Knockdown of the germ cell factor Dead end induces multiple transcriptional changes in Atlantic cod (Gadus morhua) hatchlings

Abstract

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.