Protruding Vulva Mutants Identify Novel Loci and Wnt Signaling Factors That Function During Caenorhabditis elegans Vulva Development

Corresponding author: David M. Eisenmann, Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250., eisenman{at}umbc.edu (E-mail)

Communicating editor: R. K. HERMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Caenorhabditis elegans vulva develops from the progeny of three vulval precursor cells (VPCs) induced to divide and differentiate by a signal from the somatic gonad. Evolutionarily conserved Ras and Notch extracellular signaling pathways are known to function during this process. To identify novel loci acting in vulval development, we carried out a genetic screen for mutants having a protruding-vulva (Pvl) mutant phenotype. Here we report the initial genetic characterization of several novel loci: bar-1, pvl-4, pvl-5, and pvl-6. In addition, on the basis of their Pvl phenotypes, we show that the previously identified genes lin-26, mom-3/mig-14, egl-18, and sem-4 also function during vulval development. Our characterization indicates that (1) pvl-4 and pvl-5 are required for generation/survival of the VPCs; (2) bar-1, mom-3/mig-14, egl-18, and sem-4 play a role in VPC fate specification; (3) lin-26 is required for proper VPC fate execution; and (4) pvl-6 acts during vulval morphogenesis. In addition, two of these genes, bar-1 and mom-3/mig-14, are known to function in processes regulated by Wnt signaling, suggesting that a Wnt signaling pathway is acting during vulval development.

DURING development, polarized epithelial cells are exposed to signals from surrounding cells that cause them to modify their behavior or cellular fate. In responses to external signals, cells may undergo cell division and terminal differentiation or may undertake coordinated morphogenetic movements. One excellent model system for studying the processes of cell signaling, cell polarity, cell-fate determination, and morphogenesis is the development of the hermaphrodite vulva of the nematode Caenorhabditis elegans (reviewed in GREENWALD 1997 ).

The formation of the vulval opening has been extensively studied, and this process has been divided into four stages (Fig 2; FERGUSON et al. 1987 ; GREENWALD 1997 ): (1) the "generation" stage in which the blast cells that will give rise to the vulva are born; (2) the "fate specification" stage in which these cells are induced to adopt distinct cell fates by extracellular signals; (3) the "execution" stage in which these cells divide and differentiate according to their cell fate and generate 22 vulval cells; and (4) the "morphogenesis" stage during which these 22 cells go through extensive morphogenetic movements to form the opening connecting the uterus to the outside.

View larger version (28K): In this window In a new window Download PPT slide   Figure 1. Genetic map locations of the pvl loci. Each of the six C. elegans linkage groups is shown with the map positions of visible markers (above the line) and Tc1-linked STS markers (below the line) used in the mapping of the Pvl mutants. The three-factor mapping data upon which this figure is based are given in Table 2. Some mapping intervals are defined by deficiency mapping data, described in MATERIALS AND METHODS (deficiencies are not shown).

View larger version (30K): In this window In a new window Download PPT slide   Figure 2. Four stages of C. elegans vulval development. (A) Generation of P1.p–P12.p during the L1 stage. P3.p–P8.p make up the Vulval Equivalence Group. The remaining six cells fuse with the hypodermis of the animal during the L1 stage. (B) In the late L2 stage, the fates of the six vulval precursor cells (P3.p–P8.p) are determined in response to several extracellular signals: an inductive signal from the anchor cell, a lateral signal acting between Pn.p cells, and a general inhibitory signal acting between the Pn.p cells and the surrounding hypodermal syncytium. The outcome of these signaling events is that P6.p adopts the 1° vulval cell fate, its neighbors, P5.p and P7.p, adopt the 2° vulval cell fate, and P3.p, P4.p, and P8.p adopt the 3°, nonvulval cell fate. Note that P3.p also adopts the F fate 50% of the time in wild-type animals. (C) The vulval precursor cells execute their cell fates in the L3 stage. Shown are P3.p–P8.p with their patterns of cell division and the fate of their progeny cells (vulval or hypodermal). For P5.p–P7.p, the letters indicate the plane of the axis of the last cell division (see Table 2). (D) The 22 vulval cells go through morphogenetic movements during the L4 larval stage to form the vulval opening. A diagram of an intermediate stage in that process, referred to as the "Christmas tree" stage, is shown here. The 8 cells descended from P6.p occupy the center of the developing vulva, and the 7 cells each descended from P5.p and P7.p occupy the sides of the developing vulva. To the right of each diagram are indicated the genes examined in this work that are likely to function at that stage in vulval development.

During the generation stage, the 12 Pn.p cells, P1.p–P12.p, are born along the ventral midline of the animal in the early first larval stage (L1; SULSTON and HORVITZ 1977 ). The Pn.p cells are the posterior daughters of the 12 P cells, embryonic cells that migrate into the ventral midline from ventrolateral positions and divide to generate a Pn.a neuroblast and a Pn.p hypodermal cell (except P12, which follows a different fate). The 6 Pn.p cells in the mid-body region, P3.p–P8.p, express the Hox gene lin-39 and form the Vulval Equivalence Group (WANG et al. 1993 ; MALOOF and KENYON 1998 ). The results of genetic and laser ablation experiments have shown that all 6 Pn.p cells in the Vulval Equivalence Group are competent to generate vulval tissue, and they are therefore referred to as the vulval precursor cells (VPCs; SULSTON and HORVITZ 1977 ; SULSTON and WHITE 1980 ; STERNBERG and HORVITZ 1986 ). The cells that do not express lin-39 (P1.p, P2.p, P9.p–P12.p) do not form part of the Vulval Equivalence Group and become part of the large hypodermal syncytium of the animal.

The six VPCs are initially equivalent in developmental potential and are competent to adopt one of three distinct cell fates called 1°, 2°, and 3°. Cells adopting the 1° and 2° fates contribute to the vulva, while cells adopting the 3° do not, but rather contribute to the syncytial hypodermis (SULSTON and WHITE 1980 ; STERNBERG and HORVITZ 1986 ). Although all six VPCs are initially equivalent, the pattern of fates adopted in wild-type animals is always either 3° 3° 2° 1° 2° 3° or F 3° 2° 1° 2° 3° (in which P3.p adopts an alternate nonvulval fate described below). This invariant pattern of cell-fate specification is achieved through the use of multiple extracellular signals acting on the VPCs during the fate specification stage (reviewed in GREENWALD 1997 ; KORNFELD 1997 ). First, in the late L2/early L3 stage, a single cell of the somatic gonad called the anchor cell sends an inductive signal to the VPCs that causes the vulval precursor cell closest to the anchor cell, P6.p, to adopt the 1° cell fate. Second, P6.p then sends a lateral signal that, perhaps combined with low levels of anchor cell signal, causes the adjacent VPCs, P5.p and P7.p, to adopt the 2° vulval fate (STERNBERG 1988 ; STERNBERG and HORVITZ 1989 ; KOGA and OHSHIMA 1995 ; SIMSKE and KIM 1995 ). The remaining three VPCs, P3.p, P4.p, and P8.p, are not induced by either of these signals and consequently adopt the 3° cell fate, which is to divide once and fuse with the hypodermal syncytium. In addition, P3.p can also adopt a fourth cell fate called F, for fused, in which P3.p fuses with the hypodermal syncytium without dividing at all (FERGUSON and HORVITZ 1985 ; STERNBERG and HORVITZ 1986 ). The F fate is adopted by P3.p in 50% of wild-type animals (EISENMANN et al. 1998 ). Finally, genetic evidence exists for a third signal, an inhibitory signal from the surrounding hypodermal syncytium that antagonizes the anchor cell inductive signal, ensuring that only cells receiving high amounts of inductive signal adopt vulval fates (HERMAN and HEDGECOCK 1990 ).

During the execution stage (L3) the three induced cells, P5.p, P6.p, and P7.p, will each divide three times to generate a total of 22 progeny cells. P6.p (1° fate) will divide to generate 8 cells that form the center of the developing vulva; P5.p and P7.p (2° fate) will each divide to generate 7 cells that form the sides of the developing vulva (SULSTON and HORVITZ 1977 ; STERNBERG and HORVITZ 1986 ). During the morphogenesis stage in the L4, these 22 cells will go through specific cell fusions and short-range migrations to generate a stack of 7 mono- or multinucleate toroidal cells that form the vulval opening (NEWMAN and STERNBERG 1996 ; GREENWALD 1997 ; SHARMA-KISHORE et al. 1999 ).

Genetic analysis has identified a number of genes acting in vulval development (reviewed in EISENMANN and KIM 1994 ; GREENWALD 1997 ; KORNFELD 1997 ). These genes were identified because when mutated they generally cause a multivulva (Muv) phenotype in which more than three Pn.p cells adopt vulval fates, a vulvaless (Vul) phenotype in which no Pn.p cells adopt vulval cell fates, or because they suppress other Muv or Vul mutations. Molecular characterization of these genes has shown that the reception and transduction of the anchor cell inductive signal in the Pn.p cells is mediated by a conserved receptor tyrosine kinase/RAS/mitogen-activated protein (MAP) kinase signaling pathway utilizing the products of the let-23(RTK) (AROIAN et al. 1990 ), let-60(Ras) (HAN and STERNBERG 1990 ), and mpk-1(MAPK) (LACKNER et al. 1994 ; WU and HAN 1994 ) genes, respectively. Two transcription factors, LIN-31 and LIN-1, are known downstream targets of the Ras/MAP kinase pathway in the VPCs (MILLER et al. 1993 ; BEITEL et al. 1995 ; JACOBS et al. 1998 ; TAN et al. 1998 ). In addition to the Ras pathway mediating the anchor cell inductive signal, the lateral signal acting between Pn.p cells to specify the 2° cell fate is mediated by LIN-12, a Notch-like transmembrane receptor (YOCHEM et al. 1988 ). Therefore, vulval cell-fate specification in C. elegans utilizes two evolutionarily conserved extracellular signaling pathways.

Previous genetic screens to identify mutations affecting vulval development relied extensively on the identification or suppression of the multivulva and vulvaless phenotypes (HORVITZ and SULSTON 1980 ; SULSTON and HORVITZ 1981 ; FERGUSON and HORVITZ 1985 ). We considered it likely that genes exist that function in vulval development but which may not mutate to a Muv or Vul phenotype or that may not strongly suppress such a phenotype. To identify novel loci acting in vulval development that may have been missed in previous screens, we performed a genetic screen that relied on a novel, more subtle mutant phenotype, the protruding vulva or Pvl phenotype. In this way we have identified at least 11 loci, representing both novel and previously identified genes, which appear to act at different stages during vulval development. Here we present the preliminary genetic characterization of these loci and their vulval mutant phenotypes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genes, alleles, and general genetic procedures:
Methods for culturing, handling, and genetic manipulation of C. elegans were as described (BRENNER 1974 ). The animals described as wild type were C. elegans, variety Bristol, strain N2, which is the strain background in which all mutations were induced. The strain RW7000 was used for sequence-tagged site (STS) mapping experiments (WILLIAMS et al. 1992 ) and is C. elegans, variety Bergerac. All experiments were performed at 20° unless indicated. The genes, alleles, deficiencies, and STSs listed below were used in this work. The reference for all alleles is RIDDLE et al. 1997 , unless noted. The allele bar-1(sy324) was identified in a screen for mutations capable of suppressing the Muv phenotype of lin-1(e1777) (A. GOLDEN, unpublished results). The reference for all alleles designated "gaxx" is this work.

• LGI: lin-17(n671, ga58, ga69, ga83), unc-11(e47), unc-73(e936), lin-44(n1792), unc-74(e883), egl-34(n171), dpy-5(e61), sem-2(n1343), sem-4(n1971, ga82), lin-28(n719, ga73), unc-29(e1072), unc-59(e261, ga77, ga78), unc-54(r323), sDf4, nDf24, stP124, hP4, TCbn2.

• LGII: lin-31(n1053, n301, ga57, ga70), bli-2(e768), dpy-10(e128), unc-104(e1265), lin-26(n156, ga91), rol-6(e187), let-23(n1045), unc-4(e120), let-25(mn25), let-29/mix-1(mn29), let-243(mn226), let-244(mn97), let-245(mn185), let-268(mn189), lin-29(n333, ga93, ga94), mig-14(mu71, ga62), mom-3(or78) (THORPE et al. 1997 ), pvl-4(ga96), pvl-5(ga87), mnDf30, mnDF39, mnDf63, mnDf66, mnDf87, mnDf89, mnDf90, mnDf96, mnDf99, mnDf106, mnDf108, mnDf109, mnC1, stP100, stP196, stP101, stP50, stP36, stP98, maP1.

• LGIII: dpy-17(e164), dig-1(n1321, ga76), unc-32(e189), pvl(ga84), pvl(ga90), stP19, stP120, mgP21, stP127, stP17.

• LGIV: dpy-9(e12), egl-4(n478), egl-18(n162, n475, ga97), pvl(ga79), lin-1(e1777, n304, ga56, ga68), unc-17(e245), dpy-13(e184), let-60(n1046, ga89), dpy-20(e1282, e1362), dpy-4(e1166), stP13, stP51, stP44, stP4, stP5, stP35.

• LGV: pvl-6(ga81), unc-62(e644), unc-46(e177), unc-83(e1409, ga72), dpy-11(e224), pvl(ga88), unc-42(e270), lin-25(ga65, ga67), him-5(e1490), sDf20, stP192, bP1, stP6, stP18, stP108, stP105, stP23.

• LGX: unc-20(e112), lin-18(e620, ga75), lon-2(e678), dpy-8(e130), unc-6(e78, n102), bar-1(ga80, sy324), dpy-7(e1324, e88), unc-18(e81), unc-10(e102), dpy-6(e14), lin-14(ga54), lin-2(n768, e1309, ga59, ga60, ga61), unc-9(e101), unc-84(n1325, ga55, ga71), unc-3(e151), uDf1, nDf19, szT1, stP41, stP156, stP33, stP103, stP129, stP61, stP72, stP2.

To construct double-mutant strains, bar-1 was balanced by dpy-7 or unc-6, mom-3/mig-14 by unc-52, egl-18 by dpy-9 or unc-17, sem-4 by dpy-14, and let-60(n1046) by dpy-20. Balanced double heterozygote animals were identified and allowed to self for two generations until Egl/Pvl animals that no longer segregated either balancing marker were found. Complementation tests were performed to verify the strain genotype.

EMS mutagenesis, identification, and mapping of Pvl mutants:
EMS mutagenesis of N2 hermaphrodites was carried out as described (BRENNER 1974 ; RIDDLE et al. 1997 ). Mutagenized P0 animals were allowed to self-fertilize, F₁ progeny from these animals were picked, seven animals to a 60-mm plate, and their F₂ progeny were screened for fertile animals having a protruding vulval phenotype (Pvl). Only a single Pvl mutant animal was taken from each plate of F₂ animals. A total of 38,300 mutagenized haploid genomes were screened and 230 Pvl mutant strains were identified.To distinguish animals that displayed a Pvl phenotype due to defects in VPC generation, fate determination, or fate execution from those that were Pvl due to defects in vulval morphogenesis, we examined 30–50 L4 stage hermaphrodites from each mutant line by Nomarski differential interference contrast microscopy on a Zeiss Axioplan2 microscope. In wild-type animals at this stage, the 22 vulval cell nuclei occupy stereotyped positions, and the developing vulval structure forms a "Christmas tree"-shaped structure (Fig 2D and Fig 3C). We screened for those strains with animals that had fewer than 22 vulval nuclei, more than 22 vulval nuclei, or 22 vulval nuclei in incorrect positions. At this stage, 55 mutant strains had such defects, and of these, 36 had high or moderately penetrant Pvl or Egl (egg-laying-defective) phenotypes and were characterized further (for most Pvl mutants the Egl phenotype is more penetrant than the Pvl phenotype). At this stage, 175 mutant strains were wild type, but went on to be Pvl; therefore many of these mutants may have defects in the process of vulval morphogenesis. This class of mutants will not be described further here.

View larger version (170K): In this window In a new window Download PPT slide   Figure 3. Vulval phenotypes of Pvl mutants. Wild-type adult hermaphrodite (A) and Pvl adult hermaphrodite (B) showing the ventral mid-body region with the wild-type vulval opening (A) and the protruding vulva (Pvl) phenotype (B). Wild-type L4 stage animal showing the Christmas tree stage of vulva development (C) and similarly staged animals showing defective vulval induction in mom-3/mig-14 (D), egl-18 (E), and bar-1 (F) mutants. In these photos anterior is to the left and dorsal is to the top. Bar, 20 µm. In these mutant animals fewer than three Pn.p cells adopted induced cell fates only P6.p in D, P6.p and P7.p in E, and P5.p and P7.p in F.

Each of the 36 mutant strains was subjected to a PCR-based STS mapping strategy that relies on differences in transposon number and location between N2 and another C. elegans strain, RW7000. Briefly, for each strain pvl/+ males were crossed to RW7000 hermaphrodites and F₂ Pvl mutant animals were isolated. Two rounds of PCR were performed on individual F₂ Pvl animals to localize the mutation to one of the six chromosomes and then to localize the mutation to a smaller region of that chromosome (data not shown). The STS markers and oligonucleotides used are described in WILLIAMS et al. 1992 . Traditional three-factor mapping (Table 1) and deficiency mapping were also performed. Once each of the mutations was localized, standard complementation tests were performed with any loci in the same genetic interval that were known to perturb vulval development or egg laying when mutated (lin, let, egl, sar, sur, mig, unc, etc., genes; see GREENWALD 1997 and KORNFELD 1997 ). Complementation tests were not performed for new alleles of lin-14, lin-25, lin-28, and lin-29, but gene assignments were made based on phenotypes and map position and were subsequently confirmed by other investigators (TUCK and GREENWALD 1995 ; BETTINGER et al. 1996 ; S. EULING, personal communication). On the basis of complementation data and other criteria, we identified mutations in the following known genes: dig-1(ga76) (subsequently lost), egl-18(ga97), let-60(ga89), lin-1(ga56, ga68), lin-2(ga59, ga60, ga61), lin-14(ga54), lin-17(ga58, ga69, ga83), lin-18(ga75), lin-25(ga65, ga67), lin-26(ga91), lin-28(ga73), lin-29(ga93, ga94), lin-31(ga57, ga70), mom-3/mig-14(ga62), sem-4(ga82), unc-59(ga77, ga78), unc-83(ga72), and unc-84(ga55, ga71).

Several of the Pvl mutants may identify novel loci on the basis of their phenotypes, map positions, and complementation of mutations in known genes in the same genetic intervals. These mutants identify eight complementation groups each represented by a single allele isolated in this screen: bar-1(ga80), pvl-4(ga96), pvl-5(ga87), pvl-6(ga81), pvl(ga79), pvl(ga84), pvl(ga88), and pvl(ga90) (Fig 1). Those alleles designated pvl(gaxx) have been less well characterized and have not yet been given specific gene designations. pvl(ga90) is likely to be a heterochronic mutant and has not been studied further. All Pvl phenotypes are recessive and not affected by temperature, except that caused by let-60(ga89), which causes a partially dominant, temperature-sensitive Muv phenotype as described in EISENMANN and KIM 1997 .

Additional genetic data:
pvl-4: The deficiencies mnDf83 and mnDf66, but not mnD87, fail to complement pvl-4(ga96) for the Pvl phenotype. pvl-4(ga96) complements let-25(mn25), let-29(mn29), let-243(mn226), let-244(mn97), let-245(mn185), and let-268(mn189).

pvl-5: The deficiencies mnDf30, mnDf39, and mnDf96 fail to complement pvl-5(ga87) for the Pvl phenotype.

ga62/mom-3(or78) complementation test: N2 males were mated with rol-1(e91) mom-3(or78)/mnC1 hermaphrodites, and cross-progeny males from this cross were mated with unc-4(e120) ga62 hermaphrodites. Cross-progeny from this mating carrying the wild-type chromosome II, the mnC1 chromosome, and recombined rol-1 mom-3 chromosomes were found, but no rol-1 mom-3(or78)/unc-4 ga62 progeny could be definitively identified. A few sickly, Pvl cross-progeny were found that gave no live progeny. Examination of progeny embryos from known or78/ga62 mothers showed that while endoderm induction appeared normal, all embryos failed to hatch and exhibited morphogenesis defects similar to the 30% of embryos from or78/or78 mothers that make endoderm but also fail to hatch (A. SCHLESINGER and B. BOWERMAN, personal communication). Therefore, on the basis of their failure to complement for the zygotic Pvl phenotype and the maternal effect embryonic lethal phenotype, we believe ga62 and or78 are allelic.

egl-18: For egl-18 strains, a low percentage of animals had a strong Roller phenotype: ga97 = 8% (n = 240); n475 = 10% (n = 154); n162 = 10% (n = 283).

Characterization of Pvl mutants:
Following multiple backcrosses, each of the 12 Pvl loci in Fig 1 was characterized as follows. First, each strain was checked for embryonic and larval lethality by picking eggs to a petri plate (>200) and observing their development over sequential days. The mutants with >5% dead eggs were mom-3/mig-14(ga62) (20%), egl-18(ga97) (8%), pvl-4(ga96) (13%), pvl-5(ga87) (17%), and pvl(ga79) (7%). The only mutant displaying significant larval lethality was pvl-4(ga96) (14%). Second, the number of large hypodermal nuclei in the ventral midline of early L2 stage animals was determined to look for defects in Pn.p cell generation. Third, late L3 and early L4 stage animals were examined by Nomarski microscopy to determine the number of animals containing an abnormal vulval structure. P12.p to P11.p cell-fate transformations and defects in gonad migration were also noted. Finally, for several strains, the division patterns of P3.p–P8.p were directly determined by following vulval development in several living hermaphrodites starting in the L2 stage, using Nomarski optics. The criteria for designation of cell fate were as described in STERNBERG and HORVITZ 1986 . Cells were assigned a fate of either 1°, 2°, 3°, or F.

The F fate:
We have adopted the designation F (or fused) for the fate of P3.p in 50% of wild-type animals and for the fate adopted by other vulval precursor cells in mom-3/mig-14(ga62), bar-1(ga80), sem-4(ga82), and egl-18(ga97) mutants. A cell adopting this fate initially joins the Vulval Equivalence Group in the L1, unlike P1.p, P2.p, and P9.p–P11.p, which fuse with the hypodermal syncytium at this time. However, a vulval precursor cell adopting the F fate then fuses without dividing in the L3 stage at the same time as P4.p–P8.p begin their first round of cell division, as judged by MH27 staining (EISENMANN et al. 1998 and data not shown).

Q_L migration:
To determine if mom-3/mig-14(ga62), bar-1(ga80), egl-18(ga97), and sem-4(ga82) mutants exhibited defects in the migration of the progeny of the neuroblast Q_L, we used muIs35 (gift of C. Kenyon), an integrated array containing a mec-7::GFP reporter fusion gene that is expressed in the touch receptor neurons, including AVM (Q_R.paa) and PVM (Q_L.paa) (CHALFIE et al. 1994 ). We observed the position of green fluorescent protein (GFP) expressing cells in animals on a Zeiss Axioplan 2 using fluorescence microscopy.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Identification of protruding vulva (Pvl) mutants:
To identify genes that function in vulval development but which may have been missed in previous genetic screens that relied on the multivulva and vulvaless mutant phenotypes, we mutagenized wild-type worms with EMS and screened for mutations causing a protruding vulva or Pvl phenotype. The Pvl phenotype (Fig 3B) is characterized by the production of some vulval tissue (as opposed to a vulvaless mutant), but the inability to form a wild-type vulval structure, which results in eversion of vulval tissue and the formation of a single protrusion at the site of the vulva (as opposed to a multivulva mutant that has several ventral protrusions). Mutants exhibiting a similar mutant phenotype (Evl, for defective eversion of the vulva) have been previously described (SEYDOUX et al. 1993 ). We screened the equivalent of 38,300 mutagenized haploid genomes at 20° and identified 230 fertile mutants that display a Pvl phenotype at a penetrance >20%. Most of these mutants are also egg-laying defective (Egl) and can be either Pvl, Egl Pvl, or Egl non-Pvl. For many of the mutants, the Egl phenotype is more penetrant than the Pvl phenotype (Table 3).

In wild-type animals, the three ventral hypodermal cells P5.p, P6.p, and P7.p adopt vulval fates and divide to generate the 22 cells that make up the vulva (GREENWALD 1997 ; KORNFELD 1997 ). The nuclei of these 22 cells occupy stereotyped positions in the early L4 stage (Fig 2D and Fig 3C). We examined the 230 Pvl mutant strains at this stage using Nomarski differential interference contrast microscopy, and on the basis of the positions of the vulval cell nuclei in these mutants we classified them into two groups: (1) those with altered number or position of the vulval cell nuclei, suggesting defects in generation, fate specification, or fate execution by the VPCs and (2) those with 22 vulval cell nuclei in the proper positions, but which still become Pvl, suggesting defects in later processes such as vulval morphogenesis. Of the 230 mutants, 55 fell into the first class and we continued the characterization of 36 of these mutants that had highly penetrant mutant phenotypes.

On the basis of genetic mapping and complementation tests we have identified 26 mutations in the following previously identified genes: dig-1, egl-18, let-60, lin-1, lin-2, lin-14, lin-17, lin-18, lin-25, lin-26, lin-28, lin-29, lin-31, mom-3/mig-14, sem-4, unc-59, unc-83, and unc-84. Most of these genes were already known to function in vulval development in some manner. We have further characterized mutations in several of these genes because (1) their role in vulval development was previously not known (egl-18, mom-3/mig-14), (2) we discovered an additional role for the gene in vulval development distinct from that previously characterized (lin-26, sem-4), or (3) we identified an interesting allele of a gene (let-60(ga89)). let-60(ga89) creates a novel temperature-dependent activated Ras protein and is described in EISENMANN and KIM 1997 . Our characterization of the vulval defects in egl-18, lin-26, mom-3/mig-14, and sem-4 mutants is described below.

The remaining mutants appear to identify novel loci based on their map positions, phenotypes, and complementation of mutations in known vulval mutant loci (Fig 1). These mutants identify seven complementation groups, each represented by only a single recessive allele isolated in this screen: bar-1(ga80), pvl-4(ga96), pvl-5(ga87), pvl-6(ga81), pvl(ga79), pvl(ga84), and pvl(ga88). We have continued to characterize these pvl loci and below we present our analysis of four of them: bar-1, pvl-4, pvl-5, and pvl-6.

bar-1, mom-3/mig-14, egl-18, and sem-4 mutants have defects in vulval precursor cell fate determination:
During wild-type development P6.p adopts the 1° fate, P5.p and P7.p adopt the 2° fate, and P3.p, P4.p, and P8.p adopt the 3°, nonvulval cell fate. In addition, only P3.p can adopt another fate, the F fate (also called the 4° fate; CLANDININ et al. 1997 ), in which P3.p fuses with the hypodermal syncytium in the L3 stage without dividing (STERNBERG and HORVITZ 1986 ; FERGUSON et al. 1987 ; EISENMANN et al. 1998 ). Cell lineage analysis of animals carrying mutations in the genes bar-1, mom-3/mig-14, egl-18, and sem-4 shows that in these mutant animals vulval precursor cells other than P3.p can adopt the F fate (Table 2 and Fig 3). This F fate phenotype is most penetrant for the cells P3.p and P4.p in these mutants (Table 2). In addition, in these mutants cells that should adopt the 1° and 2° cell fates, P5.p–P7.p, can also adopt the F fate or the 3° nonvulval cell fate ("S S" in Table 2). The result of these two defects is that fewer than 22 vulval cells are formed, leading to the Pvl and/or Egl mutant phenotypes. Three of these loci were identified previously in different genetic screens: sem-4 (DESAI et al. 1988 ; BASSON and HORVITZ 1996 ), egl-18 (TRENT et al. 1983 ), and mom-3/mig-14 (HARRIS et al. 1996 ; THORPE et al. 1997 ; NISHIWAKI 1999 ), but their effects on vulval development were not characterized. The fourth gene, bar-1, was initially identified in this screen and has subsequently been further characterized (EISENMANN et al. 1998 ; MALOOF et al. 1999 ).

bar-1(ga80) X:
A single mutation in the bar-1 gene, ga80, was identified in the screen for Pvl mutants. Roughly half of bar-1(ga80) mutant animals display an Egl and/or Pvl mutant phenotype (Table 3 and Fig 3B), and analysis of individual ga80 mutant animals by Nomarski microscopy shows that this is due to defects in cell-fate specification by the cells P5.p, P6.p, or P7.p (Table 3 and Table 4, Fig 3F). In most animals, only one of these three cells is affected, although animals in which two or all three of P5.p, P6.p, and P7.p adopted an uninduced cell fate (3° or F) were seen (the latter are true vulvaless animals and are 10% of ga80 hermaphrodites; data not shown). In addition, this analysis showed that in ga80 mutants, the cells P3.p and P4.p adopt the Fused fate in the majority of animals, while the cells P5.p–P8.p adopted this fate less often (Table 2 and EISENMANN et al. 1998 ). Therefore, in bar-1(ga80) mutant animals all six vulval precursor cells can exhibit defects in adoption of their proper cell fate, indicating this gene must be required for cell-fate specification by all of the cells of the Vulval Equivalence Group. Additional alleles of bar-1 have been identified in other genetic screens (mu38, mu349, MALOOF et al. 1999 ; sy324, A. GOLDEN, unpublished results).

STS mapping and three-factor crosses indicate that the bar-1 locus is located between unc-6 and dpy-7 on linkage group X. The deficiency uDf1 fails to complement bar-1(ga80), and the Egl mutant phenotype of these animals is not substantially different from that of ga80 homozygotes (data not shown), suggesting that ga80 may be a null mutation. This result was substantiated by the cloning and sequencing of the bar-1 gene and the determination that the ga80 mutation causes a premature stop codon early in the predicted open reading frame (EISENMANN et al. 1998 ). The locus identified by the ga80 mutation was named bar-1, after the determination that the protein product is related to the ß-catenin/Armadillo family of proteins, which are known to function in Wnt signaling pathways. We have previously reported that the defect in vulval cell-fate specification in bar-1 mutants may be due to a defect in maintenance of expression of the Hox gene lin-39 in these cells (EISENMANN et al. 1998 ).

In addition to the Egl/Pvl phenotype, bar-1 mutant animals have several other phenotypes. First, bar-1 mutants have a defect in cell-fate specification by the posterior ectodermal cell P12 (Table 3). In wild-type animals, the most posterior Pn.p cell, P12.p, divides during the L1 stage to give rise to a posterior daughter, P12.pp, which undergoes a programmed cell death, and an anterior daughter P12.pa, which becomes the hypodermal cell hyp12 (SULSTON and HORVITZ 1977 ). When observed in the L2 stage, the nucleus of P12.pa is smaller than that of the other undivided Pn.p cells, which all have a large nucleus (Fig 4A). In bar-1 mutants a cell corresponding in nuclear morphology to P12.pa is not found and a second, large nucleus resembling P11.p is found in the same location (Fig 4B). This phenotype is due to a transformation of cell fate in the cell P12 to that of the cell P11 (FIXSEN et al. 1985 ). Mutations in components of a Ras signaling pathway, components of a Wnt signaling pathway, and the Hox genes mab-5 and egl-5 all affect cell-fate determination by this cell (see DISCUSSION; JIANG and STERNBERG 1998 , and references therein). Second, bar-1 mutant animals have a defect in the migration of the progeny of the neuroblast Q_L (Fig 4G). This bar-1 phenotype, in which the progeny of Q_L migrate incorrectly and behave like the progeny of Q_R, the sister cell of Q_L, has been described in detail (MALOOF et al. 1999 ). Mutations affecting Wnt signaling pathway components and the Hox gene mab-5 cause the same type of Q_L progeny migration defect as that seen in bar-1(ga80) (HARRIS et al. 1996 ; MALOOF et al. 1999 ). Third, bar-1 animals exhibit an incompletely penetrant uncoordinated phenotype (Unc). Most bar-1(ga80) animals move forward well but do not move backward as well as wild-type animals and often coil upon themselves (data not shown). Finally, bar-1(ga80) mutant males have a reduced efficiency of mating, as many more bar-1(ga80) males than wild-type males are routinely required to successfully sire cross-progeny (data not shown). Many mutations that affect cell-fate determination during vulval formation in the hermaphrodite are also known to affect cell-fate determination during formation of the male tail, leading to defects in male mating (EMMONS and STERNBERG 1997 ).

View larger version (122K): In this window In a new window Download PPT slide   Figure 4. P12, gonad migration, and QL migration phenotypes of bar-1(ga80) and mom-3/mig-14(ga62) mutants. (A–B) Posterior hypodermal cells in wild-type (A) and bar-1 mutant animals (B). (A) In wild type, P11.p has a large nucleus with a large nucleolus visible inside it, a nuclear morphology characteristic of the cells P1.p–P11.p. The cell P12.pa (anterior daughter of P12.p) has a noticeably smaller nucleus and nucleolus. (B) In bar-1(ga80) and mom-3/mig-14(ga62) mutants (not shown) two large nuclei are seen, characteristic of a transformation of the fate of the cell P12.p to that of P11.p. (C) Migration of the posterior arm of the gonad in a wild-type hermaphrodite at the L4 stage. The arrowhead points to the leading edge of the migrating gonad arm (the distal tip cell). This gonad arm began by migrating toward the posterior, then turned dorsally (indicated by the arrow), and finally turned back toward the anterior. (D) Defective posterior gonad arm migration in a ga62 mutant animal. In this animal the posterior gonad arm did not reflex dorsally and continued to migrate into the tail of the animal. (E) Defective migration of the anterior gonad arm into the head in a mom-3/mig-14(ga62) mutant animal. (F–H) Expression of the touch receptor reporter construct mec-7::GFP in wild type, bar-1, and mom-3/mig-14 mutants. The QL neuroblast is born in the posterior of the animal and migrates further posteriorly, dividing to generate three progeny neurons. One of these cells, PVM (QL.paa), is a touch receptor neuron in the posterior of the animal that expresses the mec-7::GFP fusion protein (CHALFIE et al. 1994 ). The sister of QL, the neuroblast QR, divides in the same pattern but migrates toward the anterior. QR generates the touch receptor neuron AVM in the anterior, which expresses mec-7::GFP. (F) GFP expression pattern of a wild-type animal carrying the mec-7::GFP transgene (muIs35). The cell bodies of QR- and QL-derived touch receptor neurons AVM and PVM are indicated (arrows). Also shown are ALMR and ALML (not distinguished), two other touch receptor neurons in the mid-body that also express the mec-7::GFP fusion at this time. In bar-1 (G) and mom-3/mig-14 (H) mutants at this stage, there is no large PVM-like cell body expressing GFP in the posterior, but there are now two GFP-expressing cell bodies (arrows) anterior to AVML/R (arrowheads), consistent with QL and its progeny having migrated toward the anterior as QR does.

mom-3/mig-14(ga62) II:
Mutant animals carrying the mutation ga62 display a range of phenotypes similar to those of bar-1(ga80) mutants. The majority of ga62 mutant animals have an Egl or Egl Pvl phenotype due to defects in vulval precursor cell-fate determination (Table 2 and Table 3 and Fig 3D). Specifically, the cells that give rise to the vulva can adopt either the F fate, the 3° fate, or abnormal fates causing too few vulval cells to be generated, and the cells P3.p and P4.p usually adopt the F fate. ga62 mutants also have defects in P12 cell-fate specification (Table 3) and Q_L progeny migration (Fig 4H), and ga62 males mate poorly (data not shown). In addition, ga62 animals exhibit a defect in gonad migration only rarely seen in bar-1 mutants (Table 3 and Fig 4D and Fig E).

Molecular and genetic mapping data indicate that the locus identified by ga62 is located on the right arm of linkage group II. Two previously identified loci, mig-14 and mom-3, map to the same region. A single mutation in mig-14, mu71, was identified in a screen for mutants with misplaced Q descendants (HARRIS et al. 1996 ). Our observation that mu71 mutants have a weak Egl phenotype and show defects in vulval cell and P12 fate determination (Table 3), coupled with the observation that ga62 mutants have defects in the migration of the progeny of Q_L, suggested that mu71 and ga62 might be allelic. Indeed, we found that these two mutations fail to complement for the Egl phenotype (data not shown), suggesting that ga62 is a new allele of mig-14.

Mutations in the gene mom-3 cause a maternal-effect embryonic lethal phenotype characterized by a conversion of endoderm to mesoderm (THORPE et al. 1997 ). Although the mom-3 gene product has not been identified, loss of activity of the genes mom-2 (Wnt), mom-5 (Frizzled-type receptor), wrm-1 (ß-catenin), and pop-1 (HMG box/LEF factor) also disrupts this cell-fate decision, indicating a Wnt signaling pathway is functioning in this process (ROCHELEAU et al. 1997 ; THORPE et al. 1997 ). Homozygous mom-3 mutant animals, in addition to producing dead embryos due to maternal requirements for endoderm induction, also display a protruding vulva phenotype, consistent with a zygotic requirement for mom-3 during larval development (THORPE et al. 1997 ). On the basis of the observations that 20% of ga62 homozygous animals die during embryogenesis (data not shown) and that ga62 causes phenotypes similar to those caused by mutation of the ß-catenin homolog, BAR-1, we performed a complementation test to determine whether ga62 might be a reduction-of-function mutation in mom-3. We found that rare ga62/mom-3(or78) animals were very sick, Unc, and Pvl and gave few or no viable progeny, indicating that ga62 and or78 fail to complement for both the Pvl phenotype and a maternal-effect embryonic lethal phenotype (see MATERIALS AND METHODS). Therefore ga62 is also likely to be a viable allele of mom-3. Since no deficiency exists in the ga62/mom-3/mig-14 genetic interval, we have been unable to test whether ga62/deficiency animals have more severe phenotypes, perhaps resembling those of ga62/mom-3(or78). However, our interpretation of the complementation data is that mu71, ga62, and or78 are likely to be mutations in a single locus and that mu71 and ga62 retain more wild-type function than or78. Another allele of this gene, k124, was recently identified in a screen for mutations causing a gonad migration defect (NISHIWAKI 1999 ). We refer to this gene as mom-3/mig-14 and conclude that mom-3/mig-14 gene function is necessary for the vulval precursor cells and other cells to specify their fates correctly.

egl-18(ga97) IV:
The majority of animals homozygous for the ga97 mutation display an Egl, Pvl, or Egl Pvl phenotype (Table 3). Cell lineage analysis of ga97 hermaphrodites showed that the basis for these phenotypes was similar to that for bar-1(ga80) and mom-3/mig-14(ga62); P5.p–P7.p can adopt the Fused or 3° cell fates instead of the 1° or 2° cell fate (Table 2 and Fig 3). In addition, P3.p, P4.p, and P8.p adopted the Fused fate most of the time. ga97 mutants also show another phenotype affecting the vulval precursor cells: in this strain P5.p (and less frequently P7.p) began to divide but did not complete three rounds of cell division (Table 2; N = nondivided). The cells that were generated did not participate in vulval formation and began to lose their characteristic "hypodermal" nuclear morphology (data not shown). Unlike bar-1(ga80) and mom-3/mig-14(ga62) mutants, egl-18(ga97) mutants do not display a highly penetrant defect in cell-fate determination by P12 (Table 3) or in migration of the Q_L progeny (data not shown). However, like these other mutants, ga97 males mate poorly (data not shown). In addition, ga97 mutant animals display a low penetrance Roller phenotype (Rol) at all stages (8%, n = 250). Observation of egl-18(ga97) L4 stage larvae by Nomarski microscopy showed that those animals that display the Rol phenotype have an abnormally twisted head region, suggesting a defect in the cuticle or hypodermis in this region (data not shown).

The ga97 mutation was mapped to the left arm of linkage group IV between dpy-9 and lin-1. The gene egl-18, which was identified in a screen for mutants exhibiting an Egl phenotype, also maps in this region (TRENT et al. 1983 ). Previous analysis noted that egl-18 mutants sometimes adopt a "bag of worms" phenotype; however, the nature of the vulval defect in egl-18 mutants was not determined. We found that the existing egl-18 alleles n162 and n475 fail to complement ga97 for the Egl and Rol phenotypes, and egl-18(n162) and egl-18(n475) mutant animals show the same types of defects in vulval cell-fate specification as observed for ga97 mutants (Table 2). These results indicate that ga97 is a new allele of egl-18 and that the egl-18 locus is required for vulval precursor cells to undergo proper cell-fate specification. Since no deficiency exists for the genetic interval containing egl-18, we do not know if the defects in vulval induction we have observed in ga97, n162, and n475 represent the null phenotype for this locus.

sem-4(ga82) I:
The mutation ga82 causes a highly penetrant Egl phenotype and very rarely causes a Pvl phenotype (Table 3). Cell lineage analysis of ga82 mutant animals shows that P5.p and P7.p sometimes adopted the 3° fate inappropriately, leading to fewer than three Pn.p cells adopting induced fates, and that P3.p and P4.p often adopted the F fate (Table 2). Finally, ga82 mutants also display a P12 to P11 cell-fate transformation, but at much lower penetrance than that seen in bar-1 or mom-3/mig-14 mutants (12%; Table 4). sem-4(ga82) mutants do not display an obvious Q_L descendant migration defect (data not shown).

Genetic and physical mapping placed the ga82 mutation on linkage group I between dpy-5 and unc-13, a location containing the locus sem-4, which encodes a putative zinc-finger transcription factor (BASSON and HORVITZ 1996 ). sem-4 mutant animals were identified by their sex muscle migration-defective (Sem) phenotype, but also have defects in several other cell-fate specification processes. The lack of appropriately placed sex muscles leads to a highly penetrant Egl phenotype in sem-4 mutants. In addition, it was noted that sem-4 mutants display precocious divisions of Pn.p cells, resulting in extra hypodermal cells in the ventral mid-body region (BASSON and HORVITZ 1996 ). The defect in vulval cell-fate specification described here was not noticed in the previous analysis; however, we observed the same type of defect in animals containing sem-4(n1971) (Table 2), a mutation believed to be a null mutation on the basis of genetic and molecular criteria (BASSON and HORVITZ 1996 ). We found that sem-4(n1971) and ga82 fail to complement for the egg-laying defective phenotype, indicating that ga82 is a new allele of sem-4. This suggests that sem-4 activity is required for the vulval precursor cells to adopt their cell fates correctly.

Double-mutant analysis:
We constructed several double-mutant strains containing mutations in two of the loci bar-1, mom-3/mig-14, egl-18, and sem-4 (Table 3). Since these mutations cause the same type of phenotype the utility of this type of analysis is limited; however, two observations are worth noting.

First, in double mutants containing the sem-4(ga82) mutation and either bar-1(ga80) or mom-3/mig-14(ga62), a synthetic vulvaless phenotype was observed when the double-mutant animals were examined by Nomarski microscopy. In general, animals singly mutant for bar-1, mom-3/mig-14, or sem-4 show some vulval invagination at the L4 stage because at least one cell adopts an induced fate (Table 2 and Fig 3). For each of these single mutants the percentage of animals in which none of the vulval precursor cells adopts an induced fate (a vulvaless phenotype) is <10% (Table 2 and data not shown). However, most animals display no vulval invagination in the sem-4(ga82); mom-3/mig-14(ga62) and sem-4(ga82); bar-1(ga80) double-mutant strains when observed by Nomarski microscopy (70% for sem-4; bar-1 and 84% for sem-4; mom-3/mig-14; n = 100), and most animals display the "bag of worms" phenotype characteristic of a vulvaless mutant phenotype.

Second, we also observed that each of these mutations causes a low penetrant "spewed gonad" phenotype in which bodies are found on the plate containing their gonads everted out through the vulval opening (20%; Table 3). These animals are often smaller than the living adults, suggesting that this defect may have manifested at the L4 to adult molt. We found that in the egl-18(ga97); bar-1(ga80) double mutant the penetrance of the spewed gonad phenotype was higher (63%) than expected from addition of the single-mutant phenotypes. We believe that this synergistic spewed gonad phenotype also prevented us from isolating a double-mutant strain containing egl-18(ga97) and mom-3/mig-14(ga62) (data not shown). The cellular basis for this mutant phenotype is currently not known, although it may indicate a role for these loci in later steps in vulval development.

Mutations in bar-1, mom-3/mig-14, egl-18, and sem-4 suppress the multivulva phenotype caused by an activated Ras mutation:
Since the mutations described above cause defects in cell-fate specification by the vulval precursor cells, a process known to be regulated by a Ras signaling pathway, we determined whether the activity of these genes was necessary for cell-fate specification by the Ras pathway. To address this we built double-mutant strains containing each of the four mutations in combination with an activated Ras mutation, let-60(n1046) (FERGUSON and HORVITZ 1985 ; BEITEL et al. 1990 ). let-60(n1046) causes all six vulval precursor cells to adopt the 1° and 2° induced cell fates, leading to a highly penetrant Muv phenotype (93%; Table 4). If signaling through the Ras pathway requires the activity of these four genes, then the Muv phenotype caused by the ectopic activation of that pathway by let-60(n1046) should be decreased when the pvl mutations are introduced. This was the case (Table 4), since both bar-1 and mom-3/mig-14 mutations strongly reduced the penetrance of the Muv phenotype caused by let-60(n1046) (from 93 to <10%), and egl-18 and sem-4 mutations also reduced the Muv phenotype to <50%. Since activation of let-60 Ras cannot bypass the requirement for these four genes, this suggests that bar-1, mom-3/mig-14, egl-18, and sem-4 are required for cells to respond to activation of the Ras pathway and adopt induced cell fates.

lin-26(ga91) II mutants have defects in adoption of the 2° vulval cell fate:
During the execution stage Pn.p cells divide to generate progeny cells in a manner indicative of the fate they adopted during the cell-fate specification stage (GREENWALD 1997 ). For example, a cell adopting the 2° fate divides in the manner LLTN (or NTLL), where L refers to a longitudinal, or anterior-posterior, cell division, T indicates a transverse, or left/right, axis of division, and N indicates a nondividing cell. We identified a mutation, ga91, which causes a defect in the execution stage of vulval development.

ga91 mutant animals have an almost completely penetrant protruding vulva phenotype (>98%, n = 400). Cell lineage analysis of ga91 mutant animals showed that generally in these animals the correct number of Pn.p cells adopted induced cell fates, but that the most animals displayed a subtle defect in the execution of the 2° cell fate by P5.p and/or P7.p. Only 4 of 18 cells adopting the 2° fate divided in the correct pattern. Instead P5.p and P7.p most often divided with the pattern LLLN (P5.p) or NLLL (P7.p) (Table 2 and Fig 5). This indicates that the fate of the cells that would normally adopt the 2° T fate (P5.ppa and P7.pap) has been altered in these animals. The P6.p descendant cells that divide transversely were not usually affected (Table 2). It is not clear whether this 2° cell-fate defect is the cause of the strong Pvl phenotype of these animals; however, ga91 animals have no other obvious defects.

View larger version (135K): In this window In a new window Download PPT slide   Figure 5. Phenotypes of lin-26(ga91), pvl-4(ga96), and pvl-6(ga81) mutants. (A) Diagram showing the location of the progeny of P5.p–P7.p after their third rounds of division in wild-type and lin-26(ga91) mutants. Cells located along the anterior-posterior midline [the result of a longitudinal division (L)] are in white; cells located to left or right of the midline [the result of a transverse division (T)] are shown shaded. (B) Nomarski photomicrograph of a lin-26(ga91) mutant in which both P5.p and P7.p executed the 2° cell fate incorrectly. (C) Wild-type L1 stage larva. (D) L1 stage pvl-4(ga96) larva. Note the defects in morphology at the head and in the mid-body (Vab phenotype). (E) Nomarski photomicrograph of a wild-type animal after the second round of divisions of the cells P5.p–P7.p (L3 stage). White lines indicate the four progeny of each vulval precursor cell. The cells P6.paa and P6.ppp have begun dividing, and their nuclei are no longer distinct (arrowheads). The anchor cell of the somatic gonad is visible (arrow) and has crossed the basement membrane separating the gonad from the hypodermis and is contacting the inner two daughters of P6.p, which have begun to invaginate upward. (F) pvl-6(ga81) mutant at a slightly later stage of development than the animal in E. In this animal the anchor cell has not descended and is not obvious, and the progeny of P5.p, P6.p, and P7.p have recently finished their divisions. Although 22 vulval cells are present, only one focal plane is shown. Although this animal is at a later stage than the animal in E, the VPCs have not detached from the cuticle and begun to migrate dorsally. In all photos, anterior is to the left and dorsal is to the top.

We were surprised to find that ga91, which maps to the dpy-10-unc-4 interval on LG II, failed to complement lin-26(n156). The ga91/n156 transheterozygote has a Pvl/Egl phenotype like that of ga91 homozygous animals (data not shown). Sequence analysis has verified that ga91 represents a missense mutation in the lin-26 open reading frame (DUFOURCQ et al. 1999 ). lin-26 encodes a putative zinc-finger transcription factor that is expressed in the nuclei of all hypodermal cells and is believed to be a general factor required for hypodermal differentiation. The phenotype of the previously isolated lin-26 alleles (such as n156) precluded the identification of this phenotype because these mutations cause the absence of Pn.p cells (LABOUESSE et al. 1994 ; FERGUSON and HORVITZ 1985 ). This result indicates that lin-26, which is required early in development for the generation of the Pn.p cells, is also likely to play a later role in vulval development during the execution of the 2° fate.

pvl(ga88):
Another mutation, pvl(ga88), also shows a defect in Pn.p cell-fate execution and appears to affect predominantly cells dividing along the T axis. In ga88 mutant animals, the cells that would normally divide along the transverse (left-right) axis during the third divisions for the cells P5.p–P7.p are seen to divide along the longitudinal axis or in an oblique manner (not along any of the three defined axes; Table 2).

pvl-4 II and pvl-5 II mutants have defects in the generation of the Pn.p cells:
In wild-type hermaphrodites at the L2 stage there are 11 large hypodermal nuclei along the ventral midline from anterior to posterior (the nuclei of P1.p–P11.p) and one smaller hypodermal nucleus (the nucleus of P12.pa; SULSTON and HORVITZ 1977 ). Animals carrying recessive mutations in the genes pvl-4 and pvl-5 have too few Pn.p cells present in the ventral hypodermal region at this time. pvl-4(ga96) mutants have an average of 7.7 Pn.p nuclei in the ventral cord (N = 50, range of 6–11) and pvl-5(ga87) mutants have an average of 7.4 Pn.p nuclei (N = 50, range of 4–10). Often in these mutants, fewer than three of the Pn.p cells that are generated adopt vulval fates, leading to the Pvl/Egl phenotype. To determine if the decrease in Pn.p cell numbers seen in pvl-4(ga96) and pvl-5(ga87) animals represents the null phenotype for both of these loci, we placed each mutation over a chromosomal deficiency. We found that in pvl-5(ga87)/mnDf30 animals, Pn.p cells are still generated, and the number of Pn.p nuclei in these animals is similar to that in pvl-5(ga87) animals (P. JOSHI and D. EISENMANN, unpublished results). This suggests that either loss of pvl-5 does not completely abolish Pn.p cell generation and that ga87 represents a null mutation in pvl-5 or that the ga87 mutation is a non-null mutation that, when hemizygous, still provides enough pvl-5 activity to allow the process to occur. The isolation of additional pvl-5 mutations and the molecular characterization of the pvl-5 gene should allow us to distinguish between these possibilities. We have not determined the number of Pn.p cells in pvl-4(ga96)/Df animals because these animals generally do not develop to the L2 stage (see below), suggesting that ga96 is a hypomorphic allele of pvl-4.

We do not know the reason for the decrease in Pn.p cell nuclei in pvl-4 and pvl-5 mutants. In neither of these mutants are there Pn.p-like hypodermal nuclei present in the dorsal region as in unc-83 and unc-84 mutants (FERGUSON and HORVITZ 1985 ). In addition, staining with antibodies to the transcription factor LIN-31, which is present in the nuclei of P1.p–P11.p, shows that the only LIN-31-expressing nuclei are present in the ventral hypodermal region, and the number of LIN-31-expressing nuclei is comparable to the number of hypodermal nuclei observed by microscopy (data not shown).

In addition to the defect in Pn.p cell number, pvl-4(ga96) animals also have defects in head and body morphology. In particular, 52% (N = 418) of pvl-4 L1 and L2 larvae have a bent or notched head phenotype similar to that described for Vab mutants (CHISHOLM and HORVITZ 1995 ; Fig 5D). In addition, some pvl-4(ga96) early L1 larvae have a more severe phenotype characterized by a general misshapen or lumpy appearance. These larvae generally fail to thrive (data not shown). The specific nature of these defects is not known, but may indicate defects in other hypodermal cells besides the Pn.p cells in pvl-4 mutants. Further, when pvl-4(ga96) is placed in trans to the deficiency mnDf83, only a few lumpy or misshapen ga96/mnDf83 larvae are found, which die as young L1 animals (data not shown). This suggests that the null mutant phenotype of pvl-4 may be embryonic and/or larval lethality and that ga96 is a hypomorphic allele. pvl-4(ga96) complements the known let genes located in the same genetic interval, suggesting ga96 is likely to identify a novel locus.

pvl(ga79) IV:
Unlike pvl-4 and pvl-5 mutants, pvl(ga79) animals have too many Pn.p cell nuclei present in the ventral midline region. pvl(ga79) animals have an average of 12.8 Pn.p-like nuclei in the L2 (N = 20; range 12–14), suggesting that the extra cell(s) arise prior to this stage, perhaps from the precocious division of one or more of P1.p–P11.p during the L1 or L2, as is seen in lin-25, lin-31, and sem-4 mutants (MILLER et al. 1993 ; TUCK and GREENWALD 1995 ; BASSON and HORVITZ 1996 ). In addition to this defect in Pn.p cell number pvl(ga79) animals also often have a misshapen vulval structure (data not shown); however, the exact nature of the vulval defect for pvl(ga79) has not been determined.

pvl-6 V mutants display an altered interaction between the anchor cell and the descendants of P6.p:
During wild-type vulval development, the first morphogenetic movements begin in the L3 stage when the cells P5.p, P6.p, and P7.p have each divided twice to generate a total of 12 Pn.pxx cells (SULSTON and HORVITZ 1977 ; GREENWALD 1997 ). At this time, the anchor cell, which is centered over the four P6.p descendants, moves ventrally and appears to cross over the basement membrane separating the gonad from the hypodermis. At the same time, the inner two P6.p descendants, P6.pap and P6.ppa, move dorsally toward the descending anchor cell (Fig 5E; SULSTON and HORVITZ 1977 ; SHARMA-KISHORE et al. 1999 ). As these two cells move inward from the ventral midline, the remaining progeny of P6.p and some P5.p and P7.p progeny follow. At the same time, these cells begin their third round of divisions (Fig 5E), such that at the end of this process the 22 vulval cells have been generated and the Christmas tree-shaped vulval structure is formed (Fig 3C).

In animals carrying the pvl-6(ga81) mutation, the interaction between the anchor cell and the descendants of P6.p is often abnormal, presumably leading to the protruding vulva phenotype. We directly observed vulval development in eight pvl-6(ga81) animals and found that at the time during wild-type development when the anchor cell makes contact with P6.pap and P6.ppa, in six of eight ga81 animals the anchor cell had not descended to make contacts with these cells and these cells did not move dorsally toward the somatic gonad. These cells and the other Pn.pxx cells went on to divide and generate 22 cells; however, in the absence of morphogenetic movements by the P6.p descendants no vulval invagination was seen (Fig 5F). Subsequent observation of these ga81 mutants showed that the anchor cell did descend ventrally in these animals, sometimes making the correct contacts with the P6.pxxx cells and forming a wild-type-looking vulva (data not shown). In the remaining two of eight animals we observed that the anchor cell descended ventrally at the correct time, but did not make contact with P6.pap and P6.ppa and instead was displaced anteriorly or posteriorly and made contacts with other Pn.pxx cells at the 12-cell stage. In these animals the vulval structure that was formed was often misshapen (data not shown). Therefore, in pvl-6 mutants the defect is not in the generation of the vulval precursor cells, or in the adoption and execution of cell fates by those cells, but in the later process of vulval morphogenesis.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Here we describe the results of a genetic screen designed to identify novel loci functioning during C. elegans vulval development. We chose the Pvl phenotype as the basis for a new screen in an attempt to identify genes functioning at any stage of vulval development that may have been missed in previous genetic screens. Two reasons validate the choice of Pvl as a useful phenotype for this approach. First, the 36 mutations we isolated that have defects in the number or placement of the 22 vulval cell nuclei were either mutations in previously identified genes known to function in vulval development or identified novel loci. Second, preliminary characterization of the mutants described here shows that mutations affecting all four stages in vulval development can lead to a Pvl phenotype. Additionally, the fact that we isolated only a single allele for each novel locus suggests that this screen was not saturated and that additional loci may be found by continuing to identify mutants exhibiting this phenotype. We have initially concentrated our attention on several loci identified in this screen, some of which represent known loci not previously shown to function in vulval development and others that represent loci described here for the first time.

Genes affecting generation of the Pn.p cells:
During the L1 stage, the embryonically derived P cells divide to generate the 12 Pn.p cells, P1.p–P12.p. Six of the 12 Pn.p cells, P3.p–P8.p, constitute the VPCs, so mutations affecting the generation of the Pn.p cells can lead to defects in vulval development. Previous genetic analysis has identified several genes that affect the generation of the proper number of VPCs in the ventral midline when mutated. These include mutations that affect the migration of the P cells or their nuclei (unc-83, unc-84, unc-40; FERGUSON and HORVITZ 1985 ; CHAN et al. 1996 ; MALONE et al. 1999 ), the division of the P cells (lin-26; FERGUSON and HORVITZ 1985 ; LABOUESSE et al. 1994 ), and the specification of Pn.p cells as VPCs (lin-39; CLARK et al. 1993 ; WANG et al. 1993 ). In addition, mutations in lin-25, lin-31, and sem-4 result in too many Pn.p-like nuclei in the ventral hypodermal region because some Pn.p cells divide precociously during the L1 stage (lin-25) or L2 stage (lin-31 and sem-4; MILLER et al. 1993 ; TUCK and GREENWALD 1995 ; BASSON and HORVITZ 1996 ).

We describe here two mutations, pvl-4(ga96) and pvl-5(ga87), that cause too few Pn.p nuclei to be present in the ventral midline at the L2 stage. We currently do not know the process that is defective in either pvl-4 or pvl-5 mutants. For example, defects in P cell survival, migration, or division or in Pn.p cell survival or differentiation could all result in too few Pn.p-like nuclei being present. However, in neither pvl-4(ga96) nor pvl-5(ga87) animals are obviously mislocalized P cell nuclei seen, and the only cells that express a transcription factor found in P1.p–P11.p (TAN et al. 1998 ) are in the ventral cord in these mutants, suggesting that Pn.p cells are not born in other locations in these mutants. Preliminary data suggest that the egg-laying defect of pvl-5(ga87) mutants is partially suppressed by a mutation in the programmed cell death gene ced-3, suggesting that abnormal cell death of P or Pn.p cells may occur in pvl-5 mutants (P. JOSHI and D. EISENMANN, unpublished results). This suggests a possible function for pvl-5 in keeping the Pn.p cells from undergoing inappropriate cell death.

It is likely that the vulval phenotype seen in pvl-4(ga96) animals does not represent the pvl-4 null phenotype, since pvl-4(ga96)/mnDf83 animals do not survive and have severe defects in body morphology. The defects in head and body morphology seen in pvl-4(ga96) animals [the Vab phenotype (BRENNER 1974 )] are similar to those seen in vab-1 and vab-2 mutants, which encode an Ephrin receptor and an Ephrin ligand, respectively (GEORGE et al. 1998 ; CHIN-SANG et al. 1999 ). pvl-4(ga96) complements known lethal mutations in the genetic region where it maps and does not map near other predicted Ephrin homologs (WANG et al. 1999 ), suggesting that pvl-4 may represent a novel locus that is required for the structure, function, or differentiation of hypodermal cells, including the P and/or Pn.p cells. Future experiments will help determine how these two mutants function in Pn.p cell generation.

Genes acting during vulval precursor cell-fate specification:
The majority of genes identified in previous genetic screens for vulval mutants appear to act during the stage of vulval precursor cell fate specification (reviewed in GREENWALD 1997 ; KORNFELD 1997 ). Mutations in these genes generally cause either a multivulva phenotype or a vulvaless phenotype or suppress other mutations causing a Muv or Vul phenotype. We describe here four loci, bar-1, mom-3/mig-14, egl-18, and sem-4, that have a vulval cell fate specification defect that is distinct from those described previously. In these mutants the six VPCs can adopt a fourth cell fate, called the F or fused fate, which is a fate normally adopted by only P3.p in wild-type animals (SULSTON and HORVITZ 1977 ; STERNBERG and HORVITZ 1986 ). It is unclear what causes P3.p to sometimes fall out of the Vulval Equivalence Group and adopt the F fate instead of remaining as a vulval precursor cell during wild-type development. Whatever the mechanism, the mutant phenotypes of bar-1, mom-3/mig-14, egl-18, and sem-4 suggest that the activity of these genes is required to keep P4.p–P8.p from adopting a similar F fate in wild-type animals and that these four gene products normally function to ensure that proper vulval precursor cell fate specification occurs.

A Wnt signaling pathway is likely to be acting during vulval induction:
bar-1 encodes a C. elegans homolog of vertebrate ß-catenin and Drosophila Armadillo proteins (EISENMANN et al. 1998 ). Proteins of this family are known to function in both epithelial cell adhesion and in signal transduction by wingless/Wnt signaling pathways (GUMBINER 1995 ; MILLER and MOON 1996 ). We have previously proposed, on the basis of the identity of the BAR-1 product, the bar-1 mutant phenotype, and the regulation of lin-39 by bar-1, that a Wnt signaling pathway acts in the VPCs during cell fate specification (EISENMANN et al. 1998 ). Several lines of evidence support the model that a Wnt signaling pathway is acting in the VPCs during induction and that bar-1 and mom-3/mig-14 act in that pathway. First, bar-1(ga80) and mom-3/mig-14(ga62) mutants have a defect in cell-fate specification by the posterior ectodermal cell P12 like that caused by mutations in lin-44, which encodes a Wnt protein (HERMAN and HORVITZ 1994 ; HERMAN et al. 1995 ), and lin-17, which encodes a Frizzled (Wnt receptor) homolog (SAWA et al. 1996 ; JIANG and STERNBERG 1998 ), suggesting that bar-1 and mom-3/mig-14 are functioning in a Wnt-mediated developmental process in P12. Second, bar-1(ga80) and mom-3/mig-14(ga62) mutants have a defect in the migration of the progeny of the neuroblast Q_L that is like that caused by mutations in egl-20, which encodes a Wnt protein (MALOOF et al. 1999 ) and lin-17, suggesting that bar-1 and mom-3/mig-14 function in a Wnt-mediated process in Q_L and/or its progeny. Third, in both of those processes, a mutation in the Hox gene expressed in that cell (mab-5 in Q_L and egl-5 in P12) causes a defect like that caused by mutations in bar-1, mom-3/mig-14, or other Wnt pathway component genes (CHISHOLM 1991 ; JIANG and STERNBERG 1998 ; MALOOF et al. 1999 ). Consistent with this, bar-1 and mom-3/mig-14 are required for the expression of mab-5 in Q_L (HARRIS et al. 1996 ; MALOOF et al. 1999 ), and bar-1 is required for the expression of lin-39 in the VPCs (EISENMANN et al. 1998 ). Wnt signaling pathways are known to regulate Hox gene expression in Drosophila and Xenopus (BRANNON et al. 1997 ; RIESE et al. 1997 ). Finally, disrupting the activities of three other C. elegans homologs of Wnt pathway components can cause a Pvl phenotype. Reduction of mig-5 disheveled (ANTEBI et al. 1997 ) activity by RNA-interference technology (J. WAGMAISTER and D. EISENMANN, unpublished results), production of an antisense message to apr-1 APC in the VPCs (HOIER et al. 2000 ), and mutation of the mom-1 porcupine gene (THORPE et al. 1997 ) all cause a Pvl phenotype, suggesting that these genes may function in vulval development in a manner similar to bar-1 and mom-3/mig-14.

Therefore, since the bar-1 and mom-3/mig-14 mutant phenotypes in Q_L and P12 development are identical to those caused by mutations in known Wnt pathway components, we believe the most likely hypothesis is that bar-1 and mom-3/mig-14 are acting in a Wnt pathway during vulval precursor cell development as well. Mutations affecting this pathway were not identified in previous genetic screens for vulval mutants, most likely due to their incompletely penetrant vulval defects. In fact, only when Wnt signaling and Ras signaling are both compromised do most VPCs adopt the F fate (EISENMANN et al. 1998 , and data not shown). This indicates that in the process of cell-fate specification by the six VPCs, evolutionarily conserved Ras, Notch, and Wnt pathways all play a role. Also, Wnt signaling mediated by bar-1 has been shown to regulate expression of the Hox genes lin-39, mab-5, and egl-5 in three different postembryonic developmental events (cell-fate specification by the VPCs, Q_L, and P12), which suggests that Wnt signaling may be a general mechanism used to maintain expression of Hox genes in cells that adopt their fates during C. elegans postembryonic life.

Finally, it should be noted that mom-3/mig-14 appears to be involved in almost all developmental processes known or suggested to be controlled by Wnt signaling in C. elegans: (1) specification of the VPCs (this work and EISENMANN et al. 1998 ), (2) migration of Q_L (HARRIS et al. 1996 ), (3) specification of P12 (this work and JIANG and STERNBERG 1998 ), (4) asymmetric division of the embryonic blastomere EMS (THORPE et al. 1997 ), (5) proper orientation of the EMS mitotic spindle (SCHLESINGER et al. 1999 ), and (6) asymmetric division of P7.p (A. SCHLESINGER and B. BOWERMAN, personal communication). Experiments on the role of mom-3/mig-14 in EMS fate determination in the embryo have shown that mom-3/mig-14 activity is required in P2, the cell believed to secrete the Wnt ligand, and is not required in EMS itself (THORPE et al. 1997 ). This suggests a role for mom-3/mig-14 activity in expressing, processing, or secreting the Wnt ligand. In Drosophila, mutations in the porcupine gene, which encodes a transmembrane protein associated with the endoplasmic reticulum (KADOWAKI et al. 1996 ), and in several genes encoding factors involved in proteoglycan biosynthesis can lead to decreased signaling through the wingless pathway, presumably through a decrease in the amount of Wnt ligand available to interact with its receptor on receiving cells (VAN DEN HEUVEL et al. 1993 ; BINARI et al. 1997 ; HACKER et al. 1997 ; HAERRY et al. 1997 ; LIN and PERRIMON 1999 ). Although the mom-3/mig-14 gene has not been identified, examination of the C. elegans genomic DNA sequence in the interval to which it localizes shows that there are no genes homologous to known Wnt/wingless pathway components or regulators, suggesting that mom-3/mig-14 may encode a novel, general factor involved in Wnt signaling in C. elegans.

sem-4 is a previously identified gene that encodes a putative transcription factor containing seven C₂H₂-class zinc fingers similar to those in the Drosophila gene spalt and the human transcription factor PRDII-BFI (BASSON and HORVITZ 1996 ). sem-4 mutants have defects in cell-fate specification for several mesodermal and neuronal cell types generated during larval life. Here we show that sem-4 mutants display defects in VPC fate choices like those in bar-1 and mom-3/mig-14 mutants. Together this analysis suggests that sem-4 may be a transcription factor functioning in the differentiation of several postembryonically derived cells, including the VPCs. On the basis of the result that the combination of sem-4(ga82) with either bar-1(ga80) [a likely null mutation (EISENMANN et al. 1998 )], mom-3/mig-14(ga62), or egl-18(ga97) leads to a highly penetrant synthetic vulvaless phenotype (Table 3), it is possible that sem-4 may function in a distinct, redundant pathway from these other loci and that only when both pathways or functions are compromised is vulval induction strongly inhibited. It will be interesting to determine whether sem-4 is a downstream target of the Ras pathway or the Wnt pathway or whether sem-4 regulates lin-39 expression.

lin-26 acts during the execution of the 2° cell fate:
During the "fate execution" stage of vulval development, the vulval precursor cells execute the cell fate they adopted during the previous stage by dividing and differentiating in a fate-specific manner. Although there is much pattern formation going on at this stage, only a few mutations have been identified that perturb cell-fate execution. For example, lin-11 mutations cause cells adopting the 2° fate to divide in the pattern LLLL rather than LLTN/NTLL (FERGUSON and HORVITZ 1985 ; FREYD et al. 1990 ). The lin-11 gene encodes a LIM domain transcription factor that is expressed in the cells in the "TN" branch of the 2° lineage. Also, in lin-17 and lin-18 mutants, P7.p, which should execute the NTLL fate, executes either a reversed asymmetric LLTN fate (like P5.p) or the symmetric LLLL fate, as in lin-11 (FERGUSON and HORVITZ 1985 ; STERNBERG and HORVITZ 1988 ). lin-17 encodes a member of the Frizzled family of seven-transmembrane proteins that function as receptors in Wnt signal transduction pathways, suggesting that the polarity of the P7.p lineage may be controlled by an extracellular Wnt signal (SAWA et al. 1996 ).

lin-26 encodes a zinc-finger transcription factor expressed in the nuclei of all hypodermal cells that is believed to be a general factor required for hypodermal differentiation (FERGUSON and HORVITZ 1985 ; LABOUESSE et al. 1994 ). Our analysis shows that lin-26 also functions in a more specific manner, being required for the proper differentiation of two of the 12 Pn.pxx cells that generate the vulva. Consistent with the more subtle effect of this lin-26 mutation, it has been found that the lin-26(ga91) mutation represents a missense mutation in one of the two zinc fingers of LIN-26 (DUFOURCQ et al. 1999 ).

It is possible that the 2° lineage may be defined by the function of specific transcription factors in specific sublineages. For example, the presence of the LIN-11 factor in the TN half of the lineage causes those two cells to develop differently from the LL half of the lineage. The asymmetric segregation of lin-11 activity may be regulated by a Wnt signal, mediated by lin-17. The activity of lin-26 might then serve to make the T cell different from the N cell. However, this model presumes that the activity of lin-26 is required in the vulval precursor cell descendants for proper execution of the 2° lineage. Since lin-26 is expressed in all hypodermal cells, lin-26 could be acting in a nonautonomous manner to affect the division of the cells of the 2° T sublineage. Experiments designed to determine in which cells of a lin-26(ga91) mutant the wild-type lin-26 gene must be expressed for wild-type vulval induction to occur could resolve this issue.

pvl-6 acts at an early step in vulval morphogenesis:
After the 22 vulval cells have been generated by the divisions of P5.p–P7.p, the cells go through a series of short-range migrations and cell fusions to form the vulval opening (NEWMAN and STERNBERG 1996 ; SHARMA-KISHORE et al. 1999 ). During this process the 22 cells fuse into 7 mono- and multinucleate toroidal-shaped cells that make attachments to the surrounding hypodermal syncytium, the lateral hypodermal seam cells, and uterine cells in the somatic gonad. We know little about the genes required for the morphogenetic movements of the vulval cells. Mutations in eight sqv genes caused defects in morphology of the vulval opening, and three of these genes encode enzymes involved in glycosylation (HERMAN and HORVITZ 1999 ; HERMAN et al. 1999 ). Also, mutations in the genes cog-2 (encodes a Sox domain transcription factor; HANNA-ROSE and HAN 1999 ), egl-38 (encodes a Pax transcription factor; CHAMBERLIN et al. 1997 ), and lin-11 (encodes a LIM-domain transcription factor; FREYD et al. 1990 ; BLELLOCH et al. 1999 ) cause defects in the creation of a functional connection between the vulval cells and the uterine cells. We show here that a mutation in the gene pvl-6 causes defects in an early step in vulval morphogenesis, the interaction of the anchor cell with the descendants of P6.p.

In many pvl-6 mutants, the anchor cell does not descend ventrally at the time normally observed in wild type, and after eventually descending, it sometimes interacts with P6.paa and P6.pap, or P6.ppa and P6.ppp, instead of the central two P6.p descendants P6.ppa and P6.pap. We believe these "off-center" contacts are at least one cause of the Egl and Pvl phenotypes in these mutant animals. We do not know the identity of the pvl-6 gene product, nor do we know in what cell pvl-6 functions. Given the behavior of the anchor cell in pvl-6 mutants, it is possible that there is signaling between the anchor cell and P6.pap/P6.ppa to ensure that the proper cell-cell contact is made. pvl-6 could function in such a signaling process in either the anchor cell or P6.pap/P6ppa. Alternatively, pvl-6 could function in the migration of the anchor cell. Both of these models would explain why the behavior of the anchor cell appears temporally slower than in wild type, yet often the cell ends up making a correct interaction. Further genetic and molecular analysis of pvl-6, including the determination of whether this phenotype represents the pvl-6 null phenotype, will help clarify the role of pvl-6 in this early morphogenetic process.

	ACKNOWLEDGMENTS

We thank Cynthia Kenyon, Michel Labouesse, and Bruce Bowerman for communication of unpublished results. We thank Cynthia Kenyon for providing mig-14(mu71) and muIs35 strains, and Bruce Bowerman for providing a mom-3(or78) strain. We thank the members of the Kim laboratory for helpful discussions and other support, and Suzanne Barr, Albert Candia, and Bruce Wightman for critical reading of the manuscript. We thank Elizabeth Chen for help during the beginning stages of the Pvl genetic screen, and Kirsten Rhodes and Irene Su for help at subsequent stages. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources. This research was supported by grants to S.K.K. from the Lucille P. Markey Charitable Trust, the Searle Scholars Program/The Chicago Community Trust and the National Institutes of Health. D.M.E. was supported by an American Cancer Society Postdoctoral Fellowship Award, a Basil O'Connor Starter Scholar Research Award from the March of Dimes, and by the National Science Foundation under grant no. 9817123.

Manuscript received April 5, 2000; Accepted for publication July 20, 2000.

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