Gene name - naked cuticle
Cytological map position - 75E-F
Function - signaling
Symbol - nkd
FlyBase ID: FBgn0002945
Genetic map position - 3-
Classification - EF-hand calcium-binding domain protein
Cellular location - probably cytoplasmic
|Recent literature||Wang, R., Xie, H., Yang, L., Wang, P., Chen, M. M., Wu, H. Y., Liao, Y. L., Wang, M. Y., Wang, Q., Gong, X. X., Cheng, Q., Cheng, L., Xie, F. Y., Bi, C. L. and Fang, M. (2021). Naked cuticle inhibits wingless signaling in Drosophila wing development. Biochem Biophys Res Commun 576: 1-6. PubMed ID: 34474244
Wnt signaling is one of the major signaling pathways that regulate cell differentiation, tissue patterning and stem cell homeostasis and its dysfunction causes many human diseases, such as cancer. It is of tremendous interests to understand how Wnt signaling is regulated in a precise manner both temporally and spatially. Naked cuticle (Nkd) acts as a negative-feedback inhibitor for Wingless (Wg, a fly Wnt) signaling in Drosophila embryonic development. However, the role of Nkd remains controversial in later fly development, particularly on the canonical Wg pathway. This study shows that nkd is essential for wing pattern formation, such that both gain and loss of nkd result in the disruption of Wg target expression in larvae stage and abnormal adult wing morphologies. Furthermore, it was demonstrated that a thirty amino acid fragment in Nkd, identified previously in Wharton lab, is critical for the canonical Wg signaling, but is dispensable for Wg/planar cell polarity pathway. Putting aside the pleiotropic nature of nkd function, i.e. its role in the Decapentaplegic signaling, it is concluded that Nkd universally inhibits the canonical Wg pathway across a life span of Drosophila development.
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 wgIl114/+ embryos, induction of P[Hs-nkd] before 4 h AEL results in decreased en and wg expression. wgIl114/+ embryos are patterned normally, but practically all wgIl114 /+ 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).
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 nkd7E89 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-armS10, 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 armYD35 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 nkd7H16 or nkd7E89 were crossed to females heterozygous for armYD35 and nkd7H16. 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).
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).
Wnt signals control cell fate decisions and orchestrate cell behavior in metazoan animals. In Drosophila, embryos defective in signaling mediated by the Wnt protein Wingless (Wg) exhibit severe segmentation defects. The Drosophila segment polarity gene naked cuticle (nkd) encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. Nkd antagonizes Wg via a direct interaction with the Wnt signaling component Dishevelled (Dsh). Two mouse and human proteins, Nkd1 and Nkd2, related to fly Nkd, are described. The most conserved region among the fly and vertebrate proteins, the EFX domain, includes the putative EF hand and flanking sequences. EFX corresponds to a minimal domain required for fly or vertebrate Nkd to interact with the basic/PDZ domains of fly Dsh or vertebrate Dvl proteins in the yeast two-hybrid assay. During mouse development, nkd1 and nkd2 are expressed in multiple tissues in partially overlapping, gradient-like patterns, some of which correlate with known patterns of Wnt activity. Mouse Nkd1 can block Wnt1-mediated, but not beta-catenin-mediated, activation of a Wnt-dependent reporter construct in mammalian cell culture. Misexpression of mouse nkd1 in Drosophila antagonizes Wg function. The data suggest that the vertebrate Nkd-related proteins, similar to their fly counterpart, may act as inducible antagonists of Wnt signals (Wharton, 2001).
Comparison of the fly Nkd amino acid sequence with those of the vertebrate Nkd-related proteins reveals two regions of similarity. (1) Both fly and vertebrate Nkd proteins share a histidine-rich C-terminus (7/10 amino acids in the fly protein, 12/15 and 16/19 amino acids in vertebrate Nkd1 and Nkd2 proteins). While His-rich regions have been described in many proteins, the His repeats at the C-terminus of the vertebrate Nkd proteins are interrupted by conserved glutamate residues. (2) The fly and vertebrate proteins share an approximately 66 amino acid region of similarity including the putative EF hand. This region has been termed the EFX domain. The amino acid sequences of the EFX domains are 42% identical between fly Nkd and mNkd1, and 41% identical between fly Nkd and mNkd2 (Wharton, 2001).
EF hands are modular, helix-loop-helix Ca2+ binding motifs present in many Ca2+ binding proteins, including calmodulin and recoverin. EF hand-containing proteins typically have between two and four consecutive EF hands, but proteins with single EF hands have been described. Previous sequence comparisons suggested that, among published protein sequences, Drosophila Nkd shared the greatest sequence similarity with the high-affinity Ca2+ binding EF hand (EF3) of the recoverin subfamily of EF-hand proteins. Recoverins are N-terminally myristoylated, Ca2+-dependent switch proteins that regulate the localization and activity of a variety of enzymes from yeast to mammals. The EF hand region of mNkd1 is 35% identical over 53 amino acids to Drosophila Frequenin, the recoverin subfamily EF hand protein most closely related to mNkd1. While the Drosophila Nkd EFX is about 40% identical over 66 amino acids to all of the vertebrate EFX sequences, the vertebrate EFX sequences are all 84% identical to each other over the same region. In some characterized EF hand motifs, amino acid substitutions have occurred that preclude ion binding, indicating those domains do not serve as ion sensors. Both fly and vertebrate nkd genes encode oxygen-donating residues in EF loop positions known to coordinate ion binding, although fly Nkd has an unusual pair of histidine residues at the apex of the loop that could, based on comparisons with EF hand-crystal structures, alter ion binding. It is not known whether any of the Nkd proteins bind Ca2+ or indeed whether they adopt standard EF hand conformations. Examination of all four vertebrate Nkd-related protein sequences reveals that all four vertebrate genes form a gene family with 36% overall amino acid identity. The vertebrate proteins share blocks of amino acid conservation on either side of the EF hand that are not obviously shared with fly Nkd. Supporting the idea that nkd1 and nkd2 represent distinct orthologs, the amino acid sequence of mNkd1 is 86% identical to hNkd1, but only 43% identical to hNkd2. Conversely, mNkd2 is only 43% identical to hNkd1 but 75% identical to hNkd2. Further, mNkd1 and hNkd1 have identical amino acids in 206/309 positions that are different in mNkd2 and hNkd2, excluding amino acid residues shared among all four encoded proteins. Similarly, mNkd2 and hNkd2 have identical amino acids in 164/309 positions that are different in mNkd1 and hNkd1. All four Nkd-related proteins, similar to the recoverins, have a myristoylation consensus at their N-terminus (MGKxxSK), although it is not known whether the vertebrate Nkd-related proteins are indeed myristoylated. In contrast, fly Nkd does not have an N-terminal myristoyl consensus sequence. According to high-throughput genome sequence mapping data, human nkd1 maps to 16q12, and human nkd2 maps to 5p15.3, the latter of which has been confirmed by radiation hybrid analysis (Wharton, 2001).
Genetic studies have identified Drosophila Naked Cuticle (Nkd) as an antagonist of the canonical Wnt/ß-catenin signaling pathway, but its mechanism of action remains obscure. A mammalian homolog of Naked cuticle, mNkd, has been cloned. mNkd interacts directly with Dishevelled. Dishevelled is an intracellular mediator of both the canonical Wnt pathway and planar cell polarity (PCP) pathway. Activation of the c-Jun-N-terminal kinase has been implicated in the PCP pathway. mNkd has been shown to acts in a cell-autonomous manner not only to inhibit the canonical Wnt pathway but also to stimulate c-Jun-N-terminal kinase activity. Expression of mNkd disrupts convergent extension in Xenopus, consistent with a role for mNkd in the PCP pathway. These data suggest that mNkd may act as a switch to direct Dishevelled activity toward the PCP pathway, and away from the canonical Wnt pathway (Yan, 2001).
The interaction of mNkd with mDvl was demonstrated in mammalian cells using coimmunoprecipitation experiments. mNkd was transiently expressed in Cos7 or HEK293 cells; endogenous mDvl proteins were immunoprecipitated from total cell lysates. Because the EF-hand is included in the region of mNkd that is associated with mDvl in yeast two-hybrid experiments and is highly conserved between Drosophila Nkd and mNkd, the requirement of the EF-hand in the association with mDvl was investigated. Based on the crystal structure of the third EF-hand of the Recoverin protein, mutations were made that either changed the consensus residues in the calcium-binding loop or deleted the entire calcium-binding loop together with the surrounding amino acids. These mNkd mutants were expressed in HEK293 or Cos7 cells. Coimmunoprecipitation experiments revealed that none of these mutations significantly impaired the ability of these mNkd proteins to associate with mDvl. These data show that mNkd associates with mDvl in mammalian cells and that the intact EF-hand is not required for the association (Yan, 2001).
The domain of Dishevelled that associates with mNkd was identified. Dishevelled is a highly conserved protein and contains three distinct domains. The N-terminal region has a DIX domain that is required for canonical Wnt signaling. The middle region of Dishevelled contains a PDZ domain that is known to bind GBP/Frat1 and CK1epsilon, both positive regulators of the canonical Wnt pathway. The C-terminal region contains a DEP domain that is crucial for regulating the PCP pathway. Fragments corresponding to different regions of Drosophila Dishevelled (Dsh) were expressed in E. coli as GST-fusion proteins. Equal amounts of each fragment were mixed with in vitro-translated mNkd in the binding buffer, precipitated with glutathione beads and separated by SDS/PAGE. mNkd associates with the DM fragment of Dsh that encompasses the PDZ domain with the adjoining N-terminal basic amino acid stretch. Notably, the PDZ domain alone is not sufficient for the association. Also, there is no association of mNkd with the DIX domain in the N-terminal region or the DEP domain in the C-terminal region of Dsh. Thus mNkd is associated with a region within Dsh shared with GBP/Frat 1 and CK1epsilon (Yan, 2001).
Because Dishevelled is a known positive regulator of the canonical Wnt pathway and mNkd is found here to be directly associated with Dishevelled, the role of mNkd in the canonical Wnt pathway was tested in mammalian cell culture by using a Wnt-1 ligand-responsive luciferase reporter assay. In multiple experiments, activation of the reporter by Wnt-1 was inhibited 75% by coexpression of mNkd in mammalian cells. Expression of wild-type mNkd, in the absence of Wnt-1, has no effect on the activity of the reporter. These data suggest that mNkd negatively regulates the canonical Wnt pathway and are consistent with the inhibitory effect that Drosophila Nkd has on Wingless signaling in genetic studies. Interestingly, the EF-hand mutants of mNkd show an impaired ability to inhibit the canonical Wnt pathway, although these mutants are all capable of binding to Dishevelled. These data suggest that the association of mNkd with Dishevelled alone is not sufficient to inhibit the canonical Wnt pathway, and that the intact EF-hand is required for the inhibitory function. Importantly, mNkd fails to inhibit the gene response elicited by overexpression of ß-catenin, a result that places mNkd upstream of ß-catenin in the canonical Wnt pathway (Yan, 2001).
The induction of secondary axes by ectopic expression of Xwnt-8 in the Xenopus embryo was used as an assay for the role of mNkd in the canonical Wnt pathway in vivo. Ventral blastomere injection of 5-10 pg of Xwnt-8 RNA induced secondary axes in over 50% of the embryos; most of these secondary axes contained anterior structures. Coinjection of 25 pg of mNkd RNA suppresses the effect of Xwnt-8 and results in fewer secondary axes. Coinjection of higher doses of mNkd (250 pg) resulted in even fewer secondary axes, only half of which contained anterior structures, indicating that mNkd inhibits the canonical Wnt pathway in vivo. The promoter of the Xenopus nodal-related-3 (Xnr-3) gene has been shown to be directly activated by the canonical Wnt/ß-catenin signaling in the Xenopus embryo, and expression of Xwnt-8 RNA into Xenopus animal caps activates transcription from a coinjected Xnr-3-luciferase reporter plasmid in vivo. Consistent with the results from mammalian cell culture (and the secondary axis assay), coinjection of mNkd RNA suppresses Xwnt-8 activation of the Xnr-3 promoter. These data indicate that mNkd is an inhibitor of the canonical Wnt/ß-catenin pathway both in vitro and in vivo. Together, these findings that mNkd interacts directly with Dishevelled and inhibits the canonical Wnt pathway upstream of ß-catenin suggest that mNkd is an intracellular antagonist of the Wnt pathway (Yan, 2001).
Activation of JNK seems to be an important step in the PCP pathway, and a vertebrate cognate of the Drosophila PCP pathway controls convergent extension movements during vertebrate development. In both Xenopus and Drosophila, hyperactivation of this pathway disrupts PCP signaling without affecting the canonical Wnt pathway. Consistent with its ability to activate JNK in vitro, mNkd overexpression inhibits the normal elongation of Xenopus embryos. The normal formation of anterior structures in these embryos indicates that the phenotype is not the result of ventralization, suggesting that mNkd inhibits convergent extension. To assess more directly the effects of mNkd on convergent extension, open-face Keller explants of the dorsal mesoderm were examined. Such explants made from control embryos elongate and change shape significantly, whereas explants made from embryos expressing mNkd fail to elongate. These effects are similar to those elicited by overexpression of other wild-type components of the planar cell polarity cascade, such as Xdsh and Xfz-8, indicating a role for mNkd in controlling the PCP pathway (Yan, 2001).
Because mNkd is an inhibitor of the canonical Wnt pathway, it was important to test whether the effects of mNkd on convergent extension were simply a consequence of the effects of mNkd on the canonical Wnt pathway. Expression of dominant-negative GSK-3ß, a strong activator of the canonical Wnt pathway in Xenopus, does not attenuate the inhibitory effects of mNkd on convergent extension. Taken together, these data suggest that mNkd inhibits convergent extension by overstimulating the PCP-signaling cascade, and that this effect is independent of its inhibitory role on the canonical Wnt pathway (Yan, 2001).
Recently, a number of genes have been identified that are induced by Wnt expression in mammalian cells. Whether mNkd-mRNA levels change when cells are treated with Wnt ligands was tested. BALB/c LI mouse liver epithelial cells were treated with Wnt-3A-conditioned medium for 8 h, 19.5 h, or 27 h, respectively. Levels of mNkd transcripts increase significantly in cells treated with Wnt-3a-conditioned medium for 19.5 h and 27 h compared with control treatments. Wnt-3a-conditioned medium also causes an increase in mRNA levels of mNkd in L cells. The mRNA levels of mNkd also increases in BALB/c LI mouse liver epithelial cells treated with lithium chloride, a known inhibitor of GSK-3ß. Thus, the ability of mNkd to inhibit the intracellular signaling of the canonical Wnt pathway, in conjunction with the result that mNkd is itself a downstream target of the canonical Wnt signaling, suggests that mNkd is an intracellular cell-autonomous negative-feedback regulator of the canonical Wnt pathway (Yan, 2001).
Genetic and biochemical studies have shown that Dishevelled controls cell polarity by acting as an upstream activator of the JNK pathway both in vivo and in vitro. Because mNkd is directly associated with Dishevelled, whether mNkd participates in the JNK pathway was tested. NIH 3T3 cells were transfected with expression constructs of mNkd and c-Jun, in which c-Jun served to monitor JNK activities. In this assay, expression of mNkd or mDvl alone induces a strong phosphorylation of c-Jun that was detected by blotting with an antibody specific for phosphoserine-63. These data show that mNkd has an effect similar to Dishevelled in activating the JNK pathway in mammalian cell culture assays (Yan, 2001).
The Wnt signaling cascade is a central regulator of cell fate determination during embryonic development, whose deregulation contributes to oncogenesis. Naked cuticle is the first Wnt-induced antagonist found in this pathway, establishing a negative-feedback loop that limits the Wnt signal required for early segmentation. In addition, Naked cuticle is proposed to function as a switch, acting to restrict classical Wnt signaling and to activate a second Wnt signaling pathway that controls planar cell polarity during gastrulation movements in vertebrates. Little is known about the biochemical function of Naked cuticle or its regulation. PR72, a Protein Phosphatase type 2A regulatory subunit of unknown function, interacts both physically and functionally with Naked cuticle. PR72, like Naked cuticle, acts as a negative regulator of the classical Wnt signaling cascade, establishing PR72 as a novel regulator of the Wnt signaling pathway. These data provide evidence that the inhibitory effect of Naked cuticle on Wnt signaling depends on the presence of PR72, both in mammalian cell culture and in Xenopus embryos. Moreover, PR72 is required during early embryonic development to regulate cell morphogenetic movements during body axis formation (Creyghton, 2005).
These data suggest a role for both PR72 and Naked in negative regulation of dishevelled stability. This would be in agreement with reported similarities between the phenotypes caused by ectopic Naked expression and dishevelled loss-of-function mutants in embryogenesis. Loss of Naked cooperates with loss of PR72 in disturbing cell morphogenetic movements during gastrulation, which could represent a polarity defect. Since both PR72 and Naked cuticle are negative regulators of dishevelled protein levels, it is difficult to see how either would function as an activator of the PCP pathway. Indeed, the role of Naked cuticle in PCP is still under debate, since loss of Nkd does not affect cell polarity in the Drosophila wing and Naked misexpression is indistinguishable from dishevelled loss-of-function mutants (Creyghton, 2005).
Both introducing exogenous dishevelled and interfering with endogenous dishevelled by introduction of a dominant negative mutant can cause defects in cell morphogenetic movements during gastrulation. This result not only underscores the importance of tight regulation of dishevelled levels during development but also makes it difficult to conclude whether the observed defects in gastrulation caused by Naked misexpression are the result of an overactive PCP pathway or a defect in this pathway. It is proposed that PR72 and Naked cuticle are negative regulators of both the canonical and the PCP pathways (Creyghton, 2005).
Nkd1 is an antagonist of the canonical Wnt/beta-catenin signaling pathway. The EF-hand motif of Nkd1 is required for its inhibitory function. Early studies suggested that Nkd1 might play important roles in mouse embryonic development and tumorigenesis. Nkd1-/- mice were constructed whose Nkd1 protein lacked the EF-hand and was therefore unable to inhibit Wnt/beta-catenin signaling. The homozygotes were viable and grew normally, but their fertility in males was reduced. In wild-type adult testes, Nkd1 mRNA is expressed more abundantly in the elongating spermatids than in the round spermatids. Lack of EF-hand causes reductions in the testis weight and sperm count by 30% and 60%, respectively. During testis development, Nkd1 mRNA expression starts at the 25th day after birth, coincident with the onset of Wnt1 expression. Nuclear localization of beta-catenin increases in the elongating spermatids, suggesting that the mutant Nkd1 fails to inhibit the Wnt/β-catenin pathway. These results suggest that deletion of the EF-hand from Nkd1 reduces the number of the elongating spermatids at haploid stage. In contrast, the mutant Nkd1 does not affect intestinal polyposis in ApcDelta716 mice (Li, 2005).
date revised: 23 September 2001
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