Mutant Sensory

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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



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



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).



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-



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



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



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).




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



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




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.




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).



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.




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.




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-




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.




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



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.




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




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.



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.




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-



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




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-



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



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




/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



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.



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



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).



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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