De-Li Shi
email: de-li.shi@upmc.fr

Wnt/planar cell polarity signaling controls morphogenetic movements of gastrulation and neural tube closure

Keywords: Wnt, PCP, Frizzled, Dishevelled, Vangl1, Vangl2, Prickle, Celsr1, morphogenesis, NTDs, neural tube defects

Gastrulation and neurulation are successive morphogenetic processes that play key roles in shaping the basic embryonic body plan. Importantly, they operate through common cellular and molecular mechanisms to set up the three spatially organized germ layers and to close the neural tube. During gastrulation and neurulation, convergent extension movements driven by cell intercalation and oriented cell division generate major forces to narrow the germ layers along the mediolateral axis and elongate the embryo in the anteroposterior direction. Apical constriction also makes an important contribution to promote the formation of the blastopore and the bending of the neural plate. Planar cell polarity proteins are major regulators of asymmetric cell behaviors and critically involved in a wide variety of developmental processes, from gastrulation and neurulation to organogenesis. Mutations of planar cell polarity genes can lead to general defects in the morphogenesis of different organs and the co-existence of distinct congenital diseases, such as spina bifida, hearing deficits, kidney diseases, and limb elongation defects. This review outlines our current understanding of non-canonical Wnt signaling, commonly known as Wnt/planar cell polarity signaling, in regulating morphogenetic movements of gastrulation and neural tube closure during development and disease. It also attempts to identify unanswered questions that deserve further investigations.

Introduction

Morphogenetic movements of gastrulation set up the three spatially organized germ layers (ectoderm, mesoderm and endoderm). Neurulation occurs directly after gastrulation to transform the neural plate into a neural tube, the precursor of the central nervous system. Therefore, gastrulation and neurulation are fundamental developmental events that play critical roles in shaping the basic embryonic body plan. Moreover, they operate through common as well as conserved cellular and molecular mechanisms. Convergent extension (CE) movements are mostly driven by cell intercalations that promote extensive exchange of places between neighboring cells [START_REF] Huang | Cell migration in the Xenopus gastrula[END_REF][START_REF] Keller | Convergent extension in the amphibian, Xenopus laevis[END_REF][START_REF] Sutherland | Convergent extension in mammalian morphogenesis[END_REF][START_REF] Tada | Convergent extension: using collective cell migration and cell intercalation to shape embryos[END_REF][START_REF] Yin | Convergence and extension movements during vertebrate gastrulation[END_REF]. While mediolateral intercalation occurs in a coplanar manner, radial intercalation takes place between adjacent planes. During these processes, lateral cells move toward the dorsal midline to narrow the germ layers and promote neural tube closure, while dorsal midline cells extend along the anteroposterior (AP) axis to lengthen the embryo [START_REF] Williams | Cellular and molecular mechanisms of convergence and extension in zebrafish[END_REF]. Other asymmetric cell behaviors, such as oriented cell division and apical constriction, also make an important contribution to epithelial morphogenesis during gastrulation and neurulation [START_REF] Baldwin | Assays for apical constriction using the Xenopus model[END_REF][START_REF] Gong | Planar cell polarity signalling controls cell division orientation during zebrafish gastrulation[END_REF][START_REF] Ciruna | Planar cell polarity signalling couples cell division and morphogenesis during neurulation[END_REF].

The phenomenon of planar cell polarity (PCP) is widely conserved among metazoan and is critically involved in coordinating cellular orientation within the plane of an epithelium or a tissue. In vertebrates, as in Drosophila, PCP is mostly regulated by a set of evolutionarily conserved proteins including Frizzled (Fzd), Dishevelled (Dvl), Celsr (cadherin EGF LAG sevenpass G-type receptor), Vangl, Prickle, and Ankrd6 (ankyrin repeat domain 6). These "core" PCP proteins form two separate complexes that localize to opposite cell borders [START_REF] Goodrich | Principles of planar polarity in animal development[END_REF][START_REF] Wallingford | Planar cell polarity and the developmental control of cell behavior in vertebrate embryos[END_REF][START_REF] Yang | Wnt-Frizzled/planar cell polarity signaling: cellular orientation by facing the wind (Wnt)[END_REF]. They transduce Wnt/PCP signaling to regulate cytoskeletal rearrangements through distinct downstream effectors [START_REF] Wallingford | Planar cell polarity and the developmental control of cell behavior in vertebrate embryos[END_REF][START_REF] Adler | From planar cell polarity to ciliogenesis and back: The curious tale of the PPE and CPLANE proteins[END_REF]. Several vertebrate Wnt ligands, such as Wnt5a, Wnt5b, and Wnt11, are also important for activating Wnt/PCP signaling to instruct cellular polarization, although they are generally not considered as "core" PCP proteins [START_REF] Gao | Wnt regulation of planar cell polarity (PCP)[END_REF]. Besides the "core" PCP pathway, there are also other conserved protein complexes that function as important PCP regulators. For example, the Fat and Dchs (dachsous cadherin-related) heteromeric protocadherins form ligand-receptor pairs and represent the second PCP pathway [START_REF] Blair | Big roles for Fat cadherins[END_REF]; the Scrib (Scrb1 or Scribble1) polarity system, consisting of Scrib, Dlg (Discs-large) and Lgl (Lethal-giant larvae) proteins in a complex, mostly contributes to establish baso-lateral cell identity [START_REF] Milgrom-Hoffman | Regulation of cellular and PCP signalling by the Scribble polarity module[END_REF].

The Wnt/PCP pathway functions in a wide variety of morphogenetic processes and instructs cells with positional cues for directed movements [START_REF] Koca | Wnt-frizzled planar cell polarity signaling in the regulation of cell motility[END_REF]. Particularly, Wnt/PCP signaling is required for cell intercalations during gastrulation, neurulation, and asymmetric organogenesis [START_REF] Williams | Cellular and molecular mechanisms of convergence and extension in zebrafish[END_REF][START_REF] Henderson | Planar cell polarity in organ formation[END_REF][START_REF] Matsuda | Xenopus neural tube closure: A vertebrate model linking planar cell polarity to actomyosin contractions[END_REF][START_REF] Shi | Planar cell polarity regulators in asymmetric organogenesis during development and disease[END_REF]. Although Wnt/PCP signaling mostly contributes to mediolateral cell intercalation [START_REF] Walck-Shannon | Cell intercalation from top to bottom[END_REF],

there is increasing evidence that several "core" PCP proteins also mediate radial cell intercalation and contribute to the establishment of apico-basal polarity [START_REF] Huang | Cell cortex regulation by the planar cell polarity protein Prickle1[END_REF][START_REF] Ossipova | The involvement of PCP proteins in radial cell intercalations during Xenopus embryonic development[END_REF][START_REF] Tao | Mouse prickle1, the homolog of a PCP gene, is essential for epiblast apical-basal polarity[END_REF]. Dysfunctions of PCP regulators (herein referred to as proteins involved in Wnt/PCP signaling) are closely linked to the congenital disorder of neural tube defects (NTDs), such as spina bifida and anencephaly [START_REF] Nikolopoulou | Neural tube closure: cellular, molecular and biomechanical mechanisms[END_REF][START_REF] Wang | Update on the role of the non-canonical Wnt/planar cell polarity pathway in neural tube defects[END_REF]. This review aims to provide an outline of past achievements and recent advances in deciphering the implication of Wnt/PCP signaling during gastrulation and neural tube closure, by focusing on molecular and cellular mechanisms underlying the regulatory functions of PCP-related genes in development and disease. As an outcome, it attempts to identify challenges in understanding the functional interaction between different polarity pathways as well as the interplay between cell polarity and cell fate specification during morphogenetic movements.

Planar cell polarity protein complexes

Vertebrate Wnt signaling pathways include three branches that trigger distinct biological outcomes. In canonical Wnt or Wnt/ß-catenin signaling, Wnt ligands bind to Fzd receptors and LRP5/6 (low density lipoprotein receptor-related protein 5/6) co-receptors, leading to phosphorylation of Dvl and inhibition of the ß-catenin destruction complex consisting of GSK3ß/Axin/APC. Stabilized ß-catenin associates with T-cell factor in the nucleus to activate transcription of target genes and regulate cell fate specification. Non-canonical Wnt pathways include Wnt/PCP and Wnt/Ca 2+ branches that signal independently of ß-catenin. Wnt/PCP signaling is activated by binding of non-canonical Wnt ligands to the Fzd-Ror1/2 (receptor tyrosine kinase-like orphan receptor 1/2) complex or the Fzd-Ryk (receptor tyrosine kinase) complex. As will be discussed later, there are also other co-receptors that can interact with noncanonical Wnt ligands. The Wnt/PCP pathway functions to regulate cytoskeletal organization through two classes of downstream effectors: the well-characterized planar polarity effector (PPE) proteins such as Daam1 (Dishevelled-associated activator of morphogenesis 1), Rho family of small GTPases, and Jun N-terminal kinase (JNK), and the less-studied ciliogenesis and planar polarity effector (CPLANE) proteins including Intu, Fuz and Wdpcp [START_REF] Adler | From planar cell polarity to ciliogenesis and back: The curious tale of the PPE and CPLANE proteins[END_REF]. Wnt/Ca 2+ signaling triggers phospholipase C activity through heteromeric G-proteins to induce intracellular calcium flux and calcium-dependent responses. Dvl family proteins are important scaffolds that relay signals of all three Wnt branches. They can modulate the activation of Wnt/ß-catenin and Wnt/PCP pathways through conformational changes mediated by intramolecular interaction between the PDZ domain and the extreme C-terminus PDZ-binding motif [START_REF] Lee | Conformational change of Dishevelled plays a key regulatory role in the Wnt signaling pathways[END_REF][START_REF] Qi | Autoinhibition of Dishevelled protein regulated by its extreme C terminus plays a distinct role in Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways[END_REF].

The six "core" PCP proteins display the characteristic feature of forming two separate complexes in planar polarized epithelia. Fzd, Dvl and Ankrd6 localize to the posterior side of the cell, while Vangl and Prickle reside at the anterior side; Celsr1 atypical cadherins are distributed on both sides and form homodimers between adjacent cells to propagate polarity information (Figure 1). Recent studies show that Celsr1 can also function in cis-interactions to organize Fzd6 and Vangl2 into asymmetric junctional complexes [START_REF] Stahley | Celsr1 adhesive interactions mediate the asymmetric organization of planar polarity complexes[END_REF]. The asymmetric localization of PCP protein complexes is a hallmark of planar polarization in the tissue. Fat and Dchs heteromeric protocadherins are also localized to opposite sides of adjacent cells to mediate cell-cell interaction, and their activities are positively or negatively regulated by the Golgi resident transmembrane kinase Fj (Four-jointed), respectively [START_REF] Devenport | The cell biology of planar cell polarity[END_REF]. The Scrib polarity system not only regulates baso-lateral polarity but also modulates the localization of "core" PCP proteins, thus contributing to Wnt/PCP signaling [START_REF] Milgrom-Hoffman | Regulation of cellular and PCP signalling by the Scribble polarity module[END_REF]. Therefore, through multiple biochemical, functional and genetic interactions, the "core" PCP pathway, the Fat/Dchs polarity module and the Scrib polarity complex play essential roles in regulating gastrulation cell movements and neural tube closure.

The following sections will detail their implications in these key developmental processes by emphasizing the consequences of their mutations or dysfunctions on the occurrence of NTDs.

PCP proteins in morphogenetic movements of gastrulation

Wnt/PCP signaling does not appear to regulate dorsal closure during gastrulation in Drosophila [START_REF] Wallingford | Planar cell polarity and the developmental control of cell behavior in vertebrate embryos[END_REF]. However, its requirement for CE movements during gastrulation has been well documented in vertebrates, particularly using Xenopus and zebrafish as models [START_REF] Williams | Cellular and molecular mechanisms of convergence and extension in zebrafish[END_REF][START_REF] Jussila | Zebrafish models of non-canonical Wnt/planar cell polarity signalling: fishing for valuable insight into vertebrate polarized cell behavior[END_REF][START_REF] Kühl | Non-canonical Wnt signaling in Xenopus: regulation of axis formation and gastrulation[END_REF][START_REF] Tada | Non-canonical Wnt signalling and regulation of gastrulation movements[END_REF].

The formation of the primitive streak in amniotes and mammals also involves CE movements and requires a functional Wnt/PCP pathway [START_REF] Serrano Nájera | Cellular processes driving gastrulation in the avian embryo[END_REF][START_REF] Stower | The evolution of amniote gastrulation: the blastopore-primitive streak transition[END_REF]. Mediolateral cell intercalations are major forces that drive CE movements to narrow and elongate tissues in the early embryo, while radial cell intercalations result in thinning of multilayered epithelia during large-scale morphogenesis (Figure 2A,B). Apical constriction that changes tissue curvature and folds epithelial surface also contributes to gastrulation, particularly blastopore formation and mesendoderm invagination. In addition, asymmetric cell division that leads to differential partitions of signaling proteins can contribute to cell lineage specification (Figure 2C). PCP proteins regulate these asymmetric cell behaviors for proper morphogenesis to establish the embryonic axes. Thus, the coordinated actions of embryonic patterning and gastrulation cell movements orchestrate the formation and the spatial organization of the three germ layers, thereby setting up the basic embryonic body plan [START_REF] Carron | Specification of anteroposterior axis by combinatorial signaling during Xenopus development[END_REF].

Non-canonical Wnt ligands in gastrulation

Zebrafish mutants for wnt11f2, previously known as wnt11 or silberblick (slb), show abnormal extension of the axial tissue in a cell non-autonomous manner [START_REF] Heisenberg | Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation[END_REF]. In these embryos, there is a slowed migration of hypoblast cells within the forming germ ring due to the defective orientation of cellular protrusions along the movement directions [START_REF] Ulrich | Slb/Wnt11 controls hypoblast cell migration and morphogenesis at the onset of zebrafish gastrulation[END_REF]. Analysis by in vivo live imaging also reveals a disrupted animal-vegetal polarity of cell intercalations and cell divisions in the dorsal tissues [START_REF] Gong | Planar cell polarity signalling controls cell division orientation during zebrafish gastrulation[END_REF]. Wnt11f2 regulates these polarized cell behaviors by modulating cellular cohesion through Rab5c-mediated E-cadherin endocytosis [START_REF] Ulrich | Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin[END_REF]. It also induces the asymmetric accumulation of Fzd7 receptor and the recruitment of Dvl at the plasma membrane of adjacent cells, leading to locally an increase of Celsr2-mediated cell contact persistence [START_REF] Witzel | Wnt11 controls cell contact persistence by local accumulation of Frizzled 7 at the plasma membrane[END_REF]. In Xenopus, inhibition of Wnt11 function using a dominant negative mutant blocks gastrulation by specifically disrupting Wnt/PCP signaling [START_REF] Tada | Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway[END_REF]. Interestingly, Xenopus Wnt11 regulates CE movements by promoting the formation of adhesion-modulating complexes consisting of Fzd7 and paraxial protocadherin (PAPC) or Fzd7 and C-cadherin, which function in parallel to prevent C-cadherin clustering and reduce cell adhesion [START_REF] Kraft | Wnt-11 and Fz7 reduce cell adhesion in convergent extension by sequestration of PAPC and C-cadherin[END_REF]. These observations suggest that Wnt11-mediated signaling induces cell shape changes and intercalation behaviors at least partially through regulation of cell adhesion.

It is worth mentioning that wnt11f2 mutants also show craniofacial defects and cyclopia at larval stages due to defective formation of midline structures in the gastrula. Indeed, wnt11f2 belongs to the "midline group" genes whose mutations impair the extension of axial tissues and remotely affect the development of head cartilages [START_REF] Kimmel | Specification and morphogenesis of the zebrafish larval head skeleton[END_REF]. Similarly, mutations of other components in the Wnt/PCP pathway that disrupt the proper positioning of the prechordal mesoderm at the end of gastrulation can also cause craniofacial phenotypes [START_REF] Topczewski | The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension[END_REF][START_REF] Xing | Mutational analysis of dishevelled genes in zebrafish reveals distinct functions in embryonic patterning and gastrulation cell movements[END_REF].

Wnt5a and Wnt5b display both specific and redundant roles during CE movements in different species. In zebrafish, Wnt5a can interact with the CD146 receptor, also known as melanoma cell adhesion molecule, to regulate cell motility during gastrulation [START_REF] Ye | Wnt5a uses CD146 as a receptor to regulate cell motility and convergent extension[END_REF], while Wnt5b signals through Ryk and focal adhesion kinase (Fak) to induce cellular polarization [START_REF] Hung | Wnt5b integrates Fak1a to mediate gastrulation cell movements via Rac1 and Cdc42[END_REF][START_REF] Lin | Wnt5b-Ryk pathway provides directional signals to regulate gastrulation movement[END_REF]. In addition, Wnt5b functions specifically in posterior regions of the zebrafish gastrula to regulate cell elongation and CE movements, while it shows redundant roles with Wnt11f2 in more anterior regions [START_REF] Kilian | The role of Ppt/Wnt5 in regulating cell shape and movement during zebrafish gastrulation[END_REF]. In Xenopus, Wnt5a specifically interacts with Ror2 to regulate PAPC expression and CE movements through activation of JNK signaling, suggesting that it can also elicit transcriptional responses [START_REF] Schambony | Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway[END_REF]. Importantly, Wnt5a and Wnt11 can provide directional cues to establish cellular polarization. In ectodermal cells of Xenopus midgastrula, they instruct PCP by orienting the Vangl2-Prickle3 complex to anterior cell borders, away from their source [START_REF] Chu | Wnt proteins can direct planar cell polarity in vertebrate ectoderm[END_REF]. In the avian embryo, Wnt5a and Wnt5b may function redundantly with Wnt11 to regulate cell migration through the primitive streak [START_REF] Hardy | Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation[END_REF]. In mice, Wnt5a and Wnt11 are also required for the migration of axial and paraxial mesodermal precursor cells to promote notochord formation through regulation of epithelial-mesenchymal transition [START_REF] Andre | Wnt5a and Wnt11 regulate mammalian anteriorposterior axis elongation[END_REF].

"Core" PCP proteins in asymmetric cellular behaviors during gastrulation

Extensive analyses have provided important insights into the functions of "core" PCP proteins in morphogenetic movements of gastrulation. Pioneer studies in Xenopus demonstrate that disrupting the functions of Fzd7 or Dvl impairs CE movements but not embryonic patterning [START_REF] Djiane | Role of frizzled 7 in the regulation of convergent extension movements during gastrulation in Xenopus laevis[END_REF][START_REF] Sokol | Analysis of Dishevelled signalling pathways during Xenopus development[END_REF][START_REF] Wallingford | Dishevelled controls cell polarity during Xenopus gastrulation[END_REF]. More recent studies by mutational analyses indicate that zebrafish maternal-zygotic Dvl2 and Dvl3a cooperatively regulate CE movements independently of Wnt/ß-catenin signaling, suggesting an important contribution of maternal PCP proteins to morphogenetic movements that occur after zygotic genome activation [START_REF] Xing | Mutational analysis of dishevelled genes in zebrafish reveals distinct functions in embryonic patterning and gastrulation cell movements[END_REF]. The membrane recruitment of Dvl by Fzd receptors is important for its activity in Wnt/PCP signaling [START_REF] Qi | Autoinhibition of Dishevelled protein regulated by its extreme C terminus plays a distinct role in Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways[END_REF][START_REF] Axelrod | Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways[END_REF]. In zebrafish, Celsr1 is required for the formation of Fzd-Dvl complex, at least by regulating membrane recruitment of Dvl [START_REF] Carreira-Barbosa | Flamingo regulates epiboly and convergence/extension movements through cell cohesive and signalling functions during zebrafish gastrulation[END_REF]. Prickle1 regulates Wnt/PCP signaling during gastrulation cell movements through interaction with Dvl and activation of JNK signaling [START_REF] Carreira-Barbosa | Prickle 1 regulates cell movements during gastrulation and neuronal migration in zebrafish[END_REF][START_REF] Takeuchi | The prickle-related gene in vertebrates is essential for gastrulation cell movements[END_REF][START_REF] Veeman | Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements[END_REF]. It shows asymmetric localization in cells undergoing CE movements and is enriched at the anterior edge, while Dvl is accumulated at the posterior side [START_REF] Yin | Cooperation of polarized cell intercalations drives convergence and extension of presomitic mesoderm during zebrafish gastrulation[END_REF]. This segregation of PCP proteins helps to define distinct anterior and posterior cell properties and provides bias for cell intercalations. Accordingly, inappropriate functioning of Prickle1 causes CE defects, likely by disrupting Fzd-dependent membrane recruitment of Dvl [START_REF] Carreira-Barbosa | Prickle 1 regulates cell movements during gastrulation and neuronal migration in zebrafish[END_REF]. Intriguingly, recent studies suggest that diffusely distributed cytoplasmic Prickle1 counteracts the activity of Dvl2 to increase F-actin content and cortical tension in Xenopus prechordal mesoderm cells, thereby generating mechanical forces to promote cell migration and rearrangements during radial cell intercalations [START_REF] Huang | Cell cortex regulation by the planar cell polarity protein Prickle1[END_REF].

As Prickle1, zebrafish Vangl2 (also known as Strabismus or Trilobite) displays stagedependent membrane accumulation and enrichment in highly elongated cells undergoing mediolateral intercalation [START_REF] Roszko | A dynamic intracellular distribution of Vangl2 accompanies cell polarization during zebrafish gastrulation[END_REF]. Both gain and loss of Vangl2 function disrupts Wnt/PCP signaling and affects mediolateral cell polarity necessary for effective intercalation and directed migration [START_REF] Darken | The planar polarity gene strabismus regulates convergent extension movements in Xenopus[END_REF][START_REF] Jessen | Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements[END_REF][START_REF] Park | The planar cell-polarity gene stbm regulates cell behaviour and cell fate in vertebrate embryos[END_REF]. In the Xenopus gastrula, Vangl2 is also enriched at the constricted apical surfaces of blastopore bottle cells. This asymmetric localization plays a role in blastopore formation and closure by promoting the apical accumulation of Rab11 and its associated motor protein Myosin V, suggesting that Vangl2 can also regulate apico-basal polarity during morphogenetic movements [START_REF] Ossipova | Vangl2 cooperates with Rab11 and Myosin V to regulate apical constriction during vertebrate gastrulation[END_REF].

In zebrafish, Ankrd6 (previously known as Diversin) is required for gastrulation cell movements through interaction with Dvl and activation of JNK [START_REF] Moeller | Diversin regulates heart formation and gastrulation movements in development[END_REF][START_REF] Schwarz-Romond | The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling[END_REF]. However, biochemical and functional analyses suggest that Ankrd6 represents a molecular switch to stimulate Wnt/PCP signaling but suppress Wnt/ß-catenin pathway in a dose-dependent manner [START_REF] Schwarz-Romond | The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling[END_REF]. Therefore, Ankrd6 may also function in embryonic patterning, and how this impacts on cell behavior changes needs further investigation.

In amniotes, mediolateral cell intercalation has been shown to define the primitive streak before gastrulation [START_REF] Voiculescu | The amniote primitive streak is defined by epithelial cell intercalation before gastrulation[END_REF]. This event occurs in a subdomain of the epiblast where the primitive streak will form during gastrulation and requires the function of several "core" PCP proteins including Dvl, Celsr1, Vangl2 and Prickle1 [START_REF] Voiculescu | The amniote primitive streak is defined by epithelial cell intercalation before gastrulation[END_REF]. Therefore, Wnt/PCP signaling promotes primitive streak formation before gastrulation. Interestingly, as discussed above, this pathway remains active during gastrulation to regulate cell exit from the primitive streak [START_REF] Hardy | Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation[END_REF].

Co-receptors of the Wnt/PCP pathway in convergent extension movements

Glypican 4 (Knypek in zebrafish), a member of the heparan sulfate proteoglycans (HSPGs) family, functions as a co-receptor for a variety of growth factors and regulates CE movements in zebrafish and Xenopus by promoting Wnt/PCP signaling [START_REF] Topczewski | The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension[END_REF][START_REF] Ohkawara | Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis[END_REF]. Both functional and biochemical analyses suggest that it interacts with Wnt5a and Wnt11 during gastrulation [START_REF] Ohkawara | Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis[END_REF].

Mutations that disrupt the function of Knypek prevent mediolateral alignment and elongation of ectodermal and mesodermal cells in the paraxial region, leading to CE defects and shortened AP axis [START_REF] Topczewski | The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension[END_REF]. Ror2 and Ryk are also implicated in the regulation of cell polarity and CE movements during gastrulation in vertebrate embryos by functioning as co-receptors for Wnt5a or Wnt11 [START_REF] Bai | Ror2 receptor mediates Wnt11 ligand signaling and affects convergence and extension movements in zebrafish[END_REF][START_REF] Hikasa | The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling[END_REF][START_REF] Kim | Ryk cooperates with Frizzled 7 to promote Wnt11-mediated endocytosis and is essential for Xenopus laevis convergent extension movements[END_REF][START_REF] Macheda | The Wnt receptor Ryk plays a role in mammalian planar cell polarity signaling[END_REF].

Protein tyrosine kinase 7 (PTK7) is an evolutionarily conserved transmembrane receptor that functions as a vertebrate-specific PCP regulator in many developmental processes [START_REF] Berger | PTK7 faces the Wnt in development and disease[END_REF]. In zebrafish, Ptk7 regulates Wnt/PCP signaling by potentiating the activity of Wnt5 and Wnt11; maternal-zygotic ptk7 mutants show impaired mediolateral intercalation [START_REF] Hayes | Ptk7 promotes non-canonical Wnt/PCPmediated morphogenesis and inhibits Wnt/β-catenin-dependent cell fate decisions during vertebrate development[END_REF]. However, it is not entirely clear how Ptk7 signals in the Wnt/PCP pathway to polarize cell behaviors because CE defects caused by its loss of function can be rescued by expression of a membrane-tethered extracellular domain of the protein [START_REF] Hayes | Ptk7 promotes non-canonical Wnt/PCPmediated morphogenesis and inhibits Wnt/β-catenin-dependent cell fate decisions during vertebrate development[END_REF]. Knockout of Ptk7 in mice also leads to defective mediolateral and radial intercalations, but the identity of Wnt ligands that interact with Ptk7 in these processes remains unclear [START_REF] Yen | PTK7 is essential for polarized cell motility and convergent extension during mouse gastrulation[END_REF]. Studies in Xenopus suggest that Ptk7 functions in Wnt/PCP signaling during morphogenetic movements of gastrulation at least through JNK-mediated transcriptional response. In this context, Ptk7 interacts with Wnt5a and cooperates with Ror2 to modulate PAPC expression [START_REF] Martinez | The PTK7 and ROR2 protein receptors interact in the vertebrate WNT/planar cell polarity (PCP) pathway[END_REF].

Other polarity regulators in gastrulation cell movements

Studies in zebrafish have identified additional polarity regulators that function in morphogenetic movements during gastrulation. Maternal-zygotic Dchs1b may be involved in prechordal mesodermal migration and CE movements by regulating actin or microtubule organization in a Fat-independent manner [START_REF] Li-Villarreal | Dachsous1b cadherin regulates actin and microtubule cytoskeleton during early zebrafish embryogenesis[END_REF]. Maternal but not zygotic Scrib1 shows strong genetic interaction with Vangl2 and the two proteins may form a functional complex in CE movements [START_REF] Wada | Dual roles of zygotic and maternal Scribble1 in neural migration and convergent extension movements in zebrafish embryos[END_REF]. Moreover, Scrib1 also interacts with LPP, a zyxin-related actin cytoskeleton protein, to promote the dorsal convergence of paraxial cells [START_REF] Vervenne | Lpp is involved in Wnt/PCP signaling and acts together with Scrib to mediate convergence and extension movements during zebrafish gastrulation[END_REF]. The PDZ domain protein Mcc (mutated in colorectal cancer) directly interacts with the cytoplasmic tail of Vangl2 and can function as an effector of Wnt5a/Ror2/Vangl2 signaling to activate RhoA and JNK in CE movements [START_REF] Young | The PDZ domain protein Mcc is a novel effector of non-canonical Wnt signaling during convergence and extension in zebrafish[END_REF]. Recent research shows that the E3 ubiquitin ligase Mindbomb1 (Mib1) controls the endocytosis of Ryk in gastrulating cells, and therefore, zebrafish mib1 null mutants display defective PCP-dependent CE movements [START_REF] Saraswathy | The E3 ubiquitin ligase mindbomb1 controls planar cell polarity-dependent convergent extension movements during zebrafish gastrulation[END_REF].

Wnt/PCP-regulated cytoskeletal rearrangements and adhesive changes during gastrulation

Wnt/PCP-dependent cellular polarization is mediated by dynamic rearrangements of the actin and microtubule cytoskeleton. Key regulators of the cytoskeleton architecture including Rho and Rac are activated by Fzd receptors through Dvl and the PPE protein Daam1, a formin homology protein with actin-binding activity, thereby promoting asymmetric cellular activities [START_REF] Habas | Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1[END_REF][START_REF] Habas | Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation[END_REF]. Moreover, Wnt/PCP signaling mediated by Dvl and Knypek also controls the proper location of the microtubule-organizing center in both ectodermal and mesodermal cells undergoing CE movements [START_REF] Cheng | Leucine repeat adaptor protein 1 interacts with Dishevelled to regulate gastrulation cell movements in zebrafish[END_REF][START_REF] Sepich | Wnt/PCP signaling controls intracellular position of MTOCs during gastrulation convergence and extension movements[END_REF]. This influences the polarity of the microtubule cytoskeleton, which is required for the asymmetric distribution of PCP proteins [START_REF] Sepich | Wnt/PCP signaling controls intracellular position of MTOCs during gastrulation convergence and extension movements[END_REF]. There is also evidence that a spatiotemporal clustering of Prickle2 is correlated with alternative actomyosin oscillation across cell membranes of neighboring cells, which may modulate submembranous accumulations of Factin and facilitate CE movements [START_REF] Shindo | PCP-dependent transcellular regulation of actomyosin oscillation facilitates convergent extension of vertebrate tissue[END_REF]. These observations suggest that some PCP proteins can function to set up the anisotropy of many cellular properties. Therefore, either gain or loss of their activity can disrupt asymmetric cellular behaviors and leads to CE defects [START_REF] Cheng | Leucine repeat adaptor protein 1 interacts with Dishevelled to regulate gastrulation cell movements in zebrafish[END_REF].

Polarized cellular protrusions also require Wnt/PCP-regulated organization of extracellular matrix (ECM). In Xenopus, inhibition of Wnt/PCP signaling disrupts fibronectin assembly in the blastocoel roof by reducing its mechanical tension [START_REF] Dzamba | Cadherin adhesion, tissue tension, and noncanonical Wnt signaling regulate fibronectin matrix organization[END_REF]. Fzd7, Vangl2 and Prickle1 function in mediolateral intercalation of mesodermal cells by regulating fibronectin secretion and assembly at their outer surfaces that contact the overlying ectoderm and the underlying endoderm [START_REF] Goto | Planar cell polarity genes regulate polarized extracellular matrix deposition during frog gastrulation[END_REF]. In zebrafish, "core" PCP proteins are also differentially involved in ECM organization [START_REF] Creighton | Core pathway proteins and the molecular basis of planar polarity in the zebrafish gastrula[END_REF]. Loss of Fzd7 leads to increased fibronectin assembly in the gastrula, while loss of Vangl2 or Prickle1 decreases fibronectin protein levels, thus preventing polarized membrane protrusion and directed migration [START_REF] Dohn | Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation[END_REF][START_REF] Love | Vangl2-dependent regulation of membrane protrusions and directed migration requires a fibronectin extracellular matrix[END_REF][START_REF] Williams | VANGL2 regulates membrane trafficking of MMP14 to control cell polarity and migration[END_REF]. Therefore, Wnt/PCP-dependent ECM organization and cell-substrate adhesion are important to promote polarized cell behaviors for asymmetric movements [START_REF] Prince | Dorsal convergence of gastrula cells requires Vangl2 and an adhesion protein-dependent change in protrusive activity[END_REF].

PCP regulatory genes in neural tube closure and neural tube defects

The neural plate emerges at the end of gastrulation as the neuroectodermal tissue above the dorsal mesoderm lengthens into columnar cells (Figure 3). The neural tube can be formed through two different processes, called primary and secondary neurulation, which involve fusion of the neural folds or epithelialization from a solid cord of neural tube progenitor cells at the tail bud region, respectively [START_REF] Nikolopoulou | Neural tube closure: cellular, molecular and biomechanical mechanisms[END_REF]. During primary neurulation in mammals, birds and Xenopus, neural folds arise bilaterally at the boundaries between the neural plate and the surrounding epidermis.

They elevate and converge toward the midline for fusion to form the neural tube by a "zippering" process either initiating at different closure sites along the AP axis or occurring simultaneously at all axial levels. In zebrafish and other teleost fish, the neural tube is formed through a process similar to secondary neurulation, without the formation of neural folds. As gastrulation, neurulation also involves cell intercalation and apical constriction, which generate major forces driving the AP elongation and bending of the neural plate. Disruption of these polarized cell behaviors prevents neural tube closure and are associated with varying forms of NTDs (Table 1), which are the most common congenital malformations affecting the central nervous systems. The implication of PCP genes in NTDs has been extensively reviewed recently [START_REF] Wang | Update on the role of the non-canonical Wnt/planar cell polarity pathway in neural tube defects[END_REF]. By integrating data obtained from complementary animal models with advances on the study of congenital disorders, this work focuses on the analysis of molecular and cellular mechanisms underlying the regulatory roles of PCP-related genes in neural tube closure during development and disease. It not only discusses Wnt/PCP signaling in primary and secondary neurulation but also highlights recent advances in the study of somatic mutations of "core" PCP genes associated with NTDs.

Asymmetric localization of "core" PCP proteins in the neuroectoderm

The subcellular localization of several "core" PCP proteins displays asymmetric bias in the neural epithelium. In Xenopus, Vangl2 is enriched at anterior cell boundaries, which is dependent on its interaction with Prickle and Par3 proteins [START_REF] Chuykin | Par3 interacts with Prickle3 to generate apical PCP complexes in the vertebrate neural plate[END_REF][START_REF] Ossipova | Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and Myosin II signaling[END_REF]. Wnt5a gradients can provide instructive cues to promote the anterior localization of Vangl2 by inducing its phosphorylation [START_REF] Ossipova | Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and Myosin II signaling[END_REF]. A recent study further suggests that Fzd3, which is specifically expressed in the neuroectoderm [START_REF] Shi | Expression of Xfz3, a Xenopus frizzled family member, is restricted to the early nervous system[END_REF],

promotes the anterior accumulation of Vangl2-Prickle3 complexes by phosphorylating Vangl2 on specific threonine residues [100]. In addition, the Vangl2-Prickle3 complex is also enriched near the outermost surface of deep neural plate cells that undergo radial intercalations during neurulation [START_REF] Ossipova | The involvement of PCP proteins in radial cell intercalations during Xenopus embryonic development[END_REF]. These observations indicate that Vangl2 and Prickle3 can regulate both mediolateral and radial cell intercalations in the neuroepithelium. Importantly, the asymmetric enrichment of PCP proteins is closely correlated with actomyosin-driven contractile behaviors of cell-cell junctions [101]. Moreover, Myosin II can exert a positive feedback on the asymmetric localization of Vangl2 protein, suggesting that mechanical forces contribute to establish PCP in the neural plate [START_REF] Ossipova | Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and Myosin II signaling[END_REF]. Accordingly, diffusion of locally expressed Wnt11 in the posterior neuroectoderm acts with anisotropic tension imposed by unidirectional tissue stretch to orient PCP through regulation of cell shape changes [102]. Therefore, interactions between "core" PCP proteins and actomyosin-mediated contractility regulate polarized cellular behaviors necessary for driving neural tube closure, such as mediolateral cell intercalation that elongates the neural plate along the AP axis and apical constriction that causes bending of the neural plate [START_REF] Matsuda | Xenopus neural tube closure: A vertebrate model linking planar cell polarity to actomyosin contractions[END_REF].

"Core" PCP proteins in neural tube development and disease

Functional studies using animal models and analyses of genetic mutations in humans have firmly demonstrated the contribution of PCP proteins to neural tube closure. In Xenopus, Vangl paralogs are critically involved in neural tube closure [112]. In Xenopus, an optimal level of Vangl2 is crucial for proper cellular polarization underlying normal cell intercalation.

Therefore, either an increase or a decrease of Vangl2 activity disrupts CE movements of the neuroepithelium and inhibits neural tube closure [113]. This suggests that Wnt/PCP signaling should be tightly regulated at local levels to confirm the tissue-level constraints. In zebrafish, the neural tube is essentially formed through cavitation of the neural keel, a process similar to secondary neurulation. Vangl2-mediated Wnt/PCP signaling regulates neural tube formation by promoting polarization of neural progenitors along the AP axis and re-intercalation of mitotic daughter cells into the neuroepithelium, thereby coupling cell division and neurulation [START_REF] Ciruna | Planar cell polarity signalling couples cell division and morphogenesis during neurulation[END_REF]. In mice, missense or null mutations of Vangl2 impair CE movements of the neuroepithelium and produce NTDs, with severity depending on the reduction of Vangl2 activity [114,115,116,117]. It is likely that Vangl2 regulates Wnt/PCP signaling in a dosage-sensitive manner. Thus, heterozygosity of the Looptail mutation, which results in a single amino acid substitution that is predicted to

Other PCP-related genes in neural tube morphogenesis

Wnt/PCP signaling in secondary neurulation

It is well established that Wnt/PCP signaling plays crucial roles in primary neurulation, as discussed above. However, there is also evidence that PCP genes are involved in secondary neurulation. In mice, analyses of genetic interactions between multiple heterozygous mutations of PCP genes indicate that some double heterozygous mutants exhibit NTDs that may result from impaired secondary neurulation [148]; conditional knockout of Vangl2 in the surface ectoderm during neurulation disrupts polarized cell body orientation and results in caudal spina bifida that may be partially due to secondary neurulation defects [174]. Interference with the activity of PPE proteins such as Cdc42 and Rac1 disrupts mesenchymal-epithelial transition during secondary neurulation in the chick embryo [175]. As aforementioned, secondary neurulation occurs throughout the neural axis in zebrafish, Vangl2-mediated Wnt/PCP signaling is required for neural tube formation by polarizing neural progenitors along the AP axis [START_REF] Ciruna | Planar cell polarity signalling couples cell division and morphogenesis during neurulation[END_REF]. Furthermore, it is likely that closed spina bifida occurs as a consequence of defective secondary neurulation [176]. Indeed, mutations of PCP genes can cause varying degrees of NTDs. Specifically, some heterozygous missense mutations in CELSR1, VANGL1 and VANGL2 genes have been identified in human NTDs with phenotypes reminiscent of abnormalities in secondary neurulation [124,132,177,178]. These observations suggest that there are common molecular mechanisms regulating primary and secondary neurulation. The implication of Wnt/PCP signaling in secondary neurulation may be dependent on the genetic interactions, dosage or activity, and spatiotemporal expression of different PCP genes.

Concluding remarks

Gastrulation and neurulation are highly coordinated morphogenetic processes that are regulated by common molecular and cellular mechanisms. The "core" Wnt/PCP pathway functions in cooperation with or independently of other PCP regulatory genes to control polarized cellular behaviors. Extensive studies using different animal models have greatly advanced our understanding on the implication of these PCP regulators in morphogenetic cell movements that contribute to establish the basic body plan. However, there also raise many intriguing questions that deserve future investigation.

It is well documented that "core" PCP proteins display polarized localization within the cell of various organs undergoing asymmetric morphogenesis [START_REF] Shi | Planar cell polarity regulators in asymmetric organogenesis during development and disease[END_REF]. This feature seems to be also conserved in cells undergoing mediolateral and radial intercalations during gastrulation and neurulation. In addition to instructive cues provided by Wnt gradients [START_REF] Chu | Wnt proteins can direct planar cell polarity in vertebrate ectoderm[END_REF], mechanical signals generated by cell movements also play an important role to redistribute PCP proteins. In Xenopus, it has been shown that mechanical strain acts through microtubules to determine the axis of planar polarity and transport PCP proteins in a directed manner [179,180]. Thus, it will be of interest to decipher the interplay between different polarity modules and mechanotransduction during morphogenetic movements of gastrulation and neurulation.

It becomes increasingly evident that Wnt/PCP signaling also couples embryonic polarity Since PCP proteins use similar cellular mechanisms, such as oriented cell division and cell intercalation, to regulate tissue and organ morphogenesis, it would be not surprising that their dysfunctions can cause general anomalies in multiple organs that display these PCP-dependent cell movements. As such, mutations of PCP genes may need to be taken into consideration when there is a co-existence of NTDs and other congenital disorders. Biochemical and functional interactions between different polarity pathways in NTDs also need further investigations.

Deciphering the detailed functions of PCP proteins and their interplay in regulating cell behaviors during development and disease should help to define therapeutic approaches targeting Wnt/PCP signaling.

  Dvl2 regulates CE movements in the mesoderm and neural plate to generate parallel forces for elongating the AP axis [103]. Perturbation of its function prevents fusion of nascent neural folds and closure of the neural tube [104]. In mice, all the three Dvl genes (Dvl1, Dvl2 and Dvl3) are involved in neural tube closure, but Dvl2 appears to play a predominant role [105,106,107]. Since different Dvl genes exhibit partial redundant functions and cooperatively regulate Wnt/PCP signaling in CE movements, PCP-dependent neural tube closure is extremely sensitive to Dvl dosage [108, 109]. Consistent with their importance for neurulation, missense mutations of different DVL genes have been identified in patients with NTDs or Dandy-Walker malformation characterized by abnormal cerebellar development [110, 111]. However, these missense variants likely produce complex effects, by differentially disrupting the activity of all three Wnt signaling branches.

  produce a malfunctional Vangl2 protein, can exacerbate NTDs in mice carrying heterozygous or homozygous mutations of other polarity genes, often resulting in craniorachischisis [105, 107, 118,119,120,121,122]. There is a possibility that the malfunctional Vangl2 protein aggravates PCP defects by interfering with the proper localization of other "core" PCP components. In humans, heterozygous missense mutations of VANGL1, which disrupt the interaction of VANGL1 and DVL in Wnt/PCP signaling, are linked to varying degrees of spina bifida [123]. Depending on the variants, heterozygous missense mutations of VANGL2 result in open spinal bifida and closed spinal NTDs or cause anencephaly and are lethal to the fetus [124, 125]. These observations reveal a critical role for VANGL-mediated Wnt/PCP signaling in neural tube closure and strongly implicate VANGL as a risk factor and the genetic causation of human spinal NTDs. Indeed, neural tube closure is particularly vulnerable to loss of Vangl2 activity. Recent research using mouse embryos indicates that an absence of Vangl2 function in a minority (16%) of neuroepithelial cells can non-autonomously prevent neural fold elevation by inhibiting apical constriction of neighboring cells, suggesting that tissue mosaicism generated by post-zygotic mutations of some PCP genes may lead to severe failure of morphogenesis [126]. Consistently, somatic mutations of FZD6, VANGL1 and CELSR1 have been associated with human NTDs [127], suggesting that disruption of Wnt/PCP signaling during different stages of embryonic development can contribute to defective morphogenesis of the neural tube. Fzd1 and Fzd2 genetically interact with Vangl2 in Wnt/PCP signaling to regulate various tissue fusion processes, including neural tube closure at different locations along the neural axis [122]. Combined knockout of Fzd3 and Fzd6 in mice causes craniorachischisis with almost 100% penetrance [128]. Heterozygous missense or frameshift mutations of the human FZD6 gene have been shown to cause different forms of NTDs [129]. Homozygous Celsr1 mutant mice fail to initiate neural tube closure, resulting in craniorachischisis [130], while heterozygous missense variants of CELSR1 may represent the underlying pathogenic mechanism of craniorachischisis as well as other forms of NTDs in humans [131,132,133]. In chick embryos, Celsr1 in floor plate cells recruits Fzd receptors and Dvl to regulate actomyosin-dependent mediolateral contraction of adherens junctions, promoting bending of the neural plate by causing simultaneous midline convergence and apical constriction [134]. Prickle1 is required for junctional neurulation in the chick spinal cord, which is a process involving the fusion and connection of the primary neural tube with the posterior neural tube formed by secondary neurulation from the caudal cell mass [135]. Heterozygous missense mutations of PRICKLE1 in humans have been reported to cause varying degrees of spina bifida [136]. Although it is unclear how Ankrd6 regulates neural tube closure, rare missense mutations that disrupt its activity in the balance of Wnt/PCP and Wnt/ßcatenin signaling have been identified in human NTDs [137]. The CPLANE protein Fuz, a vertebrate ortholog of Drosophila Fuzzy, is required for neural tube closure in Xenopus and mice likely by regulating ciliogenesis in the neural plate [138,139,140]. Consistently, mutations of FUZ affecting the formation of primary cilia and ciliary length have been associated with both open and closed NTDs in humans, which are spinal defects exposed at birth or covered by the skin, respectively [141].

Fat1

  and Fat4 protocadherins show genetic interaction in cranial neural tube closure, and loss of their functions in mice causes exencephaly[142, 143]. They may function as cisheterodimers to modulate cytoskeletal organization and apical constriction[142]. However, it is unclear whether they regulate polarized cell behaviors and cell-cell interaction by forming ligandreceptor pairs with Dchs protocadherins.Mutations that impairScrib function in mice severely affect neural tube closure and cause craniorachischisis by disrupting cell intercalation required for neural plate CE movements and by preventing apical constriction necessary for neural plate bending [119, 144, 145]. Scrib may regulate polarized cell behaviors by influencing the localization and expression of junctional and cytoskeletal proteins [144]. It also directly binds to Vangl2 through its PDZ domains to promote the proper localization of Vangl2 in mouse neuroepithelial cells [146]. Combined heterozygous mutations of Scrib and Vangl2 or Scrib and Celsr1 produce variable phenotypes ranging from craniorachischisis to other forms of NTDs [144, 145, 147, 148]. Further supporting its implication in neural tube closure, there are several studies linking heterozygous missense mutations of SCRIB gene with human NTDs [131, 146, 149]. Dishevelled binding antagonist of ß-catenin 1 (Dact1), also known as Dapper1, is a cytoplasmic protein that interacts with Dvl2 and Vangl2 to regulate their expression at the posttranslational level [150,151,152]. Knockout of Dact1 in mice disrupts Wnt/PCP signaling and causes malformations at the posterior primitive streak [152, 153]. Consistent with its requirement for CE movements and neural tube closure, several rare heterozygous missense mutations that lead to loss or reduction of DACT1 activity have been identified in human NTDs [154]. SEC24B is a cargo-binding protein in the coat protein complex II (COPII) and is involved in vesicle trafficking. It functions in Wnt/PCP signaling by selectively sorting Vangl2 to COPII during neural tube closure. In mice, mutations of Sec24b gene that introduce a premature stop codon disrupt PCP-dependent CE movements and cause craniorachischisis by inhibiting proper endoplasmic reticulum (ER) to Golgi transport of Vangl2 [155, 156]. Heterozygous missense mutations of SEC24B that disrupt the subcellular localization of Vangl2 have been associated with open and closed spina bifida in humans [157]. PTK7 displays conserved roles in neural tube morphogenesis. Inhibition of PTK7 function in Xenopus impairs CE movements of the neural plate by preventing Dvl membrane localization mediated by Fzd7 and the adaptor protein RACK1 [118, 158]. In mice, PTK shows strong genetic interaction with Vangl2 or Celsr1 and is required for the initiation of neural tube closure [118, 159]. Its loss of function disrupts the polarity of cell motility and intercalation in the neural plate by preventing polarized localization of Myosin IIB, leading to craniorachischisis [160]. Consistent with this importance in neural tube closure, rare heterozygous missense mutations of PTK7 gene have been associated with various forms of NTDs in human patients [161, 162]. Although LRP6 generally functions as a co-receptor in Wnt/ß-catenin signaling, there is evidence that it regulates neural tube closure through the Wnt/PCP pathway. In mice, both gain and loss of LRP6 activity affect neural tube closure and cause varying degrees of NTDs, which may be attributed to defective Wnt/ß-catenin or Wnt/PCP signaling [163,164,165,166,167,168]. Heterozygous missense mutations in LRP6 that disrupt Wnt/ß-catenin or Wnt/PCP signaling, or both, have been identified in human NTDs [169,170,171]. The mechanism by which gain and loss of LRP6 link Wnt/PCP signaling to NTDs remain elusive. It is possible that the extracellular domain of LRP6 antagonizes Wnt/PCP signaling through sequestration of non-canonical Wnt ligands such as Wnt5a and Wnt11 [172, 173]. Therefore, LRP6 may serve as a molecular switch from Wnt/ß-catenin or Wnt/PCP signaling during neural tube closure in a dosage-dependent manner.

  and patterning. The coordinated action of Wnt/PCP and Wnt/ß-catenin signaling regulates ectodermal cell fate during Xenopus development. Wnt/PCP signaling controls the apico-basal cell polarity by directing the asymmetric localization of the canonical Wnt co-receptor LRP6, leading to elevated Wnt/β-catenin signaling in the deep layer of ectoderm cells and differentiation of multiciliated cells [181]. Moreover, the asymmetric localization of PCP proteins can contribute to cell differentiation by promoting radial intercalation. The Vangl2-Prickle3 complex, for example, is enriched at the apical side of multiciliated cell progenitors, which undergo intercalation and move to the external surface during neurulation [98]. Wnt/PCP signaling also interacts with other patterning signals in morphogenetic cell movements. In zebrafish, it cooperates with Nodal signaling to regulate embryonic axis extension [182, 183]. Therefore, it will be of interest to elucidate how PCP proteins coordinate cell polarity with gene expression and how cell polarization is coupled with cell fate specification. However, since Wnt/PCP signaling can also induce transcriptional changes, specific readouts need to be applied for examination of cell behavior changes. Live imaging of cell behaviors and PCP protein dynamics should contribute to mechanistic analyses of PCP functions in morphogenesis [101, 184].

Figure 1 .

 1 Figure 1. Wnt/PCP signaling pathway and asymmetric distribution of "core" PCP protein

Figure 2 .

 2 Figure 2. Asymmetric cellular behaviors in morphogenetic movements during gastrulation and

Figure 3 .

 3 Figure 3. Neural plate formation and neural tube closure. Transverse sections in successive

Table 1 .

 1 Mutations of PCP regulatory genes linked to the congenital disorder of human NTDs. Shown here are most frequently occurred mutations. Other mutations, such as truncations and inframe deletions, have been also identified in NTDs.

	Genes	Mutations*	Phenotypes	References
	FZD6**	Heterozygous missense and frameshift	Myelomeningocele, meningocele, caudal regression syndrome	[127, 129]
	DVL1-3	Heterozygous missense	Varying forms of NTDs	[110, 111]
	VANGL1** Heterozygous missense	Cranial, open and closed*** spinal dysraphisms	[123, 127, 177]
	VANGL2	Heterozygous missense	Anencephaly, open spinal bifida, closed spinal NTDs***	[124, 125]
	PRICKLE1 Heterozygous missense	Varying degrees of open spina bifida	[136]
	CELSR1**	Heterozygous missense	Craniorachischisis, other forms of NTDs, tight filum terminale***	[127, 131,132,133]
	ANKRD6	Heterozygous missense	Lipomyeloschisis, meningocele, scoliosis, and other forms of NTDs.	[137]
	SCRIB	Heterozygous missense	Varying forms of open and closed NTDs	[131, 146, 149]
	FUZ	Heterozygous missense	Open and closed NTDs	[141]
	PTK7	Heterozygous missense	Varying forms of NTDs	[161, 162]
	DACT1	Heterozygous missense	Craniorachischisis, closed NTDs, encephalocele	[154]
	SEC24B	Heterozygous missense	Open and closed NTDs	[157]
	LRP6	Heterozygous missense	Spina bifida	[169,170,171]

***Somatic mutations of these genes have been also associated with NTDs. ***Possible consequences of secondary neurulation defects.

Acknowledgements

I would like to thank members of the laboratories for discussion and supports during the preparation of this work.

Funding

This work was supported by the National Natural Science Foundation of China (grant number 32070813), the French Muscular Dystrophy Association (AFM-Téléthon grant number 23545), and by the annual supports from the Centre National de la Recherche Scientifique (CNRS) and the Sorbonne University.

Availability of data and material

Not applicable

Declarations

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare no competing interests.

Authors' contributions

DLS conceived the project, performed literature analyses, prepared figures, and wrote, reviewed and edited the manuscript.


Frédéric Chaux-Jukic
email: frederic.chaux-jukic@sorbonne-universite.fr

Nicolas Agier

Stephan Eberhard

Zhou Xu
email: zhou.xu@sorbonne-universite.fr

Extraction and selection of high-molecular-weight DNA for long-read sequencing from Chlamydomonas reinhardtii

Keywords: Algae DNA extraction, high-molecular weight DNA, long-read sequencing, size selection

come

Introduction

In recent years, long-read sequencing technologies, such as the ones developed by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (Nanopore), have emerged as a solution to the pitfalls of short-read technologies in the detection of structural variants and in assembling repeated sequences and other complex regions [START_REF] Logsdon | Long-read human genome sequencing and its applications[END_REF]. Additionally, because native DNA is used, long-read technologies can directly detect a variety of modified bases, including the most commonly studied methylated cytosines [START_REF] Rand | Mapping DNA methylation with high-throughput nanopore sequencing[END_REF][START_REF] Feng | Detecting DNA modifications from SMRT sequencing data by modeling sequence context dependence of polymerase kinetic[END_REF]. For their applications in genome assembly and structural variant detection, these technologies typically sequence DNA molecules ranging in size from kilobases to hundreds of kilobases as a continuous read. Reads traversing repeated sequences are necessary to correctly assemble neighboring regions, with longer reads enabling more contiguous genome assemblies. Today, the major bottleneck to sequence long reads comes from the ability to extract high-quality DNA devoid of polyphenol and polysaccharide contaminants with sizes compatible with this purpose. This is especially true for most plant tissues and algae cells, because polyphenols and polysaccharides are often co-extracted with DNA and can inhibit downstream applications such as sequencing [START_REF] Porebski | Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components[END_REF][START_REF] Healey | Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species[END_REF].

Chlamydomonas reinhardtii is a unicellular green alga that is widely used as a model organism to study photosynthesis and cellular motility [START_REF] Harris | The Chlamydomonas Sourcebook[END_REF], and is an organism of choice for biotechnological application, with many synthetic biology tools being currently developed [START_REF] Scaife | Establishing Chlamydomonas reinhardtii as an industrial biotechnology host[END_REF][START_REF] Crozet | Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii[END_REF]. In C. reinhardtii, as for other plants and algae, contending with phenolic and polysaccharide contaminants while preserving HMW DNA is a major challenge and requires an optimized protocol. PacBio and Nanopore sequencing have been performed on this organism, contributing to important advances in our understanding of its genome structure and content, base modifications and evolution [START_REF] O'donnell | Highly Contiguous Nanopore Genome Assembly of Chlamydomonas reinhardtii CC-1690[END_REF][START_REF] Liu | Detection of DNA base modifications by deep recurrent neural network on Oxford Nanopore sequencing data[END_REF][START_REF] Chaux-Jukic | Architecture and evolution of subtelomeres in the unicellular green alga Chlamydomonas reinhardtii[END_REF][START_REF] Craig | An Ancient Clade of Penelope-Like Retroelements with Permuted Domains Is Present in the Green Lineage and Protists, and Dominates Many Invertebrate Genomes[END_REF][START_REF] Craig | Comparative genomics of Chlamydomonas[END_REF]. However, previous protocols did not include a size selection step, which can substantially enrich for longer molecules.

Here, we present a detailed protocol dedicated to efficiently extract and select HMW DNA from C. reinhardtii cells. The protocol minimizes DNA-shearing manipulations and comprises an additional step to enrich for HMW DNA. We validated the method by pulsefield gel electrophoresis (PFGE) and measurement of read length from Nanopore sequencing.

Materials and Methods

The protocol described here is also published on protocols.io, dx.doi.org/10.17504/protocols.io.8epv59j9jg1b/v2 and is included as supporting information file 1 with this article.

Nanopore sequencing

Sequencing libraries were prepared as per manufacturer's recommendations, using NEBNext companion module (E7180S, NEB) and Ligation Sequencing Kit SQK LSK-109 (Nanoporetech), except for the ligation time, which we increased to 30 min. For each run, 500 ng were loaded on MinION flow cells (R9.4.1, Nanoporetech) and sequenced for 6h to 16h, depending on flow-cell kinetics. Libraries were loaded at least twice, with 1h wash using the manufacturer's washing buffer (EXP-WSH004) between runs. Basecalling was performed using Guppy (version 4.3.4) with parameters set to "high accuracy".

Results

We extracted genomic DNA following the presented protocol (S1 Supplementary Protocol) and applied size selection using the Short Read Eliminator (SRE) kit (Circulomics), an easyto-use method that does not require dedicated devices which is based on a length-dependent precipitation of nucleic acids driven by polyvinylpyrrolidone crowding. Large amounts of small DNA fragments can be detrimental for long-read Nanopore sequencing [START_REF] Delahaye | Sequencing DNA with nanopores: Troubles and biases[END_REF], not only because the subsequent reads are short, but also because these molecules can outcompete the longer ones, both for adapter ligation and pore usage, thus yielding suboptimal results.

The size distribution of the extracted DNA was assessed by PFGE and Nanopore sequencing, with and without size-selection for HMW DNA. Samples were migrated in a pulse field, stained by ethidium bromide and imaged with UV light (Fig. 1a). The DNA molecules extracted without size selection migrated as a large smear spread between approximately 1.5 and 150 kb. After size selection with the SRE kit, the upper part of the distribution remained unchanged while the low-molecular-weight fragments (< 10 kb) were visibly reduced. We made a similar observation after electrophoresis and staining of the samples in a 0.3% agarose gel (Fig. 1b).

Size-selection of DNA fragments before preparation of libraries for Nanopore sequencing reproducibly led to a substantially decreased number of shorter molecules and an enrichment of longer ones (Fig. 2a-c), without negatively affecting read quality (Fig. 2d) and with no effect on genome-wide sequencing depth (S3 Supplementary ). The longest molecules we sequenced were over 100 kb, which are instrumental for genome assemblies.

  Figure). Size-selection doubled the mean read length, increased the N50 from 12 kb to 17 kb (20 kb in two other experiments), with reads in the top decile being longer than 21 kb (26 kb and 27 kb in two other experiments) (S2 Table

Figure 1 .Figure 2 .

 12 Figure 1. Visualization of extracted genomic DNA size distributions. (a) PFGE using 0.5 µg

Acknowledgements

We thank Samuel O'Donnell for his help in the initial development of this protocol.

Supporting information

S1 Protocol.

Step-by-step protocol, also available on protocols.io: dx.doi.org/10.17504/protocols.io.8epv59j9jg1b/v2 Sample C was sequenced in the presence of control DNA ("DNA CS" from Oxford Nanopore sequencing), which peaked at 3 kb.

S2