Reverse genetics and microRNAsin zebrafish

The zebrafish (Danio rerio) has become an important genetic model organism in various research areas of modern biology. Originally, it was chosen as a vertebrate model system because its excellent properties that allow the study of embryonic development could be combined with powerful genetic analysis. Forward mutagenesis screens have led to the identification of thousands of mutants that are defective in developmental processes. More recent genetic screens have yielded many additional mutants in diverse biological processes, some of which are very reminiscent of human diseases. Great effort is undertaken to identify the genes responsible for these mutant phenotypes. However, the reverse genetic discovery of gene function, by inactivation of genes through knockout technologies, has not been possible in zebrafish until recently. This thesis describes the establishment of a knockout technology in zebrafish. It describes the creation of the first zebrafish knockout, the rag1 gene, by target-selected mutagenesis. By resequencing a comprehensive library of mutagenized zebrafish, a null mutation in the rag1 gene was identified. This mutation leads to a failure in rearrangement of the immunoglobulin locus and consequently results in immunodefiency. Next, several refinements to this knockout technique are described. Instead of direct resequencing of mutagenized animals, the TILLING technique was used to pre-screen for point mutations in several genes. Using this method, thirteen potential zebrafish knockouts were identified. The current methodologies and potential future applications to do reverse genetic analysis in zebrafish are reviewed. The other subject of this thesis is 'microRNAs (miRNAs) in zebrafish development’. miRNAs are small non-coding RNA molecules that post-transcriptionally regulate gene expression. The base-pairing of miRNAs to target mRNAs results in translational inhibition or mRNA cleavage. Hundreds of miRNAs have been identified in various multicellular organisms and many miRNAs are evolutionarily conserved. Although the biological functions of most miRNAs are unknown, miRNAs are predicted to regulate up to 30% of the genes within the human genome. The recent advances in miRNA biology are reviewed. Particularly, there is a focus on the roles of miRNAs in vertebrate development and disease. The construction and analysis of the dicer knockout in zebrafish is described. Disruption of the miRNA-producing enzyme Dicer results in developmental arrest and failure to produce miRNAs, indicating that miRNAs are essential for vertebrate development. Furthermore, the miRNA expression patterns during zebrafish embryonic development are described. Most miRNA are expressed in a highly tissue-specific manner during segmentation and later stages, but not early in development, which suggests that their role is not in tissue fate establishment but in differentiation or maintenance of tissue identity