Project description:Humans and animals have problems producing eggs with high embryo developmental competence, but the causes of poor egg quality are usually unknown. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading model for vertebrate development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were used to create pooled or replicated sample sets subjected to different levels of fractionation before LC-MS/MS. Obtained spectra were searched against a custom zebrafish proteome database and detected proteins were annotated, categorized and quantified based on their normalized spectral counts. Manual and automated enrichment analyses were highly confirmative, showing that good and poor quality eggs have disparate proteomes. Proteins involved in protein synthesis, energy metabolism, and lipid metabolism, and certain vitellogenin products were strikingly underrepresented in poor quality eggs. Poor quality eggs also had significantly higher representation of proteins related to immune system and endosome/lysosome functioning, oncogenes, and apoptosis, as well as lectins and egg envelope proteins. Quantitative comparisons of highly abundant proteins revealed 9 candidate egg quality markers warranting further study. In conclusion, the zebrafish egg proteome appears to be linked to embryo developmental potential, a phenomenon that begs further investigation.
Project description:Humans and animals have problems producing eggs with high embryo developmental competence, but the causes of poor egg quality are usually unknown. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading model for vertebrate development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were used to create pooled or replicated sample sets subjected to different levels of fractionation before LC-MS/MS. Obtained spectra were searched against a custom zebrafish proteome database and detected proteins were annotated, categorized and quantified based on their normalized spectral counts. Manual and automated enrichment analyses were highly confirmative, showing that good and poor quality eggs have disparate proteomes. Proteins involved in protein synthesis, energy metabolism, and lipid metabolism, and certain vitellogenin products were strikingly underrepresented in poor quality eggs. Poor quality eggs also had significantly higher representation of proteins related to immune system and endosome/lysosome functioning, oncogenes, and apoptosis, as well as lectins and egg envelope proteins. Quantitative comparisons of highly abundant proteins revealed 9 candidate egg quality markers warranting further study. In conclusion, the zebrafish egg proteome appears to be linked to embryo developmental potential, a phenomenon that begs further investigation.
Project description:Zebrafish (Danio rerio) model system have used widespread vertebrate investigations for genetic and cell biological analyses, and is suitable for small molecular screens such as chemical, toxicity and drug in order to use for human diseases and drug discovery . Recently, These powerful zebrafish model increasingly apply to human metabolic disease such as obesity and diabetes and toxicology. Despite a lot of advantages, proteomics research at zebrafish has received little interest in comparison with genetic and biological research using histology and in situ hybridization. Protein lysine acetylation is one of the most known post-translational modifications with dynamic and reversibly controlled by lysine acetyltransferase such as histone acetyltransferases and lysine deacetylase such as histone deacetylases and sirtuins family.Also, during the past year, global lysine acetylome studies using MS-based proteomics approach was in diverse species such as human, mouse, E. coli, Yeast and plants. Based on global acetylome data, our understanding of the roles of lysine acetylation in various cellular processes has increased. . The aim of this study was to identify Lysine acetylation in zebrafish embryos and determine the homology from Human at modified site level. Here we showed the global lysine acetylation study in Zebrafish embryos using MS-based zebrafish embryos.
Project description:All vertebrates have multiple genes encoding for different CASQ isoforms. Increasing interest has been focused on mammalian and human CASQ genes since mutations of both cardiac (CASQ2) and skeletal (CASQ1) isoforms cause different, and sometime severe, human pathologies Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work expression, biochemical properties and cellular and sub-cellular localization of Danio rerio native CASQ isoforms are investigated. By quantitative PCR three mRNAs were detected in skeletal muscle and one mRNA in heart. Three zebrafish CASQs were identified by mass spectrometry and they share properties with mammalian skeletal and cardiac CASQs. Skeletal calsequestrins were found primarily, but not exclusively, at the sarcomere Z-line level where Terminal Cisternae of Sarcoplasmic reticulum are located.
Project description:Purpose: Construction of 3D zebrafish spatial transcriptomics data for studying the establishment of AP axis. Methods: We performed serial bulk RNA-seq data of zebrafish embryo at three development points. Using the published spatial transcriptomics data as references, we implemented Palette to infer spatial gene expression from bulk RNA-seq data and constructed 3D embryonic spatial transcriptomics. The constructed 3D transcriptomics data was then projected on zebrafish embryo images with 3D coordinates, establishing a spatial gene expression atlas named Danio rerio Asymmetrical Maps (DreAM). Results: DreAM provides a powerful platform for visualizing gene expression patterns on zebrafish morphology and investigating spatial cell-cell interactions. Conclusions: Our work used DreAM to explore the establishment of anteroposterior (AP) axis, and identified multiple morphogen gradients that played essential roles in determining cell AP positions. Finally, we difined a hox score, and comprehensively demonstrated the spatial collinearity of Hox genes at single-cell resolution during development.
Project description:Histidine phosphorylation is a reversible post-translational modification that is known to regulate signal transduction in prokaryotes. In an effort to help elucidate the heretofore hidden vertebrate phosphoproteome, this report presents a global phosphorylation analysis of Danio rerio (zebrafish) larvae. Phosphopeptide enrichment was performed using a TiO2 affinity technique. A total of 68 unique phosphohistidine sites were detected on 63 proteins among 1076 unique phosphosites on 708 proteins. This report provides the first phosphohistidine dataset obtained from zebrafish.
Project description:Lysine succinylation (Ksu) is a novel identified post-translational modification that conserved from prokaryote to eukaryotes. As a kind of acylation, Ksu was reported to have different functions with others acylation at lysine residue. However, recently studies on the Ksu mainly focus on the plants and bacterial, there are still very rare studies in the vertebrate. Therefore, the biological role of succinylation remains largely unknown in mammal. In this study, we performed global Ksu mapping in Danio rerio (zebrafish) using mass spectrometry-based proteomics with enrichment of Ksu peptides by immunoprecipitation technology. As a result, we totally identified 552 Ksu sites in 164 proteins. Compared with our previous studies on lysine acetylation and crotonylation, Ksu plays a major role in a diverse metabolic process, such as carbon metabolism and tricarboxylic acid circle. In addition, we defined 5 new succinylation motifs: (su)KA, (suc)KxxxxA, (su)KxxxxL, (su)KxA, (su)KxV. In conclusion, our result provides proteome-wide database for study of Ksu in zebrafish and our bioinformatics result facilitated the understanding of the Ksu in the role of central metabolism.
Project description:Methyl tert-butyl ether (MTBE) has been shown to target developing vasculature in piscine and mammalian model systems. In the zebrafish, MTBE induces vascular lesions throughout development. These lesions result from exposure to MTBE at an early stage in development (6-somites to Prim-5 stages). During this time period, transcript levels of vegfa, vegfc, and vegfr1 were significantly decreased in embryos exposed to 5 mM MTBE. We performed global gene analysis as an unbiased approach to discover possible modes of action of MTBE vascular toxicity. Embryos were exposed at 3 hours post fertilization (hpf) in triplicate to one of three concentrations of MTBE: 5mM (induces vascular lesions and significantly decreases vegfa), 0.625mM (NOAEL; no observed adverse effect level), and 0.00625mM (100-fold below NOAEL), or to embryo media (control). Samples were collected at 6-somites (~15hpf), 21-somites (~24 hpf), and Prim-5 (~30 hpf) stages of development. Embryos were meticulously staged at exposure and at the time of collection to maintain a homogeneous population. Our experimental design sought to explore the effect of three concentrations MTBE on three different stages of zebrafish embryonic development during the critical period established for the chemical. This time period also corresponds to an important time in the cardiovascular system develop of our model vertebrate.
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).