The Functional Study Of Becn1 In Zebrafish

Autophagy is an important intracellular degradation pathway. Double-membrane vesicles, autophagosomes, are formed in cells to sequester organelles, proteins and portions of cytoplasm to deliver to lysosomes and fuse with them to form autophagolysosomes. The sequestered contents are degraded in lysosomes, for producing amino acids and other biological molecules for recycling, to maintain organelles renewal and cell homeostasis. Autophagy plays important roles in the response to cell starvation, regulation of cell quality and energy homeostasis, suppression of tumorgenesis, antigen presentation and immune defense. In recent years, studies of systemic and tissue-specific knockout of ATG genes in mice have showed that autophagy is essential for preimplantation development, embryogenesis and newborn survival. Autophagy has also been proven to play important roles on the regulation of physiological metabolism in the liver, and in many liver diseases including fatty liver and hepatocarcinogenesis.Beclin1, also known as BECN1, is the yeast ATG6 homologue in mammals. It locates on human chromosome 17q21 with 12 exons, and is ubiquitously expressed in normal human tissues. It’s an essential gene for mammalian autophagy. The protein structure and functions of Beclin1 are highly conserved in a variety of species, and Beclin1 contains a Bcl-2 homology domain(BH3), a coiled-coil domain(CCD), an evolutionarily conserved domain(ECD) and a nuclear export signal(NES).These domains allow Beclin1 to recruit other autophagy-related proteins to locate in autophagic vacuoles membrane, and form a multi-protein complex to regulate the formation and maturation of autophagosomes in mammals.As a vertebrate model, zebrafish(Danio rerio) has some advantages for the study of organ development and regeneration, such as small size, easy to raise, external fertilization, rapid embryonic development, transparent embryos, and great regeneration capabilities. The homologs of many autophagy related genes, such as atg1, atg3, atg4, atg5, becn1, atg7, atg9, ambra1, atg12 atg14 and atg16 are found in zebrafish genome, and the protein structures are highly similar to their human counterparts. Owning to the above advantages, zebrafish is a useful model for studying the function and mechanism of autophagy. In recent years, some studies used MOs to knock-down ATGs in zebrafish embryos to investigate the function of autophagy in organ development and regeneration, as well as to explore the function in the defense of virus infection, innate immunity and acquired immunity. Since 2012, the TALEN and CRISPR/Cas9 technologies have been successfully applied in zebrafish, which is of a great use of gene knockout in this model organism. It also provides a powerful tool for the study of autophagy in zebrafish.In this work, we used CRISPR/Cas9 system to knockout becn1 in zebrafish, an important regulator of autophagy, to construct genetic mutants, and the mutant was used to explore the role of becn1 gene and autophagy in embryonic development and organogenesis, especially, liver development and functions in zebrafish.The preliminary results showed that we successfully knocked-out the autophagy related gene becn1 in zebrafish by CRISPR/Cas9 and obtained becn1 stable genetic mutants. Compared with wild-type fish, the becn1 mutant fish showed very normal from fertilization to 6dpf, indicating that becn1 may not be necessary for embryonic development in zebrafish. Nonetheless, subtle changes of becn1 during zebrafish embryonic development could not be ruled out, such as the differentiation and physiological environment of specific cells. At 7~8dpf, we found that the liver of becn1 mutant fish was significantly larger than that of wild-type fish, and it’s about 1.5 to 2 times bigger in size than wild-type liver. And vigorous proliferation of hepatocyte were occurred in mutant fish liver indicated by pH3 immnostaining. We also found P62, ubiquitinated proteins and protein aggregates were accumulated in the mutant liver. And the Cox4i1 protein also significantly increased indicating mitochondria were accumulated in cells of becn1 mutant. These results suggested that becn1 mutant had autophagy deficiency. Furthermore, ER stress related proteins such as, Chop, Bip, p-Eif2 were also significantly increased in becn1 mutant suggesting that the ER stress were induced. At 9~10dpf becn1 mutant fish died. Two ATG mutants, atg7 and atg5, were successfully constructed in zebrafish using CRISPR/Cas9 technology. Preliminary results showed that the embryonic development of these mutants were normal, but both of them died during 13 dpf to 25 dpf. The death reason of these mutants and phenotypes of organs before their death were to be addressed in the future.In this study, we successfully constructed three stable genetic mutants of autophagy related genes becn1, atg5 and atg7 in zebrafish. Due to the external fertilization and rapid embryonic development features of zebrafish, these mutants avoid the embryonic lethality or early death after birth which occur in mammals with systemic knockout of these genes. It provides good materials for further study of autophagy functions in development, maintenance of cell differentiation and cell physiological state, and the effect on tumorigenesis.