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Cell cultures are effective supplemental models to study specific biochemical pathways used for environmental adaption in animals. They enable isolation from system influence and facilitate control the extracellular environment. For work focusing on fish species many representative cell lines now exist, including a tilapia brain cell line (OmB) developed in our lab. CRISPR/Cas9 gene editing is an additional tool aiding these studies by allowing manipulation of specific genetic loci and evaluating their causal relationship between phenotypes of interest. However, established CRISPR/Cas9 gene targeting tools and methods often have not functioned as efficiently in fish cells as seen in other animal cell models such as mammalian cell lines, consistent with our initial attempts to apply CRISPR/Cas9 in OmB cells that failed to indicate genomic alteration at the targeted sites. Poor expression of heterologous promoters in OmB cells was hypothesized to be a primary cause for this occurrence so we constructed a custom plasmid vector based system utilizing tilapia endogenous promoters (EF1 alpha to express Cas9 and a U6 to express gRNAs). This system demonstrated substantial editing of most target sites attempted with mutational efficiency as high 80%. This work specifically highlighted the importance of phylogenetic proximity in selection of a polymerase III promoter for gRNA expression as commonly used interspecies U6 promoters (human and zebrafish) yielded no detectable gene editing when applied in this system with a common gRNA target sequence. These new tools will allow generation of knockout cell lines for gene targeting studies in tilapia and other phylogenetically close fish species. 

Euryhaline fish tolerate a wide range of environmental salinity by employing molecular mechanisms for coping with the associated osmotic stress. We have previously shown that osmotic stress transcription factor 1 (OSTF1) is part of these mechanisms. OSTF1 is transiently and rapidly upregulated in gill epithelial cells of tilapia (Oreochromis mossambicus) exposed to hyperosmolality. Hyperosmotic induction of OSTF1 in tilapia gills was reproduced in the tilapia OmB cell neuroepithelial cell line. OSTF1 shares the signature sequence of the TSC-22 family suggesting that it is a transcriptional repressor. If, in fact, OSTF1 is a transcription factor, we hypothesize that it will localize to the nucleus during hyperosmotic stress. Using standard cloning procedures, OSTF1 was tagged with enhanced green fluorescent protein (EGFP) at either the C- or N-terminus. Using fluorescent microscopy we show that the fusion proteins are retained in the cytosol under iso-osmotic conditions. To evaluate potential nuclear translocation of OSTF1 during hyperosmotic stress, we subjected OmB cells expressing the OSTF1:EGFP fusion protein to hyperosmotic media and imaged at time intervals from 5 minutes to 4 hours using a Leica Dmi8 microscope with automated scanning stage. At four hours and 650 mOsmol/kg, subcellular localization quantified by LASX image analysis (Leica) demonstrated that OSTF1:EGFP was mostly localized to the nucleus. This result supports our hypothesis that OSTF1 is indeed an osmotically inducible transcription factor. Current work evaluates influence of specific OSTF1 domains on nuclear localization. 

Histone post-translational modifications (PTMs) are epigenetic marks that modify the state of chromatin and lead to alterations in gene expression. Advances in mass spectrometry have enabled the high-throughput analysis of histone PTMs without the need for prior knowledge of individual PTMs of interest. In this study, the global histone PTM landscape was analyzed in the gills, kidney, and testes of Mozambique tilapia (Oreochromis mossambicus) through tandem mass spectrometry using data dependent acquisition (DDA-LCMS2) and PTM mapping approaches. PTM assignment to a specific amino acid was validated using A-score and localization probability scores that are based on the detection of diagnostic MSMS ions. These values signify the robustness of PTM assignment to a specific residue within the protein sequence. For PTMs that were represented by both modified and unmodified versions of the corresponding peptide, the stoichiometry was calculated and compared between tissues. We have identified multiple types of histone PTMs and assigned them to specific residues in each tissue. These PTMs include acetylation, methylation, demethylation, trimethylation, phosphorylation/ dehydration, and ubiquitination. Our results indicate that the gills, kidney, and testes each display a unique profile of histone PTMs. These data provide a strong basis for the generation of spectral libraries that enable high-throughput quantitative analyses of histone PTM stoichiometry on a global scale in tilapia exposed to diverse environmental and developmental contexts. 

Organismal physiology, morphology, and behavior are based on the function of structural proteins and enzymes. Proteins represent the central regulatory plane in the genome to phenome continuum. The protein complement of cells and tissues (the proteome) is highly dynamic and mirrors environmental and developmental influences on organismal phenotypes. Therefore, dynamic proteomes are excellent bioindicators of environmental exposure. Comprehensive blueprints of environmental exposures are reflected in specific proteome states and capturing those states is achieved by quantitative proteomics. We have developed quantitative proteomics workflows to characterize environmental influences on proteome states and proteome dynamics of euryhaline and euryhthermal fish populations in coastal areas. These workflows utilize tissue- and cell-specific assay libraries for data-independent acquisition (DIA) or Sequentially Windowed Acquisition of all THeoretically possible MSMS spectra (SWATH) mass spectrometry. Qunatitative proteome datasets generated with these workflows are highly accurate and they consistently cover precisely defined sets of proteomes. This consistent coverage renders systematic and long-term network and topological data analysis (TDA) approaches feasible. These workflows and approaches are explained and their application to coastal fish biology is discussed using selected datasets as examples. The data presented illustrate that habitat differences such as salinity and temperature changes are readily captured in state changes of tissue-specific proteomes. The overall topology of proteome states is indicative of particular tissues, species, and environmental contexts and is therefore suitable for deducing functional and phenotypic consequences of environmental changes on coastal organisms.