Axonal guidance mutants of Caenorhabditis elegans identified by filling sensory neurons with fluorescein dyes

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Axonal Guidance Mutants of Caenorhabditis elegans Identified Filling Sensory Neurons with Fluorescein Dyes EDWARD M. HEDGECOCK,*~'JOSEPHG.CULOTTI,~

J. NICHOLTHOMSON,*

ANDLIZABETHA.

by

PERKINS?'

*MRC Laboratory of Molecular Biology, Hills Road Cambridge CB2 2&H, England and j-Department of Neurobiology and Physiology Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60201 Received October 22, 1984; accepted in revised form March

and

11, 1985

Eight pairs of chemosensory neurons in Caenorhabditis elegans take up fluorescein dyes entering through the chemosensory organs. These are amphid neurons ADF, ASH, ASI, ASJ, ASK, and ADL and phasmid neurons PHA and PHB. When filled with dye, the processes and cell bodies of these neurons can be examined in live animals by fluorescence microscopy. Using this technique, we have identified five genes, uric-33, uric-& ~7~51, une-76, and unc106, that affect the growth of the amphid and phasmid axons. These genes were found to affect the axons of the mechanosensory PDE neurons as well. The uric-33 mutation specifically affects neuronal microtubules. Sensory dendrites in this mutant have a superabundance of microtubules. Moreover, many of these microtubules are abnormal in diameter, and some form hooks or multiple tubules. G 1985 Academic press, I~C. INTRODUCTION

The precision and complexity of axon outgrowth first became apparent in the 19th century with the discovery by Golgi of a method for staining a small, essentially random fraction, of neurons in their entireties while leaving the remainder of the nervous system unstained. Since then, a variety of techniques for selectively staining a subpopulation of cells have been devised. Most elegant of these are intracellular injection of fluorescent dyes or horseradish peroxidase and staining of neuronal antigens with specific antibodies. These techniques have facilitated studies of the mechanisms of axonal guidance. The small soil nematode, Caenorhabditis elegans, is increasingly used for genetic studies of behavior and development. The hermaphrodite has only 302 neurons. The cell lineages (Sulston and Horvitz, 1977; Sulston et al, 1983) and synaptic connections of these neurons (Albertson and Thomson, 1976; Hall and Russell, 1985; White et al, 1976, 1985) are now completely known. Some of these neurons can be stained by light microscopic methods. For example, Sulston et al. (1975) and Horvitz et ah (1982) have identified eight dopaminergic and two serotonergic neurons in the hermaphrodite using the formaldehyde-induced fluorescence technique. Peanut lectin and monoclonal antibodies have also been used to stain neuron processes in C.

’ Present address: Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, N. J. 07110. ’ Present address: Developmental Genetics and Anatomy, Case Western Reserve University, Cleveland, Ohio 44106. 0012-1606185 $3.00 Copyright All rights

0 1985 by Academic Press. Inc. of reproduction in any form reserved.

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elegans and Ascaris lumbricoides (H. Ellis, J. Yuan, and H. Horvitz; R. Francis and R. Waterson; E. Hedgecock; H. Okamoto; S. Siddiqui, and J. Culotti; A. Stretton and C. Johnson; personal communications and unpublished observations). Dye microinjection has been used successfully in filling motorneurons and interneurons in A. lumbricoides (J. Angstadt and A. Stretton, personal communication). This last technique is not at present practical in the smaller nematode C. elegant. In this paper we describe how certain chemosensory and mechanosensory neurons in C. elegant can be filled with fluorescein by bathing living animals in fluorescein solutions. In Perkins et al. (1985), we show that the dye enters these neurons through their exposed receptor cilia. We have used the fluorescein-filling technique to screen an existing collection of locomotory mutants (Brenner, 1974) for mutations affecting axon growth. Since dye filling is harmless, we have also isolated new mutants by searching through a mutagenized population by fluorescence microscopy and selecting individuals with abnormal processes. Here we describe five genes which affect the growth of many types of axons throughout the nervous system. MATERIALS

AND

METHODS

Strains and genetics. Brenner (1974) describes culturing and genetic manipulation of C. elegans. Strains were obtained from the collection held at the MRC Laboratory of Molecular Biology in Cambridge, England. The cat-6 (e1861) and the-14 (1960) mutations are described in Perkins et al. (1985). A new recessive

HEDCECOCK ET AL.

Axcmal

mutation, uric-106 (ev&OO), on linkage group X was induced with ethylmetha:nesulfonate. All of these mutants are available through the Caenorhabditis Genetics Center at the University of Missouri-Columbia. Fluorescein isothiocyanate (FITC) staining protocol. A stock dye solution containing 20 mg/ml 5-fluorescein isothiocyanate in dimethylformamide was stored at -20”. If kept dry, it was Istable indefinitely. For routine genetics, 50 ~1 of the stock dye solution were mixed with 200 ~1 of M9 buffer and applied evenly to the surface of a lo-ml NGM plate preseeded with a lawn of Escherichia coli (Brenner, 1974). After 2 hr to allow the dye to diffuse into the agar (final concentration 0.1 mg/ml), live animals were transferred to the plate. After staining for 2 hr or, if convenient, as long as overnight, the animals were transferred to an agar plate without dye for at least 10 min to remove free FITC from the intestine. Olnce filled, the neurons remain brightly stained for many hours. Stained animals have normal growth rate, broold size, and mating ability. For examining the phasmid or PDE neurons, it was

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helpful to reduce interfering FITC fluorescence from the intestine by staining and destaining the animals in ice-cold M9 buffer. The chilled animals do not feed and hence no FITC enters the intestine. Typically, animals were washed free of bacteria in buffer alone, stained for 4 hr in cold buffer containing 0.4 mg/ml FITC, washed three times in cold buffer, and then transferred to a seeded agar plate at room temperature. Mounting for jluwrescence microscopy. Animals were mounted on layers of 5% agar prepared as described by Sulston et al., 1980. For photography, they were anesthetised in 0.5% 1-phenoxy-2-propanol (Koch-Light Laboratories) and mounted on agar containing 0.2% lphenoxy-2-propanol. RESULTS

FITC Filling

of Amphid

and Phasmid

Neurons

When animals are placed in solutions of 5-fluorescein isothiocyanate, six pairs of neurons in the head and two pairs of neurons in the tail fill with dye (Fig. 1).

FIG. 1. FITC filled amphid and phasmid neurons in living animals. (a) Ventral view of amphid neurons. The cell bodies of ADF, ASH, and ASJ are in focus. The cell ;bodies of ASK, ADL, and AS1 are blurred below the plane of view. Processes from the sensory organs (arrowheads) run in the plane of view. Processes in the nerve ring (arrows) are seen in cross section. (b) Ventral view of phasmid neurons PHA and PHB. Processes from the sensory organs (arrowheads) and processes in the ventral nerve cord (arrows) run in the plane of view. Scale bar is 20 pm.

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AS1

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-

FIG. 3. Schematic of phasmid neurons. (a) Left lateral view. Cell bodies of PHA and PHB are in the lumbar ganglia. A process from each neuron extends backward to the phasmid sensilla (arrowhead). Second processes extend forward, gathering into a commissure with processes from other lumhar neurons on the same side (douhle arrowhead). The two lumbar commissures pass between the ventral body muscles and the hypodermis and enter the ventral nerve cord. There, both PHA and PHB neurons meet their contralateral homologs and course forward together in the nerve cord (arrows), making and receiving synapses with other cord processes (Hall and Russell, 1985; White et aZ., 1985). The rectum is indicated by an oblique line. (b) Ventral view. Scale bar is 20 Frn.

ADL \

ASK

AdF

ADL

ASH

AS1

ASJ

FIG. 2. Schematic of amphid neurons. (a) Left lateral view of the neurons that fill with FITC. Cell bodies are in the lateral ganglia behind the nerve ring. Processes from each cell run forward as a bundle (single arrowhead) from the cell bodies to the amphid sensillum. Second processes run ventrally from the cell bodies, gathering with other ring neurons into a large commissure which passes between the ventral body muscles and the hypodermis (double arrowheads), and then turn upward into the nerve ring (single arrows). Synapses are made and received in the nerve ring, and at the top (double arrows), the processes terminate in electrical synapses with their contralateral homologs (White et aZ., 1985). (b) Left lateral view of ADF neuron. (c) Left lateral view of ADL neuron. This amphid neuron is exceptional in that its axon enters the nerve ring

These were identified by their position and morphology, using simultaneous Nomarski/epifluorescence microscopy, as amphid neurons, ADF, ASH, ASI, ASJ, ASK, and ADL, and phasmid neurons, PHA and PHB, respectively (Sulston et al., 1983; White et al., 1985). Amphid neurons ASE and ASG do not appear to fill. The stained neurons are believed to be chemosensory (Perkins et aZ., 1985). Each cell, excepting ADL, has an unbranched bipolar geometry. A ciliated process extends to a sensillum and a second process extends into the neuropil, either the circumpharyngeal nerve ring for the amphids (Fig. 2) or the preanal region of the ventral nerve cord for the phasmids (Fig. 3). All synaptic inputs and outputs occur on this second process (White et al., 1985; Hall and Russell, 1985). Uncoordinated Mutants with Abnormal Chemosensory Axons Locomotion in C. elegans appears to require five classes of interneurons, seven classes of motorneurons, and 95 body muscles (White et al., 1976). Brenner (1974) by a direct route and then branches (White et al, 1985). (d) Ventral view of the neurons that fill with FITC. Cell bodies of neurons ADF, ASH, and ASJ are shown in solid black. Cell bodies of ASK, ADL, and ASI, below the plane of view, are shown as dotted circles. The processes to the amphid sensilla are indicated with arrowheads. Processes in the nerve ring, seen in cross section, are indicated by arrows. Scale bar is 20 pm.

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isolated locomotory mutants of 71 distinct uric (uncoordinate) genes. Since then, another 26 uric genes have been identified (Sw.anson et al., 1984). This is a diverse collection of genes. Mutants of some 20 of these genes have been shown, by polarized light, electron microscopy, or biochemistry, to have primary defects in muscle structure (Waterson et al., 1980; Zengel and Epstein, 1980). The behavioral defects of several more mutants are attributed to alterations in the cell lineages generating various neuron classes, including motorneurons (Sulston and Horvitz, 1981; White et al., 1982; Horvitz et al., 1983). The great remainder of uric mutants have unknown defects, presumably including abnormalities in axon outgrowth and guidance, synaptogenesis, electrical excitability, and neurotransmission. We screened alleles of a.11of the published uric genes, except those known to affect muscle structure (Waterson et al, 1980; Zengel and1 Epstein, 1980), for abnormal FITC staining. Of these, four genes, uric-33 (IV), unc44 (IV), uric-51 (V), and uric-76 (V), appear essential for the correct outgrowth and guidance of the axons of the amphid and phasmid neurons. A mutant of a fifth gene, uric-106 (ev400) (X), was selected directly for abnormally positioned phasmid axons. The amphid and phasjmid cell bodies are present and, except for small displacements in uric-51 and unc106, they are normal in position in these mutants. The processes from the sensilla are normal, but the processes that grow into the neuropil make a variety of errors. We have made only a rough description of the defects of the amphid axIons. Defects in the phasmid axons are described in detail. In uric-76, most amphid axons grow into the nerve ring through the amphid commissure. In the nerve ring, the processes often separate and terminate at abnormal ventral or lateral positions. Only rarely does a process reach the dorsal part of the nerve ring. The axon terminals usually appear somewhat swollen. The amphid axons in uric-33 and uric-44 mutants are more difficult to observe as these cells stain poorly with

FIG. 4. Premature termination of phasmid axons. Ventral view of PHA and PHB neurons in uric-7’6 (e911) mutants. The processes are somewhat enlarged where they terminate. Phasmid axons in uric-3.3, WE&, and urn-51 mutants have a similar defect. Scale bar is 20 ym.

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FIG. 5. Mispositioned phasmid axons. Left lateral views of the PHA and PHB neurons from two uric-106 (eu4oO) individuals. The axons have failed to enter the ventral nerve cord, and instead, extend anterior (a) or posterior (b) along the side of the animal in lateral (L) or subventral (SV) positions. The rectum is indicated by an oblique line. Scale bar is 20 pm.

FITC. They make mistakes similar to the uric-76 mutants and generally have swollen endings at ventral positions in the nerve ring. In uncdl mutants, a majority of amphid axons reach the dorsal part of the nerve ring where they terminate in exceptionally large swellings. Finally, the amphid processes in uric-106 mutants generally complete their course through the nerve ring. In contrast to the amphid defects, the phasmid axons share a simple and penetrant phenotype in uric-33, unc44, uric-51, and uric-76. The axons terminate prematurely, stopping just as they enter the ventral nerve cord at the posterior end of the preanal region (Fig. 4). As in the nerve ring, the terminals tend to be enlarged, most exceptionally in uric-51 mutants. The uric-106 (ev400) mutation produces a different phasmid phenotype than the mutations in the other four genes. Most commonly, the axons pass under the ventral muscles normally and enter the nerve cord where they grow forward a normal distance. In a substantial minority, however, both axons on one side fail to enter the nerve cord but extend forward, or less often, backward, along the side of the animal (Fig. 5). The contralateral phasmid neurons may enter the ventral nerve cord and extend a normal length in these animals. The mispositioned axons of PHA and PHB may stay together, or less often, separate. They often run for a somewhat greater distance than their normal counterparts. The cell bodies are sometimes displaced forward, perhaps because the misplaced processes pull on them slightly during growth. We examined these uncoordinated mutants briefly by Nomarski microscopy for possible abnormalities in cell lineages or cell positions. No defects were found

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Posterior FIG. 6. Schematic of PDE neuron. Left lateral view. The cell bodies of the two PDE neurons are situated on the sides of the animal midway between the vulva and the rectum. A short, ciliated, dendrite extends into the mechanosensillum (star). A long axon extends into the ventral nerve cord where it bifurcates and extends in both anterior and posterior directions. The anterior process normally stops before reaching the head ganglia. The posterior process stops before reaching the preanal ganglion. The processes of both PDE neurons are adjacent in the ventral cord neuropil and there make and receive en passant synapses with the axons of other neurons (White et al, 1985). The phasmid neurons are also included for reference. Scale bar is 100 pm.

in uric-33, uric-64, and me-76 animals. Variable cell displacements were observed in uric-51 animals. In particular, the neurons ALM, AVM, PVM, and SDQ are frequently found anterior to their wild-type positions along the body; the P ectoblasts, precursors to ventral cord motorneurons, occasionally fail to enter the ventral cord, and more rarely, body muscle cells extend between dorsal and ventral muscle quadrants. Cell displacements were also observed in uric-106. In these animals, the vulva1 cells frequently fail to assemble properly. The pharynx, and perhaps the body, are frequently twisted. Body muscles occasionally extend between dorsal and ventral muscle quadrants, and the lateral canal of the excretory cell is occasionally displaced, dorsally or ventrally, from its midline position.

Deirid

(PDE) Neurons

The PDE cells (Fig. 6) are a bilateral pair of mechanosensory neurons born on the sides of the animal during the second larval stage (Sulston and Horvitz, 1977). They have been shown to contain the neurotransmitter dopamine (Sulston et al., 1975). Although the PDE neurons do not normally fill with FITC, they can be stained in certain mutants, e.g., cat6 and the-14, with abnormal mechanosensilla (Fig. 7). For each of the axonal mutants, uric-33, uric-44, uncdl, uric-76, and uric-106, we constructed a double mutant with either cat-6 or the-14 and examined the PDE neurons by the FITC technique. The PDE neurons in uric-76 mutants usually appeared normal but, in about one-third of the cells, the posterior branch was shorter than normal or entirely missing (Fig. 8). Many of the PDE axons had a small spur or swelling at the point where they crossed the ventral sublateral nerve tract and passed under the body muscles. In a few cases, the ventrally directed axon split at this position and sent a branch posteriorly along the muscle edge. The PDE neurons in uric-33 mutants resembled those in uric-76 in that the ventrally directed axon, though sometimes normal, often made a spur or branch as it passed under the ventral body muscles (Fig. 9). The frequency of branching was higher than in uric-76 and in several cases the two branches grew, in opposite directions, along the muscle edge. In such cases, no process reached the ventral cord neuropil.

FIG. ‘7. FITC-filled PDE neurons in living animals (cat-6 (e1861)). (a) Lateral view of a PDE neuron. A short ciliated dendrite extends dorsally to the sensory organ (star). A longer axon extends into the ventral nerve cord (arrowheads). (b) Ventral view of PDE neurons. The cell bodies are below the plane of focus. Their axons (arrowheads) extend into the ventral nerve cord (arrows) where they bifurcate and run together both anteriorly and posteriorly. Scale bar is 20 pm.

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FIG. 8. uric-76 PDE neurons. Twenty-eight neurons were examined in uric-76 (e911) mutants. Twenty-seven were bipolar with a short ciliary process and a long major axon. In one case, a second short axon grew anteriorly from the cell body in a subdorsal position (see Fig. 9d). (a) In about half (16/B) of the cells, the major axon entered the ventral nerve cord and branched normally. (b) In the other (121 28) neurons, the major axon ent.ered the ventral nerve cord but the posterior branch was abnormally short (broken line) or missing. (c) In (3/28) cases, the major axon branched subventrally, extending one process posteriorly and another into the ventral nerve cord. In another (10/28) cells, subsumed in (a) and (b) above, the major axon had a small subventral (SV) spur or swelling.

The PDE neurons in ~~4.6 and uric-51 were invariably abnormal, extending one or more axons either ventrally, laterally, or along the subdorsal or subventral muscle edges. Free-hand drawings of individual cells are shown in Figs. 10 and 11. The axons in uric-51 mutants frequently had a large varicosity near the cell body.

a

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,

* - S”A--. SV-

FIG. 10. uric-44 PDE neurons. Thirty-nine neurons were examined in uric-44 (e36.2) mutants. Twenty-eight were bipolar with a short ciliary process and long major axon. The remaining 11 cells had, in addition to a short ciliary process, two axons (9 cases) or three axons (2 cases). The mutant axons had complex and variable defects which included growth along inappropriate lateral (L), subdorsal (SD), or subventral (SV) positions and ectopic branches. Free-hand drawings of 12 individual cells are represented. These were selected from the larger set of 39 cells to illustrate typical cases.

The axons PDE neurons one-third of mally short axons failed ran obliquely

were normal in about one-third of the in uric-106 mutants (Fig. 12). In another the cells, the posterior branch was abnoror missing. In the remaining cells, the to reach the ventral nerve cord and either to the subventral muscle edge and forward

b ,

&-

r-q3==

SD-* L

FIG. 9. uric-33 PDE neurons. Thirty-one neurons were examined in uric-33 (e.204) mutants. Twenty-three were bipolar with a short ciliary process and a long major axon. The remaining eight cells had one (7 cases) or two (1 case) additional, generally shorter, axons. (a) In about half (16/31) of the animals, the major axon entered the ventral nerve cord and branched normally. The length of the anterior branch (broken line), however, was variable and generally much shorter than in wild-type animals. In about a third (506) of these animals, the major axon had a small subventral (SV) spur or swelling. (b) In (W31) cases, the major axon branched subventrally, extending one process posteriorl,y and another into the ventral nerve cord. (c) In (5/31) cases, the major axon branched subventrally, extending one process posteriorly and another anteriorly. In these cases, no process reached the ventral nerve cord. (d) Most of the ectopic axons grew in a subdorsal (SD) position, anteriorly or posteriorly. Finally, in (2/31) cases, the major axons could not be clearly classified into one of thle three categories given in (a), (b), and (c).

.

S”sDr*S”

>

FIG. 11. uric-51 PDE neurons. Twenty-four neurons were examined in uric-51 (e369) mutants. Nineteen were bipolar with a short ciliary process and a long major axon. The remaining five neurons had, in addition to a short ciliary process, two axons. The mutant axons had complex and variable defects which included growth along inappropriate lateral (L), subdorsal (SD), or subventral (SV) positions and ectopic branches. Eight of the neurons had a prominent varicosity along a major axon. Free-hand drawings of 12 individual cells are represented. These were selected from the larger set of 24 cells to illustrate typical cases.

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similar defect was observed in the sensory neurons of uric-44, uric-51, or uric-7’6.

b DISCUSSION C

WA* FIG. 12. uric-106 PDE neurons. Thirty-six neurons were examined were bipolar with a short in uric-106 (ev400) mutants. Thirty-four ciliary process and a long major axon. In one case, a short second axon grew posteriorly from the cell body in a subdorsal position (see Fig. lid). (a) In (10136) cells, the major axon entered the ventral nerve cord and branched normally. (b) In another (10136) cells, the major axon entered the ventral nerve cord but the posterior branch was abnormally short (broken line) or missing. (c) In (11/36) cells, the major axon ran obliquely from the cell body to the subventral (SV) muscle edge and continued anteriorly in that position without branching. In the remaining (5/36) cells, the axon branched anteriorly and posteriorly at the subventral position (4 cases) or grew posteriorly in that position without branching (1 case).

along it, or less often, branched and ran in both directions. Abnormal

Neuronal

Microtubules

at the muscle edge

in uric-33 Mutants

The amphid and phasmid neurons stain less brightly with FITC in uric-33 and uric-44 mutants than in wildtype animals. The amount of residual staining varies between individuals and also depends somewhat on cell type. The neurons are generally fainter in uric-44 mutants than in uric-33 mutants. The neurons in unc51, uric-7’6, and uric-106 stain with normal intensity. Using the methods of Perkins et al. (1985), we examined the anterior 15 pm of uric-33 and uric-44 mutants by electron microscopy to determine whether a defect in the amphid chemosensilla was responsible for reduced dye uptake. The sensilla appeared essentially normal in both mutants except for a partial shortening of the cilia of the amphid neurons. This shortening of the receptor cilia reduces the amount of membrane contacting the dye and probably accounts for the reduced uptake (Perkins et al., 1985). In comparison, the chemosensory cilia in uncdl and uric-76 mutants were normal in length. While examining the head sensilla in uric-33 mutants, we discovered that dendrites of apparently all classes of sensory neurons, and also the processes of sheath and socket cells, contain a superabundance of microtubules (Table 1). Moreover, these microtubules are sometimes larger than normal in diameter or form abnormal hooks, doublets, or even triplets (Fig. 13). In comparison, the microtubules in muscle and hypodermal cells appeared normal in number and structure. No

In C. elegans, neurons are generated by largely autonomous cell lineages. With few exceptions, the cells are born very near their final positions (Sulston and Horvitz, 1977; Sulston et ah, 1980, 1983). The developmental sequence of axon outgrowth is still largely unknown but the finished form of the adult nervous system has been determined from serial electron microscopic reconstructions (Albertson and Thomson, 1976; Hall and Russell, 1985; Sulston et ah, 1980; White et aZ., 1976, 1985). The mutants described in this paper, in conjunction with the known wild-type anatomy, suggest that axon growth may occur in separable stages. They also indicate several places where cell interactions likely affect axonal geometry. It should now be possible to test for these interactions by laser ablation of the candidate cells (Sulston and White, 1980). Little is known about the products of these genes. Genetic mosaics may establish whether they must be expressed in the afflicted cells, in neighboring neurons, or in the epidermal and mesodermal cells which support the nervous system (Herman, 1984). For at least one gene, uric-33, the molecular focus appears to be the axonal cytoskeleton. Development

of the Phasmid

Neurons

The PHA and PHB neurons are located in the lumbar ganglia on the sides of the animal just behind the rectum (Hall and Russell, 1985; White et ah, 1985). The axons from most of the lumbar neurons gather into two fiber bundles, the lumbar commissures, that pass circumferentially between the hypodermis and the ventral body muscles to reach the preanal terminus TABLE

1

MICROTUBULESINAMPHIDNEURONSANDAMPHID SHEATH CELL Microtubules” Wild-type neurons (N = 21) uric-33 neurons (N = 10) Wild-type sheath (N = 2) uric-33 sheath (N = 1)

11.3 + 3.2 25.0 + 6.0 52.0 f 11.0 396

Neurofilaments” 5.1 + 1.6 3.9 f 1.4 0 0

’ The means and standard deviations of the number of microtubules and neurofilaments observed in cross sections of the amphid neurons and sheath cell about 2.5 pm posterior to the cilia. The numbers of cells counted are given in parentheses. Counts are from a single wild type and a single mutant. Adjacent sections were examined to ensure that our counting was reliable.

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FIG. 13. Abnormal neuronal lmicrotubules in uric-33 (eZ04) mutants. (a, b) Low-magnification electron micrographs of amphid neurons (n), amphid sheath (sh), and amphid socket (so) cells taken about 2.5 pm posterior to the cilia. The cytoplasm of the amphid neurons and sheath cell contain more microtubules in uric-3S mutants (b) than in wild type (a). No lamellae (lam) is found in this region of the mutant sheath cell. (c, d) High-magnification electron micrographs of amphid neurons. Some of the microtubules in uric-33 neurons have abnormal hooks, doublet or triplet tubules (solid arrowheads in (d)). Small fascicles of neurofilaments are found in both mutant and wild-type neurons (open arrowheads in (c) and (d)). Scale bars are 0.5 pm.

of the ventral nerve corld. This position (2 in Fig. 14) is a unique junction in the juvenile neuropil. There, anteriorly directed processes from eleven lumbar and two dorsorectal neurons meet and reassort with about a dozen posteriorly directed processes originating in

the head ganglia and also with axons from seven neurons with cell bodies in the preanal region itself. The lumbar axons continue anterior in the nerve cord and make synapses en passant with neighboring neurons. Some extend through the entire length of the

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I/ALN\

-0 PVR

AVA AVD A% AVH AVJ AVK AVL

D

DA8

FIG. 14. Organization of the preanal region in newly hatched larvae (after Hall and Russell, 1985; White et al., 1985; Sulston et aL, 1983). Cell bodies and processes of tail neurons are shown in cylindrical projections. The positions of nerve processes are labeled D (dorsal), SD (subdorsal), SV (subventral), and V (ventral). Axons from 11 lumbar neurons (PVQ, PHA, PHB, LUA, PVC, and PVR) gather into two fascicles, called lumbar commissures (l), which pass between the hypodermis and the ventral body muscles to join the ventral nerve cord (2). Here they reassort with processes from dorsorectal neurons (DVA and DVC), processes from interneurons with cell bodies in the head ganglia (AVA, AVD, AVG, AVH, AVJ, AVK, and AVL), and also the cell bodies and processes of neurons in the preanal ganglion itself (PVT, PVP, DD6, DA8, DA9, and PDA). The processes from PVQ, PVC, and PVR continue along the nerve cord to the circumpharyngeal nerve ring in the head. The axons from PHA, PHB, and LUA end in the preanal region after making numerous synapses. In addition to the incoming axons of the lumbar neurons, the left and right lumbar commissures contain the centrifugal axons of motorneurons DA8 and DA9, respectively, which grow into the dorsal nerve cord. The neuron PDA also contributes a centrifugal axon to the right lumbar commissure at a later stage.

ventral nerve cord and into the circumferential nerve ring in the head. Others, including the PHA and PHB axons, end in the preanal region. It is not known which lumbar axons are first to reach the ventral nerve cord. PVQ and PHA (Fig. 14) are attractive candidates since they are the closest to the preanal ganglion and are also born somewhat earlier in the embryo (Sulston et al., 1983). If the idea of marker cells (Bentley and Keshishian, 1982; Goodman et aZ., 1982) applies here, any of the seven embryonic neurons in the preanal ganglion might act to identify the preanal region to incoming lumbar axons. The motorneurons, DA8 and DA9, are attractive candidates because of their posterior position in the preanal ganglion and because they contribute centrifugal processes to the lumbar commissures (Fig. 14).

The growth of the phasmid axons appears to occur in two separable stages, finding the ventral nerve cord and growing along it. In me-106 mutants, the phasmid axons frequently fail to reach the ventral cord and continue instead in various lateral positions. In animals where these axons do enter the ventral cord, however, they appear to grow forward normally. In contrast, the phasmid axons in uric-33, uric-4.4, uric-51, and unc76 mutants accurately find the ventral cord, but arrest there without forward extension (position 2 in Fig. 14). The phasmid axons normally meet target axons in the ventral cord coming from cell bodies in the head (Fig. 14). A reasonable hypothesis for the uric-33, unc44, uric-51, and uric-76 mutants is that, the phasmid axons fail to find or adhere to these targets, and in the absence of tension conferred by such adhesion, are

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ET AL.

Axonal

unable to pull themselves forward through the neuropil (Bray, 1982, 1984; Letourneau, 1982). Two likely candidates for assisting this extension are the axons of ring neurons AVA and AVG. These are major synaptic targets of PHB and PHA., respectively, and enter the preanal neuropil from the anterior (Hall and Russell, 1985; White et aZ., 1985). The detailed anatomy of the preanal neuropil has been determined from serial electron microscopic reconstructions (Hall and Russell, 1985; White et al., 1985). Some adjacencies of lumbar axons persist and others are disrupted as they enter the nerve cord. For example, axon pairs PVQ/PHA and PHA/PHB are adjacent both in the commissures and in the ventral nerve cord. The PVQL and PVQR axons are adjacent to the outgoing axons of the motorneurons DA8 and DA9, respectively, in the lumbar commissure. They separate from the motorneurons at the motorneuron soma (Fig. 14) and move to the center of the neuropil. There, the six axons of PVQL/R, PHAL/R, and PHBL/ R “zipper” together to form a vertical chain with the PVQs on top and the PHBs on bottom. The PHB axons also associate with the AVA and PVC axons near the base of the chain. The arrangement of lumbar axons in the neuropil is reflected in the pattern of synapses observed (Hall and Russell, 1985; White et al., 1985). The PHAs and the PHBs form chemical synapses and gap junctions with their homologs. The PHAs form chemical synapses onto the PHBs and PVQs. The PHBs form dyadic synapses onto the AVAs and PVCs. In general, the left and right homologs of the lumbar neurons appear synaptically equivalent. In double mutants of uric-7’6 and uric-106, the phasmid axons frequently continue anteriorly into the preanal neuropil far beyond the position (2 in Fig. 14) where they arrest in the single uric-76 mutants. This appears to occur when the contralateral phasmid axons fail to reach the nerve cord (unpublished observations). A possible explanation for the arrest of the phasmid axons in uric-76 mutants at the junction of the lumbar commissures is that the forward tension normally provided by adhesion to ventral cord axons is ins&icient to overcome backward tension provided by adhesion to contralateral lumbar axons. Chemical synapses on the lumbar axons are confined to the ventral nerve cord and none occur more proximally in the lumbar com:missures (Hall and Russell, 1985; White et al., 1985). This is not due to an inavailability of synaptic partners. For example, the PHA axons run adjacent to target axons PVQ and PHB, and similarly, the PHB axons run adjacent to target axons PVC both proximally and dlistally. The synapses formed by lumbar axons in the preanal neuropil are nearly all

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Mutants

dyadic with at least one of the postsynaptic axons coming from the head neurons, the dorsorectal neurons, or the contralateral lumbar ganglion. For example, the principal output of PHB is a simultaneous synapse onto an AVA and a PVC axon. Two models have been proposed for the formation of dyadic synapses that can explain the absence of synapses in the commissures without invoking an intrinsic difference between the proximal and distal segments of the lumbar axons. Hall and Russell (1985b) have suggested that synapse formation requires pairwise recognition by all three members of a dyad. In their model, removal of either the AVA or the PVC neurons would be expected to eliminate PHB synapses onto the other class. In the tetradic synapses made by photoreceptor neurons in the fly, postsynaptic elements are acquired sequentially and only certain sequences are observed (Frohlich and Meinertzhagen, 1983). To explain the order of acquisition, Frohlich and Meinertzhagen (1983) proposed that some postsynaptic elements can initiate new synapses while others can only join preexisting synapses. Their hierarchical model, applied to the PHB example, suggests that AVA but not PVC can initiate new synapses. It predicts that removal of the AVA neurons would eliminate PHB synapses onto the PVCs, but removal of the PVC neurons would not affect PHB synapses onto the AVAs. A third, simple model assumes that nascent synaptic complexes are laterally mobile in the membranes and have a tendency to aggregate. Complexes between PHB and AVA axons would be confined to the region of PHB and AVA overlap in the ventral cord. Complexes between PHB axons and ipsilateral PVC axons could diffuse along the commissures and into the ventral cord provided the two axons remain continuously apposed (Hall and Russell, 1985; White et aZ., 1985). In places where PHB is simultaneously apposed to both AVA and PVC, PHB/PVC complexes could coaggregate with PHB/AVA complexes. Thus PHB/AVA complexes, themselves confined to the ventral cord, could trap PHB/PVC complexes diffusing from the commissures. This model predicts that, if the AVA axons are removed or if the PHB axons fail to reach them, as may occur in uric-33, uric-44, uric-51, uric-76, and uric-106 mutants, the PHBs will still form synapses onto the PVCs, perhaps even in the commissures.

Development

of the Post-Deirid

Neurons

The PDE neurons resemble the phasmid neurons in that they are born on the sides of the animal and

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DEVELOPMENTAL BIOLOGY

extend axons circumferentially between the hypodermis and the ventral body muscles to reach the ventral nerve cord (Fig. 15). Whereas the phasmid neurons enter the posterior terminus of the ventral cord, the PDE neurons enter the middle region and bifurcate. The anterior and posterior axon branches of two PDE neurons are adjacent in the ventral neuropil and make gap junctions with each other (White et al., 1985). The main synaptic targets of the PDE neurons are the axons of DVA and AVK with cell bodies in the dorsorectal and head ganglia, respectively. Synapses are also received from the mechanosensory neurons PVM and PLM. The growth of the PDE axons, like the phasmid axons, can be broken into two stages, circumferential growth to reach the ventral nerve cord, followed by fasciculation and extension along the nerve cord. In me-106 mutants, the PDE axons frequently fail to reach the ventral nerve cord and wander laterally instead. In cases where they do reach the ventral nerve cord, they can grow normally. Thus the uric-106 gene appears to affect circumferential growth to reach the ventral nerve cord but is probably not required for subsequent growth along it. Several classes of motorneurons with cell bodies in the ventral nerve cord extend axons circumferentially to reach the dorsal nerve cord (White et al., 1976). It would be interesting to learn if uric-106 affects dorsally directed growth of these axons. Brenner (1973) found that mutations in another gene, uric-5, prevent the motorneuron axons from reaching the dorsal nerve cord. We have found that the uric-5 mutation has no effect on ventrally directed growth of the phasmid and PDE axons (unpublished observations).

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When axons meet and fasciculate with the ventral cord, they may, in principle, grow in either direction along the nerve or bifurcate, as does the PDE neuron, and grow in both directions. The actual choice appears to depend on neuron type. For example, the axon of the PVD neuron, a lineal cousin of PDE born at the same time and place, grows anteriorly, without bifurcation, upon entering the ventral cord (Sulston and Horvitz, 1977; White et al, 1985). The simplest schemes of selective adhesion to one or more axons already in the bundle do not predict the direction of growth (Goodman et ah, 1984). It seems that some asymmetry within the bundle must be invoked. A simple suggestion is that the entering axon tip acts as a sink for adhesion molecules on the target axon. If these molecules are inserted into the target cell membrane preferentially at one end of the axon, say at the soma, the entering axon would be expected to grow toward that end. Bifurcation might occur when an entering growth cone confronts two target axons whose adhesion molecules originate at opposing ends of the nerve cord. About half of the PDE axons that reached the ventral neuropil in uric-106 mutants, either lacked a posterior branch or had an abnormally short branch. A simple explanation may be that in those cases the PDE axon fails either to find or to adhere to a target axon in the posterior nerve cord. A logical candidate is the axon of the DVA neuron with cell body in the dorsorectal ganglion (Fig. 14). This embryonic neuron is a principal synaptic target of PDE (White et ah, 1985). Recently, we have identified mutations in several more uncowdination genes which specifically eliminate the posterior branch of the PDE neuron (unpublished observations).

D

D SD L SV

izi&--f’“’

;

ALN

SD L sv

FIG. 15. Path of the PDE neurons (after White et ab, 1985; S&ton et al, 1975; Sulston and Horvitz, 1977). The PDE neurons are born in the second larval stage on the sides of the animal. The cell bodies and axons grow sandwiched between the hypodermis (stippled) and the basal lamina (not shown) secreted by the hypodermis. The axons cross a small number of lateral and subventral nerve processes, and then pass between the ventral body muscles (striped) and the hypodermis to reach the ventral nerve cord. Once there, the axons bifurcate and run, in close apposition to their homolog, anteriorly and posteriorly along the nerve cord, making and receiving synapses with other cord processes.

HEDGECOCK ET AL.

Axonal

The PDE neurons of uric-33, uric-44, uric-51, and unc7’6 have, to varying extents, more axons and branches than the wild type. Mo:st commonly, these processes extend along the subventral or subdorsal muscle edges. These positions are natural grooves in the hypodermis and are the normal tracts for a small number of longitudinal processes in the animal (Fig. 15). No PDE process in any of these rnutants has been found in the dorsal nerve cord. The most frequent defect in me-33 and uric-76 mutants is an ectopic branch or spur where the ventrally directed axon crosses the subventral muscle edge. There is a strong bias for such ectopic branches to extend posteriorly rather than anteriorly. This might be explained if the mutant a.xons are following the PLM neuron (Fig. 15), a synaptic partner of PDE with cell body in the tail. Indeed, the growth cones of normal PDE neurons may explore the PLM axon as an alternative while they search for higher affinity contacts in the ventral cord neuropil. Role of Neuronal

Cytoskeletm

Surface molecules that confer specific recognition in the nervous system are presumably confined to subsets of cells (McKay, 1983; Goodman et ah, 1984). In contrast, the cytoskeletal machinery that converts selective adhesion into directed growth may be common to all neurons (Bray, 1982, 1984.; Letourneau, 1982). The unc33 (eZ@&) mutation disrupts axon guidance in many classes of neurons (unpublished observations). The dendrites of sensory neurons, and possibly all neurons, have an abnormally large number of microtubules in these mutants. Moreover, these microtubules are frequently misassembled. Taken together, it seems likely that the uric-33 product is a component of the axonal cytoskeleton and that the guidance defects observed in uric-33 mutants are caused by the cytoskeletal defect. In particular, the normal uric-33 product may be a microtubule associated protein that limits the assembly or stability of neuronal microtubules. uric-44 mutations, and to a lesser extent, uric-33 mutations impair dye uptake by the chemosensory neurons. The cause appears to be that axonemes of the chemosensory cilia are abnormally short. It is tempting to speculate that uric-44 and uric-33 specify proteins affecting both axonal and ciliary microtubules. We thank John Sulston and *John White for help in identifying cells and for their generous help throughout this work; Jonathan Hodgkin for providing strains; Donna Albertson, Dennis Bray, Richard Durbin, Andrew Fire, David Hall, Cynthia Kenyon, Anthony Otsuka, Richard Russell, and Shahid Siddiqui for many helpful discussions;

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and Martin Chalfie, Frank Margolis, and Aaron Shatkin for comments on the manuscript. E.H. was recipient of postdoctoral fellowships from the Muscular Dystrophy Association of America and the National Institutes of Health. Part of this work was supported by NIH Grants NS16510 and NS20258 and Basil O’Connor Research Grant No. 5-411 from the March of Dimes Foundation to J.C. REFERENCES ALBERTSON, D. G., and THOMSON, J. N. (1976). The pharynx of Caenorhabditis elegans. Phil. Trans. R. Sac. London Ser. B 2X,299325. BENTLEY, D., and KESHISHIAN, H. (1982). Pathfinding by peripheral pioneer neurons in grasshoppers. Science (Washington, D. C.) 218, 1082-1088. BRAY, D. (1982). Filopodial contraction and growth cone guidance. 1n “Cell Behavior” (R. Bellairs, A. Curtis, and G. Dunn, eds.), pp. 299-317. Cambridge Univ. Press, London/New York. BRAY, D. (1984). Axonal growth in response to experimentally applied mechanical tension. Dev. Biol. 102, 379-389. BRENNER, S. (1973). The genetics of behavior. Brit. Med. Bull. 29, 269-271. BRENNER, S. (1974). The genetics of Cuenorhabditis elegant. Genetics 77,71-94. FROHLICH, A., and MEINERTZHAGEN, I. A. (1983). Quantitative features of synapse formation in the fly’s visual system. I. The presynaptic photoreceptor terminal. J. Neurosci. 3, 2336-2349. GOODMAN, C. S., RAPER, J. A., Ho, R. K., and CHANG, S. (1982). Pathfinding by neuronal growth cones in grasshopper embryos. In “Developmental Order: Its Origins and Regulation” (S. Subtelny and P. G. Green, eds.), pp. 275-316. Liss, New York. GOODMAN, C. S., BASTIANI, M. J., RAPER, J. A., and THOMAS, J. B. (1984). Cell recognition during neuronal development in insect embryos. In “Molecular Bases of Neural Development” (W. Cowan, ed.), Neuroscience Res. Prog. Press. HALL, D. H., and RUSSELL, R. L. (1985). Electron microscopic anatomy of the posterior nervous system of the nematode Caenorhabditis elegans. Submitted for publication. HERMAN, R. (1984). Analysis of genetic mosaics of the nematode Caenorhabditis elegans. Genetics 108, 165-180. HORVITZ, H. R., CHALFIE, M., TRENT, C., SLJLSTON,J. E., and EVANS, P. D. (1982). Serotonin and octopamine in the nematode Caenorhab ditis elegans. Science (Washington, D. C.) 216, 1012-1014. HORVITZ, H. R., STERNBERG, P. W., GREENWALD, I. S., FIXSEN, W., and ELLIS, H. M. (1983). Mutations that affect neural cell lineages and cell fates during the development of the nematode Caenwhab ditis elegans. Cold Sprkg Harbor Symp. Quant. BioL 48, 453-463. LETOURNEAU, P. C. (1982). Nerve fiber growth and its regulation by extrinsic factors. In “Neuronal Development” (N. C. Spitzer, ed.), pp. 213-254. Plenum, New York. MCKAY, R. D. G., HOCKFIELD, S., JOHANSEN, J., THOMPSON, I., and FREDERISKEN, K. (1983). Surface molecules identify groups of growing axons. Science (Washington, D. C.) 222, 788-794. PERKINS, L. A., HEDGECOCK, E. M., THOMSON, J. N., and CULOTTI, J. G. (1985). Mutant sensory cilia in Caenmhabditis elegans. Submitted for publication. SULSTON, J. E., ALBERTSON, D. G., and THOMSON, J. N. (1980). The Caenorhabditis elegans male: Postembryonic development of nongonadal structures. Dev. Biol. 78, 542-576. SULSTON, J., DEW, M., and BRENNER, S. (1975). Dopaminergic neurons in the nematode Caenorhabditis elegans. J. Camp. Neural. 163,215226. SULSTON, J. E., and HORVITZ, H. R. (1977). Postembryonic cell

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lineages of the nematode Caenorhabditis elegans. Dev. Biol 56, 110-156. SULSTON,J. E., and HORVITZ,H. R. (1981). Abnormal cell lineages in mutants of the nematode Caenorhabditis elegans. Dev. Biol. 82,4155. SULSTON,J. E., and WHITE, J. G. (1980). Regulation and cell autonomy during postembryonic development of Caenorhabditis elegans. Dev. Biol. 78, 577-597.

SULSTON,J. E., SCIERENBERG,E., WHITE, J. G., and THOMSON,J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64-119. SWANSON,M. M., EDGELEY,M. L., and RIDDLE, D. L. (1984). Caewrhabditis elegans. In “Genetic Maps,” (S. O’Brien, ed.) pp. 244-258. Cold Spring Harbor Laboratory, New York. WATERSON,R. H., THOMSON,J. N., and BRENNER,S. (1980). Mutants with altered muscle structure in Caewrhabditis elegans. Dev. Biol. 77, 271-302.

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WHITE, J. G., HORVITZ, H. R., and SULSTON,J. E. (1982). Neuron differentiation in cell lineage mutants of Caenorhabditis elegans. Nature

(London) 297, 584-589.

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WHITE, J. G., SOUTHGATE,E., THOMSON,J. N., and BRENNER, S. (1983). Factors that determine connectivity in the nervous system of Caenorhabditis elegans. Cold Spring Harbor Symp. &ant. Biol. 48, 633-640. WHITE, J. G., SOUTHGATE,E., THOMSON,J. N., and BRENNER, S. (1985). The nervous system of Caenerhabditis elegans. Phil. Trans. R. Sot. London Ser B, in press. ZENGEL,J. M., and EPSTEIN, H. F. (1980). Identification of genetic elements associated with muscle structure in the nematode Caenorhabditis elegans. Cell Motil. 1, 73-97.