Graduation Year

2011

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Richard S. Pollenz

Co-Major Professor

Meera Nanjundan

Keywords

Danio rerio, Embryogenesis, mdm1, Morpholino, Mosaic Eye, synbl

Abstract

The zebrafish, Danio rerio, is a type of freshwater minnow often used to model human diseases including cancer, anxiety and aging diseases. The overall biology of zebrafish is strikingly similar to that of humans, allowing these fish to be used for drug discovery and toxicology studies for preclinical trials. In this study, zebrafish embryos were used to identify and characterize several candidate genes within two known regions of genomic instability on chromosome 18 and chromosome 4. This fish that were used in this study had been previously classified as genomic instability (gin) mutants due to increased incidence of somatic mutation during the early stages of embryogenesis, that can be detected with the mosaic eye assay at 48-72 hpf. Using published genome and mapping data, several candidate genes for two of the gin mutations were identified and studied during early zebrafish development.

The gin mutations are heritable, ENU-induced, and have both maternal and zygotic effects during zebrafish development. The first aim of this project was to study the normal gene characteristics of the gin-10 candidate genes, synbl, rfx4, and sir2 that are located on chromosome 18. Semi-quantitative RT-PCR, whole-mount in situ hybridization, and gene knockdown (using morpholino oligonucleotides) techniques were utilized in both wildtype and transgenic (Tg-synbl) zebrafish lines to gain an understanding of the function of each of these genes during zebrafish embryogenesis. Additionally, the synbl paralog, ric8a, was also explored, as it has been implicated in the control of asymmetric cell division in C. elegans. Single gene knockdowns were performed for each candidate in the golden heterozygous (pigment mutant) zebrafish background to test for genomic instability activity. Genomic instability activity was not observed, however the results showed that these genes are expressed throughout zebrafish embryogenesis, and are necessary for the proper development of the central nervous system, notochord and tail, as well as metabolic functions in the early embryo. Moreover, the transgenic line used for the paralog studies of synbl and ric8a was incorrectly genotyped. Using PCR analysis and sequencing, it was found that the viral insert for the Tg-synbl fish was disrupting the cry1b gene on an adjacent contig.

The second aim focused on the gin-12 region on chromosome 4, where the mdm1 gene is located. Originally cloned from a transformed mouse cell line with mdm2, the function of the mdm1 gene in these cells or during development had not yet been identified. To allow the Mdm1 protein to be evaluated, custom antibodies targeting Mdm1 were produced and the detection of Mdm1 optimized in zebrafish embryos. This would allow us to then determine whether Mdm1 was a possible regulator of the p53-Mdm2/Mdm4 pathway. Additionally, the mdm1 gene was studied in situ and in vivo to determine the normal gene expression patterns and developmental role in the embryonic zebrafish. Moreover, this gene was also studied in the golden heterozygous zebrafish line to assess whether it had a role in modulating genomic instability activity using the mosaic eye assay. Collectively, morpholino oligonucleotides, RNA rescue, whole-mount antibody staining, and overexpression studies suggest that the mdm1 gene is involved in the development of the eye and portions of the central nervous system, but did not appear to be the gin-12 mutant.

While the genes in this study did not appear to have genomic instability activity in the embryonic zebrafish based on the mosaic eye assay in the golden heterozygotes, normal developmental gene expression patterns were identified for synbl, ric8a, rfx4, sir2, and mdm1 in wildtype zebrafish embryos. Additional information was gained by the reverse genetic studies using gene knockdowns, which identified the functional roles of these genes at various stages of embryogenesis. Notably, it was determined that the mdm1 gene may be involved in retinal degenerative diseases based on our studies and recently published data. Future research of the Mdm1 protein should identify protein interactions and the specific role during eye development and retinal diseases.

Share

COinS