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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Stuckenholz
Carsten
C
Department of Medicine, Division of Hematology/Oncology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Lu
Lili
L
Thakur
Prakash
P
Kaminski
Naftali
N
Bahary
Nathan
N
D000818:Animals; D030801:Animals, Genetically Modified; D002469:Cell Separation; D002898:Chromosomes, Human, Pair 8; D004067:Digestive System Neoplasms / Q000235:genetics; D005434:Flow Cytometry; D041981:Gastrointestinal Tract / Q000196:embryology / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization; D007814:Larva / Q000235:genetics; D035683:MicroRNAs / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D038081:Organogenesis / Q000235:genetics; D015203:Reproducibility of Results; D013997:Time Factors; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
D035683:MicroRNAs; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins