Unraveling the etiology of swim bladder defects observed in folate-deficient zebrafish embryos

Abstract

Folate a water-soluble vitamin is essential for the biosynthesis of nucleotides amino acid and S-adenosylmethionine the major methyl-donor for most methylation reactions in cells Folate deficiency (FD) causes embryonic defects and diseases including neural tube defect megaloblastic anemia cardiovascular disorders and cancers Dietary folate was reported to protect individuals against lung carcinogenesis However study on both physiological and pathological effects of folate in lung is still limited In the current study we investigate the effects of folate deficiency on the development of swimbladder the evolutionary homolog of mammalian lung in zebrafish using a transgenic line displaying inducible folate deficiency We found that the swimbladder was absent in approximately 50% of FD embryos at 5 day-post-fertilization (dpf) which was partially reversed by folate supplementation The results of Whole-mount in situ hybridization (WISH) with probes specific to developing swimbladder indicated a successful formation of swimbladder in FD embryos at early stages suggesting a post-formation defect The microarray data obtained from 5-dpf FD embryos revealed an increased expression of cathepsin L a lysosomal enzyme with strong elastinolytic activity and a down-regulation of cystatin b the inhibitor of cathepsin L in vivo The up-regulation of cathepsin L was also reversed by folate supplement supporting the causal link among folate deficiency up-regulated cathepsin L and disappeared swimbladder The distribution of cathepsin L mRNA examined by WISH was focused in embryonic hatch gland and swimbladder; whereas the cystatin b transcript was evenly distributed in zebrafish embryos before 24 hour-postfertilization and became focused in the heart and swimbladder of embryos after 1 dpf The recombinant cystatin b was purified and shown to be structurally and functionally similar to its mammalian orthologs The recombinant cathepsin L was successfully induced and undergoing purification Finally we used microinject technique to send the recombinant CytB into the 3 dpf swimbladder chamber And successfully partially rescue the swimbladder defect In conclusion our results suggested that the up-regulated cathepsin L and down-regulated cystatin b contributed to the post-formation defects of zebrafish swimbladder observed in the FD embryos

Date of Award	2014 Sept 10
Original language	English
Supervisor	Tzu-Fun Fu (Supervisor)