Research On The Expression And Function Of Three Maternal Factors In Zebrafish

Primordial germ cells (PGCs) are the progenitors of reproductive cells in metazoans and are an important model for the study of cell migration in vivo. Previous reports have suggested that the protein Nanos plays critical roles in the migration and survival of PGCs in vertebrates and invertebrates. The molecular mechanisms by which Nanos governs PGCs migration are largely unknown. In this study, we report a novel interaction between zebrafish Nanos and myosin light chainâ…¡(Mylz2). This stable complex, which is formed through the RNA-binding zinc finger domain and fourteen amino acids at the C-terminus of Nanos, was confirmed both in vitro and in vivo. Both of these two parts are required for the interaction with Mylz2, but nity-one amio acids at N-terminus of Nanos are not required. Using a phospho-myosin light chain 2 (Serl9) antibody, we demonstrate that Nanos regulate the phosphorylation of the Mylz2. We discuss the biological roles of this Nanos-regulated phosphorylation of the Mylz2 in PGCs and soma. Based on these data, we suggest that the interaction between Nanos and Mylz2 and the Nanos regulated phosphorylation of the Mylz2 may play an essential role in germ cell development and PGC migration in zebrafish.NOS1 interacting with Pumilio2 has been found in drosophila and human. Using the zebrafish EST database from NCBI, we found an EST which has more homology to pumilio2. The full-length cDNA of this transcript was obtained by 3'and 5' RACE and further confirmed by PCR and sequencing, and it was named pum-like. The full-length cDNA of this gene is 3861bp and encodes a protein of 1106 amino acids, which locating at the twenty-fifth chromosome and containing twenty-one extrons and twenty introns. It shows much more homology to pumilio2 than pumiliol by phylogenetic construction, and contains five PUF repeats at the C-terminal. RT-PCR and western blot analysis showed that pum-like's mRNA transcribed at the early stage of oocyte, and its protein located in the cytoplasm of oocyte which may be a component of the yolk. Whole-mount in situ hybridization at the 2-cell and 4-cell stage of embryos revealed the remarkable signals in the cleavage furrows which formed distinct dots. It suggested that the gene regulated the cell divisions at the early stage of embryo. Along with the development of the embryo, its expression was found mainly in caudal spinal chord and brain. So we guessed it had some functions in the nervous system.Egg envelope is a specialized extracellular matrix that surrounds and protects the oocyte and plays significant roles in animal reproductive and developmental processes. Using the NCBI digital differential display program we identified an EST sequence (XM₀01340234.1) acquired from zebrafish ovary cDNA libraries in GeneBank. The full-length cDNA of this transcript was obtained by 3'and 5' RACE and further confirmed by PCR and sequencing. The full-length cDNA of the novel gene is 2720bp and encodes a protein of 761 amino acids. RT-PCR and western blot analysis showed its expression in ovary and brain but not in other tissues. In situ hybridization demonstrates that the mRNA is transcribed in ooplasm of stage I, II and III oocytes. Interestingly, immunohistochemistry on zebrafish ovarian sections showed that protein expression in the vitelline envelope was located to two thin positive lines in the stage III oocytes. These ovarian expression patterns show that this is a new component of the vitelline envelope that is synthesized during early developing oocytes. This protein was named ZVEP (zebrafish vitelline envelope protein) and it did not have any homology with other known vitelline envelope genes. Thus, we found that zvep is a novel gene related to the vitelline envelope in zebrafish.