CTNNB1 exon 3 mutations are associated with an aggressive phenotype in several of these tumor types and may be associated with therapeutic tolerance. Desmoid tumors typically have a stable genome with β-catenin mutations as a main feature, making these tumors an ideal model to study the changes associated with different types of β-catenin mutations. Here, we show that the apoptosis mechanism is deregulated in β-catenin S45F mutants, resulting in decreased induction of apoptosis in these cells. Our findings also demonstrate that RUNX3 plays a pivotal role in the inhibition of apoptosis found in the β-catenin S45F mutants. Restoration of RUNX3 overcomes this inhibition in the S45F mutants, highlighting it as a potential therapeutic target for malignancies harboring this specific CTNNB1 mutation. While the regulatory effect of RUNX3 in β-catenin is already known, our results suggest the possibility of a feedback loop involving these two genes, with the CTNNB1 S45F mutation downregulating expression of RUNX3, thus providing additional possible novel therapeutic targets for tumors having deregulated Wnt/β-catenin signaling induced by this mutation.Wnt/β-catenin signaling is one of the key cascades regulating embryogenesis and tissue homeostasis; it has also been intimately associated with carcinogenesis. This pathway is deregulated in several tumors, including colorectal cancer, breast cancer, and desmoid tumors. It has been shown that  WNT1a as an oncogene, the canonical WNT/β-catenin pathway has been found to be deregulated in several human cancers [Since the discovery of  cancers –5. In thIn the presence of Wnt ligands, Dsh forms a complex with Axin, Frizzled (FZL) and LDL receptor-related protein 5/6 (LRP5/6). This interaction favors the translocation of Axin to the plasma membrane and disruption of the destruction complex, thereby inhibiting β-catenin degradation in the proteasome. Consequently, β-catenin can translocate to the nucleus and interfere with TCF-LEF co-transcription factors, resulting in activation of several oncogenic genes [CTNNB1 gene encoding for β-catenin have been shown to stabilize β-catenin by disrupting the phosphorylation-dependent ubiquitination, resulting in activation of the β-catenin signaling. This activation leads to tumorigenesis and it has been reported in a large number of human cancers, including endometrial carcinoma, hepatocellular carcinoma, colorectal cancer, and desmoid tumors [CTNNB1 gene and its presence has been associated with an aggressive phenotype in several types of tumors, such as endometrial, hepatocellular, and thyroid cancers [CTNNB1 mutation, but also specific CTNNB1 mutations seem to be associated with a more aggressive phenotype and with therapeutic tolerance. The CTNNB1 S45 mutation results in stronger activation of β-catenin and may be associated with malignant transformation of hepatocellular adenoma [β-catenin protein was first discovered as a component of cell adhesion processes by its binding to cadherins and by linking cadherins to the actin cytoskeleton , 8. In ad tumors –14, rend cancers –17. Inte adenoma . Murine  adenoma . In addi adenoma –23.CTNNB1 gene is a common desmoid tumors feature [CTNNB1 gene, only three β‐catenin point mutations  have been commonly found in desmoid tumors. No other significant genomic alterations other than these β-catenin mutations have been found in desmoid tumors [Desmoid tumors are rare, locally invasive mesenchymal lesions that have a high risk for local recurrence, significantly decreasing patient quality of life. A deregulated Wnt/β-catenin pathway due to mutations in the  feature . Distincd tumors –27, makiRUNX3 forms a complex with β-catenin and TCF4, and that this interaction attenuates the DNA-binding activity of β-catenin/TCF4 [RUNX3 levels. Here, we demonstrate that S45F mutant β-catenin has deregulated expression of several genes involved in apoptosis, resulting in the impairment of this cell death mechanism; moreover, this impairment is specific to the S45F mutation per se and not to the cell model utilized. We also demonstrate that downregulation of RUNX3 in S45F mutant desmoids might be a possible explanation for their lack of apoptosis induction. In addition, overexpression of RUNX3 induced apoptosis in the S45F mutant desmoids, thus providing possible novel targets therapeutic interventions in tumors with deregulated Wnt/β-catenin signaling caused by the CTNNB1 S45F mutation.Another focus of investigation has sought to determine β-catenin regulatory mechanisms to enable new therapeutic strategies versus malignancies having a deregulated Wnt/β-catenin pathway. Studies of the mechanisms that regulate β-catenin transcriptional activity have become of great interest, given that nuclear localization of β-catenin is key to Wnt/β-catenin signaling. In this context, RUNX3 has emerged as a novel protein that interacts with transcriptional complex components and regulates β-catenin transcriptional activity –30. It hnin/TCF4 , 30; howTo evaluate if there is a differential gene expression pattern between the T41A and S45F mutations, we performed a gene array of 50 desmoid tumor tissues and 10 adjacent normal tissues. Array data were deposited at the National Center for Biotechnology Information with accession number GSE61097. First, to confirm that the samples in the gene array accurately represented desmoid tumors, we compared the gene array data of these desmoid tumors with that of the normal tissues. Our results, when combined with Ingenuity Pathway Analysis (IPA), showed that the first and second most deregulated pathways in our tumor samples were related to fibrosis and the WNT/β-catenin pathway, respectively, confirming that our samples were an accurate representative of desmoid tumors Fig. , given tCTNNB1 T41A and S45F mutations in a sample set containing 12 desmoid tissues with CTNNB1 T41A mutation and 13 desmoid tissues with a CTNNB1 S45F mutation. These results, combined with IPA analysis, showed that apoptotic genes are differentially expressed in S45F mutation  to recapitulate the biology observed in the patient-derived desmoid cells Fig. . Given tCTNNB1 mutations. Our results showed that the difference between the T41A and S45F mutation was not due to differential protein expression levels, since total β-catenin is equally expressed in both mutations  coimmunoprecipitated with RUNX3 in desmoid cell lines  detected by flow cytometry in 293T cells co-transfected with RUNX3 and β-catenin S45F mutant were significantly increased after treatment with doxorubicin as compared with 293T transfected only with the S45F mutant and control vectors  might play a role in the impairment of apoptosis observed in these cells. RUNX3 expression has been shown to be lost or downregulated in several tumors, including gastric, breast and colorectal cancers [RUNX3 might be related to the more aggressive behavior and therapeutic tolerance of these cells. Interestingly, our results show that overexpression of CTNNB1 S45F mutation decreases the expression of RUNX3 in 293T cells, suggesting that there might be a feedback loop where RUNX3 regulates β-catenin activity, but is also regulated by β-catenin itself. To the best of our knowledge, this is the first study to show that a specific mutation of CTNNB1 affects RUNX3 expression. Next, we sought to investigate the effects of restoration in the apoptosis inhibition observed in β-catenin S45F mutant cells. Our results showed that overexpression of RUNX3 reversed the effect of S45F-inhibited apoptosis in 293T cells, suggesting that RUNX3 plays a pivotal role in β-catenin S45F mutant therapeutic tolerance via inhibition of apoptosis, highlighting it as a potential therapeutic approach for malignancies harboring the CTNNB1 S45F mutation.Because of its role in regulating vital normal cell functions, inhibition of β-catenin is not a suitable strategy to treat patients having tumor-associated deregulated Wnt/β-catenin pathways. For this reason, identifying and characterizing mechanisms that regulate β-catenin transcriptional activity may be more relevant as new therapies. In this context, RUNX3 emerges as an important protein that regulates β-catenin activity. It has been shown that RUNX3 is able to constrain the Wnt pathway signaling even when this pathway is aberrantly activated due β-catenin activation . Our res cancers –43. More cancers . We veriRUNX3 overcomes this inhibition in the S45F mutants via the caspase pathway. Although the regulatory effect of RUNX3 on β-catenin is already known, our results suggest that there may be a previously not described regulatory feedback loop between CTNNB1 S45F mutation and downregulated expression of RUNX3. Further investigation are needed to better understand the mechanisms underlying this possible feedback loop.In summary, we have shown that the apoptosis mechanism is impaired in the β-catenin S45F mutated cells. We have also shown that RUNX3 plays a role in the inhibition of apoptosis found in these β-catenin S45F mutants and that restoration of CTNNB1 mutational status for all cell lines and corresponding tumors to confirm that the cell lines were truly desmoid tumor cells.All DT cells included in this study were created at the MD Anderson Cancer Center and at the Sarcoma Research Lab at the Ohio State University. All cells were authenticated and tested for mycoplasma prior to the start of this study and every six months during the duration of the study. This study was conducted with approval from both the Ohio State University and the MD Anderson Cancer Center institutional review board (IRB) with written informed consent of patients. Sanger sequencing was used to identify Staurosporine (cat#: S1421) and doxorubicin (cat#: S1208) were purchased from Selleckchem .CTNNB1 T41A mutation, 17 with S45F mutation, 2 with S45P mutation and 11 with no detectable CTNNB1 mutations. Power curve was used to assess sample size and power for identifying differential expressed genes (fold change > 2). Total RNA was isolated with the RNeasy mini kit  according to the manufacturer’s instructions. HumanHT-12 v4 Expression BeadChip arrays  were used to analyze gene expression in DT tissues.For gene array purposes, we included 50 desmoid tumor tissues and 10 adjacent normal tissues. The sample set contained 20 desmoid tissues with ® Reverse Transcription Reagents  were used to generate cDNA. StepOnePlus™ Real-Time PCR System (ThermoFisher) was used to analyze the cDNA by quantitative real-time PCR. Relative expression levels were normalized against β-actin and GAPDH RNA expression.TaqManAnnexin V-PI staining  was used to measure cell cycle progression and apoptosis induction as previously described . CaspaseCTNNB1 plasmids were obtained from Origene . 293T cells were transfected with Fugene HD  and selected with 1 mg/mL Neomicin (Life Technologies).Control vector plasmid and The tissues and cells were lysed in 1× lysis buffer  containing protease inhibitor. PureProteome™ Magnetic Bead  was used to perform the immunoprecipitation, according to the manufacturer’s instructions. Briefly, the protein lysate was incubated with 1 μg of the RUNX3 antibody overnight at 4 °C. PureProteome™ Magnetic Bead washed with binding buffer using the PureProteome™ Magnetic Stand (Millipore). The antibody-antigen sample was added to the beads and incubated at room temperature for 30 min with continuous mixing. Finally, 30 μl of loading buffer was added to the beads and boiled for 10 min. Elutions were collected using the PureProteome™ Magnetic Stand and loaded into the SDS-PAGE gel for protein analysis.Western blotting analysis were performed as previously described . Brieflyt test analysis assessed statistical significance between experimental groups. P < 0.05 was considered statistically significant.Unpaired two-tailed Student Legend for supplemental materialTable S1 Dysregulated genes in desmoid tumors compared to normal tissue samples.Table S2 Dysregulated genes in S45F-mutated desmoid tumors compared to T41A-mutated desmoid tumors.Figure S1 Gene array expression analysis. Outcome of the gene expression analysis summarized for |LFC| ≥ 1 and adjusted p-value < 0.01. The heatmap represents genes differentially expressed between desmoid tumors versus corresponding normal tissue.Figure S2 Inhibition of apoptosis is not due to P21 or P53. Expression of P21 and P53 levels in desmoid tumor cells treated with doxorubicin by western blot.Figure S3 Transfection of different CTNNB1 mutations in 293T cells. Different CTNNB1 mutations were stably overexpressed in 293T cells. Actin was used as a loading control.Figure S4 Overexpression of RUNX3 in transfected 293T cells. RUNX3 was stably overexpressed in CTNNB1 transfected 293T cells. GAPDH was used as a loading control.


Furthermore, we uncovered that decreased MPC1 activated the Wnt/β-catenin pathway by promoting nuclear translocation of β-catenin to mediate the expression of MMP7, E-cadherin, Snail1, and myc. Collectively, our data suggest that MPC1 has the potential to be served as a promising biomarker for diagnosis and a therapeutic target in CRC.Accumulating evidence has pointed out that metastasis is the leading cause of death in several malignant tumor, including CRC. During CRC, metastatic capacity is closely correlated with reprogrammed energy metabolism. Mitochondrial Pyruvate Carrier 1 (MPC1), as the carrier of transporting pyruvate into mitochondria, linked the glycolysis and TCA cycle, which would affect the energy production. However, the specific role of MPC1 on tumor metastasis in CRC remains unexplored. Here, by data mining of genes involved in pyruvate metabolism using the TCGA dataset, we found that MPC1 was significantly downregulated in CRC compared to nontumor tissues. Similar MPC1 expression pattern was also found in multiple GEO datasets. IHC staining in both human sample and AOM/DSS induced mouse CRC model revealed significant downregulation of MPC1. What is more, we found that MPC1 expression was gradually decreased in normal tissue, primary CRC, and metastasis CRC. Additionally, poor prognosis emerged in the MPC1 low expression patients, especially in patients with metastasis. Following, functional tests showed that MPC1 overexpression inhibited the motility of CRC cells  Colorectal carcinoma (CRC), as one of the most common gastrointestinal malignancies, has developed the world's fourth most deadly cancer with a high rate of incidence and mortality , 2. LiveDuring cancer progression, metabolic reprograming with increasing glucose utilization is termed as Warburg affect, which is accompanied by altered pyruvate and mitochondrial metabolism . The fatExisted studies have reported that MPC1 was related with immunoregulation, stemness, metabolism, cellular morphology, etc. , 8–10. Cin vitro and in vivo. Mechanically, MPC1 suppressed CRC metastasis through mediating the Wnt/β-catenin signaling. Thus, our finding firstly revealed a critical role of MPC1 in CRC liver metastasis.In the present study, we figured out the relationship between the MPC1 expression and CRC liver metastasis. We identified that decreased MPC1 was closely correlated with patient's metastasis, as well as led to poor prognosis. Functionally, MPC1 overexpression could attenuate the migration and invasion capacities of CRC cells both https://www.cancer.gov/. The primary data for GEO datasets were downloaded at https://www.ncbi.nlm.nih.gov/geo. The OncoLnc database (http://www.oncolnc.org/) was used to detect the prognostic value of MPC1 in the TCGA cohort.Seven GEO datasets  and TCGA were used to analyze the MPC1 expression pattern in CRC. The primary data for TCGA datasets were downloaded In our study, a total of 392 patients containing paired CRC tissues and adjacent nontumor tissues were enrolled from the Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University. Among them, 30 cases of liver metastases were collected. And only 225 cases were available with complete follow-up data for survival analysis. Informed consents were signed by all patients. The research was approved by the Research Ethics Committee of Renji Hospital and carried out in accordance with ethical standards as formulated in the Helsinki Declaration.Human CRC cell lines  and mouse CRC cell line MC38 were gained from the Cell Bank of the Chinese Academy of Sciences . All cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics at 37°C in a humidified incubator with 5% CO2.μg/ml blastisidin for 14 days, which termed MC38-Luc. In addition, one short hairpin RNA (shRNA) sequence against MPC1 was packaged as lentivirus and transfected into MC38-luc cells. Lovo and SW480 were transfected with lentivirus containing full-length human MPC1 cDNA or empty vehicle control. Stable transfection cells were screened under 2 μg/ml puromycin for 14 days and verified by western blot. In those assays, all lentiviral transfections were performed in the presence of 6 μg/ml polybrene.MC38 cells were transfected with lentivirus containing a luciferase reporter plasmid. Stable transfection cells were screened with 5 The protocol of this assay and quantify the MPC1 protein expression level were performed according to previously reported . Primaryβ-catenin  and incubated with Alexa 488-conjugated secondary antibody (1 : 200). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).Assays were performed according to the previous description . Brieflyβ-catenin , lamin A/C , and GAPDH . Species-specific secondary antibodies used as follows: IRDye 680 Goat anti-Mouse IgG (LI-COR) and IRDye 800 Goat anti-rabbit IgG (LI-COR).Whole-cell lysates or nuclear protein was extracted using a protein extraction buffer  or nucleoprotein extraction kit , respectively. Proteins were resolved by SDS-PAGE and transferred onto nitrocellulose (NC) membranes using standard methods. Primary antibodies used as follows: MPC1 , △CT− method was used to analyze the data and GAPDH was used as a loading control. The primer sequences are listed in Total RNA was extracted using Trizol (Takara) according to the manufacturer's instructions. cDNA was synthesized using the PrimeScript reverse transcription-polymerase chain reaction (RT-PCR) kit (Takara). The qPCR was performed using SYBR Green Master Mix (Roche). The 2 n = 5) with a concentration of 106 cells/mouse. Two weeks later, the mice were killed and the liver metastasis tissues were harvested.This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals and relevant Chinese laws and regulations. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University. The MC38 cells transplanting luciferase-expressing were injected into the spleens of C57BL/6N mice  or 100 ng FOP reporter plasmid  and 10 ng Renilla were mixed and transfected into CRC cells using Lipofectamine 2000. Dual-luciferase reporter assay system (Promega) was used to detect the firefly and Renilla luciferase activities.The protocols of this assay were operated in accordance with previous reported . 100 ng t-test was used for comparison between groups. P < 0.05 was considered as statistically significant.Data are shown as means ± SD. SPSS 20.0  and GraphPad Prism 5 software were used to manipulate statistical analyses. Kaplan-Meier method was used to calculate cumulative survival time. The chi-square test or Student's Pyruvate is a pivotal intermediate in the process of cell metabolism, which connects glycolysis and the TCA cycle. To determine the potential maladjustment genes involved in pyruvate metabolism, which is located in mitochondria, as shown in P = 0.009), lymph node invasion (P = 0.003), and TNM stage (P = 0.001), which revealed by the analysis between the clinical significance and MPC1 expression in CRC  production [Following, in the analysis between the clinical significance and MPC1 expression in CRC, we found that MPC1 expression was especially correlated with metastasis. Inspired by this, we detected the MPC1 expression pattern in normal tissue, primary CRC, and metastasis CRC by GEO datasets and patients' tissue. All results revealed that gradually downregulated MPC1 was in patients with an increase in metastasis ability. Survival analysis indicated that worse outcome was presented in patients with lower MPC1 expression, especially in patients with metastasis. Additionally, function assays verified that MPC1 overexpression could attenuate the migration and invasion capacities of CRC cells oduction . Besidesoduction . These poduction . Howeverβ-catenin signaling. The next series of experiments also confirmed that MPC1 could mediate the WNT/β-catenin pathway by redistribution of β-catenin. Previous reports showed that cytoplasmic β-catenin phosphorylated in N-terminally localized to sites of cell-cell contact is associated with E-cadherin and was required for intact cell-cell adhesions, without any change detected in the levels of total β-catenin [β-catenin limited Wnt signals [β-catenin was reported to interact with USP9x to inhibit the degradation of β-catenin through the deubiquitination of β-catenin in breast cancer [β-catenin protein interaction activated MYC expression in Acute Lymphoblastic Leukemia Cells [β-catenin was reported to interact with Yap1 to lead to rapid tumorigenesis [β-catenin maybe crosstalk with other genes or involved in other biological processes. What is more, some downstream target genes of β-catenin, such as MMP7, E-cadherin, Snail1, and myc, were changed in expression. As known to us, MMP-7 is a member of the proteolytic enzyme family, which promotes the invasion and metastasis of tumor cells by degrading the basement membrane and extracellular matrix [Subsequently, the underlying mechanism of MPC1 in regulating metastasis was explored. For this purpose, GSEA analysis was performed. The results indicated that MPC1 was involved in the Wnt/-catenin –25. Simu signals . In addit cancer . A constia Cells . In HCC,igenesis . Hence, r matrix . And prer matrix , 31. E-cr matrix . Moreover matrix , 34. Andr matrix . The comr matrix . Inspireβ-catenin contributed to CRC cell motility. This means that MPC1 has the potential to be a diagnostic biomarker and therapeutic target for metastasis patients.In conclusion, we firstly demonstrated that decreased MPC1 was closely correlated with patient's metastasis, as well as led to poor outcome. Moreover, MPC1-driven nuclear translocation of


Paenibacillus polymyxa and adhesion mechanisms of mussel byssi and octopus tentacles, we develop hierarchical microneedles with multifunctional adhesive and antibacterial abilities. With polydopamine hydrogel as the microneedle base and a loop of suction-cup-structured concave chambers encircling each microneedle, the generated microneedles can fit the skin well; keep strong adhesion in dry, moist, and wet environments; and realize self-repair after being split into two parts. Besides, as polymyxin is loaded into both the hydrogel tips and the polydopamine base, the microneedles are endowed with excellent ability to resist common bacteria during storage and usage. We have demonstrated that these microneedles not only showed excellent adhesion when applied to knuckles and ideal antibacterial activity but also performed well in drug-sustained release and treatment for the osteoarthritis rat model. These results indicate that bioinspired multifunctional microneedles will break through the limitation of traditional methods and be ideal candidates for versatile transdermal drug delivery systems.Microneedles have attracted increasing interest among various medical fields due to their painless, noninvasive, and efficient way of drug delivery. However, practical applications of these microneedles in different epidermal locations and environments are still restricted by their low adhesion and poor antimicrobial activity. Here, inspired by the antibacterial strategy of  Microneedles (MNs) could penetrate the skin without touching blood capillaries and nerve endings, thus providing a painless, noninvasive, and effective way for drug delivery , 2. In rPaenibacillus polymyxa (P. polymyxa), we suggest novel MNs with desired functions for flexible biomedical applications, as presented in P. polymyxa have been found to strikingly restrain the growth of other bacteria and even kill them by secreting a kind of antimicrobial peptide named polymyxin, which has a wide antimicrobial spectrum and could avoid drug resistance. These bioadhesive [In this paper, inspired by the adhesion features of mussels and octopi, together with the antibacterial mechanism of adhesive –26 and aadhesive –30 stratP. polymyxa, we present the desired multifunctional MNs for practical applications. Polydopamine (PDA) hydrogel was chosen as the flexible base of the MNs, which shared similar molecular structure to the adhesive protein and imparted the resultant MNs with strong adhesion. This integrated PDA could also help the MNs fit the skin and made them self-healing. Notably, each MN was surrounded by a circle of concave chambers with dome-like protuberances located inside. This octopus tentacle-mimicked structure further improved the adhesive ability, making the MNs adhesive in dry, moist, and wet environments. In addition, as the polymyxin was loaded into both the hydrogel tips and the PDA base, the MNs were endowed with the ability to resist common bacteria, such as Escherichia coli (E. coli). It was demonstrated that these MNs not only showed excellent adhesion when applied to knuckles and ideal antibacterial activity but also behaved well in drug-sustained delivery and treatment for the osteoarthritis rat model. This indicates that the bioinspired multifunctional MNs could break through the limitation of traditional methods which had difficulty in acting on the mobile parts effectively and provide a new and promising insight into the transdermal drug delivery of any part of the body.Herein, by integrating the molecular structure of adhesive protein of mussel byssi, the microstructure of octopus tentacles, and polymyxin from In a typical experiment, the multifunctional MNs were prepared by replicating a specially designed negative mold . The negπ-π stacking, etc. , MNs only with PDA hydrogel as the base (PDA-based MNs), MNs only with the suction-cup-like structure (suction-cup MNs), and MNs with both the PDA hydrogel as the base and the suction-cup-like structure  . During E. coli were killed by adding only a small quantity of polymyxin, and the killing ratios of E. coli increased corresponding to the increasing concentration of polymyxin  rat model. The KOA rat model was established by breaking the knee joints of SD rats in the overextension position Figure . Except To further explore the curative effects of glucocorticoid-loaded MNs, tissue slices in each group were stained with Safranin O-fast green, hematoxylin-eosin (H&E), and Masson. It was seen from the Safranin O-fast green staining that in the n-MN group and the control group, the surfaces of cartilages were obviously uneven and the tidemarks were irregular and almost indiscernible. On the contrary, the d-MN group showed an evident recovery of such lesions . Similar+) have been put forward to realize their self-sterilization. However, their drug resistance, allergenicity, and ion cytotoxicity still affect the practical performances. Therefore, more efforts are still anticipated to develop new adhesive, antibacterial MNs.In summary, we have presented bioinspired adhesive and antibacterial MNs which can be employed in versatile transdermal drug delivery. Making MNs adhesive to skin and resistant to bacteria has been an ongoing issue in MN-related fields. The poor adhesion ability of most MNs requires their usage in the company of auxiliary equipment like a medical adhesive tape, which may bring about inconvenience in application and cause discomfort to skin regions. To improve their adhesion ability , attentiP. polymyxa, known for their low biological toxicity, wide antibacterial spectrum, high bacterial lethality, and no drug resistance, make the MNs bacteria-resistant and safe. The fabricated MNs also possess many other features, including flexibility, self-repair, and drug-sustained release. These characteristics enable our MNs to be used versatilely, including a long time and sustainably delivering drugs to highly mobile body parts  and susceptible body regions . Thus, it is believed that these MNs can be promising candidates in transdermal drug delivery devices, wearable biomedical systems, and so on.We have herein fabricated bioinspired multifunctional MNs with ideal adhesive and antimicrobial abilities to well solve the aforementioned problems. The adhesive PDA hydrogel base and suction-cup-mimicked microstructure impart the MNs with excellent adhesion ability, while the antimicrobial peptides from Ω·cm−1. NIH 3T3 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences . Cells were cultured in high glucose Dulbecco's Modified Eagle's Medium  with 10% (v/v) fetal bovine serum  and 1% (v/v) penicillin-streptomycin double antibiotics in the incubator  under 37°C, 5% CO2. The 280-320 g male Sprague Dawley (SD) rats were from Jinling Hospital. Animals were treated in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of China. All the animal care and experimental protocols were reviewed and approved by the Animal Investigation Ethics Committee of Jinling Hospital.Poly(ethylene glycol) diacrylate , dopamine hydrochloride, gelatin, and rhodamine B  were obtained from Sigma-Aldrich. Sodium alginate was bought from Alfa Aesar. Sodium periodate and 2-hydroxy-2-methylpropiophenone (HMPP) were purchased from Aladdin. Polymyxin B (10000 IU/ml) was provided by Hangwei Corp. Glucocorticoid was provided by Jinling Hospital. SYTO and propidium iodide (PI) were supplied by KeyGEN BioTECH Corp. The MTT reagent was obtained from J&K Scientific Ltd., Shanghai. Phosphate-buffered saline (PBS) was self-prepared in the laboratory. A Millipore Milli-Q system provided deionized water with a resistivity of 18.2 MThe optical images of MNs were observed by a stereomicroscope  and recorded by CCD . Digital images of the pork skin, the knuckle, and SD rats were captured by a digital camera (Canon 5D Mark II). The SEM images were characterized using a field scanning electron microscope . The fluorescence images were taken by a laser scanning confocal microscope .v/v), HMPP (1% v/v), and sodium alginate (2% w/v) were first mixed together in the deionized water. 200 μl of the mixed solution was then added to a custom-built negative mold and placed in a vacuum for 10 min to fully fill the cavities. After the redundant solution was sucked up using a pipettor, 450 μl solution containing 0.0127 g sodium periodate, 0.0225 g dopamine hydrochloride, 0.075 g gelatin, and a small droplet of NaOH (1 M) was added to cover the negative mold. Polymyxin was premixed with the PEGDA solution and the dopamine solution with the final concentration of 25 IU/ml, 50 IU/ml, 100 IU/ml, 200 IU/ml, 300 IU/ml, or 400 IU/ml. The PEGDA-sodium alginate tips were solidified by UV irradiation  for 15 s, and the dopamine-gelatin base was gelated under room temperature for 20 min. Notably, due to its low power and short application time, the applied UV irradiation could also be applicable to biologics. Finally, the resultant MNs (300 μm in diameter and 600 μm in length) with orderly aligned concave chambers (375 μm in the top diameter and 300 μm in the bottom diameter) were gently peeled out of the negative mold.PEGDA . Four different kinds of MNs, normal MNs, PDA-based MNs, suction-cup MNs, and multifunctional MNs, were placed on the surface of the pork skin pieces and were pressed with the same force for 5 s. It should be mentioned that for variable control, the total number of microneedles and suction cups combined per unit area, as well as the size of four kinds of MNs, was kept the same. After that, the MNs were lifted off with the initial peeling angle of about 10° and the displacement velocity at about 0.1 mm/s. During this process, the time-dependent peeling strength was recorded by a bundled software (MulForce). The peeling adhesion was calculated by dividing the peeling strength before the detachment by the contact area.Pork skin bought from the local butcher's was first cut into about 3 × 2 cmE. coli were chosen to investigate the antibacterial ability of the MNs. MNs containing different polymyxin concentrations, 25 IU/ml, 50 IU/ml, 100 IU/ml, 200 IU/ml, 300 IU/ml, and 400 IU/ml, were fabricated with the aforementioned method and immersed in PBS solution (pH = 7.4) for 2 days. Then, 1 ml of the leaching liquor of the MNs was mixed with 100 μl of the E. coli solution (104 CFU ml−1) and incubated in the 48-well plate for 6 h at 37°C. To visualize the bacteria viability, the bacteria were stained with two staining agents, SYTO for 20 min and PI for 5 min in the dark. As a result, the live E. coli were dyed green and dead ones were dyed red.The Gram-negative bacteria μl of cell suspensions (about 5 × 104 cells) was seeded into each well on the plate and cultured for 2 days. After that, 100 μl MTT solution was added into the wells and incubated for 4 h. Finally, the entire medium was removed, and 500 μl DMSO was added to totally dissolve the formazan crystals. The cell viability was obtained by reading out the OD values of the DMSO solutions using a microplate reader .MNs containing different polymyxin concentrations were cultivated in the culture medium for 2 days. Subsequently, the leaching liquor was sterilized by 200 nm syringe filters and added into a 48-well plate (1 ml per well). 100 RhB was employed as a model for water-soluble small molecular drugs. MNs containing 0.5 mg/ml RhB were immersed in PBS solution and shaken on a thermomixer (Eppendorf) at the rate of 450 r/min under 37°C. The time-varying OD values of the PBS solution were measured by the microplate reader (540 nm excitation wavelength). Based on the OD values and the standard curve of RhB, the release amount of RhB from the MNs could be calculated.280-320 g male SD rats were first anesthetized by intraperitoneal injection of 10% chloral hydrate. The right knee joints of the rats were fixed by tubular plaster in the overextension position, and the plaster was wrapped with a plastic shell to prevent the rats biting the plaster. The right knee joints were immobilized for 3 weeks. During this period, the plaster was to be immediately adjusted or changed if it got loose, fell off, or caused skin necrosis and ulcers. The rats were then equally divided into three groups, which were applied with glucocorticoid- (10 mg/ml) loaded MNs, unloaded MNs, and no treatment. An applicator was employed to apply these MNs to the skin, which had a relatively weak attachment to the back of the MNs and could be detached from the MNs after application. The MNs were changed every day. It should be mentioned that during the removal, a small quantity of water was first applied to lower the adhesion of the PDA hydrogel. Then, starting from the edge, the MNs could be gently peeled off. After consecutive treatments for 7 days, the rats were sacrificed and their right knees were sampled. The joints were fixed in 4% paraformaldehyde, decalcified, embedded in paraffin, sliced up, and stained with Safranin O-fast green, hematoxylin-eosin (H&E), and Masson, respectively. The degrees of joint lesions were statistically analyzed using a modified Mankin scoring system , 35.


Wnt8 and Wnt11 mRNA induced ectopic dorsal axis in neurulae and larvae. Finally, we demonstrated that Nodal and Wnt/β-catenin signaling cooperate to promote the dorsal-specific gene expression in amphioxus gastrula. Our study reveals high evolutionary conservation of dorsal organizer formation in the chordate lineage.Deciphering the mechanisms of axis formation in amphioxus is a key step to understanding the evolution of chordate body plan. The current view is that Nodal signaling is the only factor promoting the dorsal axis specification in the amphioxus, whereas Wnt/β-catenin signaling plays no role in this process. Here, we re-examined the role of Wnt/βcatenin signaling in the dorsal/ventral patterning of amphioxus embryo. We demonstrated that the spatial activity of Wnt/β-catenin signaling is located in presumptive dorsal cells from cleavage to gastrula stage, and provided functional evidence that Wnt/β-catenin signaling is necessary for the specification of dorsal cell fate in a stage-dependent manner. Microinjection of  One of the most intriguing queries in developmental biology is how the body plan evolved throughout the animal kingdom. In vertebrates, one of the key initial events in the early development is the formation of dorsal (Spemann) organizer, which establishes early dorsoanterior/ventroposterior asymmetry. The dorsal organizer was first described in amphibian embryo at the early gastrula stage as a group of cells from dorsal blastopore lip, which has the ability to induce the formation of secondary embryonic axis (a tween embryo) if grafted to the ventral side of the host embryo . It was Xenopus by gain-of-function experiments, in which ectopic Wnt proteins induced secondary organizer formation and duplication of the embryonic axis  is distributed in early amphioxus embryo.Branchiostoma lanceolatum at the cleavage, blastula and early gastrula stages by using homemade mouse antibodies specific for amphioxus β-catenin. As previously demonstrated, these antibodies detect nuclear β-catenin in the amphioxus embryos in the same pattern as commercial rabbit anti-human β-catenin antibodies  into the amphioxus egg or treatment of embryos with CHIR99021 and scored the phenotype and protein or mRNA expression of notochord marker Brachyury and neural markers Elav or Ac-Tub (acetylated tubulin) at the neurula or larval stage. We assumed that by microinjection of Wnt ligand mRNA we would be able not only to overactivate Wnt/β-catenin signaling, but also randomly disrupt or change the activity signaling gradient in the early embryo. Although Wnt8 is not expressed in the organizer of vertebrates and amphioxus, injected Wnt8 mRNA promotes ectopic axis formation in the vertebrates  and Nodal receptor inhibitor SB505124 (0.5 µM) separately and simultaneously. We left the embryos to develop until the cap-shaped gastrula to analyze the expression of dorsal-specific genes  pathway , or mid-ker Elav . Separatv  and transported to the Institute of Molecular Genetics . The animals were maintained in the lab and the spawning was induced as described in Branchostoma floridae were originally collected in Tampa (Florida) and subsequently maintained as a continuous breeding culture in the laboratory. For raising and maintenance, larvae and adults are provided with brown phytoplankton (Isochrysis) originally obtained from NCMA at the Bigelow Laboratory for Ocean Sciences . Animals are raised at 25°C and adults are kept on a 10 hr dark/14 hr light cycle. Ripe animals are selected and maintained at 14°C for at least 6 weeks. Then the animals are moved to 28°C for 24 hr. When the light is turned off, 20–80% of the animals usually spawn 1 hr later.Adults of Branchiostoma lanceolatum were fertilized and the embryos were transferred to 6 cm Petri dishes. The embryos were kept at 19 °C and treated for 15 min after fertilization at one-cell stage or 4.5 hr after fertilization at the blastula stage with different chemicals. The blastula stage was defined according to Collected eggs from Wnt8 mRNA at a concentration of 21 ng/µl were not able to undergo gastrulation properly and died during the gastrulation.The concentrations of injected Wnt11 mRNA were 7 ng/µl, 20 ng/µl, 50 ng/µl (low dose) and 250 ng/µl (high dose). Microinjection of Wnt11 mRNA at a concentration of 7 ng/µl and 20 ng/µl had no effect on the normal development of amphioxus embryos. The concentration of injected dnTcf mRNA was 500 ng/µl.The coding regions of amphioxus Wnt8, Wnt11 and dnTcf were subcloned into pCS2+ using primers Wnt8_fw/Wnt8_rev, Wnt11_fw/Wnt11_rev and dnTcf_fw/dnTcf_rev. RNA was prepared using the mMessage mMachine kit (Ambion) and purified by RNeasy Micro kit (Qiagen). mRNA injection was performed as previously described . The injFor in situ hybridization, the fixed embryos were transferred to 70% ethanol and stored for at least several days at −70°C. In situ hybridization was performed as previously described  and carrPartial coding regions of amphioxus Elav, Goosecoid and Brachyury (Bra1) were subcloned into pET42a using primers Elav_fw/Elav_rev, Goosecoid_fw/Goosecoid_rev and Brachyury_fw/Brachyury_rev. Expression and purification of proteins for immunization were performed as described in B. lanceolatum genomic DNA using primers Nodal_fw/Nodal_rev and Vg1_fw/Vg1_rev and subcloned into pGL3-basic vector. AmphiGoosecoid-luciferase reporter gene and expression vector encoding stabilized β-catenin were described previously , a small group of basal chordates which are of crucial importance for our understanding of deuterostome evolution. While the expression and function of Wnt signaling during embryogenesis of these animals has been described previously, there was no conclusive picture of the role of Wnt/β-Catenin signaling in axial patterning and organizer formation. In the present paper, the authors reanalyzed Wnt/β-catenin signaling using an Amphioxus-specific β-Catenin antibody and loss-of-function / gain-of-function experiments. They performed a thorough and compelling analysis that shows exciting similarities in axis formation between cephalochordates and vertebrates.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Thomas Holstein (Reviewer #2); Christof Niehrs (Reviewer #3).Thank you for submitting your article "Wnt/β-catenin signaling is an evolutionarily conserved determinant of chordate dorsal organizer" for consideration by Summary:The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The reviewers agree that you have generated new insight into the role of Wnt/ß-catenin signaling during the embryonic development of two species of amphioxus. Your main conclusion is that Wnt/ß-catenin is necessary for the formation of the dorsal organizer and acts in concert with Nodal signaling, as it occurs in vertebrates. The result is viewed as interesting for the field as it contradicts results from a previous study and thereby suggests that the last common ancestor of all chordates already used the strategy of Wnt/ß-catenin activity or the formation of the dorsal organizer.Essential revisions:Each of the reviewers has made suggestions to improve the manuscript. I attach the reviews below. It seems to me that none of the comments require new experimentation. I am looking forward to receiving an updated and improved version of the work.Reviewer #1:eLife.In this study, the authors studied the role of Wnt/ß-catenin signaling during the embryonic development of two species of amphioxus. The main conclusion of their work is that as in vertebrates, Wnt/ß-catenin is necessary for the formation of the dorsal organizer and acts in concert with Nodal signaling. This result is of interest as it contradicts a previous low-quality study on the topic and thereby strongly suggests that the last common ancestor of all chordates already used this strategy. The scope of the work is thus adapted to the broad readership of eLife article. Some inconclusive experiments are included and some claims are insufficiently substantiated. The work needs some formal, and perhaps some experimental work before it reaches these standards.As it stands, however, the work does not reach the quality standard expected from an Main comments:The authors do not show evidence that the antibodies used are specific for the proteins of interest. Some of these antibodies were previously described in Bozzo et al., 2017, but this publication does not show either that the antibodies specifically recognize a single protein, using Western blot for instance.The phenotypes obtained with JW74 are not very convincing, and this compound is less specific for Wnt signaling (it blocks tankyrases) than C59 or the overexpression of dnTcf. I am not sure adding experiments with this compound is necessary, but if the authors think they are, the stages analyzed with C59 and JW74 should be identical. What happens when C59 is applied during blastula stages? Does this still lead to a failure to form axial structures? If not, this suggests that the target Wnt gene is of maternal origin.Chordin expression at the gastrula stage  is not very conclusive. It could be interpreted as overall background staining and I am not sure whether the gene is up- or down-regulated.I would have expected CHIR treatment from the zygote stage would lead to the complete vegetalization of the embryos. Is this observed (is ß-catenin found nuclear throughout the embryo?) and could this explain the gastrulation phenotype observed by the authors? I am aware that making new experiments in this Covid period is difficult/impossible but the authors should at least comment on these issues. Figure 6 suggests that there is no major increase in endoderm as documented by the presence of both Brachyury and Elav expression. Overall, the analysis at the larval stage is made difficult by the very disturbed morphogenesis induced by the inhibitor. Finally, the analysis of Molecular phenotypes obtained following Wnt8 overexpression are very difficult to interpret. I do not think they add to the story. Overexpression of Wnt11 is more interesting, but the pattern of expression of this gene in the organizer suggests that inhibition of Wnt synthesis at this stage should lead to the repression of axis formation (see comment above on the timing of action of C59). Does Wnt11 mRNA microinjection lead to early ß-catenin nuclear localization? If it does, and this gene is in reality expressed much later, then it is a poor candidate for the endogenous Wnt starting the cascade and its overexpression does not add to the experiments carried out with CHIR. It could at best be mentioned briefly in a supp figure.Overall the manuscript is at places very technical. It would gain from a substantial shortening, removing the pieces of data that are least conclusive. It would also benefit from some reorganization, which could involve grouping together the various treatments that repress Wnt signaling  on the one hand and those that activate this pathway  on the other.Reviewer #2:This is a very interesting manuscript on the role of Wnt/β-Catenin signaling in Amphioxus , a small group of basal chordates which are of crucial importance for our understanding of deuterostome evolution. While in several papers the expression and function of Wnt signaling during embryogenesis of these animals has been described, there is no conclusive picture of the role of Wnt/β-Catenin signaling in axial patterning and organizer formation so far. In the manuscript under review, the authors reanalyzed Wnt/β-catenin signaling. Using an Amphioxus-specific β-Catenin antibody and loss-of-function / gain-of-function experiments they performed a thorough and compelling analysis that shows exciting similarities in axis formation with vertebrates. They found in embryos up to early gastrulation an asymmetrical distribution of nuclear β-Catenin, which overlaps with that of goosecoid expression, an organizer-specific gene, and P-Smad2, the crucial downstream factor of activated Nodal signaling. This suggests that Wnt/β-Catenin might have a similar function in setting up the dorsal organizer  as in vertebrates. Their main finding is supported by a number of functional experiments and specific marker genes for organizer formation , but also by microinjection experiments using wnt8/wnt11 mRNA that induced ectopic dorsal axes in Amphioxus larvae. A deeper functional analysis of the interactions between Wnt/β-catenin signaling and Nodal signaling clearly requires a genetic analysis of both pathway in Amphioxus organizer formation. However, the pharmacological experiments  presented in the current manuscript already indicate a cooperativity of Nodal and Wnt/β-Catenin signaling in establishing the organizer. This is already a significant and major step forward in our understanding of the origin of the vertebrate organizer.There are also some weaknesses in the current manuscript the authors should address in a revised version. The interaction of Nodal and Wnt/β-Catenin signaling is essential for the chordate organizer, but it is not restricted to deuterostomes as shown in Figure 10. Among others Grande and Patel, 2009, and Watanabe et al., 2014, reported a function of Nodal signaling downstream of Wnt/β-Catenin signaling in mollusks and cnidarians. While Nodal signaling might have been differentially co-opted in deuterostomes and protostomes during animal evolution its occurrence and relevance should be discussed. I'm also wondering why the authors did not mention the fact that a core-signaling cassette consisting of β-Catenin, Nodal and Pitx predated the cnidarian-bilaterian split  into Figure 10. Also, the paper of Chea et al., 2005, entitled “Nodal Signaling and the Evolution of Deuterostome Gastrulation” is missing.Reviewer #3:In vertebrate development, Wnt signaling has an early role in specification of dorso-anterior structures and a later one in antero-posterior patterning after MBT and during gastrulation/neural patterning. While the late role is conserved in Amphioxus, previous studies suggested that the early role was not, the evidence relying on b-cat immunostaining and LiCl treatments. In this study, Kozmikova and Kozmik revise this notion and demonstrate that the role of Wnt/b-catenin signaling in early dorso-ventral axis formation is indeed conserved in Amphioxus.eLife, or if it is better suited for a specialized journal, where most of these types of studies are typically found. Given how comprehensive the study is, its high technical quality, and the fact that it also nicely supports the conserved epistasis Wnt->nodal->goosecoid, I would support the manuscript being adequate for eLife.While this study is overdue, it may be questioned whether the confirmation of an early role of Wnt signaling in Amphioxus in this day and age is of sufficient novelty/conceptual advance for 1) Figure 2 Abiii shows asymmetric bcat on the dorsal side; Previously the authors  showed homogenous vegetal distribution of nuclear b-catenin with the same Ab at late blastula stage . This discrepancy is not explained.2) Figure 6A: doubling of bra expression  is only moderately convincing3) Figure 6B: shows anterior development inhibited by late wnt activation, consistent with previous findings and evolutionary conservation that late Wnt controls a-p patterning; needs to be commented on in Results and Discussion;4) The endogenous, maternal wnt, which controls the early dv signal in Amphioxus remains unexplored. The mRNA injection experiments with Wnt8 and Wnt11 are not informative in this regard. But identifying the endogenous Wnt doing the job may not be essential for the overall conclusion of the study.5) Figure 7Ac axis duplication is striking, but they should indicate the "n"6) The quote "However, overactivation of Wnt/β-catenin signaling at the mid-blastula stage does not influence the axial patterning during the amphioxus development" is misleading, since Wnt regulates amphioxus ap axial patterning, and be qualified: "However, overactivation of Wnt/β-catenin signaling at the mid-blastula stage does not influence the DORSO-VENTRAL axial patterning during the amphioxus development". Reviewer #1:In this study, the authors studied the role of Wnt/ß-catenin signaling during the embryonic development of two species of amphioxus. The main conclusion of their work is that as in vertebrates, Wnt/ß-catenin is necessary for the formation of the dorsal organizer and acts in concert with Nodal signaling. This result is of interest as it contradicts a previous low-quality study on the topic and thereby strongly suggests that the last common ancestor of all chordates already used this strategy. The scope of the work is thus adapted to the broad readership of eLife.As it stands, however, the work does not reach the quality standard expected from an eLife article. Some inconclusive experiments are included and some claims are insufficiently substantiated. The work needs some formal, and perhaps some experimental work before it reaches these standards.Main comments:The authors do not show evidence that the antibodies used are specific for the proteins of interest. Some of these antibodies were previously described in Bozzo et al., 2017, but this publication does not show either that the antibodies specifically recognize a single protein, using Western blot for instance.We understand the concerns of this reviewer, however the determination of antibody specificity is generally not a trivial task and very much depends on the application for which the antibody is used. From this point of view we appreciate the suggestion to use the Western blot with membrane-bound samples but we are certain that this will not resolve the issue of antibody specificity when applied to the whole-mount immunohistochemistry technique on PFA-fixed embryos. That is why we believe that the correlation between whole-mount in situ hybridization pattern with that obtained by wholemount immunohistochemistry represents a more appropriate  indication of antibody specificity see . We provWe would like to point out, however, that in the current study we use some of the antibodies to merely label the domains/structures of the developing amphioxus embryo such as dorsal blastopore lip (Goosecoid antibody), the notochord (Brachyury antibody), or the neural tube (Elav antibody). Although in our view the expression as determined by in situ hybridization and nuclear localization of the immunohistochemistry signal in case of transcription factors is a strong indication that the antibodies used in the current study specifically detect their presumed antigen, for the purpose of labelling embryonic structures even antibodies recognizing unknown epitopes would serve the purpose (if such antibodies existed). In case of the key ß-catenin antibody, we have shown in Bozzo et al., 2017, that there is an identical pattern using an independent antibody produced in different host species, making it highly unlikely that the antibody detects another amphioxus protein (other than ß-catenin).The phenotypes obtained with JW74 are not very convincing, and this compound is less specific for Wnt signaling (it blocks tankyrases) than C59 or the overexpression of dnTcf. I am not sure adding experiments with this compound is necessary, but if the authors think they are, the stages analyzed with C59 and JW74 should be identical. What happens when C59 is applied during blastula stages? Does this still lead to a failure to form axial structures? If not, this suggests that the target Wnt gene is of maternal origin.Given the inherent difficulty in obtaining amphioxus embryos and seasonality of spawnings this project lasted several years, with JW74 being used in initial experiments as soon as the compound became available . We agree that JW74 is not only less potent in inhibiting Wnt/ß-catenin signalling but also less specific compared to C59, and the data represent the weak part of the manuscript. Therefore, we decided to follow the suggestion of this reviewer and we removed the data using JW74 inhibitor from our revised manuscript.We believe that, while pursuing additional questions raised by this reviewer would be of interest for potential future work, experiments involved would certainly require extensive time period if done properly on seasonal amphioxus, and are beyond the focus of the current manuscript. We would like to point out that our primary aim in the current study was to find out if Wnt/ß-catenin signalling plays any role in the establishment of the dorsal organizer and DV axis formation. At the molecular level an organizer has to be set up at the end of blastula stage/onset of gastrulation. Previous data  and data in our study using CHIR99021 demonstrated that late activation of Wnt/ß-catenin signalling does not disturb DV patterning. In fact, we tried C59 treatment at 4.5h blastula stage  and we did not observe disturbance of DV patterning. Since the issue of zygotic genome activation in amphioxus is not settled it is currently impossible to claim that blastula stage treatment would discriminate between maternal and zygotic components.I would have expected CHIR treatment from the zygote stage would lead to the complete vegetalization of the embryos. Is this observed (is ß-catenin found nuclear throughout the embryo?) and could this explain the gastrulation phenotype observed by the authors? I am aware that making new experiments in this Covid period is difficult/impossible but the authors should at least comment on these issues. Figure 6 suggests that there is no major increase in endoderm as documented by the presence of both Brachyury and Elav expression. Overall, the analysis at the larval stage is made difficult by the very disturbed morphogenesis induced by the inhibitor. Finally, the analysis of Chordin expression at the gastrula stage  is not very conclusive. It could be interpreted as overall background staining and I am not sure whether the gene is up- or down-regulated.“Although the embryos treated with high concentration of CHIR99021 at 1-cell stage did not invaginate properly the blastopore ring was formed and partial invagination of vegetal plate took place. We did not observe the formation of exogastrula and strong vegetalization phenotype as described previously for sea urchin .”The above paragraph was included in the revised text.Chordin and Nodal we think that the above treatment leads to transformation into dorsal cell fate. In perturbation experiments we did not stain ß-catenin but we rather used axin expression as an indication of Wnt/ß-catenin signalling activity.Based on the expression of Chordin expression at the gastrula stage we did not claim that the gene is up- or down-regulated but we stated “the expression of Chordin and Nodal spread out over the whole embryo”. We are sure that the uniformly spread signal upon CHIR99201 does not represent a background since in both untreated and treated embryos the staining was performed in parallel for the same period of time. To mitigate concerns of this reviewer we provide in Regarding Molecular phenotypes obtained following Wnt8 overexpression are very difficult to interpret. I do not think they add to the story. Overexpression of Wnt11 is more interesting, but the pattern of expression of this gene in the organizer suggests that inhibition of Wnt synthesis at this stage should lead to the repression of axis formation (see comment above on the timing of action of C59). Does Wnt11 mRNA microinjection lead to early ß-catenin nuclear localization? If it does, and this gene is in reality expressed much later, then it is a poor candidate for the endogenous Wnt starting the cascade and its overexpression does not add to the experiments carried out with CHIR. It could at best be mentioned briefly in a supp figure.Wnt8 is not expressed in the organizer of vertebrates and amphioxus, injected Wnt8 mRNA promotes ectopic axis formation in the vertebrates”. In the Discussion of the original manuscript we wrote “Wnt11 was detected by RT-PCR at the one-cell stage embryo and expressed asymmetrically in the vegetal half of the amphioxus blastula  and dorsally at the onset of gastrulation and mid-gastrula stage .” By a mistake, within the Results section we only mentioned Wnt11 expression in the gastrula thus creating a confusion. We modified the text in the Results section.We agree that mRNA injection experiments with Wnt8 and Wnt11 are not informative in elucidating the question which of the endogenous Wnt controls the early DV signal in amphioxus. We used Wnt8 and Wnt11 primarily as tools, although only Wnt11 could possibly be a candidate molecule as correctly pointed out by the reviewer. On the other hand Wnt8 mRNA injection was used in vertebrates for induction of ectopic axis. This fact was mentioned for Wnt8 case in the original version of the manuscript by the following text: “Although In summary, we share the view with reviewer #3 that identifying the endogenous amphioxus Wnt involved in the organizer formation is not essential for the overall conclusion of the study.Overall the manuscript is at places very technical. It would gain from a substantial shortening, removing the pieces of data that are least conclusive. It would also benefit from some reorganization, which could involve grouping together the various treatments that repress Wnt signaling  on the one hand and those that activate this pathway  on the other.As described above we removed the JW74 data to help shortening the manuscript. We thank the reviewer for suggestion regarding manuscript reorganization and agree that it is better to group together the various treatments that repress Wnt signaling on the one hand and those that activate this pathway on the other.Reviewer #2:This is a very interesting manuscript on the role of Wnt/β-Catenin signaling in Amphioxus , a small group of basal chordates which are of crucial importance for our understanding of deuterostome evolution. While in several papers the expression and function of Wnt signaling during embryogenesis of these animals has been described, there is no conclusive picture of the role of Wnt/β-Catenin signaling in axial patterning and organizer formation so far. In the manuscript under review, the authors reanalyzed Wnt/β-catenin signaling. Using an Amphioxus-specific β-Catenin antibody and loss-of-function / gain-of-function experiments they performed a thorough and compelling analysis that shows exciting similarities in axis formation with vertebrates. They found in embryos up to early gastrulation an asymmetrical distribution of nuclear β-Catenin, which overlaps with that of goosecoid expression, an organizer-specific gene, and P-Smad2, the crucial downstream factor of activated Nodal signaling. This suggests that Wnt/β-Catenin might have a similar function in setting up the dorsal organizer  as in vertebrates. Their main finding is supported by a number of functional experiments and specific marker genes for organizer formation , but also by microinjection experiments using wnt8/wnt11 mRNA that induced ectopic dorsal axes in Amphioxus larvae. A deeper functional analysis of the interactions between Wnt/β-catenin signaling and Nodal signaling clearly requires a genetic analysis of both pathway in Amphioxus organizer formation. However, the pharmacological experiments  presented in the current manuscript already indicate a cooperativity of Nodal and Wnt/β-Catenin signaling in establishing the organizer. This is already a significant and major step forward in our understanding of the origin of the vertebrate organizer.There are also some weaknesses in the current manuscript the authors should address in a revised version. The interaction of Nodal and Wnt/β-Catenin signaling is essential for the chordate organizer, but it is not restricted to deuterostomes as shown in Figure 10. Among others Grande and Patel, 2009, and Watanabe et al., 2014, reported a function of Nodal signaling downstream of Wnt/β-Catenin signaling in mollusks and cnidarians. While Nodal signaling might have been differentially co-opted in deuterostomes and protostomes during animal evolution its occurrence and relevance should be discussed. I'm also wondering why the authors did not mention the fact that a core-signaling cassette consisting of β-Catenin, Nodal and Pitx predated the cnidarian-bilaterian split  into Figure 10.Hydra data for Wnt/β-catenin-Nodal cassette. The fact that Nodal signaling might have been differentially co-opted in deuterostomes and protostomes during animal evolution is discussed in the revised manuscript. Although discussed in the text, in Figure 10 we would very much like to maintain the focus on D/V patterning in deuterostomes leaving L/R axis out.We welcome these comments a lot. We are aware of the studies of Hobmayer  and Watanabe  in Hydra. However, we were afraid that we would be criticized for stretching the possible conservation of dorsal organizer of chordate embryos with head or budding zone organizer regions in Hydra polyp. Encouraged by the reviewer we were happy to modify the Discussion and Figure 10 to include Also, the paper of Chea et al., 2005, entitled “Nodal Signaling and the Evolution of Deuterostome Gastrulation” is missing.We thank reviewer for pointing this out. Reference was added into Introduction and Discussion sections.Reviewer #3:In vertebrate development, Wnt signaling has an early role in specification of dorso-anterior structures and a later one in antero-posterior patterning after MBT and during gastrulation/neural patterning. While the late role is conserved in Amphioxus, previous studies suggested that the early role was not, the evidence relying on b-cat immunostaining and LiCl treatments. In this study, Kozmikova and Kozmik revise this notion and demonstrate that the role of Wnt/b-catenin signaling in early dorso-ventral axis formation is indeed conserved in Amphioxus.While this study is overdue, it may be questioned whether the confirmation of an early role of Wnt signaling in Amphioxus in this day and age is of sufficient novelty/conceptual advance for eLife, or if it is better suited for a specialized journal, where most of these types of studies are typically found. Given how comprehensive the study is, its high technical quality, and the fact that it also nicely supports the conserved epistasis Wnt->nodal->goosecoid, I would support the manuscript being adequate for eLife.1) Figure 2 Abiii shows asymmetric bcat on the dorsal side; Previously the authors  showed homogenous vegetal distribution of nuclear b-catenin with the same Ab at late blastula stage . This discrepancy is not explained.In Bozzo et al.  we did not analyse 3D whole mounts of the embryos and the figure presented there shows a single optical section . Here we provide for the convenience of the reviewer several optical sections of the embryo 3D whole-mount of which is shown in 2) Figure 6A: doubling of bra expression  is only moderately convincingThe description “two longitudinal stripes” related to Figure 6A was deleted.3) Figure 6B: shows anterior development inhibited by late wnt activation, consistent with previous findings and evolutionary conservation that late Wnt controls a-p patterning; needs to be commented on in Results and Discussion;Commented in Results and Discussion.4) The endogenous, maternal wnt, which controls the early dv signal in Amphioxus remains unexplored. The mRNA injection experiments with Wnt8 and Wnt11 are not informative in this regard. But identifying the endogenous Wnt doing the job may not be essential for the overall conclusion of the study.We agree that our study provided limited insight into the actual endogenous Wnt molecule(s) controlling DV patterning in amphioxus. Given the inherent methodological limitations associated with amphioxus as a model the identification of such molecule(s) might take many years. We share the view that its identity is not essential for the overall conclusion of our study.5) Figure 7Ac axis duplication is striking, but they should indicate the "n"The "n" stating the numbers of embryos with phenotypes shown in Figure 7 are indicated in the top panel row in the bottom right corner.6) The quote "However, overactivation of Wnt/β-catenin signaling at the mid-blastula stage does not influence the axial patterning during the amphioxus development" is misleading, since Wnt regulates amphioxus ap axial patterning, and be qualified: "However, overactivation of Wnt/β-catenin signaling at the mid-blastula stage does not influence the DORSO-VENTRAL axial patterning during the amphioxus development".Our mistake. The sentence was corrected.


The Wnt signaling is one of the major pathways known to regulate embryonic development, tissue renewal and regeneration in multicellular organisms. Dysregulations of the pathway are a common cause of several types of cancer and other diseases, such as osteoporosis and rheumatoid arthritis. This makes Wnt signaling an important therapeutic target. Small molecule activators and inhibitors of signaling pathways are important biomedical tools which allow one to harness signaling processes in the organism for therapeutic purposes in affordable and specific ways. Natural products are a well known source of biologically active small molecules with therapeutic potential. In this article, we provide an up-to-date overview of existing small molecule modulators of the Wnt pathway derived from natural products. In the first part of the review, we focus on Wnt pathway activators, which can be used for regenerative therapy in various tissues such as skin, bone, cartilage and the nervous system. The second part describes inhibitors of the pathway, which are desired agents for targeted therapies against different cancers. In each part, we pay specific attention to the mechanisms of action of the natural products, to the models on which they were investigated, and to the potential of different taxa to yield bioactive molecules capable of regulating the Wnt signaling. Drug discovery from natural products (NP) is passing through a renaissance, for the natural treasury of biologically active compounds, which previously gave birth to such essential drugs as penicillin , taxol , vinca aSpeaking of the sources of NP as drug candidates, it is clear that plants, fungi and bacteria dominate. Marine hydrobiome is lagging behind in such exploration but is doomed to catch up, given the high (and perhaps higher) and structurally different chemodiversity it hides as compared with terrestrial organisms ,16.There are a number of signaling pathways, like Notch , TGFβ 1 or HedgeThe key signal intermediate in the canonical Wnt signaling is β-catenin, which upon continuous synthesis is degraded in the cytoplasm by a multiprotein destruction complex consisting of scaffolding proteins Axin and APC and two kinases: glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1). This complex traps and phosphorylates β-catenin, thereby targeting it for ubiquitinoylation and proteasomal degradation . With loThe physiological activators of the pathway are the lypoglycoproteins of the Wnt family (19 of which exist in humans), activating on the cell surface receptors of the Frizzled family  and LRP5/6 coreceptors . After lWnt signaling can become aberrant in different manners in different types of disease. Insufficiency in the Wnt signaling compromises tissue renewal and underlies such deficits as osteoporosis, vitiligo, or neurodegeneration. Reciprocally, when overly active, it is the triggering cause of various types of cancer, most notably in the colon, stomach, liver, breast and ovaries ,28. Two Most of the artificial Wnt cascade activators known up to date, from “classic” inorganic lithium salts  to dozenAndrographis paniculata, is a potent Wnt signaling activator acting by inhibiting GSK3β by a non-ATP-competitive, substrate-competitive mode of action. In silico analyses suggested that the compound interacts with the substrate-binding site of GSK3β [Euonymus fortunei, Amygdalus communis, and Garcinia xanthochymus. The active substance from the latter plant was isolated as the flavone fukugetin, inhibiting GSK3β with IC50 = 3.2 μM. Enzyme kinetics and molecular docking experiments further unveiled the mechanism of action, revealing fukugetin as a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK3β [NP called indirubins discovered from edible mollusks  showed eof GSK3β . A screeof GSK3β .Ricinus communis, showed the ability to stimulate the Wnt cascade, having the CK1 as the target [The inhibition of GSK3β is a powerful and robust but often unprecise instrument—some of the GSK3β inhibitors listed above can also block the action of other kinases such as GSK3α . Despitee target .Sanguisorba officinalis L. grass to activate the Wnt pathway in the classical TOPFlash transcriptional assay, and subsequently to control adipo-osteogenic differentiation, making it potentially useful for medicinal purposes in osteoporosis as well as obesity treatment [Hovenia dulcis tree to activate Wnt signaling, to induce osteogenic differentiation of murine calvarial osteoblasts without being cytotoxic, and to increase femoral bone mass without affecting mouse body weight [Recent studies tend to strive towards screening of libraries of naturally derived compounds and extracts with up-to-date Wnt-relevant cell-based assays. Such a screening of 83 Korean plant extracts revealed an ability of the extract of the reatment . Anothery weight . This sty weight —in a mody weight . The naty weight . UnfortuO-methyl-l-chiro-inositol), a naturally occurring methoxy analog of inositol found in many plants and recoverable as a byproduct from the rubber industry, could promote osteogenesis in a pre-osteoblastic MC3T3-E1 cell model. Increased expression of the Wnt pathway components LRP5, β-catenin, Wnt5a, and FZD4 was observed during this experiment. These findings cumulatively brought the authors to conclude that l-quebrachitol acted as a potential Wnt pathway activator, the exact mechanism remaining unknown [Rehmannia sp. [Salvia miltiorrhizae [S. miltiorrhizae, increasing amounts of nuclear β-catenin [Rehmannia glutinosa inhibited Wnt signaling [S. miltiorrhizae also inhibited Wnt signaling, at the level of β-catenin, and suppressed osteosarcoma in cell line models [As suppression of osteogenesis and induction of bone regeneration are controlled by the Wnt pathway activation, a large series of works link the capacity of a plant extract or its components to show positive effects in cell- or animal-based bone disease models with stimulation of Wnt signaling. For example, l-quebrachitol  from the genus Bauhinia (Leguminosae) promoted viability of chondrocytes isolated from rats, concomitant with the Wnt cascade upregulation seen as overexpression of Wnt4, β-catenin, FZD2 and cyclin D and the downregulation of GSK3β, both at the mRNA and protein levels [Escherichia coli showed the potential to activate Wnt signaling in odontogenesis in the case of the osteogenic differentiation of human periodontal ligament stem cells, where Wnt pathway in turn stimulated the action of a transcriptional osteogenic factor TAZ [Another widely used plant metabolite, rosmarinic acid, activated Wnt signaling in a LacZ-based assay in cultured calvarial osteoblastic cells and stabilized cytoplasmic β-catenin in bone marrow-derived stroma ST2 cells . An abun-catenin . Polysacn levels . Interesctor TAZ , thus prIn general, this large corpus of data obtained mostly in animal and cellular bone and cartilage models can lead us to several conclusions. Plants are a rich source of potential Wnt cascade activators, with many active compounds already isolated and characterized. Unfortunately, these publications focus mainly on preclinical studies of a therapeutic potential of compounds and extracts, typically leaving the mechanisms of action relatively unexplored. Regarding bone regeneration, the exact action mechanisms and/or targets related to Wnt signaling are in most cases unknown. Further, little can be deduced about tissue-specificity of the Wnt cascade activation by these extracts and compounds, studied in the bone-directed experiments. Activation of Wnt signaling might not be a direct consequence of a given compound, but a secondary effect caused by the cross-talk of Wnt signaling and other osteogenic pathways, suggesting that compounds increasing β-catenin levels in the bone might fail to do so in skin, liver, muscle or other tissues.Aconitum ciliare were among the most active both in the TOPFlash and hair growth assays, positioning this medicinal plant for the identification of a promising Wnt activator [Vernonia anthelmintica and their derivatives emerged as Wnt pathway activators for melanin synthesis in vitiligo—a sickness manifested as skin depigmentation [Nicotiana tabacum) metabolites [Not only bone and cartilage but also skin and hair are promising targets for the Wnt-targeting regeneration therapy. Aiming at a new hair growth-promoting drug, a screen of 800 extracts was conducted using the TOPFlash assay in HEK293 cells, and hits were tested for the ability to promote hair growth in cell-based and mouse models. Extracts from ctivator . Flavonoentation . As a poentation . In thisabolites .Cannabis sativa plant. Specifically, cannabidiol upregulated Wnt signaling in human neural PC12 cells by deactivating GSK3β, thus delivering a neuroprotective activity in a cellular model of Alzheimer’s disease [Coriolus versicolor increased dendritic length and dendritic branching, concomitant with increased cytonuclear β-catenin in mouse hippocamp [In the nervous tissue, activation of the Wnt cascade was demonstrated by cannabidiol, a non-psychotomimetic phytocannabinoid from the  disease . In anotippocamp .Herba epigenii enhanced osteogenic differentiation of human bone marrow-derived mesenchymal stem cells through the upregulation of Wnt signaling measured as increased β-catenin and enhanced expression of a set of Wnt target genes [Coptidis rhizoma, promoted osteogenic differentiation of bone mesenchymal stem cells, concomitant with accumulation of total and nuclear β-catenin; activation of the Wnt cascade was also confirmed by the TOPFlash assay. Furthermore, the Wnt signaling inhibitor DKK1 was blocked by berberine [Achyranthes bidentata used in the traditional Chinese medicine for the treatment of ostheoarthritis was shown to promote proliferation of rat chondrocytes by activation of the Wnt pathway, seen as upregulation of Wnt4, FZD2, β-catenin and cyclin D, and downregulation of GSK3β expression; nuclear translocation of β-catenin was also observed [Dalbergia odorifera promoted differentiation of mouse osteoblasts in culture, involving the activation of Wnt signaling detected at the level of increased mRNA expression of Wnts, phosphorylation of GSK3β, and nuclear accumulation of β-catenin [Genistein, an isoflavonoid isolated from soybeans, was described to interact with plasma membrane and nuclear estrogen receptors , the foret genes . A flavoet genes . Berberierberine . A polysobserved . Similar-catenin . In all A pioneering work in NP research and biology-oriented chemical synthesis gave rise to a new class of small molecule co-activators of the Wnt cascade . The autMolpadia musculus, a polychaeta Travisia sp., and different representatives of the order Actiniaria including Phelliactis callicyclus, which were able to (co)activate the Wnt cascade [Crassostrea gigas. It suppressed ovariectomy-induced osteoporosis and osteoclastogenesis in mouse models, and activated osteogenesis in cell cultures and zebrafish larvae, acting through β-catenin-dependent transcription [The classical GSK3β inhibitors indirubins were derived from mollusks. To continue the exploration of marine NP targeting Wnt signaling, recently a TOPFlash-based screening of 81 ethanol extracts from deep-sea invertebrates dwelling in the Kuril Basin of the Pacific Ocean revealed a series of Wnt modulating activities. Of special interest among the examined species are a holothurian  cascade . Given tcription .Undariopsis peterseniana, edible brown algae, were tested in hair growth models in a search for drug candidates against androgenic alopecia and found to stimulate ex vivo hair-fiber growth in rat vibrissa follicles as well as in vivo hair growth in mice. It showed an increase in β-catenin accumulation and GSK3β phosphorylation in dermal papilla cells [Capsosiphon fulvescens was able to promote growth of rat gastrointestinal IEC-6 cells, involving activation of MAPK and Wnt signaling, the latter confirmed by the analysis of the Wnt target genes cyclin D and c-myc and by nuclear translocation of β-catenin [Chlorella vulgaris promoted proliferation of IEC-6 cells, with activation of MAPK, PI3K/Akt and Wnt pathways, the latter seen as increased nuclear β-catenin and cyclin D expression [Algal metabolites, most notably polysaccharides, also displayed a Wnt-activating capacity. Extracts of la cells . A polys-catenin . Similarpression .The following conclusions can be drawn about the current state of NP activators of the Wnt pathway. Marine-derived indirubins and their derivative 6BIO remain the gold standard activators with GSK3β as the target. Alongside, a large pool of data describes activation of the Wnt signaling by plant extracts or metabolites in osteogenesis models without knowledge of the exact molecular mechanisms. In the cases where certain mechanistic investigation was conducted, GSK3β again emerged as the potential target, given the described effects of the NP on the phosphorylation status of this kinase. As discussed above, targeting this enzyme may produce undesirable side effects, thus the search for other means to activate the Wnt pathway, e.g., through specific activators of FZD receptors, is desired. With the high chemical diversity of the discovered Wnt activators , sold as a herbal supplement, cosmetics ingredient, food flavoring, and food coloring ingredient [Curcumin is another famous NP Wnt signaling inhibitor of plant origin. It is a diarylheptanoid isolated from turmeric  by downregulating the transcriptional co-activator p300 and upregulating the transcriptional repressor HBP1 . Periplong sites . Henryinaffected . Mangifetein WT1 . Ginkgetcompound .Bocquillonia nervosa and the tree Neoguillauminia cleopatra [Sanguis Draxonis, inhibited proliferation and promoted apoptosis of rat hepatic stellate cells by downregulating Wnt signaling, seen as decreased expression of Wnt1 and β-catenin. The compound also upregulated miR-148-3p, and knockdown of this microRNA reversed the effects of loureirin B [Other modes of action of plant-derived Wnt pathway inhibitors have also been described. Cardamonin, a chalcone, showed antitumor activity in breast cancer cells  and in murine breast tumor models, leading to tumor growth arrest, apoptosis and inversion of the epithelial-mesenchymal transition. The luciferase-based TOPFlash assay identified cardamonin as a Wnt pathway inhibitor acting at GSK3β: through inhibition of the deactivating phosphorylation of GSK3β by Akt, cardamonin restored the activity of the former leading to increased β-catenin destruction . A screeleopatra . Some NPleopatra , and itsleopatra , which wleopatra . Anotherleopatra . Loureirreirin B .Helianthus tuberosus) and the Far Eastern endemic liana Ampelopsis japonica inhibited Wnt signaling in cell-based GFP assays and in the TOPFlash assay [Telectadium dongnaiense’s bark and its active component periplocin inhibited Wnt signaling in the TOPFlash assay in human colon carcinoma HCT116 cells and reduced expression of a series of Wnt target genes [Scutellaria barbata plant reduced colorectal cancer cell (HT-29) xenograft growth in mice, and downregulation of Wnt signaling in the tumors was verified at the protein level of expression of Wnt target genes c-myc, survivin and APC, and β-catenin itself as well as β-catenin phosphorylation levels [Malus pumila Miller cv. ‘Annurca’ and Malus domestica cv ‘Limoncella’, decreased Wnt signaling in cells carrying APC mutations upregulating the cascade and in ex vivo biopsies of patients with colorectal cancer cells carrying APC mutations. The effect was observed using the TCF-GFP constructs and β-catenin immunostaining. The authors suggested polyphenols, particularly quercetin and its glycosides, as the active substances of the extracts. Unfortunately, apple extracts as food supplements administered to patients did not produce any effect, probably due to loss of polyphenols in the digestive tract, leading to the recommendation by the authors to use enteric coating for future applications of these extracts as food supplements [Curcuma zedoaria, was shown to slow down proliferation and migration of human cervical cancer SiHa cells, decreasing levels of β-catenin and TCF7 [Lonicera japonica, was shown to suppress Wnt signaling in human colon cancer cells SW480, remarkably increasing the cytoplasmic levels of Axin, the scaffolding protein of the β-catenin destruction complex [Gynura divaricata subsp. formosana suppressed human hepatocellular carcinoma Huh7 cell growth, as well as xenograft hepatocellular carcinoma tumor growth by downregulating Wnt signaling, as seen at a panel of Wnt target genes and β-catenin [Oryza sativa) broken seeds, rice bran, and rice germ  inhibited Wnt signaling through upregulation of CK1 and APC mRNA [Some other NP inhibit Wnt signaling by as yet unknown mechanisms. For example, the plant-derived polycyclic compound dihydroartemisinin used as an anti-malaria drug, was shown in a drug repositioning trial to suppress growth of squamous cell carcinoma A431 cells by downregulating the Wnt pathway  acting ash assay , the exaet genes . A plantet genes . Ethanoln levels . Extractplements . β-elemeand TCF7 . Gigantoand TCF7 . γ-Tocotand TCF7 . Later, and TCF7 . Honokioand TCF7 . Hydnoca complex . A sligh complex . Gynura -catenin . TreatmeAPC mRNA . Based oAPC mRNA .Saussurea lappa, dehydrocostus lactone and costunolide, inhibited the Wnt pathway and suppressed proliferation and survival of SW480 human colon cancer cells by the arrest of β-catenin translocation to the nucleus, not affecting cytoplasmic levels of β-catenin [Euphorbia dracunculoides inhibited Wnt signaling in a luciferase assay in HEK293 cells, reducing the expression of Wnt target genes Axin2, c-myc and cyclin D; phosphorylation and degradation of β-catenin were similarly observed in HEK293W cells incubated with tigliane diterpenoids [Euphorbiaceae plants are otherwise famous for phorbol esters, of which several, of the New Caledonian origin, have produced strong Wnt-inhibiting activities in triple-negative breast cancer cells [Euphorbia croton tiglium and Euphorbia peplus, respectively, inhibited Wnt signaling in colon cancer cells [Tabernaemontana divaricata, inhibited Wnt signaling in SW480 human colon cancer cells by decreasing β-catenin mRNA [Ocimum sanctum, and its apigenin form, 6,8-di-C-glucoside, inhibited viability of the human colorectal cancer HT-29 cells in the MTT assay by downregulating the Wnt pathway, monitored as decreased levels of β-catenin and cyclin D [A natural plant polyphenol rottlerin inhibited Wnt signaling in human prostate cancer PC-3 and DU145 cells and breast cancer MDA-MB-231 and T-47D cells through an interesting mechanism of reduction of phosphorylation  and protein levels of the Wnt coreceptor LRP6, not affecting LRP6 transcription . Two mai-catenin . Similar-catenin . Naturalrpenoids . Euphorber cells ,178. Simer cells . The tarer cells . Coronarnin mRNA . Vicenincyclin D .A plant-derived natural product parthenolide, a sesquiterpene lactone, inhibited Wnt signaling in the TOPFlash assay in HEK293 cells; further investigations showed that parthenolide did it by blocking the synthesis of the transcriptional factors TCF4/LEF1, probably performing it by binding the ribosomal protein L10, as siRNA knockdown of the latter also decreased TCF4/LEF1 levels and downregulated Wnt signaling . The sta1-Benzyl-indole-3-carbinol, a synthetic analogue of the natural phytochemical indole-3-carbinol derived from crucifer plants, suppressed Wnt signaling in a stronger and a more specific way than its natural precursors, shown by the downregulation of β-catenin protein levels with a concomitant increase in the levels of the β-catenin destruction complex components such as GSK3β and Axin. The compound was also active as an inhibitor in the TOPFlash assay, and the inhibitory effect was rescued by expression of a constitutively active form of LRP6, indicating that 1-Benzyl-indole-3-carbinol disrupted Wnt signaling at or upstream of LRP6 .Polygonum cuspidatum inhibited Wnt signaling in human triple-negative breast cancer cell lines, as measured by β-catenin levels and the TOPFlash assay, targeting the β-catenin destruction complex [Syzygium guineense. The tannins, compounds of a polyphenolic nature, revealed an ability to directly and selectively destabilize Wnt proteins in cell culture and in the colon cancer organoids, thus acting at the very upstream levels of the pathway. This Wnt protein destabilizing way of cascade downregulation may be very attractive for medicinal applications in tumors which arise due to the overproduction of Wnt ligands, and efficacy against the colon cancer organoids makes the Syzygium guineense plant an interesting candidate for a cancer-preventive food supplement [2-methoxystypandrone from the traditional Chinese medicinal plant  complex . An intrpplement .Penicillium vulpinum [Penicillium sp. sh18, showed Wnt inhibition in a dual luciferase assay in human colon cancer SW620 and HCT116 cells [Streptomyces griseolus, selectively suppressed proliferation of leukemia cell lines and patient samples through suppressing Wnt signaling, as verified at the level of β-catenin and expression of a series of Wnt target genes [Neosartorya pseudofischeri inhibited the growth of several Wnt-dependent colorectal cancer cell lines, downregulated Wnt signaling in the TOPFlash assay, and promoted degradation of β-catenin [Ganoderma lucidum used in traditional Chinese medicine blocked Wnt signaling through inhibiting the phosphorylation of LRP6 in human (MDA-MB-231) and mouse (4T1) breast cancer cell lines, also suppressing Wnt3a-activated expression of Axin2, a Wnt target gene [Metarhizium anisopliae, suppressed proliferation and induced cell cycle arrest in human colorectal cancer HT29, SW480 and HCT116 cells suppressing Wnt-signaling by the downregulation of β-catenin and TCF4 protein levels and β-catenin/TCF4 transcriptional activity measured in the TOPFlash assay, concomitantly with decreased expression of the target genes cyclin D, c-myc and survivin. It also acted in vivo, suppressing tumorigenesis in HT29 xenograft mice; the on-target effect was confirmed by decreased levels of β-catenin, cyclin D, and survivin in the tumors [Cordyceps sinensis, a fungus parasitizing on larvae of Lepidoptera, inhibited the growth of B16-BL6 mouse melanoma cells by stimulating adenosine A3 receptors, which caused downregulation of the Wnt pathway upstream of GSK3β [Streptomyces griseus, inhibited exogenous Wnt-stimulated, as well as DVL-stimulated signaling acting upstream of GSK3β. In HEK293 cells, it was shown that the mechanism involved the intracellular domain of LRP6 and vacuolar protein sorting protein 35, endosomal V-type ATPase, and endosomal trafficking [Inonotus obliquus used in folk anticancer medicine inhibited Wnt signaling in human colorectal cancer cell lines HCT116, HT-29, SW620 and in mouse colorectal cancer models, involving the downregulation of nuclear β-catenin [Inonotus obliquus extracts used in traditional medicine.The description above makes it clear that the major source of NP-based Wnt inhibitors  has so far been various medicinal plants. However, natural Wnt inhibitors have also been identified from other taxa, such as fungi. For example, a Wnt inhibiting activity in human lung adenocarcinoma cells was demonstrated by mycotoxin patulin isolated from the fungus vulpinum . Similar16 cells . Anisomyet genes . Gliotox-catenin . The musget gene . Destruxe tumors . Destruxe tumors . Cordyceof GSK3β . Bafilomfficking . Compoun-catenin . Compoun-catenin . Interes-catenin , while i-catenin . Such daPlatismatia glauca and Hypogymnia physodes respectively, inhibiting Wnt signaling in HCT116 and DLD-1 colorectal cancer cell lines seen by the downregulation of Wnt target genes without affecting the levels or localization of β-catenin. The authors did not reveal the exact mechanism of action but proposed that the compounds interacted with nuclear transcription cofactors [Some Wnt-inhibiting NP are not fungal- but lichen-derived, like caperatic and physodic acids isolated from ofactors .50 values of 0.6–7.4 nM [There is also information on Wnt inhibitors derived from prokaryotes: bioactive secondary metabolites of the aromatic and heterocyclic nature were isolated from terrestrial actinomycetes species and found to inhibit TCF/β-catenin transcriptional activity with the IC6–7.4 nM . Marine 6–7.4 nM .Buthus martensi scorpions. An analgesic peptide rBMK AGAP isolated from the venom was tested on breast cancer cell lines MCF-7 and MDA-MB-231, demonstrating inhibition of the cancer cell stemness, epithelial-mesenchymal transition, migration, and invasion in a series of assays as well as tumor growth reduction in xenograft mice. Downregulation of the Wnt pathway  was involved as determined by decrease of β-catenin and p-GSK3β [Some Wnt-inhibiting small molecules can be found in more unexpected sources. For example, marinobufagin and other bufadienolides, cardiotonic steroids secreted by toads, demonstrated an ability to downregulate Wnt signaling acting at two levels: via GSK3β dephosphorylation at concentrations from 500 nM to 5 µM, and by an additional mechanism downstream from β-catenin stabilization, as shown in the TOPFlash assay with constitutively active β-catenin and TCF4 mutants . However p-GSK3β .Peniagone sp. and Molpadia musculus, from crustaceans Calocarides quinqueseriatus and Munidopsis antonii, from polychaetae, and especially from brittle stars (Ophiuroidae) including Ophiura irrorata [A few reports exist on the Wnt-inhibiting activities from marine sources. Two studies by the same group identified smenospongidine, a marine sponge-derived sesquiterpenoid quinone , and aerirrorata . These dSolanaceae family of plants, were synthetized yielding a library of steroids including potent inhibitors of Wnt signaling, which acted upstream of the destruction complex, stabilizing Axin in a tankyrase-independent manner [Finally, like with the Wnt pathway activators above, the NP-inspired synthesis of small molecules yielded new inhibitors of the pathway. Analogs of withanolides, naturally occurring steroids built on the ergostane skeleton found mostly in the t manner . Anothert manner . In a set manner ,211. Thet manner .Chemical diversity of the Wnt inhibitors is as broad as that of the activators . The varConcerning the sources of natural inhibitors and activators of the Wnt cascade, currently the vast majority originates from plants , Table 1These considerations let us conclude this overview of the NP-based Wnt pathway activators and inhibitors with the following recommendations. First, the relatively poorly studied sources of NP as Wnt modulators—fungi, bacteria, and especially marine organisms—should be a major focus of future research. Second, aggressive pipelines of deconvolution of complex mixtures towards identifying the active compounds should be implemented to follow the screenings of crude extracts towards Wnt modulating activities. Finally, third, careful design and redesign of the screening platforms should be in place, in order to fine-tune the search towards identification of NP selectively acting at particular, disease- or organ-specific variants of the Wnt pathway, rather than bluntly activating or suppressing the Wnt signaling as a whole. These efforts should be accompanied with the prioritizing of R&D activities towards the NP with the highest chances to be developed as drug candidates, taking into account such parameters as in vivo potency, bioavailability, stability, and others relevant for the drug discovery, early on in the research on NP-based Wnt pathway modulators. We hope that these considerations will help the renewed interest in NP to deliver promising drug candidates targeting the Wnt signaling pathway in many diseases, from degeneration to cancer.