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 have attracted a lot of attention due to their significant roles in chilling tolerance, but little is known about their functions during embryonic development. Here we identify two reduplicated homologues of polar fish type IV antifreeze proteins (Afp4) genes from gibel carp and zebrafish, and reveal their biological functions as two key regulators in zebrafish gastrulation movement and yolk absorption. They are expressed during embryogenesis in a “temporal succession” manner and spatially restricted in the yolk syncytial layer. 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. 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 the two duplicated genes by corresponding MO injection. Zebrafish embryos lacking DrAfp4a function display severe defects in morphogenetic movements during gastrulation, and cell tracing experiment clarifies that DrAfp4a contributes to convergence movement. By contrast, blocking DrAfp4b expression does not perturb morphogenetic movements during gastrulation, but produces embryonic defects during later embryogenesis. As revealed by Oil Red O staining, in DrAfp4b morphants, the yolk lipid utilization and transportation are impaired. 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 have undergone functional divergence after duplication.