BIOLOGICAL OVERVIEW

naked cuticle (nkd), a Drosophila segment-polarity gene, encodes an inducible antagonist for the Wnt signal Wingless (Wg). nkd was identified by the inabilility of known naked mutants to complement the lethality of enhancer trap line l(3)4869, which exhibits a weak nkd phenotype. The l(3)4869 insert was used to positionally clone ndk. In fly embryos and imaginal discs nkd transcription is induced by Wg. In embryos, decreased nkd function has an effect similar to excess Wg; at later stages such a decrease appears to have no effect. nkd encodes a protein with a single EF hand (a calcium-binding motif) that is most similar to the recoverin family of myristoyl switch proteins. Nkd may therefore link calcium ion fluxes to the regulation of the potency, duration or distribution of Wnt signals. Signal-inducible feedback antagonists such as nkd may limit the effects of Wnt proteins in development and disease (Zeng, 2000).

Overproduction of Nkd in Drosophila and misexpression of Nkd in the vertebrate Xenopus laevis result in phenotypes resembling those of loss of Wg/Wnt function. When P[Hs-nkd] is used to overexpress nkd in otherwise wild-type embryos, rare cuticles with weak wg-like denticle belt fusion phenotypes are observed, similar to those seen when zw3 is overexpressed. Nkd is more potent in a sensitized wg/+ background. In wg^Il114/+ embryos, induction of P[Hs-nkd] before 4 h AEL results in decreased en and wg expression. wg^Il114/+ embryos are patterned normally, but practically all wg^Il114 /+ embryos exposed to high levels of Nkd secrete cuticles with denticle belt fusions and an excess of the predominant denticle type made by wg/wg embryos (Zeng, 2000).

Misexpressing nkd during larval development using UAS/Gal4 transgenes results in adult phenotypes that are indistinguishable from many wg loss-of-function phenotypes. The observed phenotypes include (1) wing-to-notum transformations; (2) leg truncations and duplications; (3) loss, lateral displacement and disorientation of sternite bristles; (4) haltere loss; (5) ventral eye reduction; (6) loss of wing margin (7) extra wing anterior crossveins (C. Conley and S. Blair, personal communication to Zeng, 2000); and (8) loss of antennae. The gene dosage of the Wg pathway influences the effect of ectopic Nkd: loss of one wild-type copy of porcupine (porc), wg, dishevelled (dsh) or arm enhances, and loss of zw3 and nkd suppresses, the UAS-nkd overexpression phenotypes (Zeng, 2000).

During leg development, Wg and Decapentaplegic act as mutually antagonistic determinants of dorsal and ventral identity. Dpp is expressed at high levels at the anterior-posterior (A-P) boundary dorsally, and at lower levels ventrally. Wg/Dpp juxtaposition results in leg disc eversion and outgrowth during pupal morphogenesis. Excess Nkd expressed throughout the disc results in high levels of dpp-lacZ expression along the entire A-P border of the disc, and scant wg-lacZ expression, similar to that seen when wg activity is reduced. These discs give rise to variably truncated legs, indicating that excess Nkd can antagonize the normal effects of wg. More restricted ventral Nkd misexpression results in duplicated legs, which may arise by an abnormal juxtaposition of cells still expressing Wg and cells in which excess Nkd results in decreased Wg, and hence derepressed dpp. Thus two Wg/Dpp boundaries are created and appendages are duplicated (Zeng, 2000).

Whether fly nkd can alter Wnt signaling in a vertebrate was investiged by injecting mRNA into Xenopus embryos. Dorsal blastomere injection into 4-cell embryos of RNAs encoding the Wnt antagonist FrzB, as well as dominant inhibitory forms of Xfrizzled 8 (NXfz8) Dishevelled (Xdd) and Wnt-8 (DN-Xwnt-8) results in marked truncations of the A-P axis. Injections of fly nkd RNA result in very similar effects. Injection of 0.5 ng nkd RNA causes severe A-P truncations in 52% of the embryos, with milder effects in the rest, whereas 2.0 ng nkd RNA increased the penetrance to 97%, indicating a dose-response relationship. Antagonism of Wnt function apparently blocks cell movements that drive the elongation of the gastrula and neurula. These movements drive the elongation of ectodermal explants (animal caps) induced to form mesoderm by activin. Wnt antagonists (such as NXfz8 or Xdd) block elongation of explants without inhibiting mesoderm induction. Injection of 2.0 ng nkd RNA into animal caps mimics this inhibitory effect, blocking elongation in response to activin in 90% of explants (Zeng, 2000).

Ectopic ventral expression of Wnts before the onset of zygotic transcription results in duplication of the dorsal axis, which can be blocked by Wnt antagonists such as NXfz8 or FrzB. nkd also blocks Wnt-mediated axis duplication. Ventral blastomere injection of 0.5 pg XWnt8 RNA results in ectopic dorsal axes in 56% of embryos, about half of which form complete anterior structures. Co-injection of 3.5 pg nkd RNA reduces the frequency of ectopic axes to 42%, with only 10% of embryos forming complete anterior structures. Co-injection of 35 pg nkd RNA gave only 19% partial secondary axes, with no complete axes. At these doses, nkd RNA alone has no discernible effect on development when injected into either dorsal or ventral blastomeres. Higher doses of nkd RNA injected alone into ventral blastomeres give rise to ectopic heads, complete with eyes and cement glands. This striking phenotype has been attributed to antagonism of Wnt activity (Zeng, 2000).

These data show that nkd antagonizes Wg/Wnt signaling. Does nkd affect Wnt synthesis or transport, or determine how cells respond to Wnt? Because Wg and other Wnts are autoregulatory in many contexts, Nkd could affect the quantity or distribution of Wg either directly by controlling Wg synthesis or transport, or indirectly by reducing the positive feedback of Wg activity on its own synthesis. Unfortunately, the lack of a nkd clonal phenotype in the fly precludes definitive determination of cell-autonomous function. However, the first known gene expression defect in nkd mutants is in cells distant to Wg producers, indicating that nkd may act first in Wg-receiving cells. In addition, nkd RNA injected into Xenopus embryos does not alter the accumulation of epitope-tagged XWnt8, indicating that nkd may block the response to XWnt8 (Zeng, 2000).

Inducible antagonists limit the effective duration, range of action or activity of a signal, and can act cell autonomously or non-autonomously. Near-saturating genetic screens have revealed that nkd and patched (ptc; an inducible antagonist for Hh signaling) are probably the only Drosophila Wg or Hh pathway genes, other than Wg or Hh themselves, whose expression and genetic requirement are exclusively zygotic. All other known components of both signaling pathways are maternally provided. Evolutionary selective pressure apparently resists duplications of zygotically active inducible antagonist genes. Antagonist gene dosage must be carefully regulated in flies and vertebrates to balance the effects of the signals. In Drosophila, nkd and ptc mutations have haplo-insufficient effects on cuticle pattern in combination with each other and with other segment-polarity mutants. Altered regulation of both Wnt and Hh signaling in mice and humans is implicated in precancerous and cancerous cell growth. Just as vertebrate ptc1 regulates cell fates and is a key tumor-suppressor gene, vertebrate Nkd-like proteins may be essential for restraining Wnt activity during development and possibly cancer progression (Zeng, 2000).

naked cuticle targets dishevelled to antagonize Wnt signal transduction

Using ectopic expression, it has been found that Nkd affects, in a cell-autonomous manner, a transduction step between the Wnt signaling components Dishevelled (Dsh) and Zeste-white 3 kinase (Zw3). Zw3 is essential for repressing Wg target-gene transcription in the absence of a Wg signal, and the role of Wg is to relieve this inhibition. Double-mutant analysis shows that, in contrast to Zw3, Nkd acts to restrain signal transduction when the Wg pathway is active. Yeast two hybrid and in vitro experiments indicate that Nkd directly binds to the basic-PDZ region of Dsh. Specially timed Nkd overexpression is capable of abolishing Dsh function in a distinct signaling pathway that controls planar-cell polarity. These results suggest that Nkd acts directly through Dsh to limit Wg activity and thus determines how efficiently Wnt signals stabilize Armadillo (Arm)/ß-catenin and activate downstream genes (Rousset, 2001).

The Drosophila eye is composed of mechanosensory bristles present at vertices of ommatidia. Bristle formation is suppressed near the circumferential margin of the eye, and the degree of suppression is least at the extreme dorsum of the head, typically 0-2 ommatidial diameters in width. Wg signaling, active at the circumference of the developing eye where wg is expressed, is responsible for this suppression of peripheral bristle formation. To assay the function of nkd in eye bristle formation, the EGUF/hid method was used to make homozygous mutant nkd eyes in nkd/+ animals. In this technique, Flp-mediated recombination between a chromosome mutant for nkd and a chromosome harboring both recessive and dominant cell-lethal mutations is specifically induced in the eye using the eyeless promoter. During eye development, the only cells surviving are those that have lost the cell-lethal chromosome through recombination, producing an eye homozygous mutant for nkd. Examination of eyes mutant for the strong allele nkd^7E89 reveals, at the dorsum of the eye, consistent eye bristle suppression 3-5 ommatidial diameters away from the margin, with occasional closer bristles. This result suggests that endogenous nkd regulates interommatidial bristle suppression by antagonizing the effects of endogenous Wg in cells farther than one cell diameter away from the Wg source (Rousset, 2001).

To determine how Nkd impinges on the Wg pathway, the ability of Nkd to block the action of the positive regulators Wg, Dsh, and Arm was tested. To do so, advantage was taken of a Drosophila eye misexpression system. Production of Wg in a subset of photoreceptor cells throughout the eye using a sevenless promoter transgene (P[sev-wg]) prevents formation of interommatidial bristles in a paracrine fashion; otherwise, the eye is normal. Previous Nkd misexpression experiments did not indicate whether Nkd blocks Wg synthesis, Wg distribution, or cellular responses to received Wg. To distinguish between these possibilities, the GAL4/UAS binary expression system was used to evaluate the effect of Nkd (UAS-nkd) on Wg-mediated eye bristle suppression. Misexpression of Nkd alone using multiple repeats of the eye-specific glass (gl) enhancer (GMR) to drive the yeast transcription factor GAL4 (P[GMR-GAL4]) has no visible effect on eye development. However, the combination of sev-wg with nkd misexpression results in nearly complete suppression of the P[sev-wg]-induced bristle-loss phenotype. Nkd misexpression did not alter the levels or distribution of Wg antigen, indicating that Nkd is probably blocking signaling events downstream from Wg (Rousset, 2001).

The effect of Nkd on the downstream Wg pathway components Dsh and Arm was also tested using the GMR-GAL4 system. Dsh misexpression (UAS-dsh) produces small, bristle-less eyes devoid of ommatidia. Nkd strongly suppresses the Dsh misexpression eye phenotype, restoring numerous bristles and ommatidia. If the Dsh misexpression eye phenotype is Wg-dependent, its suppression by Nkd could be due to Nkd acting on Wg rather than on Dsh or other downstream components. Previous work suggests that the Dsh misexpression eye phenotype is Wg-independent. To confirm the Wg-independence of the Dsh phenotype, a dominant-negative form of Dfz2 (UAS-GPI-Dfz2) was coexpressed with either sev-wg or UAS-dsh. UAS-GPI-Dfz2 effectively suppresses sev-wg-induced bristle loss in the eye. Coexpression of UAS-GPI-Dfz2 and UAS-Dsh results in some eye necrosis, but it has negligible effects on the UAS-dsh eye phenotype. These results confirm that the Dsh misexpression effect in the eye is Wg-independent. Therefore, rescue of the UAS-dsh phenotype by Nkd is not an indirect effect due to suppression of Wg activity (Rousset, 2001).

GMR-driven expression of UAS-arm^S10, a constitutively activated form of arm, also produces bristle loss and failure of proper ommatidial development. Nkd coexpression had no effect on the Arm misexpression phenotype. Dsh and Arm misexpression phenotypes are not affected by simultaneous expression of UAS-lacZ, indicating that suppression of the dominant eye phenotypes by Nkd was not due to GAL4 titration. The ability of Nkd to block effects of Wg and Dsh but not Arm suggests that Nkd is acting at the level of, or downstream from, Dsh but not downstream of Arm (Rousset, 2001).

The relationship between Nkd and Zw3 could not be determined by a similar suppression test because both proteins are negative regulators of Wg. In addition, the subtlety of the nkd phenotype in the eye made this tissue unsuitable for analyzing the epistasis between nkd and zw3. Instead, Zw3/Gsk3ß was overproduced in nkd mutant embryos using genetic and mRNA injection methods: Heat shock promoter (hsp70)-controlled GAL4 was used to drive Zw3 production, or injections with Xenopus gsk3ß mRNA. nkd mutants lack ventral denticle belts and are considerably smaller than wild-type embryos. Overproduction of Gsk3ß or Zw3 in nkd mutants results in partial to almost complete restoration of denticle belts and restoration of more normal embryo size. Because Zw3 restores denticles to nkd mutants, Zw3 cannot act genetically upstream of the defect in nkd mutants (i.e., by stimulating nkd function) in the linear Wg pathway. Nkd therefore is likely to act upstream of, or in a pathway parallel to, Zw3 and downstream from, or at the level of, Dsh (Rousset, 2001).

The eye misexpression results suggest that Nkd antagonizes Wg signaling at the level of, or downstream from, Dsh. Loss-of-function dsh clones reveal that Dsh acts autonomously in Wg-responsive cells, suggesting that Nkd must also act in Wg-responsive cells. Indeed, previous observations in fly embryos suggest an initial requirement for nkd in cells receiving the Wg signal. Because eye development allows fairly easy production of sharply bounded clones, the eye was chosen to assess the cell autonomy of Nkd action. Marked clones of Nkd-misexpressing cells were produced in developing eyes and the range of Nkd action on sev-wg was monitored (Rousset, 2001).

The flip-on GAL4 system was used to make random clones of cells misexpressing both Nkd and a cell-autonomous marker, green fluorescent protein (GFP), in eyes with excess Wg (sev-wg eyes). All clones examined showed suppression of bristle loss, with the suppression consistently within or immediately adjacent to GFP misexpression clones. No bristles were present outside the clones, indicating a local action of Nkd. To address whether Nkd was acting only in bristle precursor cells, and hence cell-autonomously, those cells were specifically marked with antisera against the Cut nuclear protein in pupal eye discs. In the vicinity of Nkd misexpression clones, there was a perfect correlation between GFP and Cut-labeled cells: all Cut-positive bristle precursor cells expressed GFP and hence Nkd; no GFP-negative/Cut-positive cells were found. These results suggest that Nkd acts within Cut-positive bristle precursor cells to antagonize the inhibitory effects of Wg on bristle cell differentiation (Rousset, 2001).

Cuticles derived from embryos lacking wg activity (wg, dsh, or arm) have nearly continuous fields of denticles, whereas HS-wg embryos, or those mutant for the negative regulator zw3, secrete naked cuticle. Wg misexpression and double-mutant analyses show that Wg acts sequentially through Dsh, Zw3, and Arm. Embryos doubly mutant for wg and zw3 (zw3;wg), as well as zw3;dsh embryos, resemble zw3 embryos, whereas zw3;arm embryos resemble arm embryos, indicating that zw3 acts downstream from dsh and upstream of arm. Mutations in either nkd or zw3 give rise to a naked cuticle phenotype, with posterior expansion of en expression and ectopic wg expression in the developing embryo. However, in contrast to the naked cuticle phenotype of the zw3; wg embryo, the wg;nkd embryo has a wg-like phenotype, indicating a dependence on Wg for the naked cuticle phenotype of nkd mutants (Rousset, 2001 and references therein).

To clarify the relationship between Nkd and other Wg pathway components in the embryo, embryos were made doubly mutant for nkd and dsh or arm, using both genetic means and RNA interference (RNAi). Whereas the nkd gene is strictly zygotic, dsh has a maternal contribution that must be removed via germ-line clones to obtain the embryonic dsh phenotype. Females heterozygous for nkd and carrying dsh germ-line clones were crossed to males heterozygous for nkd. Embryos derived from crosses using different combinations of nkd and dsh alleles were counted and grouped according to their cuticle phenotypes. The expected Mendelian ratios are 37.5% wild type (3/8), 37.5% dsh (3/8), 12.5% nkd (1/8), and 12.5% dsh;nkd (1/8). Only three phenotypes could be detected -- wild type, dsh, and nkd -- indicating that the dsh;nkd mutants exhibit one of these phenotypes or die before secreting cuticle. Whereas the observed percentages for the wild-type and nkd categories are very close to the expected percentages (38.2% and 14.3%, respectively), the percentage of dsh embryos is significantly higher (47.5%), suggesting that the dsh;nkdmutant resembles the dsh mutant (Rousset, 2001).

To confirm the dsh;nkd phenotype, RNAi experiments were performed. Injection of nkd double-stranded RNA (dsRNA) into wild-type embryos efficiently mimics nkd loss of function: 76% of the injected embryos develop with greatly reduced denticles compared to wild-type embryos. The majority of these mutant embryos (69%) show an intermediate to strong nkd cuticle phenotype, the others showing a weak expressivity characterized by a loss of only a few denticles. RNAi was also attempted with dsh, but, in contrast to nkd, both the penetrance and the expressivity of the dsh phenotype are very weak. Increasing the dsh dsRNA concentration has little effect, producing only a fusion between belts A4 and A5 in <5% of the injected embryos and ruling out the utility of nkd and dsh double injections. Instead nkd dsRNA was injected into dsh embryos derived from germ-line clones. Half of the collected embryos are wild type due to rescue by the paternal X-chromosome. To score only nonrescued dsh mutant embryos, females carrying germ-line clones were crossed to males carrying an X-chromosome GFP balancer and GFP-negative embryos were scored after eliminating GFP embryos. Injection of nkd dsRNA into dsh embryos had no effect on the dsh phenotype, confirming that dsh;nkd double mutants resemble dsh embryos (Rousset, 2001).

The null allele arm^YD35 was used to generate arm;nkd double-mutant embryos. Embryos homozygous for this allele have a strong arm phenotype, even without making germ-line clones. Male heterozygotes for the strong alleles nkd^7H16 or nkd^7E89 were crossed to females heterozygous for arm^YD35 and nkd^7H16. Since these crosses generate a majority of wild-type embryos (a ratio of nine wild type to seven mutants), only cuticles from unhatched embryos were counted; these unhatched embryos are expected to be mutant for arm (ratio 3:7, 42.9%), nkd (3:7, 42.9%), and arm;nkd (1:7, 14.3%). Like the dsh; nkd embryos, the arm;nkd mutants do not exhibit a distinct phenotype. The results show that the nkd phenotype is found at the expected frequency (42.6%), but the arm phenotype is over-represented (57.4% instead of 42.9%), indicating that this category also contains the arm;nkd embryos. Therefore, the arm;nkd mutant is covered with denticles and resembles arm embryos (Rousset, 2001).

The double-mutant analysis indicates that the nkd phenotype occurs only if wg, dsh, and arm genes are active, confirming the requirement for Wg signaling to generate the nkd phenotype. Zw3 constitutively represses Wg target-gene transcription, and the role of Wg is to overcome this inhibition. These results indicate that Nkd, in contrast, is required to oppose Wg signal. Removal of nkd in the absence of wg, dsh, or arm has little effect on cuticle phenotype. Accordingly, increased levels of Nkd do not modify the wg mutant cuticle. The negative influence of Nkd could be mediated by inhibition of Dsh activity, stimulation of Zw3 activity, or by interactions with unknown pathway components. To test whether Nkd can directly interact with known Wg signaling components, yeast two-hybrid and in vitro binding assays were used (Rousset, 2001).

Expression in yeast of full-length Nkd protein fused to the GAL4 DNA-binding domain (GB-Nkd) does not activate transcription by itself. When GB-Nkd was coexpressed with Dsh fused to the activation domain of GAL4 (GAD-Dsh), strong ß-galactosidase activity was detected, indicating an interaction between Nkd and Dsh. The interaction between Dsh and Nkd was confirmed using coimmunoprecipitation and glutathione-S-transferase (GST) pull-down assays. Three protein association assays indicate that Nkd and Dsh can directly interact, in keeping with the epistasis results that suggested a role for Nkd at the level of Dsh or Zw3 (Rousset, 2001).

Production of Nkd-GFP fusion protein in larval salivary glands reveals striking colocalization with endogenous Dsh, stained with an anti-Dsh antibody, indicating that the two proteins may also interact in vivo. However, an association between the two proteins was not detected using coimmunoprecipitation experiments with embryo extracts or lysates from Drosophila cell lines. The negative results may be due to protein complex dynamics, accessibility to antibodies, low levels of the complex in fly cells, as well as possible modes of regulation of the interaction, which are currently being investigated (Rousset, 2001).

The Dsh protein contains three defined domains: DIX, PDZ, and DEP. The DIX (Dishevelled, Axin) domain shares homology with the C-terminal part of D-Axin, the PDZ (PSD-95, Dlg, Zo-1) domain is a modular region involved in protein-protein interactions, and the DEP (Dsh, Egl-10, Pleckstrin) domain is usually found in signaling proteins, although its role remains unclear. In addition, a stretch of basic residues is present between the DIX and PDZ domains. Using both the yeast two-hybrid system and GST pull-down experiments, the region in Dsh that binds Nkd was defined. These results indicated that the central region of Dsh containing the basic sequence and the PDZ domain is sufficient for binding Nkd. The PDZ domain of Dsh is necessary for the interaction with Nkd but, in contrast to proteins such as casein kinase I or Frat1, it cannot efficiently bind Nkd by itself (Rousset, 2001).

Dsh is a branchpoint connecting two distinct signaling pathways in Drosophila development: the Wg pathway and the planar cell polarity pathway (PCP). nkd mutant clones have normal planar cell polarity; so there is no detectable normal role for nkd in the PCP pathway. If Nkd affects Dsh during Wg signaling, as the data suggest, then appropriately timed overexpression of Nkd might be able to specifically alter Dsh function in PCP signaling. Nkd was tested for its ability to interfere with PCP signaling during a time when Fz and Dsh are not appreciably participating in Wg signaling. Timed overexpression of Nkd at 24 h after puparium formation (APF) produces adult flies with wing hair polarity defects that are indistinguishable from those seen in dsh¹ mutant adults. dsh¹ is an adult viable allele of dsh that harbors a missense mutation in the C-terminal DEP domain. Genetic tests have shown dsh¹ to be a null allele for PCP signaling. The Nkd overexpression polarity pattern is reproducible and qualitatively distinct from that produced by complete loss of function of other known PCP mutants, including fz or prickle (pk). The Nkd overexpression defect is also different from those associated with Fz or Dsh overexpression (Rousset, 2001).

The PCP phenotype associated with Fz overexpression is sensitive to the dose of dsh. To determine whether Nkd could similarly titrate Dsh from PCP signaling induced by Fz overexpression, Fz and Nkd were simultaneously expressed. Indeed, overexpressed Nkd suppresses the effects of excess Fz. Neither excess Nkd nor decreased nkd dosage modifies the wing bristle polarity of dsh¹ mutant flies. The results suggest that Nkd can specifically interfere with Dsh function in planar cell polarity and that this effect requires wild-type Dsh protein (Rousset, 2001).

That Nkd can act through Dsh has important implications for the dynamic control of Wg/Wnt signaling. By acting upstream of the ß-catenin degradation machinery, Nkd may determine how effectively a given dose of Wnt causes ß-catenin accumulation and target-gene activation and thereby influences the sensitivity of a cell to a given amount or type of Wnt ligand. The kinetic and dynamic parameters of the feedback loop involving Wg, Dsh, and Nkd may play key roles in controlling the duration and extent of signaling activity. Tight regulation of this feedback loop is clearly important for normal Drosophila embryonic development, and in various animals it may be subject to spatial and temporal adjustments during evolution or during disease progression. Future experiments will test how the interaction between Nkd and Dsh affects responses to Wnt signals during development and may provide insight into Wnt-associated tumor progression (Rousset, 2001).

PROTEIN STRUCTURE

Amino Acids - 928

Structural Domains and

Nkd is a relatively basic (calculated isoelectric point, pI = 9.1) and largely hydrophilic protein. Single-stranded conformation polymorphism analysis and direct genomic sequencing reveals a nonsense mutation Q60stop in nkd ^7H16, predicting a truncated protein of 59 amino acids. The identity of the nkd cDNA has been further confirmed by its ability, when activated with a heat-shock promoter, to rescue the naked cuticle phenotype and the En and Wg expression abnormalities in nkd mutants. Nkd has significant similarity to the high-affinity Ca²⁺-binding EF hand of the recoverin family of myristoyl switch proteins; 39% amino-acid identity, 63% similarity with Drosophila Neurocalcin. EF hands are conserved Ca²⁺-binding motifs that usually occur in pairs, although they have been observed singly. Mouse and human expressed sequence tag clones encoding EF-hand sequences similar to fly Nkd may be vertebrate Nkd homologs, a possibility that is being tested. No obvious homolog has been identified in Caenorhabditis elegans (Zeng, 2000).

date revised: 23 September 2001

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