Excessive Polyamine Generation in Keratinocytes Promotes Self-RNA Sensing by Dendritic Cells in Psoriasis.

Psoriasis is a chronic inflammatory disease whose etiology is multifactorial. The contributions of cellular metabolism to psoriasis are unclear. Here, we report that interleukin-17 (IL-17) downregulated Protein Phosphatase 6 (PP6) in psoriatic keratinocytes, causing phosphorylation and activation of the transcription factor C/EBP-ß and subsequent generation of arginase-1. Mice lacking Pp6 in keratinocytes were predisposed to psoriasis-like skin inflammation. Accumulation of arginase-1 in Pp6-deficient keratinocytes drove polyamine production from the urea cycle. Polyamines protected self-RNA released by psoriatic keratinocytes from degradation and facilitated the endocytosis of self-RNA by myeloid dendritic cells to promote toll-like receptor-7 (TLR7)-dependent RNA sensing and IL-6 production. An arginase inhibitor improved skin inflammation in murine and non-human primate models of psoriasis. Our findings suggest that urea cycle hyperreactivity and excessive polyamine generation in psoriatic keratinocytes promote self-RNA sensation and PP6 deregulation in keratinocytes is a pivotal event that amplifies the inflammatory circuits in psoriasis.

Hippo Kinases Mst1 and Mst2 Sense and Amplify IL-2R-STAT5 Signaling in Regulatory T Cells to Establish Stable Regulatory Activity.

Interleukin-2 (IL-2) and downstream transcription factor STAT5 are important for maintaining regulatory T (Treg) cell homeostasis and function. Treg cells can respond to low IL-2 levels, but the mechanisms of STAT5 activation during partial IL-2 deficiency remain uncertain. We identified the serine-threonine kinase Mst1 as a signal-dependent amplifier of IL-2-STAT5 activity in Treg cells. High Mst1 and Mst2 (Mst1-Mst2) activity in Treg cells was crucial to prevent tumor resistance and autoimmunity. Mechanistically, Mst1-Mst2 sensed IL-2 signals to promote the STAT5 activation necessary for Treg cell homeostasis and lineage stability and to maintain the highly suppressive phosphorylated-STAT5+ Treg cell subpopulation. Unbiased quantitative proteomics revealed association of Mst1 with the cytoskeletal DOCK8-LRCHs module. Mst1 deficiency limited Treg cell migration and access to IL-2 and activity of the small GTPase Rac, which mediated downstream STAT5 activation. Collectively, IL-2-STAT5 signaling depends upon Mst1-Mst2 functions to maintain a stable Treg cell pool and immune tolerance.

Hippo cooperates with p53 to maintain foregut homeostasis and suppress the malignant transformation of foregut basal progenitor cells.

Basal progenitor cells serve as a stem cell pool to maintain the homeostasis of the epithelium of the foregut, including the esophagus and the forestomach. Aberrant genetic regulation in these cells can lead to carcinogenesis, such as squamous cell carcinoma (SCC). However, the underlying molecular mechanisms regulating the function of basal progenitor cells remain largely unknown. Here, we use mouse models to reveal that Hippo signaling is required for maintaining the homeostasis of the foregut epithelium and cooperates with p53 to repress the initiation of foregut SCC. Deletion of Mst1/2 in mice leads to epithelial overgrowth in both the esophagus and forestomach. Further molecular studies find that Mst1/2-deficiency promotes epithelial growth by enhancing basal cell proliferation in a Yes-associated protein (Yap)-dependent manner. Moreover, Mst1/2 deficiency accelerates the onset of foregut SCC in a carcinogen-induced foregut SCC mouse model, depending on Yap. Significantly, a combined deletion of Mst1/2 and p53 in basal progenitor cells sufficiently drives the initiation of foregut SCC. Therefore, our studies shed light on the collaborative role of Hippo signaling and p53 in maintaining squamous epithelial homeostasis while suppressing malignant transformation of basal stem cells within the foregut.

Mst1 is an interacting protein that mediates PHLPPs' induced apoptosis.

PHLPP1 and PHLPP2 phosphatases exert their tumor-suppressing functions by dephosphorylation and inactivation of Akt in several breast cancer and glioblastoma cells. However, Akt, or other known targets of PHLPPs that include PKC and ERK, may not fully elucidate the physiological role of the multifunctional phosphatases, especially their powerful apoptosis induction function. Here, we show that PHLPPs induce apoptosis in cancer cells independent of the known targets of PHLPPs. We identified Mst1 as a binding partner that interacts with PHLPPs both in vivo and in vitro. PHLPPs dephosphorylate Mst1 on the T387 inhibitory site, which activate Mst1 and its downstream effectors p38 and JNK to induce apoptosis. The same T387 site can be phosphorylated by Akt. Thus, PHLPP, Akt, and Mst1 constitute an autoinhibitory triangle that controls the fine balance of apoptosis and proliferation that is cell type and context dependent.

Mst-1 deficiency promotes post-traumatic spinal motor neuron survival via enhancement of autophagy flux.

The mammalian Ste20-like kinase 1 (Mst-1) is a serine-threonine kinase and a component of the Hippo tumor suppressor pathway, which reacts to pathologically relevant stress and regulates cell death. However, little is known about its role in spinal cord injury. Here, we found that p-Mst-1, the activated form of Mst-1, was induced in the post-traumatic spinal motor neurons. In vivo evidence demonstrated that Mst-1 deficiency promoted post-traumatic spinal motor neuron survival, Basso mouse scale scores, and synapse survival. Moreover, we found that autophagosome formation and autolysosome degradation enhanced by Mst-1 deficiency were crucial to attenuate the death of injured spinal motor neurons. Taken together, our findings demonstrate that Mst-1 deficiency promotes post-traumatic spinal motor neuron survival via enhancement of autophagy flux.

Hdac7 promotes lung tumorigenesis by inhibiting Stat3 activation.

BACKGROUND: Lung cancer is the leading cause of cancer death worldwide. However, the molecular mechanisms underlying lung cancer development have not been fully understood. The functions of histone deacetylases (HDACs), a class of total eighteen proteins (HDAC1-11 and SIRT1-7 in mammals) that deacetylate histones and non-histone proteins, in cancers are largely unknown. METHODS: Hdac7 +/-/K-Ras mice and HDAC7-depleted human lung cancer cell lines were used as models for studying the function of Hdac7 gene in lung cancer. Kaplan-Meier survival analysis was performed to explore the relationship between HDAC7 expression and prognosis of human lung cancers. Recombinant lentivirus-mediated in vivo gene expression or knockdown, Western blotting, and pull-down assay were applied to investigate the underlying molecular mechanism by which Hdac7 promotes lung tumorigenesis. RESULTS: The number and burden of lung tumor were dramatically reduced in Hdac7 +/-/K-Ras mice compared to control K-Ras mice. Also, in Hdac7 +/-/K-Ras mice, cell proliferation was significantly inhibited and apoptosis in lung tumors was greatly enhanced. Similarly, cell proliferation and anchorage-independent growth of human lung cancer cell lines expressing shHDAC7 were also significantly suppressed and apoptosis was dramatically elevated respectively. Mechanistic study revealed that Hdac7 mutation in mouse lung tumors or HDAC7 depletion in human tumor cell lines resulted in significantly enhanced acetylation and tyrosine-phosphorylation of Stat3 and HDAC7 protein directly interacted with and deacetylateed STAT3. The Hdac7 mutant-mediated inhibitory effects on lung tumorigenesis in mice and cell proliferation/soft agar colony formation of human lung cancer cell lines were respectively reversed by expressing dnStat3. Finally, the high HDAC7 mRNA level was found to be correlated with poor prognosis of human lung cancer patients. CONCLUSION: Our study suggests that Hdac7 promotes lung tumorigenesis by inhibiting Stat3 activation via deacetylating Stat3 and may shed a light on the design of new therapeutic strategies for human lung cancer.

PP6 controls T cell development and homeostasis by negatively regulating distal TCR signaling.

T cell development and homeostasis are both regulated by TCR signals. Protein phosphorylation and dephosphorylation, which are catalyzed by protein kinases and phosphatases, respectively, serve as important switches controlling multiple downstream pathways triggered by TCR recognition of Ags. It has been well documented that protein tyrosine phosphatases are involved in negative regulation of proximal TCR signaling. However, how TCR signals are terminated or attenuated in the distal TCR signaling pathways is largely unknown. We investigated the function of Ser/Thr protein phosphatase (PP) 6 in TCR signaling. T cell lineage-specific ablation of PP6 in mice resulted in enhanced thymic positive and negative selection, and preferential expansion of fetal-derived, IL-17-producing Vγ6Vδ1(+) T cells. Both PP6-deficient peripheral CD4(+) helper and CD8(+) cytolytic cells could not maintain a naive state and became fast-proliferating and short-lived effector cells. PP6 deficiency led to profound hyperactivation of multiple distal TCR signaling molecules, including MAPKs, AKT, and NF-κB. Our studies demonstrate that PP6 acts as a critical negative regulator, not only controlling both αß and γδ lineage development, but also maintaining naive T cell homeostasis by preventing their premature activation before Ag stimulation.

Mammalian Sterile 20-like Kinase 1 (Mst1) Enhances the Stability of Forkhead Box P3 (Foxp3) and the Function of Regulatory T Cells by Modulating Foxp3 Acetylation.

Regulatory T cells (Tregs) play crucial roles in maintaining immune tolerance. The transcription factor Foxp3 is a critical regulator of Treg development and function, and its expression is regulated at both transcriptional and post-translational levels. Acetylation by lysine acetyl transferases/lysine deacetylases is one of the main post-translational modifications of Foxp3, which regulate Foxp3's stability and transcriptional activity. However, the mechanism(s) by which the activities of these lysine acetyl transferases/lysine deacetylases are regulated to preserve proper Foxp3 acetylation during Treg development and maintenance of Treg function remains to be determined. Here we report that Mst1 can enhance Foxp3 stability, its transcriptional activity, and Treg function by modulating the Foxp3 protein at the post-translational level. We discovered that Mst1 could increase the acetylation of Foxp3 by inhibiting Sirt1 activity, which requires the Mst1 kinase activity. We also found that Mst1 could attenuate Sirt1-mediated deacetylation of Foxp3 through directly interacting with Foxp3 to prevent or interfere the interaction between Sirt1 and Foxp3. Therefore, Mst1 can regulate Foxp3 stability in kinase-dependent and kinase-independent manners. Finally, we showed that treatment of Mst1(-/-) Tregs with Ex-527, a Sirt1-specific inhibitor, partially restored the suppressive function of Mst1(-/-) Tregs. Our studies reveal a novel mechanism by which Mst1 enhances Foxp3 expression and Treg function at the post-translational level.

Mst1 regulates hepatic lipid metabolism by inhibiting Sirt1 ubiquitination in mice.

Previous study showed mammalian Ste20-like kinase (Mst1) may serve as target for the development of new therapies for diabetes. However, the function of Mst1 involved in liver lipid metabolism has remained elusive. In this study, we report that the liver of Mst1 knockout (Mst1(-/-)) mice showed more severe liver metabolic damage under fasting and high-fat diet than that of control mice. And fasting induced hepatic Mst1 expression. Mst1 overexpression inhibited Srebp-1c expression and increased the expression of antioxidant genes in primary hepatocytes. We also found that fasting-induced expression of hepatic Sirt1 was attenuated in Mst1(-/-) mice. Mst1 overexpression promoted Sirt1 expression, probably due to inhibiting Sirt1 ubiquitination. In summary, our study suggests that Mst1 regulates hepatic lipid metabolism by inhibiting Sirt1 ubiquitination in mice.

Hippo/MST1 signaling mediates microglial activation following acute cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury is a major public health concern that causes high rates of disability and mortality in adults. Microglial activation plays a crucial role in ischemic stroke-induced alteration of the immune microenvironment. However, the mechanism underlying the triggering of microglial activation by ischemic stroke remains to be elucidated. Previously, we demonstrated that the protein kinase Hippo/MST1 plays an important role in oxidative stress-induced cell death in mammalian primary neurons and that the protein kinase c-Abl phosphorylates MST1 at Y433, which increases MST1 kinase activity. Microglial activation has been implicated as a secondary detrimental cellular response that contributes to neuronal cell death in ischemic stroke. Here, we are the first, to our knowledge, to demonstrate that MST1 mediates stroke-induced microglial activation by directly phosphorylating IκBα at residues S32 and S36. We further demonstrate that Src kinase functions upstream of MST1-IκB signaling during microglial activation. Specific deletion of MST1 in microglia mitigates stroke-induced brain injury. Therefore, we propose that Src-MST1-IκB signaling plays a critical role in stroke-induced microglial activation. Together with our previous work demonstrating that MST1 is important for oxidative stress-induced neuronal cell death, our results indicate that MST1 could represent a potent therapeutic target for ischemic stroke.

Mst1/Mst2 regulate development and function of regulatory T cells through modulation of Foxo1/Foxo3 stability in autoimmune disease.

Foxp3 expression and regulatory T cell (Treg) development are critical for maintaining dominant tolerance and preventing autoimmune diseases. Human MST1 deficiency causes a novel primary immunodeficiency syndrome accompanied by autoimmune manifestations. However, the mechanism by which Mst1 controls immune regulation is unknown. In this article, we report that Mst1 regulates Foxp3 expression and Treg development/function and inhibits autoimmunity through modulating Foxo1 and Foxo3 (Foxo1/3) stability. We have found that Mst1 deficiency impairs Foxp3 expression and Treg development and function in mice. Mechanistic studies reveal that Mst1 enhances Foxo1/3 stability directly by phosphorylating Foxo1/3 and indirectly by attenuating TCR-induced Akt activation in peripheral T cells. Our studies have also shown that Mst1 deficiency does not affect Foxo1/3 cellular localization in CD4 T cells. In addition, we show that Mst1(-/-) mice are prone to autoimmune disease, and mutant phenotypes, such as overactivation of naive T cells, splenomegaly, and autoimmune pathological changes, are suppressed in Mst1(-/-) bone marrow chimera by cotransplanted wt Tregs. Finally, we demonstrate that Mst1 and Mst2 play a partially redundant role in Treg development and autoimmunity. Our findings not only identify Mst kinases as the long-searched-for factors that simultaneously activate Foxo1/3 and inhibit TCR-stimulated Akt downstream of TCR signaling to promote Foxp3 expression and Treg development, but also shed new light on understanding and designing better therapeutic strategies for MST1 deficiency-mediated human immunodeficiency syndrome.

The non-canonical Hippo/Mst pathway in lymphocyte development and functions.

The canonical Hippo/Mst pathway, originally discovered in Drosophila, is famous for its function in promoting apoptosis, inhibiting cell proliferation and tumorigenesis, and regulating tissue regeneration. However, emerging evidence shows that multiple non-canonical Hippo signaling pathways are also implicated in the regulation of various other biological processes. Recent studies have revealed that Mst1/2, the core kinases of Hippo/Mst pathway are required for T cell development, function, survival, trafficking, and homing, and also involved in regulation of autoimmunity. In this review, we discuss the roles of non-canonical Hippo/Mst signaling pathways in lymphocyte development and functions.

Ablation of vacuole protein sorting 18 (Vps18) gene leads to neurodegeneration and impaired neuronal migration by disrupting multiple vesicle transport pathways to lysosomes.

Intracellular vesicle transport pathways are critical for neuronal survival and central nervous system development. The Vps-C complex regulates multiple vesicle transport pathways to the lysosome in lower organisms. However, little is known regarding its physiological function in mammals. We deleted Vps18, a central member of Vps-C core complex, in neural cells by generating Vps18(F/F); Nestin-Cre mice (Vps18 conditional knock-out mice). These mice displayed severe neurodegeneration and neuronal migration defects. Mechanistic studies revealed that Vps18 deficiency caused neurodegeneration by blocking multiple vesicle transport pathways to the lysosome, including autophagy, endocytosis, and biosynthetic pathways. Our study also showed that ablation of Vps18 resulted in up-regulation of ß1 integrin in mouse brain probably due to lysosome dysfunction but had no effects on the reelin pathway, expression of N-cadherin, or activation of JNK, which are implicated in the regulation of neuronal migration. Finally, we demonstrated that knocking down ß1 integrin partially rescued the migration defects, suggesting that Vps18 deficiency-mediated up-regulation of ß1 integrin may contribute to the defect of neuronal migration in the Vps18-deficient brain. Our results demonstrate important roles of Vps18 in neuron survival and migration, which are disrupted in multiple neural disorders.

MST1 promotes apoptosis through regulating Sirt1-dependent p53 deacetylation.

Mammalian Sterile 20-like kinase 1 (MST1) protein kinase plays an important role in the apoptosis induced by a variety of stresses. The MST1 is a serine/threonine kinase that is activated upon apoptotic stimulation, which in turn activates its downstream targets, JNK/p38, histone H2B and FOXO. It has been reported that overexpression of MST1 initiates apoptosis by activating p53. However, the molecular mechanisms underlying MST1-p53 signaling during apoptosis are unclear. Here, we report that MST1 promotes genotoxic agent-induced apoptosis in a p53-dependent manner. We found that MST1 increases p53 acetylation and transactivation by inhibiting the deacetylation of Sirtuin 1 (Sirt1) and its interaction with p53 and that Sirt1 can be phosphorylated by MST1 leading to the inhibition of Sirt1 activity. Collectively, these findings define a novel regulatory mechanism involving the phosphorylation of Sirt1 by MST1 kinase which leads to p53 activation, with implications for our understanding of signaling mechanisms during DNA damage-induced apoptosis.

Vps18 deficiency inhibits dendritogenesis in Purkinje cells by blocking the lysosomal degradation of Lysyl Oxidase.

Dendrite development occupies a central position in the formation of nervous system. However, whether lysosomal degradative function is required for dendritogenesis of neurons remains unknown. We have recently demonstrated the critical role of Vps18 in the lysosomal degradation pathway in mice. Here, we report that Vps18 deficiency severely blocks the dendrite development of Pukinje cells but not cerebral cortical neurons. Furthermore, we also demonstrate that the lysyl oxidase (Lox) protein is degraded through lysosome and accumulated in the Vps18 deficient cerebellum but not in cerebral cortices. Our results suggest that lysosome regulates dendritogenesis of Purkinje cells though degrading Lox.

A cell-intrinsic role for Mst1 in regulating thymocyte egress.

The MST1 kinase was recently identified as playing an essential role in the promotion of lymphocyte polarization and adhesion stimulated by chemokines and TCR signaling. However, the physiological relevance of the Mst1 pathway in thymocyte development is not completely understood. In this study, we analyzed the effect of Mst1 disruption on thymocyte development and migration. Mst1-deficient (Mst1(-/-)) mice displayed an accumulation of mature thymocytes in the thymus, a dramatic reduction of lymphocytes in blood and peripheral lymphoid tissues, and a decrease of homing ability to peripheral lymph nodes. Mst1(-/-) thymocytes were impaired in chemotactic response to chemokines, such as CCL19, but not to sphingosine-1-phosphate. Further analyses of Mst1(-/-) mice revealed a severe impairment in the egress of mature T cells from the thymus. T lineage-specific knockout of the Mst1 gene demonstrates a cell-intrinsic role for Mst1 in regulating T cell development. Our study indicates that Mst1 is crucial in controlling lymphocyte chemotaxis and thymocyte emigration.

LHCGR and ALMS1 defects likely cooperate in the development of polycystic ovary syndrome indicated by double-mutant mice.

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder with evidence of polygenetic components, and obesity may be a risk factor for hyperandrogenism. Previous studies have shown that LHCGR is enriched in the ovary and LHCGR deficiency causes infertility without typical PCOS phenotypes. ALMS1 is implicated in obesity and hyperandrogenism, the common phenotypes among PCOS patients. Through whole-exome sequencing of 22 PCOS families and targeted candidate gene sequencing of additional 65 sporadic PCOS patients, we identified potential causative mutations in LHCGR and ALMS1 in a sibling-pair PCOS family and three sporadic PCOS patients. The expression of LHCGRL638P in granulosa-like tumor cell line (KGN) cells promoted cyclic adenosine monophosphate production and granulosa cell proliferation, indicating that LHCGRL638P is an activating mutation. LhcgrL642P/L642P mice showed an irregular estrous cycle, reduced follicles with dynamic folliculogenesis, and increased testosterone (T), estradiol (E2), and dehydroepiandrosterone. Lhcgr+/L642PAlms1+/PB mice displayed increased T and E2 but decreased late secondary and preovulatory follicles. We showed that activating mutation of LHCGR likely plays important roles in the pathophysiology of PCOS involving abnormal reproductive physiology, whereas ALMS1 deficiency may promote anovulatory infertility via elevated androgens, suggesting that the disturbed LHCGR and ALMS1 cooperatively induce PCOS phenotypes, characterized as anovulation and hyperandrogenemia frequently observed in PCOS patients with obesity.

Lats2 is a negative regulator of myocyte size in the heart.

Mammalian sterile 20-like kinase (Mst)1 plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Because Hippo, a Drosophila homolog of Mst1, forms a signaling complex with Warts, a serine/threonine kinase, which in turn stimulates cell death and inhibits cell proliferation, mammalian homologs of Warts, termed Lats1 and Lats2, may mediate the function of Mst1. We here show that Lats2, but not Lats1, dose-dependently increased apoptosis in cultured cardiac myocytes. Lats2 also dose-dependently reduced [(3)H]phenylalanine incorporation and cardiac myocyte size, whereas dominant negative Lats2 (DN-Lats2) increased them, suggesting that endogenous Lats2 negatively regulates myocyte growth. DN-Lats2 significantly attenuated induction of apoptosis and inhibition of hypertrophy by Mst1, indicating that Lats2 mediates the function of Mst1 in cardiac myocytes. Cardiac specific overexpression of Lats2 in transgenic mice significantly reduced the size of left and right ventricles, whereas that of DN-Lats2 caused hypertrophy in both ventricles. Overexpression of Lats2 reduced left ventricular systolic and diastolic function without affecting baseline levels of myocardial apoptosis. Expression of endogenous Lats2 was significantly upregulated in response to transverse aortic constriction. Overexpression of DN-Lats2 significantly enhanced cardiac hypertrophy and inhibited cardiac myocyte apoptosis induced by transverse aortic constriction. These results suggest that Lats2 is necessary and sufficient for negatively regulating ventricular mass in the heart. Although Lats2 is required for cardiac myocyte apoptosis in response to pressure overload, it was not sufficient to induce apoptosis at baseline. In conclusion, Lats2 affects both growth and death of cardiac myocytes, but it primarily regulates the size of the heart and acts as an endogenous negative regulator of cardiac hypertrophy.

Tbx20 Induction Promotes Zebrafish Heart Regeneration by Inducing Cardiomyocyte Dedifferentiation and Endocardial Expansion.

Heart regeneration requires replenishment of lost cardiomyocytes (CMs) and cells of the endocardial lining. However, the signaling regulation and transcriptional control of myocardial dedifferentiation and endocardial activation are incompletely understood during cardiac regeneration. Here, we report that T-Box Transcription Factor 20 (Tbx20) is induced rapidly in the myocardial wound edge in response to various sources of cardiac damages in zebrafish. Inducing Tbx20 specifically in the adult myocardium promotes injury-induced CM proliferation through CM dedifferentiation, leading to loss of CM cellular contacts and re-expression of cardiac embryonic or fetal gene programs. Unexpectedly, we identify that myocardial Tbx20 induction activates the endocardium at the injury site with enhanced endocardial cell extension and proliferation, where it induces the endocardial Bone morphogenetic protein 6 (Bmp6) signaling. Pharmacologically inactivating endocardial Bmp6 signaling reduces expression of its targets, Id1 and Id2b, attenuating the increased endocardial regeneration in tbx20-overexpressing hearts. Altogether, our study demonstrates that Tbx20 induction promotes adult heart regeneration by inducing cardiomyocyte dedifferentiation as well as non-cell-autonomously enhancing endocardial cell regeneration.

DVL mutations identified from human neural tube defects and Dandy-Walker malformation obstruct the Wnt signaling pathway.

Wnt signaling pathways, including the canonical Wnt/ß-catenin pathway, planar cell polarity pathway, and Wnt/Ca2+ signaling pathway, play important roles in neural development during embryonic stages. The DVL genes encode the hub proteins for Wnt signaling pathways. The mutations in DVL2 and DVL3 were identified from patients with neural tube defects (NTDs), but their functions in the pathogenesis of human neural diseases remain elusive. Here, we sequenced the coding regions of three DVL genes in 176 stillborn or miscarried fetuses with NTDs or Dandy-Walker malformation (DWM) and 480 adult controls from a Han Chinese population. Four rare mutations were identified: DVL1 p.R558H, DVL1 p.R606C, DVL2 p.R633W, and DVL3 p.R222Q. To assess the effect of these mutations on NTDs and DWM, various functional analyses such as luciferase reporter assay, stress fiber formation, and in vivo teratogenic assay were performed. The results showed that the DVL2 p.R633W mutation destabilized DVL2 protein and upregulated activities for all three Wnt signalings (Wnt/ß-catenin signaling, Wnt/planar cell polarity signaling, and Wnt/Ca2+ signaling) in mammalian cells. In contrast, DVL1 mutants (DVL1 p.R558H and DVL1 p.R606C) decreased canonical Wnt/ß-catenin signaling but increased the activity of Wnt/Ca2+ signaling, and DVL3 p.R222Q only decreased the activity of Wnt/Ca2+ signaling. We also found that only the DVL2 p.R633W mutant displayed more severe teratogenicity in zebrafish embryos than wild-type DVL2. Our study demonstrates that these four rare DVL mutations, especially DVL2 p.R633W, may contribute to human neural diseases such as NTDs and DWM by obstructing Wnt signaling pathways.