Stable anterior anchoring of the oocyte nucleus is required to establish dorsoventral polarity of the Drosophila egg

Wnt signals control cell fate decisions and orchestrate cell behavior in metazoan animals. In the fruit fly 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). Here we describe two mouse and human proteins, Nkd1 and Nkd2, related to fly Nkd. 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.     

Viable mice with compound mutations in the Wnt/Dvl pathway antagonists nkd1 and nkd2

Gradients of Wnt/beta-catenin signaling coordinate development and physiological homeostasis in metazoan animals. Proper embryonic development of the fruit fly Drosophila melanogaster requires the Naked cuticle (Nkd) protein to attenuate a gradient of Wnt/beta-catenin signaling across each segmental anlage. Nkd inhibits Wnt signaling by binding the intracellular protein Dishevelled (Dsh). Mice and humans have two nkd homologs, nkd1 and nkd2, whose encoded proteins can bind Dsh homologs (the Dvl proteins) and inhibit Wnt signaling. To determine whether nkd genes are necessary for murine development, we replaced nkd exons that encode Dvl-binding sequences with IRES-lacZ/neomycin cassettes. Mutants homozygous for each nkd(lacZ) allele are viable with slightly reduced mean litter sizes. Surprisingly, double-knockout mice are viable, with subtle alterations in cranial bone morphology that are reminiscent of mutation in another Wnt/beta-catenin antagonist, axin2. Our data show that nkd function in the mouse is dispensable for embryonic development.     

A targeted mutation of Nkd1 impairs mouse spermatogenesis

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. We constructed Nkd1(-/-) mice whose Nkd1 protein lacked the EF-hand and was 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 was expressed more abundantly in the elongating spermatids than in the round spermatids. Lack of EF-hand caused reductions in the testis weight and sperm count by 30 and 60%, respectively. During testis development, Nkd1 mRNA expression started at the 25th day after birth, coincident with the onset of Wnt1 expression. Nuclear localization of beta-catenin increased in the elongating spermatids, suggesting that the mutant Nkd1 failed to inhibit the Wnt/beta-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 did not affect intestinal polyposis in Apc(Delta716) mice.     

Zebrafish Naked1 and Naked2 antagonize both canonical and non-canonical Wnt signaling

Wnt signaling controls a wide range of developmental processes and its aberrant regulation can lead to disease. To better understand the regulation of this pathway, we identified zebrafish homologues of Naked Cuticle (Nkd), Nkd1 and Nkd2, which have previously been shown to inhibit canonical Wnt/beta-catenin signaling. Zebrafish nkd1 expression increases substantially after the mid-blastula transition in a pattern mirroring that of activated canonical Wnt/beta-catenin signaling, being expressed in both the ventrolateral blastoderm margin and also in the axial mesendoderm. In contrast, zebrafish nkd2 is maternally and ubiquitously expressed. Overexpression of Nkd1 or Nkd2a suppressed canonical Wnt/beta-catenin signaling at multiple stages of early zebrafish development and also exacerbated the cyclopia and axial mesendoderm convergence and extension (C&E) defect in the non-canonical Wnt/PCP mutant silberblick (slb/wnt11). Thus, Nkds are sufficient to antagonize both canonical and non-canonical Wnt signaling. Reducing Nkd function using antisense morpholino oligonucleotides resulted in increased expression of canonical Wnt/beta-catenin target genes. Finally, reducing Nkd1 function in slb mutants suppressed the axial mesendoderm C&E defect. These data indicate that zebrafish Nkd1 and Nkd2 function to limit both canonical and non-canonical Wnt signaling.     

Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling

Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle (pk) and strabismus (stbm) are thought to antagonize Fz signaling. We show that they act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. We demonstrate that Stbm and Pk interact physically and that Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus our data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling.     

Naked1 antagonizes Wnt signaling by preventing nuclear accumulation of β-catenin

Cyto-nuclear shuttling of β-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of β-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and β-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to β-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of β-catenin.     

Nkd1 Functions as a Passive Antagonist of Wnt Signaling

Wnt signaling is involved in many aspects of development and in the homeostasis of stem cells. Its importance is underscored by the fact that misregulation of Wnt signaling has been implicated in numerous diseases, especially colorectal cancer. However, how Wnt signaling regulates itself is not well understood. There are several Wnt negative feedback regulators, which are active antagonists of Wnt signaling, but one feedback regulator, Nkd1, has reduced activity compared to other antagonists, yet is still a negative feedback regulator. Here we describe our efforts to understand the role of Nkd1 using Wnt signaling compromised zebrafish mutant lines. In several of these lines, Nkd1 function was not any more active than it was in wild type embryos. However, we found that Nkd1’s ability to antagonize canonical Wnt/β-catenin signaling was enhanced in the Wnt/Planar Cell Polarity mutants silberblick (slb/wnt11) and trilobite (tri/vangl2). While slb and tri mutants do not display alterations in canonical Wnt signaling, we found that they are hypersensitive to it. Overexpression of the canonical Wnt/β-catenin ligand Wnt8a in slb or tri mutants resulted in dorsalized embryos, with tri mutants being much more sensitive to Wnt8a than slb mutants. Furthermore, the hyperdorsalization caused by Wnt8a in tri could be rescued by Nkd1. These results suggest that Nkd1 functions as a passive antagonist of Wnt signaling, functioning only when homeostatic levels of Wnt signaling have been breached or when Wnt signaling becomes destabilized.     

Wnt ligand–dependent activation of the negative feedback regulator Nkd1

Misregulation of Wnt signaling is at the root of many diseases, most notably colorectal cancer, and although we understand the activation of the pathway, we have a very poor understanding of the circumstances under which Wnt signaling turns itself off. There are numerous negative feedback regulators of Wnt signaling, but two stand out as constitutive and obligate Wnt-induced regulators: Axin2 and Nkd1. Whereas Axin2 behaves similarly to Axin in the destruction complex, Nkd1 is more enigmatic. Here we use zebrafish blastula cells that are responsive Wnt signaling to demonstrate that Nkd1 activity is specifically dependent on Wnt ligand activation of the receptor. Furthermore, our results support the hypothesis that Nkd1 is recruited to the Wnt signalosome with Dvl2, where it becomes activated to move into the cytoplasm to interact with β-catenin, inhibiting its nuclear accumulation. Comparison of these results with Nkd function in Drosophila generates a unified and conserved model for the role of this negative feedback regulator in the modulation of Wnt signaling.     

Nkd2, a negative regulator of Wnt pathway, delays mitotic exit in Hela cell

Frequent amplification and abundant expression of Nkd2 has been identified in malignant peripheral nerve sheath tumors (MPNSTs), dominant for genomic instability, who is involved in both Wnt pathway and EGFR signaling pathway. As a negative regulator of Wnt pathway, Nkd2 suppresses Wnt signaling by binding to Dvl1 and causing its ubiquitination followed by 26S proteasome degradation. On the other hand, it interacts with TGF-α for its transportation to basolateral plasma membrane in polarized epithelial cells. It is of interest to determine if Nkd2 over-expression contributes to tumorigenesis and genomic instablity. In this paper, we found that cells expressing NKD2 delayed mitotic exit stage after double thymidine block synchronization, but aneuploidy was not detected in these cells. This was further confirmed by Western blotting. In nocodazole-synchronised cells, Cyclin B1 degradation was delayed with Nkd2 over-expression compared to control group. Given many previous publications showed that Wnt pathway components are involved in mitotic progression. Further investigation on Nkd2’s function in mitosis might give more clues on MPNSTs pathological progression.     

Characterization of mouse dishevelled (Dvl) proteins in Wnt/Wingless signaling pathway.

The dishevelled (dsh) gene family encodes cytoplasmic proteins that have been implicated in Wnt/Wingless (Wg) signaling. To demonstrate functional conservation of Dsh family proteins, two mouse homologs of Drosophila Dsh, Dvl-1 and Dvl-2, were biochemically characterized in mouse and Drosophila cell culture systems. We found that treatment with a soluble Wnt-3A leads to hyperphosphorylation of Dvl proteins and a concomitant elevation of the cytoplasmic beta-catenin levels in mouse NIH3T3, L, and C57MG cells. This coincides well with our finding in a Drosophila wing disc cell line, clone-8, that Wg treatment induced hyperphosphorylation of Dsh (Yanagawa, S., van Leeuwen, F., Wodarz, A., Klingensmith, J., and Nusse, R. (1995) Genes Dev. 9, 1087-1097). Furthermore, we showed that mouse Dvl proteins affect downstream components of Drosophila Wg signaling as Dsh does; overexpression of Dvl proteins in clone-8 cells results in elevation of Armadillo (Drosophila homolog of beta-catenin) and Drosophila E-cadherin levels, hyperphosphorylation of Dvl proteins themselves, and inhibition of Zeste-White3 kinase-mediated phosphorylation of a microtubule-binding protein, Tau. In addition, casein kinase II was shown to coimmunoprecipitate with Dvl proteins, and Dvl proteins were phosphorylated in these immune complexes. These results are direct evidence that Dsh family proteins mediate a set of conserved biochemical processes in the Wnt/Wg signaling pathway.     

CKIepsilon/discs overgrown promotes both Wnt-Fz/beta-catenin and Fz/PCP signaling in Drosophila

The related Wnt-Frizzled(Fz)/beta-catenin and Fz/planar cell polarity (PCP) pathways are essential for the regulation of numerous developmental processes and are deregulated in many human diseases. Both pathways require members of the Dishevelled (Dsh or Dvl) family of cytoplasmic factors for signal transduction downstream of the Fz receptors. Dsh family members have been studied extensively, but their activation and regulation remains largely unknown. In particular, very little is known about how Dsh differentially signals to the two pathways. Recent work in cell culture has suggested that phosphorylation of Dsh by Casein Kinase I epsilon (CKIepsilon) may act as a molecular "switch," promoting Wnt/beta-catenin while inhibiting Fz/PCP signaling. Here, we demonstrate in vivo in Drosophila through a series of loss-of-function and coexpression assays that CKIepsilon acts positively for signaling in both pathways, rather than as a switch. Our data suggest that the kinase activity of CKIepsilon is required for peak levels of Wnt/beta-catenin signaling. In contrast, CKIepsilon is a mandatory signaling factor in the Fz/PCP pathway, possibly through a kinase-independent mechanism. Furthermore, we have identified the primary kinase target residue of CKIepsilon on Dsh. Thus, our data suggest that CKIepsilon modulates Wnt/beta-catenin and Fz/PCP signaling pathways via kinase-dependent and -independent mechanisms.     

The prickle-related gene in vertebrates is essential for gastrulation cell movements

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.     

Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway

Large‐scale proteomic approaches have been used to study signaling pathways. However, identification of biologically relevant hits from a single screen remains challenging due to limitations inherent in each individual approach. To overcome these limitations, we implemented an integrated, multi‐dimensional approach and used it to identify Wnt pathway modulators. The LUMIER protein–protein interaction mapping method was used in conjunction with two functional screens that examined the effect of overexpression and siRNA‐mediated gene knockdown on Wnt signaling. Meta‐analysis of the three data sets yielded a combined pathway score (CPS) for each tested component, a value reflecting the likelihood that an individual protein is a Wnt pathway regulator. We characterized the role of two proteins with high CPSs, Ube2m and Nkd1. We show that Ube2m interacts with and modulates β‐catenin stability, and that the antagonistic effect of Nkd1 on Wnt signaling requires interaction with Axin, itself a negative pathway regulator. Thus, integrated physical and functional mapping in mammalian cells can identify signaling components with high confidence and provides unanticipated insights into pathway regulators.