F BRENNER (1973, 1974), many single-gene mutants

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1 Copyright by the Genetics Society of America Mosaic Analysis of Two Genes That Affect Nervous System Structure in Caenorha bditis elegans Robert K. Herman Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota Manuscript received February 6, 1987 Accepted April 2, 1987 ABSTRACT The mutation mec-4(e161 I), identified by M. Chalfie, leads to the degeneration and death of the six neurons, called the microtubule cells, that mediate the response of wild-type animals to light touch. The fates of two of these cells, PLML and PLMR, which are responsible for response to light touch in the tail of the animal, have been monitored in animals mosaic for the mec-4(e1611) mutation. The results are consistent with the view that the mutation behaves cell autonomously in its killing effect; in particular, none of the neurons that make either chemical synapses or gap junctions to PLML or PLMR is responsible for the deaths of PLML or PLMR. The results of gene dosage and dominance tests suggest that the mec-4(+) gene product, which is required for wild-type microtubule cell function, is altered by the e1611 mutation into a novel product that kills the microtubule cells. Mutation in the gene unc-3 leads to the derangement of the processes of the motor neurons of the ventral cord. Mosaic analysis strongly suggests that unc-3(+) expression is required only in the motor neurons themselves for normal neuronal development. In particular, the hypodermis surrounding the ventral cord is not the primary focus of unc-3 action (body muscle was excluded in earlier work). Finally, the mosaic analysis supports an earlier suggestion that a sensory defect caused by a daf-6 mutation is localized to a non-neuronal cell called the sheath cell. OLLOWING a strategy outlined and initiated by F BRENNER (1973, 1974), many single-gene mutants affecting the behavior of the nematode Caenorhabditis elegans have been identified and are being characterized by a variety of methods. A major goal of this work is to elucidate the genetic basis of nervous system development and function. Not all mutants affected in behavior are expected to have nervous system defects; the primary defects in certain paralyzed mutants, for example, are in proteins of the body wall muscle (for review, see WATERSTON and FRANCIS 1985). Many behavioral mutants, however, have been shown to have structural aberrations in particular sets of neurons (BRENNER 1973; LEWIS and HODCKIN 1977; CHALFIE and SULSTON 1981 ; ALBERT, BROWN and RIDDLE 1981; HEDGECOCK et al. 1985; PERKINS et al. 1986; J. WHITE, personal communication). These mutants are being used to address two general problems: the first is the relationship between nervous system structure and function, and the second is the role of the wild-type genes in normal nervous system development, Regarding the second problem, an important question about a gene known to affect neuronal structure is whether it exerts its effect cell autonomously or, alternatively, through cell-cell interaction. In this paper I use genetic mosaics to ask this question for two genes, mec-4 and unc-3. Each of these genes was originally identified through the isolation of a behavioral mutant and was subsequently shown to affect neuronal structure. It has proved useful for technical reasons to study the expression of mec-4 and unc-3 (as well as a third gene-daf-6) in identical sets of mosaic animals. The mutation mec-4(e1611) leads to the degeneration and death of six sensory neurons, the microtubule cells, a few hours after they are born (CHALFIE and SULSTON 1981). CHALFIE and SULSTON (1981) have shown by laser ablation of the microtubule cells or their precursors that the microtubule cells mediate the response of wild-type animals to light touch. Animals carrying e1611, which is dominant to its wildtype allele, are touch insensitive (CHALFIE and SULS- TON 1981). M. CHALFIE (personal communication) has assigned e1611 to the gene mec-4, which is also represented by recessive alleles that confer touch insensitivity without having any apparent effect on microtubule cell structure (CHALFIE and SULSTON 1981). Additional mec-4 alleles with properties like those of e161 1 have also been identified (M. CHALFIE, personal communication). All of the synaptic connections made by the microtubule cells have been deduced from reconstructions derived from serial section electron micrographs (CHALFIE et al. 1985). In this paper I have focused on the left and right posterior lateral microtubule cells, PLML and PLMR, which are responsible for response to light touch in the tail. I have then asked whether the deaths of PLML and PLMR conferred by mec-4(e1611) might be the result of murder Genetics (July, 1987)

2 378 R. K. Herman by one or more neurons to which the microtubule cells synapse; the results rule out all of these potential murderers. The hypodermis has not been cleared of all suspicion, but microtubule cell death by suicide seems very likely. Mutations in unc-3 confer a severe uncoordination; animals move their heads normally but are unable to propagate along their bodies the normal dorsoventral bends that are necessary for smooth movement. In earlier work making use of unc-3 mosaic animals (HER- MAN 1984), I showed that unc-3 has a diffuse focus of action that is at least primarily localized among the descendants of two sister cells, called AB.pl and AB.pr: when all the descendants of both AB.pl and AB.pr lacked unc-?(+), the animal showed a complete Unc-3 phenotype; but when the descendants of just one of the two cells, either AB.pl or AB.pr, lacked unc-?(+), then the animal was semi-unc-3. Inspection of the completely known C. eleguns cell lineage (SULS- TON et al. 1983), led to the suggestion that prime candidates for the focus of unc-3 action were the motor neurons, or a subset of the motor neurons, of the ventral and dorsal nerve cords. Body muscle cells were shown not to be involved in unc-3 expression; only one body muscle cell derives from the sisters AB.pl and AB.pr, and the absence of unc-?(+) from the other 94 body muscle cells did not result in uncoordinated movement. More recently, the ventral cord of unc-3 animals has been studied by serial section electron microscopy (J. WHITE, personal communication). It was found that all classes of motor neuron in the ventral cord had highly disorganized processes. Most did make neuromuscular junctions but often at ectopic sites. The processes of interneurons were relatively undisturbed from their wild-type locations, but the motor neurons did not receive their appropriate interneuron synaptic inputs because of the misplacement of their processes along the cord U. WHITE, personal communication). A possible explanation for the rather general disorganization of the motor neuron processes in the ventral cord that would be consistent with the previous mosaic analysis is that the focus of action of unc-3 is in the hypodermis surrounding the cord rather than in the motor neurons themselves; according to this view, the "?(+) gene product might be needed in the hypodermis to direct normal outgrowth of motor neuron processes. Results presented below make this explanation very unlikely and make stronger the likelihood that unc-3(+) expression is required only in the motor neurons themselves for normal neuronal development, MATERIALS AND METHODS Genes, alleles, genetic metbods and nomenclature: The Bristol strain N2 was the wild-type parent for all strains used in this work. The following genes and mutations were used: Linkage group (LG) I: dpy-5(861) and unc-54(e190) (BREN- FIGURE I.-Linkage used in this work. map of the right end of the X, showing loci NER 1974). LG 111: unc-93(e1500) (GREENWALD and HORV- ITZ 1980). LG X: unc-3(e151), unc-t(e5) and unc-9(elol) (BRENNER 1974), duf6(e1377) (ALBERT, BROWN and RIDDLE 198 I), sup-lo(mn219) (HERMAN 1984), osm-l(p808) (CU- LOTTI and RUSSELL 1978), and mec-#(e1611) and mec- 4(e1497) (CHALFIE and SUUTON 1981), which will be referred to as mec-#(d) and mec-4(r), respectively, where d and r stand for dominant and recessive, respectively. The e1611 mutation, previously assigned to mec-13 (CHALFIE and SUS- TON 1981), has been assigned to the mec-4 gene because of its very tight linkage to mec-4(r) (M. CHALFIE, personal communication) and because reversion of the dominant touch sensitive phenotype of mec-4(d)/+ animals has yielded a new recessive mec-4 allele (M. CHALFIE, personal communication). A genetic map of the right end of the X chromosome showing the loci used in this study is given in Figure 1. Media, culture and mating techniques were as described by BRENNER (1974) and HERMAN (1978). Unless otherwise noted, animals were grown at. Genetic nomenclature follows the guidelines described by HORVITZ et al. (1979). Duplications: The X duplications carrying mec-4(d) were induced, identified and characterized by C. KARI (personal communication) by methods that have been generally described (HERMAN, ALBERTSON and BRENNER 1976). In brief, mec-#(d)/o males were gamma-irradiated by I3'Cs in a Shepherd irradiator (model ). Doses of 7,200 roentgens (r) were used at a dose rate of 600 r/min. The irradiated males were mated to unc-? or unc-7 hermaphrodites, and the progeny were screened for non-unc males. Duplicationbearing strains were outcrossed at least three times before the constructions of the duplication-bearing stocks used in this work were begun. The average percentage nulloduplication self progeny of mndp7(x;f)-bearing hermaphrodites was 56% (1282 total progeny), and the average percentage of nullo-duplication self-progeny of mndp2l (X;I)/+ hermaphrodites was 24% (990 total progeny). About one-third of the duplication-bearing progeny of mndp21/+ hermaphrodites were mndp21 homozygotes, which segregated virtually no nullo-duplication-bearing self-progeny (HER- MAN, MADL and KARI 1979); mndp2l was shown to be loosely linked to dpy-5 and very tightly linked (less than 0.2% recombination) to unc-54. Both mndp7 and mndp21 complement unc-3, daf-6, sup-10 and osm-1 and do not complement unc-9. Strain constructions: Most of the strains were constructed by standard methods that need not be described. The generation of an unc-93; unc-? sup-10 osm-1 stock, which was used in the construction of the mndp21/+; unc- 93; unc-3 sup-10 osm-1 and unc-93; unc-3 sup-10 osm-1; mndp7 strains, has been described (HERMAN 1984). The Unc-93 and Unc-3 phenotypes are clearly distinguishable, and sup-io is a recessive suppressor of unc-93 (GREENWALD and HORVITZ 1980). The two strains referred to in RESULTS as mndp21/+; unc-3 daf-6 osm-1 and unc-3 daf-6 osm-1 ; mndp7 actually had the following genotypes, respectively: mndp2 I/+; unc-3 daf-6 sup-10 osm-1 and unc-3 daf-6 sup-10 osm-1; mndp7. The sup-10 mutation in these strains confers no detectable phenotype; its presence was a residue of the method of strain construction that made use of sup-io in conjunction with unc-93 to select a recombinant chromosome carrying

3 Neural Mutant Mosaics of C. eleguns 379 both duf-6 and osm-1, each of which abolishes FITC staining of amphid and phasmid neurons (see below). The strains were thus derived from an unc-3 daf-6 sup-10 osm-1 strain, which was constructed as follows. N2 males were mated with unc-93; sup-10 osm-1 hermaphrodites. Wild-type male progeny were mated with unc-3 duf-6 hermaphrodites, and wildtype hermaphrodite progeny were picked. Those of genotype unc-93/+; sup-10 osm-llunc-3 daf-6 were identified on the basis that they segregated Unc-93 animals among their self progeny. Unc-93 non-unc-3 offspring were picked (putative genotype: unc-93; sup-io osm-llunc-3 duf-6). Many non-unc-93 non-unc-3 self progeny of these animals were picked individually. A small fraction yielded Unc-3 segregants, one of which was picked to establish a strain having the putative genotype unc-93; unc-3 daf-6 sup-10 osm-i. The presence of the duf-6 mutation in this strain was confirmed by mating with daf-6 males, picking wild-type hermaphrodite cross progeny and ascertaining their FITC staining pattern. Next, the unc-93; unc-3 daf-6 sup-io osm-1 strain was mated with N2 males; wild-type hermaphrodite progeny were picked and allowed to self. Wild-type hermaphrodite progeny were picked to identify a brood containing Unc-3 animals but no Unc-93 animals. An Unc-3 animal was picked from such a brood to establish a putative unc-3 daf-6 sup-10 osm-1 strain. The presence of both daf-6 and osm-1 in this strain was confirmed by FITC staining of wild-type progeny arising from the mating of the strain with daf-6 and osm-1 males, respectively. Identifying PLM cells: When present, PLMR and PLML are readily identifiable in live animals by Nomarski differential interference contrast microscopy (CHALFIE and SULS- TON 1981). Late L4 or young adult hermaphrodites were mounted on 5% agar slabs on microscope slides, prepared as described by SULSTON and HORVITZ (1977). Animals to be scored for the presence or absence of PLM cells were picked with a 32-gauge platinum wire and placed in a small drop of 5 times-diluted M9 buffer on an agar slab and covered with a coverslip; both the drop of buffer and the agar contained 0.5% 1-phenoxy-2-propanol as anesthetic. Zeiss Nomarski optics were used. As described by CHALFIE and SULSTON (198 l), the PLM cell bodies have a characteristic spindle shape and are located posterior to the anus, ventral to the lateral midline and close to the outer surface of the animal. The thinness of the animal in the region of the PLM cell bodies makes it possible, by focusing up and down, to score for the presence or absence of both PLM cells without having to turn the animal over (the animal lies on its side on the agar surface). Micrographs showing both PLM cells of a living animal are shown in Figure 2, A and B. FITC staining procedure and identifying stained and unstained neurons: The fluorescein isothiocyanate (FITC) staining protocol on agar plates described by HEDCECOCK et al. (1985) was used. Live animals were exposed to FITC for at least 3 hr, mounted as described above for Nomarski microscopy and observed with a Zeiss microscope equipped with epifluorescence. Most screening was done using a 40X Neofluor objective. Micrographs and diagrams showing the relative positions and cell names of the amphid and phasmid neurons that are normally stained by FITC have been published by HEDGECOCK et ue. (1985). For a brief discussion of cell nomenclature, see the legend to Figure 3. When only one of the two neurons of a phasmid stained and it was important to ascertain whether it was PHA or PHB, I used Nomarski optics (1 OOX Plan objective) to locate the PHA and PHB nuclei. PHA is immediately posterior to the middle of the rectum and anterior to PHB; both cells tend to be closer to the cell surface than other neurons in the neigh- borhood. An eyepiece micrometer disc was used to mark the position of one of the nuclei, and without moving the microscope stage or objective, the optics were switched to epifluorescence to see which cell body was stained. Figure 2, C-E, shows two phasmid neurons by both Nomarski and fluorescence microscopy. Micrographs showing both neurons of both phasmids of one animal are shown in Figure 2, F-G. The three more dorsal (ASK, ADL and ASI) of the six FITC-stained cell bodies of an amphid are situated fairly close to one another so that I could not always be confident that all three, rather than just two, neurons were staining. The other three cell bodies are well spaced, however. The position, and hence FITC staining status, of ASH was determined with reference to its stained neighbors, ADF and ASJ. ADF is the most dorsal and anterior of the three; ASJ is the most ventral and posterior; ASH is the nearest to the outer surface of the animal (HEDGECOCK et al. 1985). Figure 2H illustrates the relative position of ASH among the FITC stained cell bodies of an amphid. No attempt was made to locate any of the amphid neurons by Nomarski microscopy. The results of various controls on FITC staining are given in Table 1. The incidences of nonstaining found in the mndp21 strain are taken as indicative of background levels of nonstaining, unrelated to somatic duplication loss, because mndp21 is a translocated duplication. The mndp21 strain showed some nonstaining phasmids (both neurons nonstaining) and some nonstaining single neurons (one neuron nonstaining, other neuron staining) of phasmids. The incidences of such nonstaining seem to have been enhanced by the mec-l(d) mutation carried by mndp21, since the mec- 4(d) mutation by itself showed similar frequencies of nonstaining. The results with N2, showing a low but significant phasmid nonstaining (possibly due to blockage of the phasmid channel) and virtually no single phasmid neuron nonstaining, confirm previous findings (HERMAN 1984). No nonstaining amphids were observed in any of these three control strains. At least some of the nonstaining observed with the mndp7(x;f)-bearing strains is expected to be due to somatic loss of the free duplication (see RESULTS). The data given in Table 1 were obtained with animals displaying wildtype movement, whereas the data given in RESULTS were obtained with animals displaying a semi-unc-3 phenotype. Touch sensitivity tests: Nematode touch sensitivity was tested by the method Of CHALFIE and SULSTON (198 I), using the light transverse touch of an eyebrow hair. Only posterior touch responses were tested, and only animals that were temporarily stationary were tested. If an animal responded by moving forward after each of three successive light touches across its posterior half, it was deemed tail touch sensitive. Touch insensitive animals, although unresponsive to the light touch, responded to a heavier touch, from a platinum wire, for example. The Unc-93 phenotype seemed to make animals more touch sensitive; thus, unc-93 and unc- 93; mec-4(d) controls were important references for touch tests on other Unc-93 animals. RESULTS Phenotypes conferred by mec-l(d) and mec-4(r) : In this work I have focused on just two of the six microtubule cells found in adult animals: PLML and PLMR, which are responsible for the tail touch response (CHALFIE and SULSTON 1981). Homozygous young adult me~-4(e1611)-referred to hereafter as mec- 4(d)-animals raised at were touch insensitive in the tail, and both their PLM cells were invariably

4 380 R. K. Herman 1 f FIGURE 2.-Photomicrographs of N2 animals showing PLML, PLMR and phasmid and amphid neurons stained with FITC. A and B show the two PLM cells of a single animal by Nomanki optics; the right side of the animal is at a higher plane of focus than the left, and the anterior of the animal is to the right in both pictures. C is a Nomarski picture, with PHAR and PHBR marked with arrows. That the arrows do in fact identify the cells that take up FITC is illustrated by D and E. D is a fluorescence picture of the anesthetized animal in C, and the print E was produced by superimposing the negatives used to make C and D. F and G show both phasmid neurons of both phasmids of a single animal by Nomanki optics; the anterior of the animal is to the right in both pictures. H is a fluorescence picture showing the ASHR cell body situated between the cell bodies of ADFR and ASJR; the anterior of the animal is to the right. The bar in each picture is 10 Pm. TABLE I control. on FITC d dng Fraction of Fraction of single Fraction of Fraction of single phasmids phasmid neurons amphie ASH neurons -ype nonstaining nonstaining nonstaining nonstaining Wild type (N2) 2/108 (0.02) 0/2 12 (0.00) 0/1OS (0.00) w ~ ~ - 4 ) ( d 6/98 (0.06) 6/184 (0.03) 0/98 (0.00) w mndppl(x;i)/+; unc-3 om-1' 19/204 (0.09) 3/370 (0.01) 0/204 (0.00) 1/204 (0.01) unc-3 om-i; mndp7(x& 15/140 (0.1 1) 24/250 (0.10) 0/140 (0.00) 1/38 (0.03) unc-3 daf4 om-1; "h47 10/102 (0.10) 6/184 (0.03) 3/102 (0.03) 6/99 (0.06) ND = not determined. ' The animals screened were the non-unc-3 self progeny of mndp21/+; unc-3 om-1 hermaphrodites; tw-thirds of such animals are mndp21 heterozygotes and one-third are mn421 homozygotes. absent and presumed killed; see Table 2. Table 2 also shows that animals bearing a single copy per cell of mcc-#(d) were often touch insensitive in the tail and missing PLM cells, but the penetrance of a single copy

5 Neural Mutant Mosaics of C. elegans TABLE 2 Effeets of mec4(d) and mec4(r) on tail touch sensitivity and survival of PLM ells 38 1 Genotype. mec-4(d)/mec-4(d) mec-4(d)/unc-3 mec-4(d)/unc-3 unc-3 mc-4(d)/+ mcc-l(d)/unc-3 unc-3 mcc-4(r)/mcc-l(r) unc-3 mcc-#(r)/+ unc-3 mcc-#(r)/mec-4(d) mndp2l(xl)/mndppl; unc-3 osm-1 mndp21/+; unc-3 osm-1 mndp21/+; unc-3 daf-6 osm-1 mndp2ll-k; unc-9r unc-3 sup-10 osm-1 unc-3 osm-1; mndp7(xfi unc-3 daf-6 osm-1; mndp7 unc-93; unc-3 sup-10 osm-1; mndp7 Fraction of an- Temperature imals tail touch insensitive Fraction of PLM cells kill& 16" O 20 O 20 O 20 O 20 O 20/20 (1.O) 27/27 (1.0) 13/19 (0.68) 29/40 (0.73) 15/33 (0.45) 20/20 (1.O) 0/20 (0.0) 40/40 (1.O) 58/58 (1.0) 9/13' (0.69) 17/20' (0.85) 83/88b (0.94) 31/44 (0.70) 53/70 (0.76) 83/86 (0.97) 30/30 (1.O) 78/80 (0.98) 30/38 (0.79) 25/32 (0.78) 28/54 (0.52) 0/30 (0.0) 0/28 (0.0) 22/34 (0.64) 32/32 (1.O) 40/57' (0.70) 37/40' (0.93) 51/60' (0.85) 77/108 (0.71) 83/102 (0.81) 94/96 (0.98) a The duplications mndp7 and mndp21 carry mc-4(d). ' The animals picked for these determinations were non-unc-3 self-progeny of heterozygous mndp2l hermaphrodites; because one-third of such animals are in fact mndp21 homozygotes (yielding no Unc-3 self progeny), which give 100% penetrance for tail touch insensitivity (and PLM cell death), one-third of the total animals (or cells) scored were subtracted from both numerator and denominator. The statistical errors in these determinations are thus somewhat greater than the simple fractions given would otherwise imply. of the mutation was not loo%, whether ascertained by effect on tail touch insensitivity or killing of the PLM cells. Note that both the translocated duplication mndppl(x;i) and the free duplication mndp7(x;f) carry mec-l(d). Among animals carrying a single copy of mec-l(d), those determined to be tail touch sensitive were virtually always found to have present either PLML or PLMR or occasionally both; for example, among 12 tail touch sensitive unc-3 osm-1; mndp7 animals, five had PLML only present, four had PLMR only present and three had both PLML and PLMR present. This result is consistent with the finding of CHALFIE and SULSTON (1981) that the ablation of either PLML or PLMR (but not both) does not abolish the tail touch response. Single copy mec-4(d) animals judged to be tail touch insensitive, however, were sometimes found to have a PLM cell present; for example, among 19 tail touch insensitive unc-3 osm-1 ; mndp7 animals, three had PLML only present, one had PLMR only present, and the remaining 15 had neither PLM cell present. This result suggests that some surviving PLM cells are nonfunctional. A few times in the course of these experiments, a PLM cell observed to be present in a young adult heterozygous mec-l(d) animal was subsequently observed to undergo cell death; the dying cell became rounded and the cytoplasmic region took on the appearance of a vacuole, with the nucleus or remnant of the nucleus apparent for a time within the vacuole (CHALFIE and SULSTON 1981). Clearly, in these animals the process of cell death occurred much later than it occurs in mec-l(d) homozygotes. The effect of a single copy of mec-l(d) on PLM cell death and the touch response was temperature sensitive: the penetrance of the mutation decreased with increasing temperature (Table 2). The penetrance of the mutation seemed also to depend on genetic background; a homozygous unc-93 gene, for example, seemed to enhance the penetrance of a single copy of mec-4(d) (Table 2). This is probably an indirect effect (conceivably due to reduced growth rate), since unc- 93 appears to confer a muscle defect (see below for references). Animals carrying two mec-4(d) genes per cell, on mndp21, in addition to two mec-4(+) genes, on the X chromosomes, showed 100% penetrance for the mutant phenotype; thus the wild-type alleles seemed to have no counteracting effect on the survival of the PLM cells. Animals homozygous for the mec-4(r) mutation were invariably tail touch insensitive, but the PLM cells were always present; mec-4(r) was completely recessive to mec-4(+) with respect to its effect on touch sensitivity (Table 1). The mec-4(d)/mec-4(~) heterozygotes were all tail touch insensitive; however, the frequency of PLM cell death in mec-4(d)/mec-4(r) animals was characteristic of that found in animals carrying a single copy of mec-4(d) and either one or two meel(+) alleles. These results lead to two suggestions: first, that the mec-4(+) gene is essential for the proper functioning in the touch response of those few surviving PLM cells that support a touch response in mec(d)/mec-4(+) heterozygotes; and second, that the death of the PLM cells is determined solely by the product of the mec- 4(d) gene, two copies of the gene giving a greater

6 382 R. K. Herman PO AB PI ASJL ASKL AB.pla 32 Body AB.plp AB.pra AB.prp Muscle Cells D p4 probability of cell death than one copy and ma-#(+) and mec-#(r) having little or no effect on the killing of PLM cells. The second suggestion is also supported by the results with mndp21 homozygotes already noted. Genetic mosaics and cell lineage: The mec-#(d) mutation carried by mndp7, as shown above, leads to the death of about 70% or more of the PLM cells in mndp7-bearing animals grown at 20. To ask about the cell autonomy of mec-#(d), I have identified animals in which mndp7 was lost somatically during embryogenesis; the question is whether or not the absence of mec-#(d) from a particular PLM cell (and not any other cell) invariably rescues the PLM cell from death. I emphasize that the great majority of the surviving PLM cells that are found in mndp7-bearing animals are not surviving as a consequence of free duplication loss. Animals carrying a single copy of a translocated duplication, mndp21, which should not be subject to loss (HERMAN 1984), showed comparable frequencies of survival of PLM cells (Table l), and the frequency of PLM cell survival was much too high to be accounted for by spontaneous mndp7 loss (see below). The approach, then, was to make use of other genetic markers carried by mndp7 to select for mosaics in which I could specify at which cell division or divisions duplication loss must have occurred and then to see whether such losses invariably led to the rescue of one or both PLM cells. Figure 3 gives the lineages (SULSTON et al. 1983) of PLML, PLMR and other cells that are relevant to the action of the other genetic markers used. Loss of mec-d(d) at PI: I selected directly for duplication loss at PI (Figure 3) using hermaphrodites of genotype unc-93; unc-3 sup-io osm-i; mndp7. The unc-93 mutation confers an uncoordinated phenotype easily distinguishable from Unc-3 (GREENWALD and HORVITZ 1980). It also makes hermaphrodites egglaying deficient; as a consequence, the eggs hatch internally, killing the parent and limiting the brood size to about 30. The sup-10 mutation is a recessive suppressor of both the uncoordination and egg-laying deficiency conferred by unc-93 (GREENWALD and HORVITZ 1980). It has been concluded that the actions of both unc-93 and sup-io are specific to muscle cells, both body wall muscles, which affect overall body coordination, and egg-laying muscles, which affect egg-laying (GREENWALD and HORVITZ 1980; HERMAN 1984). The phenotype of unc-93; unc-3 sup-io osm-i; mndp7 hermaphrodites is Unc-93 because of the presence of the dominant sup-io(+) allele on mndp7, which also carries unc-3(+) and osm-i(+). Self-progeny lacking the duplication are Unc-3 Osm-1 non-unc-93. The unc-93 mutation is suppressed in the great majority of body muscle cells owing to a single event of duplication loss in unc-93; unc-3 sup-io osm-i; mndp7 zygotes if the loss occurs at PI but not later in the lineage (HERMAN 1984); this is because PI is precursor cell to 94 of the 95 body muscle cells, and its two daughter cells, EMS and PP, are precursors to 42 and 52 body muscle cells, respectively (Figure 3). An animal that has suffered duplication loss at PI is also egg-laying proficient, because the egg-laying muscles all derive from PI, and non-unc-3, because the action of the unc-3 gene is specific to the AB (sister of PI) lineage (the osm-1 gene was ignored in this set of experiments). Finally, because P1 is precursor to the germ line, the non-unc-3 non-unc-93 duplication mosaic gives only nullo-mndp7 self-progeny, which are therefore all Unc-3 non-unc-93. I found five animals satisfying all of these criteria, at a frequency of about one per 400 duplication-bearing animals.

7 Neural Mutant Mosaics of C. elegans 383 Three of the five were missing both PLM cells, and the other two had PLML present and PLMR absent. Four (including the two animals with PLML present) of the five animals were tested for tail touch sensitivity; all were judged to be touch insensitive. I conclude that the absence of mec-4(d) from all the descendants of PI does not save either PLML or PLMR from the killing effect of the mec-l(d) mutation. Loss of me44 at AB.pl or AB.pr: In previous experiments I showed that the loss of an unc-3(+) duplication at AB or AB.p (posterior daughter of AB) in an otherwise homozygous unc-3 animal leads to an Unc-3 phenotype and that duplication loss at AB.pl or AB.pr (the daughters of AB.p) gives rise to a semi- Unc-3 phenotype (HERMAN 1984). Unc-3 animals are unable to back up when touched on the head with a platinum wire (to which even touch insensitive animals normally respond); semi-unc-3 animals do back up but in an uncoordinated fashion, characteristic of a weak unc-3 allele. Not all semi-unc-3 animals arise from duplication loss at AB.pl or AB.pr; later duplication losses can also produce the semi-unc-3 phenotype. The osm-l gene can be used to identify those animals in which duplication loss occurred at AB.pl or AB.pr. Wild-type animals exposed to the fluorescent dye FITC incorporate the dye into sixteen chemosensory neurons: twelve in a pair of sensilla in the head called amphids and four in a pair of sensilla in the tail called phasmids (HEDGECOCK et al. 1985). The osm-1 mutation abolishes FITC staining (PERKINS et al. 1986) in what appears to be a cell autonomous fashion (HERMAN 1984). The lineages of the neurons stained by FITC in wild-type animals are shown in Figure 3. Loss of an osm-1 (+) duplication at AB.pr in an otherwise homozygous osm-1 mutant, assuming cell autonomous expression, generates an animal in which none of the neurons of either the right amphid or right phasmid stain, but all the neurons of the left amphid and left phasmid stain normally; see Figure 3. On the other hand, loss of osm-i(+) at AB.pl will generate an animal in which neither neuron of the left phasmid stains but only two neurons (ASIL and ASHL) of the six of the left amphid are nonstaining because the other four descend from AB.a. I have screened the self progeny of unc-3 osm-i; mndp7 hermaphrodites for young adult semi-unc-3 animals; they were found at a frequency among mnd7- bearing animals of roughly 0.5%. The semi-unc-3 animals were exposed to FITC and scored both for patterns of FITC staining and for survival of the PLM cells. Because of their defective movement, response to light touch was not determined. Among 96 semi- Unc-3 animals scored, 12 fit the expected FITC staining pattern for duplication loss at AB.pr (Table 3); in all 12 cases PLMR was present, but in only one case was PLML present. As explained in MATERIALS AND TABLE 3 Semi-Uno3 animals derived from unc-3 osm-1; mndp7 zygotes No. with No. with Proposed point same-side opposite-side of duplication No. of PLM PLM FITC nonstaining loss animals* present present RA and RP AB.pr ASHL and LP AB.pl /6 RA and PHBR AB.pra PHBL only AB.pla or descendant ASHR and PHAR AB.prp ASHL and PHAL AB.pIp a RA refers to all six normally stainable neurons of the right amphid 5/6 RA refers to all six neurons of the right amphid except a ventrally placed one, presumed to be ASHR; RP and LP refer to both neurons of the right and left phasmids, respectively. * The 48 occurrences of the nonstaining patterns listed here were found in 47 animals; one severely semi-unc-3 animal showed nonstaining patterns characteristic of duplication loss at both AB.pl and AB.pra. An additional 49 semi-unc-3 animals did not show any of the six listed patterns of nonstaining; most of these showed some nonstaining but all of the patterns corresponded to duplication loss (or losses) later in the AB.p lineage. Within this sec of 49 animals, the fates of 86 PLM cells were ascertained: 24 were found to be present. METHODS, the FITC staining status of ASIL was not determined; therefore, the criteria for duplication loss at AB.pl were nonstaining of ASHL and nonstaining of the left phasmid. Seventeen semi-unc-3 animals satisfied these criteria (Table 3); in 16 of the 17 animals, PLML was present, but in only eight was PLMR present. I presume that the one exceptional animal in which PLML was not present is a case in which the pattern of nonstaining was produced by two separate events rather than by duplication loss at AB.pI. Each of the separate events could be a duplication loss or simply background nonstaining. Controls on FITC staining of non-unc-3 unc-3 osm-i; mndp7 animals (Table l), indicate that such excep tions should be expected at a frequency of roughly 0.11 X 0.03 = 0.3% of the semi-unc-3 animals, assuming the two separate events are independent. Double duplication losses may be somewhat more frequent than expected for independent events (E. HEDGE- COCK, personal communication), but not markedly so (KENYON 1986). I conclude that duplication loss at AB.pr saves PLMR but not PLML from death, and duplication loss at AB.pl saves PLML but not PLMR from death. The conclusion just drawn was confirmed by genetic mosaics generated by duplication loss in unc-3 daf-6 osm-i; mndp7 animals. The daf-6 mutation also abolishes FITC staining (PERKINS et al. 1986), but in this case it appears to be the genotype of a single nonneuronal supporting cell, the sheath cell, that determines whether the neurons of a given sensillum stain-in all-or-none fashion (HERMAN 1984; ALBERT, BROWN and RIDDLE 1981; also see results below for

8 384 R. K. Herman TABLE 4 Semi-Unc-3 animals derived from unc-3 daf-6 osm-i; mndp7 zygotes Proposed No. with No. with oppoint of same-side posite-side FITC duplication No. of PLM PLM nonstaining loss animalsb present present RA and RP AB.pr LA and LP AB.pl RAandPHBR AB.pra 0 LA and PHBL AB.pla ASHRandRP AB.prp 2 0' 0" ASHL and LP AB.plp a Abbreviations are the same as for Table 3 with the addition that LA refers to all six normally-stainable neurons of the left am hid 'The 28 occurrences of the nonstaining patterns listed here were found in 27 animals; the one animal showing a nonstaining pattern characteristic of duplication loss at AB.pla also showed nonstaining of RA and RP (and was the exceptional case, with PLMR present). An additional 17 animals did not show any of the six listed patterns of nonstaining; most of these showed some nonstaining but all of the patterns corresponded to duplication loss (or losses) later in the AB.p lineage. Within this set of 17 animals, the fates of 25 PLM cells were ascertained: two were present. The fate of only one PLM cell (of two) was determined. further justification of this assumption). If mndp7, which carries duf-6(+), is lost at AB.pr in an unc-3 duf- 6 osm-1; mndp7 animal, all right amphid and right phasmid neurons should be nonstaining, both because of the absence of osm-l(+) from the neurons and because of the absence of duf-6(+) from the right amphid sheath and right phasmid sheath (Figure 3). If the duplication is lost at AB.pl, the left amphid and left phasmid neurons are expected to be nonstaining because of the absence of duf-6(+) from the left amphid sheath and left phasmid sheath (the absence of osm-l(+) from ASIL, ASHL and both left phasmid neurons would also promote their nonstaining). Among 44 semi-unc-3 animals derived from unc-3 duf-6 osm-1; mndp7 zygotes, 16 FITC staining patterns were consistent with duplication loss at AB.pr (Table 4). All but one of these had PLMR present, but only four had PLML present. I again presume that the one exceptional animal, with PLMR absent, was a case in which the pattern of nonstaining was produced by two separate events rather than by duplication loss at AB.pr. Seven animals exhibited an FITC staining pattern characteristic of duplication loss at AB.pI (Table 4); in all seven cases PLML was present, but in only one case was PLMR present. Combining the data of Tables 3 and 4, I have seen 52 examples of FITC nonstaining characteristic of duplication loss at either AB.pl or AB.pr; in 50 of the 52 cases the same-side PLM was present, but in only 14 cases was the opposite-side PLM present; the latter frequency is that expected simply from the incomplete penetrance of the single copy of the mec-l(d) mutation. In summary, it seems clear that loss of mec-4(d) at AB.pr leads to rescue of PLMR but not PLML, and loss of mec-4(d) at AB.pl rescues PLML but not PLMR. Loss of mec-4(d) at AB.pra or AB.pla: Duplication loss at AB.pra in an unc-3 osm-1 ; mndp7 animal should lead to nonstaining of five of the six right amphid neurons (the exception being ASHR), which descend from AB.praa, and nonstaining of PHBR, which descends from AB.prap (Figure 3). I have seen five examples of this pattern, and in all five cases PLMR was present (Table 3). The criteria for recognizing duplication loss at AB.pla were less stringent. In this case, ASIL and PHBL should be rendered nonstaining (Figure 3). As already noted, however, it is difficult to be confident of the status of ASIL staining in individual animals. This leaves a nonstaining PHBL as the only indicator of duplication loss at AB.pla. Later duplication losses in the lineage leading to PHBL would also be expected to lead to PHBL nonstaining, but selection of semi- Unc-3 animals appears to select for duplication loss early in the AB.pl (or AB.pr) lineage (HERMAN 1984). In any case, three of the semi-unc-3 animals showed nonstaining PHBL neurons as the only apparent abnormality in the staining pattern, and in all three animals PLML was present (Table 3). Thus, among the total of eight cases of duplication loss at AB.pra or AB.pla (or descendant of AB.pIa), the same-side PLM was present in all eight. The probability of this happening by chance, assuming a 30% chance of PLM survival in each case, is less than I conclude that mec-4(d) loss at AB.pra rescues PLMR and mec- 4(d) loss at AB.pla rescues PLML. Among the 44 semi-unc-3 animals derived from unc-3 duf-6 osm-1; mndp7 zygotes, only one gave an FITC staining pattern expected for duplication loss at AB.pra or AB.pla. It showed FITC nonstaining of the left amphid and PHBL, which is the pattern expected for duplication loss at AB.pla because the left amphid sheath, a focus of action of duf-6, descends from AB.plaa, and PHBL, which is cell autonomously affected by osm-i, descends from AB.plap (Figure 3). In this one animal, PLML was present, as expected (Table 4). Loss of mec-4(d) and uno3(+) at AB.prp or AB.plp: Duplication loss at AB.prp in an unc-3 osm-1; mndp7 animal should lead to nonstaining of both ASHR, which descends from AB.prpa, and PHAR, which descends from AB.prpp (Figure 3). Similarly, duplication loss at AB.plp should lead to nonstaining of both ASHL and PHAL. Among the 96 semi-unc-3 animals found as offspring of unc-3 osm-1 ; mndp7 hermaphrodites, four fit the expected pattern for duplication loss at AB.prp, and in all four cases PLMR was absent (Table 3). Seven other animals fit the expected pattern for duplication loss at AB.plp;

9 Neural Mutant Mosaics of C. eleguns 385 PLML was present in four cases and absent in the other three. Duplication loss at AB.prp in an unc-3 daf-6 osm-1 ; mndp7 animal should lead to nonstaining of ASHR, owing to the absence of osm-l(+) from that neuron (which descends from AB.prpa) and nonstaining of both neurons of the right phasmid, owing to the absence of daf-6(+) from the right phasmid sheath (which descends from AB.prpp; Figure 3). Similarly, duplication loss at AB.plp should lead to nonstaining of ASHL and both neurons of the left phasmid. Two examples of each pattern were found among semi- Unc-3 animals derived from unc-3 daf-6 osm-1 ; mndp7 zygotes, and in no case (among the three that were determined) was the same-side PLM present (Table 4)- The results with both unc-3 osm-1; mndp7 and unc- 3 daf-6 osm-1 ; mndp7 semi-unc-3 animals thus support two conclusions: first, loss of mec-l(d) at AB.prp or AB.plp does not lead to the rescue of the same-side PLM cell, and second, loss of unc-3(+) at AB.plp or AB.prp tends to produce a semi-unc-3 animal. These conclusions obviously hinge on the correctness of my presumption that the FITC staining patterns just described do in fact reflect duplication losses at AB.prp and AB.plp, at least in the majority of cases. An alternative possibility is that most of the patterns were the result of double events. I present two arguments against this alternative interpretation. First, controls on FITC staining patterns obtained with non-unc-3 animals (Table 1) indicate that the expected frequency of double events mimicking duplication loss at either AB.prp or AB.plp in either unc-3 osm-1; mndp7 or unc-3 daf-6 osm-1; mndp7 animals is less than 1%; among a total of 140 semi-unc-3 animals, 15 patterns fitting duplication loss at AB.prp or AB.plp were found. Second, among the same group of semi-unc-3 animals, there were, as already discussed, 61 FITC staining patterns consistent with duplication loss at AB.pr, AB.pl, AB.pra or AB.pla, and in 59 of the 61 cases, the same-side PLM was present; the high degree of consistency of these results suggests that in only two cases among 140 animals were double events responsible for any of these four patterns. I conclude that most of the patterns attributed to duplication loss at AB.prp or AB.plp have been attributed correctly. Although there were fewer examples among the semi-unc-3 animals of duplication loss at AB.pra and AB.pla, it appears that duplication loss at AB.pra or AB.pla also tends to produce a semi-unc-3 phenotype. This would explain my observation (data not shown) that animals that have undergone duplication loss at AB.pr or AB.pl tend, on average, to be more severely semi-unc-3 than those that have undergone duplication loss one cell division later, i.e., the focus of unc- 3(+) action appears to be distributed among the de- scendants of both daughters of AB.pr and both daughters of AB.pl. This may also explain why examples of duplication loss at AB.pr or AB.pl are so heavily represented among semi-unc-3 animals; animals with weaker semi-unc-3 phenotypes may sometimes be missed. Absence of duf-6(+) from socket cells: I have assumed that the focus of daf-6 action is the sheath cell of each sensillum. The basis for this assumption is twofold: first, FITC staining patterns of animals homozygous for daf-6 and carrying daf-6(+) on a free duplication indicated that all neurons of a given sensillum are either staining or nonstaining, and nonstaining for a given sensillum is attributable to a single event of duplication loss (HERMAN 1984); second, electron microscopic investigation of the amphids of daf-6 mutants has shown that the sheath cell is filled with vesicles and enlarged in such a way that the amphidial channel that normally opens to the external environment is pinched off and closed (ALBERT, BROWN and RIDDLE 1981). I now consider the possibility, raised earlier (HERMAN 1984), that the other type of supporting cell, the socket, is the focus of action of daf-6. The amphidial socket cell was not seen by electron microscopy to be abnormal (ALBERT, BROWN and RIDDLE 1981), but it is conceivable that the abnormality observed in the daf-6 sheath cell is due to an interaction with a mutant socket. I note, incidentally, that although each phasmid has two socket cells (SULSTON, ALBERTSON and THOMSON 1980), they are sisters and therefore behave in the cell lineage essentially as if they were a single cell; each amphid has a single socket cell (WARD et al. 1975; WARE et al. 1975). If daf-6 exerted its effect through the sockets rather than the sheaths, the expected patterns of FITC staining following duplication loss in unc-3 daf-6 osm-1; mndp7 animals would be different from those given in Table 4. For example, duplication loss at AB.pra would be expected to lead to nonstaining of all right amphid neurons except ASHR, owing to the absence of osm-l(+) from AB.praa, and nonstaining of both right phasmid neurons, owing to absence of daf-6(+) from AB.prap (Figure 3). Duplication loss at AB.prp would be expected to lead to nonstaining of the right amphid, owing to the absence of daf-6(+) from AB.prpa, and nonstaining of PHAR, owing to the absence of osm-l(+) from AB.prpp. And similarly, duplication loss at AB.plp would be expected to lead to nonstaining of the left amphid and PHAL. None of these patterns was in fact observed. (The pattern expected for loss at AB.pla is less diagnostic since it would only lead to nonstaining of ASIL, the status of which I did not determine, and the left phasmid.) Furthermore, the expected patterns of FITC staining listed in Table 4 for duplication loss at AB.pla, AB.prp

10 386 R. K. Herman and AB.plp, which were found in five animals, could only be explained by assuming double events in every case. I conclude that duf-6 does not act solely through the sockets. It was concluded from previous work that the action of duf-6 in blocking FITC staining by a given sensillum cannot require that both socket and sheath be mutant (HERMAN 1984), but I next consider the possibility that staining is blocked if either socket or sheath is mutant. The consequence of this would be that duplication loss at AB.pra or AB.prp would have the same effect as duplication loss at AB.pr: nonstaining of right amphid and right phasmid (Figure 3). And duplication loss at AB.pla or AB.plp would lead to nonstaining of left amphid and left phasmid. One difficulty with this picture, however, is that duplication loss at AB.prp or AB.plp would not be expected to rescue PLMR and PLML, respectively; but in fact, in 22 of 23 cases of nonstaining of the same-side amphid and phasmid, the same-side PLM was present (Table 4). Another difficulty is that the patterns of nonstaining for the five animals listed in Table 4 for duplication loss at AB.pla, AB.prp and AB.plp could only be explained by assuming double events for each. It thus seems very unlikely that a duf-6 socket, particularly one of the amphid sockets, which derive from AB.plp and AB.prp, leads to FITC nonstaining. DISCUSSION The analysis of animals genetically mosaic for the mec-4(d) gene indicates that the effect of mec-4(d) in killing PLML and PLMR is localized among the descendants of AB.pla and AB.pra (Figure 3), respectively. The evidence for this claim is the following: loss of mec-#(d) at AB.pr or AB.pra leads to the survival of PLMR but not PLML, and loss of mec-4(d) at AB.pl or AB.pla leads to the survival of PLML but not PLMR; furthermore, loss of mec-4(d) at PI or at AB.plp or at AB.prp does not rescue either PLML or PLMR. The results are consistent with the action of mec-4(d) being cell autonomous, since PLML and PLMR themselves descend from AB.pla and AB.pra, respectively. Other cells also descend from AB.pla and AB.pra, however, so the conclusion of cell autonomy is not foolproof. It will be useful to consider what cells can be excluded as potential murderers and what other possibilities remain open.. Both the intestine and body muscle are easily excluded because all intestinal cells and 94 of 95 body muscle cells descend from PI. Perhaps more likely candidates are to be found among those neurons that make synapses, either chemical synapses or gap junctions, with PLML and PLMR. All such cells, as determined by CHALFIE et al. (1985), are listed in Table 5, along with their lineages (SULSTON et al. 1983). Among this group, the cells PDEL and PDER can be TABLE 5 Synaptic partners of posterior lateral microtubule cells Synaptic Type of PLM cell partnep synapse" Cell lineageb PLMR AVAL C AB.alppaaapa AVAR C AB.alaappapa AVDL C AB.alaaapalr AVDR C AB.alaaappx-l DVA C AB.PrpPPPaPP HSNR C AB.PraPPPaPPa LUAR J AB.PrpPPaaPaP PDEL C AB.plapapaappaaa PDER C AB.prapapaappaaa PVCR AB. prpppaapaa PVR J C.aappa PLML HSNL C *B.PlaPPPapa LUAL J AB.PlPPPaaPaP PVCL J AB.plpppaapaa a This information from CHALFIE et al. (1985); J = gap junction, C = chemical synapse. * From SULSTON et al. (1983); a = anterior, p = posterior, 1 = left, r = right; C.aappa refers, for example, to the anterior daughter of the posterior daughter of the posterior daughter of the anterior daughter of the anterior daughter of the cell C shown in Figure 3. excluded because they are generated in a postembryonic lineage long after the PLM cells have degenerated and died (SULSTON and HORVITZ 1977). The pair of hermaphrodite specific neurons (HSNL and HSNR) are also unlikely candidates because they are programmed to die during male embryogenesis before the PLM cells are born (SULSTON et al. 1983), and yet the PLM cells die in mec-4(d) males. None of the remaining neurons listed in Table 5 descends from either AB.pla or AB.pra; thus none of the neurons that make either chemical synapses or gap junctions to either PLML or PLMR can be a focus of mec-4(d) killing action. More difficult to exclude as killers are hypodermal cells, which surround each PLM cell body as well as the posteriorly directed process and the anteriorly directed process extending from each PLM cell body (CHALFIE and SULSTON 1981). The hypodermis consists of a series of cylindrical syncytia linked together by desmosomes (SULSTON et al. 1983). At hatching the cell bodies of the PLM cells are surrounded by a portion of the large cylinder called hyp7, the nuclei of which derive from various parts of the lineage; the two hyp7 nuclei that appear to be closest to PLML and PLMR descend from AB.pla and AB.pra, but they are both situated in the hypodermal ventral ridge approximately equidistant from PLML and PLMR (see Figures 13 and 14 of SULSTON et al. 1983), thus making it difficult to see how the nucleus derived from AB.pla could be responsible for the death of PLML and the other nucleus could be responsible for the death of PLMR. None of the nuclei of the more posterior hypodermal cylinders, which contact the posteriorly directed processes of PLML and PLMR,

11 Neural Mutant Mosaics of C. eeeguns 387 descend from AB.pla or AB.pra. The hypodermal nuclei nearest the anteriorly-directed processes of PLML and PLMR, however, do descend from AB.pla and AB.pra, respectively, so I cannot exclude the possibility that these hypodermal nuclei and not those in other hypodermal regions are responsible for the deaths of the PLM cells. The nature of the mec-4 gene product is unknown, but some insight may be gained by considering the dominance and gene dosage studies. It seems likely that most recessive mec-4 mutations lead to complete or partial loss of function; animals of genotype mec- 4(r)/Df have the same touch-insensitive phenotype as homozygous mec-4(r) animals, and mec-4(+)/df animals are touch sensitive (MENEELY and HERMAN 1981). The dominance or semi-dominance of mec-4(d) to its wild-type allele suggests that it is a gain-offunction mutation. M. CHALFIE S finding (personal communication) that the touch-insensitive phenotype of mec-$(d)/+ animals can be reverted by mutation of the mec-4(d) gene to a mec-4(r) allele is further evidence that mec-4(d) and mec-4(r) are gain-of-function and loss-of-function mutations, respectively. The gene dosage experiments reported here suggest that mec- 4(d) leads to the production of a novel gene product rather than the overexpression of a wild-type gene product. Thus, the penetrance of a single copy of mec- 4(d) in causing PLM cell death was not significantly enhanced by the addition of one or even two wildtype copies of mec-4(+). Additional evidence that the products of mec-4(d) and mec-4(+) are qualitatively different is the following: at least some of the surviving PLM cells in mec-4(d)/mec-4(+) animals promote a touch-sensitive response; the PLM cells survive at a comparable frequency in mec-4(d)/mec-4(~) animals but they are all nonfunctional. It thus appears that the wild-type gene product is required for normal microtubule cell function; the mec-4(d) gene does not provide the normal function but produces a novel product that leads to the degeneration and death of the microtubule cells. The temperature-sensitive effect of a single copy of mec-4(d) in killing the microtubule cells suggests that the action of the novel product is partially heat labile. Unlike several other dominant mutations that also appear to lead to the production of novel gene products (PARK and HORVITZ 1986), the action of the mec-4(d) novel product appears not to be antagonized by wild-type gene product, i.e., the addition of wild-type alleles, whether to one or two copies of mec-4(d), does not reduce the penetrance of the mec-4(d) mutation. It seems likely on the basis of the apparent cell autonomous behavior of mec-4(d) that the contribution of the mec-4(+) gene to wildtype microtubule cell function may also be cell autonomous. I turn now to discussion of the unc-3 gene. The mosaic results indicate that the absence of unc-3(+) from either AB.plp or AB.prp gives rise to a semi- Unc-3 animal. Loss of an unc-3(+) duplication at AB.pla or AB.pra also appears to produce semi-unc- 3 animals. These results are clearly consistent with the view that it is the absence of unc-3(+) function in the motor neurons themselves that is responsible for their deranged process anatomy, which in turn is presumed to be responsible for the uncoordinated phenotype. Ten of the 22 embryonically generated cord motor neurons descend from AB.plp, and 11 descend from AB.prp (one descends from AB.a). Fifty-three additional cord motor neurons develop post-embryonically; they descend, about equally, from AB.pla and AB.pra. The idea that the hypodermis surrounding the ventral cord is solely responsible for the aberrant nervous system structure is inconsistent with the results. Many hypodermal nuclei descend from AB.pla and AB.pra, but the only hypodermal nuclei that descend from AB.plp and AB.prp are situated in positions posterior to the anus and the ventral cord (SULSTON et al. 1983). It is conceivable that unc-3(+) expression is functionally important in both hypodermis and neurons; it is obviously simpler to suppose that the gene exerts its effect through neurons only, however. It is worth considering the possibility that the focus of action of unc-3(+) is within the interneurons that make synapses with the motor neurons rather than the motor neurons themselves. There are four pairs of such interneurons. Although two of the pairs (AVBL, AVBR, PVCL and PVCR) descend from AB.plp and AB.prp, the other two pairs (AVAL, AVAR, AVDL and AVDR), which provide all of the input to the VA, DA and AS motor neurons, descend from AB.a. Furthermore, the DD and VD motor neurons receive their input from other motor neurons only (WHITE et al. 1976). But the anatomies of all classes of cord motor neuron appear to be affected in unc-3 animals (J. WHITE, personal communication); it thus seems unlikely that the focus of unc-3 action could be solely among the synaptic partners of the motor neurons. Finally, I note that even if the focus of action of unc-3(+) were limited to the cord motor neurons, the effect on neuroanatomy might not be strictly cell autonomous, since interactions between different motor neurons are possible; this question could be addressed by electron microscopic reconstruction of cord neuroanatomy in genetically mosaic animals. When the DNA sequences of the mec-4 and unc-3 genes are isolated, it may be possible to test the predictions for transcript patterns that follow from the mosaic analyses, viz., mec-4 would be expected to be transcriptionally active in microtubule cells and unc-3 in cord motor neurons. According to the simplest view, transcription might be expected not to

12 388 R. K. H [erman occur in other than these specifically identified cell types. On the other hand, mosaic analysis addresses the problem of cell function rather than transcription per se. It is certainly possible that mec-4, for example, is transcribed in many neurons but that its primary functional effect is limited to the microtubule cells. I thank MARTY CHALFIE for much generous help, especially for suggesting the mosaic analysis of e1611 in the first place and for teaching me how to identify the microtubule cells by Nomarski microscopy. I thank CLAIRE KARI for her customarily able technical help, particularly in identifying mndp7 and mndp21. This work was supported by National Institutes of Health grant GM LITERATURE CITED ALBERT, P. S., S. J. BROWN and D. L. RIDDLE, 1981 Sensory control of dauer larva formation in Caenorhabditis elegans. J. Comp. Neurol BRENNER, S., 1973 The genetics of behaviour. Br. Med. Bull BRENNER, S., 1974 The genetics of Caenorhabditis elegans. Genetics 77: CHALFIE, M. and J. E. SULSTON, 1981 Developmental genetics of the mechanosensory neurons of Caenorhabditis elegans. Dev. Biol. 82: CHALFIE, M., J. E. SULSTON, J. G. WHITE, E. SOUTHGATE, J. N. THOMSON, AND S. BRENNER, 1985 The neural circuit for touch sensitivity in Caenorhabditis elegans. J. Neurosci CULOTTI, J. G. and R. L. RUSSELL, 1978 Osmotic avoidance defective mutants of the nematode Caenorhabditis elegans. Genetics GREENWALD, I. S. and H. R. HORVITZ, 1980 unc-93(e1500): a behavioral mutant of Caenorhabditis elegans that defines a gene with a wild-type null phenotype. Genetics HEDGECOCK, E. M., J. G. CULOTTI, J. N. THOMSON and L. A. PERKINS, 1985 Axonal guidance mutants of Caenorhabditis elegans identified by filling sensory neurons with fluorescein dyes. Dev. Biol. 111: HERMAN, R. K., 1978 Crossover suppressors and balanced recessive lethals in Caenorhabditis elegans. Genetics 88: HERMAN, R. K., 1984 Analysis of genetic mosaics of the nematode Caenorhabditis elegans. Genetics HERMAN, R. K., D. G. ALBERTSON and S. BRENNER, 1976 Chromosome rearrangements in Caenorhabditis elegans. Genetics 83: HERMAN, R. K., J. E. MADL and C. K. KARI, 1979 Duplications in Caenorhabditis elegans. Genetics 92: HORVITZ, H. R., S. BRENNER, J. HODGKIN and R. K. HERMAN, 1979 A uniform genetic nomenclature for the nematode Caenorhabditis elegans. Mol. Gen. Genet. 175: KENYON, C., 1986 A gene involved in the development of the posterior body region of C. elegans. Cell LEWIS, J. A. and J. A. HODGKIN, 1977 Specific neuroanatomical changes in chemosensory mutants of the nematode Caenorhabditis elegans. J. Comp. Neurol. 172: MENEELY, P. M. and R. K. HERMAN, 1981 Suppression and function of X-linked lethal and sterile mutations in Caenorhabditis elegans. Genetics 97: PARK, E.-C. and H. R. HORVITZ, 1986 Mutations with dominant effects on the behavior and morphology of the nematode Caenorhabditis elegans. Genetics 113: PERKINS, L. A., E. M. HEDGECOCK, J. N. THOMSON and J. G. CULOTTI, 1986 Mutant sensory cilia in the nematode Caenorhabditis elegans. Dev. Biol. 117: SULSTON, J. E., D. G. ALBERTSON and J. N. THOMSON, 1980 The C. elegans male: postembryonic development of nongonadal structures. Dev. Biol SULSTON, J. E. and H. R. HORVITZ, 1977 Post-embryonic cell lineages of the nematode Caenorhabditis elegans. Dev. Biol. 56: SULSTON, J. E., E. SCHIERENBERG, J. G. WHITE AND J. N. THOMSON, 1983 The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol WARD, S., N. THOMSON, J. G. WHITE and S. BRENNER, 1975 Electron microscopic reconstruction of the anterior sensory anatomy of the nematode Caenorhabditis elegans. J. Comp. Neurol WARE, R. W., D. CLARK, K. CROSSLAND and R. L. RUSSELL, 1975 The nerve ring of the nematode Caenorhabditis elegans: sensory input and motor output. J. Comp. Neurol. 162: WATERSTON, R. H. and G. R. FRANCIS, 1985 Genetic analysis of muscle development in Caenorhabditis elegans. Trends Neurosci. 8: WHITE, J. G., E. SOUTHGATE, J. N. THOMSON and S. BRENNER The structure of the ventral nerve cord of Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. (Biol. Sci.) 275: Communicating editor: R. L. METZENBERG