Role of Fyn kinase in signaling associated with epiboly during zebrafish development

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.07.018 Byline: Dipika Sharma, Lesya Holets, Xiaoming Zhang, William H. Kinsey Keywords: Zebrafish; Development; Epiboly; Fyn Kinase; Calcium Abbreviations: BSA, bovine serum albumin; CHAPS, 3[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate; CSK, src specific kinase; DAPI, 4'6-diamidino-2phenylindole dihydrochloride; DEPC, diethyl pyrocarbonate; DIC, differential interference contrast; EDTA, ethylenediaminetetraacetic acid; EVL, enveloping cell layer; FGF, fibroblast growth factor; HEPES, [2-hydroxyethyl]piperazine-N'-[2-ethane-sulfonic acid]; IP3, Ins 1,4,5 P.sub.3; PAGE, polyacrylamide gel electrophoresis; PTK, protein tyrosine kinase; SDS, sodium dodecyl sulfate; SH2 and SH3, Src homology domain 2 and 3, respectively; PDGF, platelet-derived growth factor; PBT, (PBS + 0.1% Tween 20); YSL, yolk syncitial layer Abstract: The function of Fyn kinase during zebrafish development through the blastula stage was investigated through the use of dominant-negative constructs designed to suppress the function of zebrafish c-Fyn. Microinjection of SH2 domain-containing fusion protein or mRNA encoding the mutated, catalytically inactive Fyn at 45 min post-insemination had no significant effect during cleavage and did not inhibit formation of the yolk syncitial layer. Smoothing of the enveloping cell layer at the midblastula transition occurred normally and expression of bon/mixer and mezzo, zygotic transcription factors indicated that activation of the zygotic genome did occur. Signaling pathways involved with axis determination such as [beta]-catenin, activin, and nodal appeared to function normally as evidenced by expression of boz, goosecoid, and mezzo. However, while formation of the yolk syncitial layer was normal, the marginal blastomeres failed to migrate toward the vegetal pole and epiboly did not occur, a phenotype similar but distinct from that resulting from suppression of c-Yes kinase. The block to development was prevented by co-injection of c-Fyn mRNA with the dominant-negative construct indicating that it was a specific effect. Injection of the dominant-negative mRNA into individual blastomeres indicated that the effect was exerted on the intrinsic ability of the individual blastomeres to respond to signals directing epiboly and not on the signals themselves. Analysis of the pattern of calcium signaling in experimental and control embryos demonstrated that the elevated [Ca.sup.2+].sub.i characteristic of the marginal blastomeres was suppressed. Together, these observations indicate that Fyn kinase plays an important role in epiboly, possibly through its effects in calcium signaling. Author Affiliation: Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160, USA Article History: Received 3 March 2005; Revised 8 July 2005; Accepted 18 July 2005