OBJECTIVETo screen the wnt and fibroblast growth factor (FGF) ligands involved in palatogenesis and cleft palate, and to study the dynamic expression of them in the different stages of palatal development and cleft palate formation.

METHODSMouse model of retinoic acid (RA)-induced cleft palate was set up. At embryo day (ED) 14.5, the palatal tissues of RA-treated group and wild type were collected and prepared for gene-chip analysis. According to the gene-chip results, wnt3, wnt8a, fgf9 and fgf10 were selected and their expression level was detected at ED13.5-15.5 by using semi-quantitative reverse transcription-PCR (RT-PCR).

RESULTS(1) Gene-chip analysis showed that in RA-induced cleft palate group wnt8a and fgf9 were down-regulated, wnt3 and fgf10 were up-regulated in conversely. (2)During the different stage of the control group palatogenesis, intense expression of wnt3, wnt8a, fgf9 and fgf10 were detected with a continuous dynamic pattern. (3)Compared with the control group, the expression level of wnt3, wnt8a, fgf9 and fgf10 in RA-induced cleft palate showed significant difference, respectively (P < 0.05).

CONCLUSIONwnt and FGF signaling molecules participate in the palatogenesis, and RA pathway may interact with wnt and FGF signaling pathway.

Animals ; Cleft Palate ; Fibroblast Growth Factor 10 ; Fibroblast Growth Factors ; Genotype ; Ligands ; Mice ; Tretinoin ; Wnt3 Protein

OBJECTIVETo investigate the effects of adenoviral vector-mediated over-expression of Wnt3 on the apoptosis of hepatic progenitor cells in vitro.

METHODSHepatic progenitor cells transfected with Ad-GFP-Wnt3 vector or the control vector Ad-GFP were examined for cell apoptosis under fluorescence microscopy with Hoechst 33342 staining, and the proportion of apoptotic cells were determined by flow cytometric analysis with Annexin-PE/7-ADD staining. The mRNA and protein expressions of Bax, Bcl-2 and Bcl-xl in the cells were detected by real-time PCR and Western blotting, respectively.

RESULTSReal-time PCR and Western blotting showed a high expression of Wnt3 in Ad-GFP-Wnt3-transfected hepatic progenitor cells, which exhibited significantly decreased cell apoptosis as compared with the control group. The expressions of Bcl-2 and Bcl-xl mRNA and proteins increased significantly while Bax expression decreased obviously in Ad-GFP-Wnt3-transfected cells (P<0.05).

CONCLUSIONSAdenoviral vector-mediated over-expression of Wnt3 can suppress apoptosis of hepatic progenitor cells possibly through the Bcl-2 pathway.

Apoptosis ; Cells, Cultured ; Genetic Vectors ; Hepatocytes ; cytology ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stem Cells ; cytology ; Transfection ; Wnt3 Protein ; metabolism ; bcl-X Protein ; metabolism

As a member of the hox gene family, hoxB4 gene encodes a class of DNA-dependent homeobox domain nucleoprotein, which is a specific transcription factor, playing an important role in regulating the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Therefore, it is important to understand the mechanisms involved in regulating expression of hoxB4 in the HSC. Previous studies have suggested that some hoxB4 upstream regulatory factors, such as USF-1 (upstream activating factor -1), USF-2 (upstream activating factor -2) and NF-Y complex, as well as hematopoietic cytokines, such as platelet growth factor (TPO) and Wnt3a protein, play important regulatory roles in the expression of hoxB4 in hematopoietic stem cells. In this review the structure and biological characteristics of hoxB4, mechanisms involved in regulating expression of hoxB4 in the HSC are summarized.
CCAAT-Binding Factor ; metabolism ; Gene Expression Regulation ; Genes, Homeobox ; genetics ; physiology ; Hematopoietic Stem Cells ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; physiology ; Humans ; Transcription Factors ; genetics ; metabolism ; physiology ; Upstream Stimulatory Factors ; metabolism ; Wnt Proteins ; metabolism ; Wnt3 Protein ; Wnt3A Protein

OBJECTIVETo investigate the expression of wingless-type MMTV integration site family, member 3 (Wnt3) in rat dental follicles and its protein level in dental follicle cells (DFC) undergoing osteogenic induction and to discuss the effects of Wnt3 on the differentiation of DFC.

METHODSRats at postnatal days 1, 3, 5, 7, 9, 11 and 13 were executed, then the mandibles were immediately removed and immunohistochemistry was performed to detect the expression of Wnt3 in dental follicles of postnatal rats. The expression and distribution of Wnt3 in DFC were determined by immunofluorescence. Alizarin red-S staining was performed to assess the mineralization of DFC. Western blotting was used to evaluate Wnt3 and β-catenin protein levels after stimulated by osteogenic medium for 1, 2 and 3 weeks, respectively.

RESULTSImmunohistochemistry revealed that the expression of Wnt3 in rat dental follicles began at day 5 and sustained to day 13. On day 1 and 3, the expression of Wnt3 in dental follicles was negative.Wnt3 was expressed in the cytoplasm of DFC. Alizarin red-S staining indicated that the osteogenic medium stimulated the differentiation of DFC into osteoblastic lineage.Western blotting demonstrated that the Wnt3 protein levels were significantly up-regulated after stimulated with osteogenic medium for 1 weeks compared with the control (2.60 ± 0.04 vs.1.00 ± 0.00, P < 0.05). Then the levels of Wnt3 protein were declined, and at the 3rd week, no significant difference was observed between osteo-induced group and the control (1.00 ± 0.05 vs.1.00 ± 0.00, P > 0.05). The levels of β-catenin were increased in osteo-induced groups compared with the control (1.95 ± 0.05 vs.1.00 ± 0.00, P < 0.05; 9.77 ± 0.65 vs.1.00 ± 0.00, P < 0.05;1.75 ± 0.21 vs.1.00 ± 0.00, P < 0.05). Furthermore, the expression of β-catenin reached to a peak on the 2nd week (9.77 ± 0.65), and then declined.

CONCLUSIONSWnt3 was expressed in the rat dental follicles both in vivo and in vitro and up-regulated during early phase of osteoblast differentiation in DFC.Wnt3 may be involved in early phase of osteoblast differentiation.

Animals ; Cell Differentiation ; Cells, Cultured ; Dental Sac ; cytology ; metabolism ; Female ; Gene Expression Regulation, Developmental ; Male ; Osteoblasts ; cytology ; metabolism ; Osteogenesis ; physiology ; Rats ; Rats, Sprague-Dawley ; Serum Albumin, Bovine ; pharmacology ; Up-Regulation ; Wnt3 Protein ; metabolism ; beta Catenin ; metabolism

OBJECTIVEPrevious studies suggest that hyperbaric oxygen (HBO) treatment promotes the proliferation of neurocytes in neonatal rats following hypoxic-ischemic brain damage (HIBD). The Wnt signaling pathway is associated with neurogenesis. This study examined whether HBO promoted neural stem cells (NSCs) proliferation after HIBD, and whether that the proliferation correlated with Wnt-3 protein expression.

METHODSSeven-day-old Sprague-Dawley rats were randomly divided into three groups: normal control, hypoxia-ischemia (HI), and HI-HBO. HI was induced by the ligation of left common carotid artery, followed by a 2-hr exposure to 8% O2 in the latter two groups. HBO was administered 3 hrs after HI in the HI-HBO group for continuous 7 days (2 atmospheres absolute, once daily). The proliferating NSCs in the subventricular zone (SVZ) was examined by BrdU/nestin immunofluorescence and the expression of Wnt-3 protein in NSCs was examined by nestin/Wnt-3 immunofluorescence at 6 and 24 hrs and at 3, 7 and 14 days of HI. The cellular expressions of nestin and Wnt-3 protein were analyzed by laser scanning confocal microscopy. The linear regression analysis was used to evaluate the correlation between cellular Wnt-3 and nestin protein. The expressions of nestin and Wnt-3 protein in the ischemic cerebral hemisphere were analyzed with Western blotting.

RESULTSThe number of BrdU/nestin positive cells in the SVZ increased 3 hrs after HBO therapy, peaked at 7 days and remained at a higher level until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of Wnt-3 protein in NSCs increased significantly 3 hrs after HBO therapy, peaked at 3 days and remained at high levels until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of cellular nestin protein was closely correlated with the level of cellular Wnt-3 protein (r = 0.893, P < 0.05). The Western blotting analysis demonstrated increased Wnt-3 and nestin protein expressions in the ischemic cerebral hemispheres.

CONCLUSIONSHBO treatment promotes the proliferation of NSCs in HIBD neonatal rats, which is correlated with the activation of Wnt signaling.

Animals ; Animals, Newborn ; Blotting, Western ; Bromodeoxyuridine ; metabolism ; Cell Proliferation ; Female ; Fluorescent Antibody Technique ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Intermediate Filament Proteins ; Male ; Nerve Tissue Proteins ; Nestin ; Neurons ; cytology ; Rats ; Stem Cells ; cytology ; Wnt Proteins ; analysis ; Wnt3 Protein

Neuroplastin 65 (Np65) is an immunoglobulin superfamily cell adhesion molecule involved in synaptic formation and plasticity. Our recent study showed that Np65-knockout (KO) mice exhibit abnormal cognition and emotional disorders. However, the underlying mechanisms remain unclear. In this study, we found 588 differentially-expressed genes in Np65-KO mice by microarray analysis. RT-PCR analysis also revealed the altered expression of genes associated with development and synaptic structure, such as Cdh1, Htr3a, and Kcnj9. In addition, the expression of Wnt-3, a Wnt protein involved in development, was decreased in Np65-KO mice as evidenced by western blotting. Surprisingly, MRI and DAPI staining showed a significant reduction in the lateral ventricular volume of Np65-KO mice. Together, these findings suggest that ablation of Np65 influences gene expression, which may contribute to abnormal brain development. These results provide clues to the mechanisms underlying the altered brain functions of Np65-deficient mice.
Affective Symptoms ; metabolism ; Animals ; Brain ; diagnostic imaging ; metabolism ; pathology ; Cognition Disorders ; metabolism ; Gene Expression ; Magnetic Resonance Imaging ; Membrane Glycoproteins ; deficiency ; genetics ; physiology ; Mice, Knockout ; Microarray Analysis ; Organ Size ; Real-Time Polymerase Chain Reaction ; Wnt3 Protein ; metabolism