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Apical constriction is a cell shape change critical to vertebrate neural tube closure, and the contractile force required for this process is generated by actin-myosin networks. The signaling cue that instructs this process has remained elusive. Here, we identify Wnt4 and the transmembrane ephrinB2 protein as playing an instructive role in neural tube closure as members of a signaling complex we termed WERDS (Wnt4, EphrinB2, Ror2, Dishevelled (Dsh2), and Shroom3). Disruption of function or interaction among members of the WERDS complex results in defects of apical constriction and neural tube closure. The mechanism of action involves Wnt4 binding the non-canonical Wnt receptor, Ror2, and enhancing an interaction with ephrinB2, through the main Wnt scaffold protein, Dsh2. This complex in turn recruits the adaptor protein Shroom3, which activates Rho-associated kinase (Rock) to induce apical constriction. Remarkably, we also discovered that a key part of this mechanism is that ephrinB2 antagonizes Wnt/β-catenin signaling through the interaction with Dsh2, which switches Dsh2 function from canonical to non-canonical Wnt signaling that is required for proper neural tube closure.

Lee et al. identify Dyrk1a as a new interaction partner that phosphorylates CEP97, which in turn promotes recruitment of Plk1. The complex of CEP97–Dyrk1a–Plk1 plays a substantive role in the maturation and location of centrioles/basal bodies and proper ciliogenesis in multiciliated cells., Proper cilia formation in multiciliated cells (MCCs) is necessary for appropriate embryonic development and homeostasis. Multicilia share many structural characteristics with monocilia and primary cilia, but there are still significant gaps in our understanding of the regulation of multiciliogenesis. Using the Xenopus embryo, we show that CEP97, which is known as a negative regulator of primary cilia formation, interacts with dual specificity tyrosine phosphorylation regulated kinase 1A (Dyrk1a) to modulate multiciliogenesis. We show that Dyrk1a phosphorylates CEP97, which in turn promotes the recruitment of Polo-like kinase 1 (Plk1), which is a critical regulator of MCC maturation that functions to enhance centriole disengagement in cooperation with the enzyme Separase. Knockdown of either CEP97 or Dyrk1a disrupts cilia formation and centriole disengagement in MCCs, but this defect is rescued by overexpression of Separase. Thus, our study reveals that Dyrk1a and CEP97 coordinate with Plk1 to promote Separase function to properly form multicilia in vertebrate MCCs.

Our study reveals Drg1 as a new binding partner of Dishevelled. The Drg1–Dishevelled association regulates Daam1 and RhoA interactions and activity, leading to polymerization and stability of the actin cytoskeleton, a process that is essential for proper multiciliation., Cilia are critical for proper embryonic development and maintaining homeostasis. Although extensively studied, there are still significant gaps regarding the proteins involved in regulating ciliogenesis. Using the Xenopus laevis embryo, we show that Dishevelled (Dvl), a key Wnt signaling scaffold that is critical to proper ciliogenesis, interacts with Drg1 (developmentally regulated GTP-binding protein 1). The loss of Drg1 or disruption of the interaction with Dvl reduces the length and number of cilia and displays defects in basal body migration and docking to the apical surface of multiciliated cells (MCCs). Moreover, Drg1 morphants display abnormal rotational polarity of basal bodies and a decrease in apical actin and RhoA activity that can be attributed to disruption of the protein complex between Dvl and Daam1, as well as between Daam1 and RhoA. These results support the concept that the Drg1–Dvl interaction regulates apical actin polymerization and stability in MCCs. Thus, Drg1 is a newly identified partner of Dvl in regulating ciliogenesis.

The Zic family of zinc finger transcription factors plays a critical role in multiple developmental processes. Using loss-of-function studies, we find that Zic5 is important for the differentiation of retinal pigmented epithelium (RPE) and the rod photoreceptor layer through suppressing Hedgehog (Hh) signaling. Further, Zic5 interacts with the critical Hh signaling molecule, Gli3, through the zinc finger domains of both proteins. This Zic5-Gli3 interaction disrupts Gli3/Gli3 homodimerization, resulting in Gli3 protein stabilization via a reduction in Gli3 ubiquitination. During embryonic Hh signaling, the activator form of Gli is normally converted to a repressor form through proteosome-mediated processing of Gli3, and the ratio of Gli3 repressor to full-length (activator) form of Gli3 determines the Gli3 repressor output required for normal eye development. Our results suggest Zic5 is a critical player in regulating Gli3 stability for the proper differentiation of RPE and rod photoreceptor layer during Xenopus eye development.

Rab11 family-interacting protein 5 (Rab11fip5) is an adaptor protein that binds to the small GTPase Rab11, which has an important function in endosome recycling and trafficking of cellular proteins to the plasma membrane. Rab11fip5 is involved in many cellular processes, such as cytoskeleton rearrangement, iron uptake and exocytosis in neuroendocrine cells, and is also known as a candidate gene for autism-spectrum disorder. However, the role of Rab11fip5 during early embryonic development is not clearly understood. In this study, we identified Rab11fip5 as a protein that interacts with ephrinB1, a transmembrane ligand for Eph receptors. The PDZ binding motif in ephrinB1 and the Rab-binding domain in Rab11fip5 are necessary for their interaction in a complex. EphrinB1 and Rab11fip5 display overlapping expression in the telencephalon of developing amphibian embryos. The loss of Rab11fip5 function causes a reduction in telencephalon size and a decrease in the expression level of ephrinB1. Moreover, morpholino oligonucleotide-mediated knockdown of Rab11fip5 decreases cell proliferation in the telencephalon. The overexpression of ephrinB1 rescues these defects, suggesting that ephrinB1 recycling by the Rab11/Rab11fip5 complex is crucial for proper telencephalon development., Summary: Rab11fip5, genetic mutations of which are associated with autism spectrum disorders in humans, plays a role in regulating telencephalon development via recycling of the crucial cargo ephrinB1.

poster 1869C at TAGC 2020 Online

Presentation of poster 1675A at TAGC 2020 Online. Files include a PDF of the poster (Filename.PDF)

Fine-control of Wnt signaling is essential for various cellular and developmental decision making processes. However, deregulation of Wnt signaling leads to pathological consequences including cancer. Here, we identify a novel function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells, and required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing secondary axis with β-catenin target gene upregulation. Upon Wnt signaling activation, PAF is dissociated from PCNA, and directly binds to β-catenin. Then, PAF recruits EZH2 to β-catenin transcriptional complex, and specifically enhances Wnt target gene transactivation, independently of EZH2's methyltransferase activity. In mouse, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling, and propose a novel regulatory mechanism of Wnt signaling during tumorigenesis.

The Wnt pathways contribute to many processes in cancer and development, with β-catenin being a key canonical component. p120-catenin, which is structurally similar to β-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ- and zinc-finger-domain-containing transcription factor Kaiso. We have identified the kinase Dyrk1A as a component of the p120-catenin–Kaiso trajectory of the Wnt pathway. Using rescue and other approaches in Xenopus laevis embryos and mammalian cells, we found that Dyrk1A positively and selectively modulates p120-catenin protein levels, thus having an impact on p120-catenin and Kaiso (and canonical Wnt) gene targets such as siamois and wnt11. The Dyrk1A gene resides within the Down's syndrome critical region, which is amplified in Down's syndrome. A consensus Dyrk phosphorylation site in p120-catenin was identified, with a mutant mimicking phosphorylation exhibiting the predicted enhanced capacity to promote endogenous Wnt-11 and Siamois expression, and gastrulation defects. In summary, we report the biochemical and functional relationship of Dyrk1A with the p120-catenin–Kaiso signaling trajectory, with a linkage to canonical Wnt target genes. Conceivably, this work might also prove relevant to understanding the contribution of Dyrk1A dosage imbalance in Down's syndrome.

δ-Catenin is an Armadillo protein of the p120-catenin subfamily capable of modulating cadherin stability, small GTPase activity, and nuclear transcription. From yeast two-hybrid screening of a human embryonic stem cell cDNA library, we identified δ-catenin as a potential interacting partner of the caspase-3 protease, which plays essential roles in apoptotic as well as non-apoptotic processes. Interaction of δ-catenin with caspase-3 was confirmed using cleavage assays conducted in vitro, in Xenopus apoptotic extracts, and in cell line chemically induced contexts. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo repeat 6 of δ-catenin, was identified through peptide sequencing. Cleavage thus generates an amino-terminal (residues 1–816) and carboxyl-terminal (residues 817–1314) fragment, each containing about half of the central Armadillo domain. We found that cleavage of δ-catenin both abolishes its association with cadherins and impairs its ability to modulate small GTPases. Interestingly, 817–1314 possesses a conserved putative nuclear localization signal that may facilitate the nuclear targeting of δ-catenin in defined contexts. To probe for novel nuclear roles of δ-catenin, we performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating interaction with an uncharacterized KRAB family zinc finger protein, ZIFCAT. Our results indicate that ZIFCAT is nuclear and suggest that it may associate with DNA as a transcriptional repressor. We further determined that other p120 subfamily catenins are similarly cleaved by caspase-3 and likewise bind ZIFCAT. Our findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120-catenin subfamily members, facilitating the coordinate modulation of cadherins, small GTPases, and nuclear functions.

Although the canonical Wnt pathway and β-catenin have been extensively studied, less is known about the role of p120-catenin (also known as δ1-catenin) in the nuclear compartment. Here, we report that p120-catenin binds and negatively regulates REST and CoREST (also known as Rcor1), a repressive transcriptional complex that has diverse developmental and pathological roles. Using mouse embryonic stem cells (mESCs), mammalian cell lines, Xenopus embryos and in vitro systems, we find that p120-catenin directly binds the REST–CoREST complex, displacing it from established gene targets to permit their transcriptional activation. Importantly, p120-catenin levels further modulate the mRNA and protein levels of Oct4 (also known as POU5F1), Nanog and Sox2, and have an impact upon the differentiation of mESCs towards neural fates. In assessing potential upstream inputs to this new p120-catenin–REST–CoREST pathway, REST gene targets were found to respond to the level of E-cadherin, with evidence suggesting that p120-catenin transduces signals between E-cadherin and the nucleus. In summary, we provide the first evidence for a direct upstream modulator and/or pathway regulating REST–CoREST, and reveal a substantial role for p120-catenin in the modulation of stem cell differentiation.

Members of the plakophilin-catenin sub-family (Pkp-1, -2, and -3) facilitate the linkage of desmosome junctional components to each other (e.g. desmosomal cadherins to desmoplakin) and the intermediate-filament cytoskeleton. Pkps also contribute to desmosomal stabilization and the trafficking of its components. The functions of Pkps outside of the desmosome are less well studied, despite evidence suggesting their roles in mRNA regulation, small-GTPase modulation (e.g. mid-body scission) during cell division, and cell survival following DNA damage. Pkp-catenins are further believed to have roles in the nucleus given their nuclear localization in some contexts and the known nuclear roles of structurally related catenins, such as beta-catenin and p120-catenin. Further, Pkp-catenin activities in the nuclear compartment have become of increased interest with the identification of interactions between Pkp2-catenin and RNA Pol III and Pkp1 with single-stranded DNA. Consistent with earlier reports suggesting possible nuclear roles in development, we previously demonstrated prominent nuclear localization of Pkp3 in Xenopus naïve ectoderm ("animal cap") cells and recently resolved a similar localization in mouse embryonic stem cells. Here, we report the association and positive functional interaction of Pkp3 with a transcription factor, Ets variant gene 1 (ETV1), which has critical roles in neural development and prominent roles in human genetic disease. Our results are the first to report the interaction of a sequence-specific transcription factor with any Pkp. Using Xenopus laevis embryos and mammalian cells, we provide evidence for the Pkp3:ETV1 complex on both biochemical and functional levels.

The p120-catenin family has undergone a significant expansion during the evolution of vertebrates, resulting in varied functions that have yet to be discerned or fully characterized. Likewise, members of the plakophilins, a related catenin subfamily, are found throughout the cell with little known about their functions outside the desmosomal plaque. While the plakophilin-3 (Pkp3) knockout mouse resulted in skin defects, we find larger, including lethal effects following its depletion in Xenopus. Pkp3, unlike some other characterized catenins in amphibians, does not have significant maternal deposits of mRNA. However, during embryogenesis, two Pkp3 protein products whose temporal expression is partially complimentary become expressed. Only the smaller of these products is found in adult Xenopus tissues, with an expression pattern exhibiting distinctions as well as overlaps with those observed in mammalian studies. We determined that Xenopus Pkp3 depletion causes a skin fragility phenotype in keeping with the mouse knockout, but more novel, Xenopus tailbud embryos are hyposensitive to touch even in embryos lacking outward discernable phenotypes, and we additionally resolved disruptions in certain peripheral neural structures, altered establishment and migration of neural crest, and defects in ectodermal multiciliated cells. The use of two distinct morpholinos, as well as rescue approaches, indicated the specificity of these effects. Our results point to the requirement of Pkp3 in amphibian embryogenesis, with functional roles in a number of tissue types.

In common with other p120-catenin subfamily members, Xenopus ARVCF (xARVCF) binds cadherin cytoplasmic domains to enhance cadherin metabolic stability or, when dissociated, modulates Rho-family GTPases. We report here that xARVCF binds and is stabilized by Xenopus KazrinA (xKazrinA), a widely expressed conserved protein that bears little homology to established protein families, and which is known to influence keratinocyte proliferation and differentiation and cytoskeletal activity. Although we found that xKazrinA binds directly to xARVCF, we did not resolve xKazrinA within a larger ternary complex with cadherin, nor did it co-precipitate with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin, suggesting a potential means by which xKazrinA localizes to cell–cell borders. This was supported by the resolution of a ternary biochemical complex of xARVCF–xKazrinA–xβ2-spectrin and, in vivo, by the finding that ectodermal shedding followed depletion of xKazrin in Xenopus embryos, a phenotype partially rescued with exogenous xARVCF. Cell shedding appeared to be the consequence of RhoA activation, and thereby altered actin organization and cadherin function. Indeed, we also revealed that xKazrinA binds p190B RhoGAP, which was likewise capable of rescuing Kazrin depletion. Finally, xKazrinA was found to associate with δ-catenins and p0071-catenins but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin subfamily. Taken together, our study supports the essential role of Kazrin in development, and reveals the biochemical and functional association of KazrinA with ARVCF-catenin, spectrin and p190B RhoGAP.

Recently, the livestock industry in Korea has been threatened by many challenges such as low productivity due to labor intensiveness, global competition compelled by the Free Trade Agreement (FTA), and emerging animal disease issues such as BSE or foot-and-mouth. In this paper, we propose a smart farm management system, called uFarm, which would come up with such challenges by automating farm management. First, we automate labor-intensive jobs using equipments based on sensors and actuators. The automation subsystem can be controlled by remote user through wireless network. Second, we provide real-time traceability of information on farm animals using the radio-frequency identification (RFID) method and embedded data server with network connectivity.

Dishevelled (DVL) proteins serve as crucial regulators that transduce canonical Wnt signals to the GSK3β-destruction complex, resulting in the stabilization of β-catenin. Emerging evidences underscore the nuclear functions of DVLs, which are critical for the Wnt/β-catenin signaling. However, the mechanism underlying DVL nuclear localization remains poorly understood. Here, we discovered two Forkhead box (FOX) transcription factors, FOXK1 and FOXK2, as bona-fide DVL-interacting proteins. FOXK1 and FOXK2 positively regulate Wnt/β-catenin signaling by translocating DVL into the nucleus. Moreover, FOXK1 and FOXK2 protein levels are elevated in human colorectal cancers and correlate with DVL nuclear localization. The conditional expression of Foxk2 in mice induced intestinal hyper-proliferation that featured with enhanced DVL-nuclear localization and up-regulated Wnt/β-catenin signaling. Together, our results not only reveal a mechanism by which DVL is translocated into nucleus, but also suggest unexpected roles of FOXK1 and FOXK2 in regulating Wnt/β-catenin signaling.