Mammalian liver fatty acid binding protein (L-FABP) is a small cytosolic protein in various tissues including liver, small intestine and kidney and is thought to play a crucial role in intracellular fatty acid tra⁄cking and metabolism. To better understand its tissue-speci¢c regulation during zebra¢sh hepatogenesis, we isolated 5P P-£anking sequences of the zebra¢sh L-FABP gene and used a green £uorescent protein (GFP) transgenic strategy to generate liver-speci¢c transgenic zebra¢sh. The 2.8-kb 5P P-£anking sequence of zebra¢sh L-FABP gene was su⁄cient to direct GFP expression in liver primordia, ¢rst observed in 2 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. F2 inheritance rates of 42^51% in all the seven transgenic lines were consistent with the ratio of Mendelian segregation. Further, hhex and zXbp-1 morphants displayed a visible liver defect, which suggests that it is possible to establish an in vivo system for screening genes required for liver development. ß

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Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of src in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and src transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential in vivo platforms for drug screening for therapies for human liver cancer.

Summary The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated and how energy sensor regulates Hh pathway is not clear. AMP-activated Protein Kinase (AMPK) is an important sensor of energy stores that controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This in turn leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.

MicroRNA (miRNA) are a class of small, single-stranded, non-coding RNA that regulate mRNA expression at the post-transcriptional level and play important roles in many fundamental biological processes. There is emerging evidence that miRNA are critical regulators of widespread cellular functions, such as differentiation, proliferation, and migration. At present, little is known about miRNA expression profiles related to skeletal muscle growth in aquatic organisms. This study aimed to investigate the phenotypic variation in the body growth of the Nile tilapia (Oreochromis niloticus) and to identify and quantify the differential expression levels of selected growth-related transcriptomic miRNA in the skeletal muscle of this fish. To this end, we performed next-generation sequencing to define the full miRNA transcriptome in muscle tissue from Nile tilapia and to detect differentially expressed miRNA between 2 strains of Nile tilapia. These tilapia strains exhibited significant (P < 0.05) phenotypic variation with respect to growth-related traits (body length and BW), mitochondrial DNA (mtDNA) haplotype diversity, and the differential expression of selected growth-related genes. The results obtained from the transcriptome analysis and real-time quantitative reverse transcription PCR (qRT-PCR) revealed significant differences in miRNA expression between fast-growing and control strains of tilapia. Digital gene expression (DGE) profiling was performed based on the obtained read abundance, and we identified down-regulated miRNA, including let-7j, miR-140, miR-192, miR-204, miR-218a, miR-218b, miR-301c, and miR-460, and up-regulated miRNA, including let-7b, let-7c, miR-133, miR-152, miR-15a, miR-193a, miR-30b, and miR-34, associated with body growth in tilapia. These results were further validated using real-time qRT-PCR and microarray profiling. In summary, the up- and down-regulation of miRNA involved in the GH/IGF-1 axis signaling pathway suggests that the differential expression levels of growth-related miRNA may serve as molecular markers that are predictive of specific functional and diagnostic implications. The obtained data on genetic polymorphisms in miRNA-target interactions are particularly useful for Nile tilapia breeding programs.

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