Lonchocarpus sericeus as a Wnt/β-catenin pathway inhibitor, both in vitro and in vivo. Lonchocarpin impairs β-catenin nuclear localization and also inhibits the constitutively active form of TCF4, dnTCF4-VP16. Xenopus laevis embryology assays suggest that lonchocarpin acts at the transcriptional level. Additionally, we described lonchocarpin inhibitory effects on cell migration and cell proliferation on HCT116, SW480, and DLD-1 colorectal cancer cell lines, without any detectable effects on the non-tumoral intestinal cell line IEC-6. Moreover, lonchocarpin reduces tumor proliferation on the colorectal cancer AOM/DSS mice model. Taken together, our results support lonchocarpin as a novel Wnt/β-catenin inhibitor compound that impairs colorectal cancer cell growth in vitro and in vivo.The deregulation of the Wnt/β-catenin signaling pathway is a central event in colorectal cancer progression, thus a promising target for drug development. Many natural compounds, such as flavonoids, have been described as Wnt/β-catenin inhibitors and consequently modulate important biological processes like inflammation, redox balance, cancer promotion and progress, as well as cancer cell death. In this context, we identified the chalcone lonchocarpin isolated from  APC (adenomatous polyposis coli) that is a β-catenin destruction complex component. APC mutation occurs in 81% of non-hypermutated colorectal cancers cases and in 51% of hypermutated colorectal cancer cases, triggering tumorigenesis in intestinal polyps of patients with familial adenomatous polyposis [Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second most common cause of cancer death. According to World Health Organization (WHO), it is expected that there were 1.8 million cases and 862,000 deaths in 2018 [olyposis . The Wntolyposis ,5. In tholyposis . Wnt intolyposis . Due to Xenopus laevis stands out for its liability and efficiency. Wnt/β-catenin signaling plays a key role in two fundamental steps during the Xenopus early development that can be exploited for the screening of new drug candidates: the dorso-ventral and the antero-posterior axis patterning [Addressing normal and pathological Wnt/β-catenin signaling functioning requires multidisciplinary experiments combining in vitro and in vivo approaches. Among different models for studying Wnt/β-catenin signaling in vivo, tterning ,11,12. Itterning .Lonchocarpus sericeus (as known as Derris sericeu) by Baudrenghien and colleagues in 1949 [In addition, the AOM/DSS mouse model stands as a relevant preclinical inflammation-associated CRC model with histologic and phenotypic features that recapitulates the aberrant crypt foci-adenoma-carcinoma found in the human CRC . Consist in 1949 , and its in 1949 , howeverXenopus laevis embryonic assays. In addition, acute administration of lonchocarpin in a preclinical CRC mouse model reduced cell proliferation in adenocarcinomas. Altogether, our data show lonchocarpin as a potent Wnt/β-catenin inhibitor that impairs cancer cell proliferation both in vitro and in vivo, and a promising compound for further antitumor clinical investigation and development.In the present work, we describe lonchocarpin ability of inhibiting Wnt/β-catenin both in vitro, in colon cancer cell lines, and in vivo, with Wnt specific Lonchocarpus sericeus, is able to inhibit the Wnt signaling pathway in human colorectal cancer cell lines RKO pBAR/Renilla stimulated with Wnt3a CM (conditioned medium) for 24 h and non-stimulated SW480 pBAR/Renilla , 10 or 20 µM lonchocarpin. L-cell CM treated cells displayed 34% β-catenin positive nuclei E–E”, whiSW480 harbors a mutation in APC that deletes its carboxyl-terminus domain, preventing the destruction complex assembly ,29. ConsOur results showed that lonchocarpin inhibits Wnt/β-catenin signaling activation when pathway activation is triggered by wild-type β-catenin, β-catenin S33A, or dnTCF4 VP16 overexpression A–D. InteTogether, these data suggest that lonchocarpin inhibits Wnt/β-catenin signaling by impairing β-catenin nuclear localization while also hindering TCF activity.Xenopus laevis embryonic development has been successfully used to validate compounds targeting Wnt/β-catenin signaling through the interpretation of embryonic phenotypes and Wnt signaling overactivation [Our in vitro data suggest that lonchocarpin inhibits Wnt/β-catenin by impairing β-catenin nuclear localization E–L whiletivation . In thisIt is well established that Wnt/β-catenin signaling overactivation in the Xenopus embryo 4-cell stage ventral side induces ectopic axis containing head and dorsal structures ,12,31. Wstage Xenopus laevis embryos  was injected intraperitoneally for four days, and the mice were assessed 3 h after the last injection  in comparison to the lower dosage (50 mg·kg−1·day−1), in which no statistical relevance was found /dextran sulfate sodium (DSS)-induced model of colon cancer. After three cycles of DSS, when colon tumors were expected in most animals, lonchocarpin  in the liver. After excretion into the bile, it induces mutagenesis of the colonic epithelium. Colonic tumors are accelerated by a heparin-like polysaccharide DSS, which causes colonic epithelial damage, mirroring some of the features of inflammatory bowel disease . Acute ltothecin . First dtothecin . Togethetothecin . Interestothecin , and thetothecin . Thus, MThere are currently 55 clinical trials aiming to inhibit Wnt signaling pathway in cancers, in which 21 are CRC studies . Among the 21 CRC clinical trials, only two tested compounds inhibit canonical Wnt signaling at the transcriptional level, PRI-724 and resveratrol. In this context, in comparison with these compounds, the in vitro IC50 of lonchocarpin Wnt signaling inhibition is noticeable. PRI-724 inhibited the Wnt signaling pathway at 25 μM in vitro  . All cell lines were maintained in DMEM-F-12  enriched with 10% fetal bovine serum (Gibco). The chalcone lonchocarpin was purified by Nascimento and Mors, 1972  and kind4 cells/well RKO-pBAR/Renilla and SW480-pBAR/Renilla cells were cultured on 96-well plates in DMEM/F-12 containing 10% fetal bovine serum (Gibco). Then, 24 h later, cells were treated with lonchocarpin at the concentrations of 1, 3, 5, 10, 20, and 30 µM in the presence of Wnt3a conditioned medium for an additional 24 h. L-cell CM was used as negative control, and 0.3% DMSO was used as vehicle control. After 24 h of treatment, Firefly and Renilla luciferase activities were detected according to the manufacturer’s protocol .First, 1.2 × 104 cells/well HEK293T cells were cultured on 96-well plates in DMEM/F-12 containing 10% fetal bovine serum (Gibco). At 70% confluence, each well was transfected with 100 ng TOP-Flash plasmid, 10 ng Renilla-luciferase plasmid, and 100 ng wild type β-catenin or 100 ng β-catenin S33A using Lipofectamine 3000 . Then, 18 h after transfection, cells were treated with 1, 5, and 10 µM of lonchocarpin. After 24 h, Firefly and Renilla luciferase activities were detected according to the manufacturer’s protocol .The 1.2 × 104 cells/well in DMEM-F12 media (Gibco) containing 10% fetal bovine serum (Gibco). Cells were fixed in 4% paraformaldehyde, washed with phosphate buffered saline, and permeabilized with 0.3% Triton X-100. Then, each well was blocked for 1 h with 5% bovine serum albumin. The rabbit anti-β-catenin primary antibody (Sigma Aldrich) was diluted in PBS containing 1% bovine serum albumin (1:200) and incubated overnight. The secondary antibody anti-rabbit Alexa Fluor 546 (Sigma Aldrich) was diluted (1:500) with 1% bovine serum albumin (1:500) and incubated for 2 h at room temperature. 4,6-diamidino-2-phenylindole staining  was performed for 15 min, and then slides were mounted with Fluoromount (Sigma Aldrich). Images were captured using the confocal microscope Leica TCS SP5.SW480 cells were cultured in 24-well plates with 4.0 × 105 cells/well RKO-pBAR/Renilla cells were cultured on 12-well plates and treated with 20 μM lonchocarpin for 24 h. Then, the cells were harvested in ice-cold PBS 1X, followed by cytosolic lysis in digitonin buffer with protease inhibitors  for 5 min. Then, the lysate was centrifugated for 10 min at 2000 G at 4 °C and the supernatant was collected and considered as the cytosolic fraction. Following digitonin extraction, the remaining cell pellets were washed in ice-cold PBS 1X and lysed in NP-40 buffer  with protease inhibitors for 15 min and centrifuged for 10 min at 7000 G at 4 °C. The supernatant was collected and considered as the membrane fraction. After that, the cell pellets were washed again in ice-cold PBS 1X and lysed in RIPA buffer with protease inhibitors for 20 min and centrifugated after at 16,000 G at 4 °C. The supernatant was collected and considered as the nuclear fraction. Whole cell lysates were prepared using Triton buffer . Finally, cell lysates were denatured with Laemmli buffer at 95 for 5 min, and the protein samples were subjected to SDS-PAGE and transferred into PVDF membranes . The membrane was then blocked with 2% PVP (Sigma Aldrich) in TBS-Tween-20, incubated with primary monoclonal antibodies β-catenin , β-actin ; lamin A/C  and α-tubulin  overnight at 4 °C. After three washes with TBS-T, the membranes were incubated for 1 h with HRP-conjugated secondary antibodies (CST). The immunoblots were visualized by chemiluminescence using SuperSignal West Pico and West Femto .First, 2 × 10Xenopus laevis embryos were obtained through in vitro fertilization and staged according to Nieuwkoop and Farber [3; 0.08 mM MgSO4·7H2O; 1 mM Hepes; 0.03 mM Ca(NO3)2·4H2O; 0.04 mM CaCl2·2H2O; pH 7.7) until sibling control embryos reached stage 10. Triplicates of four embryos were lysed using 50 µL of 1× Passive Lysis Buffer (Promega). Then, 10 µL of embryo lysate was used for gene reporter activity detection. Firefly and Renilla luciferase activities were detected according to the manufacturer’s protocol .Frog experiments were carried out according to the guidelines granted by the Animal Care and Use Ethic Committee from the Federal University of Rio de Janeiro and were approved by this committee under the permission number 152/13. Female adult frogs  were stimulated with 1000 IU human chorionic gonadotropin . d Farber . All expIn order to modulate the maternal wave, 4-cell stage embryos dorsal blastomeres were injected in the animal pole with 4 nL containing 200 µM of lonchocarpin (0.8 pmol/embryo) or DMSO each, for a total of 1.6 pmol of lonchocarpin per embryo. In order to modulate the zygotic wave, stage 9 embryos were injected with 50 nL containing 200 µM of lonchocarpin (10 pmol/embryo) or DMSO into the blastocoel. After injection, embryos were maintained in 0.1X Barth, until stage 35, when the phenotypes were analyzed.4 cells/well in 96-well tissue culture plates in DMEM/F-12 containing 10% fetal bovine serum (Gibco) and cultured for 24 h. Cells were treated for 24 h with 10, 20, 30, 40, or 50 µM of lonchocarpin and 0.5% DMSO was used as the vehicle control. MTT was added to each well at a final concentration of 0.25 mg/mL for 1 h. The formazan reaction product was dissolved with 100% DMSO and quantified spectrophotometrically at 570 nm .3--2,5-diphenyltetrazolium bromide (MTT) was used to assay mitochondrial activity in viable cells. Cells were plated at a concentration of 1.2 × 10HCT116, SW480, DLD-1, and IEC-6 were cultured on 12-well plates in DMEM/F-12 containing 10% fetal bovine serum (Gibco). Confluent wells were scratched in the center of each well with a pipette tip and treated with the proliferation inhibitor Ara-C at 10 µM. After scratch, cells were treated with 5, 10, or 20 μM lonchocarpin for 24 h. Images were taken at 0 h and the wound areas were measured at 0 and 24 h after treatment. Each experiment was carried out in triplicate, and three fields were counted per well.4 cells/well in DMEM/F-12 containing 10% fetal bovine serum (Gibco). Then, 24 h later, cells were treated with 20 or 30 μM lonchocarpin. DMSO 0.3% was used as vehicle control. Then, 18 h later, we added EdU to the cells, and 6 h later cells were fixed with paraformaldehyde 4% and the experiment was conducted according to Click-iT EdU  manufacturer protocol.HCT116, SW480, DLD-1, and IEC-6 were cultured on 24-well plates with 4.0 × 10−1·day−1, of vehicle (30% polyethylene glycol 400 (PEG400) with 0.9% saline); and i.p. injections of 2.5 mg/mL lonchocarpin 50 or 100 mg·kg−1·day−1 for four days. Due to limitation of the lonchocarpin solubility, to reach 100 mg·kg−1·day−1 i.p was performed every 12 h with 50 mg/kg. Euthanasia was performed after the treatment and the animals received an i.p. BrdU injection (100 mg/kg) 1 h before euthanasia.Animal procedures were approved and carried out according to the guidelines by the Animal Care and Use Ethic Committee from the Federal University of Rio de Janeiro under register 85/15. Male and female 129SvJxC57BL6 mixed mice (8–12 weeks) were housed in microisolator cages and maintained at 23 °C with a 12/12-h light/dark cycle and free access to food and water. A total of 26 experimental mice were pre-treated with vermifuge (vetmax plus 0.04% and ivermectin 0.4%) in quarantine for 5 days. All mice were intraperitoneally injected with AOM  once. Five days later, all animals received the first cycle of DSS treatment composed by 2% DSS  in drinking water for five days, followed by a 2-week rest period without DSS. The 2% DSS treatment cycle was repeated once and followed by a last cycle of 1.5% DSS for 4 days. Mice were monitored every day. Any mouse that lost greater than 20% body weight, demonstrate hunched posture, or moved in a limited fashion was euthanized. Four weeks after the last DSS cycle the animals were randomized into five groups as follows: no treatment group (only AOM/DSS); i.p. injections of 50 or 100 mg·kgAfter euthanasia, the colons were removed, longitudinally opened, cleaned with phosphate-buffered saline (PBS) and fixed in 4% buffered paraformaldehyde for 24 h at 4 °C. The swiss-rolls were processed in sequential ethanol and xilol for paraplast inclusion and the tissues were sectioned in Leica RM2125 RTS microtome and stained with hematoxylin and eosin (H&E) for microscopic identification of lesions, adenomas, and adenocarcinomas. Indirect immunofluorescence was performed after serial deparaffinization in xilol and ethanol. Heat induced epitope retrieval was performed in sodium citrate buffer  in a steamer and nonspecific binding sites were blocked with bovine serum albumin 3% in PBS. The sections were incubated with monoclonal antibodies rat anti-Ki-67  and mouse anti-BrdU  overnight at 4 °C. Anti-rat biotinylated and anti-mouse Alexa 488-conjugated secondary antibodies (Invitrogen) were used to visualize Ki-67 and BrdU, respectively and the nuclei were stained with DAPI. Images were captured using the Olympus Light Microscope BX53 with a LUCPLFLN 20XPH objective and a SC50 color camera . Only the adenocarcinoma areas confirmed in H&E staining were considered in immunofluorescence analysis. All images were manually and independently counted by at least two authors . The proportion of positive-stained nuclei in the epithelial crypt cells found in the adenocarcinoma area were calculated and compared between the groups.p < 0.05 ** p < 0.01 *** p < 0.001.For MTT assays statistical analysis we used a two-way ANOVA following a Bonferroni post-test (GraphPad Prism version 6.0), error bars represent standard error. For all other results, we used a one-way ANOVA (GraphPad Prism version 6.0). Figures show the mean of three replicates performed three times; standard deviation and statistical significance was set at * In summary, our data describes lonchocarpin, a flavonoid from the chalcone class, as a potent inhibitor of the Wnt/β-catenin pathway that acts downstream to β-catenin stabilization level and impairs TCF mediated transcription. In addition, lonchocarpin presents anti-tumor growth properties in vitro and inhibits adenocarcinoma proliferation in an in vivo CRC model. Further studies should be conducted in order to improve its activity and perhaps propose lonchocarpin as an alternative in CRC treatment.


TPC/glycogen synthase kinase 3β (Gsk‐3β) complex to the response. Coimmunoprecipitation assays showed that wild‐type Zbed3 bound to Axin but a Zbed3 mutant lacking the Axin binding site did not. In A549 and H1299 lung cancer cells, Zbed3 overexpression promoted cancer cell proliferation and invasiveness, as well as Wnt signalling and expression of downstream mediators, including β‐catenin, cyclin D1 and MMP7 (P < 0.05). In contrast, the Zbed3 mutant failed to enhance β‐catenin expression (P > 0.05), and its ability to promote cancer cell proliferation and invasiveness was much less than wild‐type Zbed3 (P < 0.05). The ability of Zbed3 to increase β‐catenin levels was abolished by Axin knockdown in A549 cells (P > 0.05). Similarly, treating the cells with a GSK‐3β inhibitor abolished Zbed3's ability to increase β‐catenin levels and Wnt signalling. These results indicate that Zbed3 enhances lung cancer cell proliferation and invasiveness at least in part by inhibiting Axin/adenomatous polyposis coli/GSK‐3β‐mediated negative regulation of β‐catenin levels.Our previous work showed that Zbed3 is overexpressed in nonsmall cell lung cancer and that down‐regulation of Zbed3 inhibited β‐catenin expression and cancer cell proliferation and invasiveness. Here, we investigated Zbed3's ability to promote lung cancer cell proliferation and invasion and the involvement of the Axin/ At present, it is predicted that there are about 2000 BED‐zinc finger proteins in the creature, but most of them have not been verified at the protein level, and the related research is still in an initial stage. We previously observed that Zbed3 is overexpressed in nonsmall cell lung cancer (NSCLC) and acts as a positive regulator of β‐catenin to promote NSCLC malignancy.22.1The A549 and NCI‐H1299 human lung carcinoma cell lines were cultured as described previously.2.2Western blotting was performed as described previously.2.3g for 5 minutes at 4°C, and the supernatant was removed. The precipitate was resuspended in a sample buffer after washing three times using ice‐cold radioimmunoprecipitation assay buffer, and then boiled for 5 minutes. The sample was centrifugated and the supernatant was collected to be examined by Western blotting.Immunoprecipitation assays were performed as described previously.2.45 cells was added to the upper chamber after transfection for 48 hours. The cells were incubated for 48 hours and then the filters were stained with haematoxylin. Cells on the lower surface of the filter were counted in five random high‐magnification microscopes. Each experiment was performed at least three times independently.To investigate the invasiveness of cancer cells, Transwell assays were performed using polycarbonate membrane inserts  and Matrigel (BD Bioscience) as described previously.2.5To investigate lung cancer cell proliferation, colony formation assays were performed as described previously.2.6The MTT assays were performed as described previously.2.7Dual‐luciferase assays were performed as described previously.2.86 tumour cells in the axilla. The mice were examined for tumour growth every week. The mice were killed 3 weeks after inoculation and autopsied for examination of tumour growth. The experiments were approved by the Institutional Animal Research Committee of China Medical University and according to experimental animal ethics guidelines issued by China Medical University.Four‐week‐old female BALB/c nude mice were  subcutaneously inoculated with 5 × 102.9t test for densitometry measurements, Matrigel invasion assays, colony formation assays, and MTT assays. Values of P < 0.05 were considered statistically significant.Statistical analysis was performed using the SPSS statistical software package version 22.0 . In vitro experiments were performed at least three times. Differences between groups were analysed using Student's 33.1We first assessed expression of several Zbed3 and Wnt signalling molecules, including Axin, GSK‐3β, and β‐catenin in A549 and NCI‐H1299 lung cancer cells. Western blotting showed that all of these molecules were expressed in both A549 and NCI‐H1299 cells (Figure 3.2We prepared a mutant Zbed3 plasmid that lacked the Axin binding site. Transfection of wild‐type Zbed3 into A549 and NCI‐H1299 cells significantly up‐regulated β‐catenin expression, whereas transfection of the Zbed3 mutant failed to affect β‐catenin expression (Figure 3.3Colony formation and MTT assays showed that transfecting lung cancer cells with wild‐type Zbed3 significantly enhanced their proliferation, but that effect was largely inhibited by transfection of the Zbed3 mutant lacking an Axin binding sited Figure A and B. 3.4Western blotting showed that Axin is endogenously expressed in A549 cells. Transfection of Zbed3 into A549 cells did not affect Axin expression, but significantly increased β‐catenin expression Figure . However4Wnt signalling plays important roles during the development and progression of cancer, including NSCLC.Here, we found that Wnt signalling molecules, including Axin, GSK3β, and β‐catenin, are all expressed in both A549 and NCI‐H1299 cells. Overexpression of Zbed3 significantly increased β‐catenin expression and Wnt signalling in these cancer cells, as well as their proliferation and invasiveness. This is consistent with our previous findings in cancer tissues, which showed that Zbed3 expression was associated with advanced TNM stages and lymph node metastasis of NSCLC.None.


Knowledge about the curing behavior of a thermosetting resin and its composites includes the determination of kinetic parameters and constitutes an important scientific and technological tool for industrial process optimization. In the present work, the differential scanning calorimetry (DSC) technique was used to determine several curing parameters for pure epoxy and its composite reinforced with 20 vol % mallow fibers. Analyses were performed with heating rates of 5, 7.5, and 10 °C/min, as per the ASTM E698 standard. The kinetic related parameters, that is, activation energy (E), Avrami’s pre-exponential factor (Z), and mean time to reach 50% cure (t½), were obtained for the materials, at temperatures ranging from 25 to 100 °C. Response surfaces based on the mathematical relationship between reaction time, transformed fraction, and temperature were provided for optimization purposes. The results showed that the average curing time used for the production of diglycidyl ether of bisphenol A/triethylenetetramine (DGEBA/TETA) epoxy systems or their composites reinforced with natural mallow fibers can be considerably reduced as the temperature is increased up to a certain limit. Epoxy resins are thermosetting polymers characterized by the presence of glycidyl groups in their macromolecules. By reacting the glycidyl group with a suitable crosslinking agent (hardener), a three-dimensional structure is formed. Epichlorohydrin  is the universal agent bearing the epoxy group that reacts with chemical species that have active hydrogens. Bisphenol A is the most common chemical species that possesses these active hydrogens [Epoxy resins can be used for several applications. High-performance adhesives are some of the most popular. These are polymers applied to join other polymers, metals, ceramics, or composites. In this particular case, the resin is classified as chemically reactive adhesive, in which the epoxy is composed of a two-component system, going through the curing process when the components are mixed ,2.Some epoxy characteristics that are worth mentioning include excellent combination of mechanical properties, good electrical properties, dimensional stability, effective adherence, excellent corrosion resistance, and low cost. Typical applications are as adhesives , electrical molds, components for the automotive and aerospace sectors, protective coatings, sports articles, as well as a polymer matrix for composites with fiber reinforcement .Investigation on the kinetics of the curing reaction of plain epoxy resin or as the polymer matrix in composites has been conducted by different areas ,6,7,8,9.Urena lobata Linn.) stand out due to their high strength-to-weight ratio and very low cost. These fibers are produced in local communities in the Brazilian part of the Amazon forest [Currently, with the growing demand for sustainable materials, natural fiber-reinforced composites are being considered to replace traditional synthetic materials, such as fiberglass, in several applications ,22,23,24In the production of epoxy matrix composites reinforced with natural fibers ,12,13,14In the present work, the differential scanning calorimetry (DSC) analysis was performed to evaluate if the total curing time of the epoxy resin could be less than the previously reported time of 24 h ,22,23,24Thus, the objective of the present work was to obtain the curing kinetic parameters for an epoxy DGEBA resin, in order to understand, for the first time, the influence of the addition of natural mallow fibers (20 vol %) on the curing behavior of the epoxy DGEBA/TETA system.Commercial epoxy resin diglycidyl ether of bisphenol A (DGEBA) was investigated in the present work. It was hardened for the experiments using the stoichiometric ratio of triethylenetetramine (TETA). Both substances were acquired from the Brazilian company Epoxyfiber Chemical Industry .The mallow fibers were generously provided by researchers from the State University of North Fluminense (UENF), with the indication that the fibers were acquired from the Brazilian Textile Company Castanhal . E, kJ/mol), Avrami’s pre-exponential factor , rate constant for each temperature , and the mean cure time  for the pure epoxy and the mallow fiber-reinforced composite, at temperatures of 25 to 100 °C. Using the times of 30, 60, 120, 180, 240, and 300 min, and a transformed fraction interval ranging from 50% to 95%, the cure temperatures were found for each specific situation. Based on the mathematical relationships between reaction time, transformed fraction, and temperature, curves with respect to the pure epoxy and the epoxy composite reinforced with 20 vol % of mallow fibers were plotted.For the differential scanning calorimetry (DSC) analysis, the resin and hardener were mixed with 13 parts of hardener for each 100 parts of resin (phr = 13), and the mixture was placed in an aluminum crucible. Mallow fibers were added to prepare composite specimens with a 20 vol % fraction, to investigate the fiber influence on the epoxy curing kinetics. The reason for selecting that fraction to produce the composite samples was due to the adhesion between the matrix and fiber, which could be impaired with higher volume fractions of reinforcement. This best value has obtained during preliminary testing discussed elsewhere . In thatE, kJ/mol), pre-exponential factor  and mean time to reach 50% cure  for plain epoxy and its composite reinforced with mallow fibers, at temperatures ranging from 25 to 100 °C.½ is reduced. This result was already expected, since the cure of thermosetting resins is a thermally activated process and thus can be modeled by the Arrhenius equation [½. This fact can be justified by the fibers acting as nucleating sites on the curing process [The results in equation . Despite process .transfX. is the transformed fraction; k is the constant rate for each temperature (min−1); t is the transformation time (cure) in minutes; and n is Avrami’s exponent (n = 1 for ASTM E698).According to the ASTM E698 standard and others ,5,26, thk) and the pre-exponential factor (Z), the following equation is used:−1); Z is the pre-exponential factor (min−1); T is the cure temperature in Kelvin; E is the activation energy (J/mol); and R = 8.314 J/mol.K.In order to adjust a mathematical relationship combining time, transformed fraction, and temperature, Equation (2), which correlates the constant rate (By combining Equations (1) and (2), the final equation for reaction time, transformed fraction, and temperature is obtained:Similar to The most striking difference between the response surface in In the present work, the curing kinetics of a diglycidyl ether of bisphenol A/triethylenetetramine (DGEBA/TETA) epoxy system and of its composite reinforced with 20 vol % of mallow fibers was studied, following procedures indicated by the ASTM E698 standard.Z (1.08 × 108 min−1 and 8.04 × 105 min−1), and the times to reach 50% of the curing reaction (t½), for temperatures ranging from 25 to 100 °C were calculated.Kinetic parameters, such as the activation energies (58.971 kJ/mol and 44.536 kJ/mol), the pre-exponential factors ½ 138.68 and 55.26 min for curing were found for epoxy resin and the composite reinforced with 20 vol % mallow fibers, respectively.Particularly for the commonly used room temperature (25 °C), the values of tThe curves’ response surfaces obtained for both plain epoxy and 20 vol % mallow fiber-reinforced composites have practical application in the determination of the appropriate combination of time, transformed fraction, and temperature, according to the actual case.Curing temperatures for the composite reinforced with 20 vol % mallow fibers are lower than that for pure epoxy resin, for a particular time. This possibly means that natural fibers act as catalysts for the curing reaction, a finding which is reported for the first time for a natural fiber-reinforced epoxy composite.


Development of the metanephric kidney is a complicated process regulated by reciprocal signals from the ureteric bud and the metanephric mesenchyme that regulate tubule formation and epithelial branching morphogenesis. Over the past several years, several studies have suggested that Wnt signaling is involved in multiple aspects of normal kidney development as well as injury response and cancer progression. We will review these data here. During embryonic development, three sets of kidneys form within the mammalian intermediate mesoderm . The thrThe metanephric kidney serves an essential role in tissue homeostasis by regulating the balance of water and electrolytes in the plasma. It also excretes metabolic waste products and regulates the production of certain hormones. As defects in the development of the urinary system constitute some of the most common human birth defects, a better understanding of the genes required for formation of this organ is of great interest. Here, we will review data demonstrating that the Wnt pathway plays critical roles in multiple cellular events that occur during the development of the mammalian kidney.Development of the metanephros begins on embryonic day (E) 10 in mice (approximately E 32 in humans), when a caudal portion of the Wolffian duct adjacent to the hindlimbs branches dorsally and invades a population of pre-specified mesenchyme known as the metanephric mesenchyme (MM)  [The Wnts make up a family of secreted glycoproteins that have been implicated in embryonic induction, cell polarity generation, and cell fate specification in metazoan species from hydra to humans , 14. Then flies) –17. UponThe canonical pathway utilizes β-catenin as a transcriptional regulator. In the absence of ligand/receptor interactions, β-catenin levels are regulated by a destruction complex consisting of two scaffolding proteins, axin and adenomatous polyposis coli (APC), and two serine/threonine kinases, glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1) Fig. a 18, 1919. β-CatIt should be noted that, in addition to its crucial role in mediating canonical Wnt signaling, β-catenin also plays a role in establishment of the adherens junctions and interacts with the actin cytoskeleton by binding membrane bound E-cadherin and cytoplasmic α-catenin. It has been suggested that Wnt signaling, in part, functions through maintaining a balance between cytoplasmic/nuclear and junctional β-catenin , 22.2+-releasing pathway and the planar cell polarity/convergent extension (PCP/CE) pathway. In the Ca2+-releasing pathway, Wnt binding stimulates calcium flux and activates calcium-sensitive factors such as protein kinase C (PKC) and calmodulin-dependent protein kinase II (CamKII) [Wnt signaling also appears to trigger β-catenin-independent signaling events. The two most frequently discussed non-canonical/β-catenin-independent pathways are the Ca(CamKII) –25. This(CamKII) . There iAnother non-canonical pathway identified is the planar cell polarity (PCP)/convergent extension (CE) pathway, so-named because of the cellular processes the pathway is thought to mediate Fig. c. PCP deDrosophila mutants and affected the orientation of cuticular bristles, wing hairs and the ommatidia in the eye. Some of the molecules identified had previously been implicated in Wnt signal transduction, including Fz and Dvl, thus leading to the speculation that this may be a Wnt-mediated process. In flies, a molecular readout of PCP is the localization of the proteins strabismus/van gogh (Stbm/Vang), and prickle (Pk) on the proximal side of the cell and diego (Dgo), Fz and Dvl on the distal side. Another protein, flamingo/starry night (Fmi/Stan) is also required for PCP, but its localization is not polarized.Components of the PCP/CE pathway were first identified in Over the past several years, it has become clear that orthologs of the fly PCP/CE genes are essential for various aspects of vertebrate embryogenesis, including gastrulation movements, pelage development and inner-ear formation. Importantly, within the sensory epithelium of the mouse inner ear, Vangl2 (a flamingo homolog), Fz-3 and Fz-6 proteins are asymmetrically localized within the plane of the epithelium, suggesting that the sub-cellular localization, and perhaps function, of at least some of the core PCP components is conserved in mammals , 30.Signaling through the PCP/CE pathway activates alternative downstream elements of Dvl, such as the GTPases Rho, Rac and Cdc42 and the c-Jun N-terminal kinase, JNK , 27, 31.Drosophila, causing some speculation that this is a Wnt-independent event. However, there is evidence of Wnt involvement in PCP establishment in both C. elegans and vertebrates [C. elegans early embryo, the Wnt Mom-2 is both necessary and sufficient for spindle orientation and Fz localization in a transcription-independent manner, and the relative location of the Wnt appears to establish the orientation of the polarity, suggesting that the ligand may play an instructive role in establishing planar cell polarity [Xenopus embryos, although the evidence suggests that, in this system, the ligands may play permissive roles [How Wnt signaling mediates PCP is unclear. No mutation of a Wnt ligand has been associated with PCP defects in tebrates . In the polarity . Wnt5a ave roles , 34, 35.Xenopus embryos or induce tubule formation in isolated kidney mesenchyme. Wnts that could act positively in these assays were considered canonical Wnts, while those that could not were considered non-canonical. Although these rough classifications were used for many years, it was soon apparent that not all Wnts could be neatly grouped into one class or the other. Although Wnt1, Wnt3a and Wnt8 always seemed to trigger the canonical event, and Wnt5a always appeared to act non-canonically, other Wnts acted in a canonical manner in some assays and a non-canonical manner in others. For instance, Wnt4 cannot transform C57s or induce a secondary axis in Xenopus , but it does induce tubule formation in isolated mouse mesenchyme. Wnt11 does transform C57s but does not induce a secondary axis or induce tubules. Much of this discrepancy could be explained if there was binding specificity between ligands and various receptors and that either affinity for the receptor determined pathway usage, or that different receptors signaled specifically through the canonical or non-canonical pathways. In support of the former hypothesis, He et al. showed that Wnt5a was capable of acting canonically (inducing a secondary axis in Xenopus) if its mRNA was co-injected with that of Fz5. They hypothesized that the inability of Wnt5a to act canonically was due to the absence of its “canonical receptor” from the Xenopus early embryo.What determines pathway usage in vivo is still not well understood. Historically, it was thought that pathway specificity was determined by the ligand itself. The Wnts were grouped into canonical or non-canonical classes based on their ability to transform C57MG cells, induce secondary axes in Subsequent studies have shown that, in fact, Wnt5a, and other so-called non-canonical Wnts, actively inhibit canonical signaling . In someA further level of regulation is most likely determined by the cytoplasmic environment in which the signal is received. Various intracellular inhibitors of the canonical pathway have been identified, and several of these appear to work on the Dvl protein, acting as switches that divert signaling through the non-canonical pathway , 41. In Analysis of mouse embryos by in situ hybridization has revealed the expression of six Wnts in the developing kidney: Wnt2b, Wnt4, Wnt6, Wnt7b, Wnt9b, and Wnt11 –45. AnotAn interest in Wnt signaling in the process of renal vesicle formation arose in 1994, when it was discovered that Wnt1 could substitute for the UB as an inducer of tubulogenesis . Wnt1 isThe expression of these ligands in the developing ureteric bud suggested that they could be playing a role in tubule induction. To test the sufficiency of each of these factors to induce tubules, cells expressing the individual ligands were co-cultured with isolated E 11.0 metanephric mesenchyme. Wnt6, Wnt7b and Wnt9b can all induce tubulogenesis, while Wnt11 cannot . Of the Wnt9b−/− mice die within 24 h of birth due to agenesis of the kidneys [Wnt9b−/− mutants, the UB invades the MM, but the UB fails to induce the expression of several pre-tubular aggregate markers, including Wnt4, in the adjacent MM. Wnt4 has also been shown to play an essential role in renal vesicle formation [Wnt4 mRNA is expressed in the aggregates as early as E 11 and continues to be expressed in the RVs and the comma- and S-shaped bodies  mutant kidneys through at least E 14.5 and there is no significant change in the rate of apoptosis in E 11.5 Wnt9b mutants, a defect in survival in either of these mutants seems highly unlikely  and transforming growth factor beta (TGFβ) , 49. PreGata3 and Gdnf mutants, respectively) and, therefore, have never been exposed to any other duct/bud derived signals [Another possibility is that Lif and/or TGFβ function by inducing survival and/or proliferation in cells that have already been weakly induced and that this is sufficient to trigger tubulogenesis. Yang et al. showed that isolated rat mesenchymes already expressed Wnt4 prior to their treatment with Lif, suggesting that Lif was either necessary to maintain the Wnt4 expressing cells in the absence of the bud or was working through a parallel pathway that is required along with Wnt signaling . The fac signals . These dSeveral Wnts are expressed in or around the developing ureteric bud. As mentioned, the expression patterns of Wnt6, Wnt7b, Wnt9b and Wnt11 are all consistent with roles in development of this structure. In addition, at later stages of development, Wnt4 and Wnt11 are both expressed in the medullary stroma, possibly signaling back to the adjacent collecting ducts, although it appears that, at least for Wnt4, the medullary expression is actually mediating the formation of smooth muscle cells . HoweverWnt11 mutant phenotype in mice is variably penetrant, with some mutants dying in utero and others dying shortly after birth [Gdnf expression is significantly down-regulated in the mutant mesenchyme. GDNF is the ligand for the Ret receptor tyrosine kinase, which is expressed in the ureteric bud tips. Both Gdnf and Ret are required for normal branching of the UB [Ret+/− mice are crossed with Wnt11+/− mice, the resulting kidneys of the double heterozygotes are 52% smaller than those of wildtype. Ret+/−;Wnt11−/− mice have kidneys 67% the size of those of Ret+/−;Wnt+/− mice. These studies indicate that Wnt11 acts in parallel with the Ret/Gdnf pathway to regulate branching of the ureter [The er birth . In nullf the UB , 55. Whee ureter .Wnt9b−/− mutants, expression of the branching regulators Wnt11 and GDNF is down-regulated prior to the appearance of any morphological signs of branching defects. In addition, the ureteric buds of Wnt9b mutants branch significantly less than those of Wnt4 mutants, suggesting that the phenotype is not a secondary effect from failure to form the renal vesicles but instead a direct role for Wnt9b in regulation of secondary branching of the ureteric bud , as well as examination of the mRNA for a general β-catenin target gene, Axin2, showed little, if any, β-catenin activity in the mesenchyme, while there was high activity in the UB and collecting ducts –62 Fig.. These oThese results were seemingly in direct opposition to those in the previous studies on canonical pathway activity in tubule formation. This contradiction could be rectified if one assumed that Wnt9b acted in a canonical manner to stimulate Wnt4 expression, which subsequently induced tubulogenesis through the PCP pathway. However, studies using Madin–Darby canine kidney (MDCK) cells have shown that Wnt4 can activate a β-catenin/LEF/TCF luciferase reporter and Wnt4 can stabilize β-catenin in isolated MM, suggesting that it signals through the canonical branch , 57. TheL1Cam gene has been identified as a direct transcriptional target of β-catenin, and L1Cam has been implicated in mediating normal branching of the UB [Examination of both Bat-Gal and axin2 expression indicates high levels of canonical activity in the branching tips of the ureteric bud and lower levels in the stalk, suggesting that this branch of the pathway may play an important role in ureteric bud/collecting duct development. This fits with the data showing that lithium, an agonist of the canonical pathway, supports branching of isolated buds . What exf the UB , 67. Anof the UB –71.The ligand mediating canonical signaling in the bud tips is unknown. It could be one of the mesenchymally expressed Wnts or one of the bud-expressed Wnts. Candidates for a mesenchymally expressed Wnt include Wnt2b acting redundantly with another Wnt such as Wnt4 . Wnt9b or Wnt11 would seem to be good candidates for the bud-expressed Wnts, given their expression patterns and their role in branching morphogenesis, but neither appears to be necessary for axin2 or Bat-gal expression on its own (our unpublished observations). A third possibility is that reporter activation is Wnt independent. Recent data suggest that a number of non-Wnt factors are able to activate the β-catenin pathway, including the secreted molecules norrin, dickopf2, Sfrp1 and the R-spondins –76. AlthThere is no direct evidence indicating a role for the non-canonical pathway in the development of the ureteric bud/collecting duct system. As mentioned, in the Bat-Gal mouse β-galactosidase is expressed throughout the bud/ducts through birth. If this reporter represents a faithful readout of pathway activity and if only one pathway is activated in a specific cell type at a particular point in time, then one can conclude that the non-canonical pathway is not playing a major role in the prenatal development of the collecting duct system. However, if axin2 transcripts represent a more faithful readout, then the canonical pathway is not active in the distal collecting ducts, and it is possible that any Wnts expressed in or around this tissue  or the Wnts expressed in the medullary stroma (Wnt4 and Wnt11) may be signaling to the distal collecting ducts through the non-canonical pathway. However, conclusive proof of the involvement of the non-canonical branch will require identification of the receptors for each of the kidney-expressed Wnts as well as a more careful examination of the expression and functional roles of the canonical and non-canonical pathway determinants during renal development. It is tempting to speculate that the processes of convergent extension and/or planar cell polarity are involved in the directional growth of the collecting ducts, and evidence will be presented below suggesting that this is indeed the case.Inv) gene in mice. Inv encodes an ankyrin repeat domain encoding protein that has homology to diversin, the vertebrate ortholog of the fly PCP protein, diego. Mutations in Inv cause nephronophthisis type II (NPNII), an autosomal recessive disorder characterized by extensive renal cysts [inv in mice results in renal cysts and situs inversus [For years it has been known that improper regulation of the canonical Wnt pathway correlated with and caused various human diseases, including cancers. More recently it has been suggested that a fine balance between the canonical and non-canonical branches of the Wnt pathway is essential for the development and homeostasis of multiple tissues, including the kidney. One of the clearest examples of such a balance comes from functional analysis of the inversin  in Xenopus embryos, suggesting that Inv binds to and inhibits the ability of Dvl to mediate canonical Wnt signaling [Inv in zebrafish resulted in increased expression of canonical Wnt targets, and defects in CE movements, a process dependent on non-canonical Wnt signaling [Inv mutants, the canonical pathway is enhanced, while the non-canonical pathway is abrogated, leading to cyst formation.Studies performed by Simons et al. suggest that inversin plays an important role in regulating Wnt signaling and the choice between the canonical and non-canonical signaling pathways during kidney development . Misexprignaling . This stignaling . Thus, IIt appears that perturbation of the Wnt pathway may play a general role in kidney cystogenesis. Increased levels of β-catenin are strongly correlated with cyst formation in humans and over-expression of β-catenin or c-Myc, a downstream target of β-catenin, in mice gives rise to cystic kidneys –87. FurtDrosophila wing becomes shorter and wider [pck rat models of polycystic kidney disease (PKD). Fischer et al. showed that the orientation of cell division is randomized in these mutants prior to cyst formation, suggesting a causal role in cystogenesis [As the canonical and non-canonical pathways are antagonistic toward each other, over-expression of β-catenin should also antagonize the non-canonical pathway . Planar cell polarity/convergent extension processes have been shown to be involved in orienting the plane of cell division and/or cell intercalation during the growth of organs such as the heart and the pancreas . Defectsnd wider . By extrogenesis . AlthougAnother process shown to be dependent upon planar cell polarity that could contribute to cystogenesis is the intercalation of cells during convergent extension movements , 90–92. Given the extensive data implicating mis-regulation of the Wnt pathway in human disease, there has been intense investigation into the molecular nature of the factors necessary for regulation in vivo. Some of this regulation occurs at the level of the ligand, affecting its ability to interact with its receptors or co-receptors . Such annkd), daple (Dpl) and dapper/frodo (dact/frd) proteins, although, with the exception of Inv, no role in kidney development has been established for any of these factors [Inv mutant kidneys are slightly smaller than normal and cystic ones, much of early nephrogenesis occurs normally. This suggests that either the shift from one branch to the other is not essential during early embryonic stages or that there is another factor that fulfills this role at earlier time points.A second type of regulation appears to be through the production of intercellular antagonists of the signal transduction pathways. As one might expect, several proteins have recently been identified that appear to interact with Dvl to regulate pathway or branch utilization, including the previously discussed Inv, naked cuticle ( factors , 78, 104And, finally, there are several proteins that affect the ability of β-catenin to function as a transcriptional activator including groucho, kaiso, cited1 and specific isoforms of the Lef/Tcfs , 105. OuWithin the past several years we have made tremendous advances in our understanding of the molecular regulation of kidney development, and the Wnt pathway appears to play multiple crucial roles. However, we are still a very long way from comprehending the molecular and cellular function of this pathway during nephrogenesis. In fact, a number of very basic questions remain unanswered.Much of our current thinking on the role of Wnts is based on mRNA expression and knockout phenotypes. However, recent studies suggest that, in addition to regulating the transcription of the Wnt mRNA, pathway activity may be regulated at the level of ligand secretion. Studies performed on flies and worms have indicated that secretion of Wnts from the producing cells is regulated and that this level of regulation may be conserved in mammals –108. ThuAnother unanswered question is the role of some of the alternative pathway components during kidney development. In situ hybridization analysis has shown the expression of the canonical ligands R-spondin1 and R-spondin3 in the peripheral metanephric mesenchyme or stroma . What roA final unexplored frontier is the role for Wnts in patterning or maintaining the kidney. Wnt9b is necessary for MET but its mRNA continues to be expressed long after the last tubules have been induced . Wnt4 is


CTNNB1 gene encoding β-catenin. HCC-associated CTNNB1 mutations stabilize the β-catenin protein, leading to nuclear and/or cytoplasmic localization of β-catenin and downstream activation of Wnt target genes. In patient HCC samples, β-catenin nuclear and cytoplasmic localization are typically patchy, even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in β-catenin activation are not well understood. To define mechanisms of β-catenin-driven HCC initiation, we generated a Cre-lox system that enabled switching on activated β-catenin in (1) a small number of hepatocytes in early development; or (2) the majority of hepatocytes in later development or adulthood. We discovered that switching on activated β-catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/β-catenin signaling heterogeneity later in hepatocarcinogenesis, we performed RNA-seq analysis of zebrafish β-catenin-driven HCC. At the single-cell level, 2.9% to 15.2% of hepatocytes from zebrafish β-catenin-driven HCC expressed two or more of the Wnt target genes axin2, mtor, glula, myca and wif1, indicating focal activation of Wnt signaling in established tumors. Thus, heterogeneous β-catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis.Up to 41% of hepatocellular carcinomas (HCCs) result from activating mutations in the  Summary: Here we show that expression of activated β-catenin in a subset of early hepatocytes is sufficient to drive liver tumorigenesis. Heterogeneity in β-catenin activation continues during HCC progression. CTNNB1)  .β-catenin is a multifunctional protein with two cellular pools: one pool acts in the canonical Wnt signaling pathway, while the other pool functions independently of Wnt, connecting cadherins to the actin cytoskeleton as part of adherens junctions . ActivatCTNNB1 or TP53 ] leads to HCC in 78% of animals by 6 months post fertilization (mpf) (Tg(fabp10a:pt-β-cat) zebrafish contain the transgene encoding activated β-catenin in all hepatocytes, β-catenin nuclear and/or cytoplasmic staining is only observed in scattered cells in these zebrafish HCC  . These zon (mpf) . Althougfish HCC . This hestaining . The cliOne possible explanation for this variable β-catenin localization is that only very low levels of activated β-catenin, undetectable by immunohistochemistry, are required to drive target gene expression. Supporting this hypothesis is that diverse β-catenin mutations and β-catenin nuclear/cytoplasmic localization correlate with expression of the β-catenin target gene glutamine synthetase (GS) and strong, diffuse immunohistochemical staining for GS . HoweverA related question to the significance of heterogeneous β-catenin in HCC progression is the role of β-catenin in HCC initiation: is activated β-catenin required in most or all hepatocytes to initiate HCC, or is its presence in a subset of hepatocytes sufficient for tumor initiation? In most transgenic animal models of HCC , includiIn this study, we tested two related hypotheses: (1) activated β-catenin in a subset of early hepatocytes is sufficient to initiate HCC; and (2) β-catenin activity is heterogeneous during HCC progression. We used zebrafish to develop a vertebrate model of HCC that allows spatial and temporal control of β-catenin activation and performed single-cell and bulk RNA-sequencing of β-catenin-driven HCC. Our results support the hypothesis that activated β-catenin expression in a small subset of hepatocytes is sufficient to drive HCC initiation and indicate the presence of diverse populations of hepatocytes with heterogeneous Wnt target gene expression during HCC progression.Tg(fabp10a:CreERT2) line is used as a driver (Table S1). This transgenic line consists of T2CreER cDNA, encoding Cre recombinase fused to the modified ligand-binding domain of the human estrogen receptor (ERT2) (fabp10a promoter (Tg(fabp10a:loxP-BFP-loxP-Xla.Ctnnb1) line, hereafter referred to as Tg(fabp10a:flox-pt-β-cat), wherein the fabp10a promoter drives expression of activated β-catenin preceded by a blue fluorescent protein (BFP)-STOP cassette flanked by loxP sites ; Tg(fabp10a:flox-pt-β-cat) system is expected to have spatial control, as activated β-catenin is switched on only in hepatocytes due to the use of the fabp10a promoter to drive both components of the switch. We also predicted the system to be temporally controlled by tamoxifen treatment.To define mechanisms of β-catenin-driven hepatocarcinogenesis, we sought to turn on activated β-catenin with temporal and spatial control by developing a system of modular switches. We chose a Cre-lox system because this switch produces an irreversible, all-or-none event that occurs on a cell-by-cell basis depending on whether or not Cre-mediated recombination has taken place within that cell or its predecessors. In our system of modular switches, the r (ERT2) , downstrpromoter . The loxxP sites A. In theTg(fabp10a:flox-pt-β-cat)], examining the effect of Cre recombinase on BFP expression and Wnt reporter activity using the previously characterized Tg(fabp10a:Cre) zebrafish (Tg(7xTCF-Xla.Siam: mCherry), wherein cells with active Wnt signaling show nuclear mCherry expression (Tg(fabp10a:Cre); Tg(fabp10a:flox-pt-β-cat) zebrafish livers compared to control siblings without Cre recombinase (Tg(fabp10a:flox-pt-β-cat) zebrafish express functional activated β-catenin in their livers in the presence of Cre recombinase.We first tested the efficiency of the lox-switch  (Tg(fabp10a:CreERT2);Tg(ubi:switch) zebrafish than in vehicle-treated and untreated control siblings (P=0.3736). In all zebrafish, non-hepatocyte tissues including intestine showed only EGFP expression  zebrafish increases the percentage of switched hepatocytes by 25% (96% minus 69%), enabling switching on β-catenin in a subset of adult hepatocytes.To characterize TAM-dependent and TAM-independent switching in adult male and female zebrafish, brafish) F,G. We fiblings] F. The pepression G, confirTg(fabp10a:CreERT2);Tg(fabp10a:flox-pt-β-cat) zebrafish, hereafter referred to as CreLox) that enables switching on activated β-catenin in (1) the majority of larval hepatocytes in response to TAM or (2) a subset (about 9%) of larval hepatocytes in the absence of TAM. We used this CreLox model to test two related hypotheses: (1) switching on activated β-catenin in the majority of larval hepatocytes is sufficient to drive liver tumorigenesis; and (2) switching on activated β-catenin in a subset of larval hepatocytes is sufficient to drive liver tumorigenesis. CreLox zebrafish, along with sibling controls lacking either the Cre driver or the lox-switch transgene, were treated with 10 μM TAM or vehicle from 3 dpf to 6 dpf and then maintained under standard conditions until 6 mpf. In parallel under the same husbandry conditions, we maintained Tg(fabp10a:pt-β-cat) zebrafish, which carry activated β-catenin under direct control of the fabp10a promoter, and non-transgenic sibling controls.We developed a CreLox system (Tg(fabp10a:pt-β-cat) zebrafish exhibited significantly increased liver-to-body mass ratios (P<0.0001) and had significantly more HCC by histologic analysis than non-transgenic control siblings  , and HCC was diagnosed in accordance with previously defined criteria . In addi=0.0002) A–C.Fig.P=0.0032) and had significantly more HCC histologically than TAM-treated control siblings  (P=0.0029) (TAM-treated CreLox zebrafish exhibited significantly increased liver-to-body mass ratios (=0.0006) A–C. TAM-=0.0029) B,C. ThesP=0.0569). Liver-to-body mass ratios in CreLox zebrafish were similar in the presence and absence of TAM (P=0.1559). We found that 13% of vehicle-treated CreLox zebrafish showed HCC by histologic analysis, significantly more than in vehicle-treated control siblings  and similar to TAM-treated CreLox zebrafish  (P=0.0001) and similar to the rate in TAM-treated CreLox siblings (P=0.5959)  B,C. The =0.5959) B,C. The Tg(fabp10a:CreERT2);Tg(ubi:switch) zebrafish as well. The absence of significant switching by immunofluorescence analysis of Tg(fabp10a:CreERT2);Tg(ubi:switch) larvae at 6 dpf  could be due to limitations in our detection and analysis strategy and/or the time required for degradation of EGFP.Taken together, these results show that TAM treatment in CreLox zebrafish, which leads to switching on β-catenin in most hepatocytes beginning at 3 dpf, did not significantly increase HCC burden compared to vehicle treatment. The finding that the number of hepatocytes switched on from 3 dpf onward does not significantly affect hepatocarcinogenesis suggests that the critical time point for switching on β-catenin to initiate HCC occurs at or before 3 dpf. This finding also implies that switching occurs before 3 dpf in P=0.0034) (P=0.0374) and similar to TAM-treated CreLox siblings (P=0.5530) (P>0.9999) (P=0.4936)  A. Vehicl=0.5530) A. With e>0.9999) B. Additi=0.4936) B. The si=0.4936) F during P=0.0082, Fisher's exact test). This finding provides additional support for the hypothesis that switching on β-catenin in a small number of early larval hepatocytes is sufficient to initiate HCC.Independent of TAM treatment, 22% of CreLox zebrafish had HCC at 9 mpf, significantly more than control siblings lacking either the Cre driver or the lox-switch transgene (Tg(fabp10a:pt-β-cat) zebrafish HCC (Tg(7xTCF-Xla.Siam: mCherry) (Tg(fabp10a:pt-β-cat), CreLox, and control sibling livers.Having demonstrated that switching on activated β-catenin in a subset of larval hepatocytes is sufficient to initiate HCC, we next wanted to test the related hypothesis that β-catenin-dependent gene transcription is heterogeneous during HCC progression. We compared Wnt pathway activity in adult male and female fish HCC  and non-mCherry) . In paraTg(fabp10a:pt-β-cat) zebrafish HCC showed Wnt reporter expression, which was mostly heterogeneous, involving less than 10% of cells in 60% of HCCs and greater than 10% of cells in 10% of HCCs . As we reported previously, all non-transgenic non-HCC zebrafish livers exhibited only membrane localization of β-catenin (Tg(fabp10a:pt-β-cat) zebrafish showed at least some cytoplasmic β-catenin staining  (Tg(fabp10a:pt-β-cat)] or in a subset of hepatocytes (CreLox) during early development, as adults these zebrafish develop HCC showing a similar heterogeneous pattern of β-catenin localization. Together, these experiments show that β-catenin-driven transcriptional activity is heterogeneous in zebrafish β-catenin-driven HCC, implying that the heterogeneous nuclear and cytoplasmic β-catenin staining observed in these tumors is not due to limitations in detection.We noted similar findings in HCC from CreLox zebrafish: 50% of zebrafish showed cytoplasmic β-catenin in less than 10% of hepatocytes, 25% showed cytoplasmic β-catenin in 10–50% of hepatocytes, and 25% showed cytoplasmic β-catenin in greater than 50% of hepatocytes D,F. All ct test) D,F. ThesTg(fabp10a:pt-β-cat) zebrafish  of differentially expressed genes revealed that cancer was the most significantly affected ‘Diseases and Disorders’ in CreLox zebrafish HCC (Table S2), indicating cancer-related pathways are significantly altered in this model. We found that 1752 (88%) of dysregulated transcripts in CreLox zebrafish with HCC were not dysregulated in CreLox zebrafish without HCC (Tables S3–S5). These data indicate genes and pathways that are HCC-specific and may be important in driving hepatocarcinogenesis.We identified 1984 significantly dysregulated genes in CreLox zebrafish with HCC and 288 significantly dysregulated genes in CreLox zebrafish without HCC compared to sibling controls [Tg(fabp10a:pt-β-cat) model] are transcriptionally similar. We performed RNA-seq on adult male Tg(fabp10a:pt-β-cat) zebrafish HCC and non-transgenic sibling controls and identified 6717 significantly dysregulated genes (Tg(fabp10a:pt-β-cat) HCC  (Tables S6–S8) (We next tested the hypothesis that HCCs initiated by switching on activated β-catenin in a subset of hepatocytes (CreLox model) or ubiquitously in hepatocytes  of hepatocytes from zebrafish β-catenin-driven HCC had detectable expression of two or more of the Wnt/β-catenin target genes axin2, mtor, glula, myca and wif1 (Table S16), and 0.8% (CreLox HCC) to 2.2% [Tg(fabp10a:pt-β-cat) HCC] of hepatocytes had detectable expression of three or more of these genes. One Wnt/β-catenin target, jun, was widely expressed in non-HCC hepatocytes (52.6%), suggesting that the presence of jun mRNA is not indicative of aberrant Wnt/β-catenin signaling (Table S17). The level of jun expression in non-HCC hepatocytes expressing jun was significantly lower than in jun-expressing Tg(fabp10a:pt-β-cat) HCC and CreLox HCC hepatocytes  (Table S17). This analysis of Wnt/β-catenin target gene expression at the single-cell level indicates focal activation of Wnt/β-catenin signaling in established tumors, supporting the hypothesis that β-catenin-driven transcriptional activity is heterogeneous in zebrafish β-catenin-driven HCC.We examined the level and distribution of selected Wnt/β-catenin target genes across hepatocyte and non-hepatocyte clusters, focusing on genes that were significantly upregulated in male zebrafish HCC from our bulk RNA-seq analysis  . We obsea, myca) C, indicaTg(fabp10a:CreERT2); Tg(fabp10a:flox-pt-β-cat), CreLox], in which activated β-catenin is switched on using tamoxifen-inducible CreERT2 recombinase under control of the hepatocyte-specific fabp10a promoter. This tool enables switching on activated β-catenin in a subset (∼9%) of larval hepatocytes or in the majority of larval hepatocytes with 4-hydroxytamoxifen (TAM) treatment. Switching rates were estimated using the Tg(ubi:switch) reporter line to visualize both pre- and post-switch states with fluorescent proteins. We discovered that switching on activated β-catenin in the majority of hepatocytes by TAM administration from 3 to 6 dpf was sufficient to produce HCC in 28% of zebrafish by 6 mpf. Switching on activated β-catenin in a subset of larval hepatocytes led to similar HCC penetrance, indicating that activated β-catenin in a small number of developing hepatocytes is sufficient to initiate HCC and suggesting that the critical time window for turning on activated β-catenin occurs before 3 dpf.Here we report generation of a zebrafish model of β-catenin-driven HCC   (T2),Tg(fabp10a: CreER and Tg(fabp10a:flox-pt-β-cat). Embryos and larvae were cultured in egg water (2.33 g Instant Ocean in 1 l Milli-Q water with 0.5 ml Methylene Blue) and stored in a 28.5°C incubator. Adult zebrafish were housed on a recirculating system and fed brine shrimp, flakes, and powdered food. Animals were euthanized by tricaine methanesulfonate (0.02%) and/or ice water immersion (rapid chilling).Zebrafish (idelines . In addit-β-cat) , Tg(7xTCmCherry) , Tg(ubi:switch)] , Tg(fabp10a:Cre) , Tg with tamoxifen-inducible T2CreER (T2CreER insert (TP1:CreERT2-Tol2-delta (a kind gift from Nikolay Ninov of the Center for Regenerative Therapies TU Dresden), adding NheI and NotI restriction enzyme sites at the 5′ and 3′ ends, respectively, and placed downstream of the fabp10a promoter  by replacing the insulin promoter and diphtheria toxin portions with the fabp10a promoter Tg founder [hereafter referred to as Tg(fabp10a:flox-pt-β-cat)]. Transgenic descendants of T2)Tg(fabp10a:CreER and Tg(fabp10a:flox-pt-β-cat) founders were differentiated from control siblings at 3 dpf or later based on cryaa:Venus and cryaa:mCherry expression, respectively. For experiments where it was not possible to distinguish cryaa:Venus expression due to the presence of Ubi:GFP, T2)Tg.One-cell-stage embryos were microinjected with escribed . InjecteFor genotyping of larvae or adult fish, genomic DNA was extracted by immersing whole larvae or fin-clip in 100 μl of genomic extraction buffer with 25 μl Proteinase K per ml of genomic extraction buffer. Samples were cycled as follows: 55°C 120′, 95°C 10′ and 10°C hold. 1 μl of this liquid was used in a 12 μl total volume PCR reaction. For amplifying CreER, 5′-AACGAGTGATGAGGTTCGCA–3′ forward primer and 5′–TGATCCTGGCAATTTCGGCT–3′ reverse primer were used. Temperature cycles were as follows: 95°C for 2′; 40 cycles of 95°C for 30″, 54°C for 30″, 72°C for 2′30″; 72°C for 5′; and 10°C hold. PCR samples were loaded into a 3% agarose gel in PBS and a band size of 488 bp was observed to establish Cre positivity. For Cre genotyping from frozen or paraffin-embedded sections, we used zebrafish material from the embedded blocks as input for genomic DNA extraction.At 3 dpf, hatched larvae were anesthetized in tricaine methanesulfonate for fluorescence sorting based on fluorophore expression in the eyes. Sorted larvae were distributed into nine- or six-well culture plates in 3 or 4 ml solution of egg water with either 10 µM 4-hydroxytamoxifen  and 0.1% ethanol or 0.1% ethanol alone from 3 to 6 dpf. A maximum of 5 larvae/ml solution were treated. Untreated larvae were kept in egg water only with no additions.Adult zebrafish (3–4 mpf) were immersed in system water alone; system water plus 0.1% ethanol; or system water plus 0.1% ethanol and 2.3 µM TAM overnight for 3 consecutive nights. During the day, fish were fed and exposed to normal water flow. The fish were euthanized by rapid freezing either 1 week post treatment for assessing switching or 6 months post treatment for assessing HCC induction.Tg(fabp10a:flox-pt-β-cat) zebrafish, larvae were blocked for at least 1 h with PBS+4% BSA+0.3% Triton X-100 (PBT), and then incubated with chick anti-GFP primary antibody  for at least 12 h followed by AlexaFluor goat anti-chick 488 secondary antibody  for at least 12 additional hours. Larvae for all confocal imaging experiments were mounted in 1% low-melt agarose plus SlowFade Diamond Antifade Mountant (Invitrogen) and cover-slipped. All samples in each experiment were imaged on the same day on an Olympus confocal microscope using the same parameters for all zebrafish.Larvae were euthanized, rinsed in PBS, and then fixed in 4% paraformaldehyde (PFA) in PBS for at least 12 h at 4°C. Post-fixation, larvae were rinsed in PBS and dissected under a Leica dissecting microscope using forceps to reveal the liver. For assessing BFP expression in Tg(fabp10a:flox-pt-β-cat); Tg(7xTCF-Xla.Siam:nlsmCherry) larvae was done on the same zebrafish. The second z-slice from the ventral surface of the liver  was selected for quantification. Image files were blinded and randomized. To quantify BFP expression (anti-GFP antibody recognizes BFP), the mean fluorescence in each liver was determined using FIJI/ImageJ (Quantification of BFP expression and Wnt reporter activity in 5-dpf I/ImageJ . To quanTg(ubi:switch) 6-dpf larvae, whole-mount imaging was performed twice (two different clutches of zebrafish), with similar results each time; representative images from one experiment are shown while data analysis was performed on the pooled data set of the two experiments.For assessing switching in Zebrafish at 10 or 20 dpf were fixed in 4% PFA for at least 12 h at 4°C, rinsed in PBS and then decalcified in 0.5 M pH 8 Ethylene Diamine Tetra Acetic Acid (EDTA) overnight at room temperature. Adult zebrafish (3–4 mpf) were euthanized 1 week post TAM treatment. Their livers and intestines were dissected from the body cavity and fixed in 4% PFA for at least 12 h at 4°C. Whole zebrafish larvae (10 or 20 dpf) and adult liver/intestines were immersed in 30% sucrose in PBS at 4°C, allowed to equilibrate, and cryosectioned into 5 μm-thick sections. On the day of imaging, frozen sections were air-dried for 30 min to 1 h; Slowfade Diamond Antifade Mountant was applied followed by cover-slipping and imaging on an Olympus IX81 Confocal microscope, using Olympus Fluoview version 4.1 software.For quantification, the images were blinded using a randomization software and analyzed with ImageJ software. Hepatocytes were assigned and counted as either mCherry positive (switched) or EGFP positive (unswitched). Switching was assessed as the percentage of mCherry positive hepatocytes over total number of hepatocytes (mCherry positive plus EGFP positive). Genotyping for CreER was performed as described above using residual fish from the frozen block.The 10-dpf experiment was performed three times (three different clutches of zebrafish), and data from three experiments were pooled. The 20-dpf experiment was performed once. The adult experiment was performed two times (two different clutches of zebrafish), and data from two experiments were pooled.T2); Tg(fabp10a:flox-pt-β-cat), Tg(fabp10a: CreERT2)Tg(fabp10a: CreER and Tg(fabp10a:flox-pt-β-cat) zebrafish were treated with 10 µM TAM at 3 dpf or 2.3 µM TAM at 3 mpf and housed under standard conditions for 6 months post switching . Tg(fabp10a:pt-β-cat) and non-transgenic control siblings were raised in parallel.Fish were euthanized and weighed. Livers were dissected using a Leica dissecting microscope and weighed. These livers were fixed using 4% paraformaldehyde (PFA) in PBS for maximum 48 h at 4°C followed by immersion in 70% ethanol in embedding cassettes for paraffin embedding, sectioning (5 μm-thick sections) and H&E staining.H&E-stained slides were blinded and scored by a board-certified pathologist (K.J.E.) based on established criteria for zebrafish HCC . SamplesT2); Tg(fabp10a:flox-pt-β-cat), Tg(fabp10a: CreERT2)Tg(fabp10a: CreER and Tg(fabp10a:flox-pt-β-cat) zebrafish. It was performed twice (two clutches of zebrafish) for Tg(fabp10a:pt-β-cat) and non-transgenic control siblings. After each experiment, H&E slides were blinded and scored in a preliminary round. After all experiments were complete, H&E slides from all experiments were blinded, randomized, and scored again. Any slides with a substantial difference in score  were blinded again and scored a third, definitive time.This experiment was performed five times (five clutches of zebrafish) for Tg(fabp10a:pt-β-cat) and Tg(7xTCF-Xla.Siam: mCherry) zebrafish were raised to 6 mpf. The fish were weighed and their livers dissected for calculating liver-to-body mass ratios and cryosectioned for immunofluorescence imaging of antibody-stained β-catenin and reporter mCherry presence. Cryosections were air-dried for 30 min in the dark and washed in PBS, stained with mouse anti-β-catenin antibody  (1:200 in 4% BSA-PBS) overnight at 4°C, washed five times with PBS, stained with AlexaFluor donkey anti-mouse 647 secondary  at room temperature, and stained for 3 min with 5 µg/ml DAPI solution. Slides were then washed with PBS and mounted with SlowFade Diamond Antifade, cover-slipped, and imaged on an Olympus IX81 Confocal microscope and Olympus Fluoview version 4.1 software using a 405 nm laser for nuclear DAPI, 488 nm laser for β-catenin and 568 nm laser for the mCherry Wnt reporter. One field of view and z-slice was quantified per liver. Images in TIF format were analyzed using ImageJ after blinding image identity. Wnt reporter expression was scored manually as: (1) absent (no mCherry expression); (2) low (mCherry expression in less than 10% of cells); (3) high (mCherry expression in greater than 10% of cells). In terms of β-catenin localization, the samples were scored using the following matrix: 0, membrane only, wherein staining of membrane is distinct on all hepatocytes and much brighter than cytoplasm; 1+, focal (less than 10% of hepatic parenchyma) weak to moderate staining of hepatocyte cytoplasm with loss of distinct membrane staining; 2+, focal (less than 10% of hepatic parenchyma) strong cytoplasmic staining or patchy (10–50% of hepatic parenchyma) weak to moderate cytoplasmic staining; 3+, diffuse (more than 50% of hepatic parenchyma) staining of hepatocyte cytoplasm with loss of distinct membrane staining.For quantifying Wnt reporter activity and β-catenin localization in adult zebrafish, progeny from a cross of Tg(fabp10a:pt-β-cat) zebrafish and non-transgenic control siblings were only examined for β-catenin localization and not Wnt reporter activity. In these cases, immunofluorescence staining for β-catenin was performed on paraffin-embedded liver samples from one of the experiments that was used to assess HCC incidence in 6 mpf zebrafish. Paraffin-embedded sections were de-paraffinized by serial-immersion in three containers of Xylene for 5 min each, rehydrated by serial-immersion in two containers of 100% EtOH each, followed by 95% EtOH and 70% EtOH treatments, for 2 min each, ending with a 5 min ddH2O wash. Antigen retrieval was performed by treating the slides in a microwave oven (100% power setting) for 10 min in 10 mM citrate buffer pH 6.1. Before staining, sections were washed for 5 min in ddH2O. Pap pen was used to encircle the liver tissue fragment and blocking was done using PBT. Slides were stained for β-catenin using mouse anti-β-catenin antibody (1:200 in PBT) overnight at 4°C, washed four times with PBS+0.3% Triton X-100 (PT) for 15 min each, and stained with AlexaFluor donkey anti-mouse 647 secondary  overnight at 4°C. Slides were then washed four times with PT and allowed to dry completely for 30–60 min on benchtop. They were then mounted with SlowFade Diamond Antifade, cover-slipped, and imaged on an Olympus IX81 Confocal microscope and Olympus Fluoview version 4.1 software using a 647 nm laser for β-catenin. Two to five fields of view were taken per fish and all of them were analyzed using one z-slice each per field of view. Images in TIF format were blinded, analyzed and scored for β-catenin localization using the matrix described above. In cases where the diagnosis was different for multiple field of views from the same fish, the majority diagnosis was taken as final diagnosis for that sample.A subset of Tg(fabp10a:CreERT2); Tg(fabp10a:flox-pt-β-cat) zebrafish treated with TAM from 3–6 dpf (CreLox), with HCC (n=3); Tg(fabp10a:CreERT2); Tg(fabp10a:flox-pt-β-cat) zebrafish treated with TAM from 3–6 dpf (CreLox), without HCC (n=2); and Tg(fabp10a:CreERT2) zebrafish treated with ethanol from 3–6 dpf (sibling controls), without HCC (n=4). All these fish were male, and were euthanized at 11 mpf by rapid chilling. Their livers were dissected for assessment of liver-to-body mass ratios, and half of each liver was submitted for histologic analysis as described above. The other half of each dissected liver was flash-frozen on dry ice and stored in −80°C prior to RNA extraction. RNA was isolated using TriReagent  and Direct-Zol RNA Mini prep kit  including DNase treatment following the manufacturer's protocols. Samples were eluted in RNase-/DNase-free water. Sample quality control, library preparation, sequencing and alignments were performed by the Huntsman Cancer Institute (HCI) High Throughput Genomics and Bioinformatic Analysis Shared Resource. Paired-read, 150 base pair sequencing was performed using Illumina's NovaSeq instrument. Three or four pooled libraries were run on each lane of the flow cell.Zebrafish of the following genotypes/treatment groups were used as tissue source: Tg(fabp10a:pt-β-cat) (HepABC) zebrafish with HCC (n=5) and their non-transgenic siblings without HCC (n=5). Three to four pooled libraries were run on each lane of the flow cell.We performed a similar RNA sequencing experiment using livers isolated and prepared as described above from 6-mpf Tg(fabp10a:pt-β-cat); Tg(7xTCF-Xla.Siam: mCherry) [HCC(HepABC)], Tg zebrafish treated with TAM from 3–6 dpf [HCC(CreLox)], and Tg(fabp10a:flox-pt-β-cat) treated with TAM from 3-6 dpf (no HCC). Zebrafish were euthanized by rapid chilling and their livers were dissected. Half of each liver was submitted for histologic evaluation to confirm the diagnosis (HCC or no HCC). The remaining half of each liver was dissociated into single-cell suspensions and prepared for single-tube single-cell RNA sequencing (scRNA-seq) based on the 10× Genomics platform. Dissected liver tissue was immersed in 5% Fetal Bovine Serum (FBS) in Hank's Buffered Saline Solution (HBSS) without Phenol Red, with calcium and magnesium and chopped finely. The cells were homogenized in 1 ml of 0.25% trypsin+EDTA for 5 min at room temperature and re-suspended in 1 ml of 5%FBS+5 mM EDTA in HBSS-Free solution. Cells were then filtered through a 40 μm membrane filter, and the filtrate was centrifuged at 1200 RPM, 4°C for 5 min. The cell pellet was then re-suspended in phosphate buffered saline with 0.04% bovine serum albumin. The cell suspension was then filtered again through 40 μm cell strainers to obtain a liver single-cell suspension.The following 6-mpf male zebrafish were used for this experiment: Samples were submitted to the HCI High-Throughput Genomics Shared Resource, where viability and cell count were assessed on Countess I (Thermo Fisher Scientific). The Chromium Single Cell Gene Expression Solution with 3′ chemistry, version 2 (PN-120237) was used to barcode individual cells with 16 bp 10× Barcode and to tag cell specific transcript molecules with 10 bp Unique Molecular Identifier (UMI) according to the manufacturer’s instructions. Equilibrium to targeted cell recovery of 6000 cells along with 10× Gel Beads and reverse transcription reagents were loaded to Chromium Single Cell A Chip (PN-120236) to form Gel-Bead-In EMulsions (GEMs), the micro-droplets. Within individual GEMs, cDNA generated from captured and barcoded mRNA was synthesized by reverse transcription at the setting of 53°C for 45 min followed by 85°C for 5 min. Subsequent A tailing, end repair, adaptor ligation and sample indexing was performed in bulk according to the manufacturer's instructions. The resulting barcoding libraries were qualified on Agilent D1000 ScreenTape on Agilent Technology 2200 TapeStation system and quantified by quantification PCR using KAPA Biosystems Library Quantification Kit for Illumine Platforms (KK4842). Multiple libraries were then normalized and sequenced on NovaSeq 6000 with 2×150 PE mode.https://support.10xgenomics.com/single-cell-gene-expression.All protocols to generate scRNA-seq data on 10× Chromium platform including library prep, instrument and sequencing setting can be found on: https://github.com/smkalasekar/Heterogeneous-beta-catenin-activation-is-sufficient-to-cause-hepatocellular-carcinoma-in-zebrafish).For the CreLox and corresponding control zebrafish, optical duplicates were filtered out with BBMap's clumpify utility (v38.34). Sequence adapters were removed with cutadapt (v1.16). Raw FASTQ data was aligned with STAR (v2.6.1b) to the zebrafish genome (build GRCz11) with splice junctions annotated from Ensembl release 94. Gene expression was quantified with featureCounts (v1.6.3). Differential gene expression analysis was performed with DESeq2 (v1.22.2). Pipeline specifics and code are documented on GitHub .For the HepABC and corresponding control zebrafish, sequence adapters were removed with cutadapt (v1.16). Raw FASTQ data was aligned with STAR (v2.6.1b) to the zebrafish genome (build GRCz11) with splice junctions annotated from Ensembl release 94. Gene expression was quantified with featureCounts (v1.6.3). Differential gene expression analysis was performed with DESeq2 (v1.20.0). Pipeline specifics and code are documented on GitHub  CreLox(HCC)=CreLox with HCC versus control siblings (Cre driver only) without HCC; (2) NoHCC=CreLox without HCC versus control siblings (Cre driver only) without HCC; and (3) HepABC(HCC)=HepABC with HCC versus non-transgenic siblings without HCC.P<0.05) altered gene expression as inputs. Canonical Pathways Analysis was used to identify the pathways from the IPA library of Canonical Pathways that were most significant to the data set. Functional Analysis was used to identify the biological functions and/or diseases that were most significant to the data set and were associated with biological functions and/or diseases in Ingenuity's Knowledge Base. IPA calculates P-values using Fisher's exact test. For Table S2, all three datasets were fed into the Comparison Analysis feature of IPA. Heatmaps from the comparison analyses were used to identify pathways commonly dysregulated in three samples. The P-values were derived from each individual dataset's Summary or Functional Analysis outputs generated by the IPA software.Data from the aforementioned comparisons were analyzed through the use of Ingenuity Pathway Analysis (IPA), using the log ratios of molecules with significantly (http://eulerr.co/). P-values for the Venn overlap significance were calculated using the dhyper tool in R (https://rdrr.io/bioc/GeneOverlap/man/GeneOverlap.html). For calculations, the total number of genes in the genome was considered as 25,903 (Ensembl Zebrafish GRCz10).Proportional Venn diagrams in Danio rerio genome reference build GRCz11 with annotations from Ensembl release 94. The Ensembl gene annotations were filtered with ‘cellranger mkgtf’ for gene_biotypes matching ‘protein_coding’, ‘lincRNA’ and ‘antisense’ tags. All samples had additional transgenic sequence/annotations added: Xenopus β-catenin and EBFP sequences were added for HCC(CreLox) (X2) and no HCC(X4) samples, and Xenopus β-catenin sequence was added for the HCC(HepABC) sample (X1).10× Genomics' Cell Ranger software (v2.2.0) executed primary data analysis through a series of bioinformatics pipelines using a combination of custom and third-party tools. Pipeline ‘cellranger mkfastq’ converted raw sequencing data into de-multiplexed FASTQ files with Illumina's bcl2fastq software. Two custom transgenic genomic references were built with ‘cellranger mkref’, but shared the common P-value null distribution was confirmed to be approximately uniform.Each sample was processed with ‘cellranger count’ pipeline with their respective transgenic genome build with parameter ‘--expect-cells=3000’. Quality control (QC) reports from 10x Genomics reported warnings regarding the low fraction of reads in cells because its cell versus background algorithm assumes the cells' captured RNA content is within a single order of magnitude. However, the total UMI count versus ranked barcode plots (not shown) exhibited a more gradual sloping behavior than expected, which means the default Cell Ranger algorithm (v2.2.0) classifying cells versus background GEMs left out potential cell-like GEM partitions with much less RNA content due to either biological or technical reasons. In an attempt to recover those  GEM partitions, the raw gene-barcode matrices from ‘cellranger count’ (located in ‘outs/raw_gene_bc_matrices’) were processed with the EmptyDrops algorithm (R package DropletUtils v1.2.2) to discriminate cells from background GEM partitions at a false discovery rate (FDR) of 1% . GEM parTable S18. No cells exhibited extreme low UMI or gene count, while some sickly cells were dominated by extremely high mitochondrial expression. Many putative cells were removed because PCA analysis of several QC metrics indicated unusual characteristics. However, more rigorous filters based with more stringent UMI and gene count cutoffs were applied via custom R algorithms . While the PCA-based outlier filter is quite aggressive, the number of cells passing QC still far exceeds the default from 10× Genomics naïve classification of cells.Cell-based QC metrics were calculated with R package scater (v1.10.1) using the calculateQCMetrics function . Principhttps://github.com/smkalasekar/Heterogeneous-beta-catenin-activation-is-sufficient-to-cause-hepatocellular-carcinoma-in-zebrafish). Single-cell gene expression data is also publicly accessible on the Gene Expression Omnibus (GSE137788).Seurat V3 software preprint Following QC filters and data transformation to regress out variations driven by cells with high mitochondrial transcriptional activity, transcripts from a total of 1077 cells from no HCC control, 2112 cells from HCC(CreLox), and 2866 cells from HCC(HepABC) were used for subsequent analysis. After gene expression normalization, we performed PCA on the 2000 most highly variable genes in each sample as recommended by the Seurat V3 pipeline preprint.https://github.com/smkalasekar/Heterogeneous-beta-catenin-activation-is-sufficient-to-cause-hepatocellular-carcinoma-in-zebrafish) were used to identify the markers used to predict cluster composition of t-SNE plots derived from No HCC, HCC(CreLox), HCC(HepABC) zebrafish, and all three samples combined (Tables S19–S22), as well to identify upregulated genes within specific clusters in these plots .The graph-based t-Distributed Stochastic Neighbor Embedding (t-SNE) approach was executed for linear dimensional reduction of the chosen principal components (PCs) . In the Tables S13, S15, S17). Custom R scripts were also used to compute the percentage cluster-wise representation of cells from each sample (Table S11), percentage of cells from each sample expressing 0, 1, 2, 3 or 4 Wnt targets axin2, mtor, glula, myca and wif1 (Table S16) and jun expression in cells isolated from livers of Non-HCC, CreLox HCC, and HepABC HCC (Table S17).The DAVID Genome Browser ,b was usFor each experiment involving tumor formation in adult zebrafish, an entire clutch of zebrafish was raised, so sample size was dependent on the size of the clutch. Animals that died before the experimental endpoint were excluded from analysis; this exclusion criteria was pre-established. All other animals were included in analysis. For each larval imaging experiment, we typically included five to ten zebrafish per genotype/group, based on the presence of fluorescent marker(s). Zebrafish with livers that were disrupted or lost during processing were excluded from analysis; this exclusion criteria was pre-established.Immunofluorescence imaging was performed using an Olympus IX81 Confocal microscope with Olympus Fluoview version 4.1 software. Within each experiment, the same parameters  were maintained for all images. Images were blinded and randomized using R , and theP-values were less than 0.05. Mean values with standard deviations are reported for each experiment, unless otherwise mentioned.Data were analyzed and graphs were generated using GraphPad Prism software (version 7.04 or 8.0). To compare the extent of Cre-mediated switching among different genotypic and treatment groups and liver-to-body mass ratios an ordinary one-way ANOVA with Sidak's multiple comparisons test was performed. Alternatively, when standard deviations of groups were significantly different, non-parametric Kruskal–Wallis test followed by Dunn's multiple comparisons test was performed. To compare cytoplasmic β-catenin localization, Wnt reporter activity, and incidence of HCC and intermediate changes, Fisher's exact test was performed. Differences were considered statistically significant if


The purpose of this project was to use an in vivo method to discover riboswitches that are activated by new ligands. We employed phage-assisted continuous evolution (PACE) to evolve new riboswitches in vivo. We started with one translational riboswitch and one transcriptional riboswitch, both of which were activated by theophylline. We used xanthine as the new target ligand during positive selection followed by negative selection using theophylline. The goal was to generate very large M13 phage populations that contained unknown mutations, some of which would result in new aptamer specificity. We discovered side products of three new theophylline translational riboswitches with different levels of protein production.We used next generation sequencing to identify M13 phage that carried riboswitch mutations. We cloned and characterized the most abundant riboswitch mutants and discovered three variants that produce different levels of translational output while retaining their theophylline specificity. Although we were unable to demonstrate evolution of new riboswitch ligand specificity using PACE, we recommend careful design of recombinant M13 phage to avoid evolution of “cheaters” that short circuit the intended selection pressure.The online version of this article (10.1186/s13104-018-3965-6) contains supplementary material, which is available to authorized users. An important goal of microbial metabolic engineering is the development of improved methods for the integration of host and orthogonal metabolism –5. Our rThe experimental design we developed was intended to be a proof-of-concept for using PACE to discover RSs with new ligand specificity Fig. . Our goaOur adaptation of PACE placed a RS between the gene three (gIII) promoter (PIII) and the T7 RNA polymerase (RNAP) gene so that the RS was the target of directed evolution . This is a detailed explanation of how to build your own chemostats for PACE experiments. This document also contains all parts numbers and suppliers so users can purchase the same materials we used.