ABSTRACTS: Oral Presentation Abstracts
Introduction: Mice deficient in the transcription factor hnf-6 fail to develop normal bile ducts. We are using the zebrafish as a model organism to study biliary development. Hnf-6 has not previously been identified in zebrafish, and thus its role in zebrafish biliary development is unknown. Conservation of the mechanism of biliary development between mammals and teleosts will legitimize further studies of biliary development in zebrafish involving hnf-6 and other genes.
Methods: Hnf-6 was cloned from adult zebrafish liver and sequenced. Expression was determined by in situ hybridization of developing embryos and larvae. A morpholino antisense oligo-nucleotide to hnf-6 was injected into oocytes and the morphant larvae were stained for biliary-specific cytokeratin. RNA (hnf-6 and vhnf1) was injected similarly and the larvae were also stained for bile ducts. Real-time quantitative PCR was performed on cDNA derived from morphants.
Results: Sequence analysis shows that zebrafish hnf-6 is highly homologous to mammalian hnf-6. Closely related alternatively spliced transcripts are generated in zebrafish and rodents. Zebrafish hnf-6 mRNA is expressed in the developing retina and neural tube prior to the initiation of liver development; later it becomes limited to the developing liver, pancreas, and proximal intestine. Zebrafish injected with an antisense morpholino against hnf-6 have a characteristic appearance. Bile ducts in these larvae are abnormal. Overexpression of hnf6 RNA also produces abnormal bile ducts. Overexpression of a downstream target of hnf-6, vhnf1, produces bile ducts with a similar appearance. When vhnf1 RNA is coexpressed in larvae also injected with the hnf-6 morpholino, the bile ducts appear normal. Studies of gene expression by quantitative PCR in hnf-6 morphants demonstrate a decrease in expression of vhnf1 as well as other liver transcription factors.
Conclusion: We have established that the sequence and general function of hnf-6 is conserved between mammals and teleosts. We have extended the understanding of the role of hnf-6 and vhnf1 in biliary development by demonstrating abnormalities of bile duct formation when these factors are overexpressed, and that vhnf1 is sufficient to rescue decreased expression of hnf-6. Further experiments in this system should provide greater understanding of the development of the vertebrate biliary system, and may provide insight into human biliary diseases.