Sorting nexins (SNXs) are phosphoinositide-binding proteins implicated in the sorting of various membrane proteins in vitro, but the in vivo functions of them remain largely unknown. We reported previously that SNX10 is a unique member of the SNX family genes in that it has vacuolation activity in cells. We investigate the biological function of SNX10 by loss-of-function assay in this study and demonstrate that SNX10 is required for the formation of primary cilia in cultured cells. In zebrafish, SNX10 is involved in ciliogenesis in the Kupffer's vesicle and essential for left-right patterning of visceral organs. Mechanistically, SNX10 interacts with V-ATPase complex and targets it to the centrosome where ciliogenesis is initiated. Like SNX10, V-ATPase regulates ciliogenesis in vitro and in vivo and does so synergistically with SNX10. We further discover that SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a, which is a critical regulator of ciliary membrane extension. These results identify an SNX10/V-ATPaseregulated vesicular trafficking pathway that is crucial for ciliogenesis, and reveal that SNX10/V-ATPase, through the regulation of cilia formation in various organs, play an essential role during early embryonic development.

The class III PI3-kinase (PIK3C3) is an enzyme responsible for the generation of phosphatidylinositol 3-phosphate (PI3P), a critical component of vesicular membrane. Here, we report that PIK3C3 deficiency in zebrafish results in intestinal injury and inflammation. In pik3c3 mutants, gut tube forms but fails to be maintained. Gene expression analysis reveals that barrier-function-related inflammatory bowel disease (IBD) susceptibility genes (e-cadherin, hnf4a, ttc7a) are suppressed, while inflammatory response genes are stimulated in the mutants. Histological analysis shows neutrophil infiltration into mutant intestinal epithelium and the clearance of gut microbiota. Yet, gut microorganisms appear dispensable as mutants cultured under germ-free condition have similar intestinal defects. Mechanistically, we show that PIK3C3 deficiency suppresses the formation of PI3P and disrupts the polarized distribution of cell-junction proteins in intestinal epithelial cells. These results not only reveal a role of PIK3C3 in gut homeostasis, but also provide a zebrafish IBD model.

Many organisms in extremely cold environments such as the Antarctic Pole have evolved antifreeze molecules to prevent ice formation. There are four types of antifreeze proteins (AFPs). Type-IV antifreeze proteins (AFP4s) are present also in certain temperate and even tropical fish, which has raised a question as to whether these AFP4s have important functions in addition to antifreeze activity. Here we report the identification and functional analyses of AFP4s in cyprinid fish. Two genes, namely afp4a and afp4b coding for AFP4s, were identified in gibel carp (Carassius auratus gibelio) and zebrafish (Danio rerio). In both species, afp4a and afp4b display a head-to-tail tandem arrangement and share a common 4-exonic gene structure. In zebrafish, both afp4a and afp4b were found to express specifically in the yolk syncytial layer (YSL). Interestingly, afp4a expression continues in YSL and digestive system from early embryos to adults, whereas afp4b expression is restricted to embryogenesis. Importantly, we have shown by using afp4a-specific and afp4b-specifc morpholino knockdown and cell lineage tracing approaches that AFP4a participates in epiboly progression by stabilizing yolk cytoplasmic layer microtubules, and AFP4b is primarily related to convergence movement. Therefore, both AFP4 proteins are essential for gastrulation of zebrafish embryos. Our current results provide first evidence that AFP such as AFP4 has important roles in regulating developmental processes besides its well-known function as antifreeze factors.

Apo-14 is a fish-specific apolipoprotein and its biological function remains unknown. In this study, CagApo-14 was cloned from gibel carp (Carassius auratus gibelio) and its expression pattern was investigated during embryogenesis and early larval development. The CagApo-14 transcript and its protein product were firstly localized in the yolk syncytial layer at a high level during embryogenesis, and then found to be restricted to the digestive system including liver and intestine in later embryos and early larvae. Immunofluorescence staining in larvae and adults indicated that CagApo-14 protein was predominantly synthesized in and excreted from sinusoidal endothelial cells of liver tissue. Morpholino knockdown of CagApo-14 resulted in severe disruption of digestive organs including liver, intestine, pancreas and swim bladder. Moreover, yolk lipid transportation and utilization were severely affected in the CagApo-14 morphants. Overall, this data indicates that CagApo-14 is required for digestive system organogenesis during fish embryogenesis and larval development.