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A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC.

ABSTRACT: The intestine of fish is a multifunctional organ: lined by only a single layer of specialized epithelial cells, it has various physiological roles including nutrient absorption and ion regulation. It moreover comprises an important barrier for environmental toxicants, including metals. Thus far, knowledge of the fish intestine is limited largely to in vivo or ex vivo investigations. Recently, however, the first fish intestinal cell line, RTgutGC, was established, originating from a rainbow trout (Oncorhynchus mykiss). In order to exploit the opportunities arising from RTgutGC cells for exploring fish intestinal physiology and toxicology, we present here the establishment of cells on commercially available permeable membrane supports and evaluate its suitability as a model of polarized intestinal epithelia. Within 3 weeks of culture, RTgutGC cells show epithelial features by forming tight junctions and desmosomes between adjacent cells. Cells develop a transepithelial electrical resistance comparable to in vivo measured values, reflecting the leaky nature of the fish intestine. Immunocytochemistry reveals evidence of polarization, such as basolateral localization of Na+/K+-ATPase (NKA) and apical localization of the tight junction protein ZO-1. NKA mRNA abundance was induced as physiological response toward a saltwater buffer, mimicking the migration of rainbow trout from fresh to seawater. Permeation of fluorescent molecules proved the barrier function of the cells, with permeation coefficients being comparable to those reported in fish. Finally, we demonstrate that cells on permeable supports are more resistant to the toxicity elicited by silver ions than cells grown the conventional way, likely due to improved cellular silver excretion.

SUBMITTER: Minghetti M

PROVIDER: S-EPMC5658468 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC.

Minghetti Matteo M Drieschner Carolin C Bramaz Nadine N Schug Hannah H Schirmer Kristin K

Cell biology and toxicology 20170301 6

The intestine of fish is a multifunctional organ: lined by only a single layer of specialized epithelial cells, it has various physiological roles including nutrient absorption and ion regulation. It moreover comprises an important barrier for environmental toxicants, including metals. Thus far, knowledge of the fish intestine is limited largely to in vivo or ex vivo investigations. Recently, however, the first fish intestinal cell line, RTgutGC, was established, originating from a rainbow trout ...[more]

PMID: 28251411

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Project description:BackgroundGenetic maps characterizing the inheritance patterns of traits and markers have been developed for a wide range of species and used to study questions in biomedicine, agriculture, ecology and evolutionary biology. The status of rainbow trout genetic maps has progressed significantly over the last decade due to interest in this species in aquaculture and sport fisheries, and as a model research organism for studies related to carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. We constructed a second generation genetic map for rainbow trout using microsatellite markers to facilitate the identification of quantitative trait loci for traits affecting aquaculture production efficiency and the extraction of comparative information from the genome sequences of model fish species.ResultsA genetic map ordering 1124 microsatellite loci spanning a sex-averaged distance of 2927.10 cM (Kosambi) and having 2.6 cM resolution was constructed by genotyping 10 parents and 150 offspring from the National Center for Cool and Cold Water Aquaculture (NCCCWA) reference family mapping panel. Microsatellite markers, representing pairs of loci resulting from an evolutionarily recent whole genome duplication event, identified 180 duplicated regions within the rainbow trout genome. Microsatellites associated with genes through expressed sequence tags or bacterial artificial chromosomes produced comparative assignments with tetraodon, zebrafish, fugu, and medaka resulting in assignments of homology for 199 loci.ConclusionThe second generation NCCCWA genetic map provides an increased microsatellite marker density and quantifies differences in recombination rate between the sexes in outbred populations. It has the potential to integrate with cytogenetic and other physical maps, identifying paralogous regions of the rainbow trout genome arising from the evolutionarily recent genome duplication event, and anchoring a comparative map with the zebrafish, medaka, tetraodon, and fugu genomes. This resource will facilitate the identification of genes affecting traits of interest through fine mapping and positional cloning of candidate genes.

Project description:BackgroundMaternal transcripts are accumulated in the oocyte during oogenesis to provide for protein synthesis from oocyte maturation through early embryonic development, when nuclear transcription is silenced. The maternal mRNAs have short poly(A) tails after undergoing post-transcriptional processing necessary for stabilizing them for storage. The transcripts undergo cytoplasmic polyadenylation when they are to be translated. Transcriptome analyses comparing total mRNA and elongated poly(A) mRNA content among eggs of different quality can provide insight into molecular mechanisms affecting egg developmental competence in rainbow trout. The present study used RNA-seq to compare transcriptomes of unfertilized eggs of rainbow trout females yielding different eyeing rates, following rRNA removal and poly(A) retention for construction of the libraries.ResultsThe percentage of embryos to reach the 32-cell stage at 24 h post fertilization was significantly correlated to family eyeing rate, indicating that inviable embryos were developmentally compromised before zygotic genome activation. RNA sequencing identified 2 differentially expressed transcripts (DETs) from total mRNA sequencing comparing females with low-quality (< 5% eyeing), medium-quality (30-50% eyeing), and high-quality (> 80% eyeing) eggs. In contrast, RNA sequencing from poly(A) captured transcripts identified 945 DETs between low- and high-quality eggs, 1012 between low- and medium-quality eggs, and only 2 between medium- and high-quality eggs. The transcripts of mitochondrial genes were enriched with polyadenylated transcript sequencing and they were significantly reduced in low-quality eggs. Similarly, mitochondrial DNA was reduced in low-quality eggs compared with medium- and high-quality eggs. The functional gene analysis classified the 945 DETs between low- and high-quality eggs into 31 functional modules, many of which were related to ribosomal and mitochondrial functions. Other modules involved transcription, translation, cell division, apoptosis, and immune responses.ConclusionsOur results indicate that differences in egg quality may be derived from differences in maternal nuclear transcript activation and cytoplasmic polyadenylation before ovulation, as opposed to accumulation and storage of maternal nuclear transcripts during oogenesis. Transcriptome comparisons suggest low-quality eggs suffered from impaired oxidative phosphorylation and translation. The DETs identified in this study provide insight into developmental competence in rainbow trout eggs.

Dataset Information

A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC.

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A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC.

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