Project description:we report a transgenic zebrafish line using destabilized fluorescent protein, Venus-NLS-PEST (VNP), driven by the promoter of a key circadian clock gene, nr1d1. This system allows us to monitor the development of single-cell circadian rhythm in live zebrafish larva in a cell-type specific manner. To identify the cell types expressing nr1d1:VNP in the whole brain, we conducted single cell RNA-seq (scRNA-seq) of ~15,000 cells dissociated from the brain of Tg(nr1d1:VNP) larval fish at 6.5dpf. Among them, 6514 cells were identified with number of genes > 500 and used for the following analysis. 26 cells clusters were classified from scRNAseq, and manually annotated by comparing the marker genes with the adult zebrfiash whole brain single cell RNA-seq data. The mRNA of nr1d1:VNP was enriched in photoreceptors in pineal gland, granule cells and purkinje cells in cerebellum, habenula cells as well as non-neuron cell.
Project description:To investigate the role of NR1D1 in the progression of breast cancer, mammary gland tumor tissues were obtained from 14 weeks old FVB Nr1d1+/+;PyMT and Nr1d1-/-;PyMT mice and the gene expression patterns were analyzed by RNA-seq.
Project description:To understand the cellular basis of the immune compartment of the zebrafish brain we have established reliable protocols for dissociation and prospective isolation of brain leukocytes, using fluorescent transgenic lines. By combining this approach with single-cell RNA sequencing, we have generated a gene expression atlas composed of the distinct immune cells present in the homeostatic brain. These analyses revealed the presence of subpopulations of mononuclear phagocytes and other leukocytes, including cell types that have not been -or have been poorly- characterized so far. Here, we present the characterization of a new mononuclear phagocyte population that represents an important fraction among all brain leukocytes. Adult brain single-cell suspensions were prepared from adult Tg(p2ry12:GFP; cd45:DsRed) fish (n=3) and a total of 14,000 cd45:DsRed+ cells were processed for single-cell profiling using the 10x Genomics platform.
Project description:The zebrafish pineal gland (epiphysis) is an autonomous clock organ. In addition to being a site of melatonin production, it contains photoreceptor cells and functions as a circadian clock pace maker, making zebrafish a useful model system to study the developmental control of expression of genes associated with melatonin synthesis and photodetection, and the circadian clock. Here we have used DNA microarray technology to study the zebrafish pineal transcriptome. Analysis of gene expression at five different developmental stages (three embryonic and two adult) has revealed a highly dynamic transcriptional profile, revealing many genes that are highly expressed in the pineal gland. Statistical analysis of the data based on Gene Ontology (GO) annotation indicates that many transcription factors and cell cycle genes are highly expressed during embryonic stages, whereas genes dedicated to visual system signal transduction are preferentially expressed in the adult. Furthermore, several genes were identified that exhibit day/night differences in expression. Our data provide a rich source of candidate genes for distinct functions at different stages of pineal gland development. Experiment Overall Design: Adults and embryos were kept under a 14-hr-light/10-hr-dark cycle. Pineal glands were isolated manually, guided by GFP fluorescence, from embryonic (3d, 5d, and 10d) and adult (3 month and 1-2 yr) transgenic zebrafish in which expression of the GFP gene is driven by the pineal-specific aanat2 promoter. For comparison, brain tissue from which the pineal gland and eyes had been removed was also collected (referred to as “brain”). Altogether, we collected 20 types of samples: five time points (3d, 5d, 10d, 3 mo, and 1-2 yr), two organs (pineal gland and brain), and two sampling times (day and night). For each type of sample, tissue was obtained and processed three to five times. Total RNA was prepared from each sample using the RNeasy Lipid Tissue Mini Kit (Qiagen) and biotin-labeled cDNA was generated using the Ovation Biotin system kit (NeuGen). The Affymetrix GeneChip® Zebrafish Genome Array was hybridized and processed using the standard Affymetrix protocol.
Project description:We have developed and tested the efficiency of the Tg(myo6b:GFP-2A-rpl10a-3xHA) zebrafish to specifically enrich for and evaluate the translatome of inner ear and lateral line sensory hair cells (IP) compared to the whole fish transcriptome (IN). We show through RNA-seq that HA-tagged ribosome immunoprecipitation significantly enriches for RNA transcripts of known zebrafish hair cell expressed transcripts.
Project description:Adult zebrafish (Tübingen strain, sex not specified) at approximately 1 year of age were analysed. For experiments conducted under low oxygen conditions, nitrogen gas was bubbled through water to deplete oxygen before exposure of individual fish to the medium. Oxygen concentrations were measured using a dissolved oxygen meter (DO 6+, EUTECH instruments, Singapore). The dissolved oxygen level for hypoxia treatment was measured to be 1.20 ± 0.6 mg/l, whereas normal ambient oxygen levels were 6.6 ± 0.45 mg/l. Zebrafish were exposed to the hypoxic medium for 3 hours. Briefly, after each hypoxia trial, the animals were euthanized by hypothermic shock and then decapitated to remove the brain. Total RNA was extracted from samples mentioned above using the QIAGEN RNeasy mini kit (QIAGEN, GmbH, Hilden, Germany) and stored at â??80°C before further analysis. RNA concentration was determined with a NanoVueâ?¢ UVâ??vis spectrophotometer (GE Healthcare Life Sciences, Fairfield, USA). RNA integrity and quality were then estimated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and the RNA integrity number (RIN) index was calculated for each sample. Only RNAs with a RIN number >7.0 were processed further. Microarray analysis of gene expression was performed using the Zebrafish Gene 1.0 ST Array (Affymetrix Inc. Santa Clara, CA). Briefly, 300ng of total RNA derived from a single adult brain was converted to amplified sense strand cDNA using the Ambion WT Expression Kit (Life Technologies, Carlsbad, CA). The resulting sense cDNA was fragmented and Biotin end-labelled using the Affymetrix Genechip WT Terminal Labeling Kit prior to hybridisation to the array at 45 °C for 16 hours. Two treatments including hypoxia and normoxia were studied. Each treatment had three biological replicates (i.e. three fish exposed to hypoxia and three fish exposed to normoxia). Six samples were analysed. Microarray analysis of gene expression was performed using the Zebrafish Gene 1.0 ST Array. The signal intensity of the chip was scanned using a GeneChipR Scanner 3000TG and analysed using Expression Console software (www. Affymetrix.com). CEL files were imported and intensities adjusted by RMA background correction and quantile normalization.
Project description:The interaction between neurogenesis and angiogenesis after traumatic brain injury is a complex and dynamic process. To resolve this, we chose the zebrafish model organism for studying brain wound healing via systems biology approach. Transcriptome microarray data and histological analysis of injured fish were sampled at different time points during recovery process. Time-course microarray data following wound healing of zebrafish were obtained. From this set of data, we constructed two intracellular proteinM-bM-^@M-^Sprotein interaction (PPI) networks for the traumatic brain injury healing mechanism. Each fish in each group was injured by a 1.5 mm, 27G needle tip from day 0 to 28, respectively. These injured fish were collected at 0, 0.25, 1, 3, 6, 10, 15, 21, 28 dpi (day post injury). 0.625M-NM-<g of Cy3 cRNA for C. albicans array and 1.65 M-NM-<g of Cy3 cRNA for zebrafish array was fragmented to an average size of about 50-100 nucleotides by incubation with fragmentation buffer at 60M-BM-0C for 30 minutes. Each time point contain two biological repeats.
Project description:We generate whole-transcriptome RNA-seq profiles of microglia subpopulations in the adult zebrafish brain (4-month-old) to study their heterogeneity. Cells were isolated as ccl34b.1+mpeg1+ populations or ccl34b.1-mpeg1+ populations from the whole brain of double transgenic TgBAC(ccl34b.1:eGFP);Tg(mpeg1:DsRedx) fish. ccl34b.1+mpeg1+ and ccl34b.1-mpeg1+ microglia were grouped into two distinct clusters in the Principal component analysis (PCA), and we identified substantial differentially-expressed genes between these two microglial populations using the DESeq2 package. To further unveil their differential roles during inflammation, we injected E. coli into the brain ventricles of adult zebrafish and isolated both populations for whole-transcriptome RNA-seq. This RNA-seq profile provides valuable information for dissecting their respective functions in vivo.