The other murine monocyte subset expresses CX3CR1 and lower Ly6C levels, and contributes to repair in other disease models. We hypothesized that the Ly6Clo CX3CR1+ monocytes would contribute to recovery after intracerebral hemorrhage. Intracerebral hemorrhage was modeled by blood injection in WT and CX3CR1-null bone marrow chimeras. Neurological outcomes and leukocyte recruitment were quantified at various time points. Functional outcomes were equal at 1, 3, 7, and 14 days after intracerebral hemorrhage in both genotypes. No differences were observed in leukocyte recruitment between genotypes on either 3 or 7 days after intracerebral hemorrhage. A few hundred Ly6Clo monocytes were found in the ipsilateral hemisphere in each genotype and they did not change over time. Peripherally derived CX3CR1+ monocytes were observed in the perihematomal brain 7 and 14 days after intracerebral hemorrhage. Our data suggests CX3CR1 signaling on monocytes does not play an influential role in acute injury or functional recovery after intracerebral hemorrhage and therefore CX3CR1 is not a therapeutic target to improve outcome after intracerebral hemorrhage.Intracerebral hemorrhage is a subset of stroke for which there is no specific treatment. The Ly6C Based on these other models, we hypothesized that the Ly6Clo, CX3CR1+ monocytes would migrate into brain towards CX3CL1 and play an important role in functional recovery after ICH at sub-acute time points (days 3–14). Our results suggest that CX3CR1 on monocytes does not play an influential role in acute inflammation or functional recovery after ICH.The ligand for CX3CR1, CX3CL1, is constitutively expressed by neurons and soluble CX3CL1 is increased after brain injury GFP/GFP) mice were purchased from The Jackson Laboratory and then were bred in house. All mice were housed in standard conditions with a 12 hour light/dark cycle (lights on from 7∶00 am to 7∶00 pm), 22.2°C, and ad libitum access to food and water. All mouse procedures were completed with approval of the University of Connecticut Health Center Animal Care and Use Committee and were in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (Protocol number: 2010-0658).Male B6.SJL-Ptprca Pep3b/BoyJ (CD45.1), C57/BL6J (WT), and B6.129P–Cx3cr1tm1Litt/J  chimeras were created in order to specifically study the role of CX3CR1 on monocytes Mice were anesthetized and maintained with 1–5% isoflurane and all efforts were made to minimize pain and suffering. ICH was modeled by injecting 20 µl of whole blood from a WT donor into the right striatum 2.5 mm lateral to bregma, 3 mm deep, at a 5° angle as previously described Mice were behaviorally tested for 14 days after ICH to examine neurological recovery as previously described +, Ly6Chi, Ly6G−, CD11b+ (inflammatory monocytes), CD45.2+, Ly6Clo, CD11b+ (resident monocytes), CD45.2+, CD3+ (T cells), CD45.2+, Ly6G+, Ly6C+ (neutrophils), CD45.1+, CD11b+ (microglia). For samples used for intracellular cytokine stain, cells were stained with surface markers as described above and then fixed and permeabilized using a commercially available kit  for 20 minutes at 4°C. Cells were then stained with monoclonal anti-TNF-α  for 30 minutes at 4°C. Counting beads were added to samples prior to running and the sample recorded until event numbers dropped to few events/second. Cell population numbers were calculated by adjusting for counting beads recorded. To determine chimerism, flow cytometry was performed on blood samples. Blood was collected into tubes with 200U/mL heparinized-PBS and lysed twice prior to staining with cell surface markers described above. Percent chimerism was calculated by: Percent CD45.2+ cells/(percent CD45.2+ + percent CD45.1+ cells)  .Mice were intracardially perfused with 150 µg of Texas Red-tomato lectin (Vector Labs) at a concentration of 1 mg/ml Mice were sacrificed 7 days after ICH and brains were harvested, hemispheres were separated and were immediately flash frozen. Hemispheres were homogenized with 1 mm glass beads and RIPA buffer  containing phosphatase inhibitors (Roche) in a bead beater. Samples were then spun at 8,000 rcf for 30 seconds followed by sonication and an additional spin at 14,000 rcf for 15 minutes. BCA assay was performed to quantify protein concentration and 80 µg of total protein taken from brain tissue was used per well for the IL-6 and TNF ELISA assays (eBioscience).Based on means and standard errors of cylinder testing from prior work, an n = 7 is required to achieve 80% power to detect a 25% difference in laterality between groups at α = 0.05 lo monocytes in the ipsilateral hemisphere (n = 6–8). We have previously reported that the Ly6Chi, CCR2+ monocytes contribute to early injury after ICH lo, CX3CR1+ monocytes in other injury models, we sought to determine whether these cells were contributing to recovery.WT mice subjected to autologous blood injection showed a right forelimb preference twenty-four hours after ICH, indicating a left hemiparesis. By 72 hours, ICH mice have recovered equally to mice given sham surgery . Flow cylo, CX3CR1+ monocytes over time. Based on the reports that the Ly6Clo, CX3CR1+ monocytes aid in repair after spinal cord injury and myocardial infarction after 7 days, we expected the monocytes to increase in numbers in the perihematomal brain during the second week after ICH. Immunohistochemistry was performed 3, 7 and 14 days after ICH, time points when the CX3CR1+, Ly6Clo monocytes may be playing a role in tissue repair, to identify the location of the CX3CR1+ cells in the brain.Since CX3CR1 deficiency on microglia has been implicated in enhanced neuroinflammation + vasculature, CX3CR1+ peripherally-derived monocytes, and Ly6B.2+ inflammatory Ly6Chi monocytes and neutrophils (n = 5). Amoeboid, CD11b+, CX3CR1+ peripherally-derived monocytes were in the tissue in the perihematomal region 3–14 days after ICH. While Ly6B.2+ cells (Ly6Chi monocytes or neutrophils) were seen at day 3   . The CX3lo, CX3CR1+ monocyte population infiltrates injured tissue after the Ly6Chi. CCR2+ monocytes, peaking at 7 days in models of spinal cord injury lo, CX3CR1+ monocytes would increase after the peak of inflammation, therefore, we examined leukocyte populations in the ipsilateral hemisphere at day 7. In the blood, we again observed no differences in percentages of T cells, neutrophils, and Ly6Chi monocytes, with a similar reduction in the percentage of the Ly6Clo, CX3CR1+ monocytes (n = 9). Between days 3 and 7, both genotypes showed a decrease in neutrophil and Ly6Chi monocyte numbers and an increase in T cells in the ipsilateral hemisphere. However, the numbers of Ly6Clo, CX3CR1+ monocytes in the ipsilateral hemisphere did not increase between 3 and 7 days. Interestingly, we did not observe a difference in the numbers of Ly6Clo, CX3CR1+ monocytes between the WT BM chimeras and the CX3CR1-null BM chimeras, despite the lack of CX3CR1 signaling. This suggests that CX3CR1 is not the chemokine receptor involved in their recruitment into the brain.The Ly6Clo or Ly6Chi monocyte populations 14 days after ICH. We did not observe microglial TNF-α production 14 days after ICH in either genotype. We then measured phagocytic cell surface markers SIRPα and CD36. We did not observe differences in either SIRPα  or CD36 .In order to determine whether CX3CR1-deficiency on monocytes affects cytokine production and cell surface marker expression 14 days after ICH, we then performed intracellular cytokine stain (ICS). By ICS, we could not detect either the Ly6CCX3CR1 has been shown to regulate microglial neurotoxicity At baseline, there were no differences in behavior on cylinder test, open field, or top running speed between naïve, non-chimeric WT mice and CX3CR1-null mice, suggesting that the genetic background of the mice does not contribute to behavioral phenotype in the absence of injury (n = 3–9) . Twenty-lo, CX3CR1+ monocytes would be found in the brain after ICH. Monocytes have been shown to enter the ipsilateral hemisphere as early as 12 hours after ICH hi, CCR2+ monocytes contribute to acute injury after ICH lo, CX3CR1+ monocyte subset has been associated with wound repair and better functional outcome in models of spinal cord injury, myocardial infarction, and excitotoxic brain injury lo, CX3CR1+ monocytes remain in the blood and do not enter inflamed tissues lo CX3CR1+ monocytes do not enter the brain after experimental autoimmune encephalomyelitis and that tissue remodeling is regulated by microglia +, Ly6B.2−, peripherally-derived monocytes were in the perihematomal tissue, not in the vasculature, from 3–14 days after ICH  their recruitment is independent of CX3CR1 and (2) that CX3CR1 signaling does not affect functional recovery after ICH. We conclude that CX3CR1 is not a target to improve outcomes after ICH.Taken together, our results indicate that Ly6CFigure S1Method to calculate percent chimerism from blood samples. Leukocytes are gated on live and singlets prior to being located by forward and side scatter. Samples are then gated CD45.1 by CD45.2. Chimerism is calculated by dividing quadrant 1  by the sum of quadrant 1  and quadrant 3 . Percent chimerism = (Q1/(Q1+Q3)). Quadrant 1 and quadrant 3 are outlined in red boxes.(EPS)Click here for additional data file.Figure S2CX3CR1 deficiency does not affect functional outcomes at baseline.A) WT mice and CX3CR1 mice have similar initial weights. Means graphed with s.d. n = 8 B) WT mice and CX3CR1 mice do not have a forelimb preference at baseline. Means graphed with s.e.m., n = 8–9 C) Open field test shows CX3CR1 deficiency on monocytes does not impact spontaneous locomotor activity at baseline. n = 3–9 D) CX3CR1-null BM chimeras perform equally well on the forced run test at baseline. 7 and 14 days after ICH. Line represents median speed. n = 9.(EPS)Click here for additional data file.


Background. To investigate Wnt/β-catenin signaling pathway expression and its regulation of type I collagen by TGF-β1 in scleral fibroblasts from form-deprivation myopia (FDM) guinea pig model. Methods. Wnt isoforms were examined using genome microarrays. Scleral fibroblasts from FDM group and self-control (SC) group were cultured. Wnt isoforms, β-catenin, TGF-β1, and type I collagen expression levels were examined in the two groups with or without DKK-1 or TGF-β1 neutralizing antibody. Results. For genome microarrays, the expression of Wnt3 in FDM group was significantly greater as confirmed in retinal and scleral tissue. The expression of Wnt3 and β-catenin significantly increased in FDM group and decreased significantly with DKK-1. TGF-β1 expression level decreased significantly in FDM group and increased significantly with DKK-1. Along with morphological misalignment inside and outside cells, the amount of type I collagen decreased in FDM group. Furthermore, type I collagen increased and became regular in DKK-1 intervention group, whereas it decreased and rearranged more disorder in TGF-β1 neutralizing antibody intervention group. Conclusions. The activation of Wnt3/β-catenin signaling pathway was demonstrated in primary scleral fibroblasts in FDM. This pathway further reduced the expression of type I collagen by TGF-β1, which ultimately played a role in scleral remodeling during myopia development. Myopia has emerged as a major global public health issue, and it can increase the risk of irreversible visual impairment that would be a serious threat to human health and quality of life . Myopia β-catenin pathway is currently recognized as particularly important in embryogenesis and tissue regeneration [β-catenin pathway has functional links to the retina, choroid, and sclera through multiple cytokines, which play an important role in maintaining eye development after birth [β-catenin pathway and other three pathways, BMPs, Hedgehogs, and FGFs, played key roles during development of body tissues and organs [β-catenin in mRNA and protein from retina were significantly greater in FDM eyes than in control eyes [β-catenin pathway in regulating the process of myopia development. Moreover, Wnt/β-catenin signaling pathway is associated with several conditions related to ECM, such as organ fibrosis [β-catenin pathway for regulating the sclera ECM need to be discussed in order to suggest potential strategies for therapeutic purposes in myopia.The Wnt/neration . Researcer birth –12. The d organs , 13–15. d organs –20. A prrol eyes . Howeverfibrosis , 23, scafibrosis , and diafibrosis . This sifibrosis , 26. Furβ1 is a key regulator in fibrosis diseases and can induce the expression of components of ECM, especially type I collagen [β-catenin signaling pathway could affect the expression of TGF-β1 in fetal and postnatal fibroblasts [β1 and Wnt/β-catenin signaling pathway has an important role in regulating the expression of collagen [β1 is also reported to be involved in the regulation of scleral remodeling [β-catenin signaling pathway and TGF-β1 in scleral remodeling related to myopia remains unclear.Transforming growth factor- (TGF-) collagen . The Wntroblasts . Moreovecollagen , 30. TGFmodeling . Howeverβ-catenin signaling pathway in the expression of collagen by TGF-β1 in scleral fibroblasts from FDM to provide evidence for seeking new intervention targets in the pathogenesis of scleral remodeling in myopia.Our study aims to investigate the regulatory mechanism of Wnt/Two-week-old pigmented guinea pigs were obtained from the Shanghai Laboratory Animal Center . All animals in our study were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Ethics Committee of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China (Permit Number: 2009-0086). All animals were clinically examined to confirm corneal transparency of each eye and lack of injuries or infections in the eyes. Guinea pigs were reared on a cycle of 12 h illumination and 12 h darkness daily during the experimental period with freely available food and water.Thirty pigmented guinea pigs were randomly divided into two groups: the FDM group and the normal control (NC) group. In the FDM group, animals wore a translucent diffuser on one eye as previously reported , 33. TheAfter the successful establishment of the FDM guinea pig model, the retinal tissues in both the FDM group and the SC group were retrieved using microscissors. RNA was isolated using Trizol  according to the manufacturer's instructions. Whole genome microarrays in both the FDM group and SC group were used by the Gene Expression Microarray  according to the manufacturer's instructions. The extracted data were analyzed using the Agilent Feature Extraction Software.µg/mL streptomycin . Cells of 80% confluence were passaged by 0.25% trypsin-EDTA . The cells primary cultured were identified with vimentin  and keratin antibodies . Cells of 80% confluence were washed and cultured with DMEM (High Glucose) without FBS for 24 h and then incubated with the basal medium plus 100 ng/mL DKK-1 (Wnt/β-catenin pathway antagonist)  or 1 µg/mL TGF-β1 neutralizing antibodies  for 48 h to extract proteins or collect the supernatant.After myopia induction, eyes in both the FDM group and the SC group were immediately removed from each guinea pig under sterile conditions and rinsed with saline that contains gentamicin and phosphate-buffered saline (PBS) containing 10% penicillin and streptomycin three times. The scleral tissues were collected using microscissors, cut into 1 mm × 1 mm pieces, and carefully placed into separate flasks in DMEM (High Glucose)  containing 10% fetal bovine serum (FBS) , 100 U/mL penicillin, and 100 2O2 and washed three times (5 min each time) using PBS. The cells were blocked in 5% BSA for another 20 min. To stain the vimentin and keratin, the cells were incubated overnight with primary antibodies in PBS at 1 : 100 and 1 : 50 dilutions at 4°C. After being washed three times with PBS for 5 min each time, the samples were incubated for 20 min with secondary antibodies at 37°C and then washed three times with PBS for 2 min each time. The cells were then incubated for 30 min with streptavidin-biotin complex at 37°C and washed three more times with PBS for 5 min each time. After staining the cells with diaminobenzidine for approximately 3 min, distilled water was added to stop the reaction. PBS was used instead of the primary antibody for the blank control.Scleral fibroblasts grown on coverslips were immersed in 4% paraformaldehyde for 30 min. The coverslips were washed for 5 min in PBS three times. Subsequently, 0.1% Triton X-100 was added, and the cells were incubated at room temperature for 20 min. The cells were washed three times with PBS, for 5 min each time. They were then incubated for 15 min with 3% Hβ-catenin antibody , and type I collagen antibody  at a 1 : 250 dilution. After a rinse in TBST, the membranes were incubated for 1 h with a horseradish peroxidase-conjugated secondary antibody against rabbit or mouse IgG  in a 1 : 5000 dilution. Then, they were rinsed with TBST followed by SuperSignal West Pico Chemiluminescent Substrates  for detecting the blots. The densities of the bands were analyzed by the Gel-Pro Analyzer. The expression of β-actin was used as an internal control.Cells or tissues were sonicated in RIPA buffer containing protease inhibitors. The supernatants were collected after centrifugation. Proteins of an equal concentration were separated on 10% SDS-PAGE and transferred to a PVDF transfer membrane . The membranes were blocked in TBS containing 0.1% Tween-20 and 5% nonfat dry milk for 90 min, followed by an overnight incubation at 4°C with Wnt3 antibody  at a 1 : 1000 dilution, β1 ELISA kit . The optical density (OD) was measured at 450 nm on a microtiter plate reader. All measurements were performed twice, and the mean values were computed.Cell supernatant concentrations were measured by a TGF-Scleral fibroblasts grown on coverslips were immersed in 4% paraformaldehyde for 30 min. The coverslips were washed three times for 5 min each time in PBS. The cells were incubated overnight with type I collagen antibody  at a 1 : 500 dilution at 4°C. After being washed three times with PBS for 5 min each time, the samples were then incubated for 1 h with secondary antibodies at a 1 : 100 dilution at room temperature and then washed three times with PBS for 2 min each time. The cells were stained with PI for approximately 5 min and then observed with immunofluorescence microscopy.t-test, which was also used to compare the right eye and the left eye in the NC group. One-way ANOVA  was used to make comparisons among the groups after intervention. P < 0.05 was considered statistically significant. All statistical routines were used as implemented in SPSS version 19.0.All results were expressed as the mean ± standard error (SE). Comparisons between the FDM and the SC groups were made using an independent P > 0.05). In the third week, axial length in the FDM group increased significantly (P < 0.05) and refraction errors in the FDM group decreased significantly (P < 0.01) compared with those in SC group. In the fourth week, axial length in the FDM group was still longer than that in the SC group (P < 0.05), and refraction errors were still reduced (P < 0.01) (P > 0.05)  . In the  > 0.05) .The genome microarray result showed that the expression of Wnt3 was 2.13 times greater in the FDM group than in the SC group. Wnt3 also increased in the retina and scleral tissue in the FDM group observed by Western blot, consistent with the result of genome microarray .Starting on the third day, scleral fibroblasts grew out of scleral tissue pieces, which were stellate-shaped, and became spindle-shaped after six days of being cultured. On the ninth day, the tissues were removed, and the cells were fused 80%–90%. On the fifth day at the second passage, cell fusion was approximately 80%–90%.Through immunocytochemical staining, scleral cells cultured showed brownish yellow, which means positive staining for vimentin and negative staining for keratin and PBS. These results confirmed that the cells cultured were fibroblasts  Figure .β-catenin significantly increased in the FDM group compared with SC group (P < 0.05). The expressions significantly decreased in the FDM group with DKK-1 added (P < 0.05)  .β1 in cell supernatant decreased more significantly in the FDM group than in the SC group (P < 0.05). However, its expression increased significantly with DKK-1 (P < 0.05) but decreased further with the TGF-β1 neutralizing antibody in the FDM group (P < 0.05)  .P < 0.01). After adding DKK-1, type I collagen in the FDM group significantly increased (P < 0.05). After adding the TGF-β1 neutralizing antibody, the expression of type I collagen further reduced (P < 0.05)  .β1 neutralizing antibody (As observed by immunofluorescence, the arrangement of type I collagen was more disordered in the FDM group than in the SC group, and its expression in the FDM group also decreased. After adding DKK-1, the expression of collagen increased, and the arrangement was more regular. However, the arrangement of type I collagen was still disordered with the TGF-antibody .β-catenin pathway plays a critical role in the development of body tissues and organs. This pathway has a functional relationship with the cytokines in the retina, choroid, and sclera, which play an important role in maintaining eye development [β-catenin pathway may be involved in the process of FDM [β-catenin pathway in experimental myopia still remains unclear. Moreover, the downstream sites of Wnt/β-catenin pathway, which ultimately influences the scleral collagen expression, need to be further investigated.Myopia is a common ocular condition, characterized by the excessive elongation of the ocular globe . Experimelopment –12. Alths of FDM , the regβ-catenin signaling pathway was mainly induced by Wnt3 to regulate experimentally induced myopia. Wnt3 is one of canonical Wnts for activating Wnt/β-catenin signaling pathway [β-catenin signaling pathway in myopia.In this study, Wnt/ pathway . Researc pathway , 37. Wnt pathway , 39. Ourβ is a key factor in regulating ECM expression [β1 has a regulatory effect on scleral remodeling related to myopia [β are involved in a balanced network to regulate the optic vesicle development, neuron differentiation, and retinal stem cell survival [β-catenin signaling pathway can also affect the expression of TGF-β1 in fetal and postnatal fibroblasts [β1 produced in the FDM group was significantly lower than that in the control group, but it significantly increased after the addition of DKK-1, Wnt/β-catenin pathway antagonist. Therefore, Wnt3/β-catenin signaling pathway, as the upstream regulatory pathway, regulated the expression of TGF-β1 in scleral fibroblasts in experimental myopia.TGF-pression , and TGFo myopia . Moreovesurvival . Wnt/β-croblasts . Our stuβ1 mediates the communication among cells to make a regulatory effect in scleral remodeling related to myopia [β1 interacts with Wnt/β-catenin signaling pathway, which has a certain regulatory effect on the expression of collagen in human airway and skin cells [β-catenin signaling pathway is involved in the regulation of type I collagen expression with TGF-β1 in the development of FDM.Scleral remodeling is a dynamic process that involves continual synthesis and degradation of ECM produced by scleral fibroblasts. Type I collagen-dominated ECM is important in scleral remodeling in myopia . In addio myopia , 31. In in cells , 30. Howβ-catenin signaling pathway was presumed to regulate the pathological scleral remodeling process by affecting the expression of type I collagen. Second, in the FDM group treated with the TGF-β1 neutralizing antibody, the expression of type I collagen was further reduced, and the arrangement of collagen was more cluttered. We considered that TGF-β1, as the downstream factor of the Wnt3/β-catenin signaling pathway, could be involved in transmembrane bridges to mediate the internal and external communication between cells and ECM for regulating scleral remodeling.First, our study found that, along with the morphological misalignment inside and outside cells, type I collagen decreased significantly in the FDM group. After the addition of DKK-1, the expression of type I collagen increased, and the intracellular and intercellular collagen arrangements became orderly. Therefore, the Wnt3/β-catenin signaling pathway was activated during the development of FDM and that it could reduce the expression of TGF-β1 to mediate type I collagen-dominated ECM in the sclera. Furthermore, our results on the potential relationship between FDM and Wnt3/β-catenin signaling pathway clearly suggest a potential target for preventing and treating scleral remodeling in myopia.In summary, our study found that Wnt3/


Neurogenin1 (Neurog1) and repressed its expression. Consistent with this, Tcf3 repressed neuronal differentiation and increased the self-renewal activity of NPCs. We also found that Wnt signal stimulation reduces the level of Tcf3, and increases those of Tcf1  and Lef1, positive mediators of Wnt signaling, in NPCs. Together, these results suggest that Tcf3 antagonizes Wnt signaling in NPCs, thereby maintaining their undifferentiated state in the neocortex and that Wnt signaling promotes the transition from Tcf3-mediated repression to Tcf1/Lef1-mediated enhancement of Wnt signaling, constituting a positive feedback loop that facilitates neuronal differentiation.During mouse neocortical development, the Wnt–β-catenin signaling pathway plays essential roles in various phenomena including neuronal differentiation and proliferation of neural precursor cells (NPCs). Production of the appropriate number of neurons without depletion of the NPC population requires precise regulation of the balance between differentiation and maintenance of NPCs. However, the mechanism that suppresses Wnt signaling to prevent premature neuronal differentiation of NPCs is poorly understood. We now show that the HMG box transcription factor Tcf3  contributes to this mechanism. Tcf3 is highly expressed in undifferentiated NPCs in the mouse neocortex, and its expression is reduced in intermediate neuronal progenitors (INPs) committed to the neuronal fate. We found Tcf3 to be a repressor of Wnt signaling in neocortical NPCs in a reporter gene assay. Tcf3 bound to the promoter of the proneural bHLH gene  The canonical Wnt–β-catenin signaling pathway has a variety of roles in stem cell regulation during development and throughout adult life, ranging from maintenance of multipotency to induction of fate commitment Activation of the Wnt–β-catenin pathway results in the stabilization of β-catenin, which in turn associates with members of the Tcf/Lef family of DNA binding proteins and induces transcription of their target genes All animal experiments were performed in accordance with the protocol approved by the Animal Care and Use Committee of the University of Tokyo. Pregnant ICR mice were obtained from CLEA Japan  and Oriental Yeast Co. . Nestin-d4-Venus transgenic mouse line was kindly provided by Drs. Sunabori T. and Okano H. The plasmids pMX-EGFP (pMX-GFP) and pMXs-IRES-EGFP (pMXs-IG) were kindly provided by Dr. T. Kitamura. The plasmids Super8×TOP-FLASH (8×TOP-FLASH) and Super8×FOP-FLASH (8×FOP-FLASH) were kindly provided by Dr. Randall T Moon Antibodies used in this study were: goat antibodies to Tcf3  1∶1000, Neurog1  1∶200, Neurog2 (Santa Cruz) 1∶500, mouse antibodies to βIII-tubulin  1∶1000, Tcf3/4  1∶1000, Sox2 (R&D) 1∶100, chick antibody to GFP (Abcam) 1∶2000, and rabbit antibodies to GFP (MBL) 1∶1000, Pax6 (Chemicon) 1∶1000, Tbr2 (Chemicon) 1∶1000. Alexa-labeled secondary antibodies, TO-PRO-3 and Hoechst 33342 (for nuclear staining) were from Molecular Probes.Mouse embryos were fixed at 4°C in 4% paraformaldehyde in PBS for 0.5–12 h for immunohistochemistry (IHC) or overnight for in situ hybridization (ISH). Samples were cryoprotected overnight in 30% sucrose in PBS, embedded in OCT (Tissue-Tek), and frozen on dry ice. Frozen embryos were sectioned on a cryostat at 10–14 µm. Sections were processed for IHC or ISH as described Neocortical NPCs were isolated from the dosal cerebral cortex of mouse embryos at E11.5 and cultured in DMEM-F12 (Gibco) supplemented with 2% B27 (Gibco) and with or without FGF2  and EGF . Mouse Wnt3a protein, purified as described previously Premix Ex Taq (Takara). Primers are listed in RNA extraction was carried out using RNAiso (Takara) following the instructions of the manufacturer. Extracted RNAs were treated with DNaseI (Takara) to remove contaminating genomic DNA, and further purified using RNAiso. Total RNA was further purified by poly-A selection in some experiments. Total RNA and poly-A selected RNA were analyzed by Northern blotting using DIG-labeled cRNA probe according to the manufacturers’ instructions (Roche). Reverse transcription (RT) was performed with 2 µg of total RNA, oligo dT (20 mer) and ReverTra Ace (TOYOBO). The resulting cDNA was subjected to qPCR in a Roche LightCycler with SYBR t-test between control and experimental conditions. A P value of <0.05 was considered statistically significant. *P<0.05, **P<0.01, ***P<0.001 for all figures.A statistical analysis was performed using the unpaired two-tailed Student’s We investigated the expression patterns of Tcf3 in the developing neocortex as a candidate regulator of Wnt signaling. First we performed in situ hybridization analysis. We found Tcf3 mRNA was specifically expressed in the VZ but not in the intermediate zone (IMZ) or the cortical plate (CP) of the mouse neocortex at E11.5 and E14.5 ’, consisWe then asked whether Wnt signaling is active in neocortical NPCs. NPCs, acutely prepared from E11.5 mouse neocortices, were plated on poly-D-lysine (PDL) coated dishes and transfected with the reporter plasmids, and then cultured in the presence of FGF2 for 2 d. We found that the 8×TOP-FLASH reporter (TOP-FLASH), which harbors 8 repeats of the consensus binding sequence for Tcf/Lef family proteins (ATCAAAGC) upstream of the basal promoter, exhibited significantly higher activity than the 8×FOP-FLASH reporter (FOP-FLASH), in which each Tcf binding sequence is replaced by a mutant sequence (gcCAAAGC) . ImportaWnt signaling acts in the developing neocortex in a stage, cell type and context-dependent manner. Whereas it has been established that Wnt signaling promotes proliferation of NPCs, the role of Wnt signaling in promoting or suppressing neuronal differentiation of neocortical NPCs has been controversial. A recent report showed that overexpression of Tcf3 by in utero electroporation suppressed neuronal differentiation of neocortical NPCs, and knockdown of Tcf3 had an opposite effect We then examined whether endogenous Tcf3 also functions as a repressor of Wnt signaling in NPCs. Expression of two different short hairpin RNA (shRNA) constructs targeting Tcf3 (shTcf3#1 and #2) reduced the level of Tcf3 mRNA in NPCs (data not shown). This knockdown of Tcf3 significantly enhanced the activation of TOP-FLASH by Wnt treatment . This su+) cells that were positive for the neuronal marker βIII-tubulin, under a differentiation-inducing condition (culturing in the absence of growth factors) (+) cells  . Interesfactors) , possiblfactors)  [39]. Im+) cells . These r+) cells . These sTo investigate whether Tcf3 maintains the undifferentiated state of NPCs by suppressing neuronal differentiation, we carried out a neurosphere assay, which is commonly used for monitoring the amount of undifferentiated NPCs. We infected E11.5 neocortical cultures with retroviruses harboring Tcf3, ΔN-Tcf3 or Tcf1 and cultured them for 4 days in suspension (primary neurospheres). These cells were then dissociated completely and cultured in suspension at a very low  density (1000 cells/well in a 96 well dish) (secondary neurospheres). We found that the number of secondary neurospheres formed from 1000 cells obtained from primary neurospheres was significantly higher in Tcf3 or ΔN-Tcf3-expressing cultures compared with control cultures . These rNeurog1 gene   . Lef1 2n0072425) ’. We fou0072425) ’. Furthe0072425) . These rThe roles of the canonical Wnt pathway in early patterning of the cortex The functions of the full-length Tcf3 in suppressing Wnt signaling and neuronal differentiation were similar to those of a Tcf3 mutant that lacks the N-terminal β-catenin binding domain, suggesting that the full-length Tcf3 acts as a repressor of Wnt signaling independently of its β-catenin binding. This feature of Tcf3 is commonly observed in other systems + cells but becomes low in Neurog2+ cells in the neocortical VZ  Dissociated cells from E14.5 neocortices of wild-type mice (above) or Nestin-d4-Venus transgenic mice (bellow) were analyzed by FACS and sorted into Nestin-d4-Venus−, Nestin-d4-Venus +, Nestin-d4-Venus ++, and Nestin4-Venus +++ fractions.(TIF)Click here for additional data file.Figure S2Tcf3 does not so much affect the mRNA level of Cyclin D1 in NPCs. (Supplementary to) A, NPCs were infected with a retrovirus encoding control, Tcf3 shRNA #1 or Tcf3 shRNA #2 and incubated with FGF2 for 3 d. Cells were cultured for another 6 h in the absence of FGF2 (differentiated condition). The mRNA levels of Cyclin D1 was determined by qPCR analysis. B, E11.5 NPCs were infected with a retrovirus encoding control or Tcf3 and incubated with FGF2 for 3 d. The level of Cyclin D1 mRNA was determined by qPCR analysis. A,B, Data represents mean ± SEM.(TIF)Click here for additional data file.Text S1Primers used in this study.(DOCX)Click here for additional data file.


Wnt signaling was evaluated by measuring levels of β-catenin and expression of its target genes using immunoblotting. Retinal vascular permeability was measured using Evans blue as a tracer. Retinal NV was visualized by angiography and quantified by counting pre-retinal nuclei. Retinal pericyte loss was evaluated using retinal trypsin digestion. Electroretinography was performed to examine visual function. No abnormalities were detected in the β-catenin KO mice under normal conditions. In OIR, retinal levels of β-catenin and VEGF were significantly lower in the β-catenin KO mice than in littermate controls. The KO mice also had decreased retinal NV and vascular leakage in the OIR model. In the STZ-induced diabetic model, disruption of β-catenin in Müller cells attenuated over-expression of inflammatory cytokines and ameliorated pericyte dropout in the retina. These findings suggest that Wnt signaling activation in Müller cells contributes to retinal NV, vascular leakage and inflammation and represents a potential therapeutic target for DR.Retinal Müller cells are major producers of inflammatory and angiogenic cytokines which contribute to diabetic retinopathy (DR). Over-activation of the Wnt/β-catenin pathway has been shown to play an important pathogenic role in DR. However, the roles of Müller cell-derived Wnt/β-catenin signaling in retinal neovascularization (NV) and DR remain undefined. In the present study, mice with conditional  Retinal vascular leakage and retinal neovascularization (NV) are the major pathological changes leading to vision loss in diabetic retinopathy (DR) Recent studies have shown that the Wnt/β-catenin signaling pathway, which is known to regulate multiple biological and pathological processes including angiogenesis and inflammation, is involved in the pathogenesis of retinal vascular leakage, NV and inflammation in DR β-catenin out in Müller cells to study the impacts of interrupted Wnt signaling in Müller cells on ischemia-induced retinal NV and retinal inflammation in DR.Müller cells are the principal glial cells of the retina and the major producer of inflammatory and angiogenic factors such as VEGF, TNF-α and ICAM-1 in DR. It has been shown that Müller cell-derived VEGF is a key contributor to retinal NV, retinal inflammation and vascular leakage in DR 5′- CAC CAT GTC CTC TGT CTA TTC -3′) were used for PCR analysis to generate a 221-bp product for wild-type (WT) allele and a 324-bp product for β–catenin floxed allele, while primers C (5′- AGG TGT AGA GAA GGC ACT TAG C-3′) and D (5′-CTA ATC GCC ATC TTC CAG CAG G-3′) were used to detect a 411-bp product for CreAll animal procedures followed the Guidelines of the National Institutes of Health for the Use of Animal in Research and were approved by the Institutional Animal Care and Use Committees of the University of Oklahoma Health Sciences Center. The conditional KO mice were generated by cross-breeding β-catenin floxed mice with mice expressing Cre in retinal Müller cells. The Cre expressing mice were a gift from Dr. Yun Le at the University of Oklahoma Health Science Center. The cassettes of human PVMD2-rtTA and TRE-cre were used to generate the Cre transgenic mice. Cre expression in retinal Müller cells was confirmed by a Cre-activatable lacZ reporter mouse line (R26R) and a floxed interleukin six signal transducing receptor (gp130) mouse line, as described previously Oxygen-induced retinopathy (OIR) was generated by placing mice in 75% oxygen from P7 to P12, then returning them to room air, as described previously 2). The fragments were cultured in DMEM with 10% fetal bovine serum (FBS) and 1/100 penicillin/streptomycin. The retinal aggregates and debris were then removed. Attached cells were trypsinized and cultured in DMEM containing 10% FBS for another 5 days to obtain a purified population. Cultured cells of the 4th–6th passages were utilized, and serum free DMEM medium was applied before further analysis.Müller cell enrichment was conducted according to a previously established protocol Primary Müller cells were infected with an adenovirus expressing Cre  for 48 hr. An adenovirus expressing GFP (Ad-GFP) was used as control.Western blot analysis was performed as described previously Immunofluorence staining was performed according to a procedure described previously Retinal vascular permeability was measured using Evans blue as tracer following a documented procedure Retinal angiography was performed following a documented procedure with minor modifications Retinal morphometric analysis was performed as described previously Pre-retinal NV was quantified as described previously Retinal trypsin digestion assay was performed according to a previously published protocol ERG was performed as described previously The quantitative data were analyzed and compared with the littermate control. Quantitative data was expressed as mean ± SEM or mean ± SD. Statistical difference was analyzed by Student’s t test with statistical significance difference set at p<0.05.β-catenin disruption in the β-catenin KO mice, primary Müller cells were isolated and cultured from the retina of newborn β-catenin KO mice. Immunostaining of GS, a Müller cell marker, showed that more than 90% of the isolated primary cells were GS-positive , compared to WT/OIR mice under the same conditions  were determined by Western blot analysis using 50 µg total protein. Quantification of β-catenin, VEGF and TNF-α (B) by densitometry, normalized by β-actin levels and expressed as percentage of the control. All values are mean ± SD (n = 3). *p<0.05.(TIF)Click here for additional data file.


Cyclopia intermedia are used to brew honeybush tea, a herbal tea indigenous to South Africa. The aim of this study was to evaluate the protective effect of fermented honeybush extracts (FH ex) and scale-up fermented honeybush extracts (SFH ex) against ultraviolet B (UVB)-induced damage in HaCaT keratinocytes. To this end, we examined UVB-induced cell viability, antioxidant enzymes, and inflammatory mediators in HaCaT cells.The fermented leaves and stems of UVB significantly decreased HaCaT cell viability, whereas FH ex and SFH ex did not exhibit cytotoxic effects and increased the viability of the HaCaT cells. To further investigate the protective effects of FH ex on UVB-induced oxidative stress in HaCaT cells, the activities of superoxide dismutase (SOD), catalase (CAT), matrix metalloproteinases (MMPs), pro-inflammatory cytokines and skin barrier function in terms of involucrin, filaggrin, and loricrin were analyzed.UVB-induced treatment reduced the activity of antioxidant enzymes and skin barrier function, while FH ex or SFH ex increased their activity. These results suggest that FH ex exerted cytoprotective activity against UVB-induced oxidative stress in HaCaT cells through stimulation of antioxidant enzymes activities. Furthermore, FH ex and SFH ex suppressed the UVB-induced expression of inflammatory mediators, such as IL-1β, IL-6, and IL-8, at mRNA level together with down regulation of matrix metalloproteinase (MMPs). In addition, FH ex and SFH ex reversed the phosphorylation of mitogen-activated protein kinase (MAPK) induced by UVB-irradiation. Notably, FH ex and SFH ex markedly inhibited UVB-induced activation of ERK, p38, and JNK. Thus, this agent exhibits anti-oxidative and -inflammatory effects via lowering ROS production, suppressing p38, ERK, and JNK activation, and down-regulating expression of MMPs.These findings suggest that FH ex and SFH ex can be used as a skin anti-photoaging agent. Skin aging has been shown to an intrinsic component and a variable environmental component, termed photoaging. Clinically, photodamaged skin is characterized by loss of elasticity, increased roughness and dryness, irregular pigmentation, and deep wrinkling . Many ofHuman skin is directly exposed to the air, solar radiation, environmental pollutants, and other mechanical and chemical insults, which are capable of inducing the generation of free radicals, in addition to the reactive oxygen species (ROS) produced by during metabolism , 5. In eUVB-induced ROS activates matrix metalloproteinases (MMPs) in both the epidermis and dermis, and degrades collagen and elastin in the dermal extracellular matrix . Skin fi2-terminal kinase (JNK), and p38 MAPK.UVB exposure of the skin stimulates inflammatory responses via upregulation of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-8 . UVB-indFragments of collagen, a main constituent of the extracellular matrix (ECM), released as a result of MMP-induced collagen cleavage, are bioactive and participate in the regulation of the fibroblast phenotype during photoaging . Collage2O2 is converted to H2O and O2 either by catalase (CAT), located in the peroxisome as well as in the cytoplasm [Hytoplasm . In addiytoplasm .Cyclopia intermedia, or honeybush, is used as a herbal tea indigenous to South Africa [The fermented leaves and stems of h Africa . Honeybuh Africa . In prevh Africa . In our h Africa . FH ex wIn this study, we investigated the signaling pathway accounting for the cytoprotective effects of FH ex and SFH ex against UVB-induced oxidative stress in HaCaT cells. We found that FH ex or SFH ex protected cells against UVB-induced oxidative stress through stimulation of antioxidant enzyme activities. Furthermore, FH ex and SFH ex suppressed UVB-induced expression of inflammatory mediators, such as IL-1β, IL-6, and IL-8, at mRNA level, suppressing p38, ERK and JNK activation together with MMPs expression.Cyclopia intermedia was purchased from Rooibos.Ltd (www.rooibosltd.co.za), and dried samples (1.0 kg) were extracted with distilled water (8 L) two times under reflux for 1 h. The filtrate was evaporated in vacuo to afford the extract (200 g). To prepare fermented honeybush (FH ex), the method was slightly modied from the previous study [Streptococcus thermophilus) was inoculated in 100 mL of water with 5 % of the honeybush extract, 12.6 % of whole milk powder, and 3 % of sucrose. The mixture was fermented in a 37 °C incubator for 24 h. For the preparation of scale-up fermented honyebush (SFH ex), honeybush was extracted with water 2 times under reflux and then filtered. The filtrate was evaporated to give 20 brix extract. To prepare SFH ex, 10 % of lactic acid bacteria (Streptococcus thermophilus) was inoculated in 0.5 % of whole milk powder with 3 % of lactose, 23 % of the honeybush extract (20 brix), and 63.5 % of purified water. The mixture was fermented in a 37 °C incubator for 48 h. After sterilization of fermented mixture, final product was obtained by mixing dextrin (DE12) and spray drying. DMEM media and fetal bovine serum (FBS) were purchased from Gibco BRL . A CellTiter Aqueous One Solution Cell Proliferation Assay kit (MTS) was purchased from Promega Co. . Human total MMP-1 and -9 ELISA kits were obtained from R&D Systems, Inc. .us study . To prep2 in a humidified incubator. HaCaT cells were seeded and allowed to adhere for 24 h. The cells were treated with various concentrations of FH ex and SFH ex and exposed to UVB radiation at a dose of 20 mJ/cm2. Cells that received no pretreatment and were not exposed to UVB irradiation served as the control.HaCaT cells, an immortalized non-tumorigenic human keratinocyte cell line obtained from Huons Co. Ltd, , were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum and 1 % antibiotics at 37 °C and 5 % CO4) were seeded in 96-well culture plates and treated with various concentrations of FH ex and SFH ex for 24 h. Before UVB treatment, the cell culture medium was replaced by phosphate-buffered saline (PBS) and exposed to 20 mJ/cm2 of UVB. Immediately after UVB irradiation, cell viability was assessed after 1 h by reduction of [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) to formazan, according to the manufacturer’s instruction. Samples were then assayed at 490 nm using a microplate fluorimeter .HaCaT cells  according to the manufacturer’s protocol. Cells were treated with FH ex for 24 h. After UVB exposure, cells were homogenized in cold lysis buffer. The absorbance was measured at 450 nm to determine SOD activity, and at 540 nm to determine CAT activity, by using a plate reader .4) were determined using human total MMP-1 and -9 ELISA kits according to the manufacturer’s instructions. HaCaT cells were seeded in 96-well plates and treated with FH ex and SFH ex. After exposure to UVB, the culture supernatant was collected and centrifuged at 13,000 rpm for 5 min. The levels of MMP-1 and MMP-9 in culture supernatants were quantified by colorimetric analysis.After UVB irradiation, matrix metalloproteinases-1 and -9 levels in the culture media of HaCaT cells  method. Each sample was run in triplicate and the gene encoding β-actin was used as a control to normalize for differences in the amount of total RNA in each sample.Total RNA was extracted from each sample treated with UVB using TRIzol reagent . Real-time PCR was performed using TaqMan Gene assays  specific for the genes encoding MMP-1, MMP-9, tissue inhibitor of metalloproteinase (TIMP), involucrin, filaggrin, loricrin, IL-1β, IL-6, IL-8, on a QuantStudioProtein was extracted from HaCaT cells. Twenty microgram of protein lysates from each sample were electrophoresed on a 10 % sodium dodecyl sulfate (SDS)-polyacrylamide gel and were then transferred to PVDF membranes. Membranes were blocked for 1 h at room temperature in 5 % skim milk solution. Blots were incubated overnight at 4 °C with a 1:1000-diluted monoclonal antibody. The blots were washed three times for 10 min each time with Tris-buffered Saline (TBST). The membranes were then incubated for 2 h with secondary antibody. Proteins were detected using an enhanced chemiluminescence.p < 0.05 was considered statistically significant. GraphPad Prism 5 was used for statistical analyses.Results were compared using Tukey’s multiple comparison test, where a We investigated the effect of FH ex and SFH ex on the proliferation of HaCaT cells after exposure to UVB. Cell viability was reduced to 59.14 % by UVB irradiation in the absence of FH ex, but cell proliferation was increased up to 96.78 % Fig.  in its pTo investigate whether the radical scavenging activity of FH ex was mediated by antioxidant enzymes, the activities of antioxidant enzymes were examined in HaCaT cells after UVB exposure. SOD activity decreased to 0.7 U/ml in UVB-induced cells compared to the untreated control Fig. , SOD actWe evaluated the effect of FH ex and SFH ex on UVB-induced MMP expression in HaCaT cells. UVB irradiation of HaCaT cells resulted in a marked increase in MMP-1 and -9 levels Fig. . ELISA aThe mRNA expression of MMP-1 and TIMP genes in HaCaT cells after UVB irradiation was measured by RT-PCR Fig. . UVB cauWe showed that the expression of involucrin, filaggrin, and loricrin was decreased by UVB. As shown in Fig. To evaluate the effects of FH ex and SFH ex on the UVB-induced production of inflammatory mediators, cells were treated with FH ex and SFH ex for 24 h prior to UVB exposure. UVB significantly increased the levels of IL-1β, IL-6, and IL-8 mRNA as compared with those in the normal cells (Fig. We examined whether FH ex and SFH ex could suppress UVB-induced phosphorylation of MAPKs, including ERK, MEK, p38, and JNK. As shown in Fig. Herbal ingredients are used for dermatological purposes, as oral dietary supplements or are incorporated into topical formulations . Their pHuman skin is equipped with a network of antioxidant enzymes that play an important role in the balance of intracellular ROS levels . ExposurMMPs are a large family of degradative enzymes that play important roles in tissue destruction during pathological processes, such as arthritis, skin aging, tumor invasion, and metastasis . MMPs deFilaggrin and profilaggrin are essential for maintaining homeostasis in the epidermis and filaggrin also undergoes further processing in the upper stratum corneum to release free amino acids that assist in water retention . InvolucKeratinocytes respond to the major changes in inflammation and immunomodulation observed after UVB exposure, at least in part via UVB-induced expression of inflammatory enzymes and release of pro-inflammatory mediators . ChronicROS are known to play a role in UV-induced skin damage and aging. Exposure to UV irradiation induces the production of ROS in skin cells; this ROS activates cellular signaling pathways that activate kinases such as p38, JNK, and MAPK. Previous studies have shown activation of the three MAP kinases, ERK, JNK, and p38, within 1 h of acute UV radiation . We obseIn conclusion, these agents exhibit anti-oxidative and inflammatory effects that are mediated by lowering ROS production, suppressing p38, ERK, MEK and JNK activation, and down-regulating expression of MMPs. These findings suggest that FH ex and SFH ex can be used as a skin anti-photoaging agent.CAT, catalase; DMEM, Dulbecco's modified eagle medium; FH ex, fermented honeybush extracts; MAPK, mitogen-activated protein kinase; MMPs, matrix metalloproteinase; mRNA, messenger RNA; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; qRT-PCR, quantitative real-time polymerase chain reaction; RNA, ribonucleic acid; ROS, reactive oxygen species; SFH ex, scale-up fermented honeybush extracts; SOD, superoxide dismutase; TBST, Tris-buffered Saline; TIMP, tissue inhibitor of metalloproteinase; UVB, ultraviolet B.


Localized Wnt/β-catenin signaling maintains this progenitor population toward the distal tip of the regenerative blastema. As they become proximally displaced, preosteoblasts upregulate sp7 and subsequently mature into re-epithelialized Runx2−/sp7+ osteoblasts that extend preexisting bone. Auto-crine bone morphogenetic protein (BMP) signaling promotes osteoblast differentiation by activating sp7 expression and counters Wnt by inducing Dickkopf-related Wnt antagonists. As such, opposing activities of Wnt and BMP coordinate the simultaneous demand for growth and differentiation during bone regeneration. This hierarchical signaling network model provides a conceptual framework for understanding innate bone repair and regeneration mechanisms and rationally designing regenerative therapeutics.Zebrafish fully regenerate lost bone, including after fin amputation, through a process mediated by dedifferentiated, lineage-restricted osteoblasts. Mechanisms controlling the osteoblast regenerative program from its initiation through reossification are poorly understood. We show that fin amputation induces a Wnt/β-catenin-dependent epithelial to mesenchymal transformation (EMT) of osteoblasts in order to generate proliferative Runx2 Among mammalian organs, bones have unusually effective repair mechanisms, as demonstrated by continuous bone remodeling throughout life and the scarless healing of some fractures. Still, traumatic injuries or disease frequently exceed the innate regenerative capacities of human bone . EnvisioRunx2-null mice lack all ossification  pathways, both central components of bone developmental regulatory networks , are key determinants of the osteoblast lineage . Runx2-nfication . Similarrentiate . Runx2 fpression . Transcrnetworks .Runx2 and Sp7 expression and a corresponding failure of bone formation, suggesting osteogenesis requires Wnt/β-catenin signaling . BMPRs phosphorylate Smad1/5/8 transcription factors, inducing their translocation to the nucleus where they activate downstream genes. BMPs are required for osteoblast-lineage commitment of cranial mesenchyme , and loslate Sp7 . These alate Sp7 .Runx2 and Sp7 the master regulators of osteogenesis, are coordinated during the repair process  of osteoblasts lining the damaged bone. Distal Wnt signaling continuously maintains Runx2+ preosteoblasts, whereas their differentiation into mature, sp7+ osteoblasts is promoted by autocrine BMP signaling. By inducing secreted Wnt antagonists, including dkk1b, BMP facilitates negative crosstalk between differentiating osteoblasts and preosteoblasts. This straightforward signaling network produces a hierarchically and spatially organized osteoblast lineage that balances a simultaneous need for bone growth and differentiation until fin regeneration is complete.Here, we examine mechanisms of regenerative osteogenesis in the zebrafish caudal fin. Upon fin amputation, Runx2+ mesenchymal cells , osteoblasts lining preexisting bone adjacent to the amputation site upregulated expression of Runx2. Further, Runx2, but not sp7, expressing cells were found distal to the amputation site distributed in the mesenchymal tissue of the forming blastema . At 32 hal cells . By 48 hion site . At 72 hion site . Runx2+ ectively .+ and Runx2+/sp7+ cells were found in equal proportion (+ cells (−5). More Runx2+ cells incorporated EdU compared to sp7+ cells (16% versus 7%)  to label cells in S phase 6 hr prior to harvesting fins at 72 hpa. In stained section fins, we observed robust EdU labeling in the distal blastema mesenchyme but only rarely in the epidermis , in agreoportion , ~45% wi+ cells were derived from preexisting osteoblasts. We generated adult zebrafish with caudal fins containing isolated fin rays lined with permanently labeled mCherry+ osteoblasts  of osteoblasts, which display an underappreciated epithelial-like organization , to geneth zns-5 , α-catenth zns-5  and β-cath zns-5 , which wTg(sp7:EGFP)b1212 animals, which express EGFP in mature and differentiating osteoblasts  animals showed robust nuclear β-catenin primarily in distal Runx2+ cells and not in proximal osteoblasts that expressed only sp7:EGFP w25 transgenic fish that express destabilized GFP under the control of a synthetic Wnt-responsive promoter  animals with IWP-2 and monitored regeneration in individual animals from 0 to 8 dpa. At 2 and 4 dpa, IWP-2-treated fish arrested regeneration after forming a wound epidermis and lacked +sp7:EGFP osteoblasts beyond the amputation site  requires Wnt activity. Sections of fins from fish treated with IWP-2 from 0 to 24 hpa lacked both nuclear-localized β-catenin and Runx2-expressing blastema cells . Howeverpression . These rWe tested if Wnt has an ongoing role maintaining preosteoblasts during bone regrowth by blocking Wnt production from 48 to 72 hpa using IWP-2. This IWP-2 regimen stunted overall regeneration and depleted osteoblast-lineage cells distal to the amputation site . The few+ preosteoblasts and adjacent nonosteoblast distal mesenchymal cells. Therefore, the loss of osteoblasts after 24 hr IWP-2 treatment could represent direct and/or indirect Wnt signaling roles. To distinguish between these possibilities, we inhibited Wnt for brief periods (8 hr) during regeneration and monitored β-catenin, Runx2, and sp7 by immunostaining. Administration of IWP-2 or Wnt-C59 from 64 to 72 hpa was sufficient to inhibit nuclear localization of β-catenin . To test if Wnt directly promotes fin osteoblast proliferation, we treated these cultures with Wnt3A and monitored EdU incorporation in Runx2Tg(sp7:EGFP) fish with antibodies against phosphorylated, active Smad1, 5, and 8 (pSmad1/5/8), Runx2, and EGFP. We observed pSmad1/5/8 in differentiating +sp7:EGFP cells but not Runx2+/sp7:EGFP− preosteoblasts or other cell types (+ maturing osteoblasts (BMPs have been implicated in osteoblast differentiation  and are ll types . pSmad1/eoblasts . The disTg(sp7:EGFP) animals using the BMPR inhibitor (BMPRi) LDN193189  and decreased the Runx2+/sp7+ and the sp7+ populations (p < 0.007 for the Runx2+/sp7+ cells and p < 0.02 for the sp7+ population). EdU incorporation   . We recoqRT-PCR) , which wdkk1a, dkk1b, dkk2, and dkk3b, among which dkk1a, dkk1b, and dkk3b expression were reduced upon BMPRi treatment, as was sp7 w32 fins, subjected them to a heat shock regimen from 48 to 72 hpa, and examined β-catenin localization and Runx2 expression. Dkk1b induction decreased nuclear-localized β-catenin and produced a pronounced deficiency in Runx2+ preosteoblasts  generate dedifferentiated progenitors from mature osteoblasts, (2) maintain this preosteoblast population until regeneration is complete, and (3) spatially and temporally restrict redifferentiation to a subset of preosteoblasts that progressively reform lost bone. The latter two processes are opposing activities; a fine balance between progenitor expansion and terminal differentiation must be maintained to sustain bone regeneration over 2 or more weeks. Our studies suggest a model for bone regeneration in the fin  whereby ion site . As osteion site .+ preosteoblast proliferation during fin regeneration is consistent with known functions for the Wnt/β-catenin pathway in maintaining progenitor cells in diverse biological settings  and Hedgehog b1212 (Tg(hsp70l:dkk1b-GFP)w32  b1230 (Tg(TOP:GFP)w25  vu295a b1212 , Tg(hsp7-GFP)w32 , Tg(duspP) b1230 , Tg(TOP::GFP)w25 , and Tg vu295a  lines we) vu295a .Sections were rehydrated and antigen retrieval was performed for 10 min in a pressure cooker in retrieval buffer . Antibodies were diluted in PBST containing 10% nonfat dry milk and applied to slides overnight at 4°C, followed by staining with Alexa-conjugated secondary antibodies (Invitrogen) and Hoechst nuclear staining. Antibody staining details are provided in the Wnt pathway inhibitor IWP-2 and the BMPR inhibitor, LDN193189, referred to in the text and figures as BMPRi, were purchased from StemRD; Wnt-C59 was from Biovision; all were dissolved in DMSO. At the indicated times after fin amputation, IWP-2, Wnt-C59, BMPRi, or DMSO was added to fish water  at 10 μM, 100 nM, or 5 μM, respectively. For each drug treatment (n = 3), cohorts of three or four animals were used, each was analyzed at the completion of the study, and images shown are representative examples of each cohort.For combination fluorescent in situ hybridization immunostaining, sections on slides were sequentially antigen retrieval treated, hybridized with DIG-labeled probes, stained with α-DIG peroxidase-conjugated antibody (Roche), developed using the TSA system (PerkinElmer), and finally immunostained. Protocol details and probe synthesis are described in the These methods are presented in the + cells between osteoblast subtypes, combining osteoblasts scored in 12 rays from at least four fish . To assess changes in EdU incorporation between drug-treated fish, comparable stained sections were scored for the fraction of EdU incorporating cells of each osteoblast subtype. Student’s t tests assessed differences between drug-treated groups using the means of each subtype’s percentage of EdU+ incorporating cells across individual rays. For nuclear β-catenin quantitation in cultured osteoblasts, one-way ANOVA and post hoc Tukey’s tests were used to determine significant differences in the ratio of nuclear to total β-catenin in individual cells across and between treatments. To determine differentially expressed gene in qRT-PCR studies, two-tailed Student’s t tests used raw ΔCts from three or four independent control and experimental samples.Statistically significant differences between osteoblast subtypes were determined by scoring their fractional representation on comparable immuno-stained sections (for 12


Sema3f is expressed in the epidermis and Sema3g expression is restricted to arteries, whereas their receptor Nrp2 is preferentially expressed by lymphatic vessels. Both Sema3f;Sema3g double mutants and Nrp2 mutants exhibit increased LEC growth in the skin. In contrast, Sema3f;Sema3g double mutants display increased lymphatic branching, while Nrp2 mutants exhibit reduced lymphatic branching. A targeted mutation in PlexinA1 or PlexinA2, signal transducers forming a receptor complex with NRP2 for SEMA3s, exhibits an increase in LEC growth and lymphatic branching as observed in Sema3f;Sema3g double mutants. Our results provide the first evidence that SEMA3F and SEMA3G function as a negative regulator for dermal lymphangiogenesis in vivo. The reciprocal phenotype in lymphatic branching between Sema3f;Sema3g double mutants and Nrp2 mutants suggest a complex NRP2 function that regulates LEC behavior both positively and negatively, through a binding with VEGFC or SEMA3s.The development of a patterned lymphatic vascular network is essential for proper lymphatic functions during organ development and homeostasis. Here we report that class 3 semaphorins (SEMA3s), SEMA3F and SEMA3G negatively regulate lymphatic endothelial cell (LEC) growth and sprouting to control dermal lymphatic network formation. Neuropilin2 (NRP2) functions as a receptor for SEMA3F and SEMA3G, as well as vascular endothelial growth factor C (VEGFC). In culture, Both SEMA3F and SEMA3G inhibit VEGFC-mediated sprouting and proliferation of human dermal LECs. In the developing mouse skin,  VEGFC s in vivo . In Vegfculature .in vitro  acts as a co-receptor to modulate VEGFC/VEGFR3 signaling in lymphatic vessel development. The interaction of NRP2 with VEGFR3 enhances VEGFC-induced LEC survival and migration in vitro . Nrp2 mu vessels . Further vessels . Selecti vessels .in vitro , a family of ligands first identified for its role in axon guidance . SEMA3B in vitro . SEMA3F in vitro . Recentlin vitro . AlthougIn this study, we provide the first evidence demonstrating that SEMA3F and SEMA3G negatively regulate lymphatic vessel development. The NRP2 receptor has a dual function that regulates lymphatic sprouting and LEC proliferation both positively and negatively, through a binding with VEGFC or SEMA3s.in vitro. VEGFC stimulates sprouting and proliferation of human LECs in a dose-dependent manner (data not shown). We found that both SEMA3F and SEMA3G inhibit VEGFC-mediated human LEC sprouting in 3D collagen gel  respond to SEMA3F and SEMA3G in culture, dermal-derived human lymphatic microvascular ECs  were treated with AP-SEMA3F and AP-SEMA3G. Both SEMA3F and SEMA3G dramatically induced cell contraction A-D. We nagen gel E-H. We aagen gel I-L. ThesSema3b, Sema3c, Sema3f and Sema3g in E15.5 skin by in situ hybridization, stages when lymphatic network is actively developing in the skin. We found distinct expression patterns of Sema3c and Sema3f . Expression of Sema3c was detectable in the layer adjacent to the boundary between the dermis and hypodermis . To clarify the role of NRP2 on the LEC behavior, we therefore turned to in vitro LEC sprouting assay and examine what happens to SEMA3s-mediated inhibition of LEC sprouting in the absence of NRP2.The abovementioned results suggest that Sema3F/PlexinA1/NRP2 signaling controls LEC growth in lymphatic vessel development. However, phenotypic differences in lymphatic vessel branching between Nrp2-deficient LECs failed to respond to SEMA3G such that the cells did not show SEMA3G-induced cell contraction . One possible explanation is that the reduction of lymphatic sprouting in Nrp2 mutants may reflect the lack of VEGFC/VEGFR3/NRP2-mediated positive signaling. This possibility is supported by previous studies demonstrating that selectively inhibiting VEGFC binding to NRP2 with a function-blocking antibody results in the reduction of VEGFC-induced LEC migration but not LEC proliferation  or postnatal stage, and no apparent defective lymphatic network and valve formation was detectable in the adult skin. The restoration of defective lymphatic vessels has been observed in Nrp2 mutants . Genotyping for the Sema3g-lacZ allele was carried out with the following primer pairs: 5′-GCGGCTCTCCTACAGAGGTACTG-3′ and 5′-TGTCCTAGAGCCACGGACATTC-3′ as primers to detect wild-type allele; 5′-TAGCTGCACGGGCATTGAGC-3′ and 5′-GTCTGTCCTAGCTTCCTCACTG-3′ as primers to detect mutant allele. The plexinA1, plexinA2, and Sema3f mice have been reported elsewhere (rp2 mice  were purlsewhere . All aniIn situ hybridization using digoxigenin (DIG)-labeled riboprobes was essentially as previously described  with fibronectin coating, then treated with 1 nM of alkaline phosphatase (AP) control protein, mouse AP-Sema3F with cleavage resistant point mutations (R580A/R582A), or mouse AP-SEMA3G for 30 min. Sprouting assays were modified as previously reported  or AP-Sema3G, for 3 days. RNAi experiment of HMVEC-dLy-Neo, 5×105 of the cells were cultured on 6 cm dish for 2 days. Then, siRNA (Ambion) were transfected 2 times by lipofectamine RNAiMAX (Life Technologies) each 2 days. The siRNAs for Nrp2 and negative control are obtained from Silencer Select Validated/Pre-designed siRNA (Ambion); Nrp2#1 (ID#s16840), Nrp2#2 (ID#s16841) and Silencer Select Negative Control No.1 (cat#4390843). Nrp2 knockdown was detected by immunoblot analysis with anti-NRP2 antibody  and anti-β-actin antibody .Dermal-derived human lymphatic microvascular ECs  were cultured in EBM-2 medium with growth supplements. About 75% of HMVEC-dLy-Neo cells are positive for PROX1 (data not shown). For cell contraction assays, 5×10reported . To makeNrp2, Vegfr3 and Plexins were from PrimerBank (Nrp2 (ID#116686133c2); 5′-GGCAAATGGTTACTCCATGACC-3′ and 5′-ACAACCCGTGTGTCTTCACTG as primers for Vegfr3 (ID#226874801c1); 5′-GGTTGGACGATCTCTTCAAGC-3′ and 5′-GGGGAAGTATTCTGATTTGCCAT-3′ as primers for PlexinA1 (ID#253683485c3); 5′-ACTCTGAGAATCGTGACTGGA-3′ and 5′-TGTTGGTGAGTGTAAGCACTTC-3′ as primers for PlexinA2 (ID#113722112c2); 5′-CAGATACCACTCTGACTCACCT-3′ and 5′-GGCCCGTAGCTCAGTTAGG-3′ as primers for PlexinA3 (ID#6679391a1); 5′-ACAGGGCACATTTATTTGGGG-3′ and 5′-CACTTGGGGTTGTCCTCATCT-3′ as primers for PlexinA4 (ID#171543898c1); 5′-GAGCAAGCGCAACATACAGC-3′ and 5′-GGCTACAGTCAGCACTCGC-3′ as primers for PlexinD1 (ID#153792703c2).FACS isolation of LECs from embryos , and subimerBank : 5′-GACT


Wnt/β-catenin signaling is relatively understudied in immunity and autoimmunity. β-catenin blocks inflammatory mediators and favors tolerogenic dendritic cell (DC) phenotypes. We show here that leukocytes from lupus-prone mice and SLE patients express diminished β-catenin transcriptional activity, particularly in myeloid cells, although other leukocytes revealed similar trends. Serum levels of DKK-1, an inhibitor under transcriptional control of Wnt/β-catenin, were also decreased in lupus-prone mice. Surprisingly, however, preemptive deletion of β-catenin from macrophages appears to have no effect on lupus development, even in mice with varying genetic loads for lupus. Although myeloid-specific loss of β-catenin does not seem to be important for lupus development, the potential role of this transcription factor in other leukocytes and renal cells remain to be elucidated. Drosophila as homolog Armadillo, β-catenin functions in cell adhesion as well as in downstream control of transcription [The Wnt/β-catenin or “canonical” Wnt pathway is a ubiquitous, conserved signaling pathway. First characterized in cription –3. β-catcription , 5. In tcription .Drosophila and embryonic lethality in mice [This transcriptional activity is involved in multiple cell fate decisions in the embryo and adulthood. Armadillo/β-catenin knockout leads to gross malformation in  in mice , 8. Brea in mice , 10. Abe in mice , 11–13.The Wnt/β-catenin pathway also plays roles in immunity, contributing to hematopoietic stem cell (HSC) maintenance, thymocyte differentiation, pre-B cell proliferation, and the type I interferon response –17 In maWe and others have screened lupus-prone mice and SLE patient serum and find increased β-catenin-related markers . Given tCongenic control (C57BL/6 or B6), Mrl-lpr, Ctnnbfl/fl , and LyzPatient samples were collected from consecutively seen patients at a Rheumatology outpatient clinic at UT Southwestern Medical Center, without any pre-selection. All patients fulfilled ACR classification criteria for the diagnosis of SLE. Patient demographics are summarized in Splenocytes or PBMCs were isolated by centrifugation alone or on a Ficoll gradient, lysed, and prepared for Western Blot analysis in sample buffer by boiling for 10 minutes. Samples were spun down, subjected to SDS-PAGE, and transferred using standard procedures. Blots were probed for β-catenin , pSer33 β-catenin , Axin-2 , GAPDH , and β-actin . Secondary HRP-conjugated antibodies  were selected against primary antibody epitopes as appropriate. For analysis, bands were quantified using ImageJ®.Kidneys and spleens were isolated and laterally hemisected and either fixed in OCT and frozen for immunofluorescence (IF) or embedded in paraffin for immunohistochemistry (IHC). Sections comprised 3-5mm and antigens were exposed with a sodium citrate buffer. Sections were blocked in 5% milk prior to staining with rabbit anti-β-catenin , rabbit anti-pSer33 β-catenin , and APC-conjugated anti-F4/80 . Anti-Rabbit HRP conjugate antibodies were used as a secondary stain RNA was extracted from tissue and reverse-transcribed to produce cDNA using standard protocols. In brief, splenocytes were harvested, filtered through a fine mesh, depleted of red blood cells by hypotonic solution, and pelleted. Alternatively, PBMCs were harvested by centrifugation over a Ficoll gradient and isolated from the buffy coat layer. Each sample was then resuspended and sonicated in 1ml TRIzol® Reagent (Life Technologies #15596–026) and left at room temperature for 5 minutes. 200μl chloroform was then added to each sample and each sample was vortexed 15 seconds and left at room temperature for 3 minutes. Samples were then centrifuged at 12,000 x g for 15 minutes and the top (aqueous) layer was transferred to a new tube. 500μl isopropanol were added to each tube and left at room temperature for 10 minutes. Samples were then centrifuged at 12,000 x g for 10 minutes and supernatant was aspirated. Pellets were washed with 500μl 80% ethanol (w/v) and centrifuged at 7,500 x g for 5 minutes. Supernatant was removed carefully by pipette. Samples were left to air dry 2–3 minutes followed by incubation at 70°C for 2–3 minutes. Resultant RNA was resuspended in 80μl and quantified using NanoDrop® 1000 (Thermo Fisher).cDNA was produced from the foregoing RNA using a High Capacity cDNA Kit . In brief, a master mix was prepared with 2μl 10X RT-PCR buffer, 2μl10x RT primers, 0.8μl 100μM dNTP mix, and 1μl transcriptase per sample. Water and isolated RNA were added to 14.2μl and 1μg RNA for a final volume of 20μl in each sample. Samples were treated with thermocycler at 25°C for 10 minutes, 37°C for 120 minutes, and 85°C for 5 minutes.The resultant cDNA was then detected using Q-PCR with SybrGreen® using the following probes: Cyclophilin A for 5’-cattatggcgtgtaaagtcacc-3’, Cyclophilin A rev 5’- gcagacaaagttccaaagacag-3', Ctnnb for 5’- aaaatggcagtgcgtttag -3', Ctnnb rev 5’- tttgaaggcagtctgtcgta -3', Axin2 for 5’- ctggctccagaagatcacaa -3’, Axin2 rev 5’- aggtgacaaccagctcactg -3’, MMP7 for 5’- gtatggggaactgctgacatcatg -3’, MMP7 rev 5’- ctgaatgcctttaatatcatcctg -3’. Each sample was normalized to Cyclophilin A control using ΔCt to compute relative expression.Anti-dsDNA antibodies were detected using dsDNA-coated plates and alkaline phosphatase-conjugated anti-IgG antibodies . In brief, wells are pre-coated with mBSA, coated with dsDNA from calf thymus, blocked with 3% BSA and 3mM EDTA in 0.1% gelatin PBS, probed with sera, and analyzed using HRP-conjugated anti-mouse IgG or IgM antibodies or by colorimetric dye. Serum creatinine  was detected by ELISA per the manufacturers’ protocols.Cells were isolated from spleens as above, washed with PBS plus 1% BSA, and stained intracellularly with A555-conjugated anti-β-catenin antibody  or A555-conjugated isotype control using methanol intracellular staining buffer. Cells were further stained with cell-specific markers for CD11b, CD19, CD4, and CD8. Data were analyzed using FlowJo® (Treestar. Stanford) flow cytometry software.ELISA quantifying serum DKK-1 and sFRP were performed using kits from R&D  and MyBioSource , respectively. In brief, sera were prepared from individual mice by centrifugation of whole blood for 20 minutes at 4 C. Diluted samples and standards were added to antibody-coated 96-well plates, washed per protocol, and assayed using conjugated HRP with TMB substrate, and detected by absorbance at 460nm.P values where not otherwise noted were determined using a student’s t test using GraphPad® Prism software. Significance cutoffs for p values were set at 0.05. For Western Blot analysis, bands were quantified using ImageJ®.To determine whether leukocyte β-catenin activity may contribute to serum β-catenin markers we observed in lupus, we isolated splenocytes from age-matched healthy B6 control and lupus-prone Mrl-lpr mice. We analyzed cell lysates by Western blot and probed for total β-catenin, phosphorylation-inactivated (pSer33) β-catenin, β-catenin transcription target Axin-2, and loading controls . Lupus-pTo determine whether leukocyte β-catenin activity is altered in patients with SLE, we isolated peripheral blood mononuclear cells (PBMCs) from healthy controls and SLE patients. We analyzed cell lysates by Western blot and probed for total β-catenin, β-catenin transcription target Axin-2, and loading controls . SLE PBMCtnnb1 (the gene encoding β-catenin) and Axin2 mRNA  loci onto lupus-prone male B6.Sle1.Yaa and control B6.Sle1 mice and confirmed knockdown of expression by RT-PCR Click here for additional data file.S2 FigCtnnb and Axin2 expression by RT-PCR. Older lupus-prone mice exhibited significantly lower Ctnnb and Axin2 transcripts than healthy age-matched controls . In contrast, young lupus-prone mice expressed Ctnnb and Axin2 transcripts at levels similar to those in young B6 controls.Splenocytes from age-matched young (6 week) and old (6 month) healthy control B6 and lupus-prone B6.Sle1.Yaa mice were isolated and tested for (TIF)Click here for additional data file.S3 FigAxin2 and MMP7 were compared by RT-PCR in 4 month-old B6 healthy control and B6.Sle1.Sle3 lupus-prone kidneys. Transcription of these genes is elevated in B6.Sle1.Sle3 lupus-prone mice.Beta catenin expression and that of its transcriptional targets (TIF)Click here for additional data file.S4 Fig(A) Kidneys were isolated, prepared, and stained for β-catenin (red) and macrophage marker F4/80 (blue). (B) Kidneys were isolated, prepared, and stained for total β-catenin (top) and inactivated/structural pSer33 β-catenin (bottom).(TIF)Click here for additional data file.S5 FigCtnnb in each mouse. Due to animal size, there was insufficient blood extracted for a subset of samples to perform RT-PCR.Peripheral blood was taken from young (2–4 wk) B6.Sle1.Yaa.Ctnnbfl/fl and B6.Sle1.Yaa.LyzM-cre mice, RNA was isolated, and RT-PCR was performed to determine gross levels of leukocyte (TIF)Click here for additional data file.S1 TableGender, race, age, and SLEDAI scores are given for healthy controls and SLE patients from whom samples were obtained for (TIF)Click here for additional data file.


In this study, we took advantage of transgenic mice to specifically knockout or overactivate the canonical Wnt signaling mediator β-catenin in HCs, which allowed us to investigate the role of Wnt/β-catenin signaling in protecting HCs against neomycin-induced damage. We first showed that loss of β-catenin in HCs made them more vulnerable to neomycin-induced injury, while constitutive activation of β-catenin in HCs reduced HC loss both in vivo and in vitro. We then showed that loss of β-catenin in HCs increased caspase-mediated apoptosis induced by neomycin injury, while β-catenin overexpression inhibited caspase-mediated apoptosis. Finally, we demonstrated that loss of β-catenin in HCs led to increased expression of forkhead box O3 transcription factor (Foxo3) and Bim along with decreased expression of antioxidant enzymes; thus, there were increased levels of reactive oxygen species (ROS) after neomycin treatment that might be responsible for the increased aminoglycoside sensitivity of HCs. In contrast, β-catenin overexpression reduced Foxo3 and Bim expression and ROS levels, suggesting that β-catenin is protective against neomycin-induced HC loss. Our findings demonstrate that Wnt/β-catenin signaling has an important role in protecting HCs against neomycin-induced HC loss and thus might be a new therapeutic target for the prevention of HC death.Recent studies have reported the role of Wnt/ Lgr5-positive HC progenitors can self-renew to regenerate HCs after isolation in vitro and can spontaneously regenerate HCs after HC damage in the neonatal mouse cochlea in vivo.10 Recent studies have also shown that Wnt signaling has dual roles in controlling the proliferation and differentiation of HC progenitors;4 however, the role of Wnt/β-catenin signaling in HC survival and damage protection in the mouse cochlea remains unclear.Inner ear hair cells (HCs) are responsible for hearing. Aminoglycosides can be ototoxic and induce caspase-mediated apoptosis in HCs. During mammalian inner ear development, canonical Wnt signaling is critical for otocyst induction and directs the formation of the vestibular organs.β-catenin signaling pathway has been shown to function in various cell processes, including cellular protection.15 The pro-survival activity of the Wnt pathway has been reported in many tissues, and is believed to be mediated by the induction of specific anti-apoptotic genes.17 For example, in retinal ganglion cells Wnt activation reduces apoptosis by increasing the expression of protective growth factors including NT3, BDNF, and NGF.11 In the intestine, overexpression of Wnt2a glycoprotein ligand of the Wnt proteins decreases bacterial-induced intestinal epithelial cell death. In the liver, Wnt/β-catenin signaling acts as a transcriptional co-activator of hypoxia inducible factor-1α signaling and has a protective role against hypoxia-induced liver injury. Forkhead box O3 transcription factor (Foxo3) and Bim, which belong to the BCL-2 family members and are the downstream target gene of Foxo3,18 have been reported to regulate the expression of stress-response proteins and to be involved in apoptosis in multiple organs.21 Overactivation of Wnt signaling inhibits Foxo3-induced apoptosis through upregulation of serum and glucocorticoid-inducible kinase 1 (SGK1),22 and overexpression of Wnt/β-catenin signaling inhibits Foxo3 signaling in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury.23 However, the protective role of Wnt/β-catenin signaling against neomycin-induced HC loss in the mouse inner ear has been unclear.In other organs, the Wnt/β-catenin signaling in protecting HCs against aminoglycoside-induced ototoxicity in the mouse cochlea both in vivo and in vitro. We found that β-catenin regulates Foxo3 and Bim expression and controls reactive oxygen species (ROS) levels, thus protecting HCs against caspase-mediated apoptosis after neomycin injury.In this study, we used loss-of-function and gain-of-function mouse models to investigate the role of Wnt/β-Catenin loss-of-function and gain-of-function experiments were performed using Gfi1-Cre/β-cateninflox(exon2–6) and Gfi1-Cre/β-cateninflox(exon3) mice to specifically knockout or overactivate β-catenin in HCs. Consistent with a previous report,24 we found that Cre is activated in 98.76±0.33% of the HCs and Cre activity is similar among the three turns by using Gfi1-Cre/Rosa26-tdTomato mice (flox(exon2–6) mice and increased in Gfi1-Cre/β-cateninflox(exon3) mice /Rosa26-tdTomato and Gfi1-Cre/β-cateninflox(exon3)/Rosa26-tdTomato mice and isolated the tdTomato-positive HCs using flow cytometry (β-catenin in HCs was significantly decreased in Gfi1-Cre/β-cateninflox(exon2–6) mice and increased in Gfi1-Cre/β-cateninflox(exon3) mice  results showed that the mRNA expression of Axin2 and Lgr5 in HCs were both significantly decreased in Gfi1-Cre/β-cateninflox(exon2–6) mice and increased in Gfi1-Cre/β-cateninflox(exon3) mice (β-cateninflox(exon2–6) and Gfi1-Cre/β-cateninflox(exon3) mice (ato mice . Immunohn3) mice . To furtytometry . Westernn3) mice . The actn3) mice . Finallyn3) mice .β-catenin and Wnt target genes in the cochlear HCs after neomycin treatment. Both immunofluorescence and western blot results revealed increased β-catenin expression in the HCs after neomycin injury  and control mice were cultured in vitro and treated with neomycin. Without neomycin, there were no reductions in HCs either in control cochleae or in Gfi1-Cre/β-cateninflox(exon2–6) cochleae (β-cateninflox(exon2–6) cochleae had significantly greater HC loss than control cochleae in the apical and middle turns  2 Gfi1-Cre/cochleae . With nele turns . This reβ-catenin against neomycin-induced HC damage, we used the Wnt agonist Bio and β-catenin overexpressing transgenic mice in two independent in vitro experiments. First, cochlear sensory epitheliums from P2 wild-type (WT) mice were cultured with Bio (5 μM) for 48 h  transgenic mice. Compared with control cochleae, Gfi1-Cre/β-cateninflox(exon3) cochleae had significantly reduced HC loss in all three turns after neomycin treatment  mice using pure-tone auditory brainstem response (ABR) and then dissected out the cochlear sensory epithelium for immunohistochemistry staining at P30 and P60 (β-cateninflox(exon2–6) mice, we observed a 5–10 dB threshold shift at P30 and a 5–15 dB threshold shift at P60 compared with controls  transgenic mice were given daily subcutaneous injections of neomycin from P7 to P14, which is the ototoxic-sensitive period in the cochlea.25 At P30 and P60, we measured hearing function and then dissected out the cochlear sensory epithelium for immunohistochemistry staining (β-cateninflox(exon3) mice, the ABR threshold shifts were significantly lower at all frequencies compared with the control littermates at both P30 and P60 (β-catenin protects against neomycin-induced hearing loss in vivo. Immunohistochemistry results showed that Gfi1-Cre/β-cateninflox(exon3) mice had significantly reduced outer hair cell (OHC) loss compared with the control littermates at both P30 and P60  loss in either Gfi1-Cre/β-cateninflox(exon3) mice or control littermates at P30 or P60  and Gfi1-Cre/β-cateninflox(exon2–6) mice were treated with neomycin and were killed 3 days after the last injection (β-cateninflox(exon3) cochleae had no parvalbumin/caspase-3 double-positive cells in all three turns, while Gfi1-Cre/β-cateninflox(exon2–6) cochleae had significantly more parvalbumin/caspase-3 double-positive cells in the middle turn compared with the control littermates (β-cateninflox(exon3) mice had significantly lower expression of the pro-apoptotic genes Casp3, Casp9, Bax, p53, and Apaf1 (β-cateninflox(exon2–6) mice had significantly higher expression of pro-apoptotic genes  cochleae had significantly fewer TUNEL/Myosin7a double-positive cells compared with the control littermates, while Gfi1-Cre/β-cateninflox(exon2–6) cochleae had significantly more TUNEL/Myosin7a double-positive cells in the apical and middle turns  cochleae compared with controls and was significantly increased in Gfi1-Cre/β-cateninflox(exon2–6) cochleae  cochleae, HCs had significantly reduced Foxo3 staining intensity (β-cateninflox(exon2–6) cochleae, HCs had significantly greater Foxo3 staining intensity (β-cateninflox(exon3) cochleae and significantly increased in Gfi1-Cre/β-cateninflox(exon2–6) cochleae compared with the controls  cochleae and increased in Gfi1-Cre/β-cateninflox(exon2–6) cochleae  cochleae and had significantly higher Foxo3 expression in Gfi1-Cre/β-cateninflox(exon2–6) cochleae (β-cateninflox(exon3) cochleae and significantly increased in Gfi1-Cre/β-cateninflox(exon2–6) cochleae  cochleae and significantly decreased in Gfi1-Cre/β-cateninflox(exon2–6) cochleae  cochleae had significantly fewer MitoSOX/Myosin7a double-positive cells in all three turns compared with control, and Gfi1-Cre/β-cateninflox(exon2–6) cochleae had significantly more MitoSOX/Myosin7a double-positive cells in the apical turn (β-cateninflox(exon3) mice and significantly increased in Gfi1-Cre/β-cateninflox(exon2–6) mice. To find out how Wnt/β-catenin regulates ROS levels in β-catenin overexpressed/knockout HCs, qPCR was used to investigate the expression levels of several antioxidant enzymes . Results showed that antioxidant genes expression, including Sod2, Cat, and Gsr, were significantly increased in Gfi1-Cre/β-cateninflox(exon2–6) mice but significantly decreased in Gfi1-Cre/β-cateninflox(exon3) mice  mice . All of β-catenin-deficient HCs, the antioxidant N-acetylcysteine (NAC), which is a reduced glutathione provider and a direct scavenger of reactive oxygen intermediates,34 was used to treat the explant cultured cochlea with neomycin injury. After NAC treatment, HC loss dramatically decreased in both Gfi1-Cre/β-cateninflox(exon2–6) and control mice, and the number of surviving HCs was not significantly different between Gfi1-Cre/β-cateninflox(exon2–6) and control mice (β-cateninflox(exon2–6) mice after NAC treatment (β-catenin deficiency-induced HC loss in Gfi1-Cre/β-cateninflox(exon2–6) mice after neomycin injury, and demonstrated that ROS accumulation was the major cause of the high injury sensitivity to aminoglycosides in β-catenin-deficient HCs.To further investigate whether the increase in ROS levels contributes to the increased injury sensitivity to aminoglycosides of rol mice . Moreovereatment , suggestβ-catenin signaling in cochlear development and HC regeneration has been extensively studied in the mouse inner ear. Recently, Wnt/β-catenin has been reported to be required for HC differentiation in the mouse cochlea. Knockout of β-catenin inhibits prosensory cells from differentiating into HCs, but β-catenin is not required to maintain HC fate once it is specified.4 However, whether Wnt/β-catenin signaling is required for HC survival has not been investigated. In this study, we observed increased susceptibility of HCs to neomycin treatment in Gfi1-Cre/β-cateninflox(exon2–6) mice, which indicated that β-catenin might have an important role in protecting the sensory HCs.The role of Wnt/β-catenin deficiency-induced cell death, including increased apoptosis of hepatic progenitor cells due to enhanced expression of cleaved caspase-9 and caspase-3 when β-catenin expression is blocked,16 increased apoptosis in cisplatin-resistant lung adenocarcinoma cells when DKK3 is used to inhibit the Wnt/β-catenin pathway,35 and increased si-LGR5-induced apoptosis when the mitochondrial membrane potential is disrupted in colorectal cancer cells.36 In our study, significantly greater HC loss was observed in β-catenin knockout cochleae compared with controls after neomycin treatment  protects HCs from aminoglycosides by upregulating growth-associated protein 43 and netrin 1.40 In many organs and cell lines, Wnt/β-catenin has been reported to have a protective function against apoptosis. In human HCT116 colon cancer cells, Wnt/β-catenin negatively regulates the pro-apoptotic transcription factor Foxo3 and inhibits Foxo3-induced apoptosis.28 In the liver, Wnt/β-catenin protects against hepatotoxin DDC-induced liver injury and inhibits Foxo3 expression thus inhibiting oxidative stress-induced apoptosis.14 In the rat sensory epithelium OC1 cell line, Wnt/β-catenin protects the OC1 cells against cisplatin-induced cell death.41 Here, we found that after neomycin treatment overexpression of β-catenin in mouse HCs significantly inhibits the expression of Foxo3 and Bim  pathways.44 In the zebrafish lateral line, after exposure to aminoglycosides, dying HCs undergo a transient increase in intracellular Ca2+ that occurs shortly after mitochondrial membrane potential collapse. Inhibition of intracellular Ca2+ elevation mitigates toxic effects of aminoglycoside exposure.45 Under physiological conditions, calcium and ROS act as signaling molecules inside the cell and their pathways can interact. However, under pathological conditions dysfunction in either of the systems might affect the other system thus potentiating harmful effects that might contribute to cell death.46 The role of non-canonical Wnt signaling in cochlear HC survival and the role of calcium overload in β-catenin deficiency-induced increased HC susceptibility to neomycin need to be investigated in the future.Aminoglycosides are widely used in clinics to treat bacterial infections, but all aminoglycosides have ototoxic side effects, which limit their clinical use. Mammalian sensory HCs have many mitochondria and high oxygen consumption, which makes them very sensitive to oxidative stress, especially when challenged by external stimulation such as noise or aminoglycosides.reatment  and 7. Bβ-catenin is protective against oxidative stress-induced apoptosis through inhibition of Foxo3 in the liver, bones, and SH-SY5Y cells.47 There are also other papers that suggest that Wnt has the opposite effect on mitochondria. Yoon et al.48 reports that increased Wnt signals are a potent activator of mitochondrial biogenesis and ROS generation, leading to DNA damage and acceleration of cellular senescence in primary cells.48 This might be because Wnt signaling has diverse functions in different environments and stages, and sometimes these effects are in opposition to each other. For example, promoting proliferation and promoting differentiation are usually two opposing effects, and during embryonic stages Wnt/β-catenin signaling promotes proliferation during early mitotic phases of development and also promotes HC differentiation in the differentiating organ of Corti.3 Here, we found that neomycin-induced HC damage was accompanied by Foxo3 upregulation and mitochondrial ROS accumulation. Knockout of β-catenin in HCs upregulated Foxo3 expression and increased the accumulation of ROS even more, while overexpression of β-catenin in HCs inhibited Foxo3 expression and decreased the accumulation of ROS after neomycin injury. This finding indicates that Wnt/β-catenin protects HCs against neomycin injury by regulating Foxo3 expression and controlling ROS levels.Previous studies reported that Wnt/β-catenin in HCs increases neomycin-induced HC loss. Next, we reported that overexpression of β-catenin in HCs protects against neomycin-induced HC loss. Last, we demonstrated that Wnt/β-catenin signaling in HCs regulates Foxo3 expression, antioxidant enzymes, and ROS levels, thus protecting HCs against caspase-mediated apoptosis after neomycin injury. Our data suggest that Wnt/β-catenin signaling is essential for HC protection against neomycin-induced HC loss, and thus might be a new therapeutic target for the prevention of aminoglycoside-induced HC death.In summary, we showed that deletion of β-Cateninflox(exon3) mice49 were generously provided by Mark Taketo , β-cateninflox(exon2–6) mice50 were ordered from The Jackson Laboratory , Rosa26-tdTomato mice51 were ordered from The Jackson Laboratory (JAX number 007914), and Gfi1-Cre mice24 were generously provided by Lin Gan . For β-cateninflox(exon2–6) mice, Cre-induced recombination at the LoxP sites flanks exons 2–6 of β-catenin, and Cre activation results in a β-catenin-null allele.50 For β-cateninflox(exon3) mice, β-Catenin:exon3 encodes the GSK3 phosphorylation sites for degradation, and exon 3 is not functionally required for β-catenin transcriptional activity. Deletion of β-catenin:exon3 therefore results in increased levels of β-catenin.We used C57BL/6 J WT mice and transgenic mice in the C57BL/6 J background. Mice were housed with open access to food and water at the Experimental Animal Center, Shanghai Medical College of Fudan University, China. Postnatal day (P) 0 was defined as the day of birth. Mice received a daily subcutaneous injection of neomycin (200 mg/kg) or sterile saline from P7 to P14. This study was carried out in strict accordance with the 'Guiding Directive for Humane treatment of Laboratory Animals' issued by the Chinese National Ministry of Science and Technology in September 2006. All experiments were approved by the Shanghai Medical Experimental Animal Administrative Committee (Permit Number: 2009-0082). All efforts were made to minimize suffering and reduce the number of animals used.52 The explanted cochleae were treated with 1 mM neomycin  and/or 5 μM Bio (Sigma-Aldrich) or 20 mM NAC (Sigma-Aldrich). PBS was used as the vehicle control.The mice were killed at P2, then the cochlear sensory epithelium was isolated and seeded intact on a glass coverslip coated with Cell-Tak .53 All ABR tests were performed on mice older than P21.The hearing thresholds of the mice were examined with the ABR test. In this test, changes in the electrical activity of the brain in response to sound were recorded via electrodes that were placed on the scalp of the mice. Animals were anesthetized with ketamine (100 mg/kg) and xylazine (25 mg/kg) and placed on a thermostatic heating pad in a sound-attenuating chamber to maintain their body temperatures at 38 °C. Frequency-specific auditory responses were measured using the Tucker-Davis Technology system III  as previously described.μg total RNA by reverse transcription using the GoScript Reverse Transcription System  following the manufacturer's protocols. qPCR was performed using GoTaq qPCR Master Mix (Promega) on a Bio-Rad 7500 detection system . GAPDH was used as a housekeeping gene for control purposes. Primer sequences are listed in μl RIPA lysis buffer with 1% PMSF, sonicated, incubated on ice for 30 min, and stored at −80 °C. Extracts were boiled with 5 × loading buffer, subjected to PAGE , transferred onto an Immobilon-P membrane , probed with anti-Foxo3 antibody , anti-β-catenin (BD Biosciences), and anti-GAPDH  and finally incubated with HRP-conjugated secondary antibodies. Signal was detected with the Supersignal West Femto Trial Kit  on a FluorChem M system .After killing the mice, the otic capsule was immediately isolated, rapidly frozen in liquid nitrogen, and stored at −70 °C until further processing. To obtain the total RNA, 10 cochleae were pooled in TRIzol  following the manufacturer's instructions. The RNA concentration was measured with a Bio-Rad spectrophotometer . cDNA was synthesized from 1 M phosphate-buffered saline  with 0.3% Triton X-100 for 1 h at room temperature and then incubated with primary antibody overnight at 4 °C. The next day, the tissues were incubated for 2 h at 4 °C with 488- or 594-conjugated donkey secondary antibody (Invitrogen) and DAPI (Sigma-Aldrich). Omission of primary antibody served as the negative control. The following primary antibodies were used: anti-β-catenin (BD Biosciences), anti-myosin VIIA (Myosin7a) , anti-cleaved caspase-3 , anti-Foxo3 , anti-Parvalbumin (Sigma-Aldrich), TUNEL , and MitoSOX Red . Cochleae were dissected into apical, middle, and basal turns, and images were taken using a Leica SP5 confocal fluorescence microscope .After fixation, cochlear samples were blocked with 10% normal donkey serum in 10 mβ-cateninflox(exon2–6)/Rosa26-tdTomato or Gfi1-Cre/β-cateninflox(exon3)/Rosa26-tdTomato mice (P2) were dissected out to isolate ∼20–40 cochleae, and then trypsinized with prewarmed 0.125% trypsin/EDTA (Invitrogen) at 37 °C for 8 min. Soybean trypsin inhibitor  was added to terminate the reaction followed by mechanical trituration with blunt tips and pipetting up and down up to 80–100 times. Suspended cells were percolated through a 40-μm cell strainer before fluorescence activated cell sorting (FACS). tdTomato-positive HCs were sorted out on a BD FACS Aria III (BD Biosciences) using the tdTomato channel.Gfi1-Cre/n represents the number of mice examined.For HC quantification in the neomycin-treated samples, we imaged the entire cochlea using a 40 × 3 objective and counted the Myosin7a+ HCs that remained. The same procedure was used to quantify cleaved caspase-3+/Myosin7a+, TUNEL+/Myosin7a+ cells, and myosin7a+/MitoSOX Red+ cells. For all experiments, only one cochlea from each mouse was used for immunofluorescence and quantification. Thus, post hoc test. Immunofluorescence analysis was performed with a two-tailed, unpaired Student's t-test when comparing two groups or with a one-way ANOVA followed by a Dunnett's multiple comparisons test when comparing more than two groups. P<0.05 was considered as statistically significant.Statistical analyses were conducted using Microsoft Excel and GraphPad Prism software . Data were expressed as mean±S.E.M. ABR thresholds were analyzed by two-way ANOVA followed by a Newman–Keuls