| Object: The infants experience the most rapid physical growth and development rate,so they need a lot of nutrients to ensure their normal growth and development.Breast milk is the main source of nutrients in this period and the lipid supply in human milk is critical for the process of growth and development.The Carboxyl ester lipase(Cel)is a lipolytic enzyme secreted by the pancreas and is capable of hydrolyzing cholesteryl esters,triglycerides,diglycerides,monoglycerides,phospholipids,and ceramides.Cel is also secreted by mammary gland during lactation and exists in breast milk.Previous studies have shown that breast milk-derived Cel facilitates dietary fat digestion and absorption,thus contributing to normal infant development.The cel gene and its protein structure are highly conserved in vertebrates,including zebrafish.Zebrafish,as a model organism,has its uniqueness in the study of developmental biology and lipid research.Nevertheless,Cel related research has not been carried out in zebrafish.This study aimed to examine whether Cel in zebrafish embryos has a similar role of maternal lipid utilization as in human infants,and how Cel contributes to the utilization of yolk lipids in zebrafish.Methods: Zebrafish embryos at different developmental stages were collected,reverse transcription-polymerase chain reaction(RT-PCR)and whole-mount in situ hybridization(WISH)were performed to detect the spatiotemporal expression of zebrafish cel1 and cel2 genes.Zebrafish embryos were then injected by morpholino(MO)to knockdown cel1 and cel2 genes,the phenotype of developmental retardation in control MO,cel1 morphants,cel2 morphants and cel(cel1/cel2)morphants were analyzed.The fat distribution of each group were also measured.Cholesterol,oleic acid,as well as ATP were microinjected into the cel morphants to further assess whether they can rescue the developmental retardation phenotype.BODIPY-CE was microinjected into the yolk sac of embryos and the ratio of cholesterol fluorescence in body to that in yolk were detected to evaluate the involvement of Cel in the utilization of cholesterol(in its ester form)stored in the zebrafish yolk.To explore the role of Cel in zebrafish central nervous system(CNS)development,the morphological analysis of head was performed and the expression pattern of shh and its target genes nkx2.2,ptch1 were detected by WISH in control embryos,cel morphants and cel MO +cholesterol embryos.Results: The cel1 and cel2 genes were expressed ubiquitously in the blastodisc and yolk syncytial layer before 24 hpf,and in the exocrine pancreas after 72 hpf.The cel1 and cel2 morphants exhibited developmental retardation and yolk sac retention.The total cholesterol,cholesterol ester,free cholesterol,and triglyceride were reduced in the morphants’ body while accumulated in the yolk(except triglyceride).The free fatty acid content of whole embryos was much lower in morphants than in standard controls.Moreover,the delayed development in cel(cel1/cel2)double morphants was partially rescued by oleic acid and cholesterol supplementation.Delayed and weakened cholesterol ester transport to the brain and eyes was observed in cel morphants.Correspondingly,shrunken midbrain tectum,microphthalmia,pigmentation-delayed eyes as well as down-regulated shh target genes,nkx2.2,ptch1 were observed in the CNS of double morphants.Interestingly,cholesterol injections reversed these CNS alterations.Conclusion: cel genes expressed in the early stage of zebrafish embryogenesis.They participate in the lipid releasing from yolk sac to developing body,thereby contributing to the normal growth rate and CNS development in zebrafish. |
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