Identification Of Genes Related To Early Development Of Zebrafish With Phage Display Technique

During the development of vertebrates, it is during gastrulation that the fundamental body plan is brought into existence-the blastoderm is initially fated into three germ layers with obvious dorsal-ventral and anterior-posterior polarities. In our previous study of fish cloning, whether a cloned embryo could pass gastrulation is considered as one critical index to evaluate the further development of it. To rapidly characterize the zebrafish gastrulation at proteomic level, phage display technique was engaged in the present study.1) With some modified subtractive screening approaches,6types of single chain antibodies were selected from a mouse scFv library. These antibodies showed higher binding affinity to the homogenates of zebrafish tailbud stage embryos, when compared to those of30%-epiboly embryos.2types of scFvs, scFvl and scFv2, which took the highest percentages among the6types of scFvs, were taken as target molecules for subsequently screening. As shown by whole mount immunostaining, the scFv-binding proteins mainly present in cell membrane and cytosol, but absent in the nucleus.2) A T7phage display cDNA library was constructed from zebrafish embryos at tailbud stage. The primary titer is1.72×107pfu/ml and the amplified library titer is5.16×1011pfu/ml. Clony PCR analysis showed that99.1%of the clones contained insert DNA, ranging from150bp to1500bp. Specifically,32%of the inserts showed a size of500bp or over,59%between250bp and500bp, and9%lower than250bp. All these indicate that the library is of high-quality and is usable for further screening.3) We directly isolated the binders of scFv1and scFv2from the above T7phage display cDNA library with anti Etag-scFv complexes and non-purified target scFvs alternatively. We obtained the binders of two types of scFvs and all cDNA inserts were sequenced. According to the analysis of amino acid sequences translated from cDNA sequences, we found that, of the8proteins binding to scFvl,2sequences did not show any hits in databases, other6sequences were identified as proteins with known functions, such as Casein kinase1, beta-Catenin, Chromatin Assembly Factor1Subunit A, Polyadenylate-binding protein2, Nucleolin, Ribosomal protein L10and Myristoylated alanine-rich C-kinase substrate (MARCKS). The binders of scFv2shared high identities with2types of ribosomal protein L35and L7a. According to the results derived from affinity of scFvs and theoretical physicochemical properties of scFv-binding sequences, we presumed that scFvs and its binders interacted each other mainly via complementary three-dimensional structures, in which the electrostatic bonds were involved. Whole-mount in situ hybridization (WISH) approach was utilized to analyze the expression pattern of all the identified cDNA clones. All of them showed a ubiquitous expression during gastrulation except clone scFvl-21.4) Since scFvl-21clone showed a territory-specific expression pattern during gastrulation and later stages, we started to perform a molecular and functional characterization of its corresponding gene. Its cDNA sequence encodes the zebrafish Marcks protein of227amino acids with a calculated molecular weight of23.2kDa. This gene locates on chromosome17and contains two exons spaced by an intron of820bp. Zebrafish Marcks has approximately37.3,37.2,41.3, and42.5%identical to the human, mouse, chick and Xenopus MARCKS proteins, respectively. Comparison of zebrafish Marcks with the previously published MARCKS sequences revealed three conserved domains:an N-terminal membrane-binding domain, an intron splice site, and a phosphorylation domain that contains calmodulin and actin binding sites. WISH analysis showed the specific expression of marcks gene during early developments, and its transcripts could be finding prominently in neural ectoderm at gastrula and the neural tube in later developmental stages, suggesting that marcks would participate in early development of zebrafish.5) Previous studies in other animals have shown that, MARCKS is implicated in the regulation of brain development, cellular migration, cellular adhesion, tumor suppression, and neurosecretion. In Xenopus, MARCKS-like proteins might participate in pattern formation of the embryonic axis and the central nervous system. In our experiments, the marcks overexpressed embryos showed so-called ventralized phenotype, such as enlarged blood islands and decreased head size, while the embryos injected with a morpholino oligo complementary to zebrafish marcks ATG site leaded to dorsalization. WISH analysis of several marker genes identified that the induced phenotypes were accompanied by altered expression of dorsal and ventral specific genes at the levels of mesoderm and ectoderm. Moreover, several canonical Wnt targets were down-regulated in marcks morphants or up-regulated in marcks overexpressed embryos, suggesting that marcks positively regulates Wnt/beta-Catenin transcription activity. Specifically, the marcks knockdown embryos showed loss of blood tissue, while overexpressed embryos showed massive enhancement of blood development. These indicate that zebrafish marcks can not only participate in axial development and gastrulation, but also blood development as a positive regulator of Wnt signal pathway.To our knowledge, this is the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates. Fortunately we found some proteins showing difference in tailbud stage and30%epiboly stage, which may participated in early development or organogenesis of zebrafish.