PCP Pathway Research Articles

The Type 3 Deiodinase Is a Critical Modulator of Thyroid Hormone Sensitivity in the Fetal Brain.

Thyroid hormones (TH) are critical for the development and function of the central nervous system (CNS). Although their effects on the rodent brain peak within 2–3 weeks postnatally, the fetal brain has been found largely insensitive to exogenously administrated TH. To address this issue, here we examined gene expression in brains from mouse fetuses deficient in the type 3 deiodinase (DIO3), the selenoenzyme responsible for clearing TH. At embryonic day E18.5 qPCR determinations indicated a marked increase in the mRNA expression of T3-responsive genes Klf9 and Nrgn. The increased expression of these genes was confirmed by in situ hydridization in multiple areas of the cortex and in the striatum. RNA sequencing revealed 246 genes differentially expressed (70% up-regulated) in the brain of E18.5 Dio3−/− male fetuses. Differential expression of 13 of these genes was confirmed in an extended set of samples that included females. Pathway analyses of differentially expressed genes indicated enrichment in glycolysis and signaling related to axonal guidance, synaptogenesis and hypoxia inducible factor alpha. Additional RNA sequencing identified 588 genes differentially expressed (35% up-regulated) in the brain of E13.5 Dio3−/− male fetuses. Differential expression of 13 of these genes, including Klf9, Hr, and Mgp, was confirmed in an extended set of samples including females. Although pathway analyses of differentially expressed genes at E13.5 also revealed significant enrichment in axonal guidance and synaptogenesis signaling, top enrichment was found for functions related to the cell cycle, aryl hydrocarbon receptor signaling, PCP and kinetochore metaphase signaling pathways and mitotic roles of polo-like kinase. Differential expression at E13.5 was confirmed by qPCR for additional genes related to collagen and extracellular matrix and for selected transcription factors. Overall, our results demonstrate that the rodent fetal brain is sensitive to TH as early as E13.5 of gestational age, and suggest that TH distinctly affects brain developmental programs in early and late gestation. We conclude that DIO3 function is critical to ensure an adequate timing for TH action in the developing brain and is probably the main factor underlying the lack of effects on the fetal brain observed in previous studies after TH administration.

Elucidate growth control mechanisms using reconstructed spatiotemporal distributions of signaling events

A fundamental biological question is how diverse and complex signaling and patterning is controlled correctly to generate distinct tissues, organs, and body plans, but incorrectly in diseased cells and tissues. Signaling pathways important for growth control have been identified, but to identify the mechanisms their transient and context-dependent interactions encode is more difficult. Currently computational systems biology aims to infer the control mechanisms by investigating quantitative changes of gene expression and protein concentrations, but this inference is difficult in nature. We propose it is desirable to explicitly simulate events and orders of gene regulation and protein interactions, which better elucidate control mechanisms, and report a method and tool with three examples. The Drosophila wing model includes Wnt, PCP, and Hippo pathways and mechanical force, incorporates well-confirmed experimental findings, and generates novel results. The other two examples illustrate the building of three-dimensional and large-scale models. These examples support that reconstructed spatiotemporal distributions of key signaling events help elucidate growth control mechanisms. As biologists pay increasing attention to disordered signaling in diseased cells, to develop new modeling methods and tools for conducting new computational studies is important.

Dkk1 Controls Cell-Cell Interaction through Regulation of Non-nuclear β-Catenin Pools.

SummaryDickkopf-1 (Dkk1) is a secreted Wnt antagonist with a well-established role in head induction during development. Numerous studies have emerged implicating Dkk1 in various malignancies and neurodegenerative diseases through an unknown mechanism. Using zebrafish gastrulation as a model for collective cell migration, we unveil such a mechanism, identifying a role for Dkk1 in control of cell connectivity and polarity in vivo, independent of its known function. We find that Dkk1 localizes to adhesion complexes at the plasma membrane and regions of concentrated actomyosin, suggesting a direct involvement in regulation of local cell adhesion. Our results show that Dkk1 represses cell polarization and integrity of cell-cell adhesion, independently of its impact on β-catenin protein degradation. Concurrently, Dkk1 prevents nuclear localization of β-catenin by restricting its distribution to a discrete submembrane pool. We propose that redistribution of cytosolic β-catenin by Dkk1 concomitantly drives repression of cell adhesion and inhibits β-catenin-dependent transcriptional output.

Transcriptome analysis reveals the role of the PCP pathway in fipronil and endotoxin-induced lung damage

BackgroundPesticide residues in food and environment along with airborne contaminants like endotoxins pose a tangible threat to living beings. Fipronil, a broad-spectrum insecticide at acute doses in combination with endotoxin has been linked to airway inflammation. Wnt/PCP pathway plays important role in the pathogenesis of chronic inflammatory conditions by contributing to the release of downstream cytokines. However, there is no data on the PCP signaling pathway during fipronil-induced lung damage. This study aims to understand the molecular mechanisms underlying the pulmonary toxicity induced by fipronil alone or in combination with endotoxin.MethodsWe used a microarray approach to obtain a global view of the transcriptional responses of the lungs exposed to fipronil and endotoxin in a mice model. Lung tissues were harvested from male Swiss albino mice (n=42) following long term oral exposure to high and low doses of fipronil alone or in combination with endotoxin. The differentially expressed genes were analyzed by Ingenuity pathway analysis software to predict the top canonical pathways. The microarray data for selected genes was validated using quantitative PCR and immunohistochemistry.ResultsBoth BAL fluid and histopathology analysis suggested lung damage and altered transcriptomic profile of lung following exposure to high and low dose of fipronil with or without endotoxin. PCP pathway was among the top upregulated canonical pathways following exposure to both doses of fipronil individually or with LPS. Low dose of fipronil increased the expression of WNT-6, MAPK-8, IL-4 and IL-17C while higher dose decreased the expression of MAPK-8 and IL-17C. The expression data verified by realtime PCR was in concordance with microarray data.ConclusionLong-term exposure to low (4.75 mg kg-1) and high (9.50 mg kg-1) dose of fipronil alone or in combination with endotoxins alters the histoarchitecture and transcriptome profile of lungs with the involvement of the PCP pathway. PCP pathway showed higher enrichment in low dose group of fipronil alone or in combination with LPS. The data from the study provides the insights of the potential damage on lungs caused by fipronil and endotoxin interaction and helps to better understand the mechanism of this complex relation.

First evidence of DAAM1 localization in mouse seminal vesicles and its possible involvement during regulated exocytosis

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a protein belonging to the formin family, which regulates, together with the small GTPase RhoA, the nucleation and the assembly of actin fibres through Wnt-Dishevelled PCP pathway. Its role has been investigated in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. In this work, we studied the expression of DAAM1 mRNA and protein by PCR and Western blot analyses and its co-localization with actin in adult mouse seminal vesicles by immunofluorescence. We show that both proteins are cytoplasmic: actin is evident at cell–cell junctions and at cell cortex; DAAM1 had a more diffused localization, but is also prominent at the apical plasmatic membrane of epithelial cells. These findings support our hypothesis of a role of DAAM1 in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular concerning actin filaments. We were also able to detect DAAM1 and actin association in the smooth muscle cells that surround the epithelium too. In this case, we could only speculate the possible involvement of this formin in muscular cells in the maintenance and the regulation of the contractile structures. The present results strongly suggest that DAAM1 could have a pivotal role in vesicle exocytosis and in the physiology of mouse seminal vesicles.

Abstract 1045: Understanding breast cancer metastasis through circulating tumor cells

Abstract Due to the advance in technologies for rare cell isolation, circulating tumor cells (CTC) have recently received vast interest. During tumor progression tumor cells invade the primary tumor microenvironment and intravasate into blood vessels, where they are referred as CTCs. These CTCs disseminate to other organs and a subset of these cells will be able to form metastasis. The growing interest for CTC is confronted with the difficulty associated with their isolation and characterization. To address this challenge, our lab has recently optimized the ex vivo culture condition and was able to establish CTC lines from breast cancer patients. These CTC lines constitute a unique cell population in the metastatic process and gave us a rare opportunity to investigate the signal pathways involved in each step of the metastatic cascade. To assess the metastatic potential of breast cancer patient-derived CTC lines we utilized an experimental mouse model for metastasis by injecting CTCs directly into the left ventricle of the heart in female immuodeficient NSG mice. The ability of 4 CTC lines to arrest and colonize in organ was monitored by bioluminescence imaging once every 2 weeks for 5 months. CTC lines were capable of generating brain, lung, bone and ovary metastases. Most of those organs are common sites of metastases in breast cancer patients. Two patient-derived CTC lines have a high metastatic potential (over 80% of mice had metastases after 3 months) with generation of simultaneous metastases in multiple organs such as bone, lung and Ovary. These mice remained brain-metastases free for up to 8 months. However, two other patient-derived CTC lines demonstrated the brain as preferential site of metastasis despite their overall low metastatic potential. Interestingly, of 4 breast cancer patients where CTC lines were generated, one developed a metastatic brain tumor and her CTC line has the highest risk of brain metastases in our mouse model. We further investigated genetic and epigenetic determinants that regulate the organotropism of CTCs. We isolated metastatic variants corresponding to a subpopulation of CTCs with a preferential tropism for the brain, lung and bone. Gene expression analysis (RNA-seq) of these variants identified potential gene signatures of breast cancer metastasis. The pathways with the highest enrichment scores were glioblastoma multiform, ceramide biosynthesis and PCP pathway for the brain metastatic variants and were interferon and mTOR signaling for the lung metastatic variants. Next we used a method for assaying chromatin accessibility (ATAC-seq) and identified potential regulatory regions mediating the organ tropism in breast cancer. Together our data provide the evidence of a promising role of CTC as an early prognostic factor in metastasis. Additionally, we expect to develop novel organ tropism associated markers, which can be considered for potential therapeutic targets in breast cancers. Citation Format: Remi Klotz, Thomas Amal, Alan Wang, Matthew Mackay, Kathleen Heller, Lin Li, Maxwell Serowoky, Grace Lee, Jane Han, Andrew Smith, Min Yu. Understanding breast cancer metastasis through circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1045. doi:10.1158/1538-7445.AM2017-1045

Abstract IA25: Therapies directed at the Wnt pathway

Abstract The Wnt pathway represents a family of glycoproteins involved in proliferation, morphology, and migration, and dysregulation of the pathway is present in many cancers.1 Activating mutations, epigenetic events and autocrine activation of the Wnt pathway exists in many tumor types, and these alterations converge into sustained proliferation, increased tumorigenicity, and enhanced metastatic potential. The name “Wnt” comes from combining the drosophila patterning gene wingless and the homologous oncogene int1. There are three branches to the Wnt signaling pathway: the beta-catenin pathway (“canonical”); and the nonconical pathways Wnt/Ca2+ and Wnt/planar cell polarity (PCP). The canonical pathway becomes activated when Wnt ligand binds receptors which regulate nuclear beta-catenin, which in turn regulates critical transcription factors. The PCP pathway is mediated through the GTPases Rho and Rac involved in invasion, metastasis, and angiogenesis, whereas the Ca2+ pathway affects intracellular Ca2+ levels important for Ca2+ homeostasis, cell adhesion, and apoptosis. The focus of this discussion will be on the canonical beta-catenin pathway, which is more commonly implicated in cancer. The canonical pathway is driven by WNT ligands (WNT), which are a family of 19 glycoproteins that bind to multiple transmembrane receptors, including 10 members of the frizzled (FZD) family of G-protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) ROR1 and ROR2, and RTK-like protein kinases (RYKs) triggering downstream activation.2 The pathway is somewhat unusual in that it is regulated largely through a destruction complex containing adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β), and Axin. The destruction complex ubiquitinates β-catenin and prevents its accumulation. Wnt stimulation leads to disruption of the destruction complex and β-catenin accumulation and localization to the nucleus. Cellular response is induced via gene transduction alongside the TCF/LEF (T-cell factor/lymphoid enhancing factor) transcription factors.3 Beyond the destruction complex, negative regulators of the WNT pathway include secreted Dickkopf (Dkk), WNT inhibitory Factor (WIF), and soluble frizzled-related proteins (sFRP). Porcupine (PORCN), a membrane bound O-acyltransferase that is required for WNT palmitoylation, secretion, and biologic activity acts as a positive regulator of the pathway. Pharmacologic strategies targeting the Wnt pathway include modulation of the ligand/receptor interface, stabilization of the degradation complex, or interference with β-Catenin dependent gene transcription.4-6 Phase I studies of several agents have been completed, with mostly acceptable patient tolerance but limited single-agent activity.5 Phase Ib/II studies, many including combination strategies, are underway in several tumor types. In this presentation, the current status and future directions of WNT inhibitors in solid tumors will be reviewed.

Impairment of Wnt11 function leads to kidney tubular abnormalities and secondary glomerular cystogenesis.

BackgroundWnt11 is a member of the Wnt family of secreted signals controlling the early steps in ureteric bud (UB) branching. Due to the reported lethality of Wnt11 knockout embryos in utero, its role in later mammalian kidney organogenesis remains open. The presence of Wnt11 in the emerging tubular system suggests that it may have certain roles later in the development of the epithelial ductal system.ResultsThe Wnt11 knockout allele was backcrossed with the C57Bl6 strain for several generations to address possible differences in penetrance of the kidney phenotypes. Strikingly, around one third of the null mice with this inbred background survived to the postnatal stages. Many of them also reached adulthood, but urine and plasma analyses pointed out to compromised kidney function. Consistent with these data the tubules of the C57Bl6 Wnt11−/− mice appeared to be enlarged, and the optical projection tomography indicated changes in tubular convolution. Moreover, the C57Bl6 Wnt11−/− mice developed secondary glomerular cysts not observed in the controls. The failure of Wnt11 signaling reduced the expression of several genes implicated in kidney development, such as Wnt9b, Six2, Foxd1 and Hox10. Also Dvl2, an important PCP pathway component, was downregulated by more than 90 % due to Wnt11 deficiency in both the E16.5 and NB kidneys. Since all these genes take part in the control of UB, nephron and stromal progenitor cell differentiation, their disrupted expression may contribute to the observed anomalies in the kidney tubular system caused by Wnt11 deficiency.ConclusionsThe Wnt11 signal has roles at the later stages of kidney development, namely in coordinating the development of the tubular system. The C57Bl6 Wnt11−/− mouse generated here provides a model for studying the mechanisms behind tubular anomalies and glomerular cyst formation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0131-z) contains supplementary material, which is available to authorized users.

First Evidence of DAAM1 Localization During the Post-Natal Development of Rat Testis and in Mammalian Sperm.

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a formin-family protein involved in nucleation of unbranched actin filaments and in cytoskeletal organization through Wnt-Dishevelled PCP pathway, which participates in essential biological processes, such as cell polarity, movement, and adhesion during morphogenesis and organogenesis. While its role has been investigated during development and in somatic cells, its potential association with the germinal compartment and reproduction is still unexplored. In this work, we assessed the possible association of DAAM1 with the morphogenesis of rat testis. We studied its expression and profiled its localization versus actin and tubulin, during the first wave of spermatogenesis and in the adult gonad (from 7 to 60 dpp). We show that, in mitotic phases, DAAM1 shares its localization with actin in Sertoli cells, gonocytes, and spermatogonia. Later, during meiosis, both proteins are found in spermatocytes, while only actin is detectable at the forming blood-testis barrier. DAAM1, then, follows the development of the acrosome system throughout spermiogenesis, and it is finally retained inside the cytoplasmic droplet in mature gametes, as corroborated by additional immunolocalization data on both rat and human sperm. Unlike the DAAM1, actin keeps its localization in Sertoli cells, and tubulin is associated with their protruding cytoplasm during the process. Our data support, for the first time, the hypothesis of a role for DAAM1 in cytoskeletal organization during Mammalian testis morphogenesis and gamete progression, while also hinting at its possible investigation as a morphological marker of germ cell and sperm physiology. J. Cell. Physiol. 231: 2172-2184, 2016. © 2016 Wiley Periodicals, Inc.

Abstract A30: Frizzled-7 (FZD7)-mediated non-canonical Wnt-Planar Cell Polarity (PCP) signalling pathway as a novel molecular driver for the C5/Proliferative/Stem-A molecular subtype of ovarian cancer.

Abstract Ovarian cancer (OC), in particular high grade serous carcinoma (HGSC), has been shown to exhibit diverse molecular heterogeneity based on gene expression profiling by the Australian and the TCGA cohorts. This molecular heterogeneity has been demonstrated to be very robust and reproducible by a large-scale meta-analysis study consisting of 1,538 samples from our group. At least 5 distinct gene-expression based molecular subtypes (GEMS) of EOC have been identified. The C5 subtype from the Tothill dataset corresponds to the Proliferative subtype from the TCGA dataset and the Stem-A subtype from the 1,538 meta-analysis dataset. These GEMS have been correlated with patient survival. The C5/Proliferative/Stem-A GEMS is associated with poorer survival outcomes. From our own study, Stem-A OC cells tend to display preferential chemosensitivity towards microtubule depolymerizing agents affecting the plus-end dynamics of microtubule such as vincristine and vinorelbine. Interestingly, Stem-A as well as non-Stem-A cells are equally sensitive to microtubule stabilizing agents such as paclitaxel. The biology of this C5/Proliferative/Stem-A-subtyped OC is still elusive and requires in-depth study. Here, we demonstrate that a Wnt receptor Frizzled-7 (FZD7) is crucial for the aggressiveness of the C5/Proliferative/Stem-A GEMS. Silencing FZD7 in the Stem-A-subtyped OC induces decrease in cell proliferation and cell cycle arrest. Silencing FZD7 also induced compaction of OC cell colony and strengthening of actin cytoskeleton at cell-cell junctions. Stable FZD7 knockdown clones further demonstrate significant decrease in anoikis resistance, spheroid forming ability, and tumorigenesis in xenografts. We further show that these functional changes acts via the Wnt-planar cell polarity (PCP) pathway downstream of FZD7. A subset of PCP pathway genes is also enriched in Stem-A tumors. Transient silencing of three downstream PCP pathway genes, CELSR3, PRICKLE4, PTK7, gave rise to similar functional phenotypes as silencing FZD7. These FZD7-PCP silenced Stem-A OC cells also demonstrate enhanced sensitivity to the microtubule depolymerizing agent vinorelbine. We conclude that the FZD7-PCP pathway drives the aggressiveness of the C5/Proliferative/Stem-A-subtyped OC and can be potentially utilized as the marker to identify the Stem-A subgroup. Molecularly stratifying OC patients and targeting C5/Proliferative/Stem-A subtype with therapeutics against FZD7-PCP pathway would be a novel strategy for OC. Citation Format: Mohammad Asad, Meng Kang Wong, Tuan Zea Tan, David Virshup, Jean Paul Thiery, Ruby Yun-Ju Huang. Frizzled-7 (FZD7)-mediated non-canonical Wnt-Planar Cell Polarity (PCP) signalling pathway as a novel molecular driver for the C5/Proliferative/Stem-A molecular subtype of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A30.

Autocrine Signaling by Wnt-5a Deregulates Chemotaxis of Leukemic Cells and Predicts Clinical Outcome in Chronic Lymphocytic Leukemia.

ROR1, a receptor in the noncanonical Wnt/planar cell polarity (PCP) pathway, is upregulated in malignant B cells of chronic lymphocytic leukemia (CLL) patients. It has been shown that the Wnt/PCP pathway drives pathogenesis of CLL, but which factors activate the ROR1 and PCP pathway in CLL cells remains unclear. B lymphocytes from the peripheral blood of CLL patients were negatively separated using RosetteSep (StemCell) and gradient density centrifugation. Relative expression of WNT5A, WNT5B, and ROR1 was assessed by quantitative real-time PCR. Protein levels, protein interaction, and downstream signaling were analyzed by immunoprecipitation and Western blotting. Migration capacity of primary CLL cells was analyzed by the Transwell migration assay. By analyzing the expression in 137 previously untreated CLL patients, we demonstrate that WNT5A and WNT5B genes show dramatically (five orders of magnitude) varying expression in CLL cells. High WNT5A and WNT5B expression strongly associates with unmutated IGHV and shortened time to first treatment. In addition, WNT5A levels associate, independent of IGHV status, with the clinically worst CLL subgroups characterized by dysfunctional p53 and mutated SF3B1. We provide functional evidence that WNT5A-positive primary CLL cells have increased motility and attenuated chemotaxis toward CXCL12 and CCL19 that can be overcome by inhibitors of Wnt/PCP signaling. These observations identify Wnt-5a as the crucial regulator of ROR1 activity in CLL and suggest that the autocrine Wnt-5a signaling pathway allows CLL cells to overcome natural microenvironmental regulation.

Abstract POSTER-THER-1403: FZD7 drives aggressiveness in stem-A subtype of ovarian cancer via regulation of non-canonical Wnt/PCP pathway

Abstract Ovarian cancer (OC) can be classified into five biologically distinct molecular subgroups—Epi-A, Epi-B, Mes, Stem-A and Stem-B. Among them, Stem-A expresses genes relating to stemness and is correlated with poor clinical prognosis. In this study, we show that FZD7, a receptor for Wnt signalling, is overexpressed in the Stem-A subgroup. To elucidate the functional roles of FZD7, we used an RNA interference (RNAi) gene knockdown approach in three Stem-A cell lines: CH1, PA1 and OV-17R. Si-FZD7 OC cells showed reduced cell proliferation with an increase in the G0/G1 subpopulation, with no effect on apoptosis. The cells also displayed a distinctive morphological change by colony compaction to become more epithelial-like and polarised with smaller inter-nuclear distances and increased z-axis height. Immunofluorescence (IF) staining patterns of pan-cadherin and β-catenin suggested an increase in cadherin-based cell-cell adhesion in si-FZD7 cells. We also observed a significant rearrangement in the actin cytoskeleton and an increase in tensile contractility in si-FZD7 OC cells, as evident by the loss of stress fibres and the redistribution of phospho-myosin light chain (pMLC) from the sites of cell-cell contacts to the periphery of cell colonies. Furthermore, there was reciprocal regulation of RhoA and Rac1 activities upon FZD7 knockdown, with a significant reduction in RhoA activity and a concomitant up-regulation in Rac1 activity. These changes in pMLC and RhoA as well as the increased TOPFLASH reporter activities in si-FZD7 cells suggested involvement of the non-canonical Wnt/planar cell polarity (PCP) pathway. Selected PCP pathway genes (CELSR3, PRICKLE4, DAAM1, PFN2, PCDH9, PCDHA1, PCDHB17, PCDHB3, SPRY1, and PTK7) were found to be more highly expressed in Stem-A than non Stem-A subgroup of OC. Taken together, our results suggest that FZD7 might drive aggressiveness in Stem-A OC by regulating cell proliferation, cell cycle progression, maintenance of the mesenchymal phenotype, and cell migration via CK1ϵ-mediated non-canonical Wnt/PCP pathway. Citation Format: Mohammad Asad, Wong Meng Kang, Tan Tuan Zea, Mahesh Choolani, Jeffery Low, Seiichi Mori, Jean Paul Thiery, Ruby Yun-Ju Huang. FZD7 drives aggressiveness in stem-A subtype of ovarian cancer via regulation of non-canonical Wnt/PCP pathway [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1403.

Asymmetry of VANGL2 in migrating lymphocytes as a tool to monitor activity of the mammalian WNT/planar cell polarity pathway.

BackgroundThe WNT/planar-cell-polarity (PCP) pathway is a key regulator of cell polarity and directional cell movements. Core PCP proteins such as Van Gogh-like2 (VANGL2) are evolutionarily highly conserved; however, the mammalian PCP machinery is still poorly understood mainly due to lack of suitable models and quantitative methodology. WNT/PCP has been implicated in many human diseases with the most distinguished positive role in the metastatic process, which accounts for more than 90% of cancer related deaths, and presents therefore an attractive target for pharmacological interventions. However, cellular assays for the assessment of PCP signaling, which would allow a more detailed mechanistic analysis of PCP function and possibly also high throughput screening for chemical compounds targeting mammalian PCP signaling, are still missing.ResultsHere we describe a mammalian cell culture model, which correlates B lymphocyte migration of patient-derived MEC1 cells and asymmetric localization of fluorescently-tagged VANGL2. We show by live cell imaging that PCP proteins are polarized in MEC1 cells and that VANGL2 polarization is controlled by the same mechanism as in tissues i.e. it is dependent on casein kinase 1 activity. In addition, destruction of the actin cytoskeleton leads to migratory arrest and cell rounding while VANGL2-EGFP remains polarized suggesting that active PCP signaling visualized by polarized distribution of VANGL2 is a cause for and not a consequence of the asymmetric shape of a migrating cell.ConclusionsThe presented imaging-based methodology allows overcoming limitations of earlier approaches to study the mammalian WNT/PCP pathway, which required in vivo models and analysis of complex tissues. Our system investigating PCP-like signaling on a single-cell level thus opens new possibilities for screening of compounds, which control asymmetric distribution of proteins in the PCP pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0079-1) contains supplementary material, which is available to authorized users.

Planar cell polarity aligns osteoblast division in response to substrate strain.

Exposure of bone to dynamic strain increases the rate of division of osteoblasts and also influences the directional organization of the cellular and molecular structure of the bone tissue that they produce. Here, we report that brief exposure to dynamic substrate strain (sufficient to rapidly stimulate cell division) influences the orientation of osteoblastic cell division. The initial proliferative response to strain involves canonical Wnt signaling and can be blocked by sclerostin. However, the strain-related orientation of cell division is independently influenced through the noncanonical Wnt/planar cell polarity (PCP) pathway. Blockade of Rho-associated coiled kinase (ROCK), a component of the PCP pathway, prevents strain-related orientation of division in osteoblast-like Saos-2 cells. Heterozygous loop-tail mutation of the core PCP component van Gogh-like 2 (Vangl2) in mouse osteoblasts impairs the orientation of division in response to strain. Examination of bones from Vangl2 loop-tail heterozygous mice by µCT and scanning electron microscopy reveals altered bone architecture and disorganized bone-forming surfaces. Hence, in addition to the well-accepted role of PCP involvement in response to developmental cues during skeletal morphogenesis, our data reveal that this pathway also acts postnatally, in parallel with canonical Wnt signaling, to transduce biomechanical cues into skeletal adaptive responses. The simultaneous and independent actions of these two pathways appear to influence both the rate and orientation of osteoblast division, thus fine-tuning bone architecture to meet the structural demands of functional loading. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

FZD7 drives in vitro aggressiveness in Stem-A subtype of ovarian cancer via regulation of non-canonical Wnt/PCP pathway

Ovarian cancer (OC) can be classified into five biologically distinct molecular subgroups: epithelial-A (Epi-A), Epi-B, mesenchymal (Mes), Stem-A and Stem-B. Among them, Stem-A expresses genes relating to stemness and is correlated with poor clinical prognosis. In this study, we show that frizzled family receptor 7 (FZD7), a receptor for Wnt signalling, is overexpressed in the Stem-A subgroup. To elucidate the functional roles of FZD7, we used an RNA interference gene knockdown approach in three Stem-A cell lines: CH1, PA1 and OV-17R. Si-FZD7 OC cells showed reduced cell proliferation with an increase in the G0/G1 sub-population, with no effect on apoptosis. The cells also displayed a distinctive morphologic change by colony compaction to become more epithelial-like and polarised with smaller internuclear distances and increased z-axis height. Immunofluorescence (IF) staining patterns of pan-cadherin and β-catenin suggested an increase in cadherin-based cell–cell adhesion in si-FZD7 cells. We also observed a significant rearrangement in the actin cytoskeleton and an increase in tensile contractility in si-FZD7 OC cells, as evident by the loss of stress fibres and the redistribution of phospho-myosin light chain (pMLC) from the sites of cell–cell contacts to the periphery of cell colonies. Furthermore, there was reciprocal regulation of RhoA (Ras homolog family member A) and Rac1 (Ras-related C3 botulinum toxin substrate 1 (Rho family, small GTP-binding protein Rac1)) activities upon FZD7 knockdown, with a significant reduction in RhoA activity and a concomitant upregulation in Rac1 activity. These changes in pMLC and RhoA, as well as the increased TopFlash reporter activities in si-FZD7 cells, suggested involvement of the non-canonical Wnt/planar cell polarity (PCP) pathway. Selected PCP pathway genes (cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3), prickle homolog 4 (Drosophila) (PRICKLE4), dishevelled-associated activator of morphogenesis 1 (DAAM1), profilin 2 (PFN2), protocadherin 9 (PCDH9), protocadherin α1 (PCDHA1), protocadherin β17 pseudogene (PCDHB17), protocadherin β3 (PCDHB3), sprouty homolog 1 (SPRY1) and protein tyrosine kinase 7 (PTK7)) were found to be more highly expressed in Stem-A than non Stem-A subgroup of OC. Taken together, our results suggest that FZD7 might drive aggressiveness in Stem-A OC by regulating cell proliferation, cell cycle progression, maintenance of the Mes phenotype and cell migration via casein kinase 1ɛ-mediated non-canonical Wnt/PCP pathway.

Prickle1 is necessary for the caudal migration of murine facial branchiomotor neurons.

Facial branchiomotor neurons (FBMs) of vertebrates typically develop in rhombomere 4 (r4), and in mammals and several other vertebrate taxa, migrate caudally into r6 and subsequently laterally and ventrally to the pial surface. How similar or dissimilar these migratory processes between species are at a molecular level remains unclear. In zebrafish and mouse, mutations in certain PCP genes disrupt normal caudal migration of FBMs. Zebrafish prickle1a (prickle-like 1a) and prickle1b, two orthologs of Prickle1, act non-cell-autonomously and cell-autonomously, respectively, to regulate FBM migration. Here, we show that, in Prickle1 (C251X/C251X) mice which have reduced Prickle1 expression, the caudal migration of FBMs is affected. Most FBM neurons do not migrate caudally along the floor plate. However, some neurons perform limited caudal migration such that the neurons eventually lie near the pial surface from r4 to anterior r6. FBMs in Prickle1 (C251X/C251X) mice survive until P0 and form an ectopic nucleus dorsal to the olivo-cochlear efferents of r4. Ror2, which modifies the PCP pathway in other systems, is expressed by the migrating mouse FBMs, but is not required for FBM caudal migration. Our results suggest that, in mice, Prickle1 is part of a molecular mechanism that regulates FBM caudal migration and separates the FBM and the olivo-cochlear efferents. This defective caudal migration of FBMs in Prickle1C251X mutants resembles Vangl2 mutant defects. In contrast to other developing systems that show similar defects in Prickle1, Wnt5a and Ror2, the latter two only have limited or no effect on FBM caudal migration.

Atmin mediates kidney morphogenesis by modulating Wnt signaling.

The DNA damage protein and transcription factor Atmin (Asciz) is required for both lung tubulogenesis and ciliogenesis. Like the lungs, kidneys contain a tubular network that is critical for their function and in addition, renal ciliary dysfunction has been implicated in the pathogenesis of cystic kidney disease. Using the Atmin mouse mutant Gasping6 (Gpg6), we investigated kidney development and found it severely disrupted with reduced branching morphogenesis, resulting in fewer epithelial structures being formed. Unexpectedly, transcriptional levels of key cilia associated genes were not altered in AtminGpg6/Gpg6 kidneys. Instead, Gpg6 homozygous kidneys exhibited altered cytoskeletal organization and modulation of Wnt signaling pathway molecules, including β-catenin and non-canonical Wnt/planar cell polarity (PCP) pathway factors, such as Daam2 and Vangl2. Wnt signaling is important for kidney development and perturbation of Wnt signaling pathways can result in cystic, and other, renal abnormalities. In common with other PCP pathway mutants, AtminGpg6/Gpg6 mice displayed a shortened rostral-caudal axis and mis-oriented cell division. Moreover, intercrosses between AtminGpg6/+ and Vangl2Lp/+ mice revealed a genetic interaction between Atmin and Vangl2. Thus we show for the first time that Atmin is critical for normal kidney development and we present evidence that mechanistically, Atmin modifies Wnt signaling pathways, specifically placing it as a novel effector molecule in the non-canonical Wnt/PCP pathway. The identification of a novel modulator of Wnt signaling has important implications for understanding the pathobiology of renal disease.

SEC14 and Spectrin Domains 1 (Sestd1), Dishevelled 2 (Dvl2) and Dapper Antagonist of Catenin-1 (Dact1) co-regulate the Wnt/Planar Cell Polarity (PCP) pathway during mammalian development

We previously reported that Sestd1 KO phenocopies Dact1 KO in mice, consistent with a model in which Sestd1 and Dact1 act together to form a crucial functional complex that regulates Vangl2 in the Wnt/Planar Cell Polarity (PCP) pathway. Here, we show that Dvl2, a binding partner of Dact1, also forms complexes with Sestd1, and does so independently of both Dact1 and Vangl2. In cell-based assays, whereas Sestd1 does not alter Dvl2 activation of the Wnt/β-catenin signaling pathway, Dvl2 enhances activation of Rho family GTPases by Dact1 and Sestd1, consistent with a role in the PCP pathway. In mice, although Dvl2 KO is recessive in an otherwise wild type background, it leads to dominant embryonic lethality in either the Sestd1 or Dact1 KO background. This genetic synergy stands in contrast to the epistasis we have previously reported between Sestd1 and Dact1 KO, and suggests independent or semi-independent functions for Dvl2 vs. Sestd1/Dact1 in the regulation of the PCP pathway during development. In conclusion, biochemical and genetic interactions between Dvl2, Sestd1, and Dact1, in addition to prior reported interactions between these same molecules and Vangl2, suggest that all these gene products can form complexes together and regulate the PCP pathway during mammalian development. However, Sestd1 and Dact1 have a closely allied function in the post-translational regulation of Vangl2 that is at least partially distinct from the functions of Dvl2 in this pathway.

Prickle1 stunts limb growth through alteration of cell polarity and gene expression

Wnt/PCP signaling plays a critical role in multiple developmental processes, including limb development. Wnt5a, a ligand of the PCP pathway, signals through the Ror2/Vangl2 or the Vangl2/Ryk complex to regulate limb development along the proximal-distal axis in mice. Based on the interaction between Van Gogh and Prickle in Drosophila, we hypothesized the vertebrate Prickle1 has a similar function as Vangl2 in limb development. We show Prickle1 is expressed in the skeletal condensates that will differentiate into chondrocytes and later form bones. Disrupted Prickle1 function in Prickle1(C251X/C251X) mouse mutants alters expression of genes such as Bmp4, Fgf8, Vangl2, and Wnt5a. These expression changes correlate with shorter and wider bones in the limbs and loss of one phalangeal segment in digits 2-5 of Prickle1C251X mutants. These growth defects along the proximal-distal axis are also associated with increased cell death in the growing digit tip, reduced cell death in the interdigital membrane, and disrupted chondrocyte polarity. We suggest Prickle1 is part of the Wnt5a/PCP signaling, regulating cell polarity and affecting expression of multiple factors to stunt limb growth through altered patterns of gene expression, including the PCP genes Wnt5a and Vangl2.

Planar Cell Polarity Pathway Regulates Nephrin Endocytosis in Developing Podocytes

The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/β-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate.