Oxidized low-density lipoprotein (ox-LDL) can induce endothelial injury and plays a vital role in the procession and development of atherosclerosis. Little is known regarding whether Wnt/β-catenin pathway is involved in ox-LDL-induced endothelial injury or whether it further promotes atherosclerosis via increased oxidative stress. This study aimed to investigate the role of Wnt/β-catenin pathway in ox-LDL-induced vascular endothelial injury and determine whether pigment epithelium-derived factor (PEDF) could alleviate ox-LDL-induced endothelial injury by inhibiting Wnt/β-catenin pathway.Injury of human umbilical vein endothelial cells (HUVECs) was evaluated with an MTT assay, by monitoring lactate dehydrogenase (LDH) release and determining the apoptotic ratio. The expression of β-catenin (non-phosphorylated-β-catenin), disheveled-1 (Dvl-1) and Cyclin D1 was analyzed with western blotting and quantitative real-time PCR. Oxidative stress status was assessed by measuring the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO).Exposure of HUVECs to ox-LDL led to a decrease in cell viability and an increase in LDH release and apoptosis with concomitant enhancement of oxidative stress, as assessed by increased ROS and MDA generation, as well as decreased SOD activity and NO levels. Similar to lithium chloride , ox-LDL up-regulated the expression of β-catenin, Dvl-1 and Cyclin D1, markers of Wnt/β-catenin pathway activation. However, ox-LDL-induced activation of Wnt/β-catenin pathway, as well as ox-LDL-induced cell injury and oxidative stress, were synergistically promoted by LiCl and mitigated by Dickkopf 1 (DKK-1), an inhibitor of Wnt/β-catenin pathway. Additionally, ox-LDL-induced HUVEC injury and apoptosis, oxidative stress and activation of Wnt/β-catenin pathway were suppressed by PEDF, while they were further strengthened by a small interfering RNA of PEDF.Wnt/β-catenin pathway may mediate ox-LDL-induced endothelial injury via oxidative stress, and PEDF ameliorates endothelial injury by suppressing Wnt/β-catenin pathway and subsequently reducing oxidative stress.The online version of this article (doi:10.1186/s12944-017-0407-8) contains supplementary material, which is available to authorized users. Cardiovascular disease (CVD) is ranked first in morbidity and mortality, accounting for nearly a third of deaths worldwide . AtherosWnt/β-catenin signaling pathway, a canonical Wnt pathway, is thought to be essential in a number of developmental and physiological processes . Many liPigment epithelium-derived factor (PEDF) belongs to the serine protease inhibitor supergene family. As previously confirmed, PEDF is a type of endogenously produced multifunctional protein with anti-oxidative, anti-inflammatory, anti-angiogenic, anti-thrombotic, neuroprotective and neurotrophic properties , 27. In Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco . Ox-LDL was purchased from Union-Biology . DKK-1 and PEDF were obtained from PeproTech . Lithium chloride (LiCl), 3--2,5- diphenyl-2-H-tetrazolium bromide (MTT) dye and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma . Annexin V-FITC/propidium iodide (PI) apoptosis detection kits were obtained from KeyGEN Biotech . SYTO-13/PI dye was purchased from Life Technologies . Anti-β-catenin (non-phosphorylated β-catenin) polyclonal antibody, anti-disheveled-1 (anti-Dvl-1) polyclonal antibody and anti-PEDF polyclonal antibody were obtained from Santa Cruz Biotechnology . TRIZOL reagent, OPTI-MEM I reduced-serum medium and Lipofectamine 2000 were purchased from Invitrogen . QuantScript RT kits and RealMaster-Mix (SYBR Green) kits were obtained from Tiangen Biotech . Lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) and NO assay kits were purchased from Nanjing Jiancheng Bioengineering Institute .2 atmosphere in DMEM supplemented with 5 mM glucose, 10% FBS and 1% penicillin-streptomycin until subconfluent, and then, the medium was replaced with DMEM containing 1% FBS.A human umbilical vein endothelial cell (HUVEC) line was obtained from the Type Culture Collection of the Chinese Academy of Sciences . Cells were cultured at 37 °C in a humidified 5% COHUVECs were treated with ox-LDL at different concentrations  for 24 h. In addition, cells were pretreated with 20 nM DKK-1 and 20 mM LiCl for 24 h, or with different concentrations  of PEDF for 24 h, followed by exposure to 100 mg/L ox-LDL for 24 h.4 cells/well. Following treatment, 20 μL of MTT dye was added to each well at a final concentration of 0.5 mg/mL and cells were incubated for 4 h at 37 °C. After removing the supernatant, 150 μL of dimethylsulfoxide (DMSO) was added and the optical density was measured at 490 nm using a microplate reader . Cell viability is displayed as the percentage of the optical density of treated cells relative to that of the untreated control cells (100%).Cell viability was determined using an MTT assay. HUVECs were seeded into 96-well plates at a density of 1 × 10To further determine the degree of cell injury, LDH activity in the medium was assessed using an assay kit according to the manufacturer’s instructions. LDH activity was measured at 450 nm and expressed as units per liter (U/L).The quantification of apoptosis was performed with an Annexin V-FITC/PI double-staining assay. Treated cells were centrifuged, harvested and washed with phosphate-buffered saline (PBS). After resuspension in 500 μL of binding buffer, cells were double-stained with 5 μL of Annexin V–FITC and 5 μL of PI at room temperature in the dark for 15 min. Then, the samples were analyzed using Cell-Quest software on a Becton Dickinson FAC Scan flow cytometer . The total apoptotic cells (early and late-stage apoptosis) were represented by Annexin V-FITC staining alone or together with PI.Morphological changes of cell apoptosis were observed using SYTO-13/PI dye staining. Cells cultured on glass coverslips were fixed in 4% paraformaldehyde for 30 min and were washed 3 times. Subsequently, coverslips were stained with 0.99 μM SYTO-13 dye and 6.56 μg/mL PI dye diluted with PBS at room temperature in the dark for 15 min. Cells were visualized using an Olympus BX51 microscope  and were analyzed with Image-Pro Plus software .Treated cells were lysed with Radio Immunoprecipitation Assay (RIPA) lysis buffer containing 1% protease inhibitors to obtain the total protein. Equal amounts of protein were separated by 8 to 12% SDS-PAGE gels and were transferred onto polyvinylidene fluoride (PVDF) membranes by electroblotting. After being blocked with 10% fat-free dried milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 4 h at room temperature, the membranes were incubated with primary antibody overnight at 4 °C. Antibody-treated membranes were washed with TBS containing 0.1% Tween-20 and then were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Specific bands were visualized with an electrochemiluminescence (ECL) reagent. The integrated optical density of protein bands was assessed with Image-Pro Plus software and normalized to β-actin.–△△Ct method and normalized against GAPDH.Total RNA was isolated using TRIZOL reagent. Complementary DNA (cDNA) was synthesized from 2 μg of total RNA using a QuantScript RT kit according to the manufacturer’s protocol. Subsequently, a quantitative real-time PCR reaction was performed with a RealMaster-Mix (SYBR Green) kit, and products were amplified using a Rotor-Gene quantitative real-time PCR machine . Primers used in this study were synthesized by Sangon Biotech . For human β-catenin, the primers were 5′-GTGTGGCGACATATGCAGCT-3′ (forward) and 5′-CAAGATCAGCAGTCTCATTC-3′ (reverse). For human Cyclin D1, the primers were 5′-GTGCATC TACACC GACAACTCCA-3′ (forward) and 5′-TGAGCTTGTTCACCAGGAGCA-3′ (reverse). For human GAPDH, the primers were 5′-CCTCCCGCTTC GCTCTCT-3′ (forward) and 5′-GCTGGCGACGCAAAAGA-3′ (reverse). For human PEDF, the primers were 5′-ATCGTCTTTGA GAAG-3′ (forward) and 5′-CAAACTTTGTTACCCACTGC-3′ (reverse). The data were analyzed using Rotor-Gene Q software . Relative mRNA levels were calculated using the 2Intracellular ROS generation was measured with the peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) using flow cytometry. HUVECs cultivated in 6-well plates were incubated with 10 mM DCFH-DA in FBS-free DMEM at 37 °C for 30 min. Then, the cells were collected and analyzed on a FACScan flow cytometer using CellQuest software.Intracellular SOD activity, MDA content and NO levels in the medium were detected using assay kits according to the manufacturer’s instructions and expressed as units per milligram of protein (U/mgprot), nanomoles per milligram of protein (nmol/mgprot) and micromoles/liter (μM).HUVECs were cultured in antibiotic-free growth medium to reach 30–50% confluence. PEDF-specific siRNA oligomers (200 pmol) and control siRNA oligomers (200 pmol) were diluted in 250 μL of OPTI-MEM I reduced-serum medium and combined with 5 μL of Lipofectamine 2000 pre-diluted in 250 μL of OPTI-MEM I. After incubation for 20 min at room temperature, the complexes were added to the cells in a final volume of 2.5 mL of medium. The human PEDF-siRNA sequences  were as follows: 5′-CCAUCAAGCU GACUCAGGUdTdT-3′ (sense) and 5′-ACCUGAGUCAGCUUGAUGGdTdT-3′ (antisense). The negative control sequences  used were 5′-UUCUCCGAACGUGUCACGUT T-3′ (sense) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense). After transfection for 48 h, cells were treated with 100 mg/L ox-LDL for 24 h. The silencing of PEDF was detected by western blotting and quantitative real-time PCR.P-values less than 0.05 were considered significant.Results are presented as the mean ± SD. Statistical analysis was performed using one-way analysis of variance followed by Student-Newmann-Keuls multiple comparison tests with SPSS17.0 software. For all analyses, As shown in Fig. Next, we investigated the oxidative stress generation in ox-LDL-treated cells. As illustrated in Fig. The effect of ox-LDL on the expression levels of β-catenin, Dvl-1 and Cyclin D1, the hallmarks of Wnt/β-catenin pathway activation, was examined by western blotting and quantitative real-time PCR. As seen in Fig. To further confirm the activation effect of ox-LDL on Wnt/β-catenin pathway, an exogenous Wnt/β-catenin signaling activator LiCl, and DKK-1, an inhibitor of the pathway , were utGiven that ox-LDL up-regulated the expression of β-catenin and Dvl-1 in injured HUVECs, we hypothesized that Wnt/β-catenin pathway activation may be one of the underlying mechanisms of ox-LDL-induced endothelial injury. To verify this hypothesis, we measured the changes in cell viability and apoptosis in ox-LDL-incubated cells after activating or inhibiting the pathway. As seen in Fig. There is evidence suggesting that activation of Wnt/β-catenin pathway is sufficient to induce retinal oxidative stress in age-related macular degeneration . TherefoPEDF, a multifunctional glycoprotein, has been suggested to have anti-inflammatory, anti-oxidative, anti-thrombogenic, anti-angiogenic and anti-tumor properties. To further confirm the protective effect of PEDF on endothelial injury, HUVECs were incubated with exogenous PEDF for 24 h. As shown in Fig. Because PEDF can be secreted by many types of cells, including RPE cells, VSMCs, macrophages, ECs and cardiomyocytes, we investigated the effect of gene silencing of PEDF on ox-LDL-induced injury. PEDF-siRNA was transfected into HUVECs before incubating the cells with ox-LDL. Both protein and mRNA levels of PEDF were down-regulated by PEDF-siRNA Fig. . SignifiConsidering that PEDF has been shown to inhibit the activation of the Wnt ligand-induced Wnt pathway in retinal cells , we furtBecause PEDF can down-regulate intracellular production of ROS and inhibit oxidative stress, we investigated the levels of oxidative stress to determine the downstream mechanism of PEDF alleviating ox-LDL-elicited HUVEC injury by inhibiting Wnt/β-catenin pathway. Pretreatment with PEDF for 24 h observably depressed ox-LDL-induced increase in the levels of ROS and MDA and the decrease in SOD activity and NO levels, while the ox-LDL-induced oxidative stress was significantly increased by PEDF-siRNA Fig. . AltogetIn the present study, our results indicated that Wnt/β-catenin signaling pathway mediated ox-LDL-induced HUVEC injury via oxidative stress, and PEDF alleviated ox-LDL-elicited HUVEC injury by inhibiting Wnt/β-catenin signaling pathway and subsequently attenuating oxidative stress, which was supported by the following observations. First, ox-LDL activated Wnt/β-catenin signaling pathway in HUVECs. Second, similar to LiCl, ox-LDL resulted in cytotoxicity and oxidative stress in HUVECs, effects which were promoted synergistically by Wnt/β-catenin pathway activator LiCl, while they were mitigated by DKK-1, a Wnt/β-catenin pathway inhibitor. Third, pretreatment with PEDF ameliorated ox-LDL-induced cytotoxicity and oxidative stress in HUVECs and reduced the up-regulation of β-catenin, Dvl-1 and Cyclin D1 induced by ox-LDL, while this effect of ox-LDL on HUVECs was enhanced by PEDF-siRNA.Atherosclerosis occurs in the arterial wall and is characterized by chronic lipid-triggered inflammation . The incWnt/β-catenin signaling pathway is initiated once the Wnt protein simultaneously interacts with its receptor and co-receptor, frizzled (FZD) and LRP5/6. Immediately after binding, phosphorylation of Dvl inhibits the activity of a degradation complex consisting of adenomatous polyposis coli, axin, glycogen synthase kinase-3β and casein kinase1. As a result, β-catenin can avoid being phosphorylated and degraded, and accumulate in the cytoplasm. It then translocates into the nucleus where it interacts with the transcription factor T cell-specific transcription factor/lymphoid enhancer-binding factor (TCF/LEF). Consequently, activated Wnt/β-catenin pathway regulates the expression of downstream target genes including Cyclin D1 and c-myc, leading to many changes of pathophysiological reactions . It has PEDF is currently receiving more attention due to its anti-oxidation, anti-inflammation, and anti-thrombotic activity , especiaThe external administration of antioxidants has been demonstrated to exert the protective effects on cardiovascular disease. It has been reported that carotenoids by dietary supplementation play a protective role in atherosclerosis and CVD by promoting the activity of antioxidant system, scavenging ROS, inhibiting LDL oxidation, and protecting cellular membranes and lipoprotein against oxidative damage . SimilarThe present study revealed that Wnt/β-catenin signaling pathway may mediate ox-LDL-induced endothelial injury via oxidative stress, and PEDF ameliorated endothelial injury by suppressing Wnt/β-catenin pathway and subsequently reducing the oxidative stress response. This study will add novel pieces of information regarding the mechanism of ox-LDL-induced endothelial dysfunction and application of PEDF to prevention and treatment in vascular complications. Nevertheless, there are some limitations in the present study. All data are from in vitro experiments using HUVECs and they are limited in the actual pathological process of atherosclerosis in vivo. Moreover, other test indicators closely associated with atherosclerosis, including inflammatory factors, angiogenesis and thrombosis, need be evaluated. Accordingly, further experiments need be performed to confirm these promising results.


MusaVND6 and MusaVND7) for tissue specific expression and presence of 19-bp secondary-wall NAC binding element (SNBE)-like motifs. Transgenic banana plants of Musa cultivar Rasthali harboring either PMusaVND7::GUS or PMusaVND6::GUS showed specific GUS (β-D-Glucuronidase) activity in cells of the xylem tissue. Approximately 1.2kb promoter region of either MusaVND6 or MusaVND7 showed presence of at least two SNBE-like motifs. This 1.2kb promoter region was retarded in a gel shift assay by three banana VND protein . The banana VND1-VND3 could also retard the mobility of isolated SNBE-like motifs of MusaVND6 or MusaVND7 in a gel shift assay. Transcript levels of MusaVND6 and MusaVND7 were elevated in transgenic banana overexpressing either banana VND1, VND2 or VND3. Present study suggested a probable regulation of banana VND6 and VND7 expression through direct interaction of banana VND1- VND3 with SNBE-like motifs. Our study also indicated two promoter elements for possible utilization in cell wall modifications in plants especially banana, which is being recently considered as a potential biofuel crop.Deposition of secondary cell wall in the xylem elements is controlled by a subgroup of NAC  family, known as vascular-related NAC transcription factors (VNDs). In the present study, we analyzed the 5’ upstream regulatory region of two banana NAC transcription factors ( NAC transcription factors, VNDs (vascular related NAC transcription factors) are most important. Secondary cell wall deposition in tracheids is accompanied with programmed cell death, hence regulation of the activity of secondary cell wall associated genes in specialized cells is of prime importance for proper functioning and homeostasis of plants.Evolution of secondary cell wall deposition was a prominent feature which not only provided the mechanical strength required for the vertical growth of plants but also helped in the long distance transport of water and minerals. This secondary cell wall is laid in cells of xylem tissue and is composed of cellulose, hemicelluloses, lignin and xylan. Water transport in plants from roots to other organs is through the channels of xylem tissue composed of tracheids and vessel elements. These tracheids develop an array of secondary wall depositions in the form of reticulate, pitted, helical and annular thickenings. Secondary cell wall deposition is regulated and highly coordinated for systematic deposition of multiple components by many genes among which a subgroup of VND (VND1-VND7) genes from banana genomic database and analyzed their expression pattern during the time course of invitro tracheary element differentiation from banana embryogenic cells in a brassinolide supplemented medium  followed by final extension of 72°C(20min). SNBE-like sites identified from the PMusaVND7 and PMusaVND6were synthesized as complementary oligonucleotides and annealed together at 37°C after heating at 90°C in a thermal cycler machine. The oligonucleotides containing SNBE-like site from PMusaVND7 are TGGCGTGCACTCAAAGAAATTGGT-3’5’-AATTCGATAGCCTT; and 5’-ACCAATTTCTTTGAGTGCACGCCAAAGGCTATCGAATT-3’. Complementary oligonucleotides with SNBE-like site from PMusaVND6 are AGTTGTAGGGCTCAAGATTTTGGT-3’5’-AATTCGATAGCCTT and 5’-ACCAAAATCTTGAGCCCTACAACTAAGGCTATCGAATT-3’ .The annealed oligonucleotides or the PCR amplified promoter fragments was incubated with either banana VND1, VND2 or VND3 protein in a EMSA buffer  for 20 minutes. The reaction was later resolved on a 1.5% agarose-gel to visualize the DNA-protein interaction. Mutated SNBE-like(mSNBE) oligonucleotides were annealed as mentioned above and utilized in EMSA reaction as discussed before. The sequences of oligonucleotides for mSNBE were CGGAAAGCACTCATTTAGGTTGG -3’5’-CCTT and 5’- CCAACCTAAATGAGTGCTTTCCGAAGG -3’. Sequence of oligonucleotides used for mutated SNBE –like site of PMusaVND6 were CGTAAAAGGGCTCTTTAGGTTGG-3’5’-CCTTand 5’- CCAACCTAAAGAGCCCTTTTACGAAGG-3’ (consensus residues of SNBE site were mutated and are underlined).A 250bp DNA fragment (-287 to -37 upstream of transcription start site) spanning the SNBE-like site (-186 to -167) was PCR amplified from PMusaVND7 using oligonucleotides 5’-GAGCATGTGGAAACCATAGC-3’ and 5’-AAGAACATCCCATACTTGGAA-3’ and was utilized in gel shift assay with the VND1-VND3 proteins. Similarly a 224bp DNA fragment (-388 to -164 upstream of transcription start site) encompassing the SNBE-like sites in PMusaVND6 was PCR amplified (using oligonucleotides 5’-CATCACCTTCTGTGGTATTCTTC -3’ and 5’- AGAGGAGCAACAAGTGATTGA -3’)for gel shift assay with VND1-VND3 proteins. EMSA reaction with these DNA was carried out as discussed above.The MusaVND1(KT356872),MusaVND2 (KP335047) or MusaVND3 (KP666170) have been described earlier by us followed by final extension of 72°C(20min).Binary vectors (n EHA105  by electeviously . Banana  earlier . Develop earlier . PutativPMusaVND7 and PMusaVND6in different organ and tissue was carried out by staining for GUS (β-D-Glucuronidase) activity. GUS staining buffer was prepared as follows: 1mM X-Gluc (5-Bromo-4-chloro-3-indolyl Glucuronide), 0.1% triton X-100, 0.5mM potassium ferricyanide and 0.5mM potassium ferrocyanide prepared in 100mM sodium phosphate buffer with pH 7.0. GUS staining was carried out at 37°C for maximum of 12 hours. Before recording the observations, chlorophyll of green tissue was removed by repeated incubation in 70% ethanol at 37°C till satisfactory resultwas obtained. Image of GUS stained sections of different organ was observed and recorded in Eclipse 80i, Nikon microscope.Estimation of GUS activity was carried out as picomoles 4-MU per min per μg soluble protein according to protocoldescribed earlier using 4-methylumbelliferyl-b-D-glucuronide (MUG) and soluble extract of plant  reported earlier [PMusaVND7 first SNBE-like site  was positioned at -186 to -167 while the second SNBE-like site  was positioned at -926 to -907  and second SNBE-like site was positioned at -372 to -353 relative to transcription start site  proteinsuggesting that PMusaVND7 is a potential target gene for regulation by banana VND1- VND3  containing the SNBE–like site present at -186 to -167 (PMusaVND6 fragment (-388 to -164 relative to transcription start site) encompassing the SNBE–like sites was retarded by banana VND1- VND3 protein in gel shift assay  are reported to regulate the expression of genes by binding to the er region. Moreovegulation . We alsoD1- VND3 . SimilarD1- VND3 . We also to -167 . Similarft assay . These rSNBE-like sitewas detected in both PMusaVND7 and PMusaVND6and mobility of PMusaVND7 and PMusaVND6was retarded by VND1- VND3, we analyzed whether VND1- VND3 could bind to these SNBE-like sites. A 38bp double stranded DNA containing SNBE-like site of PMusaVND7 (TGGCGTGCACTCAAAGAAA positioned at -186 to -167) was incubated with either banana VND1- VND3 protein and the interaction was analyzed on a gel. All the three VND1- VND3 strongly retard the mobility of SNBE-like site of PMusaVND7 indicating that banana VND1- VND3 recognize the SNBE-site in PMusaVND7 in PMusaVND7 and PMusaVND6 indicating that VND1-VND3 specifically interact with PMusaVND7 and PMusaVND6 and the interaction is mediated by SNBE-like sites  remarkably elevated the GUS activity , which can be improved through increased mechanical strength of pseudostem by elevatingsecondary cell wall deposition [VNDs in plants like Arabidopsis, poplar and Brachypodium among otherhave been generated suggesting that these transcription factors regulate a number of downstream genes for coordinated regulation of secondary cell wall deposition [Arabidopsis seven members have been included in VND subgroup of NAC family and VND6 and VND7are suggested to be master regulators for secondary wall synthesis [VNDs is indispensable [PMusaVND7 and PMusaVND6) with specific activation in xylem tissue element which can be utilized in future for biomass engineering. Some examples of utilization of xylem specific promoter element have been reported in the past. In Arabidopsis, the expression of C4H (cinnamate 4-hydroxylase), an important gene for lignin synthesis was restricted under the activation of VND6 promoter (a vessel specific promoter) which reduced the lignin content (without plant growth reduction), thus increasing the hydrolysis of cellulose for sugar release [3-dehydroshikimate dehydratase (QsuB) under the PC4Hand emphasized the need of fiber cell specific promoter for higher stringency in lignin reduction [VND6 and VND7 in native banana indicated differential expression of MusaVND6 and MusaVND7 in different organs. Transcript levels of both VND6 and VND7was high in corm and pseudostem compared to other organs  into ethanol through fermentation process is deterred by high lignin content . Hence rposition ,20. Infoposition –24. In Aynthesis ,6. Attemynthesis ,7. Hencepensable . In this release . Anothereduction . Moreoveeduction –28. Quanr organs . Though VNDgenes during tracheary element differentiation was different suggesting that these genes may function in different aspects of tracheid development [VNDs and SND1 regulate downstream genes by binding to a 19bp imperfect palindrome known as SNBE-like motif and its consensus sequence was worked out to be (T/A)NN(C/T) (T/C/G)TNNNNNNNA(A/C)GN(A/C/T) (A/T) suggesting a common mode of regulation of a set of genes involved in tracheids differentiation [ArabidopsisSND1 and VND7 have been shown to regulate the expression of multiple class of genes like MYB transcription factors , cell wall associated genes like IRX genes (irregular xylem) and programmed cell death related genes like XCP1/2 (xylemcysteine peptidase) through SNBE-like motifs [VND genes is relatively unknown, however, a recent work on analysis of SNBE-like motif in promoter of ArabidopsisVND7 and its regulation by VND1-VND7 proteins suggested potential regulation of VND7throughSNBE-like cis-elements besides other known and unknown mechanisms [VND7 have been carried out suggesting multiple level of regulation of its expression.VND-INTERACTING2 (VNI2), is a transcriptional repressor which binds with VND proteins including VND7 protein and repress the expression of the secondary wall associated genes directly controlled by VND7 [Arabidopsis VND7 in cells is regulated by proteasome mediated degradation as accumulation of VND7 protein was observed upon treatment of cells with MG-132,which is a potent proteasome inhibiting chemical [ASYMMETRIC LEAVES2 (AS2)/LATERALORGANBOUNDARIESDOMAIN (LBD) gene family members ASL19/LBD30 and ASL20/LBD18were reported to regulate the expression of VND6 and VND7 via a positive feedback loop as the expression of ASL19/LBD30 and ASL20/LBD18 is dependent on VND6 and VND7 and plants expressing PASL20:ASL20-SRDX showed reduced expression of VND6 and VND7 [GATA5, GATA12, ANAC075 LBD15/ASL11are shown to induce the VND7 promoter suggesting additional regulations over VND7 expression [Expression pattern of the seven elopment ,6. Multintiation . Arabidoe motifs –31. Croschanisms . Some st by VND7 . Amount chemical . Two ASYand VND7 . RecentlVND6 and VND7 expression is positively regulated by banana VND1- VND3 protein through direct binding withSNBE-like motif in the PMusaVND7 and PMusaVND6.Our report is in line with study on ArabidopsisVND7 wherein its promoter was identified and induced by VND1-VND7 proteins [VND genes among themselves. In our previous work, we had successfully purified banana VND1-VND3 proteins and demonstrated their regulation over multiple downstream target genes including MYBtranscription factors and IRX genes by binding to SNBE-like sites in their regulatory region [SNBE-like sites in the regulatory regions of banana VND6 and VND7 prompting us to analyze whether these genes can also be regulated by other banana VNDs. Here we show that banana VND1- VND3 could also bind to PMusaVND7 and PMusaVND6 as well as SNBE-like motif in them using a agarose based gel shift assay [SNBE consensus residues indicating that the interaction of these three transcription factors with the PMusaVND7 and PMusaVND6 is specific and is governed by the presence of SNBE-like motifs. Elevated transcript levels of VND6 or VND7was detected in the corm but not in leaves tissue of transgenic banana overexpressing either VND1, VND2 or VND3. Moreover results obtained from transcriptional activation assay indicated that banana VND1-VND3 could activate transcription from the PMusaVND6 and PMusaVND7 confirming that these transcription factors not only bind but also could activate the expression of both MusaVND6 and MusaVND7.Our work showed that banana proteins  suggestiy region . Our worft assay . The binPMusaVND6 and PMusaVND7 in sclerenchymatous cells (xylem tissue) suggesting that these promoters are inactive in non-sclerenchymatous cells. Overexpression of VND1-VND3 in banana transdifferentiate many non sclerenchymatous cells into sclerenchymatous type and our previous work has indicated that compared to other organs, corm tissue showed remarkably higher transdifferentiation [VND6 and VND7 in VND1-VND3 overexpression plants probably occurred specifically in native sclerenchymatous as well as transdifferentiated sclerenchymatous cells of transgenic corm. Besides, there is a possibility of tissue specific regulation of VND6 and VND7 by VND1-VND3 as suggested by differential up regulation in transgenic banana overexpressing VND1-VND3. Previous work on regulation of ArabidopsisVND7 by VND1-VND7 through SNBE motifs has suggested possible presence of some unknown inhibitory factors regulating the expression of VND7 and possibly its transcript stability in non-sclerenchymatous cells of plants overexpressing VND1-VND7 and thus restricting its expression [VND6-VND7 transcript are warranted.Besides studies on regulation of transcript levels of VND6 and VND7 due to expression of VNDs in non-xylem tissue need to be carried out in detail. Nonetheless this study identified two potential xylem specific promoter regionswhich will be useful for crop improvement and presented an interesting mechanism for regulation of VND6 and VND7expression in banana.Present work showed the specific activation of ntiation ,3. Moreopression . FurtherS1 FigMusaVND7. The PMusaVND7was amplified from genomic DNA of Musa cultivar Rasthali and analyzed for the presence of SNBE-like sites. SNBE–like sites in the PMusaVND7are boxed in green. Putative TATA box is shown in red and the transcription start site (+1 TSS) is underlined.Sequence of 5’ upstream regulatory region of (PDF)Click here for additional data file.S2 FigMusaVND6 from Musa cultivar Rasthali. TATA box is boxed in red and transcription start site (+1 TSS) is indicated by an underline. SNBE–like sites detected in the sequence are boxed in green.Sequence of 5’ upstream regulatory region of (PDF)Click here for additional data file.S3 FigpCAMBIA1301- PMusaVND7:: GUS and growing on banana embryo development medium supplemented with hygromycin (5mg/l). (B) Emergence of putatively transformed embryos on hygromycin supplemented medium after 2 months of growth. (C) Close-up of embryos showing growth in various stages of development as well as emergence of secondary embryos. (D) Multiplication of transgenic shoots on banana shoot multiplication medium. (E) Rooting of transgenic banana plants on banana rooting medium containing NAA(1mg/l). (F) Rooted banana plants were hardened in a green house for GUS analysis.(A) Embryogenic cells of banana cultivar Rasthali transformed with (PDF)Click here for additional data file.S4 FigpCAMBIA1301- PMusaVND6:: GUS. (B) White and opaque embryos developed from continuous culturing of the transformed embryogenic cells on embryo development medium. (C) Developing embryos appeared globular to torpedo shaped in close-up. (D) Transformed banana shoots were multiplied on shoot multiplication medium to generate multiple lines. (E) Rooting of transgenic banana plants on banana rooting medium containing NAA(1mg/l). (F) Rooted banana plants were hardened in a green house for GUS analysis.(A) Embryogenic cells of banana cultivar Rasthali growing on banana embryo development medium supplemented with hygromycin (5mg/l) after transformation with (PDF)Click here for additional data file.S5 FigPMusaVND7::GUS. (B) PCR analysis of banana plants harboring PMusaVND6::GUS. Two different primer pairs were utilized in PCR analysis. 1: FP in either PMusaVND7 or PMusaVND6 and RP in GUS; 2–4:FP and RP in either PMusaVND7 or PMusaVND6 2–4: analysis of three independent transgenic banana lines. M: 1 KB DNA ladder. 1kb and 3 kb band size are indicated. .(A) PCR analysis of banana plants harboring (PDF)Click here for additional data file.S6 FigMusaVND6 and MusaVND7 in tissue of different organs of wild type banana was analyzed by quantitative RT-PCR. Expression of VND6 and VND7 in different tissue types is shown after normalization of the data by the expression of banana EF1α. Data was represented as mean±SD of three replications.Transcript level of (PDF)Click here for additional data file.


Considerable evidence has shown that the Wnt/β-catenin pathway is involved in osteogenic differentiation in various stem cells. However, the role of Wnt/β-catenin pathway in regulating the osteogenic differentiation of rat ectomesenchymal stem cells (EMSCs), which are considered to be the progenitors of dental mesenchymal stem cells, remains unknown. In this study, we demonstrated that nuclear β-catenin was upregulated during EMSC osteogenic differentiation. The Wnt signalling inhibitor IWR-1-endo inhibited EMSC osteogenic differentiation, while the Wnt signalling agonist SKL2001 promoted it. Moreover, nuclear β-catenin was further upregulated by the overexpression of low-affinity nerve growth factor receptor (LNGFR) during EMSC osteogenic differentiation. Further experiments demonstrated that LNGFR overexpression enhanced EMSC osteogenic differentiation, while LNGFR silencing decreased it. Additionally, IWR-1-endo attenuated LNGFR-enhanced EMSC osteogenic differentiation. Collectively, our data reveal that LNGFR targets the Wnt/β-catenin pathway and positively regulates EMSC osteogenic differentiation, suggesting that Wnt/β-catenin pathway may be involved in the development of teeth and that the targeting Wnt/β-catenin pathway may have great potential for applications in dental tissue engineering regeneration. At an early stage of embryogenesis, cranial neural crest-derived cells migrate to the maxillary and mandibular processes and are then defined as EMSCs2. After migration, EMSCs interact with the dental epithelium and subsequently differentiate into several mesenchymal stem cells, such as dental papilla and dental follicle cells, which in turn form the dentin, pulp, cementum and periodontal ligaments3. Therefore, EMSCs derived from the cranial nerve crest are considered to be the progenitors of dental mesenchymal stem cells2. Recently, emerging studies have reported that EMSCs are capable of osteogenic differentiation in vitro. Yan et al. reported that mouse EMSCs isolated from eight-week-old BALB/c mice embryos are capable of extensive multilineage differentiation in vitro, including osteogenic differentiation4. Bruno et al. reported that EMSCs isolated from human olfactory mucosa originated from neural crest origins and have the potential for osteogenic differentiation5. Our previous studies showed that EMSCs had multi-directional differentiation potential and could be used as a powerful cell model for studying tooth development in vitro1. Investigating the mechanisms involved in the differentiation of EMSCs is of great significance for improving our understanding of tooth development and may have potential applications in periodontal/dental regeneration and tissue-engineering methods to repair orofacial structures.Ectomesenchymal stem cells (EMSCs), which are isolated from rat embryonic facial processes, are derived from the cranial neural crest6. Without Wnt signalling, β-catenin cannot accumulate in the cytoplasm because of the destruction complex. Once Wnt signalling is activated, the destruction complex function is destroyed, which allows β-catenin to accumulate and localize to the nucleus and ultimately upregulate the expression of Wnt/β-catenin target genes7. The Wnt/β-catenin pathway plays key roles in embryonic development, including in body axis patterning, cell fate specification, cell proliferation and cell migration8. Emerging studies have reported that the canonical Wnt/β-catenin pathway is closely associated with osteogenic differentiation in tooth development. For example, Han et al. reported that activation of the canonical Wnt signalling pathway significantly increased mineralization, alkaline phosphatase activity, and protein expression of bone and cementum markers in human periodontal ligament cells9. Liang et al. reported that the Wnt/β-catenin signalling pathway contributes to the ET-1-enhanced differentiation of periodontal ligament stem cells into osteoblasts under an inflammatory microenvironment10. Mao et al. reported that the canonical Wnt signalling inhibitor dickkopf1 repressed the mnHA bioceramics-induced upregulation of ALP activity and osteogenic and cementogenic gene expression in human periodontal ligament stem cells11. Moreover, Liu et al. reported that the telomerase reverse transcriptase/Wnt/β-catenin cascade is upregulated by acetylsalicylic acid treatment, leading to improved stem cells from exfoliated deciduous tooth-mediated bone regeneration12. Furthermore, Yang et al. reported that Wnt3a, a representative canonical member of the Wnt family of ligands, upregulates the expression of β-catenin and enhances the osteogenic differentiation of dental follicle cells, while dickkopf1, a Wnt/β-catenin signalling inhibitor, decreases the expression of β-catenin and attenuates the osteogenic differentiation of dental follicle cells13. However, it remains unknown whether the Wnt/β-catenin pathway also participates in the osteogenic differentiation of EMSCs, which are the progenitor cells of dental mesenchymal stem cells.The Wnt/β-catenin pathway is the canonical Wnt signalling pathway, and results in the accumulation of β-catenin in the cytoplasm and its final translocation into the nucleus as a transcriptional coactivator of transcription factors belonging to the TCF/LEF familyIn the present study and for the first time, we demonstrated that the Wnt/β-catenin pathway positively regulates EMSC osteogenic differentiation. Moreover, the Wnt/β-catenin pathway was targeted by low-affinity nerve growth factor receptor (LNGFR) during EMSC osteogenic differentiation. Additionally, LNGFR promoted EMSC osteogenic differentiation, which was decreased by the Wnt signalling inhibitor IWR-1-endo. Taken together, our data revealed that LNGFR targets the Wnt/β-catenin pathway and positively regulates EMSC osteogenic differentiation, suggesting that the Wnt/β-catenin pathway may be involved in tooth development and that targeting the Wnt/β-catenin pathway may have great potential for applications in dental tissue engineering regeneration.1. The bud stage is the initiation stage of tooth germ development, at which tooth germ cells are not yet differentiated and the tooth germ is in morphogenesis; the late bell stage is the maturation stage of tooth germ development, at which the tooth germ was undergoing tissue differentiation and morphological differentiation. The bud and late bell stages represent the early and late stages of tooth germ development, respectively, which is why we chose these two time points to complete our experiments. Subsequently, we detected EMSC cell surface markers; the mesenchymal stem cell markers CD29, CD44, CD90 and CD146 were highly expressed in both EMSCs separated at embryonic day 12.5 (E12.5d) and embryonic day 19.5 (E19.5d)  pregnant rat Fig. . Our pre5d) Fig. , while t5d) Fig. . We also5d) Fig. . For thein vitro osteogenic differentiation of EMSCs separated at E19.5d, which were used in the following experiments. We found that the mRNA levels of the early osteogenic markers ALP and RunX2 were upregulated during osteogenic induction by qPCR assay  is a receptor for neurotrophins and belongs to the tumour necrosis factor receptor (TNFR) superfamilysay Fig.  and Westsay Fig. . LNGFR esay Fig. . qPCR assay Fig.  also revsay Fig.  increasesay Fig.  showed msay Fig. . These rWe next silenced LNGFR during EMSC osteogenic differentiation. Immunofluorescence assays showed that LNGFR expression was significantly downregulated when LNGFR was silenced in EMSCs Fig. . qPCR asTo further study the role of the Wnt/β-catenin pathway in LNGFR-enhanced EMSC osteogenic differentiation, we inhibited the Wnt/β-catenin pathway using IWR-1-endo while overexpressing LNGFR during EMSC osteogenic differentiation. The results showed that without IWR-1-endo treatment LNGFR increased ALP and RunX2 mRNA level, while upon the treatment of IWR-1-endo, LNGFR no longer increased ALP and RunX2 mRNA levels Fig.  and b. SIn this study, we demonstrate that nuclear β-catenin levels are higher in EMSCs separated at E19.5d compared to EMSCs separated at E12.5d and upregulated during EMSC osteogenic differentiation. Moreover, the Wnt signalling inhibitor IWR-1-endo inhibits, while the Wnt signalling agonist SKL2001 promotes EMSC osteogenic differentiation. Further experiments showed that nuclear β-catenin is further upregulated by LNGFR overexpression during EMSC osteogenic differentiation. Moreover, LNGFR promotes EMSC osteogenic differentiation, which is decreased by IWR-1-endo. Collectively, our data show that the LNGFR targets the Wnt/β-catenin pathway targeted and positively regulates osteogenic differentiation in EMSCs.21. In this study, we found that LNGFR expression is higher in EMSCs separated at E19.5d compared to EMSCs separated at E12.5d. Based on this result, we speculated that LNFGR might be involved in the regulation of osteogenic differentiation in EMSCs. We then constructed EMSCs stably overexpressing LNGFR and found that their potential for osteogenic differentiation was higher than control EMSCs. Prior to our study, emerging studies had reported that LNGFR is positively associated with osteogenic differentiation in other cell types. Alexander et al. reported that LNGFR is expressed at higher levels in mineralizing isolated jaw periosteum-derived cells (JPCs) compared with non-mineralizing JPCs22. Moreover, Akiyama et al. reported that LNGFR overexpression induced alkaline phosphatase activity and the mRNA expression of osteoblast-related genes such as osterix and bone sialoprotein, thus promoting osteoblast differentiation in the human MG63 osteoblast cell line23. Furthermore, Mikami et al. reported that LNGFR overexpression enhanced proliferation and osteogenic differentiation in the MC3T3-E1 pre-osteoblast cell line18. Regarding tooth development, current studies mainly focus on the role of LNGFR as a neural stem cell marker26. It remains to be determined whether LNGFR can regulate osteogenic differentiation in other dental mesenchymal stem cells.Low-affinity nerve growth factor receptor (LNGFR) is the receptor of neurotrophins, a family of protein growth factors that stimulate survival and differentiation in neuronal cells27. These sites include the binding of extracellular ligands and cell membrane receptors, the stability of the destruction complex, and the co-transcriptional activation of β-catenin33. In this study, we used two small molecules (IWR-1-endo and SKL2001) to regulate the Wnt/β-catenin pathway. IWR-1-endo and SKL2001 both affect the function of the destruction complex, which contains Axin, protein phosphatase 2A (PP2A), adenomatosis polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α). We used IWR-1-endo to inhibit the Wnt/β-catenin pathway and found that IWR-1-endo decreased nuclear β-catenin levels and subsequently inhibited EMSC osteogenic differentiation. IWR-1-endo was first identified as an inhibitor of Wnt/β-catenin in 200934 and reported to abrogate the destruction of Axin proteins, thus suppressing Wnt/β-catenin pathway activity31. Recent studies have used IWR-1-endo to inhibit the activity of Wnt/β-catenin37. Consistent with our research on cell differentiation, Frith et al. have reported that IWR-1-endo decreases the expression levels of key osteogenic marker genes and inhibits osteogenesis in mesenchymal precursor cells38. Moreover, Gao et al. have reported that IWR-1-endo indeed reduced the amounts of β-catenin in the nucleus, as well as TCF1, a β-catenin target gene, thus activating Wnt signalling, which is required for hepatocyte differentiation39. In this study, we also used a novel agonist of the Wnt/β-catenin pathway, SKL2001, during EMSC osteogenic differentiation. We found that SKL2001 increased nuclear β-catenin levels and subsequently enhanced EMSC osteogenic differentiation. SKL2001 is reported to increase the intracellular β-catenin protein levels by downregulating the interactions between Axin and β-catenin40. Consistent with our study, Gwak et al. reported that the treatment of mesenchymal stem cells with SKL2001 promoted osteoblastogenesis by activating the Wnt/β-catenin pathway28. However, the roles of these two small molecules in the development of teeth require further study.The Wnt/β-catenin pathway can be regulated at several points in its signalling pathwaysCTNNB1 gene, is a dual function protein41. On one hand, β-catenin is a subunit in the cadherin protein complex and is involved in regulating cell–cell adhesion. On the other hand, β-catenin is a downstream component of the Wnt signalling pathway and serves as a transcriptional co-activator44. The activation of Wnt signalling leads to a decrease in β-catenin degradation and a subsequent accumulation of β-catenin45. In our experiments, nuclear β-catenin levels were further upregulated by LNGFR overexpression during EMSC osteogenic differentiation, suggesting that the Wnt/β-catenin pathway was targeted by LNGFR. However, targeting LNGFR in the Wnt/β-catenin pathway during EMSC osteogenic differentiation is not fully understood. A previous study showed that LNGFR bound to nerve growth factor (NGF) activates casein kinase II (CK2), which subsequently phosphorylates the phosphatase and tensin homologue deleted on chromosome 10 (PTEN)46. Phosphorylated PTEN becomes inactive and allows phosphatydilinositol 3-phosphate [PI(3)P] levels to increase, thus favouring GSK-3β inactivation and axon outgrowth in hippocampal neurons46. Another study showed that NGF fails to activate GSK3b phosphorylation of MAP1B in PC12 cells and Chick ciliary ganglion neurons that lack TrkA receptors but express LNGFR47. These two studies reveal that LNGFR is capable of promoting GSK-3β inactivation, which subsequently leads to the accumulation of β-catenin. However, in our stem cell model, LNGFR is unlikely to target the Wnt/β-catenin pathway and subsequently upregulate nuclear β-catenin by inactivating GSK-3β. We found that LNGFR-enhanced EMSC osteogenic differentiation was attenuated by IWR-1-endo, which promotes β-catenin degradation by abrogating Axin protein turnover and thereby enhances the function of the β-catenin destruction complex36. Considering the mechanism by which IWR-1-endo inhibits the Wnt/β-catenin pathway, we speculate that the points in the Wnt/β-catenin pathway targeted by LNGFR are located upstream of the β-catenin destruction complex. More studies are needed to understand a role of LNGFR in targeting the Wnt/β-catenin pathway during EMSC osteogenic differentiation.β-catenin, a protein that is encoded by the EMSCs are the progenitor cells of dental mesenchymal stem cells. Therefore, it is important to investigate the mechanism of EMSC differentiation, which is helpful to improve our understanding of tooth development and provide a potential strategy for dental tissue engineering. In this paper, we found that the Wnt/β-catenin pathway positively regulates osteogenic differentiation in EMSCs, suggesting that the Wnt/β-catenin pathway is very important in early tooth development and that targeting the Wnt/β-catenin pathway may have great potential in tooth repair and dental tissue engineering regeneration.2 incubator. E19.5d EMSCs were obtained by directly culturing the tissue blocks in DMEM/F12 containing 10% FBS and antibiotics.E19.5d EMSCs and E12.5d EMSCs were separated during an abdominal operation on the same pregnant SD rat . This study was approved by the Ethics Committee of the Third Military Medical University, and all experiments were performed in accordance with relevant guidelines and regulations. The operation was strictly performed under sterile operating conditions. E12.5d EMSCs were separated from the embryonic facial processes using 1% trypsin/1 mM EDTA  at 37 °C for 10 min. E19.5d EMSCs were separated from the dental papilla using 1% collagenase I for digestion at 37 °C for 30 min. The digestion was terminated using DMEM/F12  containing 10% FBS . E12.5d EMSCs were filtered to remove the tissue blocks using a 75 µm screen mesh . After centrifugation at 800 rpm for 5 min, E12.5d EMSCs were cultured in DMEM/F12 containing 10% FBS and antibiotics (100 µg/ml penicillin and 100 µg/ml streptomycin) in a 37 °C and 5% CO48. Cell surface molecular markers, including CD29, CD44, CD45, CD90, and CD146, were detected in 3rd generation cells. A total of 5 × 105 cells were fixed in 4% paraformaldehyde for 30 min and incubated in primary antibodies, including mouse anti-rat CD29, CD44, CD45, CD90 and CD146 , at 4 °C overnight. The secondary antibody anti-mouse IgG-FITC (1:1000) was then added, and the results were detected using a FACS Calibur flow cytometer (BD Biosciences).The properties of the E19.5d EMSCs and E12.5d EMSCs were detected using flow cytometry as previously described49. The primer sequences are listed in Supplementary Table The expression of LNGFR, ALP, and RunX2 in E19.5d EMSCs and E12.5d EMSCs was detected using qRT-PCR as previously described50. The primary antibodies included anti-GAPDH (YM1038) purchased from Immunoway; anti-LNGFR (ab8874) and anti-RunX2 (ab23981) purchased from Abcam; and anti-β-catenin (8480) and anti-Lamin B1 (13435) purchased from CST.Cells were collected and the total protein and nuclear protein were extracted according to the instructions . The protein concentration was measured using a BCA protein determination reagent kit . Western blotting was performed as previously describedEMSCs were cultured in DMEM/F12 containing 10% foetal bovine serum (FBS). Third generation cells were used in the experiment. The osteogenic induction solution contained 10% FBS α-MEM . The cell culture medium was changed every 3 days.On day 7 of osteogenic induction, the cells in each well were washed twice with PBS and fixed in 4% paraformaldehyde for 30 min. Staining was performed using an ALP staining kit  according to the manufacturer’s instructions.51. After staining, the cells were washed 3 times with double-distilled water and the staining levels were observed under a phase-contrast microscope.On day 21 of osteogenic induction, the cells in each well were washed twice with PBS and fixed in 4% paraformaldehyde for 30 min. Alizarin red staining was performed as previously describedThe 3rd generation E19.5d EMSCs were plated onto 6-well plates at 70–90% confluence and were cultured for 24 h. Two hours before transfection, the culture medium was changed to DMEM/F12 . siRNA-LNGFR and siRNA-NC  were transfected into E19.5d EMSCs using Lipofectamine 2000 in OPI-MEM . The siRNA sequences are listed in Supplementary Table 52 were co-transfected into HEK-293 cells using Lipofectamine 2000 (Invitrogen) as previously described53. Twenty-four hours after transfection, the supernatant containing packaged viruses was collected and filtered before use. The 3rd generation EMSCs were infected using 8 μg/mL polybrene. After 24 h, the cells were cultured in DMEM/F12 containing 2 μg/mL puromycin for one week.The pLJM1-LNGFR plasmid and psPAX2 and CMV VSV-G packaging plasmidsThe EMSCs stably overexpressing LNGFR, control EMSCs, EMSCs transfected with siRNA-LNGFR and EMSCs transfected with siRNA-NC were inoculated onto cover slips overnight. The cells were fixed in 4% paraformaldehyde and incubated with rabbit anti-rat LNGFR (1:100) and goat anti-mouse or anti-rabbit IgG-TRITC (red fluorescence) secondary antibodies. The cells were then stained with DAPI (Sigma) and observed under a confocal laser scanning microscope .- and ≤1.5-fold changes as threshold values and a significance level of <5% FDR. Signalling pathway analyses were performed for the genes with significant differences using the DAVID online analysis tool.Total RNA was extracted from the cells using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Any contaminating deoxyribonucleic acids were removed via DNase I digestion. The Affymetrix GeneChip Rat Genome 230 2.0 Array was used for the microarray analysis. Three independent cell samples were used in the microarray analysis (n = 3). Hybridization, data acquisition, and analyses were performed by the CapitalBio Corporation . The microarray results were analysed using the robust multiarray average (RMA) method. Significance analysis of microarrays (SAM) was used to identify differentially expressed genes. SAM-identified genes with statistically significant expression changes using a set of gene-specific t-tests and a permutation process provided an estimated false discovery rate (FDR) from the generated data. Genes with scores higher than the threshold value or genes with FDR values lower than the threshold value were considered significant. Additionally, fold-change analysis was performed to calculate the ratios of the geometric means of expression intensities. To select differentially expressed genes, we used ≥1.5-tests and one-way analysis of variance (ANOVA). All the experiments were repeated in triplicate to ensure that the results were real and reliable. P < 0.05 indicated statistical significance.All the data are expressed as the means ± standard deviations. Statistical significance was evaluated using the Prism 5 software. Comparisons were performed using tSupplementary Information


Embryonic stem cells (ESC) are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons (MNs) has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC (mESC) that constitutively produce glial cell line-derived neurotrophic factor (GDNF) and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic MNs. After lentiviral transduction, ESC lines integrated and expressed the human GDNF (hGDNF) gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study MN induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal MNs, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of MNs in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant hGDNF was added to control ESC, also resulting in enhanced MN differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, MNs were selected for electrophysiological recordings. MNs differentiated from GDNF-ESC, compared to control MNs, showed greater electrophysiological maturation, characterized by increased numbers of evoked action potentials (APs), as well as by the appearance of rebound APs, sag inward rectification, spike frequency adaptation and spontaneous synaptic potentials. Upon challenge with kainate, GDNF-overexpressing cells are more resistant to excitotoxicity than control MNs. Together these data indicate that GDNF promotes proliferation of MN-committed precursors, promotes neuronal differentiation, enhances maturation, and confers neuroprotection. GDNF-expressing ESC can be useful in studies of development and disease. Neural induction of mouse ESC (mESC) has been used as a cellular model to understand basic mechanisms of differentiation and physiology of postmitotic neurons. Importantly, ESC have been differentiated using neural medium and stimulation with morphogens such as sonic hedgehog and retinoic acid for ventralization and caudalization of neural precursors, respectively; this protocol yields spinal motor neurons  with retinoic acid and Sonic Hedgehog, producing MN positive for Beta-Tubulin III, GFP (reflecting Hb9 expression) and Islet1  complementary DNA (cDNA) was a kind gift from Dr. Martha C. Bohn. The plasmid containing the GDNF cDNA (pAAV-MCS-hGDNF) was digested and the fragment containing the cDNA was separated by agarose electrophoresis and column-purified. The 639 bp fragment was ligated into the lentiviral vector pLVX-EF1α-IRES-Puro (Clonthech 631988) to produce the pLVX-EF1α-hGDNF-IRES-Puro plasmid. The resulting plasmid was sequenced and verified to contain hGDNF.Lentiviral particles were produced accordingly to the manufacturer’s instructions (Clontech protocol PT5135-1). Empty vector  or hGDNF containing plasmids, together with lenti-X HTC packaging mix and Xfect reaction buffer were mixed with Xfect polymer and then poured into a previously seeded HEK294FT cells. Forty-eight hours later, supernatants were collected and added to the R1 mESC. Puromycin treatment (2 μg/ml) was initiated 24 h later, and 4 days after treatment, several resistant colonies were obtained.Selected colonies were lysed and genomic DNA was extracted; end-point PCR for hGDNF detection was performed using a 30-cycle program at 59°C. Primers  for GAPDH were: Forward, ATCACCATCTTCCAGGAGCG and Reverse, CCTGCTTCACCACCTTCTTG and for transgenic hGDNF: Forward, AACAAATGGCAGTGCTTCCT and Reverse, AGCCGCTGCAGTACCTAAAA. The same protocol and primers were used for mRNA using Trizol extraction protocol after DNase treatment  and supplemented with protease inhibitors (Roche). Proteins were obtained by centrifugation at 13,800 g at 4°C for 15 min, and quantified by Bradford assay (Bio-Rad). Proteins (30 μg) were resolved on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham Bioscience). The membranes were blocked with 5% non-fat milk and incubated overnight with primary antibodies. Pre-stained markers (Invitrogen) were included for size determination. The following antibodies were used: rabbit anti-GDNF  and anti-GAPDH . Membranes were washed and incubated with corresponding horseradish peroxidase-coupled secondary antibodies  for 2 h. Immunoreactive bands were detected using the enhanced chemiluminescence method (Amersham Bioscience) and developed on photographic film (Kodak).To quantify secreted GDNF, 24 h-conditioned media were obtained from CTRL-ESC and GDNF-ESC. The media were collected from undifferentiated mESC and from day-6 EBs, which already contained differentiated MNs. The kit GDNF Emax Immunoassay system was purchased from Promega and used as indicated by the manufacturer’s booklet.CTRL-ESC or GDNF-ESC were electroporated with HB9::GFP plasmid (a kind donation from Dr. Hynek Wichterle) and selected with neomycin (500 μg/ml) or hygromycin B (150 μg/ml), respectively, to create CTRL-ESC/HB9::GFP or GDNF-ESC/HB9::GFP.MNs were differentiated as reported . Primary antibodies were incubated overnight at 4°C in phosphate buffer saline (PBS) plus 10% normal goat serum. Cells were washed three times and secondary antibodies incubated for 2 h in the same solution, washed three times and incubated with Hoechst for nuclei staining. Primary antibodies were used with the following dilutions: Oct 3/4, 1:200 (BD 611202); Sox2, 1:1000 (Millipore AB5603); GFP, 1:1000 (Invitrogen A11122); Olig2, 1:1000 (Millipore MANB50); Islet1, 1:10 ; TUJ1 (recognizing Tubulin β III), 1:1000 (Covance MRB-435P); Microtubule Associated Protein (MAP2), 1:1000 (Chemicon MAB378); Phospho Histone H3, 1:100 . Alexa Fluor secondary antibodies were used at 1:1000. EBs cultured for 5 days were harvested by gravity, washed with PBS and paraformaldehyde-fixed, cryo-protected by sucrose, tissue tek-embedded, cut in 20 μm slices and mounted in slides for immunofluorescence protocol.Oct4: Forward (Fwd), TTG GGC TAG AGA AGG ATG TGG TT; Reverse (Rev), GGA AAA GGG ACT GAG TAG AGT GTG G. For Sox2: Fwd, GCA CAT GAA CGG CTG GAG CAA CG; Rev, TGC TGC GAG TAG GAC ATG CTG TAG G. For Nanog: Fwd, TAT CTG GTG AAC GCA TCT GG and Rev, GAA GTT ATG GAG CGG AGC AG. The house keeping gene Actin was used to normalize the expression of the former genes in CTRL-ESC HB9::GFP and compare them with GDNF-ESC HB9::GFP. Results are expressed as fold-change relative to CTRL-ESC.Total RNA was isolated from CTRL- and GDNF-ESC. Reverse transcription was performed with random hexamers to produce cDNA, as previously reported  and whole cell input resistance (RN) were measured from the I-V plots built from the recordings: after identification, neurons were visualized using infrared differential interference microscopy . Micropipettes for whole cell recordings were pulled (Sutter Instruments) from borosilicate glass tubes  for a final DC resistance of 4–6 MΩ when filled with an internal solution with the following composition (in mM): 120 KMeSO3, 10 NaCl, 0.5 EGTA, 10 HEPES, 2 Mg2+-ATP, 0.4 Na+-GTP and 1% biocytin  for a pCa2+ of about 6.5 . Recordings were made at room temperature (~25°C) and obtained with an Axopatch 200B amplifier (Axon Instruments) while monitored with an oscilloscope  relationships at −60 mV. The latter were obtained with either depolarizing or hyperpolarizing rectangular voltage steps (current-clamp) or voltage commands (voltage-clamp). RMP was measured in current-clamp at zero current. Percentages of neurons exhibiting diverse signs of excitability suggesting various differentiating stages were obtained in both samples and compared (see “Results” Section).The expression of GFP under the HB9 promoter enables the visual identification of differentiated MNs. Because the main interest of several previous studies was the expression of the MN phenotype, such neurons were co-cultured with muscle fibers  and calcium potential differences were analyzed by Chi-square test. P < 0.05 was used as significance level.All experiments were performed in duplicate. Results were subjected to one-way analysis of variance (ANOVA) and Bonferroni analysis or gdnf gene. End-point PCR confirmed that several colonies integrated the hGDNF coding sequence, and we designated these as GDNF-ESC; empty vector-transduced cells were named CTRL-ESC  or simultaneously allows expression of hGDNF and puromycin resistance Figures . LentiviC Figure . HoweverC Figure . To analC Figure . In agreGDNF is known for having several neuronal targets, which includes MNs. We decided to study MN differentiation by using a reporter construct that expresses GFP under control of the HB9 promoter, which directs its expression only in postmitotic MNs. This reporter appears early during EB formation and allows the identification of MNs. The HB9::GFP plasmid was co-transfected with either neomycin or hygromycin B resistance cassettes in CTRL- or GDNF-ESC, respectively, and selected with the appropriate antibiotic to produce CTRL-ESC/HB9::GFP or GDNF-ESC/HB9::GFP Figure . SeveralPostmitotic neurons were evaluated after dissociation of EBs and replating. Islet1 (Isl1) is a transcription factor that is normally expressed early during MN differentiation, and together with HB9 comprise pan-MN markers  at specific periods of differentiating CTRL-ESC/HB9::GFP. These control cells were differentiated without rhGDNF Figure , or withN no differences were noted, although the distribution of GDNF-expressing cells was compacted and with less variability  blocked the slower plateau-like calcium component  discover that GDNF in EBs induced higher proliferation resulting in more committed MN precursors (pMN); (2) report that GDNF induced electrical maturation in MNs in the absence of exogenous factors; and (3) observe self-protection against injury in MN cultures. Further, the addition of exogenous rhGDNF demonstrates that this trophic factor enhances MN differentiation when added at the pluripotent state.The GDNF-overexpressing ESC lines were capable of robust MN differentiation, but moreover, pluripotency-associated factors such as Oct4 and Sox2 were not altered suggesting that stemness is intact. In differentiation protocols of pluripotent stem cells towards MNs, GDNF is commonly included as a trophic factor for postmitotic MNs. However, whether neural precursors are responsive to GDNF remained an open question. In such case, GDNF would not only impact neuronal survival, but also enhances cell commitment. Our findings suggest that GDNF might be directing the commitment of neurons towards specific phenotypes and not only increasing the survival of neurons; the same question might be addressed for other neurotrophic factors important for MN differentiation or survival such as VEGF.GDNF and its receptors are expressed in proliferating zones during spinal cord development, and thus GDNF might have additional roles to neuronal survival  GDNF specifies the differentiating cells to become MNs; for instance, GDNF is necessary for the specification of neurons in the dorsal root ganglia , channelrhodopsin-2 and GDNF were differentiated to MNs. Notably, MNs expressing GDNF survived longer than the parental cell line. GDNF MNs co-cultured with ESC-derived astrocytes for 7 days were not able to sustain a high frequency of APs after electrical stimulation, similar to MNs derived from CTRL-ESC/HB9::GFP. Only MNs co-cultured with glia for 21–35 days showed a sustained response of APs frequency with increasing depolarizing current, similar to the 9 day-old MNs differentiated from our GDNF-ESC/HB9::GFP, which did not require astrocytes. Such dissimilarities might be explained by the different methods used to produce the transgenic ESC: electroporation  by local GDNF overexpression, preventing its crossing through the injury site to reach the distal portion, which results in aggravation of the condition, as assessed both morphologically and functionally  to IV and Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (Papiit IN208713 and IN213716 to IV and IN202814 to JB). DC, YR-A, RH-M and IE-A received graduate fellowships from CONACyT and data in this work is part of DC doctoral dissertation in the Posgrado en Ciencias Bioquímicas de la Universidad Nacional Autónoma de México.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These findings indicate that BCAR4 facilitated colon cancer progression by enhancing cell proliferation and inhibiting apoptosis via BCAR4/β-catenin axis. BCAR4 may be a useful new target for treatment of patients with colon cancer.BCAR4 (Breast Cancer Anti-Estrogen Resistance 4) is a long noncoding RNA that was identified as an oncogene in breast cancer. In our research, we found that the expression level of BCAR4 was upregulated in colon cancer tissues compared to paired normal tissues. What's more, higher BCAR4 expression was correlated with lower survival rate in patients with colon cancer. Mechanistically, we showed that BCAR4 activated Wnt/β-catenin signaling in colon cancer by protecting β-catenin from degradation. We also showed that BCAR4 overexpression promoted cell proliferation and migration in colon cancer. However, silencing BCAR4 inhibited cell growth and promoted apoptosis. Besides, BCAR4 knockdown decreased tumor growth  Colon cancer (CC) is one of the most common cancers around the world, which leads to large amounts of deaths every year , 2. Up tLong noncoding RNAs are transcripts of more than 200 nt in length and have no coding potential . LncRNAsIn this study, we revealed that the expression levels of BCAR4 were upregulated in colon cancer tissues compared to adjacent normal tissues. Overexpressing BCAR4 in colon cancer cell line HCT8 and SW480 promotes cell proliferation and migration while BCAR4-silenced cells showed enhanced apoptosis and impaired proliferation. In mechanism, BCAR4 interacts with β-catenin to prevent its degradation. Then more activation of Wnt/β-catenin promotes cell proliferation and migration. Collectively, our results reveal a new function of BCAR4 in colon cancer and may provide a new sight on treatment of patients with colon cancer.in situ, and found that more BCAR4 existed in colon cancer tissues than in adjacent normal tissues  associated β-catenin, and deletion of nt 1000-1314 in BCAR4 abrogated its interaction with β-catenin Figure  and 6B. To further confirm the relationship between BCAR4 and Wnt/β-catenin signaling, we detected the expression levels of BCAR4 and target genes of Wnt/β-catenin signaling by RT-qPCR in 30 colon cancer tissues. We found that BCAR4 was positively correlated with the expression levels of these target genes of Wnt/β-catenin signaling Figure , which sin vivo, we inoculated WT or BCAR4-silenced HCT8 cells into nude mice. 4 weeks later, the weights of formed tumors was measured. We found that WT HCT8 cells formed larger and heavier tumors than BCAR4-silenced cells  were obtained from ATCC and maintained in DMEM medium supplemented with 10% fetal bovine serum , 100 μg/ml penicillin and 100U/ml streptomycin. Normal human colon epithelial cells CCD 841 CoN (ATCC) were cultured in Eagle's Minimum Essential Medium and supplemented with 10% FBS. Cells were incubated at 37°C in a humidified atmosphere with 5% COBCAR4 full-length was cloned into pCDNA3 plasmid. shBCAR4 (5′-GGGACTTGAGTTATGTTGGTGGCTA-3′) was synthetized by invitrogen and cloned into pGPH1/Neo  as described before . BCAR4 oAnti-BCL2 (4223), anti-BID (8762), anti-BAX (5023), anti-COX IV (4850), anti-GAPDH (5014), anti-β-catenin (9562), anti-phospho-β-catenin , anti-MYC (9402) and anti-CYCLIN D1 (2922) were purchased from Cell Signaling Technology.Apoptosis analysis was performed through Annexin V-FITC/PI apoptosis detection kit (eBiosciences) by FACS.6BCAR4-silenced cells were subcutaneously injected into BALB/c nude mice. Tumor weight was measured on week 5 post injection. Animal experiments were performed in accordance with relevant guidelines and regulations of the Institutional Animal Care and Use Committees at Affiliated Hospital of Zunyi Medical College, and protocols were approved by the Institutional Animal Care and Use Committees at Affiliated Hospital of Zunyi Medical College.We purchased six-week-old male BALB/c nude mice from HFK Biosciences and maintained under pathogen-free conditions with approval by Affiliated Hospital of Zunyi Medical College. For tumor propagation analysis, 2×103 cells were seeded into 96-well plates. Cell proliferation was measured at indicative time points. MTT   was added into each well and incubated for 4 h at 37°C. Then 150 μl DMSO was added to solubilize the crystals. To determine cell viability, the absorbance was read at wavelength of 570 nm and 650 nm (background reading subtracted) with a microplate reader (Bio-rad).For MTT assays, 1×103 cells were seeded into a 6-well plate and incubated at 37°C for 12 days. And then the cells were fixed in 90% ethanol and stained with crystal violet solution. The formed colonies were counted.For colony formation assays, 2×105 cells were resuspended in serum-free medium and added into the top chamber. Medium containing 10% FBS was added to the lower chamber. After incubation for 12 h, cells on the lower surface were stained, photographed, and counted by a microscope in six random fields for each group.For cell Migration assays, the transwell filter chambers  were used according to the manufacturers’ instructions. Briefly, 2×10Actb. The primer sequence information is available if requested.Total RNAs were extracted with TRIzol according to the manufacturer's protocol. Then cDNA was synthesized with the M-MLV reverse transcriptase (Promega). Then mRNA transcripts were analyzed with ABI 7300 qPCR system using specific primer pairs. Relative expressions were calculated and normalized to endogenous Protocols for Northern blot have been described before . In brie2O2) for 30 min. Biotin-conjugated probes and streptavidin-HRP conjugate were used for ISH. The samples were finally stained with haematoxylin.CC and peri-tumour samples were fixed and embedded with paraffin. Then sample sections were incubated in graded alcohols and incubated in 3% hydrogen peroxide .P< 0.05 was considered statistically significant.All statistical analyses were performed using the Statistical Package for the Social Sciences version 20.0 software . Survival curves were calculated using the Kaplan-Meier method and were analyzed using the log-rank test. For comparisons, one-way analyses of variance and two-tailed Student's t-tests were performed, as appropriate.


Wnt signaling plays crucial role in regulating melanocyte stem cells/melanocyte differentiation in the hair follicle. However, how the Wnt signaling is balanced to be overactivated to control follicular melanocytes behavior remains unknown. Here, by using immunofluorescence staining, we showed that secreted frizzled-related protein 4 (sFRP4) is preferentially expressed in the skin epidermal cells rather than in melanocytes. By overexpression of sFRP4 in skin cells in vivo and in vitro, we found that sFRP4 attenuates activation of Wnt signaling, resulting in decrease of melanocytes differentiation in the regenerating hair follicle. Our findings unveiled a new regulator that involves modulating melanocytes differentiation through a paracrine mechanism in hair follicle, supplying a hope for potential therapeutic application to treat skin pigmentation disorders. Melanocytes in the epidermis and hair follicle synthesize melanin granules to pigment skin and hairs. Melanocyte shows cyclic melanogenic activity coinciding with hair cycling, including telogen, anagen, and categen , 2. Whenβ-catenin signaling pathway [β-catenin, fail to generate melanocytes [β-catenin to the cultured mouse neural crest cells results in expansion and differentiation of melanocyte [Wnt/ pathway , 14 playanocytes , 16. On lanocyte , 18. Ourlanocyte –23. HowesFRP4 is a glycoprotein that acts as an antagonist of Wnt signaling. Previous studies show that sFRP4 is a modulator of cell differentiation and apoptosis in many tissues, such as mammary gland, ovary, and heart –26. sFRPDct-LacZ transgenic mice were kindly provided by Professor Ian Jackson . Mouse sHuman skin samples were obtained from 30–40-year-old donors. Formalin-fixed, paraffin-embedded melanoma tissues were received from Xinqiao Hospital, the Third Military Medical University. The research was approved by the ethics board at Third Military Medical University.μm sections from tissue samples. Sections were dewaxed, rehydrated, and boiled in citrate buffer solution [β-catenin , and rabbit anti-PCNA . Alexa Fluor 488  and CY3  were used as secondary antibodies. Finally, sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclei visualization.Immunostaining was performed on 5 solution , 35. AftμL recombinant sFRP4  or PBS (control) was intradermally injected into the dorsal skin after depilation. Then the treated skin samples were harvested and prepared for analysis of histology or immunostaining.To induce synchronized hair cycle, the hairs on the dorsal skin of 7-week-old mice were depilated as previously described , 37. 25 μm sections, which were then dewaxed, rehydrated, and stained with hematoxylin for nuclei visualization.For X-gal staining, the samples were prefixed in 4% paraformaldehyde at 4°C for 1 hour, then stained with X-gal staining solution  at room temperature for 24 h, and postfixed at 4°C overnight. After dehydration and embedding in paraffin, the skin samples were sliced into 5 2 in air at 37°C.The JB6Cl30-7b (JB6) cell line derived from primary cultures of neonatal mouse epidermal cells was purchased from ATCC (ATCC®CRL-2007™). The immortalized cell line of melanocyte progenitor, iMC23, was derived from neonatal mouse epidermis . The cel8 PFU/mL.Adenoviruses including AdWnt3a, AdsFRP4, and AdGFP were gifted from Dr. Tong-Chuan He (University of Chicago). The adenoviruses were propagated in HEK293 cells as previously described . After bJB6 cells were plated onto the upper chamber of transwells, infected with AdsFRP4, AdWnt3a, and AdGFP (control) separately, or coinfected with AdsFRP4 and AdWnt3a. After 12 h incubation, the lower chambers were seeded with iMC23 cells. iMC23 cells and JB6 cells were cocultured for 72 h, then iMC23 cells were harvested to perform the tyrosinase activity assay or Masson-Fontana silver staining assay.μL supernatant was transferred into 96-well plates and added with 10 μL L-DOPA  per well. The absorbance was measured at 490 nm after the samples were incubated at 37°C for 2 hours. The relative activity of tyrosinase equals (OD group X − OD blank)/(OD control − OD blank) × 100%.Cells were treated according to the aforementioned method. Tyrosinase activity assays were performed as previously described . iMC23 cCells were treated according to the aforementioned method. iMC23 cells were fixed with ice-cold acetone for 10 min and stained with a silver-ammonia solution  at 37°C for 1 h. Finally, the cells were rinsed and the images were captured using a phase-contrast microscope.β-catenin  at 4°C overnight. Blots were then incubated with HRP-conjugated secondary antibody  for 1 hour. Bands were visualized on the membranes using an ECL western blotting detection system.Western blots were done based on previous descriptions –42. Cellt-test and p < 0.05 was considered statistically significant.Data were presented as a mean ± SD of three independent experiments. Statistical analyses were evaluated using https://doi.org/10.1155/2017/2857478). sFRP4 is strongly expressed in the precursors of the inner root sheath (IRS) in hair follicles and the interfollicular dermal region during early anagen. In mid-anagen, sFRP4 is expressed in the IRS precursors, IRS, and outer root sheath (ORS) and weakly in the hair matrix. During catagen, sFRP4 is mainly expressed in the ORS above the epithelial strand and weakly in dermis. When hair follicles enter telogen, sFRP4 is expressed in the bulge of hair follicle and strongly expressed again in the dermal region surrounding the hair follicle.We first detected the expression pattern of sFRP4 in mouse dorsal skin by immunofluorescence   to deterWe then investigated the role of sFRP4 in follicular melanocytes in vivo. We depilated 7-week-old Dct-LacZ transgenic mice (in the second telogen phase) to induce synchronized anagen reentry of hair follicles, which also results in activation of McSCs , 45. ImmWe further examined the cell proliferation (PCNA) and differentiation (AE13 and AE15) after sFRP4 treatment. The result showed that the proliferated and differentiated cells are largely reduced in hair follicles after the sFRP4 treatment for 2 and 4 days , suggesting that sFPR4 also influences hair cycling, which was demonstrated by the previous study . Interesp < 0.05). These data further suggest that sFRP4 inhibits McSCs differentiation and maintains melanocytes in an undifferentiated state.To investigate the effect of sFRP4 on the differentiation of McSCs/melanocytes, we examined the expression of TRP1, which is a differentiation marker of McSCs/melanocytes . Two dayβ-catenin which is a key effector of Wnt signaling. We observed that nuclear β-catenin was present in TRP2-positive melanocytes in the PBS-treated hair follicles, but not in the sFRP4-treated hair follicles 2 days after depilation. Then, 4 days after depilation, β-catenin positive cells were increased in hair matrix and TRP2-positive melanocytes in the PBS-treated hair follicles. However, the number of β-catenin-positive melanocytes was significantly reduced in the hair follicles showing a delayed anagen reentry 4 days after sFRP4 treatment . The melanin production was also directly observed through the pigmented cell pellets due to melanogenesis. The formation of melanin was reduced in Wnt3a + sFRP4-treated group compared to Wnt3a-treated group (β-catenin in different experimental groups by western blots. The results showed that the expression of these differentiation-related genes was decreased in Wnt3a + sFRP4-treated group compared to the Wnt3a-treated group (Wnt ligands expressed by epithelial cells can activate Wnt signaling in neighboring melanocytes in early anagen hair follicle . To furted group . This waed group . We furted group .β-catenin signaling in vitro.Taken together, these results suggest that sFRP4 inhibits the differentiation of melanocytes through abrogating Wnt3a-induced Wnt/β-catenin signaling pathway (One of the most important theories in hair follicle biology is the indirect regulation of cell behaviors by the secreted factors from the microenvironment niche and macroenvironment tissues –49. Acti pathway .β-catenin, sFPR4 further regulates melanocytes differentiation. Second, unlike the inner bulge cells, which are not responsive for the local or environmental stimuli [Previous studies showed that sFRP4 mRNA is mainly expressed in the extrafollicular dermal region . By dete stimuli , it look stimuli , 52. Thi stimuli . For exa stimuli . Thus, uβ-catenin in melanocytes even causes melanoma formation in mice [The melanocytes that are differentiated from McSCs migrate out from bulge region in anagen phase and maintain their melanogenesis ability, which is reported to be largely regulated by other factors, such as Wnts . However in mice . Thus, pβ-catenin signaling pathway activation, leading to less differentiation of melanocytes. This suggests that sFRP4 inhibits melanocytes differentiation through inhibiting Wnt/β-catenin signaling pathway. What is the exact mechanism in which sFRP4 inhibits Wnt/β-catenin signaling pathway to influence melanocytes differentiation? Our recent work showed that Wnt3a contributes to promoting melanocytes differentiation through increasing Wnt/β-catenin signaling pathway [β-catenin to modulate the mammary differentiation in vitro [β-catenin signaling pathway, resulting in a decrease of Wnt3a-induced differentiation.sFRP4 is also largely involved in other pathways . In the  pathway , 22. It in vitro . By applβ-catenin signaling pathway. Importantly, our results provide future directions to investigate signaling mechanisms involving regulation between melanocytes and keratinocytes and also supply a hope for a potential clinical usage to treat skin pigmentation disorders.In conclusion, our findings demonstrate a paracrine mechanism that is involved in melanocytes differentiation during hair cycling. As a Wnt antagonist, sFRP4 is expressed by keratinocytes and inhibits the differentiation of neighboring melanocytes via inhibiting Wnt/In Supplementary Figure 1 and 2, the expression pattern of sFRP4 in mouse dorsal skin was detected by immunofluorescence. In Supplementary Figure 3, the effect of sFRP4 on the pigmentation of hair follicle was detected. The result showed that the skin eventually turned black when sFRP4 administration was ceased. In Supplementary Figure 4, the effect of sFRP4 on the differentiation of hair follicle cells was examined. The result showed that the differentiated cells were reduced after sFRP4 treatment. In Supplementary Figure 5, the effect of sFRP4 on the proliferation of hair follicle cells was examined. The result showed that the proliferated cells were reduced after sFRP4 treatment.


Drosophila Hox gene Ultrabithorax (Ubx) acts in the late embryo to establish target specificity of ventrally projecting RP motoneurons. In abdominal segments A2 to A7, RP motoneurons innervate the ventrolateral muscles VL1-4, with VL1 and VL2 being innervated in a Wnt4-dependent manner. In Ubx mutants, these motoneurons fail to make correct contacts with muscle VL1, a phenotype partially resembling that of the Wnt4 mutant. We show that Ubx regulates expression of Wnt4 in muscle VL2 and that it interacts with the Wnt4 response pathway in the respective motoneurons. Ubx thus orchestrates the interaction between two cell types, muscles and motoneurons, to regulate establishment of the ventrolateral neuromuscular network.Hox genes are known to specify motoneuron pools in the developing vertebrate spinal cord and to control motoneuronal targeting in several species. However, the mechanisms controlling axial diversification of muscle innervation patterns are still largely unknown. We present data showing that the  Summary: Ultrabithorax controls correct innervation of ventrolateral muscles by coordinating Wnt4 ligand expression in muscles with the signalling pathway response in motoneurons. Drosophila, they regulate, among other processes, survival and identity of leg MNs (Deformed (Dfd) is required in both establishment of the larval neuromuscular feeding unit and later in regulation of specific motoneuronal effector genes such as that encoding Ankyrin2-XL (Ultrabithorax (Ubx), which specifies the first abdominal segment, results in thoracic segments exhibiting an abdominal peristaltic pattern  muscles of embryonic abdominal segments A2 to A7 as well as their innervation pattern are well-described . To explHb9-Gal4 line, which is active in all postmitotic RP MNs and a limited set of interneurons and dorsal MNs -positive MNs. These MNs cross the posterior segment border and project into the intersegmental nerve branch b (ISNb) of the next segment, so that the MNs of segment A1 innervate VL muscles in segment A2 . Because Hox genes have generally been shown to be involved in late events of CNS maturation . In abdominal segments A2 to A7, however, we found that innervation of VL1 was either lost or strongly reduced  fall into this category (Table S1). The other connections were either reduced , implying a specific and novel role for Ubx in this process.To test whether  reduced B. We ass reduced A. In UbxUbx and the abdominal Hox gene abdominal A (abdA) have been shown to function redundantly in several contexts .contexts . AlthougUbx mutants is due to loss or temporal mis-specification of RP MNs, we performed anti-Hb9 staining . All RP MNs were present from A1 to A7 in 1Ubx mutants. Specifically RP5, which co-innervates VL1, could clearly be visualised with anti-Hb9 and anti-Cut double staining . Furthermore, the same study revealed differential Wnt4 expression between VL1 and VL2, with higher expression levels in VL2. Sulf1, a sulfatase implicated in the regulation of Wnt and BMP gradients at the neuromuscular junction (NMJ)  , was alson (NMJ) . As the  embryos , we wondWnt4 shows a graded expression within the VNC, having the highest levels in the most posterior segments   A (Inaki uscle 1)  and is sreported   and Derailed 2 (Drl-2). Both receptors exhibit mutant phenotypes similar to Wnt4 (Drosophila β-catenin homologue armadillo (arm), a key transducer of Wnt signalling. We used 8arm, a weak temperature-sensitive allele that lacks arm function in epidermal and neuronal Wingless (Wg) signalling but retains it in adherens junctions  of analysed hemisegments (Table S1). Instead, the contacts on VL2 were often strongly expanded. These defects were highly specific to VL1-MNs, as we were unable to find obvious phenotypes in the rest of the motoneuronal system or in muscle morphology. Loss of arm function in the canonical Wnt signalling pathway thus causes phenotypes similar to those of Ubx and Wnt4 mutants, suggesting that Wnt4 secreted from muscles activates the canonical Wnt signalling pathway in the VL1-MNs. To address more precisely whether activation of Wnt4 signalling is in fact required in these MNs, we performed an arm RNAi knockdown using Hb9-Gal4. Correct VL1 innervation was significantly reduced to 29.6% (n=142) in these embryos (Table S1), whereas projections of other MNs were not affected. We also analysed the role of further canonical Wnt signalling pathway components in VL1-MNs by overexpressing the negative regulator Glycogen Synthase Kinase-3  and a dominant-negative construct (dTCF.DN) of the Wnt effector TCF (pan – FlyBase) (Table S1). Indeed, with only 20.2% (n=94) and 59% (n=105) of correct contacts on VL1, GSK3 and TCF.DN, respectively, both induced significant defects specifically in targeting of VL1 (Table S1). Together, these results indicate that canonical Wnt signalling is likely required cell-autonomously in VL1-MNs for correct targeting of this muscle.The aforementioned study on control of VL1 innervation showed that Wnt4 requires two Wnt receptors in this context,  to Wnt4 . Howeverunctions . Because product . The obseratures B. MN conUbx interacts with Wnt4 and arm at the genetic level in this specific developmental context. Heterozygotes of 1Ubx, EMS23Wnt4, 8arm and the null mutant 4arm showed only a slight decrease in correct innervation of VL1 compared with wild type (Table S1). This changed considerably in EMS23Wnt4/+; 1Ubx/+, 4arm/X;;1Ubx/+ or 8arm/X;;1Ubx/+ double heterozygotes, in which correct contacts on VL1 were significantly decreased to 29.1% (n=103), 38.3% (n=107) and 21.4% (n=70), respectively (Table S1). Taken together, these data suggest that Ubx interacts with the canonical Wnt4 signalling pathway for correct innervation of VL1 by VL1-MNs.We then tested whether arm is clearly required in the MNs  resembles the Ubx mutant phenotype and can be rescued by providing Ubx in the same cells (Table S1). Moreover, a simultaneous RNAi knockdown of Ubx and arm enhances the effect of single knockdowns with only 10.5% (n=95) of correct contacts compared with 27.7% (UAS-Ubx RNAiIIn=83) and 29.6% , further supporting the notion that Ubx and the Wnt4 signalling component Arm interact in VL1-MNs to ensure proper VL1 innervation. Together, our results show that Ubx is necessary both in muscles (to activate Wnt4 ligand expression) and in MNs  and therefore strongly suggest that Ubx coordinates Wnt4 signalling between these two cell types to establish proper neuromuscular connections.As  the MNs  and it h the MNs , we wondUbx function is indeed required in both muscles and neurons, we performed rescue experiments, restoring Ubx expression in a tissue-specific manner in the Ubx mutant background. We first tested whether expression in muscles alone would be enough to rescue the VL1 innervation phenotype. Neither of the two mesodermal drivers we used, 24B-Gal4 and Mef2-Gal4, was able to restore normal VL1 innervation (Table S1). Expression of Ubx only in postmitotic MNs using the Hb9-Gal4 driver was also not sufficient for correct VL1 innervation (Table S1). To examine whether Ubx is needed earlier during the specification process of NB3-1, we used the scabrous-Gal4  driver, which drives in the early neuroectoderm and remains active in most cells of the VNC until late developmental stages. However, this driver line could also not rescue aberrant VL1-MN projections in the Ubx mutant (Table S1).To test whether Actin5C-Gal4 driver line  to restore Ubx expression in both tissues. Here, we observed a significantly higher rate of correct contacts on VL1  compared with 1Ubx mutants  (Table S1). The relatively low rescue rate is likely to be due to Act5C-Gal4 not being active at exactly the required developmental time points in muscles and MNs. In addition, the levels of Ubx expressed under its control, especially in the CNS, were lower than the endogenous ones  and were thus unlikely to be sufficient for a more pronounced rescue of the Ubx phenotype. Nevertheless, partially restoring Ubx expression in both tissues shows significant rescue rates, highlighting a role for Ubx in coordination of the ligand trigger from muscles and the response in MNs to ensure proper innervation of VL muscles.Finally, we employed the ubiquitous Drosophila embryos reported segment-specific differences in the morphology of VL muscles  and of the RYK family (Derailed 2) on the VL1-MNs . Howeverhila NMJ  and durihila NMJ . Howeverguidance . We now in vivo bimolecular fluorescent complementation assay , 6.28Ubx/TM6B, Tb, Sb, Dfd-lacZ, MX1abdA/TM3, Sb, Kr-Gal4, UAS-GFP , Mef2-Gal4, UAS-CD4::td-tom.FP/TM6B , Act5C-Gal4/CyO, EMS23Wnt4, bw/CyO, hb-lacZ, 4arm/FM7, grh-lacZ, 8arm/FM7c,Dfd-GMR-nvYFP, UAS-mCherry RNAi, UAS-Ubx RNAi, UAS-arm RNAi, UAS-sgg.B (UAS-GSK3), UAS-dTCFΔN (UAS-dTCF.DN), UAS-CD4::tdGFP and UAS-CD8::GFP . 8/Xarm or 8/X;; Ubx1/+arm animals were identified by the anti-Sex lethal signal.The following fly stocks were used: Oregon R,  UAS-GFP , Hb9-Galftz-lacZ , 24B-GalUbx RNAi insertion on the second chromosome (attP40) was generated using the shUbx RNAi (HMS01403) construct in pValium20 , rabbit anti-Beta-Gal , mouse anti-GFP , rabbit anti-GFP , mouse anti-Fas2 1D4 (1:10), mouse anti-Cut 2B10 (1:20), mouse anti-Sxl M18 (1:10), mouse anti-Ubx FP3.38 (1:20) , rat anti-Myosin , rabbit anti-Hb9  and guinea pig anti-Ubx .As fluorescent secondary antibodies we used anti-guinea pig Dylight 405, anti-chicken Alexa Fluor 647 (both Jackson ImmunoResearch), anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 488, anti-mouse Alexa Fluor 568, anti-rabbit Alexa Fluor 568, anti-rat Alexa Fluor 633  at 1:500. All secondary antibodies were used according to the manufacturer's protocols.in situ hybridisation, the Sulf1 probe was generated by PCR as reported previously (Wnt4 probe was made from EST clone RE26454 (BsgI (New England Biolabs). Primer sequences are: Dsulf1-fwd, 5′-GCCTTATAATTGGCGGCC-3' and Dsulf1-rev-SP6, 5′-ATTTAGGTGACACTATAGAAGAGTTGAGGAGCGGAGGAAGG-3′. Both probes were labelled using the DIG-RNA Labelling Kit (Roche). The hybridisation on embryos was carried out as described before (For eviously . The Wnt RE26454  upon digd before . Probes The non-fluorescent staining was documented using a Zeiss Axioplan. Fluorescent confocal images were acquired on a Leica TCS SP5 microscope. Laser intensities were kept constant between experiments and controls. Image processing was carried out using ImageJ, Adobe Photoshop CS4 and Adobe Illustrator CS4.2 tests for pairwise comparisons with controls. Correct contacts were tested against the combination of wrong contacts. Operators were not blind to treatment groups.Statistical analysis of categorical data was performed using χ