ST6Gal-I overexpression promoted proliferation, migration and invasion of Huh-7 cells, whereas its knockdown restricted these abilities in MHCC97-H cells. Additionally, in a mouse xenograft model, ST6Gal-I-knockdown MHCC97-H cells formed significantly smaller tumors, implying that ST6Gal-I overexpression can induce HCC cell malignant transformation. Importantly, enhanced HCC tumorigenesis and metastasis by ST6Gal-I may be associated with Wnt/β-catenin signaling promotion, including β-catenin nuclear transition and upregulation of downstream molecules. Together, our results suggest a role for ST6Gal-I in promoting the growth and invasion of HCC cells through the modulation of Wnt/β-catenin signaling molecules, and that ST6Gal-I might be a promising marker for prognosis and therapy of HCC.Abnormal sialylation due to overexpression of sialyltransferases has been associated with tumorigenesis and tumor progression. Although ST6Gal-I influences cancer persistence and progression by affecting various receptors, the underlying mechanisms and mediators remain largely obscure, especially in hepatocellular carcinoma (HCC). We found that ST6Gal-I expression was markedly upregulated in HCC tissues and cells, high levels being associated with aggressive phenotype and poor prognosis. Furthermore, we examined the roles and mechanisms of ST6Gal-I in HCC tumorigenesis and metastasis  Approximately 80–90% of cases occur in Asia and Africa, and in China, the large majority of which are associated with chronic hepatitis B virus infection.3 Currently, most of HCC patients cannot be diagnosed in the early stage and they therefore lost the best opportunity for treatment.5 Despite tremendous advances in early diagnosis and surgery, HCC incidence continues to increase worldwide. Demonstrations of the efficacy of targeted molecular therapies have triggered the search for additional molecules capable of improving patient survival.Hepatocellular carcinoma (HCC) is the fifth most frequently diagnosed malignant tumor and the second leading cause of growing life-threatening cancer worldwide.7 In cancer cells, normal glycosylation is altered8 by various causes such as glycosyltransferase dysregulation, changes in the tertiary conformation of the peptide backbone, and alterations of sugar nucleotide donors and cofactors.10 Dall’olio F et al.11 reported that the expression of glycosyltransferase can be regulated by the genetic and epigenetic changes. Abnormal sialylation is a distinctive feature of tumor cells associated with malignant phenotypes including invasiveness and metastatic potential.13 For example, gastric and colorectal cancers present high levels of sialyl-Lewis A (SLeA) and sialyl-Lewis X (SLeX) as terminal epitopes of protein O-glycans, N-glycans and glycolipids.14 There are 20 sialyltransferases to be identified, and they were divided into β-galactoside α2,3-sialyltransferases , β-galactoside α2,6-sialyltransferases , GalNAc α2,6-sialyltransferases  and α2,8-sialyltransferases (ST8SIAI–VI) families.15Glycosylation is a post-translational modification that affects protein folding, stability and activity, and plays important roles in many biological and pathological processes such as immune recognition and tumor progression.18 Lu et al.19 showed that α2,6-sialylated glycans can promote the malignant phenotypes of many tumors by regulating the related signaling pathways. ST6Gal-I is known to regulate the interaction between galectin-1 and α2,6-sialylated N-glycans, and further prevents angiogenesis and tumor progression.21 The Rabinovich lab showed that ST6Gal-I overexpression increased in the α2,6-sialylation levels of glycoproteins, and inhibited the binding of galectin-1 and promoted the cellular apoptosis.23 In our previous study, we found that upregulation of ST6Gal-I increased the adhesive capability of mouse hepatocarcinoma cell to lymph node.24 However, the predictive and prognostic values of ST6Gal-I and its effect on HCC progression remained unclear.It has been reported that the high expression of ST6Gal-I increases in the levels of α2,6-sialylation, and correlates with tumorigenesis and tumor progression.in vitro and in vivo of HCC. Moreover, its association with HCC clinical characteristics and the potential underlying mechanisms were explored. Surprisingly, examination of ST6Gal-I levels in HCC and paired normal samples revealed high expression in the former and a close correlation with survival rate. We found that ST6Gal-I upregulation promoted the proliferation, migration and invasion ability of HCC cells. Furthermore, of the pathways tested, ST6Gal-I had the largest influence on Wnt/β-catenin signaling. Therefore, ST6Gal-I might be a promising marker for prognosis and therapy of HCC.In this study, we aimed to unravel the functions and mechanisms of ST6Gal-I in modulating the growth 26 In HCC and normal liver cell lines, ST6Gal-I was expressed in all tested cells as indicated by qPCR and western blot assays. The highest expression was observed in MHCC97-H cells, compared to which Huh-7 cells exhibited low expression  in representative pairs of cancerous and matched non-tumor liver sections from HCC patients . Pearsonpression . ST6Gal-pression .in vitro, the mobility and invasiveness were dramatically increased in ST6Gal-I-overexpressing cells as compared to control groups  and parental cells (Huh-7) . Immunofl groups . Taken tTo further verify the role of ST6Gal-I in HCC, we analyzed the effects of ST6Gal-I knockdown on the malignant phenotypes of MHCC97-H cells. ST6Gal-I mRNA and protein expression in MHCC97-H/shST6Gal-I cells was substantially lower than that in those transfected with the control construct and non-transfected MHCC97-H cells as indicated by qPCR, western blotting and immunofluorescence . Lectin in vivo, nude mice were inoculated with ST6Gal-I-knockdown or control cells. Both tumor size and growth rate were dramatically decreased in the MHCC97-H/shST6Gal-I group  can induce the secretion of matrix metalloproteinases (MMPs) in the tumor–stroma interaction. MMPs are also considered to play major roles in cell processes such as migration, differentiation, angiogenesis and cancer metastasis.34 Additionally, the Wnt/β-catenin pathway is also involved in the modulation of tumor angiogenesis and metastasis by affecting the expression of MMP-2 and MMP-9. Our discoveries partly corroborate the supposition that ST6Gal-I can regulate Wnt/β-catenin signaling.Hepatocellular carcinogenesis is a complicated process resulting from multiple molecular events leading to the initiation, promotion and progression of tumors.36 Binding of the Wnt ligand to the receptor Frizzled-7 promotes the nuclear translocation of cytoplasmic β-catenin, which then associates with TCF/LEF transcription factors, and Frizzled receptors are also N-glycosylated.37 These studies indicate that N-glycoproteins play essential roles in Wnt/β-catenin signaling. Our study revealed that ST6Gal-I upregulation led to a significant increase in cytoplasmic β-catenin and release into the nuclear. Furthermore, western blotting demonstrated that related molecules  were significantly decreased in MHCC97-H/shST6Gal-I cells, but increased in Huh-7/ST6Gal-I cells. These findings indicate that ST6Gal-I regulates the Wnt/β-catenin pathway which might be attributed to the modulation of N-glycosylation of Wnt receptors, and Wnt/β-catenin signaling appears to be involved in ST6Gal-I-induced malignant transformation of HCC cells. However, the detailed molecular mechanisms how ST6Gal-I modulates the Wnt signaling pathway are still needed to elucidate in further researches.Wnts are N-glycoproteins, and modification of some of them, such as Wnt3a, activates the Wnt/β-catenin canonical pathways.In conclusion, our data implicate that ST6Gal-I overexpression in human HCC is associated with carcinoma progression and poor clinical prognostic. In addition, ST6Gal-I may accelerate HCC development through Wnt/β-catenin signaling. Our findings not only reveals the pathological roles of ST6Gal-I in HCC, but also provide a potential new marker for HCC.39 After antigen retrieval and blocking, the slides were incubated with a rabbit anti-ST6Gal-I primary antibody  overnight at 4 °C. Negative control slides were processed in the same manner, without addition of the primary antibody. For detection, diaminobenzidine (DAB)  and hematoxylin were used. Two certified pathologists blinded to the clinical data independently assessed ST6Gal-I immunostaining, scoring the results depending on the positive-staining percentages and staining intensity, as follows: (0) 0%, no staining (–); (1) 1–29%, weak positive (+); (2) 30–59%, moderate positive (++); and (3) >60%, intense staining (+++). For statistical analysis, cases were categorized as either negative (no and weak staining) or positive (moderate and intense staining). Pearson’s chi-squared test was used to assess the association between ST6Gal-I and HCC, to determine its clinical significance.ST6Gal-I levels in hepatoma carcinoma and normal hepatic tissues were evaluated by IHC using anti-ST6Gal-I on commercial tissue arrays  as previously described.2), they were tested regularly for mycoplasma contamination in the laboratory.L02 normal liver cells and Huh-7 and MHCC97-H human HCC cells were purchased from the Cell Bank of Chinese Academy of Sciences . All cell lines were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under standard culture conditions  was constructed as described previously.24 These cells were transfected with a mixture of plasmids and Lipofectamine 2000  depending on the manufacturer’s instructions. To select stably transfected cells, the culture medium was replaced with complete medium containing 800 mg/ml of G418  after 48 h. Modified expression was confirmed by qPCR and western blotting.Huh-7 cells were transfected with the recombinant pcDNA3.1/ST6Gal-I vector or a control vector.−ΔΔCT method. Sequences of the forward (F) and reverse (R) primers used for qPCR are shown previously.16Total RNA was extracted using TRIzol reagent  and was employed to synthesize cDNA using a PrimeScript RT Reagent Kit  and subsequently mixed with qPCR SuperMix . Relative changes in gene expression were analyzed using the 2Proteins were extracted from cells using lysis buffer  containing protease inhibitors. Protein concentrations in lysates were measured with a BCA kit (Beyotime). Aliquots of protein extracts were subjected to 10–15% SDS–PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes , which were blocked for 2 h with 5% skim milk in tris buffered saline, with tween-20 (TBST). The membranes were incubated with primary antibodies of ST6Gal-I , PI3K , Akt , p-Akt , GSK-3β , phosphorylated Ser9 GSK-3β , β-catenin , p-β-catenin , Erk , p-Erk , JNK , p-JNK , Cyclin D1 , c-Myc , TCF1 , TCF3 , TCF4 , MMP-2 , MMP-7 , MMP-9 , EMMPRIN  and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  antibodies. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody . Following further washing with TBST, blots were visualized using enhanced chemiluminescence reagents . After exposure, protein band densitometry was conducted with the Image Lab software .Cells were plated on slips for 24 h, then these cells were fixed with 4% paraformaldehyde for 30 min, and permeabilized using 0.1% Triton X-100 for 10 min in the dark. They were blocked with 10% bovine serum albumin for 30 min, and incubated overnight with rabbit anti-ST6Gal-I  and rabbit anti-β-catenin . A fluorescein-labeled secondary antibody  was subsequently incubated with the cells for 1 h at 37 °C. Golgi apparatus were identified using a rabbit anti-GM130 antibody , and 4,6-diamidino-2-phenylindole was used to stain nuclei for 10 min. Fluorescence was observed using a confocal laser-scanning microscope (BD Biosciences).4 Huh-7 or 8 × 104 MHCC97-H cells suspended in 0.36% agarose in the same medium constituted the top layer in 6-cm dishes. After 4 weeks of incubation, the number of colonies with>50 cells in each dish was counted using cellSens software Olympus, Tokyo, Japan).0.75% low gelling temperature agarose (Sigma-Aldrich) in complete medium served as the bottom layer, and approximately 4 × 104 cells from each sample analyzed by FCM.After washing thrice with phosphate-buffered saline (PBS), cells were collected in a tube, and fixed in 70% cold ethanol overnight. Then, they were blocked with 2% bovine serum albumin and incubated on ice for 30 min with 2 μg/mml fluorescein isothiocyanate (FITCc-onjugated) sambucus nigra agglutinin (SNA). After three washes with PBS, flow cytometry was carried out . The cell cycle distribution of Huh-7/MHCC97-H was analyzed by staining with 50 μg/ml propidium iodide. Finally, 1 × 104 Huh-7 or 4 × 104 MHCC97-H cells seeded in the upper chamber and incubated for 32 h. Migrated cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min, and counted in five non-overlapping fields and photographed. The invasion assay was carried out as above, except that the upper chambers of 24-well Transwell systems were coated with 40 μl Matrigel (diluted 1:8). After 30 min for Matrigel solidification, 1.6 × 105 Huh-7 or 8 × 104 MHCC97-H cells were seeded in each upper chamber and cultured for 48 h.Following the manufacturer’s instructions, serum-free Dulbecco’s modified Eagle’s medium was placed in the upper chambers of a Transwell plate , and 600 μl complete medium Dulbecco’s modified Eagle’s medium was added to the lower chambers as a chemoattractant. In each well, 8 × 10in vivo experiments were approved by and carried out according to the guidelines and regulations of the Dalian Medical University Sciences Center Animal Care and Use Committee. In addition, use of animals in the present study was consistent with Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. Athymic nude mice aged 4–6 weeks were obtained from the Animal Experiment Center of Dalian Medical University, China, for analysis of tumorigenicity. They were randomly divided into three treatment groups, each of which included 10 animals: an MHCC97-H group, a negative control group and an MHCC97-H/shST6Gal-I group. Approximately 2 × 107 cells in 100 μl of PBS were injected subcutaneously into the right dorsal flank of each mouse. Tumor diameters were measured with a vernier caliper every 3 days after 1 week, and tumor volumes being calculated using the following formula: 1/2 (length × width2). All mice were killed 30 days later, then tumor weights and volumes were determined. To ascertain the significance of results, the group size was chosen as to ensure that >5 animals remained in each group after 1 month when applying exclusion criteria or if death occurred during the experiment. No statistical method was used to estimate sample size.All ad libitum, taking into consideration the guidelines and regulations of the Dalian Medical University Sciences Center Animal Care and Use Committee. The mice were divided into DENA-treated and control groups, those in the former being injected with 1 mg/kg DENA in sterile 15 ml/kg PBS for 3 weeks, and those in the latter being injected with sterile PBS alone. Then, mice in the DENA group received intraperitone injections of 0.2 ml/kg sterile CCl4 diluted in sterile olive oil, twice per week for an additional 21 weeks, while control animals were injected intraperitoneally with PBS. Mice were killed at 0, 2, 4, 8, 9, 10, 16, 18, 20, 22 and 24 weeks after initial treatment to analyze liver neoplasm development. Histopathological examinations to identify liver tumors were performed as described previously.40 Allocation of the animals and assessment of the outcome were done without blinding.C57BL/6J mice 6–8 weeks of age weighing 15.8±1.5 g (average±s.d.) were purchased from Dalian Medical University and housed in a temperature-controlled, air-conditioned, specific pathogen-free facility, and received food and water t-test was used to compare two independent groups of data. Differences were considered significant at P-values<0.05.Data were analyzed with SPSS 16.0 , and every experiment was done three times and the results were shown as the mean s.e.±s.d. Student’s


Following the formation of a primary carcinoma, neoplastic cells metastasize by undergoing the epithelial mesenchymal transition (EMT), which is triggered by cues from inflammatory and stromal cells in the microenvironment. EMT allows epithelial cells to lose their highly adhesive nature and instead adopt the spindle-like appearance, as well as the invasive and migratory behavior, of mesenchymal cells. We hypothesize that a bistable switch between the epithelial and mesenchymal phenotypes governs EMT, allowing the cell to maintain its mesenchymal phenotype even after it leaves the primary tumor microenvironment and EMT-inducing extracellular signal.50  concentration of Slug necessary to inhibit E-cadherin production. The steady state behavior of Slug exhibits sensitivity to changes in the rate at which it is induced by β-catenin upon activation of the Wnt pathway. In the presence of sufficient amount of Wnt ligand, E-cadherin levels are sensitive to the ratio of the rate of Slug activation via β-catenin to the IC50 concentration of Slug necessary to inhibit E-cadherin production.This work presents a simple mathematical model of EMT, specifically the roles played by four key proteins in the Wnt signaling pathway: Dishevelled (Dvl), E-cadherin, β-catenin, and Slug. The model predicts that following activation of the Wnt pathway, an epithelial cell in the primary carcinoma must attain a threshold level of membrane-bound Dvl to convert to the mesenchymal-like phenotype and maintain that phenotype once it has migrated away from the primary tumor. Furthermore, sensitivity analysis of the model suggests that in both the epithelial and the mesenchymal states, the steady state behavior of E-cadherin and the transcription factor Slug are sensitive to changes in the degradation rate of Slug, while E-cadherin is also sensitive to the IC50 concentration of Slug necessary to inhibit E-cadherin production, shows how the adhesive nature of the cell depends on finely-tuned regulation of Slug. By highlighting the role of β-catenin in the activation of EMT and the relationship between E-cadherin and Slug, this model identifies critical parameters of therapeutic concern, such as the threshold level of Dvl necessary to inactivate the GSK-3β complex mediating β-catenin degradation, the rate at which β-catenin translocates to the nucleus, and the IC50 concentration of Slug needed to inhibit E-cadherin production.The sensitivity of E-cadherin to the degradation rate of Slug, as well as the ICThe online version of this article (10.1186/s12976-017-0064-7) contains supplementary material, which is available to authorized users. Epithelial tumors, or carcinomas, are the most common type of neoplasia found in humans, accounting for roughly 80% of all cancer related deaths in the Western world . CharactIn carcinomas, it is possible for neoplastic cells to undergo epithelial mesenchymal transition (EMT) . TriggerThere are a multitude of different pathways that can activate EMT, with significant crosstalk between these signaling systems. In mature multicellular organisms, as well as during development, the Wnt pathway helps regulate proliferation, differentiation, migration, and polarity , 10. HowXenopus egg extracts to support their hypothesized interactions . Using a scaffold model to expand upon the complicated mechanisms involved in each step of Wnt pathway activation, as well as pathway crosstalk, may indeed reveal intermediate steady states for the cell to occupy during the transition.IC50 value of Slug necessary to inhibit E-cadherin production. While the steady state behavior of free β-catenin was shown to be sensitive to its own rate of degradation, the steady state behavior of E-cadherin is actually sensitive to changes in Slug. In particular, E-cadherin is sensitive to the degradation of Slug and the half maximal concentration of Slug necessary to inhibit E-cadherin production, as well as the ratio of rate of Slug activation via β-catenin to the half-maximal concentration of Slug necessary to inhibit E-cadherin production once the pathway has been activated. These insights, coupled with the behavior of the system in response to changes in both the F2 parameter and Dvl (d) activation in Fig. 50 concentration of Slug necessary to inhibit E-cadherin production, as well as inhibiting the movement of β-catenin to the nucleus could prevent the activation of Slug and ultimately work to maintain the E-cadherin- β-catenin complex at the membrane. Pharmacological exploration of these components of the Wnt pathway could help prevent EMT prior to intravasation and metastasis, the primary cause of cancer-related mortality.With the theory of a switch underlying EMT, it would also be prudent to examine how the transition is affected by the presence of neighboring cells. The stabilization of these bonds could ultimately work against the loss of adhesion in the epithelial cell and the resulting transformation. Additionally, sensitivity analysis and exploration of the response of the system to changes in parameter values as Wnt signaling is activated suggests that excellent candidates for possible therapeutic intervention are the β-catenin degradation complex, the shuttling of β-catenin to the nucleus, and the


Xenopus laevis. The level of leptin mRNA was low in premetamorphic tadpoles, but increased strongly at the beginning of metamorphosis and peaked at metamorphic climax. This increase in leptin mRNA at the onset of metamorphosis correlated with increased cell proliferation in the neurogenic zones of tadpole brain. We found that intracerebroventricular (i.c.v.) injection of recombinant Xenopus leptin (rxLeptin) in premetamorphic tadpoles strongly increased cell proliferation in neurogenic zones throughout the tadpole brain. We conducted gene expression profiling of genes induced at 2 h following i.c.v. injection of rxLeptin. This analysis identified 2,322 genes induced and 1,493 genes repressed by rxLeptin. The most enriched Kyoto Encyclopedia of Genes and Genomes term was the canonical Wnt/β-catenin pathway. Using electroporation-mediated gene transfer into tadpole brain of a reporter vector responsive to the canonical Wnt/β-catenin signaling pathway, we found that i.c.v. rxLeptin injection activated Wnt/β-catenin-dependent transcriptional activity. Our findings show that leptin acts on the premetamorphic tadpole brain to induce cell proliferation, possibly acting via the Wnt/β-catenin signaling pathway.In addition to its well-known role as an adipostat in adult mammals, leptin has diverse physiological and developmental actions in vertebrates. Leptin has been shown to promote development of hypothalamic circuits and to induce mitosis in different brain areas of mammals. We investigated the ontogeny of leptin mRNA, leptin actions on cell proliferation in the brain, and gene expression in the preoptic area/hypothalamus of tadpoles of  We found that functional LepR is expressed in regions surrounding the 3V of premetamorphic tadpole brain, suggesting that leptin can act within tadpole neurogenic zones. In the current study, we investigated whether leptin can promote mitosis in developing Xenopus tadpole brain by administering recombinant Xenopus leptin (rxLeptin) to premetamorphic tadpoles by intracerebroventricular (i.c.v.) injection, then we analyzed cells in M phase of the cell cycle using immunohistochemistry (IHC) for phosphorylated histone 3 (pH3). We also conducted a gene expression screen for early (2 h after i.c.v. rxLeptin injection) leptin-induced transcriptional changes in tadpole preoptic area/hypothalamus. This screen identified the canonical Wnt/β-catenin signaling pathway as the major intracellular signaling pathway induced by leptin in premetamorphic tadpole brain. Using electroporation-mediated (EM) gene transfer of a Wnt/β-catenin-responsive reporter plasmid into tadpole brain, we provide additional evidence that leptin activates functional Wnt/β-catenin signaling.Genes for d fishes , 3, 22. etrapods , 24. By X. laevis tadpoles from in-house breeding and raised them in dechlorinated tap water maintained at 21–23°C with a 12L:12D photoperiod. Tadpoles were fed frog brittle twice daily  and developmental stages were determined using the Nieuwkoop–Faber (NF) staging table (X. laevis tadpoles (premetamorphic tadpoles) in a buffered solution of 0.002% benzocaine (Sigma) before administering i.c.v. injection of rxLeptin . MiX. laevis brain:  2 h after i.c.v. injection of 0.6% saline or rxLeptin (20 ng/g BW). We isolated total RNA from microdissected brain regions of three replicate pools (three brains per pool) per treatment. Samples were hybridized to the Affymetrix GeneChip Xenopus laevis Genome 2.0 Array (n = 3/treatment) at the Affymetrix and Microarray Core Facility at the University of Michigan. We selected a subset of the induced genes to cover a range of expression values (fold change) for validation by RTqPCR  clustered together, and that the two treatments clustered separately. We used the limma package (v3.30.2)  to apply2 fold change (log2FC) ranked list from limma, containing genes with a Benjamini and Hochberg (BH)-corrected p-value, or false-discovery rate (FDR) <0.05, as input into clusterProfiler (v3.2.4)  . This annt terms .2FC ranked list of all differential gene expression data from limma (FDR < 0.05) as input into clusterProfiler (v3.2.4) (2FC ranked list as input into clusterProfiler (v3.2.4). Gene expression values for KEGG pathways were plotted on the respective pathways using the pathview package (v1.14.0) (We conducted Kyoto Encyclopedia of Genes and Genomes (KEGG)  pathways(v3.2.4) . An addiv1.14.0) .Enriched biological processes GO terms, KEGG pathways, and KEGG modules were displayed using the Enrichment Map application (v2.2.0)  in Cytosin vivo. We divided NF stage 50 tadpoles into three groups for bipolar electroporation. Tadpoles were electroporated with a DNA mixture containing pRenilla-null , pCMV-eGFP , and one of the following firefly luciferase reporter vectors (each at 1 μg/μL): pGL4.23 empty (negative control), GAS-luciferase (positive control for leptin activity)   or pGL4.ctivity)  (gift ofctivity) . Twenty-ctivity) . We had t-test (p < 0.05). Derived values were log10-transformed before statistical analysis. Fisher’s least squares difference post hoc test was used to separate the means following ANOVA. We used the SYSTAT 13.0 computer program (SPSS Inc.).We analyzed data for gene expression by RTqPCR, pH3 cell counts and dual-luciferase assay data by one-way ANOVA or by unpaired Student’s lep mRNA increased 3.3-fold between stages 48 and 50, which is immediately before the onset of metamorphosis . By contrast, lep mRNA decreased in liver (p = 0.001) and was low and unchanged in brain throughout metamorphosis . This developmental pattern of lep mRNA paralleled changes in cell proliferation in tadpole brain during metamorphosis  in cells within the VZ/SVZ throughout the tadpole brain , plotted in red, are distributed evenly across MA expression levels, indicating an ability to detect differentially expressed genes at any range of expression. This plot also indicates that an absolute log2 fold change (log2FC) as low as ~0.5 can be detected with confidence (FDR < 0.05). This analysis identified 2,322 induced and 1,493 repressed genes . The top 20 induced and repressed genes are given in Tables To investigate the molecular basis for leptin action on tadpole brain, we conducted microarray analysis on the region of the diencephalon containing the preoptic area/hypothalamus of NF stage 54 tadpoles at 2 h following i.c.v. injection of 0.6% saline or rxLeptin injection (20 ng/g BW). The transformed log ratio (M) and mean average (A) plot (MA plot) in Figure p-value < 0.15), KEGG pathways , and KEGG modules. We validated a subset of the induced genes by RTqPCR  and highlighting several biological processes or pathways of interest. The enrichment map in Figure R Figure .The top ten GO terms involved developmental processes Figure . Also, sin vivo, we conducted EM gene transfer with different reporter plasmids, and injected saline or rxLeptin i.c.v. Injection of rxLeptin did not alter luciferase activity in pGL4.23-empty vector-transfected brain, but caused a strong increase  in luciferase driven by the GAS-luciferase vector, which reports pSTAT3 signaling induced by leptin binding to the LepR  driven by the pGL4.23-6TCF (Wnt/β-catenin pathway) reporter vector following injection of rxLeptin. Forced expression of constitutively active β-catenin confirmed the activity of the pGL4.23-6TCF reporter .To test if leptin can activate the canonical Wnt/β-catenin signaling pathway in tadpole brain R Figure  23, 24), 24in vilep expression increases strongly during tadpole metamorphosis in parallel with an increase in mitosis in tadpole brain. Also, i.c.v. injection of rxLeptin induced mitosis in neurogenic zones of premetamorphic tadpole brain. Gene expression screening identified genes regulated by leptin signaling in early premetamorphic tadpole brain, and highlighted several signaling pathways  through which leptin may effect its regulatory role in neurogenesis. We also show that leptin activates Wnt/β-catenin pathway signaling in tadpole brain in vivo, as evidenced by activation of a Wnt/β-catenin pathway reporter vector.Leptin has well-established roles in adult physiology, but its developmental actions are less understood. Here, we show that We observed a strong induction of mitosis in neurogenic zones of the premetamorphic tadpole brain following i.c.v. injection of rxLeptin. The rapid increase in pSTAT3-ir in cells of these regions 1 h after rxLeptin injection supports that they express functional LepR, and therefore that the action of leptin on mitosis is likely to be direct. In rodent embryonic/fetal brain, LepR mRNA is expressed within the VZ/SVZ of the 3V , 20. Theob/ob mice is restored by leptin injection , one of the TCFs, Tcf7l1  was induced by leptin in tadpole brain. This transcription factor is a transcriptional repressor  and was  mitosis . The cyc mitosis , 71.There is mounting evidence from different systems and tissues that leptin induction of mitosis and cell survival depends on Wnt/β-catenin signaling , 73. ForAfter the Wnt/β-catenin pathway, the next most enriched pathway activated by leptin in tadpole brain was that regulated by the TGFβ superfamily of ligands, in particular bone BMP and activin. The TGFβ signaling pathway plays central roles in animal development, including cell proliferation, cell differentiation, and apoptosis , 82. SevThe hedgehog signaling pathway was also found to be activated by leptin . A proposed mechanism of leptin-induced neurogenesis in murine transient amplifying neuroblasts involves hedgehog signaling regulation . We alsoXenopus, Melissa Cui Bender and Robert J. Denver, unpublished data). Alterations in hormone production, influenced by nutrition during critical periods of development can affect the timing of development, and exert lasting effects on the structure and function of hypothalamic feeding and neuroendocrine circuits, a phenomenon termed developmental programming  can influence adipocyte production of leptin in the fetus , 89 or lgramming –93. Thergramming , and modgramming –99. In agramming . A decregramming –103.Xenopus  to force LepR expression in stem/progenitor cells, followed by injection of rxLeptin i.c.v. to activate LepR signaling. We predict that forced expression of LepR in progenitor/stem cells in the developing brain will promote proliferation followed by differentiation of these cells into the LepR expressing lineage.Future fate mapping studies can investigate if cells born in the ependymal layer following leptin injection migrate to and populate the preoptic area and ventral hypothalamus. It will also be interesting to investigate the consequences of aberrant leptin signaling during early postembryonic development on later-life physiology and hypothalamic feeding control centers using the All procedures involving animals were conducted under an approved animal use protocol (PRO00006809) in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of Michigan.MB participated in the design of experiments, conducted the leptin mRNA analysis, immunohistochemistry for pH3, DNA microarray analyses and electroporation-mediated gene transfer, and participated in writing the manuscript. CS did the bioinformatics analyses of the DNA microarray data and participated in writing the manuscript. RD participated in the design of experiments, analysis of the data, and in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a shared affiliation, though no other collaboration, with the authors, and the handling editor states that the process met the standards of a fair and objective review.


The integration of morphogenic signals by cells is not well understood. A growing body of literature suggests increasingly complex coupling among classically defined pathways. Given this apparent complexity, it is difficult to predict where, when, or even whether crosstalk occurs. Here, we investigated pairs of morphogenic pathways, previously reported to have multiple points of crosstalk, which either do not share (TGFβ and Wnt/β-catenin) or share (TGFβ and bone morphogenetic protein (BMP)) core signaling components. Crosstalk was measured by the ability of one morphogenic pathway to cross-activate core transcription factors and/or target genes of another morphogenic pathway. In contrast to previous studies, we found a surprising absence of crosstalk between TGFβ and Wnt/β-catenin. Further, we did not observe expected cross-pathway inhibition in between TGFβ and BMP, despite the fact that both use (or could compete) for the shared component SMAD4. Critical to our assays was a separation of timescales, which helped separate crosstalk due to initial signal transduction from subsequent post-transcriptional feedback events. Our study revealed fewer (and different) inter-morphogenic pathway crosstalk connections than expected; even pathways that share components can be insulated from one another. Morphogenic signals provide extracellular information needed for cells to make decisions during development and differentiation , 2. It iHere, we examine the presence or absence of morphogenic crosstalk in a specific system, where crosstalk was measured by the ability of one morphogenic pathway to cross-activate core transcription factors and/or target genes of another morphogenic pathway. In this study, we focused on crosstalk between the well studied TGFβ, Wnt/β-catenin, and BMP5–5–15–18–30–A). We used immunofluorescence microscopy to quantify shifts in nuclear localization of transcription factors due to ligand treatment (B).Mechanisms of receptor activation and signal transduction for TGFβ, BMP, and Wnt pathways are not fully understood. However, activation of each pathway results in nuclear translocation of specific transcription factors or co-factors. To achieve quantitative readouts of signal transduction, we monitored two of the earliest cellular phenotypes: the nuclear localization of canonical transcription factors for each signaling pathway via microscopy, and subsequent levels of transcription via quantitative PCR (qPCR). We inferred crosstalk by measuring how stimulation of one pathway affects either the nuclear localization or the transcription factor response of another A. We usereatment B.C). Second, to enhance the possibility of cross-pathway interaction, we chose input ligand concentrations that yielded strong  responses . Third, to investigate potential cell type differences, we performed our assays across two pairs of pathway-responsive cell lines. The first pair consists of human colonic epithelial cells (HCECs (C–E). We verified that each input/output response pair could be blocked with a high-specificity antagonist . Importantly, we note that the commonly used recombinant Wnt3A  and Wnt5A  can activate Smad2/3, which is likely an artifact due to trace contamination by TGFβ ligands ; thus, we used the higher-purity Wnt3A (R&D Systems 5036-WNP/CF), which did not activate Smad2/3. Finally, we verified with qPCR that pathway responses to our ligands were sufficient to induce significant changes to downstream transcription .To study inter-pathway crosstalk prior to transcriptional feedback, we chose time scales that provide maximal transcription factor nuclear localization for each pathway  and rats (HCECs ), chosens (HCECs . The secs (HCECs ), chosens . This was true across cell lines tested at both 1 h and 2 h. Thus, we did not observe crosstalk induced by TGFβ3 and Wnt3A as measured by nuclear accumulation of canonical pathway transcription factors at these early response times.We first tested for evidence of signaling crosstalk induced by TGFβ3 and Wnt3A. We measured the responses at 2 h of either Smad2/3 or β-catenin to each ligand or combination of ligands. We found that Smad2/3 showed little or no response to Wnt3A input and, similarly, β-catenin showed little or no response to TGFβ3 input A. This wSmad7 (for TGFβ3) (Axin2 (for Wnt3A) , although nuclear levels of β-catenin were unaffected at the same 2-h time point . This transcriptional crosstalk was maintained in HCECs for at least 6 h (data not shown).We then tested for evidence of transcriptional crosstalk induced by TGFβ3 and Wnt3A. We measured expression of the downstream target genes r TGFβ3)  and AxinC, arrowheads). Indeed, treatment of HCECs with TGFβ3 alone for 18 h was sufficient to increase nuclear β-catenin, and co-treatment with Wnt3A yielded an even larger effect. Cross-pathway modulation at 18 h did not occur in the other cell lines. Thus, signaling crosstalk between TGFβ3 and Wnt3A/β-catenin is a (cell line-dependent) consequence of long-term transcriptional feedback.We wondered whether the HCEC-specific suppression of Axin2 mRNA expression induced by TGFβ3 would result in long-term increases of β-catenin protein, as Axin2 protein is a negative regulator of Wnt/β-catenin signaling , 43. We Fig. 2A). We confirmed this absence of inhibitory cross-regulation by Western blotting  and further found that this absence extended to the transcriptional regulation of Smad7, a prototypical readout of both pathways . Therefore, TGFβ3 and BMP4 do not exhibit inhibitory crosstalk as measured by nuclear accumulation of their prototypical transcription factors.To investigate crosstalk between the paralogous TGFβ3 and BMP4 pathways, we measured responses of downstream transcription factor readouts Smad2/3 and pSmad1/5/8 to combinatorial inputs at 2 h. In all tested cell lines, we found cross-pathway activation of pSmad1/5/8 by TGFβ3, consistent with reports in the literature , 44–47. 44–15–Fig. 3D). We therefore found no evidence that TGFβ3 and BMP4 signal transduction pathways negatively regulate one another via competition for Smad4.The absence of cross-pathway inhibition could be due to non-limiting quantities of Smad4 in the cell lines tested. Non-limiting abundance of Smad4 is consistent with previous studies –51 but m–Fig. 3Crosstalk between pathways in the larger context of the organism can be expected to occur in many specialized circumstances. A single growth factor can change the biochemical state of a cell, driving differentiation or proliferation and indirectly affecting other pathways by many routes. However, this is generally not what is meant by pathway crosstalk: pathway crosstalk is understood to be a property of the core pathway components themselves and a consequence of interactions that are proximal in space and time. As the results of more and more studies are combined, core signaling pathways are appearing to be increasingly interconnected. It is a challenge to make sense of these complex, static signaling models, which may not describe the operational wiring of any given cell and time. It is, in fact, conceivable that such apparent complexity is hiding a reality of underlying independence . For exaA and B, with Fig. 2C). Second, our finding that a commonly used recombinant Wnt3A may be contaminated with trace TGFβ ligands leads to uncertainty in the interpretation of previous crosstalk studies using this reagent . We have not tested multiple lots of commercial Wnt3a, but our results suggest caution when interpreting studies of crosstalk when using lower purity commercial Wnt3a, or any morphogenic ligand for that matter. Third, as our studies with multiple cell lines demonstrate, the TGFβ3 and Wnt3A/β-catenin pathways can show general insulation during upstream signaling while simultaneously showing cell line-dependent transcriptional crosstalk. Finally, even if protein-protein interactions between the TGFβ and Wnt pathways occur, these interactions need not necessarily carry signaling information or alter the process of signal transduction.We investigated crosstalk between Wnt3A/β-catenin, TGFβ3, and BMP pathways, as defined by changes in localization of core transcription factors and target gene expression. Our finding that the TGFβ3 and Wnt3A/β-catenin pathways are insulated during early signal transduction stands in apparent contradiction to the body of literature that shows cross-pathway protein-protein interactions –13. Ther–We had expected that the TGFβ3 and BMP4 pathways would display cross-pathway inhibition during signal transduction, especially after depletion of Smad4. Instead, we found activating crosstalk from TGFβ3 to the BMP4 pathway and no crosstalk in the other direction. Although we cannot rule out that the absence of cross-pathway inhibition was due to non-limiting Smad4 even after its reduction, the resulting decrease in Smad2/3 activity argues against this. An additional potential explanation could be that the well established nucleocytoplasmic shuttling of Smads , 55–57 r55–e.g. Notch and Hedgehog), biological contexts, and information channels.Taken together, our study revealed fewer (and different) inter-morphogenic pathway crosstalk connections than expected from the literature. It is possible that other components or measured phenotypes for the same pathways may reveal different patterns of crosstalk due to the activation of parallel pathways , or the 2 in RPMI 1640 (Corning Cellgro® 10-040) with 5% FBS  and antibiotics/antimycotics. We verified that overnight starvation (via the absence of FBS) did not result in qualitatively different study results. For imaging experiments, cells were plated at 2000 cells/well in 384-well glass-bottom plates (Thermo ScientificTMNuncTM 164586). Cells were left to adhere overnight, leading to near confluence, and treated the following day. Although confluency affected signaling, it did not qualitatively change the experimental outcomes (e.g. pathways remained insulated).HCECs were a kind gift from Dr. J. Shay , and the other cell lines were obtained from the American Type Culture Collection. Cell culture was performed under standard culture conditions. In brief, cells were maintained at 37 °C and 5% COFor treatments, recombinant proteins were diluted into the same medium and added to wells at time = 0. Concentrations  and treatment durations are indicated in the figure legends. Treatments  are as follows: Wnt3A ; Wnt3A ; TGFβ3 , 98%); BMP4 , 95%); Dickkopf-1 ; Noggin , 95%); and αTGFβ blocking antibody (R&D Systems MAB1835).All solutions were made in PBS (Life Technologies Gibco® 70013). All wash steps were performed three times using 0.1% Tween 20 (Fisher Scientific BP337) in PBS. Antibodies were diluted into 2.5% BSA  After treatment, cells were fixed in 4% paraformaldehyde  for 10 min, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich 93443) for 10 min, and then washed. Samples were incubated overnight at 4 °C with primary antibodies: β-catenin , Smad2/3 , and pSmad1/5/8 . Samples were then washed, stained with 1:1000-diluted secondary antibodies  and 2.5 μg/ml Hoechst for 2 h, and washed again. Secondary antibody alone was added to empty wells to serve as references for estimation of uneven illumination.E were taken on an IN Cell Analyzer 6000. All image corrections and analyses were performed using custom MathWorks Matlab® software. Uneven illumination correction and background subtraction were performed as described previously . G1/0 cells were further restricted to those within ±2 median absolute deviations of the median nuclear size as well as the median Hoechst coefficient of variation of intensity (a proxy for texture). The single-cell intensity values of the remaining cells were used for analysis. All observed response distributions for were unimodal, and treatments did not strongly affect distribution shapes (data not shown). Thus, in our subsequent analyses of G1/0 cells, we made use of population-level metrics, such as mean or median (B).Cellular nuclei were identified in corrected images by simple, automated threshold segmentation. Multiple features were then calculated for each nucleus , for each fluorescence channel. Analysis was restricted to Gr median B.A subset of ∼25 cell lines from the NCI, National Institutes of Health (NCI-60 Human Tumor Cell Lines Screen)  was screTM 353219), with triplicate wells per condition. An Ambion® TaqMan® Cells-to-CTTM (Life Technologies 4391848) kit was used according to the manufacturer's instructions to extract mRNA and create cDNA libraries. qPCR was performed by the University of Texas Southwestern Microarray Core Facility using TaqMan® probes for Axin2 (Hs0061034_m1), Smad7 (Hs00610344_m1), and 18S rRNA (Hs99999901_s1). Triplicate technical replicates were performed per experimental replicate, and the means of these were then used for normalization and statistics. The core facility was blinded to sample identity by obfuscation of sample positions and identifiers. Threshold cycle values were normalized against 18S rRNA values.Cells were cultured as above in 96-well plates , 150 mm NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 0.5 mm EDTA) containing protease and phosphatase inhibitors. The following antibodies were used at 1:1000 dilution: H3B , Smad4 , Smad1 , and pSmad1/5/8 . For RNAi, cells were transfected with Dharmacon Smad4 siGENOME SMARTpool (GE Life Sciences M-003902-01) using Lipofectamine® RNAiMAX (Life Technologies 13778075) for 48 h according to the manufacturer's instructions. Transfected cells were then treated as above.SDS-PAGE and Western blotting were performed using standard techniques. In brief, cells were grown and treated in 6-well plates, and then lysed with radioimmunoprecipitation assay buffer (50 mA. D. C. and C. A. T. designed and performed the experiments. A. D. C. analyzed the data. A. D. C., C. A. T., L. F. W., and S. J. A. wrote the manuscript. All authors reviewed the final manuscript.


Bioactive lipids such as sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) regulate diverse processes including cell proliferation, differentiation, and migration. However, their roles in cardiac differentiation and cardiomyocyte proliferation have not been explored. Using a 96-well differentiation platform for generating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) we found that S1P and LPA can independently enhance cardiomyocyte generation when administered at an early stage of differentiation. We showed that the combined S1P and LPA treatment of undifferentiated hiPSCs resulted in increased nuclear accumulation of β-catenin, the canonical Wnt signaling pathway mediator, and synergized with CHIR99021, a glycogen synthase kinase 3 beta inhibitor, to enhance mesodermal induction and subsequent cardiac differentiation. At later stages of cardiac differentiation, the addition of S1P and LPA resulted in cell cycle initiation in hiPSC-CMs, an effect mediated through increased ERK signaling. Although the addition of S1P and LPA alone was insufficient to induce cell division, it was able to enhance β-catenin-mediated hiPSC-CM proliferation. In summary, we demonstrated a developmental stage-specific effect of bioactive lipids to enhance hiPSC-CM differentiation and proliferation via modulating the effect of canonical Wnt/β-catenin and ERK signaling. These findings may improve hiPSC-CM generation for cardiac disease modeling, precision medicine, and regenerative therapies. Radiocarbon dating studies suggest that there is, at baseline, less than 0.5% yearly cell turnover in the adult human heart2. As such, mammalian adult heart regeneration is unable to compensate for the massive loss of cardiomyocytes following cardiac injury such as myocardial infarction, leading to adverse cardiac remodeling. This limited regenerative capability of the human heart has garnered significant interest in developing novel methodologies for both creating cardiomyocytes de novo and inducing proliferation in terminally differentiated cardiomyocytes. A major goal in human pluripotent stem cell research is to provide large quantities of cardiomyocytes suitable for cellular therapy in regenerative medicine6.Adult mammalian cardiomyocytes possess limited capacity for cell division9. The most up-to-date strategies use biphasic Wnt/β-catenin modulation for direct cardiac differentiation from human induced pluripotent stem cells (hiPSCs)11. To mimic developmental Wnt signals required for in vivo mesoderm induction, hiPSCs are initially treated with CHIR99021 (CHIR), a non-selective glycogen kinase 3 beta (GSK3β) inhibitor, followed by a Wnt/β-catenin inhibitor to promote cardiac cell differentiation. However, despite recent advances, there remains significant batch-to-batch variation in differentiation efficiency, as different hiPSCs lines, even those derived from the same individuals, can vary in their abilities to reproducibly generate cardiomyocytes. To address the challenge of in vitro hiPSC-CM generation with consistently high efficiency, we sought novel molecules that can further optimize our current cardiac differentiation protocol.Protocols for human pluripotent stem cell cardiac differentiation are vastly improved compared to a decade ago. Current protocols can obtain upwards of 90% pure cardiomyocytes during differentiation followed by metabolic selection, which can be further augmented by using CRISPR/Cas9 gene editing to introduce selectable markers into hiPSCsin vitro and cardiovascular development in vivo12. Among these bioactive lipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are cardinal members. In vivo studies have demonstrated a necessary role of S1P signaling via S1P receptor in cardiomyocytes in normal heart development in mice13. In vitro studies have shown that these signaling molecules are capable of regulating pluripotency and cell cycle activity in human embryonic stem cells16. The bioactive lipids have also been reported to play a role in cell proliferation in epithelial cells, fibroblasts, and various cancer cell lines, via their ability to stimulate important cellular signaling pathways such as the MAPK/ERK pathway, the Hippo Pathway, and the Wnt/β-catenin signaling pathway20. These diverse roles for bioactive lipids prompted us to consider whether S1P and LPA may regulate cardiomyocyte differentiation from hiPSCs and cardiomyocyte function.In recent years, growing evidence support lysophospholipids, a collection of bioactive lipids harboring multiple functions, as important regulators of stem cell differentiation In this study, we explored S1P and LPA as inducers of cardiomyocyte differentiation in a chemically-defined culture setting using multiple hiPSC lines. Furthermore, we examined lysophospholipid regulation of cardiomyocyte progeny from hiPSCs and their ability to stimulate cardiomyocyte proliferation. We found that S1P and LPA act synergistically with GSK3β inhibitor CHIR to regulate early hiPSC mesodermal differentiation through nuclear β-catenin accumulation. At later stages, the combined treatment of S1P and LPA resulted in cell-cycle activation in differentiated hiPSC-CMs, an effect mediated through ERK/MAPK signaling, and synergized with β-catenin signaling to increase cardiomyocyte proliferation. Taken together, our findings demonstrate unequivocally that bioactive lipids exhibit stage-specific effects on cardiac differentiation from hiPSCs.OCT4, SOX2, KLF4, and c-MYC). Subsequently, all hiPSC lines were differentiated using chemically-defined protocols to generate cardiomyocytes21. Since hiPSC lines 3, 4, and 5 differentiated well into beating cardiomyocytes without the addition of bioactive lipids, we investigated the effects of S1P/LPA on hiPSC lines 1 and 2 that exhibited impaired capacity to differentiate into cardiomyocytes.Five hiPSC lines were generated through reprogramming somatic tissues from five individuals by introducing viral vectors expressing the Yamanaka reprogramming factors , CHIR, S1P/LPA, or a combination of S1P/LPA and CHIR. Interestingly, when compared to the DMSO control group, treatment with S1P/LPA alone minimally increased TCF/LEF-luciferase activity (~1.25-fold)  Fig.  whereas ld) Fig. . Hence, ld) Fig. Figure 2BDKK4 and DKK1 , the latter of which is associated with an epithelial-to-mesenchymal transition during development.To uncover the key changes in gene expression after S1P/LPA treatment, we performed a genome-wide microarray expression analysis on hiPSCs at day 2 of differentiation. Interestingly, we found a significant downregulation of KK1 Fig. , Wnt inhTaken together, these results suggest that bioactive lipids S1P and LPA act synergistically with GSK3β inhibitor CHIR to increase Wnt signaling/β-catenin nuclear accumulation during hiPSC differentiation, potentially through enhancing β-catenin release from membrane-associated E-cadherin Fig. . S1P andCOL12A1 and VIM) induced by S1P/LPA treatment, suggesting a change in their biological phenotype  using chemically-defined protocols28 Movie  and trea28 Movie . We firs28 Movie , S2, a well Fig.  and nuclell Fig.  divisionell Fig. . Two-dayell Fig. . Additio34, we assessed the ability of bioactive lipids to promote nuclear accumulation of Yap  reporter. We found that while the canonical Wnt signaling, as measured by the TOPFlash reporter, was activated in the presence of CHIR alone, the treatment with S1P/LPA was unable to increase TCF/LEF-luciferase activity Fig. , suggestap Fig. S. Consequap Fig. S.Figure 535. We also observed a down-regulation in phosphorylation of HSP27 within 5 minutes of treatment with S1P/LPA and subtle alterations in GSK3β and JNK phosphorylation . However, S1P/LPA treatment did not lead to a decreased expression of Nanog at 24 hrs after treatment, suggesting a more mesodermal specific, rather than global, effect of S1P/LPA on hiPSC cardiac differentiation Fig. S. A hallmtry Fig. . Beyond While the increase in β-catenin nuclear localization could be due to stabilization of β-catenin (i.e. prevention of GSK3β-mediated degradation) or greater release of β–catenin from E-cadherin at the plasma membrane, addressing the relative contribution of these mechanisms of β-catenin increase in the nucleus is beyond the scope of the current study. However, the inability of S1P/LPA to directly induce early mesoderm markers such as Bry T Fig.  supports238. We confirmed that the MAPK/MEK/ERK pathway is required for S1P/LPA-induced ERK phosphorylation and cell cycle reentry by showing that treatment with trametinib, a MEK inhibitor, effectively abolished these effects  and reseeded on Matrigel-coated plates.To produce human cardiomyocytes from pluripotent stem cells, hiPSCs were differentiated into hiPSC-CMs with a chemically-defined cardiomyocyte differentiation protocol. These hiPSC-CMs were maintained in RPMI 1640 media supplemented with recombinant human albumin and ascorbic acid (CDM3)45. Fluorescence intensity and cell number was quantified using ImageJ software. For quantitative viability measurements, cells were treated with CellTiter-Glo 2.0 Viability Assay (Promega) or PrestoBlue reagent (Life Technologies) per manufacturer-recommended procedures. 96-well imaging and viability assays were conducted using a Cytation 5 plate reader/imager (BioTek Instruments). Prism (GraphPad) was utilized for graph generation and statistical analysis. Confocal imaging was performed using a Zeiss LSM 510Meta microscope (Carl Zeiss) using Zen software.For 96-well hiPSC-CM differentiation assays, hiPSCs were plated in Matrigel-coated 96-well plates at 1000 cells per well and allowed to adhere for 4 days. The hiPSCs were subsequently treated with bioactive lipids at the indicated concentrations and durations and assessed following day 8 of the chemically-defined hiPSC-CM differentiation protocol. Immunostaining using previously-published protocols was conducted to qualitatively assess cell viability and cardiomyocyte differentiation efficiencyS1P and LPA were obtained from Sigma Aldrich and dissolved in water at 1 mM and 10 mM stock solutions. S1P and LPA were applied in 10 μM final concentrations unless otherwise specified. C59 and CHIR99021 were obtained from Tocris Bioscience and dissolved in DMSO at 10 mM stock concentrations. S1P antagonist VPC 23019 was obtained from Tocris Bioscience and dissolved in acidified DMSO.Phosphorylation of human kinases and other phosphoproteins Table  was deteHiPSCs and day 30 hiPSC-CMs were replated in 96-well plates and cultured for 2–3 days before transfection with Lipofectamine (Invitrogen) and TOPFlash (TCF/LEF) luciferase Wnt signaling reporter plasmid  at 100 ng/well. After 48 hours, media was changed, and cells were subjected to different treatments for 2 hours before lysis and luciferase (Promega) luminescence was measured with a standard luminescence plate reader.46. For more comprehensive transcriptome analysis of hiPSCs following bioactive lipids treatment, a GeneChip® Human Gene 1.0 ST DNA Microarray was used (Affymetrix).Quantitative real-time PCR was used to assess the gene expression level of specific gene of interest following bioactive lipids treatment. RNA was isolated using an RNeasy Plus kit (QIAGEN), and cDNA was produced using the High-Capacity RNA-to-cDNA kit (Applied Biosystems). Real-time PCR was performed with CFXTM Connect Real-Time System (BIO-RAD) using the USB® HotStart-IT® SYBR® Green qPCR Master Mix (2×) (Affymetrix). qPCR reactions were performed in duplicate, normalized to the reference gene GAPDH, and assessed using the comparative Ct methodP < 0.05, unless otherwise specified. For microarray, multiple P-value comparisons were made using a one-way between-subject ANOVA (P < 0.05) using Affymetrix Transcriptome Analysis Console 2.0 software.Data presented as mean ± standard deviation unless otherwise specified. Comparisons were conducted via Student’s t-test with significant differences (*) defined by Wu and colleagues demonstrate a stage-specific role of bioactive lipids to enhance human hiPSC cardiac differentiation (early) and cardiomyocyte cell cycle activation (late). These findings will help improve hiPSC-CM generation for cardiac disease modeling, precision medicine, and regenerative therapies.Supplemental Movie 1Supplementary Information


Liver tumor initiating cells (TICs) have self-renewal and differentiation properties, accounting for tumor initiation, metastasis and drug resistance. Long noncoding RNAs are involved in many physiological and pathological processes, including tumorigenesis. DNA copy number alterations (CNA) participate in tumor formation and progression, while the CNA of lncRNAs and their roles are largely unknown.LncRNA CNA was determined by microarray analyses, realtime PCR and DNA FISH. Liver TICs were enriched by surface marker CD133 and oncosphere formation. TIC self-renewal was analyzed by oncosphere formation, tumor initiation and propagation. CRISPRi and ASO were used for lncRNA loss of function. RNA pulldown, western blot and double FISH were used to identify the interaction between lncRNA and CTNNBIP1.linc00210, which was highly expressed in liver cancer and liver TICs. Linc00210 copy number gain is associated with its high expression in liver cancer and liver TICs. Linc00210 promoted self-renewal and tumor initiating capacity of liver TICs through Wnt/β-catenin signaling. Linc00210 interacted with CTNNBIP1 and blocked its inhibitory role in Wnt/β-catenin activation. Linc00210 silencing cells showed enhanced interaction of β-catenin and CTNNBIP1, and impaired interaction of β-catenin and TCF/LEF components. We also confirmed linc00210 copy number gain using primary hepatocellular carcinoma (HCC) samples, and found the correlation between linc00210 CNA and Wnt/β-catenin activation. Of interest, linc00210, CTNNBIP1 and Wnt/β-catenin signaling targeting can efficiently inhibit tumor growth and progression, and liver TIC propagation.Using transcriptome microarray analysis, we identified a frequently amplified long noncoding RNA in liver cancer termed linc00210 is highly expressed along with liver tumorigenesis. Linc00210 drives the self-renewal and propagation of liver TICs through activating Wnt/β-catenin signaling. Linc00210 interacts with CTNNBIP1 and blocks the combination between CTNNBIP1 and β-catenin, driving the activation of Wnt/β-catenin signaling. Linc00210-CTNNBIP1-Wnt/β-catenin axis can be targeted for liver TIC elimination.With copy-number gain in liver TICs,  Liver cancer is the third leading cause of cancer related death all over the world, and 90% liver cancers are hepatocellular carcinoma (HCC) . Liver tSeveral signaling pathways participate in liver cancer and liver TICs, including Wnt/β-catenin, Notch, Hedgehog and NF-κB signaling pathways . Among tAs we know, many protein-coding genes participate in tumor formation and tumor initiation, including oncogenes and tumor suppresser genes . Recentllinc00210, is highly expressed in liver cancer and liver TICs. Linc00210 interacts with CTNNBIP1, blocks its inhibitory role for Wnt/β-catenin signaling, and thus drives the self-renewal of liver TICs.Compared with normal cells, cancerous cells have more frequent mutations and instable chromosomes . Gene colinc00210 knockdown impaired the self-renewal of liver TICs . Linc00210 depleted cells showed decreased expression levels of Wnt/β-catenin target genes, while, other detected pathways weren’t influenced . To large extent, linc00210 and CTNNBIP1 were located together in primary samples  are a small subset cells within tumor bulk that have self-renewal and differentiation capacity . Tumor ilinc00210 blocks the inhibitory role of CTNNBIP1 in Wnt/β-catenin activation, and enhances the interaction of β-catenin and TCF/LEF complex, and finally drives Wnt/β-catenin signaling pathway activation. Actually, several lncRNAs were found to participate in Wnt/β-catenin activation, including lncTCF7 [lnc β-Catm [lncRNA-LALR1 [lncTCF7 recruits NURF complex to TCF7 promoter to activate Wnt/β-catenin signaling [lnc β-Catm binds to and stabilizes β-catenin directly [LncRNAs were considered as byproducts of RNA polymerase II, while, their important roles have been emerging these years . LncRNAs lncTCF7 , lnc β-Cc β-Catm  and lncRNA-LALR1 . Mechaniignaling ; lnc β-Cdirectly ; lncRNA-directly  to Axin1Linc00210 is highly expressed in liver cancer, with frequent CNA. Many genes, especially oncogenes, gained more copy numbers along with tumorigenesis; while, lincRNA copy number gain is rare reported. PVT1, a long noncoding RNA near from c-Myc loci, has copy number gain in breast cancer, and plays a critical role in tumorigenesis [linc00210 in liver TICs. Linc00210 copy number gain is related to increased linc00210 expression, activated Wnt/β-catenin signaling and enhanced liver TIC self-renewal. Of note, we found only 16 samples with copy number gain in 72 samples examined. Actually, 22.2% (16/72) is a relatively high frequency of copy number gain . Througlinc00210 is required for Wnt/β-catenin activation. Using sphere formation assay, tumor propagation and tumor initiation, we proved that targeting linc00210-Wnt/β-catenin signaling was an efficient way to eliminate liver cancer and liver TICs, providing a new avenue for liver TIC targeting. Here we found linc00210 interacted CTNNBIP1 to modulate β-catenin activation. However, in linc00210 non-gained samples, the activation of Wnt/β-catenin can also be modulated and participate in tumorigenesis, indicating other regulatory mechanisms in Wnt/β-catenin activation  also exist. The relationship between linc00210 and other Wnt/β-catenin modulators remains further investigation.Wnt/β-catenin signaling, the key mediator for TIC self-renewal, plays a critical role in development, stemness and disease , 40–42. linc00210 is related to high expression of linc00210, which blocks the interaction of β-catenin and CTNNBIP1. The impaired CTNNBIP1-β-catenin interaction promotes β-catenin-TCF/LEF interaction, and finally drives the activation of Wnt/β-catenin signaling and liver TIC self-renewal. Linc00210 copy number gain, linc00210 expression levels, CTNNBIP1 and β-catenin interaction are related to clinical severity of liver cancer and liver TIC self-renewal, which can be served as targets for eradicating liver TICs.Above all, copy number gain of long noncoding RNA 293 T cells (ATCC CRL-3216), liver cancer cell line Hep3B (ATCC HB-8064) and PLC (ATCC CRL-8024) were obtained from ATCC. Cells were maintained in DMEM medium, supplemented with 10% FBS (Invitrogen), 100 μg/ml penicillin, and 100 U/ml streptomycin.Human liver cancer specimens were obtained from the department of hepatopancreatobiliary surgery, with informed consent, according to the Institutional Review Board approval. All experiments, involving human sample and mice, were approved by the institutional committee of Henan Cancer Hospital. Sample #1: advanced hepatocellular carcinoma, 57 years old, male, tumor size, 7.5 × 6.2 × 4.7 mm, non-metastasis. Sample #4: advanced hepatocellular carcinoma, 68 years old, male, tumor size, 8.6 × 7.3 × 5.2 mm, metastasis. Sample #5: advanced hepatocellular carcinoma, 63 years old, female, tumor size, 6.6 × 5.9 × 5.2 mm, non-metastasis. Sample #1 had 3 copies of linc00210 and relative high linc00210 expression, sample #4 and #5 modestly expressed linc00210 with 2 copy numbers.Anti-β-actin (cat. no. A1978) were purchased from Sigma-Aldrich. Anti-β-catenin (cat. no. ab32572), anti-CTNNBIP1 (cat. no. ab129011) antibodies were from Abcam. Anti-histone H3 (cat. no. 4499), anti-Axin2 (cat. no. 5863), anti-TCF3 (cat. no. 2883), anti-TCF4 (cat. no. 2565), anti-LEF (cat. no. 2286), anti-TCF7 (cat. no. 2203) antibodies were from Cell Signaling Technology. Phycoerythrin (PE)-conjugated CD133 (cat. no. 130098826) was from MiltenyiBiotec. Anti-EEA1 , anti-Myc (sc-4084) antibodies were purchased from Santa Cruz Biotechnology. Alexa594-conjugated donkey anti-rabbit IgG (cat. no. R37119) and Alexa488-conjugated donkey anti-mouse IgG (cat. no. R37114) antibodies were from Molecular Probes, Life Technologies. DAPI (cat. no. 28718–90-3) were obtained from Sigma-Aldrich. T7 RNA polymerase (cat. no. 10881767001) and Biotin RNA Labeling Mix (cat. no. 11685597910) were purchased from Roche Life Science. The LightShift Chemiluminescent RNA EMSA kit (cat. no. 20158) and Chemiluminescent Nucleic Acid Detection Module (cat. no. 89880) were from Thermo Scientific.Wnt/β-catenin TOPFlash reporter  and mutant FOPFlash reporter  were transfected into indicated cells, along with thymidine kinase (TK), antisense oligo or control oligo. 36 h later, cells were lysed and detected with dual-detection luciferase detection kit . Wnt/β-catenin activation was measured according to the fold change of TOPFlash versus the FOPFlash control.For sphere formation assay, proper cells were seeded in Ultra Low Attachment 6-well plates and cultured in DMEM/F12 (Life Technologies) supplemented with B27, N2, 20 ng/ml EGF and 20 ng/ml bFGF. The spheres were counted and sphere pictures were taken 2 weeks later. For sphere formation assay, 1000 HCC cell line  cells or 5000 primary cells were used. bFGF (cat. no. GF446-50UG) was purchased from Millipore. EGF (cat. no. E5036-200UG), N2 supplement (cat. no. 17502–048) and B27 (cat. no. 17504–044) were from Life Technologies. Ultra low attachment plates (cat. no. 3471) were purchased from Corning Company.Two weeks later, we collected medium containing spheres and non-sphere cells into an eppendorf tube and let stand for 5 min for sphere/non-sphere separation. The pellets were spheres, and supernatants were non-sphere cells. Supernatants were removed into a new eppendorf tube and collected by centrifugation at 4000 rpm for 5 min. Spheres and non-spheres were derived from the same cell lines or primary samples.5 HCC primary cells were plated onto the top chamber with Matrigel-coated membrane, and incubated in FBS-free medium. FBS containing medium was added in the lower chamber as a chemoattractant. The plate was incubated in incubator for 36 h and cells that did invade through the membrane were removed by a cotton swab, and the cells on the lower surface of the membrane were fixed with methanol and stained with crystal violet. The images were taken with Nikon-EclipseTi microscopy.For transwell invasion assays, 3 × 106 oncosphere cells were resuspended in 0.5 ml resuspension buffer  for 10 min’s incubation, followed by homogenization. The cytoplasmic fraction was the supernatant after centrifugation (400 g × 15 min). The pellet was resuspended in 0.2 ml PBS, 0.2 ml nuclear isolation buffer  and 0.2 ml RNase-free H2O, followed by 20 min’s incubation on ice to clean out the residual cytoplasmic faction. The pellet was nuclear fraction after centrifugation. RNA was extracted from nuclear and cytoplasmic fractions using RNA extraction kit . Linc00210 content was examined by real-time PCR (ABI7300).5 × 10Linc00210 content, standard reverse transcription was performed using reverse transcription kit (Promega). Notably, same amount of RNA and same volume of cDNA were required. In our experiment, 1 μg nuclear RNA and 1 μg cytoplasmic RNA were used, with the same final volume of nuclear and cytoplasmic cDNA (50 μl). Real-time PCR was performed using 1 μl nuclear cDNA or 1 μl cytoplasmic cDNA, with the same primers and ABI7300 profile. The relative Linc00210 contents were calculated using these formulas: nuclear ratio = 2-Ct(nuclear) / (2-Ct(nuclear) + 2-Ct(cytoplasmic)); cytoplasmic ratio = 2-Ct(cytoplasmic) / (2-Ct(nuclear) + 2-Ct(cytoplasmic)).For 2O2), the slides were incubated in boiled Tris-EDTA buffer  for antigen retrieval. Then the sections were incubated in primary antibodies and subsequent HRP-conjugated secondary antibodies. After detection with standard substrate detection of HRP, the sections were stained with hematoxylin and dehydration in graded alcohols and xylene.Formalin-fixed liver cancer sections were deparaffinized in xylene and rehydrated in graded alcohols. After treated in 3% Hydrogen Peroxide . For every samples, 6 mice were used.For tumor initiating assays, 10, 1 × 10analysis  and an ohttp://crispr.mit.edu/) and lentivirus was generated in 293 T cells.For Linc00210 depletion, dCas9-Krab CRISPRi strategy was used . BrieflyLinc00210 overexpressed cells was generated as described [linc00210 cDNA was cloned into pCDNA3.1 vector, and transfected cells with jetPEI-Hepatocyte reagent. Stable clones were obtained by selection with G418. All constructs were confirmed by DNA sequencing.escribed . Brieflylinc00210 silenced or copy number gained samples were crushed in RIPA buffer, followed by a 4-h’ incubation with β-catenin antibody. The precipitate was detected with Western blot.For coimmunoprecipitation, linc00210 or control ACTB enrichment was detected using real-time PCR.For RIP, oncospheres were treated with 1% formaldehyde for crosslinking, and then crushed with RNase-free RIPA buffer supplemented with protease-inhibitor cocktail and RNase inhibitor (Roche). The Supernatants were incubated with CTNNBIP1 or control IgG antibodies and then Protein AG beads. Total RNA was extracted from the eluent, and P < 0.05 was considered to be statistically significant.Two-tailed Student’s t tests were used for statistical analysis. In conclusion, this study defined a copy number gained lncRNA linc00210 in liver tumorigenesis and live TICs. With high expression in liver cancer and liver TICs, linc00210 was required for the self-renewal of liver TICs. Moreover, we found linc00210 interacted with CTNNBIP1 and modulated the activation of Wnt/β-catenin signaling. Linc00210-Wnt/β-catenin signaling can be targeted for liver TICs elimination. These findings revealed lncRNA copy number gain may be therapeutic target against liver TICs.


Aristaless Related Homeobox (ARX) gene are associated with a spectrum of structural  and functional  neurodevelopmental disorders. How mutations in this single transcription factor can result in such a broad range of phenotypes remains poorly understood. We hypothesized that ARX functions through distinct interactions with specific transcription factors/cofactors to regulate unique target genes in different cell types. To identify ARX interacting proteins, we performed an unbiased proteomics screen and identified several components of the Wnt/β-catenin signaling pathway, including β-catenin (CTNNB1), B-cell CLL/lymphoma 9 (BCL9) and leucine rich repeat flightless interacting protein 2 (LRRFIP2), in cortical progenitor cells. Our data show that ARX positively regulates Wnt/ β-catenin signaling and that the C-terminal domain of ARX interacts with the armadillo repeats in β-catenin to promote Wnt/β-catenin signaling. In addition, we found BCL9 and P300 also interact with ARX to modulate Wnt/β-catenin signaling. These data provide new insights into how ARX can uniquely regulate cortical neurogenesis, and connect the function of ARX with Wnt/β-catenin signaling.Mutations in the  ARX result in a broad range of neurologic phenotypes from severe structural anomalies of the brain with accompanying epilepsy and intellectual disabilities, to structurally normal brains having similar intellectual disabilities and epilepsy [ARX and the associated phenotype [The Aristaless related homeobox (ARX) protein, the vertebrate homolog of Drosophila Aristaless, is a paired-like transcription factor with temporally and spatially restricted expression in the developing central nervous system . Mutatioepilepsy ,3. Furthhenotype .Arx is expressed in neural progenitor cells (NPC) located in both the ventricular zone (VZ) of the pallium (neocortex) and the subventricular zone (SVZ) of the subpallium [Arx expression as they exit the cell cycle and migrate to the cortical plate. In contrast, interneurons from the subpallial SVZ continue to express ARX during their non-radial migration and expression persists in mature post-migratory interneurons [During brain development ence, GE),5. Postmrneurons –10. We hrneurons –11. ThusIn the current study, we took an unbiased proteomics approach to identify ARX interacting proteins in the embryonic mouse forebrain. We identified multiple components of the Wnt/β-catenin  signaling pathway expressed in the cortical  VZ, including β-catenin (CTNNB1), LRRFIP2, and BCL9. This pathway is known to function in NPC proliferation and differentiation, intermediate progenitor cell (IPC) generation, and cortical lamination . We founprotocol number: 04946). CD1 mice were purchased from Charles River Laboratories. In utero electroporation (IUEP) was performed as described previously on embryonic day 12.5 (E12.5) [All mouse studies were performed in accordance with the guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experiments were approved by Harvard Medical School’s Institutional Animal Care and Use Committee ( (E12.5) . One day (E12.5) . All secArx truncation and amino acid substitution mutants were made by site-directed mutagenesis. Briefly, primers were designed using Agilent QuikChange Primer Design software and PCR was performed by KOD Xtreme™ Hot Start DNA Polymerase (Novagen) using pCAG-Arx-IRES-GFP as a template [E. coli. Primers used in this study are listed in the supplemental table  [-catenin (MfeI/XbaI) into the pM (EcoRI/XbaI) vector for GAL4DB fusion protein expression. ΔN89-β-cateninΔC and ΔN89-β-catenin D164A mutants were made by site-directed mutagenesis as described above. pcDNA3-based constructs  were cloned using the pcDNA3.1 Directional TOPO Expression kit (Invitrogen). Amino acid substitution mutants in NLS2 , NLS3 , or aristaless domain  were made by site-directed mutagenesis. DNA constructs used for IUEP include: pTOP-dGFP-NLS-CAG-DsRed, pCAG-Arx or pCAG. The oligonucleotides used in this study are described in template . The PCRal table . Full-lecatenin)  were ampHEK293T cells were cultured in Dulbecco’s modified Eagle’s medium GlutaMAX  containing 10% fetal bovine serum. Transfection was performed using PEI reagent  as described .2, protease and phosphatase inhibitors; Roche Biochem), and sonicated on ice . The crude lysates were centrifuged at 13,000 x g for 15 min at 4°C, and the supernatants were pre-cleared with protein A/G agarose bead for 1 hr at 4°C with rotation. In order to isolate novel ARX-binding proteins, ARX antibody against amino acids 158–181 was generated in rabbit and immobilized on Dynabeads M-280 Tosylactivaed (Invitrogen) according to the manufacturer’s instructions, and the beads were incubated with the pre-cleared whole embryonic brain lysate for 2 hr at 4°C with rotation. The beads were washed in buffer B three times (each time for 3 min at 4°C with rotation), and bound protein were acid-eluted and immediately neutralized. The eluted proteins were analyzed at the Keck Mass Spec & Proteomics Resource  and Proteomics & Systems Biology core (Perelman School of Medicine at the University of Pennsylvania).The whole embryonic forebrains (E14.5) were triturated in buffer B  containing a protease inhibitor cocktail (Roche Biochem). For preclearance, lysates were incubated with protein G-conjugated beads (Invitrogen). Two micrograms of primary antibody (anti-ARX) were incubated at 4°C for 2 h, and then incubated with the protein G-conjugated beads for an additional hour. The beads were washed with TNE buffer twice and then two more times with TNE buffer containing 500 mM NaCl, followed by a final wash with TNE buffer. Bound proteins were eluted using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. To detect the associated proteins in the immunoprecipitates, SDS-PAGE followed by Western blotting was performed. Anti-ARX  , anti-FL5/well) in 12-well culture plate the day before transfection. Cells were transfected with 400 ng of luciferase reporter plasmid, 50 ng of pRL-TK Renilla luciferase as an internal control, and 100 ng . The cells were split into poly-D-lysine coated (Sigma) 96-well plate (4 x 104/well) 6 hr post-transfection and incubated for an additional 16 hr. The cells were then treated with recombinant Wnt3a  at 100 ng/ml for 8 hr, and luciferase activity measured by Dual-Glo Luciferase Assay System  and POLARstar Omega microplate reader .For the TOP-flash reporter assay, HEK293T cells were plated  siRNA  was transfected into HEK293T cells using Lipofectamin 2000 (Invitrogen). Cells were then resuspended in buffer A  and sonicated on ice . The lysates were spun at 13,000 x g for 15 min at 4°C. Protein concentration was measured by BCA protein assay kit (Bio-Rad) and proteins were subjected to SDS-PAGE.All statistical analyses were done in Prism software using 2-tailed unpaired Student’s t-test. All graphs are plotted as mean ± the standard error of the mean (SEM).Arx expression overlaps with that of Lrrfip2, Bcl9 and β-catenin in NPCs in the cortical VZ/SVZ  on protein complexes obtained from embryonic day 14.5 (E14.5) mouse forebrain that were immunoprecipitated with an anti-ARX antibody . Among tl VZ/SVZ .In canonical Wnt signaling pathway Wnt ligands bind Frizzled (Fzd)/low-density lipoprotein receptor-related protein (LRP) complex. This binding on the cell surface aggregates Dishevelled (Dsh) and functionally blocks the action of β-catenin degradation that normally occurs through the GSK3β-Axin-APC-CKIα destruction complex. The resulting accumulated β-catenin translocates to the nucleus where it interacts with T-cell factor (TCF)/lymphoid-enhancer factor (LEF) family member, bind to the promoters of the Wnt target genes , and modulate their expressions. The β-catenin/(TCF/LEF) transcription factor complex recruits several transcription co-factor proteins including BCL9/PYGO and P300/CBP, along with histone modification enzymes .Arx in the dorsal forebrain, we postulated ARX would participate in canonical Wnt signaling. To test this, we transfected a canonical Wnt reporter construct containing TCF/LEF binding sites (TOP-flash) or mutated TCF/LEF binding sites (FOP-flash) that drives the luciferase expression [Arx into HEK293T cells. The TOP-flash or FOP-flash reporter activity was assayed in the transfected cells that were treated with or without Wnt3a. Wnt3a was used as a ligand to induce canonical Wnt signaling since it is known to function in HEK293T cells [ΔN89-β-cat/TCF  up-regulated TOP-flash activity in the absence of Wnt3a . This construct contains a TCF binding site that drives nuclear destabilized GFP protein  expression in a Wnt/β-catenin signaling-dependent fashion, and expresses DsRed under the chick β-actin promoter as an internal control. We co-electroporated, with this reporter construct, an empty vector , Arx (WT), Arx (431-564-3XSV40-NLS) or Arx (1–220) construct, into the E12.5 embryonic mouse forebrain. Brains harvested at E13.5 showed that ARX strongly up-regulated GFP expression  when compared to the control  in each DsRed positive cell. ARX (WT) or ARX (431-564-3XSV4-NLS) showed higher GFP/DsRed ratio than that of the control or ARX (1–220). These results are consistent with our in vitro Wnt/β-catenin signaling reporter assay in HEK293T cells ; mid-portion of the protein (221-430aa); and C-terminal region (431-564aa). Surprisingly, none of these induced TOP-flash reporter activity  showed a similar level of activity to WT  construct contains nuclear localization sequences 2 and 3 (NLS2 and NLS3) ; we founty to WT .ΔN89-β-catenin or Vp16-ΔNTCF4. ΔN89-β-catenin is a constitutively active form of β-catenin, which is a stabilized form of β-catenin as it lacks the GSK3β phosphorylation site which targets β-catenin for degradation [ΔNTCF4 is a fusion protein of the transcriptional activator, VP16, and N-terminally deleted TCF4, ΔNTCF4, that lacks the β-catenin interaction domain, thus containing only DNA binding domain of the TCF4 [ΔNTCF4 is known to induce pTOP-flash reporter activity [ΔN89-β-catenin mediated Wnt/β-catenin signaling reporter activity, whereas it did not alter Vp16-ΔNTCF4 mediated activity. These data indicate that ARX functions through β-catenin but not directly through TCF4, suggesting the physical interaction between ARX and β-catenin results in a functional association.We next considered how ARX promotes Wnt/β-catenin signaling activity. Given that ARX is localized in the nucleus and this nuclear localization is essential for regulating Wnt/β-catenin signaling, we focused on known components of the transcription machinery of this signaling pathway. We first examined whether ARX affects Wnt/β-catenin reporter activity through β-catenin or through TCF4 (TCF7L2), one of the TCF/LEF family members well known to function in HEK293T cells . We measradation . Vp16-ΔNthe TCF4 ; Vp16-ΔNactivity . As showTo refine our understanding of the interaction between ARX and β-catenin, we studied a series of ARX truncation mutants using GST-pull down assays . The 431Arx expression construct was used at a low concentration that does not induce Wnt/β-catenin activity in order to maximize measurement of synergism. Given that the molecular mechanism by which BCL9 enhances Wnt/β-catenin signaling is better understood than that of LRRFIP2, we focused on BCL9. To establish the necessity of Bcl9 expression for ARX-mediated Wnt/β-catenin signal upregulation, the TOP-flash reporter assay was performed in the presence of a BCL9 siRNA or control siRNA. Knock down of BCL9 significantly reduced the level of luciferase activity, indicating BCL9 is required, at least in part, for ARX to promote Wnt/β-catenin signaling activity  activity . Given tactivity .ΔN89-β-catenin-D164A) [Arx and its deletion constructs on transactivation activity of the three β-catenin constructs. For the GAL4 activator, we fused the DNA binding domain of the yeast Gal4  to each of the three β-catenin mutants: 1) ΔN89-β-catenin, 2) ΔN89-β-catenin-D164A , and 3) ΔN89-β-catenin ΔC695 . For the GAL4 reporter, five copies of the Gal4 binding sequences were used for the minimal promoter that drives firefly luciferase expression. Each Gal4 activator construct was co-transfected with a GAL4 reporter  construct, and one of the following Arx expression constructs: mock DNA, Arx (WT), Arx (1–470), or Arx (431-564-3XSV40-NLS). The results of the Gal4-luc reporter assay, a readout of the β-catenin transactivation activity, revealed that ΔN89-β-catenin can function as a transcriptional activator on its own, as expected (Arx). Wildtype ARX or nuclear localized C-terminal ARX (431-564-3XSV40-NLS), but not ARX (1–470), promoted the activity of ΔN89-β-catenin transactivation . However, its activity was recovered by co-transfection of Arx (WT) or, to a lesser extent, Arx (431-564-3XSV40-NLS)  . The GALtivation , consisttivation . For ΔN8V40-NLS) . We postin-D164A . Further-catenin . These rWe next mapped the interaction between BCL9 and ARX. Only full-length ARX and ARX (1–470) appeared to interact with GST-BCL9. Neither ARX (221–430) or ARX (431-564-3XSV40-NLS) showed an interaction, implying that the N-terminal region of ARX (1–220) is required for this interaction . The facWhile BCL9 is a co-activator of β-catenin and it binds to the N-terminus of β-catenin, there are multiple co-activators and chromatin remodeling proteins that bind to the structurally flexible C-terminal region of β-catenin. Examples include TRRAP, p400, MLL1, SNF2H/ISW1, TIP49 and p300/CBP . Thus, wARX are associated with a spectrum of neurologic disorders and experimental studies indicate that ARX plays distinct roles in different neural progenitor populations [Arx mutant mouse brain and presumably in the patients with ARX mutations, connecting Wnt/β-catenin signaling to the pathophysiology of ARX related neurological diseases.Mutations in ulations –11,24. TArx-/y mice [Arx-/y mice [-/y and Wnt/β-catenin mutant mice show similar phenotypes; both resulting in part to a premature cessation of neurogenesis [Arx mutant mice [We previously described multiple developmental defects in the cerebral cortex of -/y mice . The dat-/y mice . Supportogenesis ,25. Of iant mice , suggestant mice –30. Toge/β-catenin signaling, we have delineated the inter-molecular domain interactions, required for this transcriptional complex to modulate Wnt/β-catenin signaling. The C-terminal region (431–564) of ARX, near the aristaless domain, was found to be crucial for up-regulating Wnt/β-catenin signaling. Interestingly, within this C-terminal region (431–564), the aristaless domain (529–544) itself, which is highly conserved from invertebrates to vertebrates and known to play a role in transcriptional activation [E) mutant that also retains Wnt/β-catenin activation activity, provides further evidence that the normal cortical development in ArxE mutant mice previously observed by our group and others is likely due to preservation of Wnt/β-catenin signaling activity in dorsal cortical progenitors [ARXE mice [To better understand how ARX regulates Wnttivation , does nogenitors ,31,32. IRXE mice . These dRXE mice Arx selectively in pallial or subpallial progenitor neurons [ARX mutations, depending on where Arx is deficient [Arx from subpallial progenitor cells (GE derived), which will eventually migrate to the cortex and differentiate into cortical inhibitory interneurons [Arx function in the subpallial cells causes epilepsy without gross structural brain defects. In contrast, loss of Arx from pallial progenitors results in both structural and behavioral deficits, but not epilepsy [The data in this report further support an emerging body of evidence that one transcription factor, in this case ARX, has unique roles in different progenitor cells and that these distinguishable functions can be associated with specific neurologic disorders ,33. This neurons ,35. Theseficient ,24,34–36rneurons . These mrneurons ,37,38, sARX mutations. We anticipate our ongoing studies will identify unique transcriptional pathways in ventral GE progenitors where ARX is known to play a distinct role from the dorsal progenitor cells.In summary, beginning with an unbiased proteomics screen to identify ARX interacting proteins, we found components of a transcriptional complex that positively modulate the Wnt/β-catenin signaling pathway in the dorsal, cortical progenitor cells. These data also provide new insights into the pathogenesis of the malformations observed in patients with S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Figin situ hybridization data from the public databases GenePaint (http://www.genepaint.org/)  or Allen Brain Atlas (http://www.brain-map.org/) (Ctnnb1as E15.5).Sagittal sections of mouse embryonic brain showing mRNA (TIF)Click here for additional data file.S2 FigThe level of luciferase induction by Arx or control in the cells treated with different concentration of rWnt3a is indicated as RLU (relative light unit). rWnt3a, recombinant Wnt3a.(TIF)Click here for additional data file.S3 FigARX (321–564), which contains nuclear localization sequence 2 (NLS2) and NLS3, is localized to the nucleus, whereas ARX (321–564) with mutated NLS3 (mtNLS3) failed to localize to the nucleus. When NLS from SV40 was added to ARX (321–564)-mtNLS3, nuclear localization of the ARX protein was restored. ARX (431–564) failed to localize to the nucleus but when three copies of the SV40 NLS were attached, this protein was successfully localized to the nucleus.(TIF)Click here for additional data file.


This cell culture method also maintains the proliferation of adult epithelial stem cells from various tissues. Both primary and adult stem cells retain their tissue-specific differentiation potential upon removal of the culture conditions. Due to the ability to modulate the proliferation and differentiation of the cells, this method is referred to as conditional reprogramming and it is increasingly being used in studies of tumor heterogeneity, personalized medicine and regenerative medicine. However, little is known about the biology of these conditionally reprogrammed (CR) cells. Previously we showed that β-catenin activation, a hallmark of stem cells in vivo, occurs in CR human ectocervical cells (HECs). Here we show that β-catenin-dependent transcription is necessary for the induction of epithelial stem cell markers, and that β-catenin is activated via a non-canonical pathway that is independent of Wnt and Akt/GSK-3. Active Akt actually decreases due to increased mTOR signaling, with a consequent increase in dephosphorylated, active GSK-3. Despite the increase in active GSK-3, β-catenin associates with protein phosphatase 2A (PP2A) and is activated. Inhibition of PP2A catalytic activity reduces both the level of active β-catenin and the acute induction of stem cell markers, suggesting an important role for PP2A in the activation of β-catenin. Moreover, we demonstrate similar results using human prostate and breast cells, indicating that these changes are not restricted to ectocervical epithelial cells and may represent a more fundamental property of conditional reprogramming.The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-267362, converts primary epithelial cells growing  GS. GS31]. Since canonical Wnt and Akt signaling pathways did not explain the activation of β-catenin in CR HECs, we used immunoprecipitation and Western blotting to quantify direct and indirect associations between GSK-3, PP2A and β-catenin in HECs cultured in KGM, or after 3 d of conditional reprogramming. While the amount of β-catenin that co-precipitated with GSK-3β was identical in both culture conditions, 25-fold more β-catenin co-precipitated with PR55α in CR cells . In addiin vitro and in vivo [in vivo [Okadaic acid (OA) is a potent and selective inhibitor of protein phosphatase 1 (PP1) and PP2A  in vivo . To supp in vivo . In cont[in vivo . As showSince β-catenin-dependent transcription was required to up-regulate expression of epithelial stem cell markers in CR HECs , OA inhiVery little is known about the biology of CR cells, however our previous  and presin vivo [β-catenin is stabilized as a result of reduced ubiquitination when phosphorylated at S675 by cAMP-dependent protein kinase (PKA) . Since Fin vivo , we examin vitro [We previously showed that β-catenin is activated during the conditional reprogramming of primary HECs to an adult stem cell-like state, which is characterized by increased expression of epithelial stem cell markers  and indein vitro . In the The ubiquitination and subsequent proteolysis of β-catenin is inhibited by PKA-mediated phosphorylation at S675 . We showSince the Akt protein kinase generally promotes cell proliferation and survival, it was initially somewhat surprising that Akt signaling was reduced in CR cells, which proliferate rapidly and can be propagated indefinitely. However, Akt activity increases during keratinocyte differentiation ,39 and tin vitro, and substitution of alanine for T1203 severely impairs the ability of ROCK to inactivate mTORC1 in vivo [Mechanistically, we determined that the phosphorylation of S473, a critical priming site on Akt, is greatly reduced in CR HECs due to inactivation of the corresponding kinase, mTORC2, by mTORC1 signaling. The reason for increased mTORC1 signaling is unknown, but several studies provide clues. mTORC1 is activated by direct association with the GTP-bound form of the small GTPase, Rheb . The rat in vivo . When P1 in vivo . Even th in vivo .in vitro [Our finding that increased β-catenin-dependent transcription is important for the conditional reprogramming of primary human epithelial cells to an adult stem-like state is consistent with the role β-catenin plays in maintaining self-renewal in isolated adult stem cells. The expansion of mammary  and neurin vitro . Modest in vitro  or GSK-3in vitro  maintainin vitro , as occuin vitro , induce in vitro .It has been reported that the inhibition of PP2A by OA promotes self-renewal in human ESCs , in contS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.


Most sacrococcygeal teratomas present between the 22nd and the 34th week of gestation. The diagnosis of sacrococcygeal teratoma on routine antenatal sonograms is associated with a greater than expected incidence of prenatal and perinatal complications. We report a premature baby with intrauterine spontaneous rupture of giant sacrococcygeal teratoma which was managed successfully. A 28-year-old, G3P2 mother delivered a female at 35-week of gestational age, without proper antenatal sonographic follow up, the newborn baby weighed 3200 gm (including the weight of the tumor) via emergency cesarean section (CS) due to fetal distress. At CS more than usual bloody amniotic fluid was noticed. The newborn had an APGAR scores of 3 and 7 at 1 and 5 min, respectively. The infant was very pale and had 18×11×16cm, well-vascularized lacerated mass at the coccygeal region (Fig.Fetuses with huge sacrococcygeal teratoma may undergo preterm delivery due to uterine over distention or associated polyhydramnios and have a high risk of perinatal complications and death -3. NeonaSource of Support: NoneConflict of Interest: None