Genome-wide profiling of G protein-coupled receptors in cerebellar granule neurons using high-throughput, real-time PCR

<p>Abstract</p> <p>Background</p> <p>G protein-coupled receptors (GPCRs) are major players in cell communication, regulate a whole range of physiological functions during development and throughout adult life, are affected in numerous pathological situations, and constitute so far the largest class of drugable targets for human diseases. The corresponding genes are usually expressed at low levels, making accurate, genome-wide quantification of their expression levels a challenging task using microarrays.</p> <p>Results</p> <p>We first draw an inventory of all endo-GPCRs encoded in the murine genome. To profile GPCRs genome-wide accurately, sensitively, comprehensively, and cost-effectively, we designed and validated a collection of primers that we used in quantitative RT-PCR experiments. We experimentally validated a statistical approach to analyze genome-wide, real-time PCR data. To illustrate the usefulness of this approach, we determined the repertoire of GPCRs expressed in cerebellar granule neurons and neuroblasts during postnatal development.</p> <p>Conclusions</p> <p>We identified tens of GPCRs that were not detected previously in this cell type; these GPCRs represent novel candidate players in the development and survival of cerebellar granule neurons. The sequences of primers used in this study are freely available to those interested in quantifying GPCR expression comprehensively.</p

Bases de données de pharmacovigilance et accès public

LYON1-BU Santé (693882101) / SudocSudocFranceF

Quantifying Genomic Imprinting at Tissue and Cell Resolution in the Brain

International audienceImprinted genes are a group of ~150 genes that are preferentially expressed from one parental allele owing to epigenetic marks asymmetrically distributed on inherited maternal and paternal chromosomes. Altered imprinted gene expression causes human brain disorders such as Prader-Willi and Angelman syndromes and additional rare brain diseases. Research data principally obtained from the mouse model revealed how imprinted genes act in the normal and pathological brain. However, a better understanding of imprinted gene functions calls for building detailed maps of their parent-of-origin-dependent expression and of associated epigenetic signatures. Here we review current methods for quantifying genomic imprinting at tissue and cell resolutions, with a special emphasis on methods to detect parent-of-origin dependent expression and their applications to the brain. We first focus on bulk RNA-sequencing, the main method to detect parent-of-origin-dependent expression transcriptome-wide. We discuss the benefits and caveats of bulk RNA-sequencing and provide a guideline to use it on F1 hybrid mice. We then review methods for detecting parent-of-origin-dependent expression at cell resolution, including single-cell RNA-seq, genetic reporters, and molecular probes. Finally, we provide an overview of single-cell epigenomics technologies that profile additional features of genomic imprinting, including DNA methylation, histone modifications and chromatin conformation and their combination into sc-multimodal omics approaches, which are expected to yield important insights into genomic imprinting in individual brain cells

Genes regulated in neurons undergoing transcription-dependent apoptosis belong to signaling pathways rather than the apoptotic machinery.

Neuronal apoptosis has been shown to require de novo RNA/protein synthesis. However, very few genes whose expression is necessary for inducing apoptosis have been identified so far. To systematically identify such genes, we have used genome-scale, long oligonucleotide microarrays and characterized the gene expression profile of cerebellar granule neurons in the early phase of apoptosis elicited by KCl deprivation. We identified 368 significantly differentially expressed genes, including most of the genes previously reported to be transcriptionally regulated in this paradigm. In addition, we identified several hundreds of genes whose transcriptional regulation has never been associated with neuronal apoptosis. We used automated Gene Ontology annotation, analysis of promoter sequences, and statistical tools to characterize these regulations. Although differentially expressed genes included some components of the apoptotic machinery, this functional category was not significantly over-represented among regulated genes. On the other hand, categories related to signal transduction were the most significantly over-represented group. This indicates that the apoptotic machinery is mainly constitutive, whereas molecular pathways that lead to the activation of apoptotic components are transcriptionally regulated. In particular, we show for the first time that signaling pathways known to be involved in the control of neuronal survival are regulated at the transcriptional level and not only by post-translational mechanisms. Moreover, our approach provides insights into novel transcription factors and novel mechanisms, such as the unfolded protein response and cell adhesion, that may contribute to the induction of neuronal apoptosis

Association between fungal detection and diagnosis of moderate equine asthma (mea) according to sampling site and methodology

International audienceIntroduction:Poor agreements were previously described between tracheal wash (TW) and bronchoalveolar lavage fluid (BALF), as well as fungal detection by cytology and mycology culture. The link between moderate equine asthma (mEA) and detection of fungal elements in the airways remains controversial.Objectives: To determine the prevalence of fungal detection in TW and BALF and its association with diagnosis of mEA.Methods:Prospective study on 120 horses in active training or referred for respiratory disease. Horses were classified as “control” or “mEA” based on clinical examination, airway endoscopy and BALF. A sample was considered positive if at least one colony was identified by culture or at least one fungal element was observed on cytology. Results:Respectively, 35 and 85 horses were classified as “control” and “mEA”. No significant difference was observed between groups for fungal detection by cytology, regardless the sampling site. Prevalence of positive mycology culture was significantly higher for TW (89.4%) and BALF (31.8%) of mEA horses compared to controls (respectively 68,6% and 8.6%). Diagnosis of mEA was significantly associated with positive mycology culture on both TW (OR = 3.9) and BALF (OR = 5.0) Mycology culture on BALF exhibited high specificity (0.90) and high positive predictive value (0.91), unlike mycology culture onTW (respectively 0.76 and 0.31).Conclusion and clinical importance:Despite a significant association with asthma diagnosis, the high prevalence of fungal detection in TW of control horses precludes its clinical relevance. However, positive mycology culture on BALF represents a significant risk-factor of suffering mEA

Association between fungal detection and diagnosis of moderate equine asthma (mea) according to sampling site and methodology

International audienceIntroduction:Poor agreements were previously described between tracheal wash (TW) and bronchoalveolar lavage fluid (BALF), as well as fungal detection by cytology and mycology culture. The link between moderate equine asthma (mEA) and detection of fungal elements in the airways remains controversial.Objectives: To determine the prevalence of fungal detection in TW and BALF and its association with diagnosis of mEA.Methods:Prospective study on 120 horses in active training or referred for respiratory disease. Horses were classified as “control” or “mEA” based on clinical examination, airway endoscopy and BALF. A sample was considered positive if at least one colony was identified by culture or at least one fungal element was observed on cytology. Results:Respectively, 35 and 85 horses were classified as “control” and “mEA”. No significant difference was observed between groups for fungal detection by cytology, regardless the sampling site. Prevalence of positive mycology culture was significantly higher for TW (89.4%) and BALF (31.8%) of mEA horses compared to controls (respectively 68,6% and 8.6%). Diagnosis of mEA was significantly associated with positive mycology culture on both TW (OR = 3.9) and BALF (OR = 5.0) Mycology culture on BALF exhibited high specificity (0.90) and high positive predictive value (0.91), unlike mycology culture onTW (respectively 0.76 and 0.31).Conclusion and clinical importance:Despite a significant association with asthma diagnosis, the high prevalence of fungal detection in TW of control horses precludes its clinical relevance. However, positive mycology culture on BALF represents a significant risk-factor of suffering mEA

The candidate tumor suppressor gene ZAC is involved in keratinocyte differentiation and its expression is lost in basal cell carcinomas.

ZAC is a zinc finger transcription factor that induces apoptosis and cell cycle arrest in various cell lines. The corresponding gene is maternally imprinted and localized on chromosome 6q24-q25, a region harboring an unidentified tumor suppressor gene for a variety of solid neoplasms. ZAC expression is lost or down-regulated in some breast, ovary, and pituitary tumors and in an in vitro model of ovary epithelial cell transformation. In the present study, we examined ZAC expression in normal skin and found a high expression level in basal keratinocytes and a lower, more heterogeneous, expression in the first suprabasal differentiating layers of epidermis. In vitro, ZAC was up-regulated following induction of keratinocyte differentiation. Conversely, ZAC expression triggered keratinocyte differentiation as indicated by induction of involucrin expression. Interestingly, we found a dramatic loss of ZAC expression in basal cell carcinoma, a neoplasm characterized by a relatively undifferentiated morphology. In contrast, ZAC expression was maintained in squamous cell carcinomas that retain the squamous differentiated phenotype. Altogether, these data suggest a role for ZAC at an early stage of keratinocyte differentiation and further support its role in carcinogenesis

Expression at the Imprinted Dlk1-Gtl2 Locus Is Regulated by Proneural Genes in the Developing Telencephalon

Imprinting is an epigenetic mechanism that restrains the expression of about 100 genes to one allele depending on its parental origin. Several imprinted genes are implicated in neurodevelopmental brain disorders, such as autism, Angelman, and Prader-Willi syndromes. However, how expression of these imprinted genes is regulated during neural development is poorly understood. Here, using single and double KO animals for the transcription factors Neurogenin2 (Ngn2) and Achaetescute homolog 1 (Ascl1), we found that the expression of a specific subset of imprinted genes is controlled by these proneural genes. Using in situ hybridization and quantitative PCR, we determined that five imprinted transcripts situated at the Dlk1-Gtl2 locus (Dlk1, Gtl2, Mirg, Rian, Rtl1) are upregulated in the dorsal telencephalon of Ngn2 KO mice. This suggests that Ngn2 influences the expression of the entire Dlk1-Gtl2 locus, independently of the parental origin of the transcripts. Interestingly 14 other imprinted genes situated at other imprinted loci were not affected by the loss of Ngn2. Finally, using Ngn2/Ascl1 double KO mice, we show that the upregulation of genes at the Dlk1-Gtl2 locus in Ngn2 KO animals requires a functional copy of Ascl1. Our data suggest a complex interplay between proneural genes in the developing forebrain that control the level of expression at the imprinted Dlk1-Gtl2 locus (but not of other imprinted genes). This raises the possibility that the transcripts of this selective locus participate in the biological effects of proneural genes in the developing telencephalon