Mutations Affecting Symmetrical Migration of Distal Tip Cells in Caenorhabditis elegans

Corresponding author: Kiyoji Nishiwaki, NEC Corporation, Miyukigaoka, Tsukuba 305, Japan., nishiwak{at}frl.cl.nec.co.jp (E-mail)

Communicating editor: R. K. HERMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The rotational symmetry of the Caenorhabditis elegans gonad arms is generated by the symmetrical migration of two distal tip cells (DTCs), located on the anterior and posterior ends of the gonad primordium. Mutations that cause asymmetrical migration of the two DTCs were isolated. All seven mutations were recessive and assigned to six different complementation groups. vab-3(k121) and vab-3(k143) affected anterior DTC migration more frequently than posterior, although null mutants showed no bias. The other five mutations, mig-14(k124), mig-17(k113), mig-18(k140), mig-19(k142), and mig-20(k148), affected posterior DTC migration more frequently than anterior. These observations imply that the migration of each DTC is regulated differently. mig-14 and mig-19 also affected the migration of other cells in the posterior body region. Four distinct types of DTC migration abnormalities were defined on the basis of the mutant phenotypes. vab-3; mig-14 double mutants exhibited the types of DTC migration defects seen for vab-3 single mutants. Combination of mig-17 and mig-18 or mig-19, which are characterized by the same types of posterior DTC migration defects, exhibited strong enhancement of anterior DTC migration defects, suggesting that they affect the same or parallel pathways regulating anterior DTC migration.

Asymmetrical left-right body plan is typical of animal development. However, the plan for anterior-posterior structures is usually asymmetrical. In this regard the Caenorhabditis elegans gonad structure is unusual in that the anterior and the posterior U-shaped gonad arms are rotationally symmetrical around the dorso-ventral axis at the center of the body (Figure 1). The C. elegans gonad is formed so that it folds around the intestine. Relative to the intestine, the anterior arm of the gonad is on the right side of the body cavity and the posterior arm is on the left. The symmetry of the gonad arms is generated by migration of the two mesodermal distal tip cells (DTCs), each located on the anterior or on the posterior ends of the gonad primordium, in a U-shaped pattern during larval development. Therefore, the shape of the gonad arm reflects the migration paths of the DTCs (KIMBLE and HIRSH 1979 ; HEDGECOCK et al. 1987 ). The two DTCs are lineal homologues and originate from the somatic gonad precursor cells Z1 and Z4 by symmetrical divisions during the first larval (L1) stage (KIMBLE and HIRSH 1979 ). However, their migration pathways are different from each other: the anterior DTC migrates on the basal lamina of the anterior-right basal surface of the body wall, whereas the posterior DTC migrates on that of the posterior-left.

View larger version (52K): In this window In a new window Download PPT slide   Figure 1. Schematic presentation of the wild-type hermaphrodite gonad structure. (A) Left lateral view. The intestine is shaded. The posterior arm of the gonad, which is on the left side of the body, is depicted by the solid line, and the anterior arm on the right side by the dotted line. Ph, pharynx; In, intestine; Vu, vulva; DTC, distal tip cell; AA, anterior arm; PA, posterior arm. (B) Dorsal view. (C) Distal tip cell migration in the hermaphrodite shown in cylindrical projection (modified from HEDGECOCK et al. 1987 ). The projection corresponds to the hermaphrodite body wall cut open at the ventral midline. Dorsal and ventral body wall muscles are shaded. The two DTCs are born around the L1 molt as descendants of two somatic gonad precursor cells, Z1 and Z4, over the ventral muscles (shown by circles) and start to migrate at the mid-L2 stage. They move in opposite directions along the ventral muscles, turn dorsally at the mid-L3 stage, and migrate along the lateral hypodermal cells. They turn again over the dorsal muscles around the L3 molt and migrate toward each other along the dorsal muscles (HEDGECOCK et al. 1987 ; ANTEBI et al. 1997 ). Position of vulva in adult is shown. V, ventral; D, dorsal; R, right; L, left.

unc-5, unc-6, and unc-40 are genes required for the circumferential migration of cells and axons in C. elegans (HEDGECOCK et al. 1990 ). UNC-6 is a guidance molecule localized to the basement membrane and homologous to the mammalian netrin (ISHII et al. 1992 ; SERAFINI et al. 1994 ; WADSWORTH et al. 1996 ). UNC-5 and UNC-40 are thought to be UNC-6 receptors expressed on migratory cells (LEUNG-HAGESTEIJN et al. 1992 ; CHAN et al. 1996 ) and they also have mammalian homologs (KEINO-MASU et al. 1996 ; LEONARDO et al. 1997 ). Interestingly, although these three genes all affect the migration of both the anterior and posterior DTCs, all the unc-5, unc-6, and unc-40 alleles examined affected migration of the posterior DTC more frequently than that of the anterior (HEDGECOCK et al. 1990 ). These findings suggest that the mechanisms controlling migration of the two DTCs may not always be identical despite the fact that the two cells are homologous to each other in cell lineage and migrate symmetrically during development.

To examine whether molecular mechanisms that control DTC migration are indeed different for each cell, I isolated mutations that cause one cell to migrate differently than the other. Seven mutations in six different genes, including four new genes, were identified in this study. Two of the mutations affected anterior DTC migration more frequently than posterior, and the other five affected the posterior DTC migration more frequently than anterior. This supports the idea that the migration of the two DTCs is regulated differently.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and culture conditions:
The media, culture, and handling of C. elegans were described by BRENNER 1974 . Mutants were first roughly mapped genetically by the PCR-STS (sequence-tagged site) method using RW7000 as an STS marker strain (WILLIAMS et al. 1992 ). They were then mapped more precisely using standard marker mutations and chromosomal deficiencies. The following STSs, mutations, and deficiencies were used:

• LGI: hP4

• LGII: stP100, stP196, stP101, stP50, stP36, stP98, maP1, dpy-10(e128), unc-4(e120), bli-1(e769), rol-1(e91) (HIGGINS and HIRSH 1977 ), mig-14(mu71) (HARRIS et al. 1996 ), unc-52(e444), mnDf66, and mnDf63 (SIGURDSON et al. 1984 )

• LGIII: stP19, stP120, mgP21, stP127, stP17, tar-1(e1099) (HODGKIN and BRENNER 1977 ), dpy-18(e364), unc-71(ay7) (CHEN et al. 1997 ), unc-25(e156), bli-5(e518), tDf8 (H. SCHNABEL and R. SCHNABEL, personal communication), eDf2 (HODGKIN 1980 )

• LGIV: sP4, unc-30(e191)

• LGV: stP192, bP1, stP6, stP108, stP105, stP128, dpy-11(e224), sma-1(e30), him-5(e1490) (HODGKIN 1980 ), unc-76(e911), ctDf1 (MANSER and WOOD 1990 ), arDf1 (TUCK and GREENWALD 1995 )

• LGX: stP41, stP40, stP156, stP33, stP103, stP129, stP61, stP72, stP2, unc-27(e155), vab-3(e648) (LEWIS and HODGKIN 1977 ), egl-15(n484) (TRENT et al. 1983 ), lin-2(e1309) (HORVITZ and SULSTON 1980 ), nDf19 (AMBROS and HORVITZ 1984 )

Mutations without a reference are described by BRENNER 1974 .

Isolation of mutants:
Mutations were generated by treating wild-type (N2) hermaphrodites at the fourth larval stage (L4) with ethyl methanesulfonate (EMS) as described by BRENNER 1974 . Mutants were isolated from animals at the F₂ or F₃ generation. HEDGECOCK et al. 1990 reported that distal tip cell migration defects in unc-5 or unc-6 mutant hermaphrodites can be observed by bright-field microscopy at low magnification: the abnormally returned arms of the gonad in unc-5 and unc-6 displace the dark intestine dorsally, resulting in the appearance of a white stripe on the ventral side. Therefore, to isolate mutants with abnormal gonad morphology, I looked for animals with dorsal or ventral white patches under a dissecting microscope, especially those with patches either on the anterior or the posterior body region (Figure 2). Animals with a ventral white stripe similar to those seen in unc-5 or unc-6 mutants were not picked to avoid isolating alleles of these genes. I screened 230 9-cm plates each of which contained F₂s or F₃s from 200 to 300 F₁s of mutagenized F₀ hermaphrodites. This corresponds to 230 x 2 x (200 to 300) = 92,000 to 138,000 genomes. About 4000 candidate animals were isolated and the genetic penetrance of their white patch phenotypes was assessed by examination of the progeny. A total of 103 fertile mutants with misshapen gonad arms were obtained. Of these, 7 were found in which one of the two gonad arms was obviously affected more frequently than the other.

View larger version (102K): In this window In a new window Download PPT slide   Figure 2. White patch phenotypes under a dissecting microscope. Anterior to the left, dorsal top. (A) Wild type, (B) an anterior type I defect in vab-3(k121), (C) a posterior type IV defect in mig-17(k113). See Figure 5 and Figure 6 for defective types. Bar, 0.1 mM.

Genetic mapping:
Mutants were backcrossed to N2 at least twice to eliminate extraneous mutations. All seven mutations were recessive to their wild-type alleles and affected DTC migration zygotically (data not shown). Genetic mapping was accomplished using the two-step polymorphic mapping strategy (WILLIAMS et al. 1992 ). Mutant alleles were first mapped to a specific chromosome using a single STS particular to each chromosome, hP4(I), maP1(II), mgP21(III), sP4(IV), bP1(V), and stP103(X). This analysis assigned mig-14(k124) and mig-19(k142) to linkage group II (LGII); mig-17(k113) to LGV; and vab-3(k121), vab-3(k143), and mig-20(k148) to LGX (data not shown). The data for mig-18(k140) were not clear. Assuming that mig-18 was on the end of a chromosome, the data for LGII, III, and X showed possible linkage. Using multiple STS markers for each of these chromosomes (listed above), mig-18 was subsequently found to be linked to stP17 near the right end of LGIII (Table 1). Similarly, mig-19 II, mig-17 V, vab-3(k121) X, and mig-20 X were further localized on each chromosome using multiple STSs (Table 1). Mutations within the same linkage group, mig-14 and mig-19, as well as vab-3(k121) and mig-20 complemented each other (data not shown).

The data for three-factor mapping experiments with morphological mutations are shown in Table 2. Some two-factor mapping experiments were done for mig-18. Of 95 Dpy segregants from + mig-18/dpy-18 +, 18 were dpy-18 mig-18/dpy-18 + and 77 were dpy-18 +/dpy-18 +. Thus, 2P = 18/95 and P = 0.095. All 205 Mig segregants from + + mig-18/+ bli-5 + segregated only non-Bli progeny. Thus, 2P < 1/205 and P < 0.002.

When nearby deficiencies were available, deficiency mapping was carried out as follows:

• mig-19(k142) II: dpy-10(e128) mig-19/+ + males were crossed with mnDf66/mnC1 dpy-10(e128) unc-52(e444) or mnDf63/mnC1 dpy-10(e128) unc-52(e444) hermaphrodites. dpy-10 and mig-19 were about 2 map units apart. The genotype of F₁ dpy-10 mig-19/Df hermaphrodites was determined by scoring F₂s: the observation that Dpy-10 non-Unc-52 but not Dpy-10 Unc-52 animals segregated with the Mig-19 phenotype and dead eggs indicates that the F₁ mother was dpy-10 mig-19/Df. Of the 46 non-Dpy F₂ segregants from the two F₁ dpy-10 mig-19/mnDf66 animals, 23 showed defects in DTC migration. Many of these DTC migration-defective animals segregated dead eggs, indicating that mnDf66 deletes mig-19. All of the four F₁ dpy-10 mig-19/mnDf63 hermaphrodites and most of their non-Dpy F₂ segregants were normal for DTC migration, but Dpy F₂ segregants often showed the Mig-19 phenotype, indicating that mnDf63 does not delete mig-19.

• mig-20(k148) X: Because the males of mig-20 did not mate, mig-20 hermaphrodites were crossed with tra-1(e1099) males, and F₂ males segregated from F₁ tra-1/+; mig-20/+ hermaphrodites; that is, pseudo-males homozygous for tra-1 were crossed with +/szT1[lon-2(e628)]I; nDf19/szT1 X hermaphrodites. Two F₃ hermaphrodites having DTC migration defects similar to those in mig-20 were picked. The genotype of these hermaphrodites was mig-20/nDf19, as shown by the fact that both of them segregated F₄s with the DTC migration defect and dead eggs, but they did not segregate Lon animals. Of the 36 F₄ hermaphrodites that segregated from one animal, 17 exhibited Mig-20 type DTC migration defects. Ten of the 17 animals segregated dead eggs, indicating that nDf19 deletes mig-20.

• vab-3(k121 and k143) X: vab-3(k121) males were crossed with +/szT1[lon-2(e628)] I; nDf19/szT1 X hermaphrodites. Three wild-type F₁ hermaphrodites segregated F₂ dead eggs and hermaphrodites with the Vab-3 type DTC migration defect, but did not segregate Lon animals, indicating that these F₁s were vab-3/nDf19. None of 24 F₂s with the Vab-3 phenotype were found to segregate dead eggs. Thus, nDf19 appeared to complement vab-3. Similarly, nDf19 was also found to complement vab-3(k143). nDf19 might be a complex deficiency, because it failed to complement unc-27 and mig-20 but not vab-3, which is between these two genes.

• mig-17(k113) V: mig-17 males were crossed with ctDf1 V/nT1[unc-?(n754)let-?](IVV) or unc-42(e270)arDf1 V/nT1[unc-?(n754)let-?](IVV) hermaphrodites. None of 41 F₁ mig-17/ctDf1 or 36 F₁ mig-17/unc-42 arDf1 animals exhibited the Mig-17 phenotype, indicating that neither ctDf1 nor arDf1 deletes mig-17.

• mig-18(k140) III: mig-18; unc-4 hermaphrodites were mated with unc-32(e189)tDf8/qC1 dpy-19(e1259)glp-1(q339) males. None of 32 wild-type F₁ mig-18(k140)/unc-32tDf1; unc-4/+ hermaphrodites exhibited the Mig-18 phenotype, indicating that tDf8 does not delete mig-18. mig-18 males were mated with unc-32(e189) ooc-4(e2078)/eDf2 III hermaphrodites. Ten F₁ hermaphrodites with the Mig-18 phenotype segregated Mig-18 animals and dead eggs, indicating that eDf2 deletes mig-18.

Figure 3 summarizes the positions of the genes on a genetic map determined by the genetic analysis described so far.

View larger version (20K): In this window In a new window Download PPT slide   Figure 3. Genetic mapping. mnDf66 deletes mig-19, but mnDf63 does not. eDf2 deletes mig-18, but tDf8 does not. Neither ctDf1 nor arDf1 deletes mig-17. nDf19 deletes mig-20, but not vab-3 (anomalous). Map position for vab-3 is from ACeDB data base.

Gene dosage experiments:
For mig-18, mig-19, and mig-20, the phenotypes over deficiencies were determined. The F₁ segregants from mig-18/eDf2, dpy-10 mig-19/mnDf66, or tra-1/+; mig-20/nDf19 animals were checked for their gonad phenotypes and cloned in separate plates. F₁s that segregated many F₂ dead eggs were scored as mig/Df animals.

Analysis of DTC migration phenotypes:
Young adult hermaphrodites, which were grown at 16° without starvation, were observed on a 5% agar pad using a Zeiss Axiophoto microscope equipped with a Plan 100 objective and Nomarski differential interference contrast optics (SULSTON and HORVITZ 1977 ). The trajectories of the DTCs were deduced from the shapes of the gonad arms. I sometimes observed mutant animals with anterior arms on the left side or those with posterior arms on the right side. These phenotypes might be caused by the mispositioning of the gonad primordium at the L1 stage (E. M. HEDGECOCK, personal communication). The migration of DTCs on arms formed on the wrong side and that of their counterparts on the normal-sided arms were similarly affected by the mutations. This type of abnormality was not seen in more than 200 wild-type animals scored. The frequencies of misplaced arms in 200 animals examined for each mutant were as follows (where R stands for cases where the posterior arm was on the right side and L where the anterior arm was on the left side): vab-3(e648), 4%(R) and 1%(L); vab-3(k143), 2%(R); mig-20, 3%(R); mig-14(k124), 3%(R) and 2%(L); mig-14(mu71), 1%(R) and 2%(L); mig-19, 3%(R); vab-3(e648); mig-14(k124), 2%(R) and 5%(L); vab-3(e648); mig-17, 6%(R) and 2%(L); vab-3(e648); mig-18, 8%(R) and 1%(L); vab-3(e648); mig-19, 11%(R) and 1%(L); vab-3(e648); mig-20, 2%(R); mig-14(k124); mig-17, 3%(R) and 2%(L); mig-14(k124); mig-18, 1%(R) and 2%(L); mig-14(k124); mig-20, 2%(R) and 3%(L); mig-20; mig-17, 1%(R); mig-20; mig-19, 6%(R) and 1%(L). Strains with vab-3(k121); mig-17; mig-18 and mig-17; mig-19 were not scored.

To determine the antero-posterior position of the first turn of the DTCs, L3 larvae whose posterior gonad arms had just turned were examined. Position was assessed relative to that of the postdeirid neurons (SULSTON and HORVITZ 1977 ).

Scoring of non-DTC cell migration:
Late L1 animals grown at 16° were examined by Nomarski microscopy. Migration of HSN, ALM, CAN, QR/L, and embryonic coelomocyte mother cells was scored by their final positions or positions of their descendants relative to those of the stationary Vn.a and Vn.p hypodermal cells as described by HARRIS et al. 1996 . Male linker cell migration was assessed by gonad morphology in a him-5(e1490) background for all mutations except for mig-17(k113).

Construction of double mutants:
To combine mutations of different linkage groups, other than vab-3(e648), recessive marker mutations were used which were both trans to and closely linked to each of the two mutations. Double mutants were isolated as clones that did not segregate marker mutations from the progeny of the double trans-heterozygotes listed below. The mig-18; mig-19 double was not able to be established as a line because it was sterile. Double mutants segregated with a reasonable frequency from the double trans-heterozygotes, that is, about 1/16 of the segregants. For each combination, at least two independently isolated double mutants were checked for their DTC phenotypes. Double mutants carrying vab-3(e648) and other unlinked mutations were generated using the abnormal head morphology of vab-3 and various mutant phenotypes described in RESULTS.

Double trans-heterozygotes are as follows:

• vab-3(k121)/egl-15; mig-14(k124)/unc-52

• vab-3(k121)/egl-15; mig-17(k113)/sma-1

• vab-3(k121)/egl-15; mig-18(k140)/unc-25

• vab-3(k121)/unc-27; mig-19(k142)/unc-4

• mig-14(k124)/unc-52; mig-17(k113)/sma-1

• mig-14(k124)/unc-52; mig-18(k140)/unc-25

• mig-14(k124)/unc-52; mig-20(k148)/unc-27

• mig-17(k113)/sma-1; mig-18(k140)/unc-25

• mig-17(k113)/sma-1; mig-19(k142)/unc-4

• mig-17(k113)/sma-1; mig-20(k148)/unc-27

• mig-18(k140)/unc-25; mig-19(k142)/unc-4

• mig-18(k140)/unc-25; mig-20(k148)/unc-27

• mig-19(k142)/unc-4; mig-20(k148)/unc-27

Construction of double mutants of the same linkage group was carried out as follows. To generate double mutants of mig-20(k148) and vab-3(k121) or vab-3(e648), unc-27 mig-20(k148) hermaphrodites were crossed with vab-3(k121) or vab-3(e648)/+; tra-1(e1099) males and wild-type F₁ unc-27 + mig-20(k148)/+ vab-3 + hermaphrodites were obtained. F₂ non-Unc hermaphrodites with the Mig-20 phenotype (posterior type III defect not seen in vab-3), which were expected to be + vab-3 mig-20/unc-27 + mig-20 or + + mig-20/unc-27 + mig-20, were isolated. F₃ hermaphrodites that segregated only non-Unc progeny exhibiting DTC migration defects characteristic both for vab-3(k121) and mig-20 or head abnormality characteristic for vab-3(e648) were selected.

To generate mig-19(k142) and mig-14(k124) doubles, rol-1 mig-14 hermaphrodites were crossed with mig-19/+ males (k142 homozygous males are sterile) and wild-type F₁ + rol-1 mig-14/mig-19 + + hermaphrodites were selected on the basis of segregation of the Mig-19 phenotype. Six F₂ non-Rol hermaphrodites with the Mig-14 phenotype (posterior type II defect not seen in mig-19), which were expected to be mig-19 + mig-14/+ rol-1 mig-14, were isolated. mig-19 mig-14 doubles were isolated as F₃ hermaphrodites that only segregated non-Rol progeny.

Double mutants carrying mig-14, -19, or -20 were further confirmed by phenotypes in addition to DTC migration, as described in RESULTS.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mutations affecting symmetrical migration of DTCs:
I isolated mutations that differentially affected the morphologies of the anterior and the posterior gonad arms. Mutants were initially selected by their white patch phenotypes, which are often due to defects in the morphogenesis of the gonad arms (see MATERIALS AND METHODS). Subsequently, the gonad morphogenesis of these mutants was analyzed by Nomarski microscopy. I identified seven mutations that asymmetrically affected the shapes of the two gonad arms. All of these mutations were recessive and fell into six different complementation groups. The mutations were designated k113, k121, k124, k140, k142, k143, and k148. k121 was mapped to the right arm of LGX, which contains vab-3, a mutation that results in a similar phenotype (A. CHISHOLM, personal communication). Both k121 and k143 failed to complement the e648 allele of vab-3, a gene previously identified by mutations affecting head morphogenesis (LEWIS and HODGKIN 1977 ) and which has been reported to have a DTC migration defect as well (HEDGECOCK et al. 1987 ; CHISHOLM and HORVITZ 1995 ; ZHANG and EMMONS 1995 ). k124 was found to be an allele of mig-14, a gene originally identified by a mutation that results in migration defects in several neurons and neuroblasts (HARRIS et al. 1996 ). The other four mutations, k113, k140, k142, and k148 seemed to define four new genes mig-17, mig-18, mig-19, and mig-20, respectively, judging from their associated phenotypes and genetic map positions (Figure 3).

Characterization of mutant DTC migration phenotypes:
DTC migration abnormalities were determined from the shape of the mutant gonad arms. As shown in Figure 4A, of the seven mutations isolated only vab-3 alleles k121 and k143 affected the anterior DTC more frequently than the posterior, although vab-3(e648) affected both similarly. Mutations in the other five genes affected the posterior DTC more frequently than the anterior. mig-14(mu71) (HARRIS et al. 1996 ) was found to affect the posterior DTC more frequently. The asymmetric influence was especially noticeable in the mig-14, mig-17, and mig-19 animals. None of the mutations were fully penetrant. I categorized the mutant DTC migration abnormalities into four distinct types, from I to IV, as shown in Figure 5. In the type I defect, DTCs initially turned twice, as seen in normal animals, but after the second, they often turned around on the dorsal muscles and migrated in the opposite direction. In the type II defect, DTCs turned with an acute angle at the first turn. The second turn on the dorsal muscles was similarly acute, but followed by a turn in the opposite direction. In the type III defect, the DTCs prematurely ceased their migration on the dorsal muscles after the second turn. The distal arm of the gonad usually swelled afterward, probably because of the proliferation of germ cells. In the type IV defect, DTCs appeared to deviate from their correct migration path after the first turn. They initially moved dorso-anteriorly after the first turn and then migrated anteriorly while meandering over the dorsal muscles or the lateral hypodermal cells, but not along the dorsal muscles as in the wild type.

View larger version (53K): In this window In a new window Download PPT slide   Figure 4. Effects of mutations on anterior and posterior DTC migration. (A) Asymmetrical effects of single mutations on DTC migration. (B) Effects of double mutant combinations on DTC migration. k121 and e648 are vab-3 alleles. The mig-14 allele is k124. Two independent sets of 100 animals were scored for each mutant. Average percentage of animals with misshapen anterior or posterior gonad arms in each of the mutants is indicated by the shaded bar and the SD by the thin line. No abnormalities were observed in more than 200 wild-type animals examined.

View larger version (49K): In this window In a new window Download PPT slide   Figure 5. Four types of DTC migration abnormalities. Defective patterns are shown for the posterior gonad arm, although the defects could be in the anterior or posterior arm depending on the mutations and individuals. The normal part of the migration path is shown by a thin line and the abnormal part by a bold line. The dotted line indicates that this section of the migration path could be variable.

Although every mutant exhibited multiple types of DTC migration abnormalities, one or two major types of abnormalities were prevalent for different mutants. On the basis of the most frequently observed abnormal patterns of DTC migration, the mutations were classified into four classes (Table 3). vab-3(k121), vab-3(k143), and vab-3(e648) caused a major anterior type I defect (plus a major posterior type I defect in the cases of k143 and e648) and they constituted the first class. mig-20(k148) resulted in a major posterior type I defect and it constituted the second class. mig-14(k124) and mig-14(mu71) caused a major posterior type II defect and constituted the third class. mig-17(k113), mig-18(k140), and mig-19(k142) all resulted in a major posterior type IV defect and they constituted the fourth class. None of the newly isolated mutants exhibited any gross abnormality in body morphology. Phenotypes other than the cell migration abnormalities are listed in Table 4.

vab-3(k121, k143): Anterior and posterior type I DTC migration defects were often seen in these mutants (Figure 6B). vab-3 encodes a transcription factor of the Pax6 family found in mammals and Drosophila (CHISHOLM and HORVITZ 1995 ). e648 is a nonsense mutation that deletes part of the paired domain and the whole homeodomain of the polypeptide (CHISHOLM and HORVITZ 1995 ), suggesting that it is a null allele. In contrast to e648, k121 did not have any abnormalities in head morphology and k143 had weak abnormalities in it. While the extra turns in DTC migration usually occurred only once in k121 or k143, one or two additional turns were often observed in e648. Although k121 and k143 asymmetrically affected the anterior and posterior DTCs (k121, 50 ± 5% for anterior and 19 ± 4% for posterior; k143, 71 ± 2% for anterior and 42 ± 6% for posterior), e648 similarly affected both and the effect was more penetrant (85 ± 4% for anterior and 81 ± 7% for posterior; Figure 4A). The score for the heterozygote k121/e648; unc-30/+ was 46% (n = 50) for anterior and 36% (n = 50) for posterior, and that for k143/e648; unc-30/+ was 87% (n = 82) for anterior and 67% (n = 82) for posterior. Therefore, k121 and k143 seemed to be weaker than e648.

View larger version (109K): In this window In a new window Download PPT slide   Figure 6. DTC migration-defective phenotypes. The photos are of wild-type and mutant animals with representative phenotypes and show the posterior left side of the animals except for C, which is posterior right. (A) Wild type. (B) Type I defect in mig-20(k148). Similar phenotypes were frequently observed in the anterior or posterior arms of vab-3 mutants. (C) Type I defect in mig-20(k148). (D) Type II defect in mig-14(k124). (E) Type III defect in mig-20(k148). This animal has branched distal arms; the arrowhead indicates the DTC on one branch, and the arrow indicates the other branch without a DTC. (F) Type IV defect in mig-18(k140). Similar phenotypes were often displayed by mig-17(k113) and mig-19(k142). Bar, 50 µm.

mig-20(k148): The posterior type I defect was most prominent. Anterior type I and posterior type III defects were also observed (Figure 6B, Figure C, and Figure E). Although both mig-20 and vab-3 exhibited the type I defect, their phenotypes were not always the same; in mig-20 DTC migration often ceased shortly after the turn on the dorsal muscles (Figure 6C), whereas in vab-3(k121) or vab-3(k143) migrations continued to move toward the head or the tail (Figure 6B). In addition to these DTC migration abnormalities, the posterior gonad arms of k148 often bifurcated after the second turn. In such cases, a DTC was found on the tip of one of the two branches while the other branch had no DTC (Figure 6E). I deduced the trajectory of the DTC from the shape of the branch bearing the DTC. The DTC-less branch extended toward the anterior to various extents. The mechanism of migration in the DTC-less branch is not known. In k148/nDf19, 19% (n = 91) exhibited defects in the anterior arm and 44% (n = 91) exhibited defects in the posterior arm. The posterior arm defect in k148/nDf19 seems to be weaker than that of k148 homozygote (14 ± 3% for the anterior arm and 60 ± 4% for the posterior), suggesting that k148 may not be a simple loss-of-function mutation.

mig-14(k124): Although k124 appeared to be stronger than mu71, both of these alleles showed similar phenotypic characteristics. The posterior type II defect was the most prominent (Figure 6D) and the posterior type I defect was also observed. The migration distances of DTCs on the ventral muscles were frequently shorter than in wild type (Table 3). Although no deficiency that deletes the mig-14 locus is available, independent isolation of two different alleles, both of which have similar phenotypic characteristics, supports the idea that k124 is a loss-of-function mutation.

mig-17(k113), mig-18(k140), and mig-19(k142): The posterior type IV defect was most frequent (Figure 6F) and the posterior type I defect was also observed. mig-18 and mig-19 also had various defects in the anterior as well as the posterior DTC migrations. Of the mig-18(k140)/eDf2 animals, 16% (n = 111) had defects in the anterior and 70% (n = 111) had defects in the posterior arms. The score does not seem to be significantly different from that of the k140 homozygote (17 ± 2% for anterior and 65 ± 4% for posterior), consistent with k140 being a complete loss-of-function (null) or a near null allele. In mig-19, the location of the first turn of the DTCs on the ventral muscles was often anterior to the wild-type position (Table 3). Of the k142/mnDf66 animals, 18% (n = 134) exhibited defects in the anterior arm and 45% (n = 134) in the posterior arm. Because this phenotype is weaker than that exhibited by the k142 homozygote for the posterior arm (4 ± 1% for anterior and 61 ± 4% for posterior), k142 does not appear to be a simple loss-of-function allele.

Migration abnormalities in other cell types:
To ask whether these mutations affecting DTC migration affected the migration of other cell types as well, I examined eight different cells that migrate during embryonic and postembryonic development (Figure 7).

View larger version (38K): In this window In a new window Download PPT slide   Figure 7. Other cell migration defects in mutants. (A) Schematic presentation of lateral view of wild-type late L1 hermaphrodite. Vn.a/p hypodermal cells used for local markers of A/P axis are shown. Positions of left and right lateral neurons and coelomocytes, which were scored in this experiment, in a representative wild-type animal are depicted together, although they are not always in the same focal plane (modified from SULSTON and HORVITZ 1977 ). (a) ccRa/p, (b) QR.paa/p, (c) ALM, (d) ccLa/p, (e) CAN, (f) HSN, and (g) QLpaa/p. Cell names with L or R except for ALM mean the cells are at the left or right side of the body and those without these letters and ALM are bilaterally symmetrical. Positional ranges of these cells in 40 wild-type animals examined are shown by bold lines beneath the drawing. An arrow next to the range stands for direction of migration of the cell(s) and/or its precursor cell. (B) Percentage of animals with cells out of the wild-type range is shown next to the number of animals examined (in parentheses). Migration of linker cells is not shown in A and was scored by examining the shape of the adult male gonads. Asterisks indicate cells and/or precursors of the cells that migrate from the posterior body region.

Penetrant cell migration defects were found in mig-14, mig-19, and mig-20. In mig-14(k124), most of the animals exhibited HSN and QL.pa and QR.pa descendants migration defects. HSN was often too posterior. In all the animals examined, the positions of QR.pa descendants were posterior to wild type and were around the hypodermal cell V3. QL.pa descendants were also found around V3, suggesting that the direction in which QL and/or its descendants migrate was reversed, because QL is the sister of V5 and is produced just anterior to it and, in the wild-type case, subsequently migrates in a posterior direction (SULSTON and HORVITZ 1977 ). Migration of the male linker cell was also affected in mig-14. In mig-19(k142), the position of HSN was often too posterior. In mig-20(k148), HSN migration was also affected, with the QR.pa descendants frequently found located between V2 and V3. In addition to these defects, ccLa/p cells in mig-20 were frequently found in a position more anterior than their wild-type counterparts.

The effect of double mutants on asymmetrical DTC migration:
Double mutants were generated using six of the seven mutations isolated in this study and vab-3(e648). Stable double mutants were successfully constructed for all the combinations except mig-18(k140); mig-19(k142), which was sterile. The frequencies of misshapen arms observed in these double mutants are summarized in Figure 4B. When vab-3(k121) was combined with each of the other five mutations, the asymmetrical migration phenotypes became less pronounced. When two of the latter five mutations were combined, the effects on anterior DTC migration were often enhanced. The enhancement of the anterior DTC migration defect was especially evident in mig-20; mig-18, mig-17; mig-18, and mig-17; mig-19 double mutants.

Phenotypic suppression was observed between the mutant classes:
A detailed analysis of the DTC migration phenotypes of double mutants is shown in Table 5. The double mutants generally exhibited more divergent phenotypes than each single mutant and were not simply additive (compare Table 3 and Table 5). However, focusing on the major phenotypes, which were observed in >20% of individuals, I observed phenotypic suppression between some of the mutant classes as follows: (1) Both vab-3(k121) and vab-3(e648) alleles suppressed the posterior type II defect of mig-14. The first dorsalward turn of the posterior DTC was anterior to the postdeirid neurons in 21% of the vab-3(k121); mig-14 animals and in 16% of the vab-3(e648); mig-14 animals (Table 5). Thus, vab-3 also seemed to partly suppress this mig-14 phenotype (Table 3). (2) mig-17, -18, and -19 suppressed the anterior type I defect associated with the weak vab-3(k121) allele, but its effect on the anterior and posterior type I defects of the strong vab-3(e648) allele was not clear. (3) The posterior type IV defect of mig-19 was suppressed, and the weak posterior type II defect of vab-3(e648) was enhanced in vab-3(e648); mig-19 mutants. vab-3(e648) seems to partly suppress the anterior shift of the dorsalward turning point of the posterior DTC in mig-19 (compare Table 3 and Table 5). (4) mig-14 suppressed the posterior type IV defect of mig-17, -18, and -19. (5) mig-14 suppressed the posterior type I defect of mig-20.

Strong enhancement of type IV defect observed in double mutants consisting of mig-17 and mig-18 or mig-19:
mig-17, -18, and -19 are mutations within the same class characterized by a posterior type IV defect. In mig-17; mig-18 and mig-17; mig-19 double mutants, this type IV defect was strongly enhanced, especially in the anterior arm. This effect was very evident for the mig-17; mig-19 double mutants, where 92% of anterior arms showed the type IV defect. I did not score the phenotype for the mig-18; mig-19 doubles, because the animals were sick and sterile, and often were found to be ruptured at the vulva, suggesting that their gonads were severely affected or that vulval development was abnormal.

Double mutants consisting of mig-20 and mig-14 or mig-19 exhibit strong uncoordinated movement phenotype:
mig-20 and mig-14 mutations exhibited slightly uncoordinated movement. However, when combined, the double mig-20; mig-14 and mig-20; mig-19 mutants displayed a strong uncoordinated movement phenotype. The mig-20; mig-19 double mutant was small, sluggish, moved very little, rarely moved backward, and often shrank. The mig-20; mig-14 mutant moved slightly better than did mig-20; mig-19 and had a normal body size. mig-14; mig-19 double mutants were not more uncoordinated than mig-14 single mutants.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Seven mutations in six genes asymmetrically affecting the migration of the two DTCs during development were isolated in this study. Two mutations in vab-3 affected migration of the anterior DTC more often than the posterior, and the other five affected the posterior DTC more often than the anterior. These results suggest that the migration of the two DTCs is regulated differently and that the symmetrical development of the gonad arms is governed by an elaborate genetic system. It is possible that there are more genes whose mutations more strongly alter posterior DTC migration than genes that have the opposite effect. In monodelphic nematodes, which develop a single-armed gonad, the single arm is always anterior, and the posterior DTC generated from Z4 often undergoes programmed cell death (STERNBERG and HORVITZ 1981 ; FELIX and STERNBERG 1996 ). Although C. elegans is didelphic, its posterior arm might have an evolutionarily conserved bias to degenerate.

The weak vab-3 alleles k121 and k143 affected the anterior DTC more frequently than the posterior, while the strong vab-3(e648) affected both similarly. Another strong allele k109 isolated in the present screening also produced a highly penetrant effect on both DTCs (K. NISHIWAKI, unpublished results). Therefore, it is possible that the asymmetrical effect of vab-3 may be visible only for weak alleles. vab-3, which encodes a Pax6 family protein, has been reported to be expressed mainly in many neurons and most of the hypodermal cells in the anterior half of the body (CHISHOLM and HORVITZ 1995 ). The stronger effect of k121 and k143 on the anterior DTC might reflect the asymmetrical distribution of the VAB-3 protein.

DTC migration defects were classified into four distinct types. The type I defect was often observed in vab-3(k121) and mig-20(k148). One characteristic feature is a radical extra turn on the dorsal muscles that is so radical that it suggests a certain repulsive activity may be generated at the region where the turn occurs. The wild-type vab-3 and mig-20 gene products may function to suppress the generation of this repulsive activity and to make DTCs move straight along the dorsal muscles. Two mutations that affect sex myoblast migration, egl-15 and egl-17, are known to change an attractive interaction to a repulsive one (STERN and HORVITZ 1991 ).

The type II defect was mostly specific to strains of a mig-14 background and in vab-3(e648); mig-19 double mutants, and this defect was observed in the posterior DTC. mig-14(k124) also had QL/R neuroblast migration defects. The same Q cell migration abnormalities have been reported in mig-14(mu71) animals (HARRIS et al. 1996 ). mig-14 is proposed to be involved in the mab-5 pathway because the mig-14(mu71) mutation represses mab-5 expression in QL, thereby affecting QL migration (HARRIS et al. 1996 ). mig-14 also acts in a mab-5-independent manner to determine the final positions of QR descendants in the antero-posterior axis: the final positions of QR descendants are shifted posteriorly in mig-14 mutants whether or not mab-5 activity is present (HARRIS et al. 1996 ). Because the mab-5 null mutant e1239 is normal for DTC migration (KENYON 1986 ; K. NISHIWAKI, unpublished results) and mab-5 does not seem to be expressed in DTCs (COWING and KENYON 1992 ; SALSER et al. 1993 ; SALSER and KENYON 1996 ), the function of mig-14 in DTC migration is likely to be independent of mab-5. Although it is not clear whether the positioning function of mig-14 proposed for Q cells also operates in DTCs, the shortened migration distance of the posterior DTC on the ventral muscles in mig-14, which is due to an anterior shift of the dorsalward turning point, suggests this possibility. It remains to be determined whether the posterior DTC migrates more slowly in mig-14 than in wild type or if migration ceases precociously, so that the migration distance becomes shortened. Although vab-3(e648); mig-19 also showed a type II defect, it was not always associated with a shortening of the migration distance on the ventral muscles. This was also the case for vab-3; mig-14 double mutants, 20% of which exhibited shortened migration, but fewer than 10% of which had type II defects. These results suggest that the two phenotypes, the type II defect and the shortened migration on the ventral muscles, may be separable.

The type III defect was most frequently exhibited by mig-20 and double mutants carrying mig-20. The type was characterized by premature halt in migration, and this phenotype was also observed in HSN, QR.pa, and ccLa/p mother cell migrations; mig-20 function may be needed for cells to migrate normal distances.

The type IV defect was often seen in mig-17, -18, and -19 mutants. It appears that the guidance cues on basal laminae of the lateral hypodermal cells and the dorsal muscles for DTC migration, or recognition of these guidance cues by the DTC, had became obscured in these mutants. I found that a combination of mig-17 and mig-18 or mig-19 strongly enhanced the type IV defect. Surprisingly, this enhancement was especially prominent in the anterior DTC, in spite of the fact that the defect could seldom be observed in any of the three single mutants. This suggests that these three mutations affect the same or parallel pathways regulating anterior DTC migration.

Phenotypic suppression was found between some of the mutant classes. mig-14 suppressed mig-17, mig-18, mig-19, and mig-20. This might reflect the chronological order in which each of the major defective events in DTC migration caused by the respective mutations occurs (Figure 8). That is, mig-14 affects DTC migration prior to the first DTC turn; mig-17, -18, and -19 affect it after the first turn; and mig-20 affects it after the second turn. However, this idea is not consistent with the fact that vab-3, which affects DTC migration after the second turn, suppresses mig-14. One model to explain this complexity is to postulate an interaction between vab-3 and mig-14. The expression of vab-3 might be partly repressed in the posterior body region by wild-type mig-14 activity, and it might be extended to the posterior body in a mutant mig-14 background. In mig-14 mutants, derepressed wild-type vab-3 expression in the posterior region may cause a type II defect in the posterior DTC. Thus, in vab-3; mig-14 double mutants, both DTCs could be similarly affected by the mutant VAB-3 protein and thus express the type I defect, exhibiting epistasis of vab-3 to mig-14. In the case of the vab-3(e648); mig-19 double mutant, vab-3(e648) suppressed mig-19, and concomitantly the slight vab-3(e648) posterior type II defect was strongly enhanced. mig-19 may affect a process partly redundant with vab-3 whose function is manifested when VAB-3 is depleted.

View larger version (36K): In this window In a new window Download PPT slide   Figure 8. Chronology of onset of major defective events. Approximate duration when defects are first visible for each mutation is plotted on the wild-type DTC path.

In mig-14 and mig-19 animals, migration of HSN neurons or Q neuroblasts (or their descendants) was also affected. HSN is born near the tail and migrates anteriorly. Q neuroblasts are born in the posterior body and migrate anteriorly (QR) or posteriorly (QL) while dividing in a stereotypical pattern (SULSTON and HORVITZ 1977 ). The effects of these mutations on such migratory cells in the anterior body region as ALM, CAN, and coelomocyte mother cells (mothers of ccLa/p and ccRa/p) were weak (Figure 7). These observations, together with the fact that mig-14 and mig-19 had stronger effects on posterior DTC migration, suggest that the mig-14 and mig-19 genes may be especially important for the migration of various cell types in the posterior body region.

mig-14; mig-20 and mig-19; mig-20 double mutants showed abnormal movement. Because various genes have been reported that are required both for cell migration and axon outgrowth (HEDGECOCK et al. 1990 ; WIGHTMAN et al. 1996 ; FORRESTER and GARRIGA 1997 ), the strong uncoordinated movement phenotypes observed in these double mutants may be caused by defects in the migration of axons required to generate the neural connectivity essential for normal animal locomotion.

	ACKNOWLEDGMENTS

I thank Ed Hedgecock and members of his lab for introducing me to the fundamentals of C. elegans postembryonic development. I am grateful to Lee Honigberg, Edward Kipreos, John Plenefisch, Bruce Vogel, Yo Tabuse, and Tohru Sano for critical reading of the manuscript. I also thank Lee Honigberg and Cynthia Kenyon for mig-14(mu71), Christina Borland and Michael Stern for unc-71(ay7) bli-5(e518), and Theresa Stiernagle for various strains. I am grateful to Ed Hedgecock, Johji Miwa, Yo Tabuse, and Tohru Sano for discussions and encouragement and Saori Gouda and Toshie Miyamoto for technical assistance. Some C. elegans strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources.

Manuscript received July 6, 1998; Accepted for publication March 12, 1999.

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