Identification and migration of primordial germ cells in Atlantic salmon, Salmo salar: characterization of vasa, dead end, and lymphocyte antigen 75 genes

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.

Figure 1

Protein domains and phylogenetic tree of Atlantic salmon vasa, dnd, and ly75 genes. A: Protein domains of Atlantic salmon vasa amino acid sequences predicted by SMART. 5′- and 3′-UTRs (black lines) and coding region (white box) are indicated. DEAD-like helicases (DEXDc) and helicase superfamily c-terminal (HELICc) domains are shown. Scale bar shows 200 amino acids. B: Phylogenetic tree of vasa and PL10 found in vertebrates. Numbers at the nodes indicate posterior probability and approximate likelihood-ratio values obtained from the Bayesian method. Species abbreviations and their GenBank accession numbers are as follows: Vasa (Bt, Bos Taurus: NM_001007819; Cc, Cyprinus carpio: AF479820; Ci, Ctenopharyngodon idella: GQ140633; Dr, Danio rerio: NM_131057; Hs, Homo sapiens: NM_024415; Mm, Mus musculus: NM_010029; Ol, Oryzias latipes: AB063484; Om, Oncorhynchus mykiss: AB032566; On, Oreochromis niloticus: AB032467; Ss, Salmo salar: JN712912; To, Thunnus orientalis: EU253482) and PL10 (Dr: NM_130941;Mm: NM_033077; Xl, Xenopus laevis, NM_001086814). C: Protein domains of Atlantic salmon Dnd amino acid sequences predicted by SMART. 5′- and 3′-UTRs (black lines) and coding region (white box) are indicated. RRM domain is shown. Scale bar shows 200 amino acids. D: Phylogenetic tree of the Dnd and A1CF family found in vertebrates. Numbers at the nodes indicate posterior probability and approximate likelihood-ratio values obtained from the Bayesian method. Species abbreviations and their GenBank accession numbers are as follows: Dnd (Bt: NM_001007819; Cf, Canis familiaris: XM_843741; Dr: NM_212795; Ga, Gasterosteus aculeatus: ENSGACT00000025998 (Ensembl); Hs: NM_194249; Ma, Misgurnus anguillicaudatus: AB531494; Mm: NM_173383; Ol, NM_001164516; Om: NM_001124661; Rn, Rattus norvegicus: NM_001109379; Ss: JN712911; Tn1, Tetraodon nigroviridis: ENSTNIT00000007156 (Ensembl); Tn2: ENSTNIT00000000153 (Ensembl); Tr, Takifugu rubripes: ENSTRUT00000022988 (Ensembl); Xl: AY321494) and A1CF (Dr: XM_680086; Hs: NM_014576; Mm: NM_001081074). E: Protein domains of Atlantic salmon Ly75 amino acid sequences predicted by SMART. 5′- and 3′-UTRs (black lines) and coding region (white box) are indicated. SP, RICIN/CysR, FN2, CTLD, and TM domains are shown. Scale bar shows 200 amino acids. F: Phylogenetic tree of Ly75 and other members of the mannose receptor family found in vertebrates. Species abbreviations and their GenBank accession numbers are as follows: Ly75 (Bt: AY264845; Cf: XM_545488; Dr: XM_690165; Gg, Gallus gallus: AJ574899; Hs: AF011333; Ma, Mesocricetus auratus: AB059273; Mm, U19271; Mm (monkey), Macaca mulatta, XM_001093552; Om, GQ468309; Rn: XM_001068965; Ss: JN712913; To: GQ468310; Tr: AB438982), MRC1 (Hs: NM_002438; Mm, NM_008625), MRC2 (Hs, AF134838; Mm, NM_008626), and PLA2R1 (Hs, NM_008867; Mm, XM_039118). [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com]

Figure 2

Distributionof vasa, dnd, and ly75 transcripts in Atlantic salmon. A: cDNA from various tissues of adult fish (blood, brain, gill, skeletal muscle, heart, liver, spleen, gall bladder, stomach, pyloric caeca, mid gut, head kidney, kidney, skin, testis, and ovary) were used for semi-quantitative RT-PCR. Actb was used as endogenous reference. Amplicon sizes, in base pairs, are indicated on the right. Expression pattern was determined using two biological replicates. B: Total RNA (400–900 ng) from early embryonic stages (two-cell, eight-cell, early-blastula, late-blastula, mid-gastrula, and 10-somite) was electrophoresed. Both 28S and 18S rRNA, stained with SYBR Safe DNA gel stain, are shown in all stages. C: The changes of both total RNA (white squares) and mRNA (black bars) amount per egg for each developmental stage. The concentration was quantified using three replicates. D: cDNA synthesized from above-mentioned developmental stages were used for semi-quantitative RT-PCR. In order to eliminate a possibility of genomic DNA contamination, −RT (without reverse transcriptase) samples of each counterpart were examined and electrophoresed. Amplicon sizes, in base pairs are indicated on the right.

Figure 3

Localization of vasa and dnd transcripts or Ly75 protein in the genital ridge of Atlantic salmon larva. In situ hybridization with vasa (anti-sense: A and C; sense: B and D) or dnd (anti-sense: E and G; sense: F and H) probes, and immunohistochemistry with Ly75 antibody (I and K) or without primary antibody (J). (A, B) and (C–K) are hatching (83 dpf) and yolk-sac resorption stages (139 dpf), respectively. (K) is a high-magnification view of genital ridge area enclosed by dashed box in (I). Embryos were fixated with PFA (A, B, E, F, and I–K) or Bouin's solution (C, D, G, and H). Arrowheads indicate the genital ridges. g, gut; m, mesonephric duct. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com]

Figure 4

Localization patterns of vasa transcripts and PGCs in Atlantic salmon embryo. Whole-mount in situ hybridization with vasa probe at different developmental stages (A–I, A′–I′). Transverse or longitudinal sections of the embryo subjected to hybridization with the vasa probe at the beginning of eye-pigmented stage (41 dpf) (J and K, J′ and K′). (A′–K′) are high-magnification views of (A–K), respectively. Arrowheads indicate the localization of vasa transcripts. Dotted lines in F and K′ indicate the edges of blastoderm and presumptive genital ridge, respectively. m, mesonephric duct; n, notochord (a–g, a′–g′). The schematic representation of the localization vasa transcripts and PGC distribution in the Atlantic salmon embryo at two-, four-, eight-cell, mid-blastula, early-gastrula, 30% epiboly, and 10-somite stages. The vasa signals and PGCs are represented in purple (line or dots) in the schematic representation. (a–g) and (a′–g′) are anterior and lateral views of embryo, respectively.

Figure 5

Sequential tracking of GFP translated from gfp-rt-vasa 3′-UTR RNA in Atlantic salmon embryo. The fluorescent views of the control embryo (non-injected embryo; A–F and G, J), the gfp-rt-vasa 3′-UTR RNA-injected embryo (A′–F′ and H, K), or the gfp-zf-nos1 3′-UTR RNA-injected embryo (I, L). Sequential GFP localization is observed in the gfp-rt-vasa 3′-UTR RNA-injected embryo throughout embryogenesis as follows: A′: Blastodisc (11 days post-injection, dpi) showing ubiquitous GFP expression at late-blastula stage. B′: GFP expression in the embryonic shield as a thickened margin (arrow) and at the edge of blastoderm (arrowhead) along with epiboly movement at pre-mid-gastrula stage (30% epiboly, 18 dpi). C′–F′: Declining GFP in the somatic cells of embryo during somitogenesis (24–55 dpi). In some cases, the yolk shows auto fluorescence. Arrows in C′–E′ show the direction of body axis a, anterior. G–I: Lateral view of head to trunk region of embryos at 60 dpi under fluorescence. J–L: High-magnification views of the genital ridge area, indicated by dashed boxes (G–I). Arrows and arrowheads indicate weak auto fluorescence in the mesonephric duct, and the positions where the GFP-expressing cells were observed on the outside of genital ridge area, respectively.