Project description:The foggy mutant zebrafish show severe deficits in dopaminergic neurons, thus is an important tool to understanding the development of these neurons, which undergo degeneration in Parkinson's disease. Molecular study reveals that the foggy gene encodes a regulator of transcription elongation. Biochemical study suggests that the repressive activity of the Foggy protein is lost in the foggy mutant. The genes whose expression is normally repressed by Foggy are likely to play important roles in dopamine neuron development. However, their identity is unknown. Finding out these target genes of Foggy will provide important insights into dopamine neuron development and regeneration. They will also serve as an entry point to elucidate the role of transcription elongation in controlling neuronal development. We will identify genes whose expression is altered in the foggy mutant. Their expression pattern and functional significance will be examined in zebrafish through in situ hybridization and morpholino antisense knockdowm methods. We hypothesize that the genes whose transcription is regulated by Foggy play a critical role in the development of dopaminergic neurons. The species noted above is Drosophila however we are using ZEBRAFISH. We will examine three stages: 24 hour post fertilization (hpf), 36 hpf, and 48 hpf. During these stages, the dopamine neuron deficits manifest in the foggy mutant. To identify genes whose expression is altered and important for dopamine neuron development, we shall dissect embryonic brain tissues from wildtype and the foggy mutant embryos at the stages mentioned above, and prepare RNA from them. Since the foggy gene is expressed ubiquitously and also required for proper retinal and cardiovascular development, we would like to compare the gene expression between the whole embryos of wildtype and foggy mutant, to identify all possible downstream targets. We are currently applying for a supplement to support the entire microarray experiment. As a pilot experiment, we would like to perform 6 array analysis as soon as possible. Thus, we will examine the 48 hours post fertilzation time point in this pilot experiment and it will be performed in triplicate. This dataset is composed of 36 hours post fertilization wild type and mutants and 12-somite stage Foggy knockdown.

Project description:The foggy mutant zebrafish show severe deficits in dopaminergic neurons, thus is an important tool to understanding the development of these neurons, which undergo degeneration in Parkinson's disease. Molecular study reveals that the foggy gene encodes a regulator of transcription elongation. Biochemical study suggests that the repressive activity of the Foggy protein is lost in the foggy mutant. The genes whose expression is normally repressed by Foggy are likely to play important roles in dopamine neuron development. However, their identity is unknown. Finding out these target genes of Foggy will provide important insights into dopamine neuron development and regeneration. They will also serve as an entry point to elucidate the role of transcription elongation in controlling neuronal development. We will identify genes whose expression is altered in the foggy mutant. Their expression pattern and functional significance will be examined in zebrafish through in situ hybridization and morpholino antisense knockdowm methods. We hypothesize that the genes whose transcription is regulated by Foggy play a critical role in the development of dopaminergic neurons. The species noted above is Drosophila however we are using ZEBRAFISH. We will examine three stages: 24 hour post fertilization (hpf), 36 hpf, and 48 hpf. During these stages, the dopamine neuron deficits manifest in the foggy mutant. To identify genes whose expression is altered and important for dopamine neuron development, we shall dissect embryonic brain tissues from wildtype and the foggy mutant embryos at the stages mentioned above, and prepare RNA from them. Since the foggy gene is expressed ubiquitously and also required for proper retinal and cardiovascular development, we would like to compare the gene expression between the whole embryos of wildtype and foggy mutant, to identify all possible downstream targets. We are currently applying for a supplement to support the entire microarray experiment. As a pilot experiment, we would like to perform 6 array analysis as soon as possible. Thus, we will examine the 48 hours post fertilzation time point in this pilot experiment and it will be performed in triplicate. Keywords: other

Project description:The foggy mutant zebrafish show severe deficits in dopaminergic neurons, thus is an important tool to understanding the development of these neurons, which undergo degeneration in Parkinson's disease. Molecular study reveals that the foggy gene encodes a regulator of transcription elongation. Biochemical study suggests that the repressive activity of the Foggy protein is lost in the foggy mutant. The genes whose expression is normally repressed by Foggy are likely to play important roles in dopamine neuron development. However, their identity is unknown. Finding out these target genes of Foggy will provide important insights into dopamine neuron development and regeneration. They will also serve as an entry point to elucidate the role of transcription elongation in controlling neuronal development. We will identify genes whose expression is altered in the foggy mutant. Their expression pattern and functional significance will be examined in zebrafish through in situ hybridization and morpholino antisense knockdowm methods. We hypothesize that the genes whose transcription is regulated by Foggy play a critical role in the development of dopaminergic neurons. The species noted above is Drosophila however we are using ZEBRAFISH. We will examine three stages: 24 hour post fertilization (hpf), 36 hpf, and 48 hpf. During these stages, the dopamine neuron deficits manifest in the foggy mutant. To identify genes whose expression is altered and important for dopamine neuron development, we shall dissect embryonic brain tissues from wildtype and the foggy mutant embryos at the stages mentioned above, and prepare RNA from them. Since the foggy gene is expressed ubiquitously and also required for proper retinal and cardiovascular development, we would like to compare the gene expression between the whole embryos of wildtype and foggy mutant, to identify all possible downstream targets. We are currently applying for a supplement to support the entire microarray experiment. As a pilot experiment, we would like to perform 6 array analysis as soon as possible. Thus, we will examine the 48 hours post fertilzation time point in this pilot experiment and it will be performed in triplicate. This dataset is composed of 36 hours post fertilization wild type and mutants and 12-somite stage Foggy knockdown.

Project description:This project aimed at identifying developmental stage specific transcript profiles for catecholaminergic neurons in embryos and early larvae of zebrafish (Danio rerio). Catecholaminergic neurons were labeled using transgenic zebrafish strains to drive expression of GFP. At stages 24, 36, 72 and 96 hrs post fertilization, embryos were dissociated and GFP expressing cells sorted by FACS. Isolated RNAs were processed using either polyA selection and libray generation or NanoCAGE. This is the first effort to determine stage specific mRNA profiles of catecholaminergic neurons in zebrafish. Catecholaminergic neurons were labeled by four different strategies: (1) 24 hrs old embryos: we used the ETvmat2:GFP transgenic line (Wen et al. 2007). Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish. Dev Biol. 2008 Vol 314 p84-92) which at this early stage labels catecholaminergic neurons in posterior tuberculum and locus coeruleus; (2) 24 hrs old embryos: we used Tg(otpb.A:egfp)zc48 transgenic line (Fujimoto et al. Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification. Dev Biol 2011, Vol 352, p393–404) which at this stage label ventral diencephalic dopaminergic neurons and some preoptic neurons. (3) For 72 and 96 hrs old zebrafish larvae we used a th:GFP BAC transgenic lines that labels catecholaminergic neurons (Tay et al., Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems. Nat Comms 2011 Vol 2, 171; also: T. Leng and W. Driever, unpublished). (4) for the 36 and 48 hrs old zebrafish larvae we used a th:Gal4VP16 driver and UAS:EGFP responder transgenic line system to label catecholaminergic cells (Fernandes et al., Deep brain photoreceptors control light-seeking behavior in zebrafish larvae. Curr Biol. 2012 Vol 22 DOI 10.1016/j.cub.2012.08.016). We used the different transgenic lines, because lines (3) and (4) do not efficiently label catecholaminergic neurons at early stages, while lines (1) and (2) also have GFP expression in several other non-catecholaminergic populations at later stages of development. Embryos were dissociated and catecholaminergic neurons were FACS sorted from GFP-tagged zebrafish (Manoli and Driever, 2012, Cold Spring Harbor Protoc. DOI 10.1101/pdb.prot069633). RNA was either processed for NanoCAGE, or mRNA was isolated and amplified. cDNA was sequenced by Illumina technique. This data submission is a series of data files consisting of three independent experiments with diffrent RNA-Seq depth: Samples 1-4 (NanoCage): Samples 5-8 (RNA-Seq high read numbers), and SAmples 9-12 (RNA-Seq low read numbers).

Project description:Loss-of-function mutations in the parkin gene can cause early onset Parkinson’s disease, a movement disorder resulting from the selective degeneration of dopaminergic neurons in the basal ganglia. Analogously, movement deficits and loss of a subset of dopaminergic neurons are observed in Drosophila melanogaster homozygous for null mutations in parkin. Parkin is an E3 ubiquitin ligase that functions with the mitochondrial localized serine/threonine kinase PINK1 in a pathway required to maintain mitochondrial integrity. We previously established that the PINK1/Parkin pathway functions in Drosophila dopaminergic and cholinergic neurons to maintain mitochondrial membrane potential. However, the mechanisms through which the PINK/Parkin pathway selectively impacts dopaminergic neuron survival remains unclear, as do the mechanisms that lead to the selective vulnerability of dopaminergic neurons in Parkinson’s disease. Because the transcriptome of a cell determines its identity we hypothesized that knowledge of the transcriptional alterations that occur in dopaminergic neurons isolated from parkin null Drosophila would provide insight into the mystery of selective vulnerability in Parkinson's disease. Results: To test our hypothesis we measured the transcriptome of dopaminergic and cholinergic neurons isolated from isogenic heterozygous and homozygous parkin null mutants using a novel flow cytometry-based method we developed. Computational analysis and experimental confirmation demonstrate that our method allows for the successful expression analysis of defined neural subsets from the Drosophila brain. In addition, our dataset implicates iron handling and dopamine signaling as being significantly dysregulated in parkin null dopaminergic neurons. Conclusions: Our flow cytometry-based method allows for the isolation and microarray analysis of neuronal subsets from the adult Drosophila brain. Our microarray analyses implicate iron handling and dopamine metabolism as contributing factors in the etiology of parkin-associated early onset Parkinson’s disease. Here we provide a novel dataset that may serve as a foundation for subsequent functional analyses of the pathways underlying neuronal selective vulnerability in Parkinson's disease.

Dataset Information

Transcription profiling of zebrafish foggy mutant

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