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: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: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.
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:Methylmercury (MeHg) is a potent neurotoxin and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. We investigated the gene expression profile in adult female zebrafish whole brain induced by acute (96 hr) MeHg exposure. Fish were exposed by injection to 0 or 0.5 M-BM-5g MeHg/g. Gene expression changes in the brain were examined using a two-color 22,000 feature zebrafish microarray. At a significance level of p<0.01, 79 genes were up-regulated and 76 genes were down-regulated in response to MeHg exposure. Individual genes exhibiting altered expression in response to MeHg exposure implicate effects on glutathione metabolism and GABA-A receptors in the mechanism of MeHg neurotoxicity. Gene ontology (GO) terms significantly enriched among altered genes included protein folding, cell redox homeostasis, and steroid biosynthetic process. The most affected biological functions were related to the nervous system development and function, as well as lipid metabolism and molecular transport. These results support the involvement of oxidative stress and effects on protein structure in the mechanism of action of MeHg in the female brain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxins and investigate responsive genes as potential biomarkers of MeHg exposure. Wild-type strain AB-1 zebrafish (Zebrafish International Resource Center, University of Oregon, Eugene, OR) were cultured at the Columbia Environmental Research Center (CERC), USGS, for MeHg exposures. Adult female zebrafish were injected with 0 M-NM-<g/g or 0.5 M-NM-<g/g MeHg in 2 M-BM-5L Na2CO3 (pH 6.98)/g body weight. After 96 hr, fish were anesthetized using ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma, St. Louis, MO). Whole brains were removed, flash frozen with liquid nitrogen and stored at 80M-BM-0C. For the microarray experiment, two zebrafish brains were pooled per sample. Four pooled samples were taken from fish treated with 0.5 M-NM-<g/g of MeHg, and the other five were taken from control fish treated with sodium carbonate. Array hybridizations were performed using a reference design, where each sample was compared to a reference sample. The reference sample consisted of equal amounts of RNA from control and treated female brains. Five replicates for the control and four replicates for the treated were analyzed. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA).
Project description:Purpose: Identify zebrafish control and csf1r-mutant brain transcriptomes Methods: RNA sequencing was performed on whole brain of control (3x), csf1ra-/- microglia (3x) and csf1ra-/-;b+/- microglia (3x) and csf1ra-/-;b-/- zebrafish. 10-20 million reads per sample were obtained. Reads were mapped to zebrafish genome GRC10. Results: We identified that microglia gene expression was reduced in csf1ra-/-;b+/- and csf1ra-/-;b-/;- mutant transcriptomes.
Project description:During development, the inherited DNA methylation patterns from the parental gametes needs to be remodeled into a state compatible with embryonic pluripotency. In Zebrafish, this remodeling is achieved by the maternal methylome becoming hypomethylated to match the paternal methylome. However, how this is achieved in medaka (another teleost fish) is currently not known. Moreover, how DNA methylation remodeling is impacted in hybrid organisms, and the effects this may have on their development, is also not known. Here we address these questions by generation whole genome bisulfite sequencing data for zebrafish, medaka and zebrafish medaka embryos.
Project description:We exposed zebrafish embryos to 0.3, 3, and 30 ppb (µg/L) of ATZ for 72 hours post fertilization. We performed whole-genome bisulfite sequencing (WGBS) to assess the effects of developmental ATZ exposure on DNA methylation in female fish brains