Functional Study Of The Role Of Sec13in Organ Ogenesis During Zebrafish Embryogenesis

As a model system which is receiving increasing attentions, zebrafish is playing a growing role in life sciences research. Early embryogenesis of zebrafish is a complex processes controlled by a large number of genes and different signaling pathways. Any defects in these processes can lead to embryonic abnormalities.In this study, we utilized genetic, cell biology and molecular biology approaches to investigate the role of sec13gene during zebrafish early embryogenesis. Through ENU-mediated mutagenesis, we obtained a zebrafish sec13mutant, sec13sq198. Our previous study showed that there is a T to A transversion in the7th intron of the sec13gene. This point mutation creates a new splicing receptor site which leads to the addition of extra eight nucleotides into the final mRNA. This mutant mRNA is predicted to encode a polypeptide with85amino acids truncated from the carboxyl-terminus of WT Secl3and the addition of34new amino acids due to frame shift in the new open reading frame. Furthermore, Our previous work also demonstrated that carboxyl-terminus-truncated Sec13loses its affinity to Sec31a. So based on these data, we decide to comprehensively investigate the role of sec13during zebrafish early embryogenesis.Sec13is a dual functional protein in eukaryotes. First, it interacts with Sec31a to form the outer coat of the COPII complex, facilitating protein transport form ER to Golgi apparatus. Second, it also interacts with (Nup145C in yeast and Nup96in vertebrates) to form NPCs to facilitate nucleo-cytoplasmic transport and regulate gene expression. Our first part efforts mainly focused on the role of sec13during zebrafish digestive organs organogenesis, namely, the intestine, liver and pancreas. We found that the carboxyl-terminus-truncated Sec13in secl3sq198loses its affinity to Sec31a, that leads to the dysfucntuion of the COPII complex. As a consequence, protein transport from ER to golgi apparatus was blocked, giving rise to the accumulation of secretory protein in the ER of chondrocytes, liver and intestinal cells. Unfolded protein response (UPR) was activated due to the sudden build up of protein mass in ER and consequently, the stressed cells underwent cell-cycle arrest and cell apoptosis, which halt the growth of developing digestive organs.Our second part is about nuclear pore function of sec13during retinal early development. In this respect, we found that, despite that all types of the retinal cells were specified, sec13sq198mutant embryos developed small eyes characterized by gradual loss of the photoreceptor cell layer (PCL) and reduction of cell numbers in other cell layers. Surprisingly, blocking COPII function either by double-knockdown of Sec31a and Sec31b or by drug treatment (Brefeldin A), though produced small eyes, did not disrupt the layered structure of the eye, suggesting that the retina lesion observed in sec13sq198was not totally due to COPII dysfunction. Our further data from immunostaining and transmission electron microscopy (TEM) analysis demonstrated that the retina of sec13sq198failed to form proper nuclear pores which led to the nuclear accumulation of total mRNA. TUNEL assay revealed that the p53-dependent apoptosis pathway was abnormally activated which finally caused the small eye phenotype by triggering cell apoptosis in sec13sq198.Our overall data not only demonstrated the importantce of Sec13in organogenesis of digestive organs and eyes but also provided the first genetic evidence to demonstrate that the two functions of Sec13, namely as a key component of COPII and NPCs can be differentially uncoupled during the process of organogenesis in a multicellular organism.