Abstract
No information exists on the identification of primordial germ cells (PGCs) in the super-order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full-length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi-quantitative RT-PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole-mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid-blastula stage, vasa-expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp-rt-vasa 3′-UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids.
Highlights
Development of primordial germ cells (PGCs) is fundamental to further gonad formation and affects individual fertility in vertebrates (Molyneaux and Wylie, 2004)
Multiple sequence alignments showed that salmon Vasa was 94% and 79% identical to Vasa of rainbow trout and zebrafish, respectively
Phylogenetic analysis using the Bayesian inference method showed that salmon Vasa clustered with other teleost Vasa protein sequences, and was closely related to rainbow trout Vasa (Fig. 1B)
Summary
Development of primordial germ cells (PGCs) is fundamental to further gonad formation and affects individual fertility in vertebrates (Molyneaux and Wylie, 2004). PGC-ablated fish develop either as sterile males, for example zebrafish (Danio rerio) (Slanchev et al, 2005), or as either sterile males or sterile females, for example loach (Misgurnus anguillicaudatus) (Fujimoto et al, 2010). The presence of a germline is required for phenotypic female sex determination in zebrafish (Siegfried and Nusslein-Volhard, 2008), but is not the primary determinant in goldfish (Carassius auratus) (Goto et al, 2012). Regardless of their role in sex determination, the presence of PGCs in the early gonad is a prerequisite for germline and gonadal development in teleosts. Germ cell marker genes exist for advanced germ cells, such as spermatogonia/oogonia, spermatocytes/ oocytes, and spermatids, and for PGCs (Xu et al, 2010)
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