Expression Characterization And Functional Analysis Of Two Regulatory Genes During Fish Embryogenesis

Two regulatory genes during fish embryogenesis, named Apo-14 and Afp4 respectively, were isolated from SMART cDNA library of gastrula-stage gibel carp embryos. The spatio-temporal expression patterns of each gene were explored in detail, and their roles during embryonic development were further investigated by morpholino knock-down approach.Apo-14 is a fish-specific apolipoprotein and its biological function remains unknown. In this study, CagApo-14 has been cloned from gibel carp (Carassius auratus gibelio) and its expression pattern has been investigated during embryogenesis and early larval development. CagApo-14 transcript and protein product are firstly expressed in yolk syncytial layer at a very high level during embryogenesis, and then restricted to the digestive system including liver and intestine in later embryos and early larvae. Immunofluorescence localization in larvae and adults indicates that CagApo-14 protein is predominantly expressed and excreted from the sinusoidal endothelial cells of liver tissue. Morpholino knockdown of CagApo-14 results in severe disruption of digestive organs including liver, intestine, pancreas, and swim bladder. And, the yolk lipid transportation and utilization have been demonstrated to be severely affected in the CagApo-14 morphants. The current study has indicated that CagApo-14 is required for digestive system organogenesis during fish embryogenesis and larval development.Antifreeze proteins (AFPs) had been found in cold-adapted organisms for over 30 years, but almost all of the past researches were only focused on the structural and biochemical properties in freezing avoidance, and little was known about the biological functions in general organisms, especially in embryogenesis. Here we identify two reduplicated homologues of polar fish type IV antifreeze proteins (Afp4) genes from gibel carp (Carassius auratus gibelio) and zebrafish, and reveal their biological functions as two key regulators in zebrafish gastrulation movement and yolk absorption. They express during embryogenesis in a"temporal succession"manner and spatially restricted in the yolk syncytial layer (YSL). The earlier transcribed gene, DrAfp4a, is first detected at 5hpf, peaks rapidly at 8hpf and attenuated thereafter, and absent in all the examined adult tissues. In contrast, DrAfp4b is transcribed later than DrAfp4a, peaks at 30hpf, persists on during the whole process of development, and is mainly synthesized in the liver and gut in adult zebrafish. Distinct developmental defects are observed when specifically knockdown each of these two duplicated genes by corresponding MO injection. Zebrafish embryos lacking DrAfp4a function displayed severe morphogenetic movement defects during gastrulation, resulting in unclosed yolk plug and numerous embryo deaths before tailbud formation. Blocking DrAfp4b expression did not perturb morphogenetic movement during gastrulation, but produced obvious embryonic defects during the later embryogenesis, such as obvious enlargement of yolk sac, severe reduction of yolk extension, and impairment of blood circulation. Co-injection of mRNA encoding DrAfp4a or DrAfp4b can restore the developmental defects caused by each corresponding MO, but fails to rescue the deficiency of each other, indicating that DrAfp4a and DrAfp4b underwent functional divergence after duplication. Cell tracing in the DrAfp4a-MO embryos clarifies that DrAfp4a contributes to convergence movement. ORO staining reveals that the transportation and utilization of yolk lipid have been severely impaired in DrAfp4b morphants. These data reveal a biological role for antifreeze proteins as two key regulators in regulating cell movements during zebrafish gastrulation and later embryogenesis.