Single-chain fragment antibody disrupting the EphA4 function as a therapeutic drug for gastric cancer.

Studies have shown that the high expression of EphA4 in gastric cancer tissues may correlate with unfavorable clinical pathological characteristics. Therefore, EphA4 may be an effective target for treating gastric cancer in addition to HER-2/neu. In this study, generated scFv S3 can bind endogenous EphA4 of gastric cancer cells and has significant membrane staining. Additionally, scFv S3 binding to EphA4 inhibits the growth and migration of cancer cells and the growth induction that ephrinA1 generates in gastric cancer cells. We found that EphA4 molecules may degrade through antibody treatment of cells, and the increase in LAMP1 and LAMP2 indicates that lysosome is involved in the degradation. The scFv S3 administration leads to the signals pAKT, pERK, and pSTAT3 decrease in cancer cells. The xenograft model of HER-2/neu low expressing gastric cancer cell SNU-16 exhibits better therapeutic effects by scFv S3 than trastuzumab scFv. The scFv S3 administration in vivo can degrade EphA4 molecules in tumor tissues, decreasing Ki67 and increasing cleaved C3 molecule expression. Furthermore, we identified and validated that scFv S3 generates essential ionic bonding with R162 on EphA4. The antibody may provide effective treatment for patients with gastric cancer and abnormal activation or overexpression of EphA4 signaling.

Identification of a dual FLT3 and MNK2 inhibitor for acute myeloid leukemia treatment using a structure-based virtual screening approach.

Fms-like tyrosine kinase 3 (FLT3) is considered a promising therapeutic target for acute myeloid leukemia (AML) in the clinical. However, monotherapy with FLT3 inhibitor is usually accompanied by drug resistance. Dual inhibitors might be therapeutically beneficial to patients with AML due to their ability to overcome drug resistance. Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) phosphorylate eukaryotic translation initiation factor 4E (eIF4E), which brings together the RAS/RAF/ERK and PI3K/AKT/mTOR oncogenic pathways. Therefore, dual inhibition of FLT3 and MNK2 might have an additive effect against AML. Herein, a structure-based virtual screening approach was performed to identify dual inhibitors of FLT3 and MNK2 from the ChemDiv database. Compound K783-0308 was identified as a dual inhibitor of FLT3 and MNK2 with IC50 values of 680 and 406 nM, respectively. In addition, the compound showed selectivity for both FLT3 and MNK2 in a panel of 82 kinases. The structure-activity relationship analysis and common interactions revealed interactions between K783-0308 analogs and FLT3 and MNK2. Furthermore, K783-0308 inhibited MV-4-11 and MOLM-13 AML cell growth and induced G0/G1 cell cycle arrest. Taken together, the dual inhibitor K783-0308 showed promising results and can be potentially optimized as a lead compound for AML treatment.

Identification of a dual TAOK1 and MAP4K5 inhibitor using a structure-based virtual screening approach.

The STE20 kinase family is a complex signalling cascade that regulates cytoskeletal organisation and modulates the stress response. This signalling cascade includes various kinase mediators, such as TAOK1 and MAP4K5. The dysregulation of the STE20 kinase pathway is linked with cancer malignancy. A small-molecule inhibitor targeting the STE20 kinase pathway has therapeutic potential. In this study, a structure-based virtual screening (SBVS) approach was used to identify potential dual TAOK1 and MAP4K5 inhibitors. Enzymatic assays confirmed three potential dual inhibitors (>50% inhibition) from our virtual screening, and analysis of the TAOK1 and MAP4K5 binding sites indicated common interactions for dual inhibition. Compound 1 revealed potent inhibition of colorectal and lung cancer cell lines. Furthermore, compound 1 arrested cancer cells in the G0/G1 phase, which suggests the induction of apoptosis. Altogether, we show that the STE20 signalling mediators TAOK1 and MAP4K5 are promising targets for drug research.

Biological Evaluation of Selected Flavonoids as Inhibitors of MNKs Targeting Acute Myeloid Leukemia.

Excessive eIF4E phosphorylation by mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (MNK1 and MNK2; collectively, MNKs) has been associated with oncogenesis. The overexpression of eIF4E in acute myeloid leukemia (AML) is related to cancer cell growth and survival. Thus, the inhibition of MNKs and eIF4E phosphorylation are potential therapeutic strategies for AML. Herein, a structure-based virtual screening approach was performed to identify potential MNK inhibitors from natural products. Three flavonoids, apigenin, hispidulin, and luteolin, showed MNK2 inhibitory activity with IC50 values of 308, 252, and 579 nM, respectively. A structure-activity relationship analysis was performed to disclose the molecular interactions. Furthermore, luteolin exhibited substantial inhibitory efficacy against MNK1 (IC50 = 179 nM). Experimental results from cellular assays showed that hispidulin and luteolin inhibited the growth of MOLM-13 and MV4-11 AML cells by downregulating eIF4E phosphorylation and arresting the cell cycle at the G0/G1 phase. Therefore, hispidulin and luteolin showed promising results as lead compounds for the potential treatment for AML.

Cell-autonomous heparanase modulates self-renewal and migration in bone marrow-derived mesenchymal stem cells.

BACKGROUND: Stem cell-fate is highly regulated by stem cell niche, which is composed of a distinct microenvironment, including neighboring cells, signals and extracellular matrix. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells and are potentially applicable in wide variety of pathological conditions. However, the niche microenvironment for BM-MSCs maintenance has not been clearly characterized. Accumulating evidence indicated that heparan sulfate glycosaminoglycans (HS-GAGs) modulate the self-renewal and differentiation of BM-MSCs, while overexpression of heparanase (HPSE1) resulted in the change of histological profile of bone marrow. Here, we inhibited the enzymatic activity of cell-autonomous HPSE1 in BM-MSCs to clarify the physiological role of HPSE1 in BM-MSCs. RESULTS: Isolated mouse BM-MSCs express HPSE1 as indicated by the existence of its mRNA and protein, which includes latent form and enzymatically active HPSE1. During in vitro osteo-differentiations, although the expression levels of Hpse1 fluctuated, enzymatic inhibition did not affect osteogenic differentiation, which might due to increased expression level of matrix metalloproteinase 9 (Mmp9). However, cell proliferation and colony formation efficiency were decreased when HPSE1 was enzymatically inhibited. HPSE1 inhibition potentiated SDF-1/CXCR4 signaling axis and in turn augmented the migratory/anchoring behavior of BM-MSCs. We further demonstrated that inhibition of HPSE1 decreased the accumulation of acetylation marks on histone H4 lysine residues suggesting that HPSE1 also modulates the chromatin remodeling. CONCLUSIONS: Our findings indicated cell-autonomous HPSE1 modulates clonogenicity, proliferative potential and migration of BM-MSCs and suggested the HS-GAGs may contribute to the niche microenvironment of BM-MSCs.

An ensemble machine learning model generates a focused screening library for the identification of CDK8 inhibitors.

The identification of an effective inhibitor is an important starting step in drug development. Unfortunately, many issues such as the characterization of protein binding sites, the screening library, materials for assays, etc., make drug screening a difficult proposition. As the size of screening libraries increases, more resources will be inefficiently consumed. Thus, new strategies are needed to preprocess and focus a screening library towards a targeted protein. Herein, we report an ensemble machine learning (ML) model to generate a CDK8-focused screening library. The ensemble model consists of six different algorithms optimized for CDK8 inhibitor classification. The models were trained using a CDK8-specific fragment library along with molecules containing CDK8 activity. The optimized ensemble model processed a commercial library containing 1.6 million molecules. This resulted in a CDK8-focused screening library containing 1,672 molecules, a reduction of more than 99.90%. The CDK8-focused library was then subjected to molecular docking, and 25 candidate compounds were selected. Enzymatic assays confirmed six CDK8 inhibitors, with one compound producing an IC50 value of ≤100 nM. Analysis of the ensemble ML model reveals the role of the CDK8 fragment library during training. Structural analysis of molecules reveals the hit compounds to be structurally novel CDK8 inhibitors. Together, the results highlight a pipeline for curating a focused library for a specific protein target, such as CDK8.

ASPM stabilizes the NOTCH intracellular domain 1 and promotes oncogenesis by blocking FBXW7 binding in hepatocellular carcinoma cells.

Notch signaling is aberrantly activated in approximately 30% of hepatocellular carcinoma (HCC), significantly contributing to tumorigenesis and disease progression. Expression of the major Notch receptor, NOTCH1, is upregulated in HCC cells and correlates with advanced disease stages, although the molecular mechanisms underlying its overexpression remain unclear. Here, we report that expression of the intracellular domain of NOTCH1 (NICD1) is upregulated in HCC cells due to antagonism between the E3-ubiquitin ligase F-box/WD repeat-containing protein 7 (FBXW7) and the large scaffold protein abnormal spindle-like microcephaly-associated protein (ASPM) isoform 1 (ASPM-i1). Mechanistically, FBXW7-mediated polyubiquitination and the subsequent proteasomal degradation of NICD1 are hampered by the interaction of NICD1 with ASPM-i1, thereby stabilizing NICD1 and rendering HCC cells responsive to stimulation by Notch ligands. Consistently, downregulating ASPM-i1 expression reduced the protein abundance of NICD1 but not its FBXW7-binding-deficient mutant. Reinforcing the oncogenic function of this regulatory module, the forced expression of NICD1 significantly restored the tumorigenic potential of ASPM-i1-deficient HCC cells. Echoing these findings, NICD1 was found to be strongly co-expressed with ASPM-i1 in cancer cells in human HCC tissues (P < 0.001). In conclusion, our study identifies a novel Notch signaling regulatory mechanism mediated by protein-protein interaction between NICD1, FBXW7, and ASPM-i1 in HCC cells, representing a targetable vulnerability in human HCC.

Identification of selective dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors and their effects on tau and microtubule.

The overexpression of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), commonly observed in neurodegenerative diseases like Alzheimer's disease (AD) and Down syndrome (DS), can induce the formation of neurofibrillary tangles (NFTs) and amyloid plaques. Hence, designing a selective DYRK1A inhibitor would result in a promising small molecule for treating neurodegenerative diseases. Developing selective inhibitors for DYRK1A has been a difficult challenge due to the highly preserved ATP-binding site of protein kinases. In this study, we employed a structure-based virtual screening (SBVS) campaign targeting DYRK1A from a database containing 1.6 million compounds. Enzymatic assays were utilized to verify inhibitory properties, confirming that Y020-3945 and Y020-3957 showed inhibitory activity towards DYRK1A. In particular, the compounds exhibited high selectivity for DYRK1A over a panel of 120 kinases, reduced the phosphorylation of tau, and reversed the tubulin polymerization for microtubule stability. Additionally, treatment with the compounds significantly reduced the secretion of inflammatory cytokines IL-6 and TNF-α activated by DYRK1A-assisted NFTs and Aß oligomers. These identified inhibitors possess promising therapeutic potential for conditions associated with DYRK1A in neurodegenerative diseases. The results showed that Y020-3945 and Y020-3957 demonstrated structural novelty compared to known DYRK1A inhibitors, making them a valuable addition to developing potential treatments for neurodegenerative diseases.

Pathogen recognition receptor signaling accelerates phosphorylation-dependent degradation of IFNAR1.

An ability to sense pathogens by a number of specialized cell types including the dendritic cells plays a central role in host's defenses. Activation of these cells through the stimulation of the pathogen-recognition receptors induces the production of a number of cytokines including Type I interferons (IFNs) that mediate the diverse mechanisms of innate immunity. Type I IFNs interact with the Type I IFN receptor, composed of IFNAR1 and IFNAR2 chains, to mount the host defense responses. However, at the same time, Type I IFNs elicit potent anti-proliferative and pro-apoptotic effects that could be detrimental for IFN-producing cells. Here, we report that the activation of p38 kinase in response to pathogen-recognition receptors stimulation results in a series of phosphorylation events within the IFNAR1 chain of the Type I IFN receptor. This phosphorylation promotes IFNAR1 ubiquitination and accelerates the proteolytic turnover of this receptor leading to an attenuation of Type I IFN signaling and the protection of activated dendritic cells from the cytotoxic effects of autocrine or paracrine Type I IFN. In this paper we discuss a potential role of this mechanism in regulating the processes of innate immunity.

Urolithin A exhibits a neuroprotective effect against Alzheimer's disease by inhibiting DYRK1A activity.

Alzheimer's disease (AD) is a devastating neurodegenerative disease with more than 50 million people suffer from it. Unfortunately, none of the currently available drugs is able to improve cognitive impairment in AD patients. Urolithin A (UA) is a metabolite obtained from ellagic acid and ellagitannin through the intestinal flora, and it has antioxidant and anti-inflammatory properties. Previous reports found that UA had neuroprotective effects in an AD animal model, but the detailed mechanism still needs to be elucidated. In this study, we performed kinase-profiling to show that dual-specific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is the main target of UA. Studies showed that the level of DYRK1A in AD patients' brains was higher than that of healthy people, and it was closely related to the occurrence and progression of AD. Our results revealed that UA significantly reduced the activity of DYRK1A, which led to de-phosphorylation of tau and further stabilized microtubule polymerization. UA also provided neuroprotective effects by inhibiting the production of inflammatory cytokines caused by Aß. We further showed that UA significantly improved memory impairment in an AD-like mouse model. In summary, our results indicate that UA is a DYRK1A inhibitor that may provide therapeutic advantages for AD patients.

Correction: Chondroitin sulfate proteoglycan 4 regulates zebrafish body axis organization via Wnt/planar cell polarity pathway.

[This corrects the article DOI: 10.1371/journal.pone.0230943.].

Identification and analysis of a selective DYRK1A inhibitor.

The dysregulation of DYRK1A is implicated in many diseases such as cancer, diabetes, and neurodegenerative diseases. Alzheimer's disease is one of the most common neurodegenerative disease and has elevated interest in DYRK1A research. Overexpression of DYRK1A has been linked to the formation of tau aggregates. Currently, an effective therapeutic treatment that targets DYRK1A is lacking. A specific small-molecule inhibitor would further our understanding of the physiological role of DYRK1A in neurodegenerative diseases and could be presented as a possible therapeutic option. In this study, we identified pharmacological interactions within the DYRK1A active site and performed a structure-based virtual screening approach to identify a selective small-molecule inhibitor. Several compounds were selected in silico for enzymatic and cellular assays, yielding a novel inhibitor. A structure-activity relationship analysis was performed to identify areas of interactions for the compounds selected in this study. When tested in vitro, reduction of DYRK1A dependent phosphorylation of tau was observed for active compounds. The active compounds also improved tau turbidity, suggesting that these compounds could alleviate aberrant tau aggregation. Testing the active compound against a panel of kinases across the kinome revealed greater selectivity towards DYRK1A. Our study demonstrates a serviceable protocol that identified a novel and selective DYRK1A inhibitor with potential for further study in tau-related pathologies.

O-methylated flavonol as a multi-kinase inhibitor of leukemogenic kinases exhibits a potential treatment for acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease with poor overall survival characterized by various genetic changes. The continuous activation of oncogenic pathways leads to the development of drug resistance and limits current therapeutic efficacy. Therefore, a multi-targeting inhibitor may overcome drug resistance observed in AML treatment. Recently, groups of flavonoids, such as flavones and flavonols, have been shown to inhibit a variety of kinase activities, which provides potential opportunities for further anticancer applications. PURPOSE: In this study, we evaluated the anticancer effects of flavonoid compounds collected from our in-house library and investigated their potential anticancer mechanisms by targeting multiple kinases for inhibition in AML cells. METHODS: The cytotoxic effect of the compounds was detected by cell viability assays. The kinase inhibitory activity of the selected compound was detected by kinase-based and cell-based assays. The binding conformation and interactions were investigated by molecular docking analysis. Flow cytometry was used to evaluate the cell cycle distribution and cell apoptosis. The protein and gene expression were estimated by western blotting and qPCR, respectively. RESULTS: In this study, an O-methylated flavonol (compound 11) was found to possess remarkable cytotoxic activity against AML cells compared to treatment in other cancer cell lines. The compound was demonstrated to act against multiple kinases, which play critical roles in survival signaling in AML, including FLT3, MNK2, RSK, DYRK2 and JAK2 with IC50 values of 1 - 2 µM. Compared to our previous flavonoid compounds, which only showed inhibitions against MNKs or FLT3, compound 11 exhibited multiple kinase inhibitory abilities. Moreover, compound 11 showed effectiveness in inhibiting internal tandem duplications of FLT3 (FLT3-ITDs), which accounts for 25% of AML cases. The interactions between compound 11 and targeted kinases were investigated by molecular docking analysis. Mechanically, compound 11 caused dose-dependent accumulation of leukemic cells at the G0/G1 phase and followed by the cells undergoing apoptosis. CONCLUSION: O-methylated flavonol, compound 11, can target multiple kinases, which may provide potential opportunities for the development of novel therapeutics for drug-resistant AMLs. This work provides a good starting point for further compound optimization.

Inducible priming phosphorylation promotes ligand-independent degradation of the IFNAR1 chain of type I interferon receptor.

Phosphorylation-dependent ubiquitination and ensuing down-regulation and lysosomal degradation of the interferon alpha/beta receptor chain 1 (IFNAR1) of the receptor for Type I interferons play important roles in limiting the cellular responses to these cytokines. These events could be stimulated either by the ligands (in a Janus kinase-dependent manner) or by unfolded protein response (UPR) inducers including viral infection (in a manner dependent on the activity of pancreatic endoplasmic reticulum kinase). Both ligand-dependent and -independent pathways converge on phosphorylation of Ser(535) within the IFNAR1 degron leading to recruitment of beta-Trcp E3 ubiquitin ligase and concomitant ubiquitination and degradation. Casein kinase 1 alpha (CK1 alpha) was shown to directly phosphorylate Ser(535) within the ligand-independent pathway. Yet given the constitutive activity of CK1 alpha, it remained unclear how this pathway is stimulated by UPR. Here we report that induction of UPR promotes the phosphorylation of a proximal residue, Ser(532), in a pancreatic endoplasmic reticulum kinase-dependent manner. This serine serves as a priming site that promotes subsequent phosphorylation of IFNAR1 within its degron by CK1 alpha. These events play an important role in regulating ubiquitination and degradation of IFNAR1 as well as the extent of Type I interferon signaling.

A novel dual HDAC and HSP90 inhibitor, MPT0G449, downregulates oncogenic pathways in human acute leukemia in vitro and in vivo.

Acute leukemia is a highly heterogeneous disease; therefore, combination therapy is commonly used for patient treatment. Drug-drug interaction is a major concern of combined therapy; hence, dual/multi-target inhibitors have become a dominant approach for cancer drug development. HDACs and HSP90 are involved in the activation of various oncogenic signaling pathways, including PI3K/AKT/mTOR, JAK/STAT, and RAF/MEK/ERK, which are also highly enriched in acute leukemia gene expression profiles. Therefore, we suggest that dual HDAC and HSP90 inhibitors could represent a novel therapeutic approach for acute leukemia. MPT0G449 is a dual effect inhibitor, and it showed cytotoxic effectiveness in acute leukemia cells. Molecular docking analysis indicated that MPT0G449 possessed dual HDAC and HSP90 inhibitory abilities. Furthermore, MPT0G449 induced G2 arrest and caspase-mediated cell apoptosis in acute leukemia cells. The oncogenic signaling molecules AKT, mTOR, STAT3, STAT5, MEK, and ERK were significantly downregulated after MPT0G449 treatment in HL-60 and MOLT-4 cells. In vivo xenograft models confirmed the antitumor activity and showed the upregulation of acetyl-histone H3 and HSP70, biomarkers of pan-HDAC and HSP90 inhibition, with MPT0G449 treatment. These findings suggest that the dual inhibition of HDAC and HSP90 can suppress the expression of oncogenic pathways in acute leukemia, and MPT0G449 represents a novel therapeutic for anticancer treatment.

EGFL6 promotes colorectal cancer cell growth and mobility and the anti-cancer property of anti-EGFL6 antibody.

BACKGROUND: The availability of a reliable tumor target for advanced colorectal cancer (CRC) therapeutic approaches is critical since current treatments are limited. Epidermal growth factor-like domain 6 (EGFL6) has been reported to be associated with cancer development. Here, we focused on the role of EGFL6 in CRC progression and its clinical relevance. In addition, an anti-EGFL6 antibody was generated by phage display technology to investigate its potential therapeutic efficacy in CRC. RESULTS: EGFL6 expression significantly increased in the colon tissues from CRC patients and mice showing spontaneous tumorigenesis, but not in normal tissue. Under hypoxic condition, EGFL6 expression was enhanced at both protein and transcript levels. Moreover, EGFL6 could promote cancer cell migration invasion, and proliferation of CRC cells via up-regulation of the ERK/ AKT pathway. EGFL6 also regulated cell migration, invasion, proliferation, and self-renewal through EGFR/αvß3 integrin receptors. Treatment with the anti-EGFL6 antibody EGFL6-E5-IgG showed tumor-inhibition and anti-metastasis abilities in the xenograft and syngeneic mouse models, respectively. Moreover, EGFL6-E5-IgG treatment had no adverse effect on angiogenesis and wound healing CONCLUSIONS: We demonstrated that EGFL6 plays a role in CRC tumorigenesis and tumor progression, indicating that EGFL6 is a potential therapeutic target worth further investigation.

The single nucleotide variant at c.662A>G in human RRM2B is a loss-of-function mutation.

BACKGROUND: Mitochondrial DNA maintenance defects (MDMDs) is one of the critical pediatric dysfunction. One of the recent report indicated that a severe patient of MDMDs carries the NP_056528.2:p.Asn221Ser (N221S) variation in the RRM2B gene (NM_015713.5). However, there is no direct evidence demonstrating the nature of the N221S variation. MATERIALS AND METHODS: This study aimed to utilize zebrafish and morpholino oligomer (MO) knockdown technique to provide direct evidence for the nature of the N221S variation in the RRM2B. RESULTS: The results showed that two distinct MOs were both able to perturb the expression of rrm2b in zebrafish and dose-dependently induced morphological defects. Furthermore, co-injection of human wild-type RRM2B mRNA with MO-e4i4 successfully rescued the developmental defects, whereas co-injection of RRM2B/N221S mRNA with MO-e4i4 did not rescue the developmental defects. CONCLUSION: In conclusion, the functional assay in this study provided the direct evidence proving that the N221S variation is a loss-of-function mutation and plausibly related to the pathogenic developmental defects found in the infants of previous clinical reports.

Cell-intrinsic Fgf signaling contributes to primordial germ cell homing in zebrafish.

Primordial germ cells (PGCs) are specified before gastrulation and migrate toward the developing gonads. Previous in vitro studies have demonstrated a cell-intrinsic requirement of fibroblast growth factors (FGFs) by PGCs; however, no evidence suggests FGFs signal directly to PGCs in vivo. Here, using zebrafish as the animal model, we identified the mRNA expressions of Fgf receptors (Fgfrs) and determined the roles of Fgf signaling in migrating PGCs. To clarify the functions of Fgf signaling, we manipulated Fgf signaling specifically in PGCs using dominant-negative (dn) and constitutively-active (ca) Fgfrs and revealed a requirement of a basal Fgf signaling level for the robust arrival of PGCs. Repression of Fgf signaling in PGCs swayed the marginal positioning of PGCs as early as 6 h post-fertilization (6 hpf) and disrupted their arrival at the gonadal ridge at 24 hpf. On the other hand, the ectopic PGC phenotypes caused by the dn-Fgfrs could be alleviated by constitutive activation of Fgf signaling. In addition, we carefully ruled out the somatic effects in mosaic embryos by injecting RNA materials into one blastomere of the four- or eight-cell stage embryos. Injection of dn-Fgfrs into one of eight blastomeres hampered the arrival of only the treated PGCs, while the other PGCs remained unaffected. Furthermore, mosaic treatment of ca-Fgfrs rescued the ectopic rates of dn-Fgfr treated PGCs, while the other PGCs remained more ectopic within the same embryos. Interestingly, PGC-specific repression of Fgf signaling did not compromise the PGC number. To our knowledge, this is the first in vivo evidence to show that Fgf signaling plays a cell-intrinsic role in the migration of vertebrate PGCs.

Chondroitin sulfate proteoglycan 4 regulates zebrafish body axis organization via Wnt/planar cell polarity pathway.

Pericellular and extracellular proteoglycans play an important role in modulating morphogen gradients and signal transductions. Chondroitin sulfate proteoglycan 4 (Cspg4) is a membrane spanning proteoglycan expressed in immature progenitor cells and cancer cells. Cspg4 participates in cellular events such as proliferation, migration and signal transduction, and these events are generally important for embryo development. In this study, we characterized Cspg4 for its roles in zebrafish embryonic development. Our results demonstrated that cspg4 was maternally expressed from 0 to 3 hours post fertilization (hpf) and expressed in the anterior and posterior embryo end after 9 hpf. Knocking-down cspg4 resulted in a shorter anterior-posterior axis than control embryo, which could be rescued by co-injecting wnt11 mRNA suggesting that Cspg4 regulates body axis organization through modulating the Wnt/planar cell polarity signaling pathway. In addition, overexpressing cspg4 caused cyclopia. The Cspg4 transmembrane domain mutant embryo phenocopied the global over-expression of cspg4 mRNA and led to cyclopia with a very low penetrance. Our results demonstrated that the quantitatively and spatially accurate distribution of Cspg4 is critical for body axis and midline development during gastrulation.