title: Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation.
abstract: In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksb is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation.
authors.affiliation: College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Yan-Wu
authors.initials: YW
authors.lastname: Wang
authors.forename: Chang-Yong
authors.initials: CY
authors.lastname: Wei
authors.forename: He-Ping
authors.initials: HP
authors.lastname: Dai
authors.forename: Zuo-Yan
authors.initials: ZY
authors.lastname: Zhu
authors.forename: Yong-Hua
authors.initials: YH
authors.lastname: Sun
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D061505:Cell Surface Display Techniques / Q000379:methods*; D004625:Embryo, Nonmammalian; D000939:Epitopes / Q000235:genetics; D054262:Gastrulation / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013623:Tail / Q000196:embryology; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000199:Actins; D000939:Epitopes; D047908:Intracellular Signaling Peptides and Proteins; C582072:Marcksb protein, zebrafish; D008565:Membrane Proteins; D057127:Single-Chain Antibodies; D029961:Zebrafish Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate