PCP Pathway Research Articles

Rare missense variants in DVL1, one of the human counterparts of the Drosophila dishevelled gene, do not confer increased risk for neural tube defects

Neural tube defects (NTDs) are severe malformations that arise when the neural tube fails to close during embryogenesis. The planar cell polarity pathway is involved in neural tube closure and has been implicated in the pathogenesis of NTDs both in animal models and human cohorts. Dishevelled (Dvl/Dsh) is a multi-module protein and a key regulator of both the canonical Wnt and the PCP pathway. In mouse, all Dvl1(-/-) ; Dvl2(-/-) double mutants display craniorachischisis, a severe form of open NTDs. Recently, we have reported a possible role for rare variants of DVL2 as risk factors for NTDs. In view of these data, we hypothesized that DVL1 mutations might increase the risk for NTDs in some cases. Resequencing of the DVL1 gene in a cohort of 473 NTDs patients and 150 ethnically matched controls was performed. Prediction of the downstream effects of the nonsynonymous variants was done using computational methods. We identified six missense variants that were absent in our ethnically matched controls group, and four of them (p.Arg153Cys; p.Glu544Arg; p.Arg568Trp; p.Val644Phe) were predicted to have a functional effect on protein structure by one or more bioinformatic programs. However, there was no difference in the overall rate of deleterious variants between the patients and controls (four in patients and three in controls; p=0.36). Our findings did not provide evidence for the implication of DVL1 in the pathogenesis of human NTDs.

Abstract 4201: miR-124 suppresses colorectal tumor progression in a mouse xenograft model via targeting KITENIN.

Abstract MicroRNAs (miRNAs) are increasingly implicated in modulating the progression of various cancers. Although there is emerging evidences that some miRNAs can function as oncogenes or tumour suppressors, the regulation of colorectal cancer (CRC) progression by miRNA is not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin, Vangl1) is not only a major component of PCP pathway but also a functional protein promoting CRC cell motility and invasiveness. Previously, we observed that KITENIN acts as a metastasis-enhancing protein in a mouse model of colon cancer and that KITENIN is highly expressed in sporadic human CRC tissues. But the underlying regulation mechanisms of aberrant expression of KITENIN in CRC are not clearly understood. Here we tried to identify the miRNAs, which modulate the expression of KITENIN, using luciferase assay and with computational prediction of miRNA targeting KITENIN. We identified several miRNAs and one of them is miR-124. miR-124 negatively regulated KITENIN expression through binding to the 3′-untranslated region of KITENIN. We further examined whether increased expression of miR-124 affects colorectal tumor progression via targeting KITENIN. Ectopic expression of miR-124 was found to suppress the migration and invasiveness of various CRC cells. miR-124 also inhibited tumor growth in a mouse xenograft model. These findings provide the experimental evidence to a possible therapeutic effect of miR-124 on suppressing colorectal tumor progression and identification of miR-124-mediated regulation of KITENIN might provide a promising therapeutic target in treating CRC. Citation Format: So-Yeon Park, Jeong A Bae, Somy Yoon, Hangun Kim, Kyung Keun Kim. miR-124 suppresses colorectal tumor progression in a mouse xenograft model via targeting KITENIN. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4201. doi:10.1158/1538-7445.AM2013-4201

Vangl2 and planar cell polarity signaling in cardiac development

Planar cell polarity pathway regulates cell polarity and polarized cell movements.Recent studies in mouse models have found mutations in several genes in this pathway and specifically in the Vangl2 gene,which results in abnormalities in cardiac development,suggesting Vangl2 and this pathway play an important role in heart development.This review mainly elucidates the mechanisms regulated by the Vangl2 gene and PCP pathway during outflow tract development and the formation of the coronary vasculature. Key words: Vangl2; Noncanonical Wnt/PCP pathway; Cardiac development; Congenital heart disease

Functional analysis of the transition zone protein Rpgrip1l during zebrafish development

We investigated the function of Rpgrip1l during zebrafish development. Rpgrip1l encodes a protein localised at the ciliary transition zone and interacts functionally with NPH and MKS for the formation and function of the ciliary gate. The human RPGRIP1L gene is a causal gene in Meckel and Joubert type B syndromes, characterised by polydactyly, kidney cysts and central nervous system malformations. Using morpholinos injection, we show that loss of Rpgrip1l function leads to several early phenotypes such as convergent-extension phenotype, randomisation of left-right asymmetry and hydrocephaly. We focussed our study on the Wnt-PCP defects and we demonstrated that in the zebrafish floorplate, Rpgrip1l is required for correct positioning of the basal body along the planar polarity axis. We confirmed Rpgrip1l function on basal body positioning in the mechanosensory hair cells of the cochlea of the murine mutant for Rpgrip1l. Our results strongly suggest that Rpgrip1l is essential for recruiting and stabilizing dishevelled, a major actor of the PCP pathway, at the basal body and/or cilium. Indeed, in two different cell types, in the zebrafish floor plate and in the murine cochlea, dishevelled proteins are enriched at the cilium and/or basal body, and this localization is severely perturbed upon Rpgrip1l depletion. Finally, we demonstrate that, in the zebrafish floor plate, the function of Rpgrip1l in basal body positioning is mediated by dishevelled. We propose that Rpgrip1l participates in a protein complex required for recruiting and stabilizing dishevelled at the cilium, a process essential for planar polarization of the basal body.

RTTN Mutations Link Primary Cilia Function to Organization of the Human Cerebral Cortex

Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals.

Roles of Planar Cell Polarity Signaling in Maturation of Neuronal Precursor Cells in the Postnatal Mouse Olfactory Bulb

Neuronal precursor cells generated by stem cells in the subventricular zone (SVZ) migrate and differentiate into mature interneurons in the olfactory bulb (OB). The mechanisms responsible for the dynamic morphological changes in cells during this process are largely unknown. Wnt/planar cell polarity (PCP) signaling regulates various developmental events, including neuronal migration and neurite formation. Here, we studied the function of two components of the PCP pathway, Dishevelled2 and Van Gogh like-2, in the newborn neurons in the postnatal mouse OB. Electroporation- or lentivirus-mediated introduction of vectors carrying a knockdown or dominant-negative construct of these genes into the SVZ altered the distribution and dendrite formation of newborn neurons in the OB, suggesting that PCP signaling is involved in regulating the maturation of new neurons in the OB.

Wnt signalling antagonizes stress granule assembly through a Dishevelled-dependent mechanism

SummaryCells often respond to diverse environmental stresses by inducing stress granules (SGs) as an adaptive mechanism. SGs are generally assembled as a result of aggregation of mRNAs stalled in a translational pre-initiation complex, mediated by a set of RNA-binding proteins such as G3BP and TIA-1. SGs may serve as triage centres for storage, translation re-initiation or degradation of specific mRNAs. However, the mechanism involved in the modulation of their assembly/disassembly is unclear. Here we report that Wnt signalling negatively regulates SG assembly through Dishevelled (Dvl), a cytoplasmic Wnt effector. Overexpression of Dvl2, an isoform of Dvl, leads to impairment of SG assembly through a DEP domain dependent mechanism. Intriguingly, the Dvl2 mutant K446M, which corresponds to an analogous mutation in Drosophila Dishevelled DEP domain (dsh1) that results in defective PCP pathway, fails to antagonize SG assembly. Furthermore, we show that Dvl2 exerts the antagonistic effect on SG assembly through a mechanism involving Rac1-mediated inhibition of RhoA. Dvl2 interacts with G3BP, a downstream component of Ras signalling involved in SG assembly, and functional analysis suggests a model wherein the Dvl-Rac1-RhoA axis regulates G3BP's SG-nucleating activity. Collectively, these results define an antagonistic effect of Wnt signalling on SG assembly, and reveal a novel role for Wnt/Dvl pathway in the modulation of mRNA functions.

Pronephric Tubulogenesis Requires Daam1-Mediated Planar Cell Polarity Signaling

Canonical β-catenin-mediated Wnt signaling is essential for the induction of nephron development. Noncanonical Wnt/planar cell polarity (PCP) pathways contribute to processes such as cell polarization and cytoskeletal modulation in several tissues. Although PCP components likely establish the plane of polarization in kidney tubulogenesis, whether PCP effectors directly modulate the actin cytoskeleton in tubulogenesis is unknown. Here, we investigated the roles of Wnt PCP components in cytoskeletal assembly during kidney tubule morphogenesis in Xenopus laevis and zebrafish. We found that during tubulogenesis, the developing pronephric anlagen expresses Daam1 and its interacting Rho-GEF (WGEF), which compose one PCP/noncanonical Wnt pathway branch. Knockdown of Daam1 resulted in reduced expression of late pronephric epithelial markers with no apparent effect upon early markers of patterning and determination. Inhibiting various points in the Daam1 signaling pathway significantly reduced pronephric tubulogenesis. These data indicate that pronephric tubulogenesis requires the Daam1/WGEF/Rho PCP pathway.

BMP7 directs epithelial cell fate choice and dorsal–ventral partitioning of the embryonic cloaca by collaborating with the PCP pathway

BMP7 directs epithelial cell fate choice and dorsal–ventral partitioning of the embryonic cloaca by collaborating with the PCP pathway

Abstract 2733: KITENIN and ErbB4 contribute to further malignant changes in colorectal cancer through c-Jun/AP-1 activation

Abstract KITENIN (KAI1 C-terminal interacting tetraspanin, Vangl1) is not only a major component of PCP pathway but also a functional protein promoting colorectal cancer (CRC) cell motility and invasiveness. Here, we report the mechanism of which KITENIN leads further malignant changes in CRC progression after loss of tumor suppressor Adenomatous polyposis coli (APC). KITENIN increases the level of AP-1 activity through interaction with Dvl2, which is subsequently being degraded. Decreasing Dvl2 via interaction with KITENIN or via treatment of si-RNA to Dvl2 significantly increases the AP-1 activity through elevating the level of c-jun and c-jun phosphorylation. EGF acts upstream of KITENIN/AP-1 signaling and induces synergic AP-1 activation in the presence of elevated KITENIN, which requires the interaction of KITENIN with ErbB4. The morphological characteristics of adenocarcinoma were observed in intestinal tumor tissues from compound villin-KITENIN transgenic/APCmin/+ mice but not from APCmin/+ mice, both of which express the elevated level of ErbB4. This result demonstrates that further malignant phenotype of intestinal tumor can be induced by elevated KITENIN and ErbB4 via promoting c-Jun/AP-1 activation within an APC loss-associated tumor microenvironment. Our present results suggest the possibility of the addition of anti-KITENIN therapy to the use of EGFR inhibitors in the treatment of CRC patients with APC mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2733. doi:10.1158/1538-7445.AM2011-2733

Frizzled Signaling: Gα o and Rab5 at the Crossroads of the Canonical and PCP Pathways?

Endocytosis modulates the activities of members of a family of receptors known as Frizzled (Fz) proteins, which control canonical β-catenin signaling and the planar cell polarity pathway; however, what determines which of the two pathways is activated is unclear. Evidence now suggests that the guanosine triphosphatase Rab5, a key regulator of endocytosis, binds directly to Fz proteins and that subsequent trafficking steps determine the outcome of Fz signaling.

C21orf59 regulates cilia motility and acts through the PCP pathway

c21orf59 regulates cilia motility and acts through the PCP pathway

Epidermal Wound Repair Is Regulated by the Planar Cell Polarity Signaling Pathway

The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3(-)(/-) mice, we identified RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair.

The role of actin dynamics and the PCP pathway in mammalian convergent extension and establishment of left–right asymmetry

The role of actin dynamics and the PCP pathway in mammalian convergent extension and establishment of left–right asymmetry

Wnt signaling and the evolution of pattern formation

Evolution of gastrulation was crucial for metazoan diversification, but the molecular origins of developmental polarity that led to the onset of gastrulation mechanisms are unknown. Nuclear ß‐catenin marks the site of gastrulation in cnidarians and several bilaterians, raising the possibility that evolution of mechanisms to asymmetrically activate Wnt signaling in embryos may have been causal in evolution of gastrulation. We have shown that localized activation of Dishevelled (Dsh), a key regulator of Wnt/ß ‐catenin signaling, plays a crucial role in germ layer segregation and gastrulation in sea urchins and in the cnidarian Nematostella vectensis. Dsh also regulates the Wnt/Planar Cell Polarity (PCP) pathway, which mediates vertebrate gastrulation movements. In Nematostella, blocking Dsh signaling in the PCP pathway, or blocking Strabismus function using morpholinos blocks gastrulation, suggesting that Wnt/PCP signaling mediated gastrulation movements in the last common ancestor to cnidarians and bilaterians. Because Dsh can regulate multiple Wnt pathways, it may have been a critical link between evolution of cell fate specification and evolution of gastrulation morphogenesis. Our results also support the hypothesis that one ancient function of PCP signaling was to mediate the developmental asymmetry that led to the evolution of gastrulation. (Supported by NSF grants IOS 0720365 and IOS 0754323)

Roles of Noncanonical Wnt/PCP Pathway Genes in Neuronal Migration and Neurulation in Zebrafish

Noncanonical Wnt/planar cell polarity (PCP) pathways regulate oriented cell movement during development in both Drosophila and vertebrates. Recent studies have revealed similarities and differences between these pathways in the tissues on which they act. In zebrafish, PCP pathway genes regulate the directional migration of a specific population of motor neurons in the hindbrain, as well as morphogenesis of the neuroepithelium. In the present review, neuronal and neuroepithelial defects in zebrafish PCP pathway mutants are compared, and the possible cellular and molecular mechanisms underlying these phenotypes are discussed. Future analyses of zebrafish PCP mutants will reveal the general mechanisms underlying the development of the neuroepithelium and provide novel insights into both conserved and diverse functions of PCP pathway genes in vertebrate development.

Importance of Wnt Signaling in the Tumor Stroma Microenvironment

Wnt signaling plays an important role in cancer. Signaling is initiated by binding of Wnt ligands to Frizzled cell surface receptors and results in signaling via one of three pathways, the canonical Wnt pathway, which is the best characterized in both normal tissues and in cancer, and two non-canonical Wnt pathways, the Ca(2+)-dependent and the PCP pathways. Canonical Wnt signaling results in beta-catenin accumulation in the cytoplasm, translocation into the nucleus and activation of transcription of Wnt target genes including the c-Myc oncogene. Some cancer types, including colorectal cancer, have mutations in APC and Axin, which are involved in beta-catenin phosphorylation, such that the canonical pathway is constitutively active. Few studies have investigated the role non-canonical Wnt signaling in cancer, or of Wnt signaling on tumor stromal cells. Wnt overexpression is observed in tumor stroma, as is overexpression of the Wnt pathway inhibitors, secreted Frizzled-related proteins and Dickkopf proteins. Interactions between epithelial cells and stromal cells have been observed to activate Wnt signaling in both cell types. Wnt signaling is also observed in tumor blood vessels and is likely to be activated by signals from tumor cells. Current cancer therapies focus on interfering with canonical Wnt signaling in the tumor cells. Future therapeutic targets for interfering with Wnt signaling include cell surface receptors such as the RYK and Ror2 receptors and secreted signaling molecules, which mediate signaling between cancer cells and the stromal environment.

Nucleoredoxin regulates the Wnt/planar cell polarity pathway in Xenopus

The Wnt signaling pathway is conserved across species, and is essential for early development. We previously identified nucleoredoxin (NRX) as a protein that interacts with dishevelled (Dvl) in vivo to negatively regulate the Wnt/beta-catenin pathway. However, whether NRX affects another branch of the Wnt pathway, the Wnt/planar cell polarity (PCP) pathway, remains unclear. Here we show that NRX regulates the Wnt/PCP pathway. In Xenopus laevis, over-expression or depletion of NRX by injection of NRX mRNA or antisense morpholino oligonucleotide, respectively, yields the bent-axis phenotype that is typically observed in embryos with abnormal PCP pathway activity. In co-injection experiments of Dvl and NRX mRNA, NRX suppresses the Dvl-induced bent-axis phenotype. Over-expression or depletion of NRX also suppresses the convergent extension movements that are believed to underlie normal gastrulation. We also found that NRX can inhibit Dvl-induced up-regulation of c-Jun phosphorylation. These results indicate that NRX plays crucial roles in the Wnt/PCP pathway through Dvl and regulates Xenopus gastrulation movements.

Perturbed desmosomal cadherin expression in grainy head-like 1-null mice

In Drosophila, the grainy head (grh) gene plays a range of key developmental roles through the regulation of members of the cadherin gene family. We now report that mice lacking the grh homologue grainy head-like 1 (Grhl1) exhibit hair and skin phenotypes consistent with a reduction in expression of the genes encoding the desmosomal cadherin, desmoglein 1 (Dsg1). Grhl1-null mice show an initial delay in coat growth, and older mice exhibit hair loss as a result of poor anchoring of the hair shaft in the follicle. The mice also develop palmoplantar keratoderma, analogous to humans with DSG1 mutations. Sequence analysis, DNA binding, and chromatin immunoprecipitation experiments demonstrate that the human and mouse Dsg1 promoters are direct targets of GRHL1. Ultrastructural analysis reveals reduced numbers of abnormal desmosomes in the interfollicular epidermis. These findings establish GRHL1 as an important regulator of the Dsg1 genes in the context of hair anchorage and epidermal differentiation, and suggest that cadherin family genes are key targets of the grainy head-like genes across 700 million years of evolution.

Coordination of Cell Polarity during Xenopus Gastrulation

Cell polarity is an essential feature of animal cells contributing to morphogenesis. During Xenopus gastrulation, it is known that chordamesoderm cells are polarized and intercalate each other allowing anterior-posterior elongation of the embryo proper by convergent extension (CE). Although it is well known that the cellular protrusions at both ends of polarized cells exert tractive force for intercalation and that PCP pathway is known to be essential for the cell polarity, little is known about what triggers the cell polarization and what the polarization causes to control intracellular events enabling the intercalation that leads to the CE. In our research, we used EB3 (end-binding 3), a member of +TIPs that bind to the plus end of microtubule (MT), to visualize the intracellular polarity of chordamesoderm cells during CE to investigate the trigger of the establishment of cell polarity. We found that EB3 movement is polarized in chordamesoderm cells and that the notochord-somite tissue boundary plays an essential role in generating the cell polarity. This polarity was generated before the change of cell morphology and the polarized movement of EB3 in chordamesoderm cells was also observed near the boundary between the chordamesoderm tissue and naïve ectoderm tissue or lateral mesoderm tissues induced by a low concentration of nodal mRNA. These suggest that definitive tissue separation established by the distinct levels of nodal signaling is essential for the chordamesodermal cells to acquire mediolateral cell polarity.