Effects of anti-androgenic compounds on zebrafish insulin mutants
Ontology highlight
ABSTRACT: Aiming to identify insulin-independent modulators of glucose homeostasis, we performed a drug screen on zebrafish insulin (ins) mutants and identified androgen receptor (AR) antagonists. To investigate how AR antagonism mediates glucose level reduction in ins mutants, we evaluated the effects of antagonist treatment using transcriptomic studies. RNA-Seq analyses were performed on 120 hours post fertilization (hpf) ins mutants treated with Flutamide or Cyproterone starting at 84 hpf compared to vehicle (DMSO) treated mutants.
Project description:Transcriptomic differences of gata1+ erythrocytes between the kdm4b-/- mutants and the WT embryos at 48 hpf were analyzed by RNA sequencing.
Project description:RNA-seq was performed to reveal the RNA expression profile at two different stages (shield and tail-bud stage) in the embryogenesis of zebrafish (wild type strain AB). Zebrafish embryos were collected at the shield stage and tail-bud stage, which correspond to 6 hours post fertilization (hpf) and 10 hpf, respectively. Total RNA was extracted with miRNeasy Mini Kit, libraries for sequencing were prepared with TruSeq stranded mRNA library prep kit (Illumina), and paired-end sequencing was performed using NovaSeq 6000 (Illumina).
Project description:To identify genes regulated by Rx3 during optic vesicle morphogenesis, adult zebrafish carriers of a null rx3 mutation were mated. Before 13 hours post fertilization (hpf), the earliest time point at which optic vesicle evagination phenotypes could be reliably detected, offspring were phenotypically separated into pools comprising of mutants with an absence of optic vesicles or siblings exhibiting a wild-type phenotype. Three replicates of pooled RNA samples from 13 hpf eyeless mutants (rx3-/-) or phenotypically wild-type siblings (rx3+/+ or rx3+/-), and one replicate of 13 hpf wild-type zebrafish larva were collected for whole transcriptome sequencing. Whole transcriptome sequencing (RNA-seq) was performed on zebrafish rx3-/- mutants, wild-type siblings and wild-type AB strains at 13 hpf
Project description:The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Teadresponsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosagedependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson’s chorioretinal atrophy and congenital retinal coloboma. 60 pooled eyes from 36 hpf wild type or vsx2:Gal4/dsRed:14xUAS:YapS87A embryos were pooled for one sample. Three wild type and three vsx2:Gal4/dsRed:14xUAS:YapS87A pools were analyzed for RNA.
Project description:We integrate zebrafish embryology with photoactivatable caged morpholinos (cMO), the photoactivatable lineage tracer caged fluorescein-dextran (cFD), fluorescence-activated cell-sorting (FACS) and RNA sequencing (RNA-seq) to identify Spadetail-regulated genes in mesodermal cells enriched for early somite progenitors. 1 to 4-cell stage zebrafish embryos were injected with either cFD alone or in combination with the spt cMO and allowed to develop until 6 hours post-fertilization (hpf). Early ventral somite progenitors were then optically targeted with ultraviolet (360 nm) light using a 100-μm diameter circular diaphragm. Embryos were permitted to develop until 9 hpf, dechorionated, and dissociated into single-cells. Fluorescein-positive cells were then isolated by FACS. Five experimental replicates were performed. Sequencing libraries were then prepared from purified cells according to the CEL-Seq protocol (Hashimshony, T et al. Cell Reports. 2012). In short, total RNA from each sample were individually barcoded and combined prior to sequencing on a HiSeq 2000. Reads were processed and aligned to zebrafish assembly Zv8 using custom CEL-seq scripts within the Galaxy bioinformatics platform. Count files were then analyzed using EdgeR statistical analysis to determine differentially expressed genes.
Project description:We sequenced a total of 16 cDNA libararies derived from fragmented total mRNA and ribosome protected mRNAs from S. aureus hpf mutant (NE838) and its parental strain JE2 grown in tryptic soy broth (TSB) or minimal medium (MM). The data represented 2 independent biological replicates. Examination of the impact of hpf on global S.aureus translatome, mRNA abundance and the ribosome density along the transcripts.
Project description:TgBAC(gata5:EGFP) transgenic line was used to isolate mesendoderm cells enriched for cardiac lineage. Wild type embryos at 6, 8, 10, 13 hours post fertilization (hpf) and morpholinos injected Gata5/6 know-down (KD) embryos at 13 hpf were pre-screened for GFP signal and dissociated into single-cell suspension in separate wells. GFP+ cells at each condition were isolated through fluorescent activated cell sorting (FACS). Single-cell cDNA libraries were constructed through the 10x Chromium Single Cell Gene Expression platform. Final sequencing was performed on an Illumina HiSeq 2500 platform (Rapid Run Model). Note that the 10h single-cell library was sequenced twice (Named as cDNA10h_1* and cDNA10h_2* separately).
Project description:We use the zebrafish embryo model to study the innate immune response against Staphylococcus epidermidis. Therefore, we injected S. epidermidis (and three controls groups) into the yolk at 2 hpf and samples at mutiple timepoints. Gene expression profiles were obtained at 6, 30, 54, 78, 102 and 126 hpi by microarrays. The results show that the gram-positive bacterium S. epidermidis induces a late immune response with a strong response at 102 hpi. This microarray study was designed to determine the gene expression profile during infection with Staphylococcus epidermidis. RNA was isolated from groups of embryos (20) at 6 timepoints during the infection. Wildtypes zebrafish embryos were micro-injected into the yolk (2hpf) with (1) 20 CFU of S. epidermdis O-47 mCherry bacteria suspended in PVP (Polyvinylpyrrolidone), (2) mock-injected with PVP as a control, (3) Needle insertion as control, (4) Non-injected as a control. After injections embryos were transferred into fresh egg water and incubated at 28M-BM-0C. At 8 hpf (6 h post infection), 32 hpf (30 h post infection), 56 hpf (54 h post infection), 80 hpf (78 h post infection), 104 hpf (102 h post infection) or 128 hpf (126 h post infection) twenty embryos per treatment group were snap-frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent. All treatment groups were analyzed using a common reference approach.
Project description:MyD88 is an adaptor protein in Toll-like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88, leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88-dependent genes. This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents (allele hu3568, van der Vaart et al., 2013, Disease models & mechanisms 6, 841-854) or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection (4 dpi). The following treatment groups were used: homozygous mutants mock-injected with PBS/2%PVP 4 dpi, (2) wildtype siblings mock-injected with PBS/2%PVP 4dpi, (3) M. marinum-infected homozygous mutants 4dpi, (4) M. marinum-infected wildtype siblings 4dpi. Embryos were grown at 28.5M-bM-^@M-^S30M-BM-0C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200 colony forming units (CFU) of Mycobacterium marinum Mma20 bacteria into the caudal vein, or were mock-injected with buffer (PBS/2%PVP) as a control. After injections embryos were transferred into fresh egg water and incubated for 4 days at 28M-BM-0C. After the incubation period, single embryos were snap-frozen in liquid nitrogen and RNA was isolated for RNAseq analysis.
Project description:To discover insulin-independent pathways controlling glucose homeostasis, we generated zebrafish insulin mutants. Aiming to characterize the transcriptional changes induced by deletion of insulin, we performed a microarray analysis comparing insulin mutant animals with their non-mutant siblings at 108 hours post fertilization. Embryos were grown at 28 degrees C until 108 hpf, and were separately collected as mutants or non-mutants to form pools of 10 animals per sample. Total RNA was isolated, sample quality was tested using a Bioanalyzer and microarray analysis was performed by Oak Labs (Germany)