Date of Degree
PhD (Doctor of Philosophy)
Val C. Sheffield
Diane C. Slusarski
Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder characterized by obesity, retinitis pigmentosa, polydactyly, mental retardation and hypogonadism. To date, twelve BBS genes have been identified, however, the precise functions of BBS genes are yet to be unraveled. To unravel the pathophysiology of BBS, we developed the zebrafish BBS model.
In the zebrafish animal model system, we performed loss-of-function analysis using antisense morpholino oligonucleotides (MO). We observed strikingly overlapping phenotypes in the MO-injected embryos (morphants) including: Kupffer's vesicle (KV) disruption, a premature loss of KV cilia leading to alterations in left-right asymmetry and altered intracellular trafficking of organelles (melanosome). These phenotypes can be rescued by co-injection of in vitro synthesized wild-type RNA demonstrating the specificity of MO knockdown. In contrast to other bbs gene knockdowns, bbs1 knockdown resulted in an additional phenotype: pericardium effusion, which was rescued by co-injection with the bbs1 wild-type RNA and was significantly suppressed by the M390R mutant RNA. In addition, immunohistochemistry revealed that bbs1 wild-type protein localizes to the centrosome and cytoplasm, while bbs1 M390R mutant protein only has cytoplasmic localization. The BBS1 M390R missense mutation is the most common mutation in all BBS cases and the methionine at amino acid position 390 is evolutionarily conserved. Our data suggest that the BBS1 M390R allele is a hypomorphic allele and that the residual function of the mutant protein is necessary for embryonic development.
Furthermore, we demonstrated the utility of the zebrafish BBS model in BBS candidate gene identification. Data from linkage analysis, expression profile correlation and mutation screen, suggested that TRIM32 is a BBS gene. Therefore, we identified the zebrafish trim32 gene and performed loss-of-function analysis. MO knockdown of zebrafish trim32 gene expression phenocopied the prototypic zebrafish BBS phenotypes: KV formation defect and a delay in intracellular transport. In addition, these phenotypes were rescued by co-injection of wild-type trim32 and LGMD2H mutant (D487N) RNA, but the RNA with the BBS-associated mutation (P130S) failed to rescue these phenotypes. The functional evidence, combined with data from linkage analysis, expression correlation and mutation screening demonstrate that TRIM32 is a BBS gene (BBS11).
xv, 151 pages
Includes bibliographical references (pages 139-151).
Copyright 2007 Hsan-jan Yen