posterior branch remains
within the sheath cell. It contains a sma ll rod and an abnormal large aggregate of dark tubule-associated material (TAM). Some of the
microtubules surrounding the dark material appear to be attached to the membrane (black arrow). (c) Section 5.1 pm posterior to (a) showing
the main dendrite (star) of the CEP neuron and an ectopic branch containing membrane-attached microtubules (black arrow) and a rod of
dark tubule-associated material (TAM). Lamellae (LAM) in the sheath c ell surround the ectopic branch. Scale bar is 0.5 pm.
osm-3 Speci&ally Required for Amphid
and Phasmid Cilia
The distal segments of the amphid channel neurons
are absent in osm-3 (~802) mutants (Fig. 21). Both the
transition zones and middle segments are normal in
length and contain a full complement of membrane-
linked doublet and central singlet microtubules. The cilia
end abruptly, however, in the region where the B subfi-
bers normally terminate. Thus the distal segments, con-
taining only A subfibers and central singlets, are entirely
truncated and the socket channel is empty of cilia.Because the channel cilia in osm-3 (~802) have normal
FIG. 15. OLQ cilia in wild-type, osm-5, and the-13 mutants. (a) Section
through w ild-type OLQ cilium showing nine doublet microtubules plus
the cross-bridges that join four of them into a central square. Inside
the square, fine radial arms join the doublet microtubules to a hub.
(b) Section through osm-5 (pX13) OLQ cilium. Cross-bridges join five
of the doublet microtubules into an irregular pentagon. (c) Section
through the-13 (el805) OLQ cilium. Cross-bridges join five of the doublet
microtubules into an irregular pentagon. Fine radial arms connect the
doublet-microtubules to two separate hubs. Scale bar is 0.5 pm.
middle segments, they are substantially longer than the
the-13, osm-1, osmd, and osm-6 cilia. Moreover, the cilia
are not displaced forward in the sheath cell as in the
mutants without middle segments. Finally, no ectopi-
tally assembled membrane-linked microtubules are
found in osm-3 (~802) dendrites.
The amphid wing cilia are essentially normal in osm-
3 (~802). Similarly, the AFD dendrites, and the various
mechanosensilla, are also normal. The only defect in osm-
3 (~802) besides the distal truncation of the amphid
channel cilia, is an accumulation of abnormal, large ma-
trix-filled vesicles in the anterior cytoplasm of the sheath
cell (Fig. 22).
the-12 Afects the Amphid Sheath Matrix
The matrix vesicles of the amphid sheath cell appear
pale or empty in the-12 (el812). The lumen of the sheath
channel and the extracellular space surrounding the
AFD fingers are devoid of matrix. The amphid wing and
channel cilia, particularly near the membrane, are ab-
normally dark (Fig. 23). The channel cilia are shorter
than normal and only extend partway through the socket
channel. Unlike other mutants with shortened cilia, no
large matrix vesicles accumulate in the sheath cyto-
plasm.
Irregular vesicles are present between the two layers
of the adult cuticle in the-12 (el812) (Fig. 23).
the-14 Affects the Joining of the Amphid Channels
The amphid channel is abnormally large in diameter
and poorly aligned at the join between the sheath and
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472 DEVELOPMENTAL BIOLOGY VOLUME 117,1986
FIG. 16. IL1 cilium in osm-1 (~808) mutant. (a) Section through transitional fibers of an IL1 cilium . Th e cilium is displace d posteriorly from
its wild-type position and is nearly at the level of the ILl/sheath junction (JN). The dark membrane-attached disc (D), normally present at
the distal tip of the IL1 cilium, is also misposition ed. (b) Section 0.15 Km posterior to (a). Ectop ic membrane-attached singlet and doublet
microtubules (arrows) extend posteriorly. A large striated rootlet (SR) is associate d with the cilium while a smaller rootlet is recruited by the
ectopic membrane-attached microtubules. (c-e) Sections 0.45, 1.1, and 1.2 pm posterior to (a). The ectopic mierotubules (arrows) and their
associate d rootlet segregate from the main dendrite and form a posterior projection within the sheath cell. The main dendrite, and the large
striated rootlet (SR), leave the sheath cell. Scale bar is 0.5 pm.
socket cells in the-1.4 (el960) mutants. The socket scaffold
is disorganized and some of the intermediate filaments
are oriented circumferentially rather than longitudi-
nally. The socket cytoplasm contains abnormal vesicles
FIG. 17. Unmodified centrioles in amphid dendrite of uhf-19 (m86)
mutant. (a) Section near the termination of a sensory dendrite in the
amphid sheath c ell. A centriole with no membrane assoc iations is
shown by an arrow. (b) Section 0.15 pm posterior to (a) showing a
second centriole (arrow), oblique to the first centriole, and the neuron/
sheath junction (JN). Sca le bar is 0.5 pm.
and the cuticle lining of the channel is abnormally thin.
The sheath scaffold is apparently stretched thin near
the join and the dark lining of the channel is absent.
More posteriorly in the sheath cell, the scaffold and dark
lining appear normal. The belt junction between the
sheath and socket cells is normal.
In some cases, the socket channel fails to connect with
the sheath channel and ends as a blind, cuticle-lined
pocket (Fig. 24). When the cilia, which form a normalfascicle in the sheath, reach an obstructed socket chan-
nel, they are either deflected sideways in the sheath cell
or invaginate the socket cell without obtaining access
to the externally open channel (Fig. 25). Matrix accu-
mulates in the sheath around the distal ends of the de-
flected fascicles.
The cuticle at the tip of the head in the-1.4 (el960) is
thin and irregular. The hypodermis, which is pale and
somewhat distended, reveals numerous aggregates of
longitudinal intermediate filaments (Fig. 26). Presum-
ably similar filaments are present in the wild-type hy-
podermis.
The cuticle-embedded specializations of certain me-chanocilia are abnormal in the-14 (e1960). The discs at
the tips of the IL1 cilia are tilted. The nubbins of theCEP and OLQ dendrites are recessed n cuticular tunnels
(Fig. 27). The joined squares of the OLQ cilia are some-
times misoriented and, even when the squares are ori-
ented normally, the dark material that normally flanks
the circumferential corners occurs in abnormally small
pieces and is positioned randomly.
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PERKINS ET AL. Sensory Cilia in Nematodes 473
FIG. 18. Amphid cilia in the-11 (el810) mutants. Section through the
anterior sheath ch annel showing the middle segmen ts of the channel
cilia and the AWC cilium . Many of the cilia are enlarged and irregular
in contour and contain abnormal dark ground material (arrowheads).
The doublet and singlet microtubules of the axoneme are present and
nearly normal. An abnormal, detached doublet (arrow) is visible in
one of the channel cilia. The AWC cilium has failed to spread into
wing-shaped sheets. Scale bar is 0.5 pm.
Like the CEP neurons, the ADE and PDE neurons fi ll
with fluorescein in the-14 (el960) mutants (Table 1). This
suggests that defects in hypodermis and cuticle may ex-
tend along the entire length of the animal.
met-8 Afects Fusciculation of the Amphid Cilia
In met-8 (e398), the amphid wing and channel den-drites invaginate the sheath cell at staggered levels,
usually posterior to normal, and their cilia, though nor-
mal in length and ultrastructure, fail to fasciculate (Fig.
28). Individual cilia and partial fascicles course sepa-
rately through the sheath cell and accrete matrix, dark
lining, and scaffold material (Fig. 29). Some cilia turn
laterally or even posteriorly and most end blindly within
the sheath cell. The belt junctions connecting the amphidsocket and sheath cells are mispositioned and the cuticle-
lined channel of the socket cell sometimes ends in a blind
pocket without opening onto a channel in the sheath
cell. Channel cilia reaching the socket cell may invagi-
nate it without obtaining access to the externally open
channel. Abnormal large matrix-filled vesicles accu-
mulate in the sheath cells.
The various mechanosensilla of the head are normal
in met-8 (e398).
cat-6 Affects the CEP Specializations
The transition zones and middle segments of the CEP
cilia in cat-6 (el861) are positioned slightly anterior of
normal but are normal in length. The distal specializa-
tions, supernumerary microtubules and associated dark
material, form normal rod-shaped aggregates. These
rods, however, are not confined to the distal segments
but assemble along the entire cilia as well as ectopically,
proximal to the cilia (Figs. 30, 31d, e). The cuticle-at-
tached nubbins may also contain rods separated from
the ciliary shaft. Such nubbins are enlarged and often
extend completely through the cuticle (Figs. 30, 31a-c).
The OLQ cilia in cat-6 (el861) may have a reduced
amount of dark material flanking the circumferential
corners of the square of doublet microtubules. The otherclasses of mechanocilia, including IL1 and OLL, and the
amphid sensilla appear normal.
ttx-1 Thermosensory Mutants Lack the AFD Fingers
The fingers of the AFD neurons in the cryophilic mu-
tant ttx-l(p767; formerly EH67, Hedgecock and Russell,
1975) are entirely missing. Instead, a fingerless sack of
membrane protrudes from the dendrites just below the
cilia (Fig. 32). The cilia are about 4 pm long, three times
their normal length, and are tilted ventrally at their
bases, away from the anteriorly projected sack (Fig. 33).
The amphid wing and channel cilia and the variousmechanosensilla of the head are normal.
DISCUSSION
Development of the Sensilla
The assembly of an individual sensillum requires in-
teractions between at least four cell types. One or moreneurons invaginate and form junctions with the sheath
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474 DEVELOPMENTAL BIOLO GY VOLUME 117, 1986
FIG. 19. Amphid channel cilia in the-10 (el809). (a) Section through the anterior amphid sheath channel. A single ciliu m (c) with fairly
normal appearance is present in the lumen as are possible remnants of other cilia . (b) Section 3.5 pm posterior to (a). An irregular belt junction
(JN) joins a dendrite to the sheath cell. Another dendrite, sectioned distal to its junction with the sheath cell, terminates in a large swelling
filled with ground material (arrow). No ciliary structure is evident in either dendrite. Scale bar is 0.5 pm.
cell , the sheath cel l forms junctions with the socket cell, the embryo, many sensilla are assembled concurrently
and the socket cell forms junctions with adjacent epi- in a small region of the head. Thus each cel l must adhere
dermal cells. Presumably a specific cell-cell adhesion is to its correct partners despite possible competit ion from
required before any permanent junction can form. In nearby cells of similar type. This specificity of attach-
FIG. 20. IL1 cilium in the-10 (e1809). (a) Section through the transition zone of an IL1 cilium (white arrow) in the-f0 (e1809). No rootlet is
seen in the center of the cilium. The IL2 dendrite (black arrow) is also visible. (b-d) Section s 0.3, 0.9, and 1.0 pm posterior to (a), respectively,
showing that the IL1 dendrite lacks a striated rootlet. Neuron/sheath junctions (JN) are present on both IL1 and IL2 dendrites. Scale bar is
0.5 pm.
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PERKINS ET AL. Sensmy Cilia in Nematodes
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.
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476 DEVELOPMENTAL BIOLOGY VOLUME 117, 1986
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-
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PERKINS ET AL. Sewwry Cilia in Nematodes 477
-cuticle
FIG. 24. Schem atic longitudinal section of an amphid sens illum in
the-14 (e1960). The cilia form a normal fascic le in the sheath cell. The
sheath and socket channels connect aberrantly or, as shown here, fail
to connect. In this case , the cuticle-lined socket channel ends as a blind
pocket. T he filamentous scaffold (FS) in both sheath and socket ce lls
is disorganized and the dark lining that surrounds the anterior sheath
channel is missin g near the join of the sheath (sh) and socket (so)
cells. Cilia either deflect sideways in the sheath cell or invaginate the
cytoplasm of the socket cell. Scale bar is 1.0 pm.
defined sheath channel, the socket channel sometimes
ends in a blind pocket in met-8 mutants. The sheathchannel appears nearly normal in the-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 (el37?‘) mutants sug-
gests they, too, may be defective in the joining of the
sheath and socket channels (Albert et al., 1981). Consis-
tent 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 mu-
tants. Vestigial centrioles, without membrane attach-
ments, were found in a few dendrites. No membrane-
linked microtubules assemble ectopically in these mu-
tants, 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 Chlamydomcmas (Goodenough and St.
Clair, 1975).
The the-13, osm-1, osm-5, and osm-6 mutations shorten
the axonemes of all classes of cilia. Microtubules, at-
tached to the membrane by Y links, assemble ectopically
in these mutants. The number and lengths of these ec-
topic microtubules vary by neuron type and roughly
parallel the normal lengths of cilia in these cells. Wesuggest these ectopic microtubules are misassembled
components of the axoneme. Thus the peripheral doub-
lets and Y links can apparently self-assemble and the
wild-type products of these four genes are needed to en-
sure they assemble only on the ciliary template. Inter-
estingly, the transition zones are fairly normal in these
mutants. Also, the OLQ axonemes are probably less af-
fected 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 the-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 the-2 and the-3 mutants
have been previously shown to have normal transition
zones and truncated axonemes (Lewis and Hodgkin, 1977;
Albert et ah, 1981). It was reported that microtubules
assemble ectopically below the transition zones in both
of these mutants. Whereas the truncated amphid cilia
in the-2, the-13, osm-1, osm-5, and osm-6 mutants are
normal in diameter, the amphid cilia in the-3 mutantshave enlarged, bulb-shaped endings filled with dark
ground material (Lewis and Hodgkin, 1977).
The amphid channel cilia of the-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 the-11 channel
cilia are nearly normal in length whereas the daf-10 cilia
are described as foreshortened (Albert et al., 1981). Both
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478 DEVELOPMENTAL BIOLOGY VOLUME 117,1986
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
recognizable nine-fold organization. Rearrangement
might be expected if, for example, the neurons under-
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PERKINS ET AL. 479
FIG. 26. Cuticle and hypodermis in wild type and c//e-U m utant. (a). ” _Section 5 Frn from tip of head in wild-type adult. Struts (S) join the
two layers of the adult cuticle. The hypodermis (hyp) is thin and dark
and is attached to the subcu ticle by hemidesm osomes (arrows). (b)
Comparable section through the-14 (e1960) mutant. The cuticle is thin
and irregular. The hypodermis is pale and possibly expanded. Numerous
aggregates of intermediate filaments (F) fill the hypodermal cytoplasm.
Scale bar is 0.5 Wm.
synthesize ciliary proteins dur ing dendrite growth. Mu-
tants which destabilize the axoneme might actually leave
more material available for maintain ing the transition
zone than in the wild type. It may be interesting to ex-
amine the IL2 cilia in embryos or Ll larvae.
Sfriated Rootlets
Striated rootlets are frequent ly associated with the
basal bodies of both sensory and motile cilia but their
function is unknown. Salisbury and Floyd (1978) haveshown that the rootlets of certain flagella te 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 Te-
truselmis striata (Salisbury et al., 1984). Striated rootlets
have also been purif ied from several other sources (see
Salisbury et ccl., 1984). In each case, only one or two pro-
teins account for most of the protein in the purified
rootlets. However, the molecular weights of these pro-
teins 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 den-
drites. Interestingly, certain sensory neurons in C. ele-
gans males contain striated rootlets but no associatedcilia (Sulston et ah, 1980). The rootlets are attached to
dark plates, resembling hemidesmosomes, at the den-
dritic tips. These observations imply that rootlet and
cilium formation can occur independently and an un-
known mechanism ensures that the distal end of the
rootlet attaches to the base of the cilium. A similar con-
clusion has been reached using basal body defective and
rootlet defective mutants of Chlamydomonas (Gooden-
ough and St. Clair, 1975; Wright et ah, 1983). Interest-
ingly, the ectopic membrane-linked microtubules in the
the-13, osm-1, osm-5, and osm-6 mutants can recruit small
rootlets. Presumably, the same affin ity exists between
the rootlets and microtubules of normal cilia.The the-10 mutation eliminates striated rootlets from
the mechanosensory cil ia 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 exp lanation, that
the strain harbors two mutations, one responsible for
the rootlet defect and one for the amphid defect, can be
resolved by isolat ing a second, independent the-10 mu-
tant 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 cutic le 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 cil ia show that
the discs can assemble normally in the absence of cil ia
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 mater ial, itself amorphous, appears
to be molded into rods by the associated microtubules.
A similar dark material is found at the tips of the OLLcil ia where it forms a ball. The difference in shape may
reflect the comparative paucity of microtubules in the
OLL cil ia. In mutants with truncated or missing cilia,
the supernumerary microtubules and dark tubule-as-
sociated material in the CEP neurons assemble ectopi-
tally 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
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480
a
CN
DEVELOPMEN TAL BIOLOGY VOLUME 117, 1986
b c d
e
FIG. 27. CEP and OLQ cilia in the-14 (e1960) mutant. (a-h) Series of sections taken at approximately O.l-pm intervals from anterior to
posterior through the distal segments of the CEP (open arrow) and the OLQ (black arrow) cilia. The cuticle-asso ciated nubbin (CN) of the
OLQ cilium completely penetrates the cuticle (a). The joined square of doublet microtubules is misoriented (c-h) and the cuticle is abnormally
thick. The cuticle-asso ciated nubbin (CN) of the CEP cilium also penetrates the cuticle (e) and ends at the base of a deep, cuticle-lined pit
(star in c, d). Scale bar is 0.5 pm.
at the distal tip of the dendrite. In the cat-6 mutants,
both the CEP cilia and their specializations appear nor-
mal but the association between them is specifically dis-
rupted.Amorphous dark material is also present in the OLQ
cilia as small lumps that flank the circumferential cor-
ners 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
circumferent ially. There is no obvious structure joining
the OLQ axoneme to the support cells or the cutic le that
might provide orientation. A simple suggestion is that
the square and associated dark mater ial 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 the-14 mutants whichhave abnormally thick subcuticle adjacent to the cilia.
Perhaps relevant, the cilia of the respiratory epithe lia,
which normally are oriented to beat in a common di-
rection, are randomly oriented, as judged by their basal
feet, in subjects with immoti le cil ia (Afzelius, 1981).
Here, it is thought that c ilia form in random orientations
and become oriented through a mechanism involving
active beating.
As the CEP, OLL, and OLQ cil ia are situated some-
what posterior to the IL1 cilia, they probably detect ra-
dial, rather than axial, displacements. The geometry of
the OLQ cil ia suggests they have substantial directiona ldiscrimination. 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 an-
chorage of the dendrite to the cuticle. They are not a
ciliary specialization as such since they persist in mu-
tants with truncated cil ia and, at least for the CEP den-
drites, in the daf-19 mutants without cilia. In the cat-6
mutants with enlarged CEP nubbins or the the-14 mu-
tants 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 atthe distal tips of the CEM cilia . Here, it penetrates the
cuticle and is believed to provide access of the CEM den-
drite to the chemical environment. As there is no cutic-
ular 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 cutic le and pore of
the inner labial sensilla appear to be formed by the inner
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PERKINS ET AL. Sensory Cilia in Nematodes 481
/z cuticle
FIG. 28. Schem atic longitudinal section through the amphid sensillum
in met-8 (e398). The wing and channel cilia fail to form a single fascic le
within the sheath (sh) cell. Instead, they course sep arately or in sma ll
fascic les, accreting matrix, and, sometime s, the dark lining and fila-
mentous scaffold (FS) material that surround the anterior sheath
channel in wild-type. The cuticle-lined channel of the socket (so) cell
may end in a blind pocket rather than connecting to any of the fascic les
in the sheath cell. S cale bar is 1.0 pm.
labial socket cell. They persist in mutants with truncated
or missing IL1 and IL2 cilia.
Chemosensory Behaviors
C. elegans has at least five distinct chemosensory be-
haviors. First, it is attracted or repelled by a variety of
small molecules at low concentrations (lop3 Mor below)
(Ward, 1973; Dusenbery, 1974, 1975, 1976a, 1980a,c).
Second, it is repelled by very high concentrations of var-
ious 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, ar-rested stage, adapted for long-term survival and dis-
persal (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 ab, 1982; Golden and Riddle, 1982; Trent et
ah, 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 phero-
mone.
The amphid and phasmid sensilla have long been sus-pected of mediating many of these chemosensory be-
haviors (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 ah, 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 ah, 1981). These mu-
tants 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, COz and H+ (Du-
senbery, 1980b). This may reflect some residual respon-
siveness of the shortened channel cilia. Alternatively,
the amphid wing neurons or the IL2 neurons may be the
principal detectors for these chemical species. Signifi-
cantly, the osm-1 (~808) mutation, which affects the as-
sembly of all classes of cilia, abolishes even these re-
sponses (Dusenbery, 1980b). Hence, all known chemical
attractants, repellants, and pheromones are apparentlysensed through ciliated receptors in C. elegant.
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
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482 DEVELOPMENTAL BIOLOGY VOLUME 117,1986
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PERKINS ET AL. Sensc.n-y Cilia in Nematodes 483
can shift decisions to enter or exit the dauer stage in
either direction.
Horvitz et al. (1982) have reported that the-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 mu-
tants is a disruption of the amphid and phasmid cilia.This suggests that these neurons either make octopa-
mine or regulate the neurons which do.
Mating Behavior
Mating by C elegans males is a complex behavior in-
volving ten classes of male-specific sensory neurons
(Ward et aZ., 1975; Sulston et al, 1980; Hodgkin, 1983).
Ten genes that affect mechanosensory receptors in the
head, the-2, the-3, the-10, the-11, the-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 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.
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bFN
CCN
FIG. 31. CEP cilia in cut-6 (el861) mutants. (a) Longitudinal section through a newly molted L4 hermaphrodite showing the cuticle-asso ciated
nubbin (CN) of a CEP cilium as it penetrates the cuticle. Unlike wild-type, microtubules and dark tubule-associated material (TAM) extend
into the nubbin. (b) Transverse section through a CEP cilium of an adult cat-6 mutant showing the cuticle-asso ciated nubbin (CN) as it
penetrates the cuticle. (c) Section 0.2 pm posterior to (b) showing that the tubules and tubule-associated material (TAM) partition into the
cuticle-asso ciated nubbin (CN) and the main shaft. (d) Section 3.8 pm posterior to (b) at the level of the neuron/sheath junction (JN). A cluster
of microtubules and dark tubule-associated material (TAM) remain within the sheath cell as the main dendrite (star) e xits. (e) Section 4.2 pm
posterior to (b). Microtubules and tubule-associated material (TAM) remain in a posterior projection within the sheath cell. The main dendrite
(star) is entirely outside the sheath cell and devoid of ciliary structures. Scale bar is 0.5 pm.
FIG. 32. AFD cilia in wild type and ttz-I mutant. (a) Section through wild type amphid sheath cell showing the AFD cilium (C) and about
25 fingers (stars). (b) Comparable section through th-I (e767) amphid sheath cell. Distal to the neuron shea th junction, the AFD dendrite has
bifurcated into a fingerless sack (black arrow) and a cilium (C). The cilium is longer than normal and tilted ventrally. The sack is surrounded
by lamellae of the sheath cell. The channel cilia are completely normal. Scale bar is 0.5 Km.
484
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PERKINS ET AL. Senscny Cil ia in Nematodes 485
When placed in a thermal gradient, wild-type animals
move toward the temperature at which they were pre-
viously raised (Hedgecock and Russell, 1975). The ttx-1
thermotaxis mutants seek the cold regardless of their
thermal history. These mutants are also hyper-respon-
sive to dauer-inducing pheromone (Golden and Riddle,
1984a,b). Elevated temperatures are known to lower thepheromone 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 sen-
sory behaviors, including chemotaxis and mating, are
normal in this strain (Hedgecock and Russell, 1975; Du-
senbery and Barr, 1980).
A reduction in the number of fingers on the AFD den-
FIG. 33. Schem atic longitudinal section of AFD dendrite in &r-ldrites has also been reported for the the-1 mutants, e1034
(~767).The cilium is tilted ventrally and is longer than normal. Below and a74 (formerly DD74) (Lewis and Hodgkin, 1977; R.
the cilium, dendritic membrane protrudes in a fingerless sack. Scale Ware, D. Clark, M. Salzay, and R. Russell, personal com-
bar is 0.5 Fem. munication). No thermotaxis defects were detected inpopulation assays of the-1 mutants (Hedgecock and
gers are entirely missing and the AFD cilia are longer Russell, 1975). However, the finger abnormality in che-
than normal in ttx-1 mutants. The other sensory recep- 1 mutants is variable and comparatively mild. About
tors in the head appear ultrastructurally normal. half of the the-1 (e1034) animals examined by Lewis and
TABLE 2
SUMMARYOF ULTRASTRUCTURAL DEFECTS
duf-19
the-P
the-13
osm-I
osm-c5
a‘m-6
the-.Pb
ch,e-II
daf-lob
the-10
Sensory cilia
All cilia are absent. Ves tigial centrioles are found
in some dendrites.
Middle and distal segments of all cilia are absent.
Transition zones are normal. Membrane-linked
microtubules assem ble ectopically.
Middle segments of amphid cilia reduced to bulb-
shaped endings filled with ground material.
Transition zones are normal. Membrane-linked
microtubules assem ble ectopically.
Mechan ocilia are also abnormal.
Amphid cilia are nearly normal in length but
have irregular contours and ground ma terial in
their centers. Mechano cilia are also abnormal.
Amphid cilia usually absent and dendrites have
bulb-shaped endings filled with ground
material. Rare amphid cilia appear normal
suggesting this defect i s degenerative.
Mechan ocilia lack striated rootlets.
the-14
the-14
daf-6 b
met-1 ‘sr
met-8
cat-6
ttx-1
Socket and sheath c ells
Matrix material is absent from sheath vesicles
and channel. Amphid cilia are nearly normal
in length but abnormally dark.
Socket and sheath sc affolds are abnormal and
channe ls often do not join. Hypodermis is
also abnormal.
Socket and sheath channe ls do not join.
Fasciculation
Amphid dendrites invaginate sheath but
partially fail to fasciculate . Nonciliated
mechanosensory neurons (ALM, PLM, AVM,
PVM) are also abnormal.
Specializations
Tubular bodies assemb le along entire CEP
dendrite. CEP axonemes are normal.
Fingers of AFD dendrites are entirely missing .
O&W-3 Distal segments of amphid cilia are absent.
Middle s egments and transition zones are
normal. Mecha nocilia are normal.
a Lewis and Hodgkin (1977).
*Albert et al. (1981).
’ Chalfie and Sulston (1981).
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486 DEVELOPMEN TAL BIOLOGY VOLUME 117. 1986
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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