Wnt/β-catenin signaling in the trabecular meshwork (TM) is required for maintaining normal intraocular pressure (IOP), although the mechanism(s) behind this are unknown. We hypothesize that Wnt/β-catenin signaling regulates IOP via β-catenin's effects on cadherin junctions.n = 8–10). Conscious IOPs were assessed for 35 days.Nonglaucomatous primary human TM (NTM) cells were treated with or without 100 ng/ml Wnt3a, 1 μg/ml sFRP1, or both for 4 to 48 hours. Cells were immunostained for β-catenin, total cadherins, or cadherin isoforms. Membrane proteins or whole-cell lysates were isolated for Western immunoblotting and probed for cadherin isoforms. RNA was extracted for cDNA synthesis and qPCR analysis of cadherin expression. Some NTM cells were cultured on electric plates for cell impedance assays. Ad5.CMV recombinant adenoviruses encoding K-cadherin, and/or sFRP1 were injected into eyes of 4- to 6-month-old female BALB/cJ mice , and Western immunoblotting showed that Wnt3a increased K-cadherin in NTM cells, which was inhibited by the addition of sFRP1. Cell impedance assays showed that Wnt3a treatment increased transcellular resistance and anti-K-cadherin siRNA decreased transcellular resistance . Our in vivo study showed that K-cadherin significantly decreased sFRP1-induced ocular hypertension . Western immunoblotting also showed that K-cadherin alleviated sFRP1-induced β-catenin decrease in mouse anterior segments.Upon Wnt3a treatment, total cadherin expression increased and β-catenin accumulated at the TM cell membrane and on processes formed between TM cells. qPCR showed that Wnt3a significantly increased K-cadherin expression in NTM cells (Our results suggest that cadherins play important roles in the regulation of TM homeostasis and IOP via the Wnt/β-catenin pathway. Primary open angle glaucoma (POAG) is the most common form of glaucoma, and a meta-analysis of 81 studies on POAG prevalence showed that in 2015, 57.5 million people globally were affected by POAG.2 This number is expected to rise to 65.5 million by 2020,2 with some 11.1 million people being bilaterally blind .2 POAG is a heterogeneous disease associated with multiple risk factors including ethnicity, family history, aging, and ocular hypertension (OHT).3 OHT is defined by an intraocular pressure (IOP) of 21 mm Hg or higher and is the main causative and currently the only clinically modifiable risk factor. In POAG patients with OHT, IOP increases when aqueous humor outflow resistance at the trabecular meshwork (TM) increases, and increased outflow resistance is due to pathological changes in the TM. However, there are no currently available therapeutics that directly target TM pathology, and the causes of these pathological changes to the TM are still under investigation. When compared with nonglaucomatous TM (NTM), glaucomatous TM tissues show decreased TM cellularity, increased extracellular matrix (ECM) in trabecular beams and juxtacanalicular tissue, increased TM stiffness, increased actin contractility, increased cross-linked actin networks, and dysregulation of TM cell signaling pathways including the transforming growth factor beta (TGFβ), bone morphogenetic protein, and Wnt/β-catenin signaling pathways.47 Many studies showed that increased expression of the growth factors including TGFβ2, gremlin, and secreted frizzled-related protein 1 (sFRP1) can cause dysregulation of these homeostatic pathways in the glaucomatous TM.815Glaucoma is a blinding disease characterized by optic nerve degeneration. Glaucoma is age-associated, and a study from the 2005–2008 National Health and Nutrition Examination Survey (NHANES) cohort shows that 2.91 million Americans over the age of 40 years suffer this disease.17 In the absence of Wnt ligands, the intracellular “Wnt/β-catenin inhibitory complex” made up of glycogen synthase kinase 3 beta (GSK-3β), Axin, APC, and casein kinase 1 (CK1) proteins tag the Wnt/β-catenin signaling mediator β-catenin for destruction through phosphorylation, ubiquitination, and proteasomal degradation. Therefore, β-catenin will be constantly degraded in the cell and will cease to translocate into the nucleus and will be unable to regulate transcription via binding at the T-cell factor/lymphoid enhancer factor promoter.18 Our previous studies showed that the expression of sFRP1 is increased in glaucomatous TM cells.10 We have shown that a number of Wnt pathway genes are expressed in the human TM (HTM), and the Wnt ligand Wnt3a activates the Wnt/β-catenin signaling pathway.19 Overexpression of sFRP1 or Dkk1  leads to increased IOP in perfusion-cultured human eyes and in mouse eyes.19 We also found that cotreatment with a small-molecule Wnt pathway activator inhibits sFRP1-induced OHT in mouse eyes. Indirect activation of Wnt/β-catenin signaling in the TM using lithium chloride (LiCl) was reported by the Rhee group to decrease the production of some ECM and matricellular proteins as well as inhibit the TGFβ pathway and TGFβ2-induced ECM production.20 Sun et al.21 showed that intraperitoneal administration of LiCl decreased IOP in a rat OHT model. However, this has been the only report showing the ocular hypotensive effect of LiCl and further studies are needed to determine the mechanism of LiCl; that is, whether LiCl functions via the Wnt/β-catenin signaling pathway and/or other signaling pathways.21 We have shown that the Wnt/β-catenin and TGFβ/Smad pathways cross-inhibit each other in NTM cells, and this cross-inhibition relies on the key pathway transcription factors β-catenin and Smad4.22 However, the mechanism by which Wnt/β-catenin signaling regulates IOP remains unclear.sFRP1 is an antagonist of the Wnt signaling pathway that sequesters extracellular Wnt ligands, thereby blocking their binding to frizzled receptors.23 The classical cytosolic domain of cadherins binds directly to β-catenin, among other accessory proteins such as α-catenin and p120-catenin.24 Cadherins are connected to and stabilized by the actin cytoskeleton through these catenin accessory proteins, and β-catenin is essential for the cadherin-actin complex. Phosphorylation of β-catenin at different sites and the localization of β-catenin determine whether β-catenin remains bound to cadherins, and the lack of β-catenin-cadherin binding disrupts the cadherin-β-catenin-actin complex.26 Because β-catenin plays major roles in both Wnt/β-catenin signaling and adherens junctions, it is likely that Wnt/β-catenin signaling regulates the anchoring of cadherins junctions to the actin cytoskeleton by regulating membrane associated β-catenin. Currently, the connection between Wnt/β-catenin signaling and cadherins junctions has not been explored in the TM.β-catenin is the key transcription factor for Wnt/β-catenin signaling, but it has another function in the cell as an accessory protein for classical cadherin junctions. Cadherins are transmembrane proteins whose extracellular domains bind homophilically to their counterparts on other cells in a calcium-dependent manner.2730 Many isoforms of cadherin proteins exist in multiple species including humans, and their expression and distribution are highly tissue specific. Different cadherin isoforms convey different physiological properties to cells. Wecker et al.31 showed that in human TM cells, N-cadherin and OB-cadherin are up-regulated by TGFβ2. However, the expression of the other cadherin isoforms and the effect of Wnt signaling on cadherins in the TM are still unclear. In addition, the role of cadherins in IOP regulation is unknown.Cadherins play key roles in cell-cell adhesion, directing tissue patterning and forming neural connections during development, epithelial to mesenchymal transition in cancers and wound healing, thereby serving as mechanosensors that relay mechanical signals from the cells' environment to the actin cytoskeleton to facilitate a tensile or migratory response.In this study, we determined the expression and localization of a subset of cadherins in primary NTM cells and the regulation of these cadherins by Wnt/β-catenin signaling. We also studied the role of cadherins and Wnt signaling on cell adhesion using real-time cell analysis (RTCA) impedance assays, as well as the role of cadherins in sFRP1-induced OHT in mice. We hypothesize that Wnt/β-catenin signaling activation increases the expression of cadherins in TM cells, contributing to TM cell adhesion, and that the inhibition of Wnt/β-catenin signaling compromises cadherin expression as well as TM cell adhesion and mechanotransduction, thereby inducing OHT.33 The cell strains used in these experiments were derived from human donor eyes and included: NTM895-03, female, age 63 years; NTM176-04, male, age 72 years; NTM875-03, male, age 77 years; NTM496-05, female, age 82 years; NTM1022-02, male, age 67 years; NTM444-05, male, age 85 years.Primary NTM cells were characterized and donated by Alcon as described previously.NTM cells were cultured on glass coverslips in DMEM-low glucose medium supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin and streptomycin . When cells were confluent, culture medium was changed to serum-free medium, and the cells were cultured for an additional 24 hours. The cells were then treated with or without 100 ng/ml recombinant Wnt3a and/or 1 μg/ml recombinant sFRP1  for 24 hours. At the end of treatment, cells were fixed with 4% paraformaldehyde at 4 °C, treated with 0.05% Triton X-100, blocked with Superblock (Thermo Fisher Scientific) and immunostained with rabbit anti-β-catenin , rabbit anti-K-cadherin  or rabbit anti-Pan-cadherin antibodies . After incubation with a secondary donkey anti-rabbit antibody conjugated with Alexafluor 488 , some cells were stained with with Phalloidin conjugated with Alexafluor 594  for 1 hour. After staining, glass coverslips were mounted on slides using the ProLong gold antifade reagent with 4′,6-diamidino-2-phenylindole . All images were captured using a Nikon Eclipse Ti-U Epifluorescent microscope  in combination with a Nuance imaging system .NTM cells were cultured in 12-well plastic dishes until they were confluent. They were serum-starved for 24 hours, then treated for an additional 24 hours with or without 100 ng/ml Wnt3a and/or 1 μg/ml sFRP1. Triplicates were used for each treatment. After treatment, total RNA was isolated using the Qiagen RNeasy Mini Kit . RNA was reverse transcribed into cDNA using iScript Reverse Transcription Supermix , and qPCR was performed using the SsoAdvance Universal SYBR Green Supermix (Bio-Rad) in a CFX96 thermocycler (Bio-Rad). The thermoprofile consisting of 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds, followed by a temperature dissociation curve. GAPDH primers were used as an internal control. All the primers used were designed to span or flank exon-exon junctions. The primer sequences  are as follows:Axin2:Forward: CAGATCCGAGAGGATGAAGAGA; reverse: AGTATCGTCTGCGGGTCTTCK-cadherin:Forward: GGCAGATCAGTTGATTCAGA; reverse: GCCGTGTTGTCTTTGTTGTCOB-cadherin:Forward: CAAAGTTTCCGCAGAGCGTA; reverse: GCTTTATCACCCCCTCCTGTN-cadherin:Forward: ATCCTGCTTATCCTTGTGCTG; reverse: CCTGGTCTTCTTCTCCTCCA34:GAPDHForward: GGTGAAGGTCGGAGTCAAC; reverse: CCATGGGTGGAATCATATTGCadherin-19 A:Forward: TGCAGGCTCTGGTCAGGTA; reverse: GAAGACAGGTTCTTCTTGAAGGTTCadherin-19 B:Forward: AGCACAAGCGTCTGTAACTCTG; reverse: GGAAACAGGACGTCACTAACAA.NTM cells were cultured in 60-mm dishes until they were confluent. The cells were serum-starved for 24 hours and treated with 100 ng/ml Wnt3a and/or 1 μg/ml sFRP1 for an additional 4, 24, or 48 hours. After treatment, the membrane protein fraction was isolated using the Eukaryotic Membrane Extraction Kit (Thermo Fisher Scientific), while total protein was isolated using the M-PER Mammalian Extraction Reagent (Thermo Fisher Scientific). Equal amounts of protein from each sample were separated using SDS-PAGE, transferred onto an Immobilon-P transfer membranes, blocked with 5% dry milk, and immunoblotted overnight at 4°C with rabbit anti-K-cadherin antibody , rabbit anti-OB-cadherin antibody , rabbit anti-β-catenin antibody , rabbit anti-GAPDH antibody , or mouse anti-Lamin A/C antibody . The blots were then washed and incubated in secondary horse radish peroxidase (HRP)-linked anti-rabbit or anti-mouse IgG antibodies . Signals were developed using the Clarity Western ECL Blotting substrate (Bio-Rad). Images were taken using the Bio-Rad ChemiDoc imaging system (Bio-Rad). For some blots, densitometry was performed using ImageJ .NTM cells were grown to 80% confluency in 8- or 12-well Acea electric plates containing copper electrodes. The transfection mix contained 1nM anti-K-cadherin, anti-OB-cadherin, or nontargeting siRNAs  along with 0.83 μl Attractene (Qiagen) per well for Acea plates and 5 μl Attractene per well for regular 12-well plates. Attractene and siRNA were incubated together in a transfection mix in OptiMEM (Thermo Fisher Scientific) for 20 minutes. Trypsinized NTM cells were resuspended in OptiMEM containing 5% FBS and 1% glutamine at a density of 2 × 105/ml and 200 uL of transfection mix was added. Eighteen to 24 hours after transfection, culture medium was changed to Dulbecco's modified Eagle medium (DMEM) low glucose containing 10% FBS, 1% glutamine, and 1% penicillin/streptomycin. NTM cells in 12-well plates were harvested 72 hours after treatment for WB. NTM cells cultured in 8-well Acea plates were placed on the Acea iCelligence system for continuous cell impedance measurements.35 Briefly, this assay measures impedance in real time of live cells cultured in specialized copper electrode-coated plates. The cell index (CI) is used to represent the maximum change in cell impedance during a specific time frame.Rcell(if) is the measured electric resistance/impedance at a certain time point in wells with cells.The RTCA iCELLigence system  was used for NTM cell impedance (electric resistance) measurements. This system has been described previously.Rb(if) is the measured electric resistance/impedance at a certain time point in wells without cells.CI was calculated every hour. “Normalized CI” represented CI values compared to a CI value chosen by the researcher and that CI was set at 1. In these experiments, we chose to normalize the CI to a time point within 2 hours before treatment, which was indicated by the black bar in the iCelligence software screenshot .n = 4–6). Baseline CI values were collected every hour for at least 48 hours. NTM cells were then either treated with recombinant proteins or transfected with siRNA, and CI values were collected every hour for 72 to 96 additional hours. Recombinant protein treatment regime included control, 100 ng/ml Wnt3a, 1 μg/ml sFRP1, or both. The averaged maximum and minimum CI values for each treatment group during this 72-hour time period were presented. For transfection experiments, NTM cells were transfected with anti-K-cadherin siRNA (n = 6), anti-OB-cadherin siRNA (n = 6), or nontargeting siRNA (n = 4), and CI values were collected every hour for 96 hours. The averaged maximum and minimum CI values during the last 72 hours of this time period were presented because siRNA treatment typically takes 24 hours to knockdown expression of the targeted mRNA.NTM cells were seeded on Acea copper electrode-coated plates in replicates (Adenovirus serotype 5 (Ad5) vectors that overexpress the human K-cadherin and mCherry (Ad5.K-cadherin), mouse sFRP1 (Ad5.sFRP1), as well as a null vector (Ad5.Null) were obtained commercially from Vector Labs . The expression of each exogenous gene was driven by its own cytomegalovirus (CMV) promoter, including mCherry in the Ad5.K-cadherin vector.2 [0.8 L/min]). After anesthesia, equal numbers of infectious units (IFU) were injected into the vitreous chamber of left eyes, 3 × 107 infectious units in 1 to 5 μl were slowly injected during a period of 1 to 2 minutes using a glass microsyringe  fitted with a 33-G needle. The uninjected right eyes served as paired controls.All mouse studies were conducted in compliance with the University of North Texas Health Science Center Institutional Animal Care and Use Committee and the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. Female BALB/cJ mice were purchased from The Jackson Laboratory . Mice aged 4 to 6 months were used for intravitreal adenoviral injection. Prior to use, all animals' eyes were examined using a hand-held ophthalmoscope  to confirm a normal appearance. Immediately before viral injection, mice were anesthetized with a cocktail of ketamine/xylazine  administered intraperitoneally. Some of the mice were instead anesthetized using inhalation anesthesia  + Ad5.sFRP1 (1.5 × 107 IFU); Ad5.K-cadherin (1.5 × 107 IFU) + Ad5.Null (1.5 × 107 IFU); Ad5.sFRP1 (1.5 × 107 IFU) + Ad5.Null (1.5 × 107 IFU); and Ad5.Null (3 × 107 IFU).The treatment groups were as follows with each group composed of 8 to 10 mice: Ad5.K-cadherin . Baseline IOP was measured before viral injection, and IOP post–viral injection was monitored on indicated days. At days 9 and 21, two mice from the Ad5.K-cadherin, Ad5.sFRP1, and Ad5.K-cadherin + Ad5.sFRP1 groups were sacrificed for subsequent analyses.The mice were euthanized using carbon dioxide. Immediately afterward, the mouse eyes were enucleated and placed in ice-cold PBS.The anterior segments of some eyes were dissected using a dissection microscope by first cutting around the equator of the eye and removing the posterior segment. The lens, ciliary body, and iris were then removed from the anterior segment, leaving only the cornea and the tissues of the conventional outflow pathway. This tissue, which we refer to as the anterior segment, was placed in the T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific) with 1:100 protease inhibitors. Protein was extracted using the Tissuelyser (Qiagen). An equal amount of protein from each sample was separated using SDS-PAGE, transferred onto an Immobilon-P transfer membranes, blocked with 5% dry milk, and immunoblotted with anti-mCherry , anti-β-catenin , or anti-GAPDH  antibodies, overnight at 4°C. The membranes were rinsed and then incubated in secondary HRP-linked antirabbit or antimouse IgG antibody . Signals were developed using the Clarity Western ECL Blotting substrate (Bio-Rad). Images were taken using the Bio-Rad ChemiDoc imaging system (Bio-Rad).The other eyes were fixed with 4% paraformaldehyde at 4°C for 2 hours. Fixed eyes were washed with PBS, dehydrated, embedded in paraffin, sectioned, and mounted on glass slides. Some sections were subjected to hematoxylin and eosin staining. The other sections were immunostained for sFRP1, mCherry, and β-catenin. These tissue sections were baked at 60°C for 30 minutes, dewaxed, and rehydrated through a series of incubations with Xylene, 100% ethanol, 95% ethanol, 50% ethanol, and water. Tris-EDTA buffer (pH 9.0) was used for antigen retrieval together with the 2100 Retriever . After antigen retrieval, the sections were treated with 0.5% Triton X-100, blocked with Superblock Blocking Buffer (Thermo Fisher Scientific), and incubated with 1:100 rabbit anti-sFRP1 antibody  overnight at 4°C. The sections were then incubated with the donkey antirabbit secondary antibody conjugated with Alexafluor 488  at room temperature for 2 hours, and mounted using the ProLong gold antifade reagent with DAPI (Life Technologies). Images were taken using the Nikon Eclipse Ti-U Epifluorescent microscope  and the Nuance imaging system .P values less than 0.05 were considered significant.One-way ANOVA was used for analysis, and 19 and harvested for ICF to study the distribution of β-catenin  that recognizes all isoforms of cadherins , so the increase of K-cadherin production may be a secondary effect of Wnt/β-catenin signaling activation.We then determined whether the expression of the three cadherins is regulated by the Wnt signaling pathway. We treated NTM cells with or without Wnt3a and/or sFRP1 for 24 hours and compared the levels of K, OB, and N-cadherins using qPCR. Axin2, whose expression is induced by the Wnt pathway, is frequently used as a marker for Wnt signaling activation.reatment . CotreatBecause the induction of K-cadherin by Wnt3a correlated with Wnt signaling activation, we further studied whether the distribution of membrane-associated K-cadherin is regulated by the Wnt/β-catenin signaling in the TM. We treated NTM cells with or without Wnt3a and sFRP1 and compared K-cadherin expression using ICF A. We fouTo quantitatively compare membrane-associated K-cadherin, we extracted the membrane protein fraction or whole-cell lysate from treated cells for WB analysis B. MembraTo determine the contribution of K-cadherin to TM cell adhesion, we performed RTCA cell impedance assays. Cell impedance is a measurement of electric resistance across cells that is affected by cell adhesion. As described in the Methods section, CI was calculated to quantify changes in impedance.P < 0.001, n = 4; P < 0.001, n = 4; First, we studied how changes in Wnt signaling affects cell impedance. NTM cells were cultured, and cell impedance baselines were established. The cells were then treated with or without Wnt3a and/or sFRP1 for 3 to 4 days. We found that Wnt3a significantly increased both maximum and minimum CI values compared to untreated controls, while the Wnt signaling antagonist sFRP1 significantly decreased CI  tissues by injecting Ad5.Null, Ad5.sFRP1, Ad5.K-cadherin, or Ad5.sFRP1+Ad5.K-cadherin virus(es) into the vitreous chamber of the left eyes of 4- to 6-month-old BALB/cJ mice. Viral particle numbers were identical between groups, with some groups also receiving Ad5.Null virus to compensate. The right eye served as an uninjected naïve control. Our published studies have shown that Ad5 has a tropism for the TM.38 Conscious IOPs of both eyes were recorded using a rebound tonometer. Baseline IOP at day 0 and postinjection IOPs at indicated days were plotted over time , which matched our previously published studies.19 We also found that Ad5.K-cadherin alone did not affect IOP (P > 0.05 compared to both controls). However, cotransduction with Ad5.K-cadherin partially but significantly inhibited Ad5.sFRP1-induced OHT from days 6 to 35 , and it completely inhibited OHT at day 35 (P > 0.05 compared to controls).We found that Ad5.sFRP1 induced OHT  and WB, respectively. We compared sFRP1 expression using IF because it was difficult to collect aqueous humor from mouse eyes. K-cadherin was coexpressed with the mCherry protein tag and was detected by WB using anterior segment tissue lysates. Using IF staining, we found that endogenous sFRP1 was expressed in multiple tissues including the cornea, sclera, iris, ciliary body, and retina A. HoweveHaving confirmed that sFRP1 and K-cadherin were expressed in the transduced mouse eyes, we then determined whether overexpressed sFRP1 inhibited Wnt/β-catenin signaling. We dissected anterior segment tissues and determined β-catenin levels using WB C. We fou39 The differences in infectious viral particles were compensated using Ad5.Null virus, and multiplicity of infection was 1:100 . We found that sFRP1 expression was only slightly affected by cotransduction of Ad5-K-cadherin , further supporting the roles of Wnt signaling in maintaining K-cadherin junctional function. K-cadherin overexpression significantly reduced the ocular hypertensive effects of sFRP1 in vivo. Our findings suggest that sFRP1 may induce OHT by disrupting K-cadherin, which effectively disrupts TM cell-cell adhesion and tissue homeostasis.40 K-cadherin is expressed in the kidney throughout adulthood, but is also expressed in other tissues such as the ventricles of the brain and central nervous system during development and adulthood.4143 Cadherins play many roles in the cell as homophilic cell-cell adhesion molecules, as cell migration promoters or inhibitors, and as mechanosensors and mechanotransducers relaying external mechanical stimulation to the actin cytoskeleton. Different cadherin isoforms may confer different physiological properties and characteristics to cells and tissues due to protein structural differences . It is necessary to further determine how K-cadherin affects TM cell behavior to understand how it regulates IOP at the molecular level. In addition to the K-cadherin expression and localization, we found that K-cadherin knockdown decreased cell impedence (CI) more than knockdown of OB-cadherin, another cadherin expressed in the TM. Interestingly, the knockdown of OB- or K-cadherin increased the expression of the other, suggesting that these two cadherins may also complement each other's function in TM cells. We need to better understand the biomechanical properties of these two highly expressed cadherins in TM cells and determine the potential functional differences between them.K-cadherin, or fetal kidney cadherin, was initially named for its high expression in the developing kidney.46 and IOP.19 We also have shown that the Wnt/β-catenin signaling pathway and the TGFβ/Smad signaling pathway, another important pro-POAG pathway, cross-inhibit each other in the TM,22 suggesting another way by which Wnt/β-catenin signaling regulates TM homeostasis and IOP.We show that active Wnt/β-catenin signaling and K-cadherin expression are important in IOP regulation, and blocking this pathway leads to IOP elevation in glaucoma . Recent 31 examined TGFβ signaling and its effects on β-catenin as well as OB-cadherin and N-cadherin in TM cells. They found that TGFβ2 increases the expression of β-catenin, N-, and OB-cadherin in HTM cells as well as enhances TM cell-substrate adhesion.31 It is interesting that K-cadherin and other cadherins may work synergistically or antagonize each other during mesenchymal to epithelial transition and/or neuronal development.4749 Based on our findings and Wecker's studies, we speculate that TGFβ signaling and Wnt signaling use OB-cadherin and K-cadherin to differentially regulate TM cell adhesion and function because they have opposing effects on IOP .Future studies will determine how this cross-inhibition affects cadherin expression, TM cell adhesion, and IOP regulation. Wecker et al.50 Changes in the actin cytoskeleton including increased TM contractility and formation of cross-linked actin networks as well as alterations in the focal adhesion integrin molecules may contribute to increased IOP in POAG.5153 Because we found that the Wnt/β-catenin signaling inhibitor sFRP1 decreased K-cadherin expression, it may affect K-cadherin mediated cell-cell adhesion, actin cytoskeleton, and focal adhesions and thereby regulate aqueous humor resistance via the TM outflow pathway.Cadherin-mediated cell-cell adhesion may contribute to TM homeostasis in multiple ways. Cadherin junctions may keep cell processes in close proximity to enable gap junction formation and paracrine cell signaling. Cadherin junctions may be part of mechanical signal transduction such as contraction/relaxation to the actin cytoskeleton and may even regulate focal adhesion.Supplement 1Click here for additional data file.


Aberrant regulation of WNT/β-catenin signaling has a crucial role in the onset and progression of cancers, where the effects are not always predictable depending on tumor context. In melanoma, for example, models of the disease predict differing effects of the WNT/β-catenin pathway on metastatic progression. Understanding the processes that underpin the highly context-dependent nature of WNT/β-catenin signaling in tumors is essential to achieve maximal therapeutic benefit from WNT inhibitory compounds. In this study, we have found that expression of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alters the invasive potential of melanoma cells in response to WNT/β-catenin signaling, correlating with differing metabolic profiles. This alters the bioenergetic potential and mitochondrial activity of melanoma cells, triggered through regulation of pro-survival autophagy. Thus, WNT/β-catenin signaling is a regulator of catabolic processes in cancer cells, which varies depending on the metabolic requirements of tumors. However, the pathways do not function in an autonomous manner, but rather exist in a signaling network where there is concomitant cross-talk and regulation between the β-catenin-dependent, and -independent pathways.2 The WNT/β-catenin pathway results in the stabilization of a cytoplasmic pool of β-catenin that would otherwise be marked for proteasomal-mediated degradation by a destruction complex, composed of (among other proteins) APC (encoded by APC), AXIN-1/2 (encoded by AXIN1/2) and GSK-3β (encoded by GSK3B). Upon binding to receptors of the Frizzled (FZD) family and the co-receptors, LRP5/6, extracellular WNT stimulation transmits a signal to the intracellular Disheveled (DVL) adaptor, which inhibits the destruction complex and allows nuclear translocation of stabilized β-catenin to activate gene transcription. In the nucleus, β-catenin acts as a cofactor with transcription factor/lymphoid enhancer-binding element transcription factors to stimulate expression of WNT target genes such as AXIN2 and MYC.5The importance of WNT signaling in metazoans cannot be underestimated; the pathways are essential in embryonic development, coordinating correct tissue patterning and for homeostasis in adult tissues. As such, aberrant WNT signaling can lead to a host of embryonic malformations, degenerative diseases and cancer. Thus, understanding how WNT signaling affects cells and tissues has far reaching implications in animal biology. WNT proteins comprise a large family of secreted glycoproteins (19 members in vertebrates) that modulate a number of signal transduction pathways in a highly tissue context-dependent manner.8 The WNT signaling network is now known to regulate oncogenesis in a diverse array of cancers including (in addition to breast), tumors derived from the blood, gastro-intestinal tract, brain, prostate, ovaries, liver and skin, to name a few. Traditionally, the WNT/β-catenin pathway has been viewed as oncogenic, where hyperactivity of the pathway promotes carcinogenesis. For example, in colorectal cancer loss-of-function mutations in APC can be detected in up to 80% of tumors,10 leading to increased β-catenin signaling and prompting researchers to develop inhibitory compounds for the pathway.11 However, it is not that simple in all tumor contexts, as high levels of nuclear β-catenin does not always correlate with poor prognosis for all tumor types, including medulloblastoma,12 ovarian cancer,13 prostate cancer14 and melanoma.15Since the 1980s researchers have known that the WNT pathways have an influential role in carcinogenesis, after WNT1 was shown to induce the formation of mammary tumors in mice.17 WNT/β-catenin signaling in melanoma tumors has previously been shown to correlate with improved survival in patients, which was corroborated by murine xenograft models where melanoma cells overexpressing WNT3A exhibit reduced tumor volumes and metastasis compared with parental cells.15 Furthermore, reduced β-catenin expression has been associated with progression of melanoma in additional clinical cohorts.21 However, other experimental work challenges the assumption that increased WNT/β-catenin signaling reduces the growth and spread of melanocytic tumors. Engineered murine models of melanoma that express melanocyte-specific phosphatase and tensin homolog deleted on chromosome 10 (PTEN) loss and the constitutively activating BRAFV600E mutation (two mutations commonly associated with melanocytic tumors in patients), exhibit highly metastatic and aggressive tumors when β-catenin is stabilized.22 These observations suggest that subsets of melanoma tumors containing distinct mutational contexts, respond to stabilized β-catenin with potentially differing effects on disease progression, highlighting the need to better understand the role of the WNT/β-catenin pathway in melanoma cell behavior.Melanoma is a malignancy derived from the pigment-producing cells, melanocytes, and alarmingly has some of the fastest growing incidence rates among human cancers worldwide.23 Indeed, metabolic reprogramming is a hallmark of cancer,24 which has been demonstrated in a number of tumor types to be regulated by WNT signaling , including in melanoma.26 We have previously shown that WNT5A  promotes aerobic glycolysis in melanoma cells,26 which may contribute to the pro-metastatic effects of this signaling pathway in melanoma. Furthermore, the WNT/β-catenin pathway has also been shown to promote aerobic glycolysis in colorectal cancer and promote cell proliferation.27 Currently it is unknown if WNT/β-catenin signaling can also reprogram melanoma cell metabolism and if so, what effect this may have on tumorigenic state.Interest in the topic of cancer metabolism has been revived in recent years as accumulating evidence has demonstrated the contribution that these metabolic alterations have on the establishment and progression of tumors.WT) with those expressing genetic deletions in this tumor suppressor and analyze the phenotypic effects of the WNT/β-catenin pathway in both subsets. We demonstrate that the WNT/β-catenin pathway has profound effects on melanoma cell invasion, metastasis and metabolic status between tumor cells depending on PTEN expression status. Moreover we provide evidence that in melanoma cells expressing PTEN, alterations in cellular metabolism are associated with the control of mitochondrial activity and remodeling. Taken together, these results support a model whereby WNT/β-catenin signaling controls subsets of melanoma cells based on PTEN expression status, which regulates metabolic behavior in the cells to alter their invasive potential.Here we compare melanomas expressing wild-type PTEN -AKT pathway) through deletion, mutation or reduced expression, occurs in approximately 5–40% of melanocytic tumors.33 We selected a panel of cell lines with known status of these common mutations in melanoma  or NRAS (approximately 20%),melanoma  to invesPTENMut; . The PTEWT lines  and highut lines .15 To confirm this finding in our melanoma panel, we ensured that recombinant WNT3A (rWNT3A) could activate a β-catenin-responsive reporter in the cells  culture of melanoma cells.43 Proliferative phenotypes form round, individual colonies and invasive phenotypes form interconnected colonies.43 We were unable to switch the 3D phenotypes of a subset of our melanoma panel (two PTENWT and two PTENMut lines) in response to rWNT3A stimulation  as a marker of active WNT/β-catenin signaling and divided the samples into two cohorts as PTEN low or high expressing  for which PTEN expression data were available, do not follow the same trend when subdivided into PTEN high (n=97) or low (n=30) cohorts  network cutaneous melanoma data set, we identified 209 patient samples with high cal data  showed tcal data . Distantta n=11; . Importa cohorts , suggest25 we decided to investigate the bioenergetic properties of PTENWT and PTENMut melanoma cells in response to WNT3A signaling. To test this, we used a Seahorse XFe96 extracellular flux analyzer to investigate whether oxygen consumption rates (OCRs) or extracellular acidification rates (ECARs) were affected by WNT3A, allowing us to study oxidative phosphorylation and indirectly also glycolytic rates in the melanoma cells, respectively. Using the overexpression melanoma cells, we found that both OCR and ECAR rates were altered in response to WNT3A signaling, which interestingly differed between the PTENWT and PTENMut lines (WT) cells, WNT3A overexpression led to reduced rates of OCR and ECAR, which when analyzed for basic parameters of cell respiratory control,47 showed significantly reduced rates of basal respiration, ATP production, maximal respiratory capacity, non-mitochondrial respiration and basal ECAR  activity, which is the initial enzyme of the tricarboxylic acid cycle, to determine mitochondrial and oxidative phosphorylation activity in the cells. In all the PTENWT cell types tested, CS activity was significantly reduced in response to hyperactive WNT3A signaling , however, stable overexpression of WNT3A led to enhanced lactate secretion in these cells  and found efficient reduction of CTNNB1 gene expression in response to treatment , which is a well-known secreted inhibitor of the WNT/β-catenin signaling pathway.51 By forming a ternary complex with LRP5/6 along with another receptor, Kremen, DKK1 stimulates the endocytic removal of LRP5/6 from the cell surface thereby preventing WNT ligand-mediated stimulation of the WNT/β-catenin pathway.52 We found that rDKK1 treatment of WNT3A-stimulated A375 (PTENWT) melanoma cells not only inhibited WNT/β-catenin signaling  of PTENt probes . Specifit probes  and TMRMt probes  fluorescWT cells  and 5 coWT melanoma cells . We investigated PINK1-Parkin accumulation in PTENWT melanoma cells in response to WNT/β-catenin signaling to determine whether impairment of either could lead to increased levels of MFN proteins and found that while differences in Parkin levels were modest , we did detected a marked difference in PINK1 expression . Importantly, the uncoupler, FCCP (which dissipates ΔΨm), also stabilizes FL-PINK1 in PTENWT melanoma cells, demonstrating that these cells are capable of proteolytic cleaving of PINK1  expression, which is a protein that targets cargos for autophagy.59 Importantly, we also detect repression of SQSTM1 expression in PTENWT melanoma cells following treatment with rWNT3A  proteins (marking them for degradation) including MFNspression . PINK1 upression , suggestof PINK1 . Furtherignaling , but notut cells  suggestima cells . WNT/β-ch rWNT3A .WT cells exhibit a significant reduction in invasion and metastasis  due to defective mitophagy . Our findings show that the effects of β-catenin signaling in melanoma cells differs depending on PTEN expression; in the absence of PTEN loss, cellular bioenergetics is compromised by β-catenin and tumor invasion/metastasis reduced, whereas cells exhibiting reduced expression of PTEN have increased invasion in response to β-catenin signaling, which does not appear to be due to a definable metabolic reprogramming event in these cells, suggesting the pro-invasive effects of WNT/β-catenin signaling are potentially independent of metabolic reprogramming in this particular context. Our findings are supported by a transgenic mouse model, where stabilized β-catenin combined with PTEN loss and activating BRAFV600E mutation in cells of melanocytic origin, resulted in increased metastasis.22 β-Catenin stabilization in this model was recently shown to block T-cell recruitment to the tumors, providing resistance to immunotherapies and showing that WNT/β-catenin signaling can promote progression of melanoma through a variety of mechanisms in this specific mutational background,77 where (unlike in melanoma cells expressing PTEN) maintenance of high metabolic activity would be essential for WNT/β-catenin signaling to promote tumor progression in this context.Hyper-activation of the mitogen-activated protein kinase signaling pathway through constitutively activating mutations in BRAF or NRAS causes oncogene-induced senescence in melanocytes,79 suggesting that in PTENWT melanoma cells, WNT/β-catenin signaling may be restricted compared with the PTENMut cells, which may explain the phenotypic differences observed between the two cell types. However, the WNT/β-catenin pathway is not blocked in PTENWT melanoma cells and can still be strongly activated by exogenous WNT treatment , which is a target of WNT/β-catenin signaling and can regulate aggressive behavior in melanoma cells.81 However, we were unable to identify differences in expression of MITF between the PTENMut and PTENWT melanoma cells (data not shown) and inhibition of cell migration by β-catenin has only been shown to be partially dependent on MITF in melanocyte lineage cells,61 suggesting that differing MITF expression levels are unlikely to account for the difference in cell migration and invasion noted here for the PTENMut and PTENWT melanoma cells in response to WNT/β-catenin signaling. Rather, we suggest that the differences in cell motility identified between these two cells types are due to a fundamental change induced by the WNT/β-catenin pathway on the metabolic status of the melanoma cells.PTEN antagonizes nuclear accumulation of β-catenin,reatment , demonst83 and we have discovered distinct mitochondrial activity between PTENWT and PTENMut cells in response to WNT/β-catenin signaling. Specifically, we found that in PTENWT melanoma cells, WNT/β-catenin signaling increases mitochondrial networking by upregulating expression of mitochondrial fusion proteins and concomitant downregulation of mitochondrial fission machinery, indicating that this pathway is an important regulator of mitochondrial dynamics at least in a subset of melanoma cells. Interestingly, this WNT pathway is not the only one that can control mitochondrial morphology, as the WNT5A/Ca2+ signaling pathway has also been found to modulate mitochondrial dynamics in normal cells by promoting fission86 (at least in part through DRP1 activation84). However, in these same cells, the WNT/β-catenin pathway was unable to regulate mitochondrial aggregation, failing to alter mitochondrial morphology nor antagonize WNT/β-catenin-independent-mediated mitochondrial fission,86 highlighting the context-dependent nature of the WNT signaling network on mitochondrial dynamics. It is tempting to speculate that the effects of the WNT/β-catenin pathway in regulating mitochondrial dynamics in PTENWT melanoma cells, may highlight a potentially novel tumor-specific role of this pathway in certain cancer contexts. It remains to be determined which additional tumor types may also be affected, however, other cancer cells that exhibit augmented metabolic profiles in response to WNT/β-catenin signaling, such as colorectal cancer,27 represent a logical extension to further such studies.In melanoma, mitochondria remain functional92 In this model, PINK1 is turned over by proteolysis in well-coupled mitochondria, but when the ΔΨm drops, FL-PINK1 is stabilized on the OMM, allowing for selective accumulation on impaired mitochondria and marking these organelles for degradation.93 However, our data contrast with this current model where we detected high expression of FL-PINK1 in response to WNT/β-catenin signaling in PTENWT cells, despite a concomitant increase in ΔΨm . Interestingly there are a couple of alternative, ΔΨm-independent mechanisms can also result in FL-PINK1 accumulation, which include overwhelming of the mitochondrial import channels with excess PINK1 and preventing PINK1 cleavage by reducing mitochondrial protease activity.94 One of these or an alternative mechanism could be responsible for the increased FL-PINK1 levels observed in PTENWT melanoma cells in response to WNT/β-catenin signaling. Mitophagy is defined as the degradation of mitochondria through the macroautophagic pathway, thus defects in the autophagic machinery inhibit mitophagy. We found an increase in p62 protein following β-catenin knockdown and β-catenin-mediated repression of the SQSTM1 transcript, as well as increased autophagosome numbers  was recently found to be a marker of autophagy in melanoma, suggesting that inhibition of WNT/β-catenin signaling could increase autophagy in melanoma cells.96PINK1-Parkin signaling represents a mitochondrial quality control pathway that is able to tag defective mitochondria for selective degradation by the autophagosomal machinery, where the current model states that PINK1 is selectively stabilized on the OMM of defective mitochondria, facilitating Parkin recruitment to poly-ubiqutinated OMM proteins (including MFN1 and MFN2) and isolate defective mitochondria in the autophagosome. numbers , highligma cells . HoweverMut melanoma cells: It has long been known that PTEN can increase autophagy by inhibiting AKT activity97 (and independently of AKT-mTOR activity98), and that loss of AKT in PTEN-null cancer cells induces autophagy,99 suggesting that tumor cells with reduced PTEN expression will have reduced autophagy in general. Indeed our data support this where the PTENMut A2058 cells exhibit low basal autophagy as seen by low expression of the autophagy marker, LC3II, which does not alter markedly in response to WNT3A signaling  for tumor cell survival, which makes them particularly susceptible to autophagy inhibition .Our model  can alsoignaling . PTENWT 100 Our results suggest that PTEN expression level could represent a potential biomarker for the implementation of WNT inhibitors in melanoma.Small-molecule WNT antagonists have been developed for cancer treatment, however, because the role of the WNT/β-catenin pathway is controversial, it is difficult to predict how such drugs could be implemented in the treatment of melanoma. Here we show that the effects of the WNT/β-catenin pathway differ vastly in melanoma depending on PTEN expression levels; when PTEN levels are high, β-catenin signaling reduces metastasis, inhibits mitophagy and reduces the bioenergetic status of the cells (none of this occurs in melanoma cells with depleted PTEN levels). Genomic subtyping in cancer is needed for the implementation of molecular therapies and recent TCGA stratification of cutaneous melanomas provides a potential guideline for therapeutic decisions.Details of all cells, culture conditions, reagents and statistical analyses used are provided in the 101 for 6 h in media without serum or antibiotics. A Renilla luciferase expression plasmid was co-transfected for normalization of transfection efficiency. Transfected cells were then stimulated for 24 h with either rWNT3A (50 ng/ml) or carrier control. Cell extracts were assayed for luciferase activity using the dual luciferase kit , according to the manufacturer's instructions. Renilla-normalized TOPFlash results were then normalized to FOPFlash expression and the data presented as actual luminescence units.Melanoma cells were plated in 24-well plates and transiently transfected with 150 ng of either Super8TOPFlash or Super8FOPFlash reporter constructsApo-TRACE Apoptotic Cell Staining Kit  was used according to the manufacturer's instructions. Briefly, cells were serum starved for 48 h, washed in phosphate-buffered saline and Apo-TRACE added at 75 μg/ml for 1 h in the dark. Cell lysates were read at ex 328 nm, em 563 nm on a POLARstar Optima microplate reader . Results were normalized to total protein concentration, as assessed by BCA assay .26 Briefly, a wound was scratched across a confluent monolayer of melanoma cells and the wells rinsed with fresh media to remove floating cells. Images were taken of the scratched area at × 10 magnification, where the wound-healing process was monitored at 24 and 48 h in the same location. Images were analyzed using Image J software  to measure the open area of cells and the data presented as fold-change in wound confluence over the control.This was done as previously described.36Melanoma cells were either stimulated in serum-free media or serum-starved for 48 h before the assay (as indicated for each experiment), resuspended in serum-free medium and added to the upper chambers of 8.0 μm pore inserts  coated with Matrigel . Growth media were added to the lower chamber and the cells incubated for 48 h. Migrating cells were quantified after crystal violet staining. Each experiment was performed in triplicate. The insert was then removed and the number of invaded cells were analyzed as previously described.5/ml cells were injected into 11-week-old, male NOD scid gamma (NSG) mice through the tail vein using a 26G needle. Six mice were injected per group  and experiments performed in a blinded manner to ensure randomization of the approach. Lungs were harvested 4 weeks post injection and fixed with 10% neutral-buffered formalin (Fisher Scientific UK Ltd). All animal studies were performed using Institutional Animal Care and Use Committee protocols, as approved by a review board at the University of Washington.Low passage, lentiviral transduced WNT3A overexpression or control cancer cells were suspended in phosphate-buffered saline at low passage. 10 × 10e96 flux analyzer  and the XF Cell Mito Stress Test kit , according to the manufacturer's instructions. In all, 8x104 cells were seeded onto poly-d-lysine-coated XF microplates. Briefly, metabolic flux measurements were assessed under basal conditions and in response to the ATP synthase inhibitor, oligomycin (2.5 μM), the electron transport chain (ETC) accelerator, FCCP (1 μM), and finally the ETC complex 1 and 3 inhibitors, antimycin A and rotenone (2.5 μM), respectively. Data were analyzed using the XF software .OCR and ECAR were measured using the XFThe CS Assay Kit (Sigma-Aldrich) was used according to the manufacturer's instructions. Cells were seeded and stimulated for 48 h before analysis and results normalized to total protein concentration for each sample.26 Melanoma cells were seeded and stimulated or not (as indicated for each experiment), 48 h before the analysis.The Lactate Assay Kit  was used according to the manufacturer's instructions and experiments conducted as previously described.M) were diluted in phenol-red-free media and incubated with the cells for 15 min at 37 °C. The stain was then removed and replaced with 1 ml phenol-red-free media and incubated at 37 °C for 10 min. Following staining, cells were fixed using 5% paraformaldehyde for 10 min, permeabilized using 0.5% Triton X-100 for 5 min and then counterstained with Phalloidin 488 (0.25 units) and Hoechst 33342 (10 nM).Cells were grown and stimulated on 18-mm sterile coverslips for 48 h. MitoTracker stains , and counterstained with Hoechst 33342. After washing, cells were mounted with Vectashield mounting medium  and analyzed (as for the MitoTracker stained cells) by LSM 510  to capture Z-stack images at optimum slice depths for each sample. Gain settings and exposure time were kept constant between samples. For Imaris (Bitplane AG) analysis, sampling was carried out using 3D images to calculate surface area staining by setting the background threshold to 0.5 μm. The area in defined size ranges was then calculated by Imaris software as a percentage of the total.YWHAZ, UBC and ACTB were used as reference genes for relative quantification of the transcripts under investigation. All oligos used for quantitative PCR are shown in The Human Mitochondrial to Nuclear DNA Ratio Kit  was used according to the manufacturer's protocol for whole-cell lysis. In all, 50 cells per well in DNase/RNase-free water were used with a 2X Fast SYBR green mastermix. For reverse transcription–quantitative PCR, RNA was purified using the GeneJet RNA purification kit (Fisher Scientific UK Ltd.) following the manufacturer's protocol. In total, 2 μg of complementary DNA was synthesized using RevertAid M-MuLV Reverse Transcriptase  and diluted to 100 ng/ml, followed by quantification using SYBR green reagent  and a LightCycler 480 system . m urea, 50 mM ammonium bicarbonate, pH 7.8, and heated to 50 °C for 20 min. Proteins were reduced with 2 mM DTT, alkylated with 15 mM iodoacetamide and digested overnight with a 1:50 ratio of trypsin to total protein. The resulting peptides were desalted on Waters Sep-Pak C18 cartridges . Peptides were measured by nano-LC-MS/MS on a Thermo Scientific Q Exactive (QE). Peptides were separated online by reverse-phase chromatography using heated 50 °C 30 cm C18 columns (75 mm ID packed with Magic C18 AQ 3 μM /100 A beads) in a 180 min gradient (1% to 45% acetonitrile with 0.1% formic acid) and separated at 250 nl/min. The QE was operated in data-dependent mode with the following settings: 70 000 resolution, 400–1600 m/z full scan, Top 10, and an 1.8 m/z isolation window. Identification and label-free quantification of peptides was done with MaxQuant 1.5 , using a 1% false discovery rate against the human Swiss-Prot/TrEMB  database. Three replicates per condition were analyzed. Peptides were searched using a 5 p.p.m. mass error and a match between run window of 2 min. Proteins that were significantly regulated between conditions were identified using a permutation-based t-test  in Perseus 1.4.1.3. Gene ontology (GO)-terms were analyzed using Protein ANalysis THrough Evolutionary Relationships (PANTHER) 9.0 (http://pantherdb.org).For sample preparation, non-glycoslyated β-catenin was removed using conA beads . Samples were pre-cleared with rabbit IgG before incubation with β-catenin-coated Dynabeads G . Immunoprecipitated proteins were solubilized in 1 M) or TMRM  for 2 h. All samples were diluted in phenol-red-free media. The stain was removed and replaced with 1 ml phenol-red-free media and incubated at 37 °C for 10 min before re-suspension in FACS buffer  and analysis on the Accuri C6 flow cytometer (BD Biosciences).Mitochondrial membrane potential measurements were made using flow cytometry protocols. Cells were grown and stimulated for 48 h before incubation with JC-1  stain (2 μ


For imaging, we developed a novel high-energy XDF system that overcomes the limitations of currently established setups. Our XDF radiographs yield sufficiently high image quality to enable radiographic evaluation of the lungs. We consider this a milestone in the bench-to-bedside translation of XDF imaging and expect XDF imaging to become an invaluable tool in clinical practice, both as a general chest X-ray modality and as a dedicated tool for high-risk patients affected by smoking, industrial work and indoor cooking.X-ray chest radiography is an inexpensive and broadly available tool for initial assessment of the lung in clinical routine, but typically lacks diagnostic sensitivity for detection of pulmonary diseases in their early stages. Recent X-ray dark-field (XDF) imaging studies on mice have shown significant improvements in imaging-based lung diagnostics. Especially in the case of early diagnosis of chronic obstructive pulmonary disease (COPD), XDF imaging clearly outperforms conventional radiography. However, a translation of this technique towards the investigation of larger mammals and finally humans has not yet been achieved. In this letter, we present the first  As clinically used conventional attenuation-based radiography of the lung cannot resolve these microstructures, its diagnostic window is mostly restricted to indirect signs caused by late-stage pathologies. In contrast, XDF radiography4, pulmonary fibrosis5, pneumothoraces6 and neonatal lung injury associated with mechanical ventilation7. Furthermore, the assessment of pulmonary carcinoma, edema, as well as pneumonia may significantly benefit from XDF imaging. These pathologies are characterized by a destruction (as in the case of COPD) or densification (by fibrotic or tumorous tissue) of the natural alveolar structure as the disease progresses. The loss of air-tissue interfaces consequently results in a reduction of the XDF signal compared to the distinct signal of healthy lung tissue. Hence, variations in the XDF lung pattern can indicate pathological changes. The combination of XDF with conventional imaging can be used for differential diagnosis8.Recent small-animal studies demonstrated that XDF imaging enhances pulmonary diagnosis, e.g. for the early detection and staging of COPD15. Among these, grating-based imaging (GBI) methods (i.e. grating interferometers13 and coded apertures15) are most promising for clinical applications, as they are compatible with conventional X-ray sources and detectors. Additionally, GBI has the advantage of simultaneously acquiring a conventional attenuation image alongside the XDF signal, providing the radiologists with both functional (XDF) and anatomical (attenuation) imaging by means of a single scan only.In order to measure the XDF signal (of lungs and other specimens), different approaches have been developedIn GBI, an arrangement of three gratings with periods in the range of micrometers is placed in the X-ray beam to create a fringe pattern on the detector. The contrast of the interference pattern is commonly referred to as the (interferometric) visibility of the system and the XDF signal is subsequently given by the reduction in visibility when a scattering specimen is introduced into the beam path.ex-vivo samples. In-vivo imaging of larger mammals still poses substantial technical challenges: first, the entire thorax has to be covered, which demands a field-of-view (FOV) of at least 30 × 30 cm2. Second, to minimize motion artifacts, the full scan has to be conducted during a single (induced) breath stop, which should not exceed one minute due to animal care regulations. Current GBI systems offer a maximal FOV of about 10 × 10 cm2, contingent on the fabrication limitations of the gratings. One simple way to increase the effective FOV is to use multiple exposures and subsequently stitch the obtained images together, which results in excessively long scan times and unfavorable movement of the specimen16. To overcome this restriction and to increase the FOV, scanning approaches18 and tiled gratings19 have been suggested. Apart from the large FOV, the images have to be acquired at parameters compliant with clinical standards. Finally, the images have to be acquired at clinically relevant energies . While first applications of high-energy XDF have been carried out as proof-of-principle investigations21, none of the proposed systems satisfied all of the above requirements at once.XDF chest imaging has until now been limited to the investigation of small animals or 2 of a living pig acquired with a novel high-energy XDF scanner  and is built in a compact system design. To achieve the necessary FOV, we combined a slot-scanning approach with linearly tiled gratings of about 40 × 2.5 cm22. Figure Porcine thorax anatomy is considered to closely resemble that of humans and pigs are therefore a widely accepted model for translational respiratory medicine2, resulting in an effective dose of approximately 80 µSv for the PA exposure. This value is compatible with conventional chest radiographs (20 µSv for PA examinations24) and equals around ten days of natural background radiation23.Figure When comparing XDF Fig.  with the7 proved that the XDF signal strength is directly correlated to the number of intact alveolar interfaces. Therefore, any pathology associated with their loss will continuously reduce the XDF signal up to the point where no interfaces are left at all. This opens up a new diagnostic window to detect early stages of respiratory diseases, which typically appear radiolucent on the attenuation radiograph. The extreme case, in which lung tissue/alveolar interfaces are completely displaced by air, would e.g. occur in a pneumothorax or bulla, for which the air-filled stomach is only considered as an educational and demonstrative model here. The scatterplot in Fig. In order to demonstrate the potential of the complementarity between XDF imaging and conventional radiography, Fig. 13. These are shown in Fig. 25. Therefore, the differential-phase images of the in-vivo pig in our experiments provide little additional information . The source grating G0 exhibits an active area of 5.0 × 2.5 cm2, shaping the extended focal spot into an array of fine slit sources, and preventing blurring of the fringe pattern. The reference grating G1 and analyzer grating G2 both have an area of 40 × 2.5 cm2, obtained by tiling 8 smaller sub-tiles of 5.0 × 2.5 cm2. Grating tiles were assembled under a microscope, with stitching gaps smaller than one pixel column. Coarse Moiré fringes for imaging were intentionally created on the detector by introducing a small periodic mismatch between the G1 and the analyzer grating G2  operated at 70 kVp and the detector is a first-generation Pixium RF 4343 . To achieve a high readout rate (currently 12 Hz), the detector is operated in a fluoroscopy, 3 × 3-binning mode with a resulting pixel size of 444 µm.The core part of the scanner consists of three gratings .The scanner achieves a visibility of 31 ± 4% over the full FOV with a clinical 70 kVp tungsten spectrum. Nevertheless, the visibility still changes when measuring a highly attenuating sample like a thorax due to an unavoidable energy dependency of the grating interferometer. Consequently, to account for beam hardening in the measured XDF signal, calibration is conducted prior to the measurements18 in which there is an intrinsic movement in the x-direction  from the measured dose area product (DAP) via: ED[mSv] = K * DAP[Gy*cm2].Assuming an adequate similarity between porcine and human thoraxes for PA projections, we used the conversion factor K given by Wall PTW NOMEX dosimeter  and the illuminated area was estimated from the FOV, resulting in a DAP of 0.5 Gy*cm2. With K = 0.16 [mSv/ (Gy*cm2)] (ICRP 103 at 120 kVp) an ED of 80 µSv was calculated for the PA exposure. The conversion factor for a tube voltage of 70 kVp will be lower and consequently our reported effective dose values are slightly higher than the actual dose.The incident air kerma at the table was measured by a n = 1; male; weight = 23 kg; age 3 months) was sedated by intramuscular application of Ketamine  and Azaperone . Anesthesia was continued by intravenous injection with Propofol  using a syringe pump  with dose adjusted to effect. The animal was kept under automated ventilation throughout the experiment. For imaging, ventilation was paused for the duration of the scan (max. 60 sec at a time) with a constant pressure of approx. 11 mbar in the airways. Heart rate and oxygenation were monitored continuously. All animal procedures were performed with permission of the local regulatory authority, Regierung von Oberbayern (ROB), Sachgebiet 54, 80534 Munich, approval number AZ 55.2-1-54-2532-61-2015. The application was reviewed by the ethics committee according to §15 TSchG German Animal Welfare Law. All experiments were performed in accordance with relevant guidelines and regulations. To terminate the experiment, the animal was euthanized under anesthesia by intravenous injection of T61®  according to the manufacturer’s instructions. No randomization or blinding was performed.One German Landrace Hybrid pig (wildtype, Institute of Molecular Animal Breeding and Biotechnology, Ludwig Maximilian University Munich breeding facility; The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Figure 1


On the other hand, it showed the phosphorylation of different Stat proteins. It was determined that cADSCs and mADSCs show the potential to differentiate into adipocytes, osteocytes, and chondrocytes. Furthermore, a difference in protein phosphorylation between undifferentiated cADSCs and mADSCs was identified.This study aimed to demonstrate single-cell phosphospecific flow cytometric analysis of canine and murine adipose-derived stem/stromal cells (ADSCs). ADSCs were obtained from clinically healthy laboratory beagles and C57BL/6 mice. Cell differentiation into adipocytes, osteocytes, and chondrocytes was observed for the cultured canine ADSCs (cADSCs) and murine ADSCs (mADSCs) to determine their multipotency. We also performed single-cell phosphospecific flow cytometric analysis related to cell differentiation and stemness. Cultured cADSCs and mADSCs exhibited the potential to differentiate into adipocytes, osteocytes, and chondrocytes. In addition, single-cell phosphospecific flow cytometric analysis revealed similar  Adipose-derived stem/stromal cells (ADSCs) reside in adipose tissues and are considered to be the progenitors of adipocytes. A recent study revealed that ADSCs can differentiate into adipocytes, osteocytes, and chondrocytes . MesenchThe expression of cluster of differentiation (CD) molecules in murine and human ADSC has been reported. Specifically, some surface antigen markers, such as CD34, CD44, and CD90, have been detected as ADSC markers . HoweverIn this study, we performed a comprehensive analysis of protein phosphorylation in mADSCs and cADSCs. We also compared protein phosphorylation between these two types of cell.The institutional animal experiment ethics committee approved the animal experiments in this study. This work was conducted in accordance with the institutional guidelines of Yamaguchi University.μg/kg buprenorphine  and 0.2 mg/kg meloxicam  were used as analgesics. Adipose tissue was washed with Dulbecco's phosphate-buffered saline  and cut into fine pieces that were incubated at 37.5°C for 1 h with shaking in high-glucose Dulbecco's modified Eagle's medium (Wako) supplemented with 10% fetal bovine serum , penicillin (100 U/mL)/streptomycin (100 μg/mL), amphotericin B (0.25 μg/mL) , and collagenase type I . A 100-μm nylon mesh  was used to filter digested tissues, followed by centrifugation at 1,800 rpm for 5 min in FACS buffer composed of 30 mL of DPBS supplemented with 1% FBS and 1 mM EDTA·3Na (Wako). The pellet was seeded on culture plates and reached 80%–90% confluence; ADSCs were replated using trypsin-EDTA (0.25%).Subcutaneous adipose tissue was collected from clinically healthy laboratory beagles  and C57BL/6J mice purchased from a private company . cADSCs and mADSCs were isolated as previously described . After washing with DPBS, cells were mounted with a solution containing 5 μg/mL Hoechst 33342  to stain nuclei.To detect adipogenic differentiation by immunocytochemistry, cells were incubated for 1 h in DPBS containing 10 μg/mL goat anti-mouse osteopontin antibody to label osteocytes. After washing with DPBS, cells were incubated for 1 h in DPBS containing PE-conjugated rabbit anti-goat secondary antibody, washed with DPBS, and mounted as described above.To detect osteogenic differentiation by immunocytochemistry, cells were then incubated for 1 h in DPBS containing 10 μm using a specimen matrix  and a cryostat . After cutting into sections and placing on a slide, the slide was incubated for 1 h in DPBS containing 10 μg/mL sheep anti-mouse collagen II antibody to label chondrocytes. After washing with DPBS, cells were incubated for 1 h in DPBS containing PE-conjugated donkey anti-sheep secondary antibody , washed with DPBS, and mounted as described above. For a negative control, we used DPBS without a primary antibody for all differentiation potentials.To detect chondrogenic differentiation by immunocytochemistry, the pellet was fixed and cut into sections with a thickness of 5–10 μL of Fc receptor-blocking reagent . Cells were stained with reagent to exclude dead cells . After washing the cells, IC fixation buffer  was added with shaking and then incubated. Permeabilization buffer (Affymetrix Japan) was diluted with purified water 10 times and added to wash the cells. After permeabilization, antibodies were used to stain each phosphorylated protein, as shown in Adherent cells from passage 4 were dissociated and cells were resuspended and incubated in FACS buffer containing 2 Both mADSCs and cADSCs passaged four times and displayed spindle-shaped morphology, as determined with phase-contrast microscopy Figures . To asseβ-catenin, β-catenin (pS45), Akt (pT308), Akt (pS473), CD140b, Stat1, Stat3, Stat4, Stat5, Stat6, p38 MAPK, Smad2/3, and ERK1/2 were measured using their respective antibodies. In mADSCs, β-catenin, β-catenin (pS45), Akt (pS473), Stat4, and Stat5 were detected, whereas Akt (pT308), CD140b, Stat1, Stat3, Stat6, p38 MAPK, Smad2/3, and ERK1/2 were not. Meanwhile, in cADSCs, β-catenin, β-catenin (pS45), Akt (pS473), and Stat4 were detected, and Akt (pT308), CD140b, Stat1, Stat3, Stat5, Stat6, p38 MAPK, Smad2/3, and ERK1/2 were not detected , and Stat4 phosphorylation and only mADSCs expressed Stat5 phosphorylation.In this study, we revealed that mADSCs and cADSCs have multipotency to differentiate into adipocytes, osteocytes, and chondrocytes. Both undifferentiated mADSCs and cADSCs expressed β-catenin signaling inhibits adipogenesis in humans and mice. For example, the overexpression of Wnt1 or glycogen synthase kinase 3-b (GSK3b) phosphorylation-defective β-catenin mutant was shown to inhibit adipogenesis in 3T3-L1 preadipocytes  by canonical pathway activation [Previous studies revealed that WNT/tivation . Anothertivation .β-catenin signaling and ADSC adipogenesis in canines. In this study, we also revealed β-catenin and β-catenin pS45 phosphorylation in mADSCs and cADSCs. This suggests that cADSCs also inhibit adipogenesis with WNT/β-catenin signaling.However, to the best of our knowledge, there have been no previous reports on WNT/Akt, also known as protein kinase B, is a serine/threonine-specific protein kinase that plays roles in glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration, among others . In thisIn this study, we detected the phosphorylation of Stat4 and Stat5 in mADSCs, but only that of Stat4 in cADSCs. A previous study revealed Stat4 expression at the mRNA level in mADSCs . HoweverMany clinical trials have been conducted for the ADSC-related treatment of cardiovascular disease, spinal cord injury, cirrhosis, renal insufficiency, and skin fistula with Crohn's disease and for breast reconstruction after mastectomy in humans . In veteβ-catenin and Akt, in contrast to that of the Stat family.mADSCs and cADSCs showed adipogenic, osteogenic, and chondrogenic differentiation potential. mADSCs and cADSCs induced similar phosphorylation of


Wnt-signalling is well understood and has been extensively described in many developmental processes. The regulation of this signalling pathway is of outstanding relevance for proper development of the vertebrate and invertebrate embryo. Axin2 provides a negative-feedback-loop in the canonical Wnt-pathway, being a target gene and a negative regulator. Here we provide a detailed analysis of the expression pattern in the development of the chicken embryo. By performing in-situ hybridization on chicken embryos from stage HH 04+ to HH 32 we detected a temporally and spatially restricted dynamic expression of Axin2. In particular, data about the expression of Axin2 mRNA in early embryogenesis, somites, neural tube, limbs, kidney and eyes was obtained.Canonical  Axin2  is a homologue of Axin protein. It shares approximately 45% of amino acids with Axin [β-catenin and for the glycogen-synthase kinase 3 beta (GSK-3β) were described [Wnt-signalling pathway molecule β-catenin, Axin and Axin2 function as negative regulators of the canonical Wnt-signalling pathway [Wnt-signalling pathway is one of the best elucidated signalling pathways. First, the canonical Wnt-pathway was described, followed by at least two non-canonical pathways. The pathway of planar cell polarity (PCP) and the Wnt/Ca2+-pathway are referred to as non-canonical pathways [Wnt/Ca2+-pathway) .Axin acts as a central scaffold protein in the degradation complex by binding and thus bringing together all important components [Axin2 contain highly conserved regions. GSK-3β phosphorylates β-catenin, which subsequently is ubiquitinated by the E3 ubiquitinase βTrCP and degraded by a proteasome [β further phosphorylates Axin itself, leading to stabilization of its interaction with β-catenin [Wnt pathway, the low density lipoprotein related receptor LRP 5/6 are also known to be substrates of GSK-3β [β is capable of phosphorylating β-catenin alone, Axin unites both proteins, facilitating and significantly accelerating this process [β-catenin in the cytoplasm to a level that prohibits its access to the nucleus. In the presence of APC, the turnover of β-catenin increases [β-catenin molecules to the environment of the destruction complex [Wnt-ligands, they bind to the seven-pass transmembrane receptor Frizzled and to its co-receptor, the low density lipoprotein related protein receptor (LRP) 5 or 6 [β-catenin-destruction complex [Wnt-binding. For instance, Wnt was described to induce Dvl, that is thought to recruit Axin-bound GSK-3β to the membrane, where the latter phosphorylates LRP 5/6 and as a consequence dissociates from Axin [β-catenin degradation complex, the protein phosphatase 1 (PP1) [β-catenin [β-catenin-destruction complex, β-catenin is not degraded any more, cytoplasmatic levels rapidly rise and it enters the nucleus [Axin2 was found, when discovering its transcriptional dependence on TCF/LEF motive [Axin2 expression therefore is initiated by canonical Wnt-signalling and provides a negative-feedback loop [Axin2 in regulating the Wnt-signalling pathway, it is important to mention the state of the art regarding the role of canonical Wnt-pathway in development and in disease. During development, canonical Wnt-signalling is described to be required for proper posterior axis formation and for the formation of the head [Wnt-signalling is known to be indispensable in the developing central and peripheral nervous system [Wnt pathway cause several cancers and Wnt-signalling is required for stem cell self renewal [Wnt-signalling via Axin and Axin2 impacts embryonic development and health in the adult, as described by many studies. Axin mutant mice failed to survive [Axin2 developed a secondary caudal body axis [Axin2 mutations [Axin2 abnormalities in mice and human is familial tooth agenesis and oligodontia [Axin2 mutations play a secondary role in familal adenomatous poliposis coli (FAP), when the causal mutation is not situated in APC and because proper function of APC requires Axin [Axin2 is described [Axin2 was previously tested on its functional redundancy [Axin2 was shown to be able to at least partially compensate for mutated Axin when expressed in the respective cells. Axin however, is expressed in small amounts in all embryonic tissues, while Axin2 expression was described to be restricted and dynamic during mouse development [Wnt regulation, referring to its low cytoplasmatic levels [Axin2 on the other hand, is highly expressed, suggesting an extensive role for Axin2 regulation in certain tissues. This observation, together with the fact that Axin2 is a target of Wnt-signalling, indicates the importance of Axin2 mediated negative regulation in certain tissues. In this study, we demonstrate the dynamic expression pattern of Axin2 in the development of the chick.ith Axin , 2. The ith Axin –5. The Nith Axin , 6. Centescribed . At its escribed , 9. Bein pathway –12. The pathways –15. Thesathway)  5 or 6 –36. This) 5 or 6  and init complex , 39. Sevrom Axin . The phorom Axin . Axin pr 1 (PP1) , 43. Unp-catenin , 40 and -catenin . Previou-catenin . As a re nucleus . A speciack loop , 37, 45.the head –48. Mores system . It is ds system  and in ts system  for revi renewal , 52. The survive , 53 and ody axis  and exhiody axis . This mautations . Anothergodontia , 56, 57.godontia , 59, ovagodontia  and to mgodontia . Axin2 mres Axin , 63. Preescribed , 63. Shedundancy . Axin2 welopment , 45. Intc levels . Axin2 o°C and 80% relative humidity. Eggs were provided by a local breeder (Sörries-Trockels Vermehrungszucht). Staging was performed according to Hamburger and Hamilton [Fertilized eggs of Gallus gallus domesticus were incubated at 37Hamilton .h. For description and analysis of the expression pattern of Axin2 during chicken development, chicken embryos in developmental stages HH 04 to HH 32 were proceeded in in-situ hybridization.The obtained chicken embryos were isolated, fixed in 4% PFA for at least 24in-situ hybridization was performed as previously described [Axin2 riboprobe for detection of Axin2 transcripts in all embryonic tissue.Whole mount escribed , using cAxin2in-situ hybridization was generated from a pCMS-EGFP plasmid containing a full length Axin2 coding sequence. It was restricted using EcoRV and SmaI to obtain a 835bp fragment binding from bp926 to bp1788 on Axin2 mRNA (NCBI Reference Sequence: NC_006105.4). The purified fragment was blunted and cloned to pJET1.2/blunt Cloning Vector. From here, the fragment was excised using XbaI and XhoI and ligated to pBluescript II KS+ Vector. The obtained plasmid was suitable for generating a riboprobe in in vitro transcription.The probe for cVibratome (Leica VT 1000 S) to 50–80μm. Sections were collected and covered with cover slips and Aquatex (Merck).The embryos were embedded in 2, 5 − 4% agarose gel and sectioned with In-situ hybridized chicken embryos were embedded in Leica tissue freezing medium®and frozen with liquid nitrogen. Obtained blocks were sectioned with Leica CM3050 S cryo-stat. Sections were collected on slides, dried and covered using Aquatex (Merck).According to German legislation, the use of embryonic vertebrates in an animal experiment needs approval only if the animal is in the last third of its embryonic development. In the case of chicken, this means that experiments done on animals before embryonic day 14 (E14) are not regarded as an animal experiment by the Tierschutzgesetz, and therefore, do not need approval or governmental permission.The chicken embryos sacrificed for this work were between developmental stages HH+04 (E1) and HH32 (E7.5). All embryos were sacrificed at the end of the study by opening the shell and tearing the allantois and amnion with forceps. Thereafter, the embryos were immersed in 4% PFA/PBS solution for fixation. No permits were required for the described study, which complied with all relevant regulations.in-situ hybridization, a dynamic expression pattern of Axin2 was found from stages HH 04 to 32. In early embryogenesis, Axin2 expression was observed in the primitive streak (ps)(Axin2 from stage HH 07+ onwards (Axin2 transcripts were detectable in the Hensen′s node (hn), posterior presomitic mesoderm (psm)    and in the notochord (nc) , E and move (ng) , D.3 andhord (nc), the devn’s node , the preAxin2 in the dorso-medial lip (dml) appears (Axin2 (Axin2 expression is visible predominantly in the mid-brain (mb), C.2.Axin2 expression has shifted to a patch in the ventral mesoderm, flanking the mid-brain (mb), C.5 and cup (oc), C.5. ThWnt-signalling during gastrulation, neurulation, axis- and head formation. In the early patterning events of the vertebrate body, canonical Wnt-signalling is believed to first act as dorsalizing and later as posteriorizing signal [Wnt-mutant mice exhibit truncated posterior axis, lost tail formation and disturbed somitogenesis [Wnt overexpression [Wnt inhibition in the anterior embryonic tissue [Wnt inhibitors was found to induce notochord formation [Axin2 during chicken embryogenesis support the idea that appropriate regulation Wnt-signalling via Axin2 influences body patterning, axis elongation and head formation. The expression of several Wnts in the chicken primitive streak and Hensen’s node reinforce this hypothesis [Previous studies have investigated the role of g signal , 68. In ogenesis , 70. Exppression . Proper c tissue –74. Ectoormation . The exaormation . Furtherormation . These fpothesis .Axin2 mRNA from their onset  is stained for Axin2 in two longitudinal stripes, at first weakly  are distinguishable, expressing Axin2 in similar regions, compared to the earlier developmental stages, with little changes. Axin2 is expressed in brain and otic vesicle (ov) throughout these stages  by stage HH 26  on the back of the farthest developed stages   [Wnt1 and Wnt3a are required for the formation of the dml [Axin2 expression in the dml and its progenitors  . Additiogenitors  indicateAxin2 is expressed from neurulation to the differentiated mature neural tube (nt) and in the roof- and floor plate (rp)(fp) are intensively stained Figs . In Fig tube (nt), D.2. By (rp)(fp), G.1. Th (rp)(fp) reveals Axin2 transcripts were also found in secondary neurulation in the tail bud , but is expressed intensively in the dorsal third, such as in the ectoderm and subectodermal mesenchyme , A.2, asWnt family are expressed in the developing limb ([aer) [Wnt genes are described to initiate the formation of the limb bud (Wnt2b) from the lateral plate mesoderm as well as the aer (Wnt3a) [aer in chicken expresses Wnt3a that, by initiating fibroblast growth factor (FGF) expression, mediates the rapid cell proliferation in the mesenchymal progress zone (PZ) underlying the aer [Wnt7a is expressed in the dorsal ectoderm of the chicken limb, being responsible for dorsalization [Wnt3a in the ectoderm during early limb growth [Wnt7a target genes are expressed in the mesenchyme underlying the dorsal ectoderm, it was suggested that their signalling ranges as far as the target gene expression [Wnt3a from early dorsal limb ectoderm as a source for early Axin2 expression in the dorsal limb mesenchyme. Mutations of Wnt3a and Wnt7a and FGFs in chicken embryo induced the expression of a gene responsible for a form of polydactyly in human, the Townes-Brock-Syndrome [Wnt-signalling is described to promote cell proliferation and the differentiation of connective tissue [Axin2 expression in accordance to our results was reported in the perichondrium of mice [Axin2 in the chicken developing limb, we want to reveal its presumable function in regulating Wnt-signals that are involved in outgrowth, proliferation and differentiation.Several members of the ng limb ( for revib ([aer) . Wnt gen (Wnt3a) . The aer the aer . Non-canlization , 95. Itsb growth . As Wnt7pression . We postSyndrome . Later ie tissue . Axin2 e of mice . By descAxin2 expression in mesonephric development is described  and coelomic epithelium (coe) is observed  and overlying coelomic epithelium (coe) invaginates to the head mesenchyme. icle (ov), B.1. AxWnt signals are required for the initiation of the eye as described in zebrafish [Wnt2b is expressed in the proliferative lens epithelium [Wnt3 and Wnt11 were found to be expressed in the outer layer of the chicken optic cup [Wnt2b was described to be responsible for maintaining the proliferative state of neural progenitors in the retina in chick [Wnt2b signalling in the chicken eye [Axin2 expression in the lens overlaps with regions of increased cell proliferation, which express Wnt-ligands as well [Wnt3a and Wnt9b [Axin2, the Gardner syndrome, includes a congenital hypertrophy of the rpe [Wnt3 mutations, exhibit optic malformations [Anteriorly expressed inhibitors of canonical ebrafish , 118. Laithelium , retinalithelium –122. Furin chick . Previoucken eye . Our obs as well , 125. Thnd Wnt9b . Interes the rpe . Additiormations .Axin2 during embryonic development. We found a dynamic, temporally and spatially restricted expression pattern in many developing structures and tissues. In the early development of the chick, Axin2 was expressed in the primitive streak and underlying mesoderm, in the neural folds and in the head fold. It was additionally expressed during secondary neurulation in the tailbud mesenchyme. Here, the pre-somitic mesoderm as well transcribes Axin2. We were able to detect such expression in the posterior psm and during the maturation of the somites in its medial epithelium and in the dml. By this developmental stage, transcripts were also detectable in the brain and differentiating neural tube. In the developing limb a dynamic expression was found. Furthermore, we detected Axin2 mRNA in the nephric duct and coelomic epithelium. Regarding the head of the chicken embryo, Axin2 was expressed in branchial arches and sensory anlagen. Later in development, expression in feather buds, interdigital spaces, external ear and scleral ossicles on the eye was observed.In the present study, we describe the expression pattern of avian Axin2 in mice was previously found in the primitive streak, head folds, neural tube, branchial arches I and II (maxillary and mandibular arch), psm and dml, tailbud, limbs, kidney and brain [The expression of nd brain , 78.Axin2 expression in the developing eye and in the otic vesicle. With this study we want to point out the often neglected impact of Axin2 in many Wnt-dependant developmental processes. While Wnt-ligands are extensively studied, investigating their regulation through Axin2 in the respective tissues might help understanding the interactions of different signalling factors.These findings are mainly consistent to the expression we found in the chick. Additionally, we were able to show