Canonical Wnt signaling, which is transduced by β‐catenin and lymphoid enhancer factor 1/T cell‐specific transcription factors (LEF1/TCFs), regulates many aspects of metazoan development and tissue renewal. Although much evidence has associated canonical Wnt/β‐catenin signaling with mood disorders, the mechanistic links are still unknown. Many components of the canonical Wnt pathway are involved in cellular processes that are unrelated to classical canonical Wnt signaling, thus further blurring the picture. The present review critically evaluates the involvement of classical Wnt/β‐catenin signaling in developmental processes that putatively underlie the pathology of mental illnesses. Particular attention is given to the roles of LEF1/TCFs, which have been discussed surprisingly rarely in this context. Highlighting recent discoveries, we propose that alterations in the activity of LEF1/TCFs, and particularly of transcription factor 7‐like 2 (TCF7L2), result in defects previously associated with neuropsychiatric disorders, including imbalances in neurogenesis and oligodendrogenesis, the functional disruption of thalamocortical circuitry and dysfunction of the habenula. ADHD, attention‐deficit/hyperactivity disorderASD, autism spectrum disorderBD, bipolar disorderCA, Cornu AmonisDG, dentate gyrusDRD2, dopamine D2 receptorDISC1, disrupted in schizophrenia 1DVL, DishevelledGSK3α/β, Glycogen Synthase Kinase 3α or βHMG, high mobility groupiOLs, immature premyelinating oligodendrocytesIPs, intermediate progenitorsiPSCs, induced pluripotent stem cellsLRP5/6, lipoprotein receptor‐related protein 5 or 6LEF1/TCFs, lymphoid enhancer factor 1 and T cell‐specific transcription factorsMD, major depressionmOLs, myelin‐producing oligodendrocytesNDD, neurodevelopmental disorderOPCs, oligodenrocyte precursor cellsRG, radial gliaSCZ, schizophreniaSNP, single nucleotide polymorphismsSVZ, subventricular zoneTCF7L2, transcription factor 7‐like 2VZ, ventricular zoneThe present view posits that the causes of mental disorders are multifactorial and involve an interplay between various innate vulnerabilities and biological and social environmental risk factors that eventually manifest as mood swings, social deficits, and alterations of the perception of reality. Accumulating evidence suggests that mental disorders that are diagnosed by psychiatrists as different entities are closely related from a biological point of view. Genetic risk factors overlap across these disorders The first demonstration of an association between the canonical Wnt pathway and mental disorders was the discovery that the classical mood stabilizer lithium activated β‐catenin by inhibiting GSK3α/β The term ‘canonical Wnt signaling’ has been used as an umbrella term to describe diverse cellular pathways that are activated by Wnt ligands and diverge at the level of Glycogen Synthase Kinase 3α or β (GSK3α/β) inhibition Fig. , Box 1, Box 12+via LEF/TCFs Wnt signaling is traditionally divided into three main branches: the canonical, planar cell polarity (Wnt/PCP) and Wnt/CaRecent reviews have broadly discussed the role of Wnt signaling in the development of pathologies that are associated with mental disorders. However, they focused more either on the role of the divergent GSK3α/β and divergent Wnt/β‐catenin pathways in dendritogenesis, synaptogenesis, and synaptic plasticity, or on upstream Wnt signaling de novo mutations in large populations of patients can help to identify molecular pathways that contribute to the pathogenesis of psychiatric disorders WNT1 and WNT2 with autism spectrum disorder (ASD), WNT2B with bipolar disorder (BD), WNT7A with ASD and BD, WNT7B with schizophrenia (SCZ), LRP5 with SCZ, attention‐deficit/hyperactivity disorder (ADHD) and major depression (MD), and LRP6 with ADHD CTNNB1 gene, which encodes β‐catenin, has also been associated with ASD and SCZ The vulnerability to mental illnesses is exacerbated by genetic factors. Although a single causal gene is unlikely to be identified, rates of common polymorphisms and the occurrence of TCF7L2 has been repeatedly associated with mental disorders in both gene candidate and genome‐wide association studies , should not be confused with transcription factor 4 , which is a SCZ risk factor itself but does not belong to the LEF1/TCF family. Several common intronic polymorphisms and polymorphisms in the proximity of the TCF7L2 locus have been associated with SCZ TCF7L2 was identified as a BD susceptibility factor in patients with elevated body mass de novo splice site mutations of TCF7L2 were found in ASD patients Among genes that encode downstream nuclear effectors of the canonical Wnt/β‐catenin pathway, only Gsk3b overexpression in a dopaminergic region Gsk3b gene knockout in dopamine D2 receptor (DRD2)‐expressing neurons, which are affected in SCZ, decreased amphetamine‐induced hyperlocomotion and prepulse inhibition, mimicking antipsychotic actions Gsk3b overexpression or genetic blocking of inhibitory phosphorylation of GSK3) increased amphetamine‐induced hyperactivity Dvl1 knockout), which is inhibitory for the GSK3α/β containing destruction complex in the canonical Wnt pathway, exhibited social deficit and stereotyped behavior, reproducing aspects of ASD Manipulations of individual components of the canonical Wnt pathway in animal models Table  resultedGsk3b knockout in DRD2 neurons, thus excluding such a contribution in this case The involvement of alterations of GSK3α/β or upstream Wnt signaling activity does not prove the involvement of β‐catenin, not to mention nuclear β‐catenin and LEF1/TCFs. For example, the expression of a stabilized form of β‐catenin that is resistant to GSK3α/β‐mediated degradation  are involved in the pathogenesis of mental disorders.The cerebral cortex is the outer layer of the brain. The main part of the cortex in mammals is the neocortex, which processes and integrates emotional and sensory information, controls problem solving and decision‐making. Development of the gyrated human neocortex requires the particularly intensive expansion of neural progenitor populations to produce sufficient numbers of neurons.Neuroimaging showed changes in cortical thickness and the cortical surface in patients who suffer from psychiatric disorders. These phenotypes may result from excessive or insufficient synaptic pruning or myelination that occurs in adolescents, but also from impaired neurogenesis, e.g., alterations of the ratio of symmetrical cell divisions and asymmetrical cell divisions of neural progenitors. In adult individuals who were diagnosed with MD, a decrease in the thickness of cortical gray matter was observed specifically in the frontal and temporal lobes Neurons in the neocortex are generated in the ventricular zone (VZ) of the dorsal telencephalon from uncommited radial glia (RG) and in the subventricular zone (SVZ) from neural intermediate progenitors (IPs) Wnt7a expression, and the presence of LEF1, TCF7L1, and TCF7L2 The canonical Wnt/β‐catenin signaling pathway is active in the cortical VZ during embryogenesis, as demonstrated by studies in Wnt reporter mice, measurement of Many studies have investigated the role of β‐catenin in different aspects of neurogenesis and neural cell migration. Much effort has been expended to differentiate its function between the regulation of cell adhesion and gene transcription (a focus of the present review), by using genetic modifications in mice Table .Ctnnb1 knockout had the opposite effect, in which it prematurely decreased the number of proliferating cells in the VZ and led to smaller cortical hemispheres at birth dm with intact adhesive function  also resulted in the depletion of progenitor cells and the precocious production of neurons, despite normal VZ architecture Enhancing canonical Wnt signaling by β‐catenin stabilization has consistently led to the expansion of a progenitor pool that starts at midgestation followed by neocortical hyperplasia Lrp6 knockout, Ctnnb1 knockout or a Ctnnb1dm mutation SVZ and postmitotic layer transiently thickened during early neurogenesis, before the depletion of RG finally led to a dramatic decrease in the number of neurons at late neurogenesis or postnatally Studies in which the uncommitted RG and committed IPs were analyzed as separate populations revealed opposite effects of canonical Wnt/β‐catenin signaling in each population Fig. . In miceTcf7l2 exhibited a decrease in progenitor proliferation at late neurogenesis and had a smaller cortex dmCtnnb1dm mutant mice, whereas development of the cortex was unaffected at this stage in Tcf7l2 knockout mice, and the ratio between RG and IPs was unaltered. This suggests that another transcription factor from the LEF1/TCF family, in addition to TCF7L2, is a mediator of the canonical Wnt/β‐catenin pathway in the developing cortex. Tcf7l1/Tcf7l2 double‐knockout did not deteriorate or rescue the phenotype of single Tcf7l2 knockout, thus excluding TCF7L1. LEF1 might compensate for the loss of TCF7L2 during neurogenesis in the neocortex, but this hypothesis needs to be tested in Lef1/Tcf7l2 double‐knockout mice.A recent study shed some light on which transcription factors of the LEF/TCF family mediate the above effects. Neither LEF1 Lrp6 knockout, Ctnnb1 knockout, and a Ctnnb1dm mutation caused a precocious switch from neurogenesis to astrogenesis in the neocortex Ctnnb1 knockout and a Ctnnb1dm mutation accelerated the production of upper‐layer neurons Further complicating the role of canonical Wnt signaling in neurogenesis is the involvement of this pathway in the regulation of the timing and duration of neurogenesis. Ctnnb1 knockout in the cortical neuroepithelium led to the early loss of RG apical adherens junctions Ctnnb1dm mutation or the knockout of Lef1, Tcf7l1, or Tcf7l2in utero‐delivered dnTCF7L2 reduced migratory speed in cortical layers, but it did not affect RG scaffolds Finally, some studies have investigated the role of nuclear β‐catenin in the establishment of ordered cortical structure. Both the expression of stabilized β‐catenin and Dvl1−/− mice), in which Wnt signaling was decreased but not entirely abolished, exhibited transient increases in brain weight and cortical thickness at mid‐gestation, accompanied by a reduction of RG and transient expansion of IP population and deep layer neurons. In adulthood, the cortex of these mice was smaller than controls (consistent with RG depletion), but the frontal cortex was thicker The association between Wnt signaling, cortical size, and adult behavioral anomalies has been demonstrated in mouse studies. Mice with deficiency in DVL activity , a chromatin remodeling factor which regulates Wnt signaling. in vitro cell differentiation, and brain organoid technologies have provided novel opportunities to model SCZ and BD. The causal role of canonical Wnt pathway downregulation was investigated in neural lineage cells that were derived from two related BD patients WNT7B downregulation compared to cells derived from healthy relatives. The treatment of these cells with the canonical Wnt agonist CHIR99021 significantly increased LEF1 levels and rescued proliferation deficits. Finally, XAV939, an inhibitor of the canonical Wnt/β‐catenin pathway (a stabilizer of Axin in the β‐catenin destruction complex), rescued morphological impairments in iPSC‐derived cerebral organoids with mutations of the disrupted in schizophrenia 1 (DISC1) gene Human induced pluripotent stem cells (iPSCs), The hippocampus is the main part of the evolutionary old allocortex that is composed of two layers of neurons: granule neurons in the dentate gyrus (DG) and pyramidal neurons in Cornu Amonis (CA) fields. This structure is involved in episodic memory formation and the consolidation of emotional memories. An interesting feature of the hippocampal DG is its ability to produce new neurons throughout life. Adult neurogenesis in the hippocampus is presumed to occur in both humans and rodents and play a role in cognitive plasticity.Neuroimaging studies have repeatedly reported the loss of hippocampal volume in individuals who are diagnosed with ADHD, BD, SCZ, ASD, and MD Despite growing interest in adult hippocampal neurogenesis in the context of mental disorders, its causative role is still controversial. Studies on animals are also inconclusive. For example, neogenin‐deficient mice exhibited depressive‐like behavior Lef1 is higher in hippocampal progenitors in both the hippocampal VZ and migratory stream compared with the neocortical VZ Lef1.In general, canonical Wnt signaling is implicated in similar processes in the hippocampus as in the neocortex; therefore we focus on differences between the two regions. The developing hippocampus is located within direct proximity to the hem, which secretes Wnt ligands Wnt3a−/− mice with conditional Ctnnb1 knockout, or mice that express LEF1 without the DNA binding domain  the hippocampal VZ was truncated at midgestation, and the hippocampus was virtually absent at the perinatal stage Dkk1, which encodes a conanical Wnt signaling inhibitor Dickkopf 1 that binds to LRP5/6, Lrp6 knockout or Lef1 knockout, which only partially inhibited the canonical Wnt pathway, had less severe effects. Nevertheless, the DG, whose progenitor domain is located in direct vicinity to the hem, was smaller Lef1−/− mice, granule cells were absent in the DG, despite a normal number of proliferating cells in the primary neurogenic niche in the VZ Lef1 expression, were normally produced in these mice, suggesting a redundant role for LEF1 and other LEF1/TCF family members in this area, similar to the neocortex. Tcf7l1 knockout did not result in any hippocampal alterations, but the size of both the granule and pyramidal cell layers of the hippocampus was markedly reduced in Tcf7l2 knockout mice Consistent with high activity of the canonical Wnt/β‐catenin pathway, the inhibition of canonical Wnt signaling at the selective level of signal transduction or LEF1/TCFs had more pronounced effects in the hippocampus than in the neocortex. Wnt3Wnt7aWnt3 or dominant negative LEF1 (dnLEF1) in the SGZ, confirmed its involvement in adult neurogenesis in the hippocampus Postnatally, the population of hippocampal progenitors becomes restricted to the subgranular zone of the DG and transforms into adult neural stem cells that continue to divide, although at a much lower rate than in embryos. Adult neurogenesis is not simply an extension of a developmental process into the adulthood because the environment in the postnatal brain is not more neurogenic Wnt2, Fz9, Frzb, Lef1, Ctnnb1, and Dvl1 expression in the hippocampus Growing evidence implicates canonical Wnt signaling‐associated defects in adult hippocampal neurogenesis in pathogenesis of psychiatric conditions. Nonetheless, much of this evidence is indirect and correlative, and, so far, no solid evidence has linked canonical Wnt‐regulated adult neurogenesis and mental disorders. Many studies reported changes in the expression of the upstream and downstream components of canonical Wnt signaling in the hippocampus of animals that were treated with antidepressant and antipsychotic drugs. For example, chronic administration of selective serotonin reuptake inhibitors increased Disc1 silencing, and the canonical Wnt agonist SB216763 (an inhibitor of GSK3β) normalized depression‐like behavior in these mice. This indicates that DISC1 acts through the canonical Wnt pathway to regulate neurogenesis and modulate mental homeostasis. However, whether the protective role of β‐catenin depends on the rescue of neurogenesis, and whether downstream Wnt signaling (nuclear of β‐catenin and LEF/TCFs) is involved has not been directly tested.The connection between adult hippocampal neurogenesis, the canonical Wnt/β‐catenin pathway, and mental disorders has been demonstrated by investigations of DISC1, which can stabilize β‐catenin through direct GSK3β inhibition. In both the developing and adult DG, DISC1 maintains neural progenitor proliferation The thalamo‐habenular region is a subcortical part of the brain derived from a common developmental progenitor domain. The thalamus relays sensorimotor information to the cortex, process this information in cortico‐thalamic network, and produce goal‐directed behaviors through cortico–basal ganglia–thalamic circuits Psychiatric disorders have been repeatedly linked with disturbances in thalamic anatomy and connectivity. Decreases in the volume of the thalamus, as well as reductions of functional connectivity between the thalamus and prefrontal region, with an increase in functional connectivity with the motor cortex are commonly detected in SCZ and BD Tcf7l2 and Lef1 was specifically observed during postmitotic development and in adulthood in the thalamus, habenula, and some midbrain structures in vertebrates, including primates Tcf7l2 in thalamic neurons prevented β‐catenin from entering the nucleus Abundant expression of Tcf7l2−/− brain, many of them specific for the thalamus and habenula Tcf7l2 , the expression of these genes was not altered, but the expression of many genes that are induced in this region postnatally was downregulated, among them ion channel and neurotransmitter genes. These results imply that both postmitotic developmental genetic program and postnatal switch in gene expression in the thalamo‐habenular region are controlled by TCF7L2. Further research should address the mechanism of differential gene expression regulation by TCF7L2 in the two developmental stages.TCF7L2 is not required for initial specification and neurogenesis of the thalamo‐habenular region but its role is important at later developmental stages Dcc, Unc5, Robo3, Epha3, Epha8, Slit3, Nrp1, and Ntng1Cntn4, Cntn6, and Reln), which potentially regulate cell migration and axon outgrowth, are another group that is affected by Tcf7l2 knockout. All of these genes have been linked with SCZ Some thalamic dysfunctions might arise from improper neuronal migration and aberrant thalamocortical circuit development, considering that many risk factor genes for psychiatric diseases are involved in this process. For example, numerous genes that are associated with SCZ and ASD encode axon guidance molecules that belong to the families of SLIT and ROBO, Ephrins and EPH receptors, DCC and UNC5, Semaphorins and Plexins, and Neuregulin Tcf7l2 knockout, are also often associated with psychiatric disorders. Among ion channels whose expression is altered in the adult TCF7L2‐deficent thalamus is CACNA1C, which encodes a Ca2+ channel. Mutations of these genes are associated with SCZ disorder with the third highest score in the largest genome wide association study to date KCNIP4 are associated with personality disorders and ASD CACNA1G was one of the most downregulated genes in neurons derived from BD patients SLC6A4 has been linked with ASD Ion channels and neurotransmitter genes, some of which were dysregulated in mice with postnatal TCF7L2 and endophenotypes of the thalamus, and more thoroughly investigate the role of this transcription factor in behavior regulation in animal models.These multiple genetic overlaps between embryonic and adult TCF7L2‐target genes in the thalamus and psychiatric conditions imply TCF7L2‐mediated thalamic alterations in the pathogenesis of mental disorders. This hypothesis has not been yet directly addressed. Future research should explain the relationship between risk variants of OLIG2 and SOX10) and genes that encode myelin proteins  with SCZ Oligodendrocytes are myelinating cells in the vertebrate central nervous system. Their role is to provide trophic support and electric insulation to axons to increase the velocity of nerve signal conduction and maintain axonal integrity. The loss of oligodendroglia and cortical demyelination, followed by the demage of axons are the primary pathology in multiple sclerosis and other leukodystrophies. Slowly accumulating evidence implicates disturbances in oligodendrogenesis and impairments in myelination also in the development of mental disorders. However, when compared to neurogenesis, the possible involvement of oligodendrogenesis in the etiology of psychiatric illnesses has been little investigated. Most of this research has focused on SCZ, but some evidence suggests similar endophenotypes in BP. The levels of oligodendrocyte‐ and myelin‐related transcripts were altered in cortical samples from SCZ Oligodendroglia is produced from RG in the VZ/SVZ of the spinal cord and several niches in the brain during embryonic development and postnatally, after neurogenesis is completed. In rodents, most of oligodendrocytes in the adult cortex are derived from the cortical VZ/SVZ of the dorsal telencephalon. OPCs are produced in this niche postnatally and migrate radially into the corpus callosum (a nerve tract beneath the cortex) and cortex. The generation of functional oligodendrocytes occurs through several intermediate stages. Proliferating OPCs generate immature premyelinating oligodendrocytes (iOLs), which finally maturate into myelin‐producing oligodendrocytes (mOLs) Fig.  132.Ctnnb1 in oligodendroglia lineage cells in utero injection of Dkk1 or a construct that encoded dnLEF1 to downregulate Wnt/β‐catenin signaling, both of which significantly increased the number of OPCs perinatally in the cortex Oligodendrocyte precursor cells begin to be produced in the cortical VZ/SVZ when the level of canonical Wnt/β‐catenin signaling activity, which is high during neurogenesis, decreases Tcf7l2 transcripts were first observed in OPCs, increased to the highest levels in iOLs, and then decreased to lower levels in mOLs Tcf7l2 knockout or TCF7L2ΔHMG mutation) and in mice with the ectopic expression of stabilized (β‐cateninΔexon3) in maturing oligodendroglia, myelin did not form properly in the corpus callosum or spinal cord during development and in demyelination lesions At subsequent stages of oligodendrogenesis that follow the generation of OPCs, the activity of canonical Wnt/β‐catenin signaling was detected in subsets of differentiating and maturating cells in vitroThese puzzling findings were resolved by the genome‐wide mapping of TCF7L2 chromatin occupancy at different stages of oligodendrocyte differentiation In summary, after the generation of OPCs, further steps of oligodendrogenesis are sequentially regulated by TCF7L2 Fig. . Wnt/β‐cSOX10 and myelin‐associated genes Possible direct links between mental disorders, Wnt, TCF7L2, and oligodendrogenesis have not been investigated. The only indication that such links exist comes from the aforementioned genetic studies that associated mental disorders with TCF7L2 are among the strongest risk factors for diabetes, and TCF7L2 has been extensively studied in the context of peripheral metabolism regulation, but studies on its role in brain development and pathologies have been relatively scarce. Future research on animals with conditional mutations of Tcf7l2 may help confirm putative links between TCF7L2, brain phenotypes, and behavioral deficits. Identifying relationships between molecular and cellular events, structural and functional alterations in the brain, and behavioral deficits might contribute to the development of personalized treatments for mental disorders.Changes in the volume of cortical areas, thalamo‐cortical dysconnectivity, and white matter microstructural alterations are commonly identified endophenotypes in psychiatric research. Evidence from animal studies strongly implicates canonical Wnt/β‐catenin signaling and TCF7L2‐dependent transcription in the development of these impairments, and human genetic studies have found associations between this pathway and major mental disorders. Variants of


Taken together, we demonstrate that LOC100129940-N promotes the activation of Wnt/β-catenin signaling, which in turn regulates the downstream target genes, thereby enhancing invasion and progression of PTC.Thyroid cancer is the most common endocrine malignancy, and its incidence has increased rapidly in recent decades worldwide. Papillary thyroid cancer (PTC) is the most common type of all thyroid cancers. The molecular mechanisms underlying the disease still need to be further investigated. Long noncoding RNAs (lncRNAs), a class of noncoding RNAs (ncRNAs) longer than 200 nucleotides, are aberrantly expressed in malignant diseases, including PTC. Here, we identified a novel isoform of LOC100129940 and designated it as LOC100129940-N. We demonstrated that the expression level of LOC100129940-N was elevated in PTC, indicating that LOC100129940-N may be involved in PTC development and progression. Moreover, our results showed that overexpression of LOC100129940-N promoted, whereas silencing of LOC100129940-N suppressed, PTC cell proliferation, invasion, and migration. Mechanistically, LOC100129940-N played an important role in activating Wnt/ Thyroid cancer is the most common endocrine malignancy, and its incidence has increased rapidly in recent decades worldwide , 2. ThyrLong noncoding RNAs (lncRNAs) are a class of noncoding RNAs longer than 200 nucleotides . Accumulβ-catenin signaling is involved in various processes that are pivotal for cancer development and progression, including cancer initiation, growth, invasion, and metastasis [β-catenin is degraded by the destruction complex, whose components include Axin, adenomatous polyposis coli (APC), glycogen synthase kinase-3 (GSK3), and casein kinase 1 (CK1). In this complex, β-catenin is phosphorylated and subsequently destructed by the proteosomal machinery [β-catenin and increases its nuclear translocation, resulting in the regulation of downstream target genes [β-catenin signaling also plays an important role in PTC [β-catenin signaling in PTC will put new insights into the etiology of the disease.Wnt/tastasis –19. Withachinery , 21. Binachinery , 23. Thiet genes . Accumule in PTC –27. Furt2.The human PTC cell line KTC1 was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences . TPC1 (human PTC cell line) was kindly provided by Prof. Haixia Guan . KTC1 and TPC1 cell lines were characterized and authenticated using short tandem repeat (STR) DNA profiling. The cells have been validated to be mycoplasma-free. KTC1 and TPC1 cells were incubated in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum  at 37°C and 5% COA total of 59 cases of fresh PTC and 11 pairs of fresh PTC and their matching noncancerous tissues were obtained from the First Affiliated Hospital of Sun Yat-sen University between 2015 and 2017. Three normal thyroid tissues were obtained from donors who died in traffic accidents, and none of them has detectable pathological conditions. All specimens were histopathologically and clinically diagnosed by two pathologists. Written informed consent was obtained from patients for use of the samples. Ethical approval was obtained from the Institutional Research Ethics Committee.The transcriptional initiation and termination sites of LOC100129940-N were determined by 5′ and 3′ RACE-polymerase chain reaction (PCR) using the SMARTer RACE 5′/3′ Kit , according to the supplier's protocol. Primers used for the RACE-PCR analysis are as follows: 5′ RACE, 5′-TCTGGGTTGCTGGGTGCTTTCTTTG-3′, and 3′ RACE, 5′- CACCTCACTGACTGCTCACCTGT-3′.−ΔΔCt method. The sequences of all primers for the indicated genes are listed in Supplementary Total RNA of cultured cells or fresh tissues was extracted using TRIzol (Invitrogen). The first-strand cDNA was obtained using Moloney murine leukemia virus reverse transcriptase  with random primers. qRT-PCR was performed using SYBR©Green PCR Master Mix  on a CFX96 real-time PCR detection system . GAPDH was used as an internal control to normalize the indicated genes. Each sample was analyzed three times in triplicate. Data are calculated by the 2A commercial cell fractionation kit  was used to isolate nucleus/cytoplasm RNA extracts in accordance with the manufacturer's instruction.μm filter. Subsequently, the supernatant was incubated with the indicated cells in the presence of 8 μg/ml polybrene. For the selection of stable cell lines, the cells were cultured with 0.5 μg/ml of puromycin for 14 days [The full length of LOC100129940-N was obtained by PCR and cloned into the retroviral expression vector pQCXIP . We used empty pQCXIP as a vector control. The indicated plasmids were transfected into packing cells , an NIH3T3-derived cell line expressing the 10Al viral envelope, using Lipofectamine 3000 (Invitrogen). The supernatant from the transfected PT67 cells was then harvested and passed through a 0.45  14 days .Specific lncRNA Smart Silencer for LOC100129940-N was designed and synthesized by RiboBio . LOC100129940-N Smart Silencer is a mixture of three siRNAs and three antisense oligonucleotides (ASOs). The target sequences of siRNAs are as follows: 5′-CCATTGGAATCTCCTCTCA-3′, 5′-ACAGTACCATTCTAGCAAA-3′, and 5′-CCACAGGGCAAATAAAGCT-3′. The target sequences of ASOs are as follows: 5′-CAGCTCACATCAAGTCACCG-3′, 5′-CCATTATGTCTCCCAGTTCT-3′, and 5′-TCCACGTCAACATTCCACCC-3′. The negative control (NC) Smart Silencer does not contain domains homologous to humans, mice, and rats. Transfection was performed using Lipofectamine 3000 (Invitrogen), in accordance with the manufacturer's instructions.4 indicated cells suspended in 200 μl of serum-free medium were added to the apical chamber of Transwell inserts  precoated with or without Matrigel . Then, the inserts were placed in 24-well plates containing medium with 10% FBS. After 24 h incubation, the noninvading and nonmigrating cells on the upper side of the inserts were removed by wiping with cotton swabs. Subsequently, the migrated and invaded cells on the undersides were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet staining solution. The number of cells in 3 randomly selected fields was counted under a microscope. Each sample was analyzed three times in triplicate.Transwell invasion and migration assays were performed as previously described . Brieflyμl pipette tip to scratch on the cell monolayer. Then, cells were washed with phosphate-buffered saline (PBS) to remove nonadherent cells, and wound closure was photographed  at 18 h under serum-reduced medium. The percentage of wound closure was calculated using the ImageJ software . Each experiment was performed in triplicate and repeated three times.The wound healing assay was performed to investigate the alteration of cell motility and migration as previously described . BrieflyThe EdU incorporation assay was carried out according to the manufacturer's instruction  as previously described . Edu-pos6 indicated cells were harvested and washed in ice-cold PBS, followed by fixation in 80% ethanol in PBS overnight at -20°C. Then, the cells were spun down and resuspended in cold PBS. The resuspended cells were incubated with 2 μg/ml bovine pancreatic RNase , at 37°C for 30 min, followed by incubating cells for 20 min at room temperature with 20 μg/ml of propidium iodide . Flow cytometry data were acquired with a flow cytometer  and analyzed using FlowJo  [The proportion of the cells  in the distinct cell cycle phase was determined by flow cytometry. At 60% confluence, cells were serum-deprived for 24 h for cell cycle synchronization and then incubated for 16 h in 10% FBS DMEM after synchronization. Thereafter, 1 × 10μg) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electroblotting to the PVDF membranes, the membranes were blocked with 5% nonfat milk for 1 h at room temperature. The membranes were then incubated with the primary antibodies anti-β-catenin antibody, anti-p84 antibody, anti-TCF4 antibody , and anti-α-tubulin antibody . An enhanced chemiluminescence (ECL) detection kit  was used to detect the bands according to the manufacturer's instructions.WB analysis was performed according to standard protocols as previously described . RIPA lyt-test. The one-way ANOVA test was used to analyze the differences between multiple comparisons. Descriptive values are expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant. The SPSS 17.0 software  was used for statistical calculations.The statistic differences between two groups were analyzed by paired, two-tailed Student's >100 amino acids) ORFs  ORFs . Moreoveds) ORFs . The RNAWe next investigated the expression of LOC100129940-N in PTC. Using the specific primer for LOC100129940-N, we analyzed its expression in 11 pairs of PTC specimens and matched nontumorous thyroid tissues. As presented in To investigate the potential biological functions of LOC100129940-N in PTC, we then established stable cell lines overexpressing LOC100129940-N, namely, TPC1/LOC100129940-N and KTC1/LOC100129940-N . InitialTo further investigate the biological role of LOC100129940-N in PTC cells, we depleted the expression of LOC100129940-N using the lncRNA Smart Silencer as described in Materials and Methods . As showκB signaling and Wnt/βcatenin signaling using specific reporters. We found that the activity of NF-κB signaling was not significantly altered in response to LOC100129940-N overexpression  cell proliferation and metastasis . Moreoveβ-catenin signaling may be involved in the biological effects of LOC100129940-N on PTC. Indeed, our data demonstrated that overexpression of LOC100129940-N in PTC cells led to the activation of Wnt/β-catenin signaling. In contrast, depletion of LOC100129940-N inhibited the Wnt/β-catenin signaling activity in PTC cells. Our findings concluded that the molecular basis for the oncogenic roles of LOC100129940-N in PTC might relate to the activation of Wnt/β-catenin signaling. Several lines of evidence are provided to support this conclusion. First, β-catenin-Tcf/Lef activity is upregulated and nuclear translocation of β-catenin is increased in response to ectopic overexpression of LOC100129940-N. Second, inhibition of Wnt/β-catenin signaling with Tcf4-specific siRNAs could abrogate the effects induced by LOC100129940-N. Third, the expression of LGR5, FN1, VEGFA, SOX9, and CCND1, which are important for cell invasion and growth, was elevated by LOC100129940-N overexpression. Fourth, silencing of LOC100129940-N suppressed the activation of Wnt/β-catenin signaling and the expression of downstream target genes.To address the molecular mechanisms involved in LOC100129940-N-induced cell proliferation, invasion, and migration, we found that Wnt/β-catenin signaling has been demonstrated in various malignant diseases, including thyroid cancer [β-catenin signaling in PTC are not well understood. Here, we found that LOC100129940-N activated the Wnt/β-catenin signaling and thereby enhanced the malignant phenotypes of PTC cells. However, the mechanisms underlying the relationship between LOC100129940-N and Wnt/β-catenin signaling need to be further explored.Hyperactivation of Wnt/d cancer , 40. Nevβ-catenin signaling. Through the activation of Wnt/β-catenin signaling and upregulation of downstream target genes, LOC100129940-N promotes PTC cell proliferation, invasion, and migration. These results implicate that targeting LOC100129940-N could be a potential therapeutic strategy.Collectively, our data demonstrate that LOC100129940-N is upregulated in PTC and promotes activation of Wnt/


Beta-catenin  signaling controls CNS angiogenesis and the blood-brain and blood-retina barriers. To explore the role of the Discs large/membrane-associated guanylate kinase (Dlg/MAGUK) family of scaffolding proteins in beta-catenin signaling, we studied vascular endothelial cell (EC)-specific knockout of Dlg1/SAP97. EC-specific loss of Dlg1 produces a retinal vascular phenotype that closely matches the phenotype associated with reduced beta-catenin signaling, synergizes with genetically-directed reductions in beta-catenin signaling components, and can be rescued by stabilizing beta-catenin in ECs. In reporter cells with CRISPR/Cas9-mediated inactivation of Dlg1, transfection of Dlg1 enhances beta-catenin signaling ~4 fold. Surprisingly, Frizzled4, which contains a C-terminal PDZ-binding motif that can bind to Dlg1 PDZ domains, appears to function independently of Dlg1 in vivo. These data expand the repertoire of Dlg/MAGUK family functions to include a role in beta-catenin signaling, and they suggest that proteins other than Frizzled receptors interact with Dlg1 to enhance beta-catenin signaling. The mammalian retina depends on two distinct vascular networks: the choroidal and intra-retinal circulations, which supply the outer third and inner two-thirds of the retina, respectively . The intMature vascular endothelial cells (ECs) of the central nervous system (CNS), including the retina, form a specialized diffusion barrier between blood and tissue known as the blood-brain barrier (BBB), and its retinal analog, the blood-retina barrier (BRB) . The speThe growth and differentiation programs of CNS ECs are controlled by multiple signaling systems, including beta-catenin  signaling . In the On the cytosolic side of the membrane, beta-catenin signaling involves ligand-dependent receptor/co-receptor regulation of the beta-catenin destruction complex . The dynDlg1 transcripts (18 FPKM) are ~7 fold more abundant than transcripts coding for the next most abundant family member, Dlg5 (2.5 FPKM); transcripts for other Dlg family members are present at even lower levels  ] .Dlg1. We provide evidence for a genetic interaction between Dlg1 and Fz4, Tspan12, and Ndp (the gene coding for Norrin), which suggests that Dlg1 stimulates beta-catenin signaling in CNS ECs. This idea is supported by the finding that Dlg1 enhances beta-catenin signaling in cultured cells and by rescue of the Dlg1 EC-specific knockout phenotype upon constitutive beta-catenin stabilization in vivo. Intriguingly, although the first two PDZ domains of Dlg1 bind in vitro to a consensus PDZ-binding motif at the Fz4 C-terminus, genetic epistasis analyses of the EC-specific Dlg1 knockout and a CRISPR/Cas9-generated mutation of the Fz4 PDZ-binding motif argues that these two proteins function independently in the context of beta-catenin signaling.In the present study, we have identified a role for endothelial Dlg1 in retinal angiogenesis and BBB/BRB development as determined by the phenotype of mice with EC-specific knockout of Dlg1 leads to neonatal lethality  time points: P7, P14, and P18 , which was undetectable in WT controls  whether the phenotype associated with the combined loss of both genes is more severe than the phenotypes associated with the loss of either gene alone , eliminating endothelial Dlg1 , or halving the dosage of endothelial Dlg1 on the background of a Tspan12 knockout  produced no loss of GLUT1 expression, no increase in PLVAP expression, and no BBB breakdown as assessed by sulfo-NHS-biotin leakage  partially compromises the cerebellar BBB, as seen by a small decrease in the fraction of GLUT1+ ECs, an increase in the fraction of PLVAP+ ECs, and localized sulfo-NHS-biotin leakage  vs. homozygous loss of Dlg1 on the background of a Ndp knockout  shows a dramatic worsening of BBB breakdown in both the cerebellum and hindbrain with loss of both Dlg1 alleles  treatment in the immediate postnatal period because constitutive EC-specific activation of beta-catenin signaling, as produced by flex3/+;Tie2-CreCtnnb1, is lethal during embryonic development.To directly test the idea that Dlg1 promotes beta-catenin signaling in CNS ECs, we have asked whether the phenotypes caused by loss of Dlg1 can be rescued by artificially activating beta-catenin signaling in a manner that likely bypasses Dlg1 function. Although the site of Dlg1 function has not been defined by the genetic interaction experiments described above, by analogy with Dlg1 function in other contexts, it is a reasonable guess that Dlg1 organizes membrane-associated proteins in ECs. In the context of beta-catenin signaling, Dlg1 could interact directly or indirectly with receptors, co-receptors, the beta-catenin destruction complex, and/or regulators of the destruction complex. With this model in mind, we have asked whether the Dlg1  produces a substantially reduced vascular density compared to WT controls  . We note that the vascular coverage is more similar in a comparison between fl/Δ;Pdgfb-CreERDlg1 vs. fl/ΔDlg1 control retinas , as seen in Strikingly, the retinal vascular defects in To further assess the role of Dlg1 in beta-catenin signaling, we began by asking whether over-production of Dlg1 enhances signaling in a Super TOP Flash (STF) luciferase reporter cell line in the presence of Norrin and Fz4, which together produce a 20-50x induction of luciferase over background . Figure Dlg1 knockout (KO) cell lines (Clone #12 and Clone #15) from the starting STF cell line using CRISPR/Cas9  or monotonic (Dlg2 and Dlg4) plasmid concentration dependence  or V5 tag (Dlg5) showed protein accumulation at the expected molecular weights in transfected HEK/293T cells . In Dlg1pendence . In contpendence . These dAs noted in the Introduction, the simplest and most attractive model for Dlg1 stimulation of beta-catenin signaling posits a direct interaction between one or more of the Dlg1 PDZ domains and the canonical PDZ-binding motif at the C-terminus of many Frizzled receptors, including Fz4. Using the yeast two-hybrid system, a previous study reported that a C-terminal peptide from Fz4, which ends with the consensus PDZ-binding motif ETVV  affected stimulation by co-transfected Dlg1. A comparison of the luciferase response of n WT Fz4 , roughlyn WT Fz4 . ImportaFz4 allele with a mutated C-terminus that is unable to bind to Dlg1. For this experiment, the C-terminal three codons of Fz4 were mutated from TVV to MGK, hereafter referred to as MGKFz4  event in an experiment in which we failed to obtain a 4AFz4 allele by homology-directed repair (HDR).] As would be predicted based on structure-function analyses of the interaction between PDZ-binding motifs and the Dlg1 PDZ domains  should produce a retinal vascular phenotype that is no more severe than that produced by MGK/MGKFz4 and (ii) EC loss of Dlg1 on a MGK/MGKFz4 genetic background  should produce a phenotype that is no more severe than that produced by homozygous loss of Dlg1 alone. Contrary to the first of these predictions, at P18 fl/Δ;Tie2-CreDlg1 retinas show mildly elevated PLVAP expression in ECs and mild sulfo-NHS-biotin leakage  EC loss of  leakage , neither leakage  – phenotDlg1 and EC-specific reductions in beta-catenin signaling, (ii) the phenotypic synergy produced by the combination of EC-specific loss of Dlg1 and loss-of-function mutations in Fz4 or Tspan12 (in the retina) and Ndp or Tspan12 (in the brain), (iii) the nearly complete rescue of EC-specific loss of Dlg1 by EC-specific stabilization of beta-catenin, and (iv) the 3–4 fold enhancement of beta-catenin signaling stimulated by multiple Wnts or Norrin in Dlg1 knockout cell lines in response to Dlg1 transfection. Taken together, these data reveal a new role for a member of the Dlg protein family and they suggest that the phenotypes associated with loss of Dlg1 in mammals (and Dlg in Drosophila) may be, at least in part, referable to defects in beta-catenin signaling. It would be interesting to explore the possibility that additional molecular changes might accompany the loss of Dlg1 in ECs; such changes might be revealed by comparing the EC transcriptomes of WT and Dlg1 conditional knockout mice.The experiments described above provide strong evidence that the scaffolding protein Dlg1 enhances beta-catenin signaling in ECs in the context of CNS angiogenesis and BBB/BRB development and maintenance . This coMGK) has no apparent retinal vascular phenotype – that is, it does not recapitulate the phenotype associated with EC-specific loss of Dlg1. It is further supported by the observation that combining Fz4MGK and EC-specific loss of Dlg1 produces a retinal vascular phenotype that is substantially more severe than that produced solely by EC-specific loss of Dlg1. While these observations do not preclude the possibility of an in vivo interaction between Dlg1 and the Fz4 C-terminus, they imply that an interaction between the Fz4 C-terminus and a different partner is of greater functional significance. The most parsimonious explanations for the genetic data are that (i) loss of the Fz4 C-terminal motif reduces beta-catenin signaling in vivo (as it does in transfected cells) but not so much as to produce a retinal vascular phenotype, (ii) loss of Dlg1, which interacts with proteins other than Fz4, reduces beta-catenin signaling in vivo to produce a mild retinal vascular phenotype, and (iii) simultaneous loss of both the Fz4 C-terminal motif and Dlg1 reduces beta-catenin signaling further than with either mutation alone, producing a more severe vascular phenotype.A surprising finding is that the binding between the Fz4 C-terminal PDZ-binding motif and the PDZ domains of Dlg1 – which is robustly observed with synthetic peptides and recombinant proteins produced in bacterial or mammalian systems – appears to play little or no role in the Dlg1-dependent enhancement of beta-catenin signaling in CNS ECs. This possibility was initially suggested by the observation that mutation of the Fz4 C-terminal PDZ-binding motif in vivo , Dlg2 (PSD-93/Chapsyn-110), Dlg3 (SAP-102), and Dlg4 (PSD-95/SAP-90) control receptor and channel localization and trafficking, and loss or over-expression of Dlg family members alters synaptic transmission and/or synaptic plasticity . In the While our data support a role for Dlg1 in stimulating beta-catenin signaling in CNS ECs, further studies will be needed to explore this function in other cell types. The STF data presented here imply that Dlg2 and Dlg4 also have beta-catenin stimulatory activity . WhetherIn the context of CNS ECs, the data presented here imply that Dlg1 enhances beta-catenin signaling by interacting with one or more proteins other than Fz4. In the paragraphs that follow, we discuss candidate interactions that might mediate this activity, with a focus on PDZ interactions. We note, however, that multiple domains of Dlg1 are likely to be relevant, based on the domain deletion experiments in One obvious candidate for interacting with Dlg1 in the context of beta-catenin signaling is the Adenomatous Polyposis Coli (APC) protein, which has a C-terminal PDZ-binding motif that binds to the PDZ1 and PDZ2 domains of Dlg1 with affinities of 18 uM and 1 uM, respectively . The corA second candidate for interacting with Dlg1 in the context of beta-catenin signaling in CNS ECs is Gpr124, a seven transmembrane protein in the adhesion GPCR family that functions in concert with the GPI-anchored protein Reck to enhance Wnt7a/b-specific signaling through Frizzled receptors and Lrp5/6 co-receptors . Gpr124 Dlg1 and Fz4 genetic interaction experiments described here, it would be of interest to examine genetic interactions between Dlg1 and other Frizzled genes. Late gestation -/-Dlg1 fetuses exhibit (i) mis-orientation of the stereociliary bundles in inner ear sensory hair cells, (ii) defects in the closure of the palate, neural tube, and eyelids, and (iii) multiple cardiac defects, including ventricular septal defect, persistent truncus arteriosus, and double outlet right ventricle  targeting exon 2 of Fz4 was selected and synthesized, as described by The flDlg1 , and Pdgfb-CreER  protocol MO16M369 of the Johns Hopkins Medical Institutions.The following mouse alleles were used: Dlg1fl , Fz4Δ ; rat anti-mouse PLVAP ; mouse anti-CLDN5, Alexa Fluor 488 conjugate (Thermo Fisher Scientific 352588); Texas Red streptavidin (Vector Laboratories SA-5006). Alexa Fluor-labeled secondary antibodies were from Thermo Fisher Scientific.The following antibodies were used for immunoblot analysis: rabbit anti-6xMyc (JH6204); rabbit mAb anti-V5 ; mouse mAb anti-RIM3F4 (ascites); mouse anti-actin (Millipore Sigma MAB1501); rabbit anti-SAP97 (Abcam ab134156). Fluorescent secondary antibodies for immunoblotting were from Li-Cor.2) + 10% normal goat serum (NGS). Retinas were washed at least 3 times with 1x PBSTC over the course of 6 hr, and subsequently incubated overnight with secondary antibodies (1:400) diluted in 1x PBSTC + 10% NGS. The next day, retinas were washed at least 3 times with 1x PBSTC over the course of 6 hr, and flat-mounted using Fluoromount G (EM Sciences 17984–25).For retinal vasculature analysis, whole-mount retinas were prepared and processed for immunohistochemical analysis as described by For brain vasculature analysis, brains were prepared and processed for immunohistochemical analysis as described by All antibody incubation and washing steps were performed at 4°C unless otherwise noted. Whole-mount retinas and brain sections were imaged using a Zeiss LSM700 confocal microscope, and processed with ImageJ, Adobe Photoshop, and Adobe Illustrator software.4-HT was prepared as described by Sulfo-NHS-Biotin (Thermo Scientific 21335) was dissolved in 1x PBS to a final concentration of 20 mg/ml. Pups were injected with 150 μl sulfo-NHS-biotin IP 1–2 hr before sacrifice.The Myc-Dlg1 expression construct  was usedThe following inserts for MBP fusion constructs were PCR amplified from the Myc-Dlg1 expression vector: PDZ1 (aa187-284), PDZ2 (aa280-374), and PDZ(1+2) (aa187-374). Inserts were sub-cloned into the pMAL-cR1 expression vector.The mouse Fz4 and Tspan12 expression constructs were used as backbones to generate the Rim-Fz4 and Rim-Tspan12 expression constructs, respectively. Specifically, a Rim epitope tag (NETYDLPLHPRTAG) was inserted after the Fz4 signal sequence and before the Fz4 CRD, and after the Tspan12 signal sequence. Mutant Rim-Fz4 constructs, in which the C-terminal four amino acids (ETVV) were converted to 4A (AAAA) or MGK, were generated by PCR.The mouse Dlg2 (Origene MR222602), mouse Dlg3 , and mouse Dlg4  cDNA sequences were cloned into the pRK5 mammalian expression vector with an N-terminal Myc epitope (EQKLISEEDL) tag. The V5-Dlg5 plasmid was a kind gift from Dr. Alex Kolodkin (Johns Hopkins).Expression plasmids for mouse Wnts and Frizzleds , mouse THEK/293T cells (ATCC CRL-3216) and Super TOP Flash (STF) cells  were useDlg1 KO STF cell lines were generated using CRISPR/Cas9 technology. An sgRNA (AAGCTCATTAAAGGTCCTAA) targeting exon 10 of human Dlg1 (hDlg1) was cloned into the pSpCasp9(BB)−2A-GFP vector (PX458) (Addgene Plasmid #48138). STF cells were plated on a 10 cm plate at a confluency of 60–70%. The following day, fresh DMEM/F-12 (Thermo Fisher Scientific 12500) supplemented with 10% fetal bovine serum (FBS) was replaced in the 10 cm plate. Three hours later, cells were transfected with the PX458 expression plasmid containing the hDlg1 sgRNA (8 μg of DNA) using FuGENE HD Transfection Reagent (Promega E2311). 48 hr post-transfection, cells were harvested in 1x PBS supplemented with 0.5% bovine serum albumin (BSA).The hDlg1 locus by genomic PCR and restriction enzyme digestion with PpuM I. All clones lacking a PpuM I cleavage site, which straddles the Cas9 cleavage site, were analyzed by immunoblotting to assess Dlg1 levels (1:1000 rabbit anti-SAP97). Two clones had no detectable Dlg1 (Dlg1 KO Clones #12 and #15). The targeted region from these two clones was PCR amplified and sub-cloned for Sanger sequencing, which revealed a frame-shift mutation in each hDlg1 allele.Single, viable GFP positive cells were sorted into a 96-well plate containing fresh DMEM/F-12 + 10% FBS using a MoFlo XDP Sorter (Beckman Coulter). Single cell clones were expanded and screened for editing of the Dual luciferase assays were performed as described by The Myc-Dlg1 expression plasmid was co-transfected either in a dose-dependent manner or at 7.5 ng per three wells. Expression plasmids for domain deletion mutants of Dlg1 were co-transfected at 7.5 ng per three wells. The Myc-Dlg2, Myc-Dlg3, Myc-Dlg4, and V5-Dlg5 were co-transfected in a dose-dependent manner. All expression plasmids for the Wnt ligands were co-transfected at 60 ng per three wells.48 hr post-transfection, cells were harvested in 1x Passive Lysis Buffer (Promega E194A) for 20 min at room temperature. Lysates were used to measure Firefly and Renilla luciferase activity using the Dual-Luciferase Reporter Assay System (Promega E1910) and a Turner BioSystems Luminometer (TD-20/20). Relative luciferase activity was calculated by normalizing Firefly/Renilla values. GraphPad Prism 7 software was used to generate plots and perform statistical analysis. The mean ± standard deviations are shown.HEK/293T cells were transfected with pRK5 expression plasmids using FuGENE HD Transfection Reagent. 48 hr post-transfection, cells were lysed in 1x RIPA buffer  containing protease inhibitor (Roche 11836170001). Cell lysates were incubated at 4°C for 30 min and subsequently centrifuged at 10,000xg at 4°C for 20 min to remove cellular debris. Epitope-tagged proteins (i.e. Myc-Dlg1 or Rim-Fz4) were captured by incubating cleared lysates with anti-Myc or anti-Rim antibodies and Protein G Dynabeads (Thermo Fisher Scientific 10004D) overnight at 4°C. Bead were washed 5–6 times with RIPA buffer, and captured proteins, along with input controls, were resolved by SDS-PAGE and transferred to PVDF membranes (Millipore Sigma IPFL00010) for immunoblotting.To detect immunoprecipitated proteins, membranes were incubated with primary antibodies  with IRDye 680RD Detection Reagent (LiCor 926–68100) diluted in Odyssey Blocking Buffer (LiCor 927–40000) overnight at 4°C. Membranes were washed 3 times with 1x PBS-T (1x PBS + 0.1% Tween 20) and developed using the Odyssey Fc Imaging System (LiCor) to detect immunoprecipitated proteins.To detect co-immunoprecipitated proteins, membranes were incubated with the reciprocal primary antibodies  diluted in Odyssey Blocking Buffer overnight at 4°C. Membranes were washed 3 times with 1x PBS-T and incubated with LiCor secondary antibodies  diluted in Odyssey Blocking Buffer for 1 hr at room temperature. Membranes were washed at least 3 times with 1x PBS-T and developed using the Odyssey Fc Imaging System.The following peptides were chemically synthesized with an N-terminal biotin tag: Rhodopsin peptide (SKTETSQVAPA), Fz4 WT peptide (WVKPGKGNETVV), and Fz4 MGK peptide (WVKPGKGNEMGK). For capturing proteins expressed in HEK/293T cells, NeutrAvidin agarose beads (Thermo Fisher Scientific 29200) were coated with 20 μg of peptide per binding reaction, washed, and incubated with cleared HEK/293T overexpression lysates overnight at 4°C. Resin was washed 5 to 6 times with 1x RIPA buffer, and captured proteins, along with input controls, were resolved by SDS-PAGE and transferred to PVDF membranes for immunoblotting and analysis, as described above.Expression of MBP fusion proteins was induced in BL21 cells with 1 mM IPTG at an optical density at 600 nm of 0.4–0.6 for 2 hr at 37°C. Cells were pelleted and lysed in 1x BugBuster Protein Extraction Reagent (Millipore Sigma 70584) for 20 min at 23°C. Cell lysates were centrifuged at 16,000xg at 4°C for 20 min to remove cellular debris.MBP fusion proteins were batch purified from cleared lysates with amylose resin (New England Biolabs E8021S) in 1x amylose binding buffer  overnight at 4°C. Resin was washed 10 times with 1x amylose binding buffer with 1% Triton X-100 and 0.5% deoxycholate. Purified MBP fusion proteins were batch eluted in 1x amylose elution buffer .Purified MBP fusion proteins were captured with biotin-peptide coated Streptavidin Dynabeads (Thermo Fisher Scientific 11047) overnight at 4°C in 1x PBS. The next day, Streptavidin Dynabeads were washed 10 times in 1x RIPA buffer, and captured proteins, along with input controls, were resolved by SDS-PAGE and visualized by Coomassie Blue staining.Cell surface biotinylation was performed as described by For quantifying relative vascular density, whole mount retinas were stained with anti-CLDN5 and anti-PLVAP. Confocal images were scanned at 5 μm intervals along the Z-axis, of which two images were Z-stacked to assess vascular density in the superficial plexus or deep plexus only, or eight images were Z-stacked to assess vascular density in superficial + intermediate + deep plexuses. Images were exported to ImageJ and converted to an 8-bit format. The background (determined with a rolling circle with a 50 pixel radius) was subtracted from all channels. Images were thresholded and skeletonized to pixelate the retinal vasculature. Quantification of relative vascular density was performed by measuring the percentage of pixels covered in a 640 × 640 μm area of the retina. For each biological replicate, four distinct regions of a given Z-stacked image were quantified, and at least three biological replicates for each genotype were analyzed.For quantifying the fraction of the retina covered by vasculature at P7, images of whole mount retinas (as described above) were exported to ImageJ. All channels were combined into a composite image. The distance from the optic nerve to the peripheral edge of the retinal vasculature was measured as a fraction of the distance from the optic nerve to the peripheral edge of the retina. For each biological replicate, four measurements were made, and at least three biological replicates for each genotype were analyzed.For quantifying the fraction of vessels that immunostained for CLDN5, PLVAP, or GLUT1, vessels in the designated volume of 320 × 320 × 40 μm (retina) or 1500 × 150 × 100 μm (cerebellum) were manually traced using a Wacom tablet and Adobe Illustrator software. Pixel coverage of the traced image was determined as a fraction of the total designated volume (as described above) for each marker. The vascular density for CLDN5+, PLVAP+, and GLUT1+ vessels was then divided by the total vascular density in the designated volume.GraphPad Prism 7 software was used to generate plots and to perform statistical analysis. The mean ± standard deviations are shown. Statistical significance was determined by the unpaired t-test, and is represented by * (p<0.05), ** (p<0.01), *** (p<0.001), and **** (p<0.0001). In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Joseph Gleeson as the Reviewing Editor and Didier Stainier as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Dlg1 activates beta-catenin signaling to regulate retinal angiogenesis and the blood-retina and blood-brain barriers" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Two reviewers found the manuscript to show strong and convincing evidence of Dlg1 participation in the canonical Wnt pathway and its interaction with fz4, Tspan12. The work provides support for a role of Dlg1 in promoting both Norrin/Fz4/Tspan12 signaling and Wnt7/Fz/Gpr124/Reck signaling, and the rescue by beta-catenin stabilization indicates that this is beta catenin-dependent. The authors provide direct evidence for this interaction in vitro and identify the domains involved. However, based on in vivo evidence obtained generating a new FZD4 mutant line, they conclude that the binding between the Fzd4 C-terminus and the PDZ domains of Dlg1 plays no role in the Dlg1-dependent enhancement of beta-catenin signaling in CNS ECs. Reviewer 2 and 3 found that the work is novel and exciting, experimental design is sound, results and conclusions are convincing and commented that the paper fits very well with the Journal's scope and wide readership. Reviewer 3 commented that the evidence from the Fz4(MGK/MGK) cross is not sufficient to conclude that the Dlg1-Fzd4 interaction plays no role in regulating beta-catenin. Reviewer 1 found that the link between Dlg1 and canonical Wnt pathway unconvincing, and while convinced by the Fz4(MGK/MGK) results, Reviewer 1 encouraged new experiments to directly link Dlg1 with the components of the Wnt pathway.Essential revisions:1) The cell culture experiments are confounding, seem to yield insignificant or minor fold change levels, and directly contradict the better experiment of testing Fz4 C terminal modification in vivo, which shows no direct connection between Dlg1 and Fz4. Can the authors reconcile these observations?2) The last experiment is a good negative result, but besides this specific non-interaction we do not learn anything concrete about Dlg1 in retinal angiogenesis or its connection to signaling pathways. Reviewer 3 encourages the authors to try new experiments to directly link Dlg1 with components of the Wnt pathway and so offer something more than speculation.3) Without a detailed characterisation of the expression profile of the EC from the mutant mice (which is clearly beyond the scope of this study), it is not possible to confidently conclude that the Dlg4-Fzd4 interaction does not play a role in these phenotypes. We recommend revising the interpretation of the findings to include this possibility in the interpretation of the results. Given the extensive section on possible alternatives to Fzd4 in the Discussion, it would be tempting to ask for some evidence for a role for APC or Gpr124 in this study; however, this is likely to be the focus of following papers. Essential revisions:1) The cell culture experiments are confounding, seem to yield insignificant or minor fold change levels, and directly contradict the better experiment of testing Fz4 C terminal modification in vivo, which shows no direct connection between Dlg1 and Fz4. Can the authors reconcile these observations?We agree that the cell culture experiments that measured the activity of various Dlg1 domain deletions were largely inconclusive since no single domain had an overwhelming effect, which is why these data were relegated to a supplementary figure . However, we disagree with the above comment in regard to the more important cell culture analyses of the activity of Dlg1 and the other Dlg family members in combination with Norrin and Wnts . In Figure 6, we observed that Dlg1 transfection of the Dlg1 KO reporter cell line produced a dose-dependent enhancement of beta-catenin signal strength that was maximally ~4-fold over baseline in the presence of Norrin and various Wnts. A 4-fold change may sound modest, but we note that (i) the reporter cell line also expresses other Dlg family members, which could contribute to the non-zero signal in the absence of Dlg1 transfection (hence lowering the fold induction), (ii) the well-established Norrin/Fz4 signaling co-activator Tspan12 gives only a several-fold induction in this and other reporter assays, and (iii) signaling in transiently transfected reporter cells (where receptors and/or ligands are over-expressed) is an imperfect proxy for signaling in the living animal where all of the signaling proteins are present at low abundance. We specifically disagree with the implication that the changes shown in Figure 6 are: (i) “insignificant” , or (ii) “minor” .Regarding the comment that the results in cell culture “contradict the better experiment of testing Fz4 C terminal modification in vivo”, our view is that both sets of experiments are technically “correct” and it is only their interpretations that potentially contradict each other. More specifically, these experiments teach the lesson that “all that glitters (in cell culture and in vitro) is not necessarily gold (in vivo)”, a lesson that is sometimes ignored in the world of biomedical research. There are many binding interactions (like the Fz4 C-terminus and Dlg1 PDZ interaction) that are biochemically “real” but still need to be tested for their biological significance, and here is one system where that has been done. This aspect of the present body of work may recommend this paper as a good one for students to read.2) The last experiment is a good negative result, but besides this specific non-interaction we do not learn anything concrete about Dlg1 in retinal angiogenesis or its connection to signaling pathways. Reviewer 3 encourages the authors to try new experiments to directly link Dlg1 with components of the Wnt pathway and so offer something more than speculation.That is a valid criticism. The current study has tested the most favored model – that the PDZ-binding motif in Fz4 interacts directly with the PDZ domain of Dlg1 in vascular endothelial cells – and ruled it out as a significant mechanism for Dlg1 action in beta-catenin signaling in vivo. Now we are left with a large number of possible targets for Dlg1 action, i.e., any other intracellular or trans-membrane protein involved in beta-catenin signaling. Taking the next step to identify and validate those target(s) is going to be a major undertaking and we think that it is beyond the scope of the current manuscript.3) Without a detailed characterisation of the expression profile of the EC from the mutant mice (which is clearly beyond the scope of this study), it is not possible to confidently conclude that the Dlg4-Fzd4 interaction does not play a role in these phenotypes. We recommend revising the interpretation of the findings to include this possibility in the interpretation of the results. Given the extensive section on possible alternatives to Fzd4 in the Discussion, it would be tempting to ask for some evidence for a role for APC or Gpr124 in this study; however, this is likely to be the focus of following papers.That is a valid point. We agree that a Dlg1-Fz4 interaction could exist in vivo in ECs and that eliminating that interaction could produce a phenotype that is so subtle that we have missed it based on our histologic analyses. A transcriptome analysis could reveal such a phenotype, if it exists. As suggested, we have modified the Discussion to include this possibility. We agree that genetic analyses with Dlg1 and APC or with Dlg1 and Gpr124 are beyond the scope of the current manuscript.


The term WNT (wingless-type MMTV integration site family) signaling comprises a complex molecular pathway consisting of ligands, receptors, coreceptors, signal transducers and transcriptional modulators with crucial functions during embryonic development, including all aspects of proliferation, morphogenesis and differentiation. Its involvement in cancer biology is well documented. Even though WNT signaling has been divided into mainly three distinct branches in the past, increasing evidence shows that some molecular hubs can act in various branches by exchanging interaction partners. Here we discuss developmental and clinical aspects of WNT signaling in neuroblastoma (NB), an embryonic tumor with an extremely broad clinical spectrum, ranging from spontaneous differentiation to fatal outcome. We discuss implications of WNT molecules in NB onset, progression, and relapse due to chemoresistance. In the light of the still too high number of NB deaths, new pathways must be considered. Int1 gene (see list of abbreviations at the end of the text) found in mice and the wingless gene from drosophila [The name WNT is an acronym combining the osophila . In mammosophila ). This oosophila ,5. The mosophila . WNT patosophila . The recosophila ,8,9. Thi2+-regulated ’WNT/Ca2+ pathway’ are both independent of β-catenin and TCF/LEF signaling. They are activated by WNT4, WNT5 and WNT11, and involve FZD receptors as well as co-receptors such as ROR1, ROR2, and RYK  and the Caiewed by ). The WNand NFAT . The maiand NFAT ,14).Drosophila Fmi (Flamingo), to orientate epithelial as well as mesenchymal cells during the development and regeneration by cell-cell interactions according to a WNT gradient [The PCP pathway is activated by the receptor tyrosine kinases ROR1, ROR2 and RYK, which may act alone, or as co-receptors in conjunction with FZDs (reviewed by ). It actgradient . This algradient ). The regradient ).The separation of WNT signaling into distinct pathways may be feasible to generate research approaches and to concentrate on functional aspects in a given cell-type or organism. However, there is emerging evidence that WNT signaling is much more sophisticated than initially expected . SevIn sum, canonical and non-canonical WNT signaling pathways have their specific roles in the regulation of cell behavior, but they also interact, mostly in a mutually inhibitory fashion . TherebyALK gene or gene amplifications of MYCN [Neuroblastoma (NB) is a pediatric tumor of the developing sympathoadrenal system. Severe, unfavorable NB acquire activating mutations in the  of MYCN ,24. Howe of MYCN ,26. Only of MYCN . PostnatEarly section studies showed that NB often exist prenatally with an incidence much higher than the clinically diagnosed cases ,29. ThisTo understand the differentiation ability of NB, its origin must be considered. NB is widely accepted as an embryonic developmental malignancy derived from progenitor cells originating from the sympathetic neural crest (reviewed by ). These Both growth and spontaneous regression of NB seem to recapitulate the developmental program of sympathoadrenal progenitor cells. Considering that many NB do not attract clinical attention and many of those that require clinical intervention regress spontaneously or respond well to mild therapy, the drivers of malignant NB, such as ALK and MYCN, seem to be acquired after the differentiation program failed. Of note, tyrosine hydroxylase promoter-targeted Mycn over-expression has been shown to induce NB in mice, indicating that it is a sufficient and independent factor that defines a highly malignant subclass of tumors . In nudeNB evolve from progenitors of postganglionic sympathetic neurons. These emigrate from the sympathetic neural crest (NC), which spans a large portion of the trunk NC. It overlaps with the adrenal NC, but is distinctly separated from the cranial, vagal and sacral NC. As the developmental aspects were extensively reviewed recently , only a The sympathoadrenal progenitors migrate ventrally via two routes , dopamine beta-hydroxylase (DBH), and also WNT5A . AutocriIn the past, WNT signaling in NB did not gain much attention, although the first studies appeared in the mid-2000s. Then, it took almost 20 more years to increase evidence and interest for WNT signaling in NB. As noted above, β-catenin-dependent  and β-catenin independent  pathways must be considered.MYCN non-amplified cell lines, enhanced WNT/β-catenin signaling was shown to increase MYCN levels [MYCN-amplified NB cell-lines reduced viability due to increased apoptosis and increased expression of DKK1, thus inhibiting WNT/β-catenin signaling [The WNT/β-catenin signaling has shown to influence NB. An early microarray study, however, did not report differentially expressed genes obviously involved in WNT signaling . NeverthN levels . TherebyN levels . Knock-dN levels . In bothignaling .Very recently, TRIM 59, a transmembrane protein of the endoplasmic reticulum, has been shown to regulate proliferation and β-catenin levels in NB cells . Knock-dLGR5 has been identified as a marker of aggressive NB and was shown to be elevated in relapsed NB . It is rWNT/β-catenin signaling is also involved in chemoresistance of NB. Comparison of doxorubicin-resistant and non-resistant LAN1 and IGRN1 NB cells revealed higher expression of MDR, FZD1 and other components of WNT/β-catenin signaling in the resistant subclones. Elimination of FZD1 by shRNA in these cells reduced MDR1 expression and restored sensitivity to doxorubicin .+ cells could be identified, and they showed increased resistance to doxorubicin compared to CD133- cells [+ with CD133- cells, each treated with doxorubicin, and detected differential expression of WNT signaling molecules. Especially APC, DVL2, FZD4, FZD6, CSK1 and LEF1 were associated with CD133+ cells under doxorubicin treatment. These cells were shown to exhibit lower sensitivity to doxorubicin, but could be sensitized by concomitant application of XAV 939, which inhibits WNT/β-catenin-mediated transcription [+ cells increased β-catenin and pGSK-3β levels as compared to CD133-. Similar results were shown for SH-SY5Y and IMR32 cells treated with XAV939. Treatment led to decreased β-catenin abundance and enhanced sensitivity to doxorubicin [In SK-N-SH a subset of CD1333- cells . Notably3- cells ). The sacription . Additioorubicin .Together these results reveal that canonical WNT signaling is a major component for chemoresistance in NB, and is associated with tumor cell stemness and expression of stem cell markers. However, all studies are focused on doxorubicin and it cannot be excluded that resistance to other chemotherapeutics differs .One of the first studies on this topic was published by Orme et al. 2003, who used the N2A mouse NB cell line and showed that GSK-3 is involved in ’neurite’ outgrowth, a widely accepted sign for NB differentiation . They alRecently SLC34A2, which is associated with lung and colorectal cancer malignancy, was show to be up-regulated in spheroids of SH-SY5Y cells and to produce direct effects by inducing MIR-25, which leads to the degradation of GSK-3β and accumulation of β-catenin, thus increasing proliferation and expression of stemness markers . Knock-dNon-canonical WNT signaling has shown to be involved in both progression and suppression of several human cancers (reviewed in: ). DecreaPCP signaling is initiated by binding of WNT5A or WNT11 to ROR1, ROR2 or RYK receptor tyrosine kinases. Wnt5a knock-out mice show severe craniofacial defects and malformation of dermal lymphatics  . In and DKK3 ,107. ForWNT2B, WNT4, WNT11, ROR1, VANGL1 and others . Loss o