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FACS-assisted microarray profiling implicates novel genes and pathways in zebrafish gastrointestinal tract development.

ABSTRACT: The zebrafish Danio rerio is an excellent model system for mammalian gastrointestinal development. To identify differentially regulated genes important in gastrointestinal organogenesis, we profiled the transcriptome of the zebrafish developing gastrointestinal tract.Embryos from a transgenic zebrafish line expressing green fluorescent protein (GFP) in the developing intestine, liver, and pancreas were dissociated at 4 developmental time points, their cells sorted based on GFP expression with fluorescence-activated cell sorting (FACS), and analyzed with microarrays. To improve our analysis, we annotated the Affymetrix Zebrafish GeneChip with human orthologs.Transcriptional profiling showed significant differences between GFP(+) and GFP(-) cells. Up-regulated genes and pathways were consistent with mammalian gastrointestinal development, such as hepatic nuclear factor gene networks and cancer. We implicate the phosphatidylinositol 3 kinase (PI3K) pathway and show that inhibition with LY294002 causes gastrointestinal defects in zebrafish. We identified novel genes, such as the microRNAs miR-217 and miR-122, the tight junction protein claudin c, the gene fam136a, and a zebrafish tetraspanin. Novel pathways include genes containing a putative transcription factor binding sequence, GGAANCGGAANY, and a nucleolar gene network. The zebrafish microarrays also identify a set of 32 genes that may mediate the effects of gain of chromosome arm 8q in human colon, liver, and pancreatic cancers.We successfully combine FACS and microarray profiling to follow organogenesis throughout development. These experiments identify novel genes and pathways that probably play a role in mammalian gastrointestinal development and are potential targets for therapeutic intervention in the management of gastrointestinal disease and cancer.

SUBMITTER: Stuckenholz C

PROVIDER: S-EPMC2785077 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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FACS-assisted microarray profiling implicates novel genes and pathways in zebrafish gastrointestinal tract development.

Stuckenholz Carsten C Lu Lili L Thakur Prakash P Kaminski Naftali N Bahary Nathan N

Gastroenterology 20090627 4

<h4>Background & aims</h4>The zebrafish Danio rerio is an excellent model system for mammalian gastrointestinal development. To identify differentially regulated genes important in gastrointestinal organogenesis, we profiled the transcriptome of the zebrafish developing gastrointestinal tract.<h4>Methods</h4>Embryos from a transgenic zebrafish line expressing green fluorescent protein (GFP) in the developing intestine, liver, and pancreas were dissociated at 4 developmental time points, their ce ...[more]

PMID: 19563808

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Project description:BackgroundThe human gastrointestinal (GI) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. Increasingly molecular profiling techniques are being employed to examine this complex microbial community. The purpose of this study was to develop a microarray technique based on 16S ribosomal gene sequences for rapidly monitoring the microbial population of the GI tract.ResultsWe have developed a culture-independent, semi-quantitative, rapid method for detection of gut bacterial populations based on 16S rDNA probes using a DNA microarray. We compared the performance of microarrays based on long (40- and 50-mer) and short (16-21-mer) oligonucleotides. Short oligonucleotides consistently gave higher specificity. Optimal DNA amplification and labelling, hybridisation and washing conditions were determined using a probe with an increasing number of nucleotide mismatches, identifying the minimum number of nucleotides needed to distinguish between perfect and mismatch probes. An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population. The sensitivity of the microarray was determined to be 8.8 x 104 bacterial cells g-1 faecal sample, which is more sensitive than a number of existing profiling methods. The short oligonucleotide microarray was used to compare the faecal flora from healthy individuals and a patient suffering from Ulcerative Colitis (UC) during the active and remission states. Differences were identified in the bacterial profiles between healthy individuals and a UC patient. These variations were verified by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing.ConclusionIn this study we demonstrate the design, testing and application of a highly sensitive, short oligonucleotide community microarray. Our approach allows the rapid discrimination of bacteria inhabiting the human GI tract, at taxonomic levels ranging from species to the superkingdom bacteria. The optimised protocol is available at: http://www.ifr.ac.uk/safety/microarrays/#protocols. It offers a high throughput method for studying the dynamics of the bacterial population over time and between individuals.

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FACS-assisted microarray profiling implicates novel genes and pathways in zebrafish gastrointestinal tract development.

Publications

FACS-assisted microarray profiling implicates novel genes and pathways in zebrafish gastrointestinal tract development.

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