Neuroligins (NLs) are postsynaptic cell-adhesion proteins that play important roles in synapse formation and the excitatory-inhibitory balance. They have been associated with autism in both human genetic and animal model studies, and affect synaptic connections and synaptic plasticity in several brain regions. Yet current research mainly focuses on pyramidal neurons, while the function of NLs in interneurons remains to be understood. To explore the functional difference among NLs in the subtype-specific synapse formation of both pyramidal neurons and interneurons, we performed viral-mediated shRNA knockdown of NLs in cultured rat cortical neurons and examined the synapses in the two major types of neurons. Our results showed that in both types of neurons, NL1 and NL3 were involved in excitatory synapse formation, and NL2 in GABAergic synapse formation. Interestingly, NL1 affected GABAergic synapse formation more specifically than NL3, and NL2 affected excitatory synapse density preferentially in pyramidal neurons. In summary, our results demonstrated that different NLs play distinct roles in regulating the development and balance of excitatory and inhibitory synapses in pyramidal neurons and interneurons.
Animals ; Cell Adhesion Molecules, Neuronal ; physiology ; Cells, Cultured ; Cerebral Cortex ; embryology ; physiology ; GABAergic Neurons ; physiology ; Interneurons ; physiology ; Membrane Proteins ; physiology ; Nerve Tissue Proteins ; physiology ; Protein Isoforms ; physiology ; Pyramidal Cells ; physiology ; Rats, Sprague-Dawley ; Synapses ; physiology

OBJECTIVEThe aim of this study was to investigate the ability of Pref-1+ adipocyte progenitor cells to mobilize into mesenteric lymph nodes (MLNs) and the dynamic expression of related chemokines during the development of rat MLNs.

METHODSImmunohistochemical analyses were used to detect the expression of Pref-1 and related chemokines. Transmission electron microscopy (TEM) was used to observe the changes in ultrastructure of MLNs.

RESULTSCells containing lipid droplets were found in all rat MLNs at embryonic day (E) 18.5, 2 and 6 weeks (w) after birth, and they were similar to fibroblastic reticular cells (FRCs) or follicular dendritic cells (FDCs) under TEM. Pref-1+ adipocyte progenitor cells were found in all MLNs. The expression level of Pref-1 was significantly increased at 2 w after birth and decreased at 6 w after birth. The tendency of Cxcl12 expression was consistent with that of Pref-1 and was positively correlated with the expression of Pref-1 (P < 0.01; r = 0.897). At E18.5, Cxcl13, and Ccr7 were significantly expressed in the MLN anlage, but the expression level of Ccl21 was low. The expression level of Cxcl13, Ccr7, and Ccl21 in MLN were significantly increased at 2 w after birth (P < 0.05), while the expression of Ccr7 and Ccl21 were significantly decreased at 6 w after birth (P < 0.05).

CONCLUSIONAdipocyte progenitor cells are involved in the rat MLNs development through differentiation into FRC and FDC. The expression of the relevant chemokines during the development of MLNs is dynamic and may be related to the maintenance of lymph nodes self-balance state.

Animals ; Chemokines ; genetics ; metabolism ; Female ; Gene Expression Regulation, Developmental ; physiology ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Lymph Nodes ; embryology ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mesentery ; embryology ; Pregnancy ; Rats

Background: Valproic acid (VPA) exposure during pregnancy has been proven to contribute to congenital heart disease (CHD). Our previous findings implied that disruption of planar cell polarity (PCP) signaling pathway in cardiomyocytes might be a factor for the cardiac teratogenesis of VPA. In addition, the teratogenic ability of VPA is positively correlated to its histone deacetylase (HDAC) inhibition activity. This study aimed to investigate the effect of the VPA on cardiac morphogenesis, HDAC1/2/3, and PCP key genes (Vangl2/Scrib/Rac1), subsequently screening out the specific HDACs regulating PCP pathway. Methods: VPA was administered to pregnant C57BL mice at 700 mg/kg intraperitoneally on embryonic day 10.5. Dams were sacrificed on E15.5, and death/absorption rates of embryos were evaluated. Embryonic hearts were observed by hematoxylin-eosin staining to identify cardiac abnormalities. H9C2 cells (undifferentiated rat cardiomyoblasts) were transfected with Hdac1/2/3 specific small interfering RNA (siRNA). Based on the results of siRNA transfection, cells were transfected with Hdac3 expression plasmid and subsequently mock-treated or treated with 8.0 mmol/L VPA. Hdac1/2/3 as well as Vangl2/Scrib/Rac1 mRNA and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. Total HDAC activity was detected by colorimetric assay. Results: VPA could induce CHD (P < 0.001) and inhibit mRNA or protein expression of Hdac1/2/3 as well as Vangl2/Scrib in fetal hearts, in association with total Hdac activity repression (all P < 0.05). In vitro, Hdac3 inhibition could significantly decrease Vangl2/Scrib expression (P < 0.01), while knockdown of Hdac1/2 had no influence (P > 0.05); VPA exposure dramatically decreased the expression of Vanlg2/Scrib together with Hdac activity (P < 0.01), while overexpression of Hdac3 could rescue the VPA-induced inhibition (P > 0.05). Conclusion VPA could inhibit Hdac1/2/3, Vangl2/Scrib, or total Hdac activity both in vitro and in vivo and Hdac3 might participate in the process of VPA-induced cardiac developmental anomalies.
Animals ; Cell Polarity ; Enzyme Inhibitors ; adverse effects ; Female ; Fetal Heart ; embryology ; Heart Defects, Congenital ; chemically induced ; physiopathology ; Histone Deacetylase Inhibitors ; Histone Deacetylases ; drug effects ; physiology ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; Pregnancy ; Rats ; Transfection ; Valproic Acid ; adverse effects

This study investigated the expression of lung surfactant proteins SP-B and SP-C, and their modulating factors TTF-1 and PLAGL2 in the fetal lung of rats with fetal growth restriction (FGR). The rat FGR model was established by prenatal hypoxia in the first stage of pregnancy, 180 rats for experiment served as hypoxia group, and 197 healthy rats served as normal control group. The FGR incidence in hypoxia was compared with that in normal control group. The histological changes in the fetal lung were observed under the light microscope and electronic microscope in two groups. The SP-B, SP-C, TTF-1 and PLAGL2 proteins were determined in the fetal lung of two groups immunohistochemically. The expression levels of SP-B, SP-C, TTF-1 and PLAGL2 protein and mRNA in the fetal lung of two groups were detected by using Western blotting and RT-PCR respectively. The FGR rat model was successfully established by using hypoxia. Pathologically the fetal lung developed slowly, and the expression levels of SP-B, SP-C, TTF-1 and PLAGL2 protein and mRNA in the fetal lung were significantly reduced in hypoxia group as compared with those in normal control group. It was suggested that maternal hypoxia in the first stage of pregnancy could induce FGR, and reduce the expression of SP-B and SP-C, resulting in the disorder of fetal lung development and maturation.
Animals ; Base Sequence ; DNA Primers ; Female ; Fetal Growth Retardation ; Lung ; embryology ; metabolism ; Peptides ; metabolism ; Pregnancy ; Pulmonary Surfactant-Associated Protein B ; metabolism ; Rats ; Real-Time Polymerase Chain Reaction

In the present study, we examined the effect of oxygen glucose deprivation (OGD) post-conditioning (PostC) on neural cell apoptosis in OGD-PostC model and the protective effect on primary cortical neurons against OGD injury in vitro. Four-h OGD was induced by OGD by using a specialized and humidified chamber. To initiate OGD, culture medium was replaced with de-oxygenated and glucose-free extracellular solution-Locke's medium. After OGD treatment for 4 h, cells were then allowed to recover for 6 h or 20 h. Then lactate dehydrogenase (LDH) release assay, Western blotting and flow cytometry were used to detect cell death, protein levels and apoptotic cells, respectively. For the PostC treatment, three cycles of 15-min OGD, followed by 15 min normal cultivation, were applied immediately after injurious 4-h OGD. Cells were then allowed to recover for 6 h or 20 h, and cell death was assessed by LDH release assay. Apoptotic cells were flow cytometrically evaluated after 4-h OGD, followed by re-oxygenation for 20 h (O4/R20). In addition, Western blotting was used to examine the expression of heat-shock protein 70 (HSP70), Bcl-2 and Bax. The ratio of Bcl-2 expression was (0.44±0.08)% and (0.76±0.10)%, and that of Bax expression was (0.51±0.05)% and (0.39±0.04)%, and that of HSP70 was (0.42±0.031)% and (0.72±0.045)% respectively in OGD group and PostC group. After O4/R6, the rate of neuron death in PostC group and OGD groups was (28.96±3.03)% and (37.02±4.47)%, respectively. Therefore, the PostC treatment could up-regulate the expression of HSP70 and Bcl-2, but down-regulate Bax expression. As compared with OGD group, OGD-induced neuron death and apoptosis were significantly decreased in PostC group (P<0.05). These findings suggest that PostC inhibited OGD-induced neuron death. This neuro-protective effect is likely achieved by anti-apoptotic mechanisms and is associated with over-expression of HSP70.
Animals ; Apoptosis ; drug effects ; Blotting, Western ; Cell Hypoxia ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; blood supply ; cytology ; embryology ; Flow Cytometry ; Glucose ; pharmacology ; HSP70 Heat-Shock Proteins ; metabolism ; Ischemic Postconditioning ; methods ; Neurons ; cytology ; drug effects ; metabolism ; Oxygen ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo bioinformatically predict and analyze target genes of miRNA-126(*), with the aim of providing certain basis for related research about target genes and regulatory mechanism in the future.

METHODSThe miRNA chip technology was applied to measure expression levels of miRNA-126(*) in 3 time points (embryo 16, 19 and 21 days) of fetal lung development. Then the target genes of miRNA-126(*) were screened through miRGen2.0 database. Subsequent bioinformatic analysis of these target genes was performed by Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes Pathway analysis (KEGG Pathway analysis).

RESULTSmiRNA-126(*) manifested continuously upregulated expression with the lung development (from embryo 16 to 21 days). There were 422 predicted target genes in total, and the gene set mainly located in glucuronosyltransferase activity, transferase activity (GO molecular function), multicellular organismal development, developmental process (GO biology process) and intracellular part (GO cellular component). The KEGG Pathway analysis demonstrated that the gene set mostly located in RNA degradation (signal transduction pathway) and prion diseases (disease pathway).

CONCLUSIONSThe results suggest that miRNA-126(*) plays a certain role in fetal lung development and provide a basis for lung development research in the future.

Animals ; Computational Biology ; Female ; Glucuronosyltransferase ; metabolism ; Lung ; embryology ; Male ; MicroRNAs ; physiology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology

OBJECTIVETo investigate the expression of Junctophilin 1 (JP1) in cardiogenesis of mammalian.

METHODSCardiac differentiation of embryonic stem cells (ESCs) was generated by hanging drop method. Fetal heart was obtained from the rats aged d 14-20 of gestation. The expression of JP1 and JP2 during cardiogenesis of ESCs and rat embryos was analyzed by RT-PCR or Western blotting. Immunofluorescence staining was employed to reveal the distribution of JP1 and JP2 in embryoid body (EB), probing for merging of JP1 and JP2 and cardiac sarcomeric α-Actinin or Troponin-T. Percentage of JP1 and JP2-positive staining cells was analyzed quantitatively by FCS on d17.

RESULTSJP1 mRNA was up-regulated at the early stage (d 5-11) and then decreased. The expression of JP1 protein was up-regulated at the early stage (d 7-9), then decreased gradually and disappeared after d 15. While JP2 gene and protein expression increased in a time-dependent manner during cardiogenesis of rat embryos. The results of immunofluorescence staining showed that there was a parallel co-localization of JP2 with Troponin-T or α-Actinin on d17, while JP1 failed to express in the sarcomeric positive area at the same time point. Furthermore, FCS analysis showed that about 16.59% of cells were JP2-positive, while no cells were stained positively for JP1 in d17 EBs.

CONCLUSIONJP1 gene is expressed during the whole process of cardiogenesis, while JP1 protein only appears on the early stage. The expression of JP1 in cardiogenesis of ESCs is consistent with that of rat embryos.

Actinin ; genetics ; metabolism ; Animals ; Cell Differentiation ; Cell Line ; Embryonic Stem Cells ; cytology ; metabolism ; Heart ; embryology ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred ICR ; Myocytes, Cardiac ; cytology ; metabolism ; RNA, Messenger ; genetics ; Rats ; Troponin T ; genetics ; metabolism

OBJECTIVETo investigate the expression and role of miRNA-126/miRNA-126(*) in the fetal lung development of rats.

METHODSTwelve pregnant Sprague-Dawley rats were randomly divided into 3 groups and the fetal rats were removed at 16, 19 and 21 days of gestation respectively. Hematoxylin and eosin staining was performed to observe lung morphology of fetal rats. Then microRNA (miRNA) microarray was used to study the expression patterns of miRNA-126/miRNA-126(*) in fetal lungs at the three time points. And miRNA-126(*) was selected for further study by real-time PCR.

RESULTSThere was no evident difference in the expression of miRNA-126 among the three groups, however the expression level of miRNA-126(*) increased gradually as the fetal lung developed. The real-time PCR result further showed that expression of miRNA-126(*) increased gradually with lung development, displaying significant differences among the three groups (P<0.05).

CONCLUSIONSmiRNA-126(*) may play an important role in development of the fetal lung in rats.

Animals ; Female ; Lung ; embryology ; Male ; MicroRNAs ; analysis ; physiology ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate whether the suppression of Wnt10b by siRNA could prevent the development of hair follicle in the cultured rat embryonic skin.

METHODSsiRNA-Wnt10b was synthesized by chemosynthesis method. The dorsal skin of SD rat at embryos were cultured in DMEM in the presence of different percentage of interfering RNA targeting Wnt10b. Wnt10b/beta-catenin expression was analyzed by real-time PCR everyday and by Western blot on the third day. The cultured embryonic skin underwent paraffin embedding, section, HE staining on the third day,in which the number of de novo hair follicle was calculated and statistically analyzed.

RESULTSWnt10b gene in the cultured embryonic skin could be knocked down with the siRNA-based method. Beta-catenin mRNA was not greatly influenced by the downregulation of Wnt10b mRNA. The number of de novo hair follicle placode in cultured embryonic skin decreased, along with the downregulation of Wnt10b and beta-catenin proteins expression.

CONCLUSIONSThe downregulation of Wnt10b mRNA and protein by siRNA reduces the number of de novo hair follicle placode in the cultured rat embryonic skin. Wnt10b may control cytoplasm beta-catenin concentration at the protein level.

Animals ; Hair Follicle ; embryology ; metabolism ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Rats ; Skin ; embryology ; metabolism ; Tissue Culture Techniques ; Wnt Proteins ; genetics ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo investigate the effect of nonylphenol (NP) exposure on the genital development of fetal male rats in pregnant rats, and to measure the mRNA and protein expression of insulin-like factor3 (Insl-3) in the testicular tissue of fetal rats.

METHODSA total of 100 pregnant SD rats were equally assigned to blank control group and four NP treated groups. Each rat in the NP treated groups received intragastric administration of NP at doses of 5, 40, 100, or 200 mg/kg/d from day 14 to 19 of gestation, and the rats in the blank control group received intragastric administration of pure peanut oil. The pregnant rats were sacrificed on day 19 of gestation. The body weight and testicular weight of each fetal rat were measured, and the descent of testis was also observed. The mRNA and protein expression of Insl-3 in the testicular tissue of fetal rats was analyzed by reverse transcription-PCR and Western blot.

RESULTSCompared with the blank control group, the 40, 100, and 200 mg/kg NP treated groups showed significantly decreased body weight and weight coefficient of testis (P < 0.05 or P < 0.01), significantly decreased testicular descent (P < 0.05), and significantly decreased mRNA and protein expression of Insl-3 (P < 0.05 or P < 0.01).

CONCLUSIONExposure to nonylphenol can lead to testicular maldevelopment, incomplete testicular descent, and Insl-3 expression downregulation of fetal male rats in pregnant rats.

Animals ; Body Weight ; Female ; Fetal Development ; drug effects ; Insulin ; metabolism ; Male ; Maternal Exposure ; adverse effects ; Organ Size ; Phenols ; toxicity ; Pregnancy ; Proteins ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Testis ; drug effects ; embryology ; pathology