Genetic Screen Of Zebrafish Mutants With Developmental Defects In Digestive Organs

Digestive system, one of the most important vertebrate systems, has always been the focus in the field of biology and medicine. The physiological function of digestive system is closely associated with health, some serious digestive diseases threat to people’s life directly. Digestive system is composed of digestive tract and digestive gland. Liver, gall bladder and intestine are very important digestive organs which play roles in food digestion, storage, nutrient absorption, excretion, and endocrine. Study of the development of digestive organs makes people better understand the molecular mechanisms of some congenital digestive organ developmental defect diseases, such as congenital liver cirrhosis, congenital intrahepatic biliary atresia, Hirschsprung’s disease and duodenal atresia psychosis. Since patients with such congenital diseases usually die in the embryonic period or infancy, clinical cases and diseases families are very lacking in the study of the molecular mechanisms of congenital digestive organs developmental defects.Zebrafish, a small sub-tropical freshwater fish, is a representative animal model of vertebrates. At the genomic level, it has high similarity with mammals and humans. The zebrafish digestive organs include the liver, gall bladder and intestine etc. They are similar to the higher vertebrates in the structure. For a long time, zebrafish has been proven to be a wonderful model system to study the vertebrate embryogenesis due to its advantaged characteristics. The zebrafish embryos are small, transparent and develop outside of the maternal body. Moreover, zebrafish is easy to raise and highly reproductive, which make it suitable for a large-scale genetic screen.The intestine, liver and gall bladder develop from endoderm, but the differences of molecular mechanism among the three organs are not clear. To explore this question, we use zebrafish as a model animal for a classical forward genetic screen with no gene bias to obtain the corresponding defective digestive organs mutants. At the same time, the second generation sequencing method was utilized to obtain the zebrafish tissue-specific genes in digestive organs. The main contents of this work are:1. forward genetic screening of zebrafish mutants with defective digestive organs, including the ENU mutagenesis, the generation of F1 fishes and F2 families, the screen in F2 families, the generation of F3 families, the screen in F3 families, and F3 putative mutants phenotype classification; 2. The analysis of digestive organ transcriptomes for the identification of tissue-specific genes, we extract total RNAs from the wild-type zebrafish digestive organs for RNA-Seq, then identify the tissue-specific genes in the digestive organs by the bioinformatics analysis. Forward genetic screening of zebrafish digestive organ mutants with developmental defects can help us understand the molecular mechanisms of zebrafish digestive organogenesis, and the differences among digestive organs; zebrafish digestive organs transcriptome analysis will help us find the specific genes in the digestive organs which provides important start points for the study of developmental processes in the digestive organs.In forward genetic screening, we used the classic ENU mutagenesis screening process and the three generations screening strategy. In the screen in F2 family, we crossed male and female individuals within the family to get F3 embryos, and used Ifabp probe for in situ hybridization and BES-H2O2-Ac staining to observe the liver, gallbladder and intestinal phenotype in F3 embryos to judge whether whose parents were heterozygous mutants or not according to Mendelian law. Then we outcrossed putative F2 families with wild-type zebrafish to generate F3 families, using the same screening methods to identify F3 putative mutants and to test whether the phenotype of F2 putative mutants are heritable. In the end, we got 23 mutant lines with defective digestive organs (originated from 14 F2 families) after screening 128 mutagenized genomes. We classified the obtained mutants into six groups according to their phenotypes:Ⅰ. "intestine-specific defective mutants ",1 mutant line belongings to this group; Ⅱ. "liver-specific defective mutants",6 mutant lines belonging to this group; Ⅲ. "gall bladder-specific defective mutant",7 mutant lines belonging to this group; Ⅳ. "liver and gall bladder common defective mutants",1 mutant line in this category; Ⅴ. " intestine and gall bladder common defective mutants",4 mutant lines in this category; VI. "liver, intestine and gall bladder common defective mutants",4 mutant lines in this category.In digestive organs transcriptomes analysis, we used the second generation sequencing to obtain the data of the zebrafish digestive organs transcriptomes in the liver, the gall bladder, the whole intestine, segments of the intestine including foregut, midgut, small intestine, rectum and anus, digestive organs in 5dpf and adults and the remaining body in 5dpf and adults. By a preliminary analysis of the data of the intestine segments and body control, we got six gene clusters with different gene expression patterns, including body specific genes, anus specific genes, small intestine specific genes, foregut and midgut specific genes, midgut and small intestine specific genes, and genes gradually decreased expression along digestive tract. Compared with the human digestive system specific gene expression and sequencing data, our results showed that zebrafish and human intestinal tracts had high similarity in gene expression, and no independent stomach structure in zebrafish. At the same time, we compared the enriched genes in the liver, gall bladder and the whole intestine and identified the tissue specific genes. The deep analysis and experimental identification will be carried out in the future.Finally, in this study we obtained 23 mutant lines with liver, gall bladder and intestine developmental defects by screening F2 & F3 families, we will select 2-3 respective mutants for mappings and gene cloning in the future; Through the analysis of transcriptome data, we picked out a number of DEGs in liver, gall bladder and intestine, which will be used to study the molecular mechanism of gene function in corresponding digestive organs by reverse genetics methods (gene knockout).