Differential Transcriptional Modulation of Duplicated Fatty Acid-Binding Protein Genes by Dietary Fatty Acids in Zebrafish (Danio reno)

In the Duplication-Degeneration-Complementation (DDC) model, subfuctionalization and neofunctionalization have been proposed as major processes driving the retention of duplicated genes in the genome. These processes are thought to occur by gain or loss of regulatory elements in the promoters of duplicated genes. Many duplicated genes exist in teleost fishes as a result of a whole-genome duplication event that occurred early in the ray-finned fish lineage 250-400 million years ago. To test the DDC model, I chose to determine whether duplicated fatty acid-binding protein (fabp) genes are retained in the zebrafish genome owing to either subfunctionalization or neofunctionalization. I first determined the spatio-temporal distribution of transcripts for the fabp1b.2 and fabp11b genes by two methods: (i) in situ hybridization of riboprobes to embryos, larvae and sections of adult zebrafish, and (ii) reverse-transcription, polymerase chain reaction (RT-PCR) using RNA extracted from adult tissues. The results of these initial studies showed that the spatio-temporal distribution of duplicated copies for the fabp1 and fabp11 genes have markedly diverged. Furthermore, comparison of the spatio-temporal distribution of the duplicated copies of the zebra fish fabp1 and fabp11 genes with that of their single-copy mammalian orthologs indicated that the zebrafish fabp duplicates had been retained in the zebrafish genome owing to either subfunctionalization or neofuctionalization. In a second experimental approach to test the DDC model, transcriptional induction of duplicated zebrafish fabp genes by fatty acids (FAs), compounds known to induce the transcription of several mammalian fabp genes, was assayed by quantitative RT-PCR. Adult zebrafish fed four diets differing in FA content exhibited different FA profiles in intestine, brain, muscle and liver depending on diet. Following quantitative RT-PCR, steady-state levels of fabp7b transcripts were induced in brain by a diet rich in linolate; fabp1b.1and fabp7b transcripts in intestine were elevated by a diet rich in linolenate; fabp7ba transcripts in liver were elevated in fish fed a low lipid diet; and fabp7 and fabp11a transcripts were elevated in muscle by diets that were either enriched in linolate, or contained low lipid content. None of the sister duplicates of these fabp genes exhibited an increase in the steady-state transcript levels in tissues of fish fed one of the four diets. Moreover, the level of heterogeneous nuclear RNA for a given fabp gene correlated with the induction of the steady-state level of mRNA transcripts indicating that up-regulation of fabp transcripts occurred at the site of transcriptional initiation. The differential transcriptional induction of duplicated zebrafish fabp genes by dietary FAs provides further evidence to support the DDC model for retention of duplicated genes in the zebrafish genome by either subfunctionalization or neofunctionalization.