Planar Cell Polarity Signaling Research Articles

Identification of novel rare mutations of DACT1 in human neural tube defects

Neural tube defects (NTDs) constitute the second most frequent cause of human congenital abnormalities. Complex multigenetic causes have been suggested to contribute to NTDs. The planar cell polarity (PCP) pathway plays a critical role in neural tube closure in model organisms and in human. Knockout of Dact1 (Dapper, Frodo) leads to deregulated PCP signaling with defective neural tube in mice. Here, we report that five missense heterozygote mutations of the DACT1 gene are specifically identified in 167 stillborn or miscarried Han Chinese fetuses with neural tube defects. Our biochemical analyses revealed that among the five mutations, N356K and R45W show loss-of-function or reduced activities in inducing Dishevelled2 (DVL2) degradation and inhibiting jun-N-terminal kinase (JNK) phosphorylation, implicating mutated DACT1 as a risk factor for human NTDs. Our findings, together with early reports, suggest that rare mutations of the PCP-related genes may constitute a great contribution to human NTDs.

Activation of Wnt Planar cell polarity (PCP) signaling promotes growth plate column formation in vitro

Disrupting the Wnt Planar Cell Polarity (PCP) signaling pathway in vivo results in loss of columnar growth plate architecture, but it is unknown whether activation of this pathway in vitro is sufficient to promote column formation. We hypothesized that activation of the Wnt PCP pathway in growth plate chondrocyte cell pellets would promote columnar organization in these cells that are normally oriented randomly in culture. Rat growth plate chondrocytes were transfected with plasmids encoding the Fzd7 cell-surface Wnt receptor, a Fzd7 deletion mutant lacking the Wnt-binding domain, or Wnt receptor-associated proteins Ror2 or Vangl2, and then cultured as three-dimensional cell pellets in the presence of recombinant Wnt5a or Wnt5b for 21 days. Cellular morphology was evaluated using histomorphometric measurements. Activation of Wnt PCP signaling components promoted the initiation of columnar morphogenesis in the chondrocyte pellet culture model, as measured by histomorphometric analysis of the column index (ANOVA p = 0.01). Activation of noncanonical Wnt signaling through overexpression of both the cell-surface Wnt receptor Fzd7 and receptor-associated protein Ror2 with addition of recombinant Wnt5a promotes the initiation of columnar architecture of growth plate chondrocytes in vitro, representing an important step toward growth plate regeneration.

Neural tube defects—disorders of neurulation and related embryonic processes

Neural tube defects (NTDs) are severe congenital malformations affecting 1 in every 1000 pregnancies. 'Open' NTDs result from failure of primary neurulation as seen in anencephaly, myelomeningocele (open spina bifida), and craniorachischisis. Degeneration of the persistently open neural tube in utero leads to loss of neurological function below the lesion level. 'Closed' NTDs are skin-covered disorders of spinal cord structure, ranging from asymptomatic spina bifida occulta to severe spinal cord tethering, and usually traceable to disruption of secondary neurulation. 'Herniation' NTDs are those in which meninges, with or without brain or spinal cord tissue, become exteriorized through a pathological opening in the skull or vertebral column (e.g., encephalocele and meningocele). NTDs have multifactorial etiology, with genes and environmental factors interacting to determine individual risk of malformation. While over 200 mutant genes cause open NTDs in mice, much less is known about the genetic causation of human NTDs. Recent evidence has implicated genes of the planar cell polarity signaling pathway in a proportion of cases. The embryonic development of NTDs is complex, with diverse cellular and molecular mechanisms operating at different levels of the body axis. Molecular regulatory events include the bone morphogenetic protein and Sonic hedgehog pathways which have been implicated in control of neural plate bending. Primary prevention of NTDs has been implemented clinically following the demonstration that folic acid (FA), when taken as a periconceptional supplement, can prevent many cases. Not all NTDs respond to FA, however, and adjunct therapies are required for prevention of this FA-resistant category.

PTK7 Regulates Myosin II Activity to Orient Planar Polarity in the Mammalian Auditory Epithelium

Planar cell polarity (PCP) signaling is a key regulator of epithelial morphogenesis, including neural tube closure and the orientation of inner ear sensory hair cells, and is mediated by a conserved noncanonical Wnt pathway. Ptk7 is a novel vertebrate-specific regulator of PCP, yet the mechanisms by which Ptk7 regulates mammalian epithelial PCP remain poorly understood. Here we show that, in the mammalian auditory epithelium, Ptk7 is not required for membrane recruitment of Dishevelled 2; Ptk7 and Frizzled3/Frizzled6 receptors act in parallel and have opposing effects on hair cell PCP. Mosaic analysis identified a requirement of Ptk7 in neighboring supporting cells for hair cell PCP. Ptk7 and the noncanonical Wnt pathway differentially regulate a contractile myosin II network near the apical surface of supporting cells. We provide evidence that this apical myosin II network exerts polarized contractile tension on hair cells to align their PCP, as revealed by asymmetric junctional recruitment of vinculin, a tension-sensitive actin binding protein. In Ptk7 mutants, compromised myosin II activity resulted in loss of planar asymmetry and reduced junctional localization of vinculin. By contrast, vinculin planar asymmetry and stereociliary bundle orientation were restored in Fz3(-/-);Ptk7(-/-) double mutants. These findings suggest that PTK7 acts in conjunction with the noncanonical Wnt pathway to orient epithelial PCP through modulation of myosin II-based contractile tension between supporting cells and hair cells.

Planar Cell Polarity Links Axes of Spatial Dynamics in Neural-Tube Closure

Neural-tube closure is a critical step of embryogenesis, and its failure causes serious birth defects. Coordination of two morphogenetic processes--convergent extension and neural-plate apical constriction--ensures the complete closure of the neural tube. We now provide evidence that planar cell polarity (PCP) signaling directly links these two processes. In the bending neural plates, we find that a PCP-regulating cadherin, Celsr1, is concentrated in adherens junctions (AJs) oriented toward the mediolateral axes of the plates. At these AJs, Celsr1 cooperates with Dishevelled, DAAM1, and the PDZ-RhoGEF to upregulate Rho kinase, causing their actomyosin-dependent contraction in a planar-polarized manner. This planar-polarized contraction promotes simultaneous apical constriction and midline convergence of neuroepithelial cells. Together our findings demonstrate that PCP signals confer anisotropic contractility on the AJs, producing cellular forces that promote the polarized bending of the neural plate.

Drosophila CK1-γ, gilgamesh, controls PCP-mediated morphogenesis through regulation of vesicle trafficking

Cellular morphogenesis, including polarized outgrowth, promotes tissue shape and function. Polarized vesicle trafficking has emerged as a fundamental mechanism by which protein and membrane can be targeted to discrete subcellular domains to promote localized protrusions. Frizzled (Fz)/planar cell polarity (PCP) signaling orchestrates cytoskeletal polarization and drives morphogenetic changes in such contexts as the vertebrate body axis and external Drosophila melanogaster tissues. Although regulation of Fz/PCP signaling via vesicle trafficking has been identified, the interplay between the vesicle trafficking machinery and downstream terminal PCP-directed processes is less established. In this paper, we show that Drosophila CK1-γ/gilgamesh (gish) regulates the PCP-associated process of trichome formation through effects on Rab11-mediated vesicle recycling. Although the core Fz/PCP proteins dictate prehair formation broadly, CK1-γ/gish restricts nucleation to a single site. Moreover, CK1-γ/gish works in parallel with the Fz/PCP effector multiple wing hairs, which restricts prehair formation along the perpendicular axis to Gish. Our findings suggest that polarized Rab11-mediated vesicle trafficking regulated by CK1-γ is required for PCP-directed processes.

Role of the planar cell polarity gene CELSR1 in neural tube defects and caudal agenesis

Neural tube defects (NTDs), including anencephaly and spina bifida, have a complex etiology. Defects in the planar cell polarity (PCP) signaling pathway have been strongly associated with NTDs in animal models and human cohorts. In this genetic study, we examined the core PCP gene CELSR1 in NTDs and caudal agenesis cases to determine whether mutations at this gene predispose to these defects. We sequenced the coding region and the exon-intron junctions of CELSR1 in a cohort of 473 patients affected with various forms of open and closed NTDs (412) or caudal agenesis (61). Novel and rare variants (<1%) were genotyped in a cohort of 639 ethnically-matched individuals. The effect of novel missense mutations absent in controls and in public databases on protein function was predicted in silico. We identified in our cohort one nonsense mutation in exon 1 of CELSR1 that truncates the majority of the protein in one patient with NTD and one in-frame 12 bp deletion that removes a putative PKC phosphorylation "SSR" motif in one caudal agenesis patient. We also detected a total of 13 novel missense variants in 12 patients (11 NTDs and 1 caudal agenesis) that were predicted to be pathogenic in silico. We detected novel CELSR1 mutations predicted to be pathogenic in 2.9% of our NTD cohort and 3.3% of our caudal agenesis cohort. Our findings implicate CELSR1 as a risk factor for NTDs or caudal agenesis and provide additional evidence for a pathogenic role of PCP signaling in these malformations.

Multi-cellular rosettes in the mouse visceral endoderm facilitate the ordered migration of anterior visceral endoderm cells.

Author SummaryThe mouse visceral endoderm (VE) is a simple epithelium in the egg cylinder stage mouse embryo. Many functions associated with epithelia require them to undergo extensive remodelling through changes in the shape and relative positions of constituent cells, a process about which we understand relatively little. The anterior visceral endoderm (AVE) is a specialized group of cells in the simple epithelium of the VE, and their stereotypic migratory behaviour is essential for establishing the orientation of the anterior-posterior axis in the early mouse embryo. We show that AVE migration is linked to changes in cell packing in the VE and an increase in “rosettes,” which are striking collections of five or more cells meeting at a central point. To probe the role of rosettes during AVE migration, we have developed a mathematical model of cell movement in the VE. Simulations suggest that rosettes are not essential for AVE migration, but are crucial for the orderliness of this migration. We also explored the role of Planar Cell Polarity (PCP) signalling, which is known to coordinate cell polarization and rearrangement in many different tissues. We find that mutants in which PCP signalling is disrupted have fewer rosettes, altered epithelial packing, and abnormal AVE migration. We suggest that rosettes in the mouse VE are a PCP-dependent arrangement of cells that act to buffer the disturbances in cell packing generated by AVE migration, thereby enabling AVE cells to migrate in an orderly manner.

Planar cell polarity signaling: coordination of cellular orientation across tissues

Establishment of Planar Cell Polarity (PCP) in epithelia, in the plane of an epithelium, is an important feature of the development and homeostasis of most organs. Studies in different model organisms have contributed a wealth of information regarding the mechanisms that govern PCP regulation. Genetic studies in Drosophila have identified two signaling systems, the Fz/PCP and Fat/Dachsous system, which are both required for PCP establishment in many different tissues in a largely non-redundant manner. Recent advances in vertebrate PCP studies have added novel factors of PCP regulation and also new cellular features requiring PCP-signaling input, including the positioning and orientation of the primary cilium of many epithelial cells. This review focuses mostly on several recent advances made in the Drosophila and vertebrate PCP field and integrates these within the existing PCP-signaling framework.

Patterning skin by planar cell polarity: the multi‐talented hair designer

In mammals, the skin can form complex global and local patterns to meet diverse functional requirements in different parts of the body. To date, the fundamental principles that underlie skin patterning remain poorly understood because of the involvement of multiple interacting processes. Genes involved in the planar cell polarity (PCP) signalling pathway, which is capable of polarizing cells within the planar plane of an epithelium, can control the orientation and differentiation of hair follicles, underlining their involvement in skin pattern formation. Here, we summarize recent progress that has been made to understand the PCP signalling pathway and its function in mammalian skin, including its role in hair follicle morphogenesis, ciliogenesis and wound healing. We argue that dissecting PCP signalling in the context of hair follicle formation might reveal many as-yet-undiscovered functions for PCP in the development, homeostasis and regeneration of skin.

VANGL2 regulates membrane trafficking of MMP14 to control cell polarity and migration

Planar cell polarity (PCP) describes the polarized orientation of cells within the plane of a tissue. Unlike epithelial PCP, the mechanisms underlying PCP signaling in migrating cells remain undefined. Here, the establishment of PCP must be coordinated with dynamic changes in cell adhesion and extracellular matrix (ECM) organization. During gastrulation, the membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) is required for PCP and convergence and extension cell movements. We report that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell-surface availability of MMP14 in manner that is dependent on focal adhesion kinase. We demonstrate that zebrafish trilobite/vangl2 mutant embryos exhibit increased Mmp14 activity and decreased ECM. Furthermore, in vivo knockdown of Mmp14 partially rescues the Vangl2 loss-of-function convergence and extension phenotype. This study identifies a mechanism linking VANGL2 with MMP14 trafficking and suggests that establishment of PCP in migrating gastrula cells requires regulated proteolytic degradation or remodeling of the ECM. Our findings implicate matrix metalloproteinases as downstream effectors of PCP and suggest a broadly applicable mechanism whereby VANGL2 affects diverse morphogenetic processes.

Inversin, Wnt signaling and primary cilia

Mutations of the ankyrin-repeat protein Inversin, a member of a diverse family of more than 12 proteins, cause nephronophthisis (NPH), an autosomal recessive cystic kidney disease associated with extra-renal manifestations such as retinitis pigmentosa, cerebellar aplasia and situs inversus. Most NPH gene products (NPHPs) localize to the cilium, and appear to control the transport of cargo protein to the cilium by forming functional networks. Inversin interacts with NPHP1 and NPHP3, and shares with NPHP4 the ability to antagonize Dishevelled-stimulated canonical Wnt signaling, potentially through recruitment of the Anaphase Promoting Complex (APC/C). However, Dishevelled antagonism may be confined towards the basal body, thereby polarizing motile cilia on the cells of the ventral node and respiratory tract. Inversin is essential for recruiting Dishevelled to the plasma membrane in response to activated Frizzled, a crucial step in planar cell polarity signaling. During vertebrate pronephros development, the Inversin-mediated translocation of Dishevelled appears to orchestrate the migration of cells and differentiation of segments that correspond to the mammalian loop of Henle. Thus, defective tubule migration and elongation may contribute to concentration defects and cause cyst formation in patients with NPH.

Mutations in the planar cell polarity genes CELSR1 and SCRIB are associated with the severe neural tube defect craniorachischisis.

Craniorachischisis (CRN) is a severe neural tube defect (NTD) resulting from failure to initiate closure, leaving the hindbrain and spinal neural tube entirely open. Clues to the genetic basis of this condition come from several mouse models, which harbor mutations in core members of the planar cell polarity (PCP) signaling pathway. Previous studies of humans with CRN failed to identify mutations in the core PCP genes, VANGL1 and VANGL2. Here, we analyzed other key PCP genes: CELSR1, PRICKLE1, PTK7, and SCRIB, with the finding of eight potentially causative mutations in both CELSR1 and SCRIB. Functional effects of these unique or rare human variants were evaluated using known protein-protein interactions as well as subcellular protein localization. While protein interactions were not affected, variants from five of the 36 patients exhibited a profound alteration in subcellular protein localization, with diminution or abolition of trafficking to the plasma membrane. Comparable effects were seen in the crash and spin cycle mouse Celsr1 mutants, and the line-90 mouse Scrib mutant. We conclude that missense variants in CELSR1 and SCRIB may represent a cause of CRN in humans, as in mice, with defective PCP protein trafficking to the plasma membrane a likely pathogenic mechanism.

Using 32-cell stage Xenopus embryos to probe PCP signaling.

Use of loss-of function (via antisense Morpholino oligonucleotides (MOs)) or over-expression of proteins in epithelial cells during early embryogenesis of Xenopus embryos, can be a powerful tool to understand how signaling molecules can affect developmental events. The techniques described here are useful for examining the roles of proteins in cell-cell adhesion, and planar cell polarity (PCP) signaling in cell movement. We describe how to target specific regions within the embryos by injecting an RNA encoding a tracer molecule along with RNA encoding your protein of interest or an antisense MO to knock-down a particular protein within a specific blastomere of the embryo. Effects on cell-cell adhesion, cell movement, and endogenous or exogenous protein localization can be assessed at later stages in specific targeted tissues using fluorescent microscopy and immunolocalization.

Waif1/5T4 Inhibits Wnt/β-Catenin Signaling and Activates Noncanonical Wnt Pathways by Modifying LRP6 Subcellular Localization

Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/β-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits β-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances β-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.

Tagged Fibrocystin Sheds Its Secrets

Tagged Fibrocystin Sheds Its Secrets

The Prorenin Receptor

The discovery of the prorenin receptor, now nearly a decade ago, altered our view of the renin-angiotensin system (RAS).[1][1],[2][2] Binding of prorenin, the inactive precursor of renin, to the prorenin receptor results in full catalytic activity of prorenin through a nonproteolytic mechanism that

When It Is Hard to Get to with Genetics—Planar Cell Polarity under a Chemical Scalpel

Planar cell polarity (PCP) has been under genetic dissection for decades. More and more fundamental developmental processes have been found relying on PCP signaling. However, mechanisms of how PCP signaling generates asymmetry is still unknown. A recent paper in Chemistry & Biology (Sundberg etal., 2011) represents the efforts to decipher the intracellular code of polarity signaling.

Disruption of Wnt Planar Cell Polarity Signaling by Aberrant Accumulation of the MetAP-2 Substrate Rab37

Identification of methionine aminopeptidase-2 (MetAP-2) as the molecular target of the antiangiogenic compound TNP-470 has sparked interest in N-terminal Met excision's (NME) role in endothelial cell biology. In this regard, we recently demonstrated that MetAP-2 inhibition suppresses Wnt planar cell polarity (PCP) signaling and that endothelial cells depend on this pathway for normal function. Despite this advance, the substrate(s) whose activity is altered upon MetAP-2 inhibition, resulting in loss of Wnt PCP signaling, is not known. Here we identify the small G protein Rab37 as a MetAP-2-specific substrate that accumulates in the presence of TNP-470. A functional role for aberrant Rab37 accumulation in TNP-470's mode of action is demonstrated using a Rab37 point mutant that is resistant to NME, because expression of this mutant phenocopies the effects of MetAP-2 inhibition on Wnt PCP signaling-dependent processes.

Functional dissection of phosphorylation of Disheveled in Drosophila

Disheveled/Dsh proteins (Dvl in mammals) are core components of both Wnt/Wg-signaling pathways: canonical β-catenin signaling and Frizzled (Fz)-planar cell polarity (PCP) signaling. Although Dsh is a key cytoplasmic component of both Wnt/Fz-pathways, regulation of its signaling specificity is not well understood. Dsh is phosphorylated, but the functional significance of its phosphorylation remains unclear. We have systematically investigated the phosphorylation of Dsh by combining mass-spectrometry analyses, biochemical studies, and in vivo genetic methods in Drosophila. Our approaches identified multiple phospho-residues of Dsh in vivo. Our data define three novel and unexpected conclusions: (1) strikingly and in contrast to common assumptions, all conserved serines/threonines are non-essential for Dsh function in either pathway; (2) phosphorylation of conserved Tyrosine473 in the DEP domain is critical for PCP-signaling — Dsh Y473F behaves like a PCP-specific allele; and (3) defects associated with the PCP specific dsh 1 allele, Dsh K417M, located within a putative Protein Kinase C consensus site, are likely due to a post-translational modification requirement of Lys417, rather than phosphorylation nearby. In summary, our combined data indicate that while many Ser/Thr and Tyr residues are indeed phosphorylated in vivo, strikingly most of these phosphorylation events are not critical for Dsh function with the exception of DshY473.