Impact of Enriched Environment on Murine T Cell Differentiation and Gene Expression Profile.
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ABSTRACT:
SUBMITTER: Rattazzi L
PROVIDER: S-EPMC5042968 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PurposeFollowing transplantation, cultured retinal progenitor cells (RPCs) integrate into the diseased host retina and exhibit morphologies and markers indicative of local cellular phenotypes. In vitro analysis of cultured RPCs allows detailed examination of marker gene expression during the initial phase of differentiation and can provide insight into the variables influencing this process.MethodsUsing cultured murine RPCs, this study compares the effects of fetal bovine serum (FBS) with those of ciliary neurotrophic factor (CNTF), individually or in combination with epidermal growth factor (EGF). Differentiation was assessed by way of the relative expression of 17 genes using quantitative PCR (qPCR) at five time points over a seven-day period.ResultsBoth CNTF and FBS rapidly altered the gene expression of RPCs, with very marked upregulation of glial fibrillary acidic protein (GFAP; FBS>CNTF) and marked down-regulation of the proliferation marker K(i)-67, consistent with the induction of differentiation. The evidence supports a preponderantly pro-glial influence for both the FBS and CNTF, however, neuronal markers were also upregulated to a lesser extent. Immunocytochemistry confirmed subpopulations labeling with neuronal markers, including rhodopsin. In the presence of sustained EGF stimulation, the differentiating influences of both FBS and CNTF remained perceptible as transient peaks of relative gene expression, but were markedly diminished overall.ConclusionsThis study shows that it is possible to compare the relative efficacy of in vitro differentiation protocols using murine RPCs and qPCR. The differentiating influences of both serum and CNTF were confirmed, but shown to be powerfully moderated by EGF. This suggests that EGF withdrawal is the dominant feature of these differentiation protocols and that exposure to either serum or CNTF is insufficient to irreversibly commit a cultured RPC population to terminal differentiation unless accompanied by concomitant cessation of mitogenic stimulation.
| S-EPMC2765240 | biostudies-literature
Project description:The mouse Hox-2.5 gene containing a Drosophila Antennapedia-type homeobox sequence is expressed in a spatially and temporally restricted manner during embryogenesis. We found that the mouse embryonal carcinoma cell line P19 expresses Hox-2.5 during differentiation by the treatment with retinoic acid (RA). Expression of the Hox-2.5 gene was not detected in undifferentiated P19 cells, but detected 72 hours after treatment with RA. In order to analyze this inductive response, we first identified the Hox-2.5 transcription initiation site and a possible promoter region. Subsequently, we prepared constructs containing various Hox-2.5 DNA fragments fused to a firefly luciferase reporter gene and transfected these into undifferentiated or differentiating P19 cells. These studies have demonstrated that a region -279 to +15 with respect to the transcription initiation site has a differentiation-responsive promoter activity. Deletion analysis suggests that the sequences responsible for this induction are located in several distinct domains within the 294 bp promoter region. Two of the possible differentiation-responsive elements were identified by analysis of DNA-protein interactions, and in vivo competition assays lend support to the notion that these regions are involved in the differential expression of Hox-2.5 promoter activity.
| S-EPMC334409 | biostudies-other
Project description:Deficiency of activity-induced expression of brain-derived neurotrophic factor (BDNF) disturbs neurotransmitter gene expression. Enriched environment treatment (EET) ameliorates the defects. However, how BDNF deficiency and EET affect the neurotransmitter gene expression differently across ages remains unclear. We addressed this question by determining the neurotransmitter gene expression across three life stages in wild-type and activity-dependent BDNF-deficient (KIV) mice. Mice received 2-months of standard control treatment (SCT) or EET at early-life development (ED: 0-2 months), young adulthood (2-4 months), and old adulthood (12-14 months) (N = 16/group). Half of these mice received additional 1-month SCT to examine persisting EET effects. High-throughput quantitative reverse transcription polymerase chain reaction measured expression of 81 genes for dopamine, adrenaline, serotonin, gamma aminobutyric acid, glutamate, acetylcholine, and BDNF systems in the frontal cortex (FC) and hippocampus. Results revealed that BDNF deficiency mostly reduced neurotransmitter gene expression, greatest at ED in the FC. EET increased expression of a larger number of genes at ED than adulthood, particularly in the KIV FC. Many genes down-regulated in KIV mice were up-regulated by EET, which persisted when EET was provided at ED (e.g., 5-hydroxytryptamine (serotonin) transporter [5HTT], ADRA1D, GRIA3, GABRA5, GABBR2). In both the regions, BDNF deficiency decreased the density of gene co-expression network specifically at ED, while EET increased the density and hub genes (e.g., GAT1, GABRG3, GRIN1, CHRNA7). These results suggest that BDNF deficiency, which occurs under chronic stress, causes neurotransmitter dysregulations prominently at ED, particularly in the FC. EET at ED may be most effective to normalize the dysregulations, providing persisting effects later in life. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the Open Science badges can be found at https://cos.io/our-services/open-science-badges/.
| S-EPMC7319906 | biostudies-literature
Project description:The global prevalence of weight loss is increasing, especially in young women. However, the extent and mechanisms by which maternal weight loss affects the offspring is still poorly understood. Here, using an enriched environment (EE)-induced weight loss model, we show that maternal weight loss improves general health and reprograms metabolic gene expression in mouse offspring, and the epigenetic alterations can be inherited for at least two generations. EE in mothers induced weight loss and its associated physiological and metabolic changes such as decreased adiposity and improved glucose tolerance and insulin sensitivity. Relative to controls, their offspring exhibited improved general health such as reduced fat accumulation, decreased plasma and hepatic lipid levels, and improved glucose tolerance and insulin sensitivity. Maternal weight loss altered gene expression patterns in the liver of offspring with coherent down-regulation of genes involved in lipid and cholesterol biosynthesis. Epigenomic profiling of offspring livers revealed numerous changes in cytosine methylation depending on maternal weight loss, including reproducible changes in promoter methylation over several key lipid biosynthesis genes, correlated with their expression patterns. Embryo transfer studies indicated that oocyte alteration in response to maternal metabolic conditions is a strong factor in determining metabolic and epigenetic changes in offspring. Several important lipid metabolism-related genes have been identified to partially inherit methylated alleles from oocytes. Our study reveals a molecular and mechanistic basis of how maternal lifestyle modification affects metabolic changes in the offspring.
| S-EPMC4335202 | biostudies-literature
Project description:BackgroundAnalysis of gene expression at the mRNA level, using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), mandatorily requires reference genes (RGs) as internal controls. However, increasing evidences have shown that RG expression may vary considerably under experimental conditions. We sought for an appropriate panel of RGs to be used in the 3T3-L1 cell line model during their terminal differentiation into adipocytes. To this end, the expression levels of a panel of seven widely used RG mRNAs were measured by qRT-PCR. The 7 RGs evaluated were ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl-transferase I (HPRT), ATP synthase H+ transporting mitochondrial F1 complex beta subunit (ATP-5b), tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide (Ywhaz), Non-POU-domain containing octamer binding protein (NoNo), and large ribosomal protein L13a (RPL).Methodology/principal findingsUsing three Excel applications, GeNorm, NormFinder and BestKeeper, we observed that the number and the stability of potential RGs vary significantly during differentiation of 3T3-L1 cells into adipocytes. mRNA expression analyses using qRT-PCR revealed that during the entire differentiation program, only NoNo expression is relatively stable. Moreover, the RG sets that were acceptably stable were different depending on the phase of the overall differentiation process (i.e. mitotic clonal expansion versus the terminal differentiation phase). RPL, ACTB, and Ywhaz, are suitable for terminal differentiation, whereas ATP-5b and HPRT, are suitable during mitotic clonal expansion.ConclusionOur results demonstrate that special attention must be given to the choice of suitable RGs during the various well defined phases of adipogenesis to ensure accurate data analysis and that the use of several RGs is absolutely required. Consequently, our data show for the first time, that during mitotic clonal expansion, the most suitable RGs are ATP-5b, NoNo and HPRT, while during terminal differentiation the most suitable RGs are, NoNo, RPL, ACTB and Ywhaz.
| S-EPMC3358259 | biostudies-literature
Project description:The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.
| S-EPMC3162409 | biostudies-literature
Project description:The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERalpha, ERbeta) are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen-dependent manner by endogenous ERs or by transiently overexpressed ERalpha. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation.
| S-EPMC1350973 | biostudies-literature
Project description:IntroductionIn vitro expansion and differentiation of mesenchymal stem cells (MSC) rely on specific environmental conditions, and investigations have demonstrated that one crucial factor is oxygen environment.ObjectivesIn order to understand the impact of oxygen tension on MSC culture and chondrogenic differentiation in vitro, we developed a mathematical model of these processes and applied it in predicting optimal assays.Methods and resultsWe compared ovine MSCs under physiologically low and atmospheric oxygen tension. Low oxygen tension improved their in vitro population growth as demonstrated by monoclonal expansion and colony forming assays. Moreover, it accelerated induction of the chondrogenic phenotype in subsequent three-dimensional differentiation cultures. We introduced a hybrid stochastic multiscale model of MSC organization in vitro. The model assumes that cell adaptation to non-physiological high oxygen tension reversibly changes the structure of MSC populations with respect to differentiation. In simulation series, we demonstrated that these changes profoundly affect chondrogenic potential of the populations. Our mathematical model provides a consistent explanation of our experimental findings.ConclusionsOur approach provides new insights into organization of MSC populations in vitro. The results suggest that MSC differentiation is largely reversible and that lineage plasticity is restricted to stem cells and early progenitors. The model predicts a significant impact of short-term low oxygen treatment on MSC differentiation and optimal chondrogenic differentiation at 10-11% pO(2).
| S-EPMC6495153 | biostudies-literature
Project description: Not available
| S-EPMC4695919 | biostudies-literature
Project description:Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children's health. Here we examined the effects of longterm (14 days) and low dose (1 ?M) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ?1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.
| S-EPMC5086726 | biostudies-literature