Novel Secretory Proteins Leg1a And Leg1b Are Essential For Normal Liver Development In Zebrafish

The process of liver organogenesis, from hepatic cell specification, liver bud initiation, bud expansion and hepatocyte differentiation, is governed by a network formed by a series of spatial and temporal morphogens and factors, including liver enriched genes. Although lots of works have been done by so far themolecular mechanisms of liver development are still poorly understood.Our lab previously reported the discovery of a novel liver-enriched gene leg1 (liver-enriched gene 1). Morpholino-mediated knock-down of Leg1 expression resulted in morphants displaying small liver phonotype. To further investigate the function of leg1 in zebrafish liver development, we analyzed its genomic structure and chromosomal location, its spatial and temporal expression patterns, its function(s) during zebrafish embryogenesis, as well as the underlying molecular mechanisms of the small liver phenotype caused by Leg1 expression knock-down.We found that leg1 is a duplicated gene in zebrafish. leg1a and leg1b, the two duplicated copies, are adjacent to each other on linkage group 20 with a gap of-6 kb. leg1a and leg1b share 95.2% identity in their coding sequence and Leg1a and Leg1b peptides share 90.6% homology. Leg1 represents a novel protein family characterized by consisting of an N-terminal signal peptide and a DUF781 (Domain of Unknown Function) domain. Expression analysis revealed that leglais the prominent form with more than 90% in embryonic stage and 60% in adult fish of total leg1 expression. We also provided both bioinformatic and experimental data to demonstrate that Leg1 is a novel secreted protein synthesized by the liver and is exported into the blood stream after biosynthesis. Based on their capability to rescue the morphant small liver phenotype, we found that both Leg1a and Leg1b are essential for early liver development and meanwhile are functionally partially redundant. Whole mount insitu hybridization (WISH) analysis with a variety of molecular markers showed that knockdown of Leg1 expression inhibits liver expansion but not the liver bud initiation. Knockdown of Leg1 expression also affects the development of the gut and exocrine pancreas. Overexpression of N-terminal signal peptide truncated Leg1 failed to rescue the small liver phenotype conferred by MOATG morphants, suggesting that a functional Leg1 relies on its secretion. In order to investigate the cellular mechanism of small liver caused by Leg1 knock-down, we carried out PH3 immunostaining and TUNEL assay in MOATG morphants and found that the small liver was caused by cell cycle arrest but not cell apoptosis during liver bud expansion stage.In addition.two independent leglamutant lines. e11 and M1 were obtained from an ENU mutagenesis population by TILLING approach. This lays the foundation for further study of Leg1's physiological function as a secretory protein in adult zebrafish. We also carried out a pioneering work of co-immunoprecipitation with the purpose to identify Leg1 interacting protein. In conclusion, the vast amount of data presented in this Thesis strongly indicate that Leg1 likely acts as a novel type of extracellular signal factor that plays an important role in organogenesis and development of digestive organs in zebrafish. No doubt, further investigations are needed to solidify this hypothesis and more efforts need to be spent to unravel the molecular mechanism of Leg1 function in liver development.