Activation of the embryonic genome during development represents a major developmental transition in all species. The history of its exploration began in the 1950s-1960s, when this idea was put forward and proven experimentally by Alexander Neyfakh. He observed the aberrant development of fish embryos upon X-ray irradiation and noted the different developmental outcomes depending on the stage when fertilized eggs were subjected to irradiation. Neyfakh also discriminated a regional difference of X-irradiation between the nucleus and the cytoplasm. By selecting the X-ray dose causing nuclear damage, he determined the beginning of zygotic transcription, which at that time became known as the morphogenetic function of nuclei.  https://www.selleckchem.com/products/tabersonine.html  His team defined the link of zygotic transcription with the asynchronization of cell division and cell migration, the two other hallmarks, which along with the morphogenetic function (or the zygotic genome activation), are at the core of the mid-blastula transition during development. Within this framework, current studies using maternal mutants and application of modern methods of whole-embryo and single-cell transcriptomics begin to decipher the molecular mechanisms of the mid-blastula transition (or the maternal-zygotic transition).Protein-protein interactions (PPIs) play a central role in all cellular processes. The discovery of green fluorescent protein (GFP) and split varieties, which are functionally reconstituted by complementation, led to the development of the bimolecular fluorescence complementation (BiFC) assay for the investigation of PPI in vivo. BiFC became a popular tool, as it is a convenient and quick technology to directly visualize PPI in a wide variety of living cells. In combination with the transparency of the early zebrafish embryo, it also permits detection of PPI in the context of an entire living organism, which performs all spatial and temporal regulations missing in in vitro systems like tissue culture. However, the application of BiFC in some research areas including the study of zebrafish is limited due to the lack of efficient and convenient BiFC expression vectors. Here, we describe the engineering of a novel set of Gateway®-adapted BiFC destination vectors to investigate PPI with all possible permutations for BiFC experiments. Moreover, we demonstrate the versatility of these destination vectors by confirming the interaction between zebrafish Bucky ball and RNA helicase Vasa in living embryos.Protein production and degradation are tightly regulated to prevent cellular structures from accumulating damage and to allow their correct functioning. A key aspect of this regulation is the protein half-life, corresponding to the time in which half of a specific protein population is exchanged with respect to its initial state. Proteome-wide techniques to investigate protein half-lives in vivo are emerging. Recently, we have established and thoroughly tested a metabolic labeling approach using 13C lysine (Lys(6)) for measuring protein lifetimes in mice. The approach is based on the fact that different proteins will incorporate a metabolic label at a rate that is dependent on their half-life. Using amino acid pool modeling and mass spectrometry, it is possible to measure the fraction of newly synthesized proteins and determine protein half-lives. In this chapter, we show how to extend this approach to zebrafish (Danio rerio), using a commercially available fish diet based on the stable isotope labeling by amino acids in cell culture (SILAC) technology. We describe the methods for labeling animals and subsequently use mass spectrometry to determine the lifetimes of a large number of proteins. In the mass spectrometry workflow proposed here, we have implemented the BoxCar data acquisition approach for increasing sample coverage and optimize machine use. To establish the proteome library used in the BoxCar approach, we recommend performing an in-solution digestion followed by peptide fractionation through basic reversed-phase chromatography. Overall, this chapter extends the use of current proteome technologies for the quantification of protein turnover to zebrafish and similar organisms and permits the study of germline changes following specific manipulations.Rapid innovations in core proteomic technologies and proteome-based bioinformatics fortified by recent genome sequencing allow the characterization and quantification of proteins on a global scale. These capabilities empower research to develop a more comprehensive understanding of how changes in protein expression and modification can affect complex signaling and regulatory networks. The consequences of these studies have significant implications for understanding how myriad activities are regulated in biological systems.Proteomic approaches have been applied to investigate the physiology, developmental biology, and impact of contaminants in fishes as model organisms. Here, we describe the use of label-free protein quantification and global proteome profiling to characterize eggs of different quality grades in the zebrafish .Transgenic zebrafish in which the germline is specifically labeled with enhanced green fluorescent protein (eGFP) can be used for continuous observation of germline development during the lifetime, from the primordial germ cells (PGCs) in the early embryo to the gametes in the mature gonad. In this chapter, we describe a procedure for the generation of transgenic fish Tg(piwil1egfp-UTRnanos3), the sample preparation for live imaging of PGCs, for high-resolution imaging of germ cells in developing gonads, and quantifying PGC numbers. The methods described in this chapter are not only applicable to the study of germ cells, but also provide general advices for researchers who are willing to generate transgenic zebrafish and do observation on live embryos as well as on fixed tissues.Zebrafish is an excellent system for the study of gonad development due to available genetic tools and its utilization as a human disease model. The zebrafish serves as an experimental system to model human disorders affecting the reproductive system, toxicological effects on fertility and sexual development, and hormonal regulation of fertility. Forward genetic screens have been used to uncover genetic causes of infertility and reverse genetic approaches have demonstrated that genes involved in germ cell development have similar functions in zebrafish and mammals. The most comprehensive picture of the gonad can be visualized by histology. There are a variety of methods that give excellent histology of zebrafish gonads. Below are methods for two staining approaches for the histology of paraffin-embedded zebrafish gonads.