Search

The importance of the aquaculture production is increasing with the declining global fish stocks, but early sexual maturation in several farmed species reduces muscle growth and quality, and escapees could have a negative impact on wild populations. A possible solution to these problems is the production of sterile fish by ablation of the embryonic primordial germ cells (PGCs), a technique developed in zebrafish. Cell-specific regulation of mRNA stability is crucial for proper specification of the germ cell lineage and commonly involves microRNA (miRNA)-mediated degradation of targeted mRNAs in somatic cells. This study reports on the functional roles of conserved motifs in the 3' untranslated region (UTR) of the miRNA target gene nanos3 identified in Atlantic cod, Atlantic salmon, and zebrafish. The 3'UTR of cod nanos3 was sufficient for targeting the expression of green fluorescent protein (GFP) to the presumptive PGCs in injected embryos of the three phylogenetically distant species. 3'UTR elements of importance for PGC-specific expression were further examined by fusing truncated 3'UTR variants of cod nanos3 to GFP followed by injections in zebrafish embryos. The expression patterns of the GFP constructs in PGCs and somatic cells suggested that the proximal U-rich region is responsible for the PGC-specific stabilization of the endogenous nanos3 mRNA. Morpholino-mediated downregulation of the RNA-binding protein Dead end (DnD), a PGC-specific inhibitor of miRNA action, abolished the fluorescence of the PGCs in cod and zebrafish embryos, suggesting a conserved DnD-dependent mechanism for germ cell survival and migration.

The RNA binding protein Dead end (DnD) is essential for maintaining viable germ cells in vertebrates and silencing of the gene has been demonstrated to cause sterility in several mammalian and fish species. Here we investigated transcriptome changes in hatched larvae of Atlantic cod induced by DnD knockdown using morpholino oligonucleotides (MO) injected in two-cell embryos. Whereas no fluorescently labeled germ cells were shown in embryos coinjected with dnd MO and nanos3 3'UTR coupled to green fluorescent protein, DnD knockdown had no visible effect on the number and location of Vasa protein positive cells in larvae. However, quantitative real-time RT-PCR (qPCR) revealed decreased vasa, nanos3 and tudor domain containing protein 7 mRNA expression and genome-wide oligonucleotide microarray analyses indicated profound suppression of genes involved in development and regulation of the reproductive system. DnD morphants showed lowered expression of genes encoding proteins involved in lipid, retinoid, cholesterol and steroid metabolism, including those with roles in sex hormone metabolism. Biotransformation of lipophilic compounds appeared suppressed too, as evidenced by down-regulation of several key genes from the phases 1 and 2 detoxification pathways. Effects of DnD silencing were highly pleiotropic and consisted of endocrine and metabolic changes, massive induction of histones and suppression of diverse developmental processes, including erythropoiesis and formation of extracellular matrix. While transient inhibition of dnd mRNA translation did not block development of primordial germ cells until hatch, results suggested that ablation of DnD might have major indirect consequences, including suppression of reproductive functions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The primordial germ cells (PGCs) giving rise to gametes are determined by two different mechanisms in vertebrates. While the germ cell fate in mammals and salamanders is induced by zygotic signals, maternally delivered germ cell determinants specify the PGCs in birds, frogs and teleost fish. Assembly of the germ plasm in the oocyte is organized by the single Buc in zebrafish, named Velo1 in Xenopus, and by Oskar in Drosophila. Secondary loss of oskar in several insect lineages coincides with changes in germline determination strategies, while the presence of buc in mammals suggests functions not associated with germline formation. Results: To clarify the evolutionary history of buc we searched for the gene in genomes available from various chordates. No buc sequence was found in lamprey and chordate invertebrates, while the gene was identified in a conserved syntenic region in elephant shark, spotted gar, teleosts, Comoran coelacanth and most tetrapods. Rodents have probably lost the buc gene, while a premature translation stop was found in primates and in Mexican axolotl lacking germ plasm. In contrast, several buc and buc-like (bucL) paralogs were identified in the teleosts examined, including zebrafish, and the tetraploid genome of Atlantic salmon harbors seven buc and bucL genes. Maternal salmon buc1a, buc2a and buc2b mRNAs were abundant in unfertilized eggs together with dnd and vasa mRNAs. Immunostained salmon Buc1a was restricted to cleavage furrows in 4-cell stage embryos similar to a fluorescent zebrafish Buc construct injected in salmon embryos. Salmon Buc1a and Buc2a localized together with DnD, Vasa and Dazl within the Balbiani body of early oocytes. Conclusions: Buc probably originated more than 400 million years ago and might have played an ancestral role in assembling germ plasm. Functional redundancy or subfunctionalization of salmon Buc paralogs in germline formation is suggested by the maternally inherited mRNAs of three salmon buc genes, the localized Buc1a in the cleavage furrows and the distribution of Buc1a and Buc2a in the Balbiani body during oogenesis. The extra-ovarian expression of salmon buc genes and the presence of a second zebrafish bucL gene suggest additional functions not related to germ cell specification. [ABSTRACT FROM AUTHOR]