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BMC Evol Biol
2016 Oct 26;161:232. doi: 10.1186/s12862-016-0809-7.
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Multiplicity of Buc copies in Atlantic salmon contrasts with loss of the germ cell determinant in primates, rodents and axolotl.
Škugor A
,
Tveiten H
,
Johnsen H
,
Andersen Ø
.
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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.
Fig. 1. Conserved synteni of buc and neighbor genes in various fish species. Spotted gar buc and bucL-(like) genes have been duplicated in teleost fish, but several paralogs are lacking. Conserved syntenic genes are shown in bold, and orthologs are given in same color
Fig. 2. Unrooted phylogenetic tree of tetrapod Buc proteins and teleost Buc and BucL (−like) paralogs. The tree was generated using the ML method based on the JTT model. To evaluate the topological stability 100 bootstrap resamplings were made. All branches with less than 30 bootstrap confidence values were collapsed. Accession numbers are given in Additional file 3: Table S1
Fig. 3. mRNA levels of salmon buc1a, buc2a and buc2b together with dnd and vasa during embryogenesis. The transcriptional levels of target genes were normalized to eEf1-a and presented as -ΔΔCt ± SE (n = 3). All stages were compared to the segmentation stage that follows late gastrulation. Significant difference is marked with * (p < 0.05)
Fig. 4. Salmon embryo at 4-cell stage with fluorescent signals in cleavage furrows from: a Immunostained endogenous Buc1a protein, b Zebrafish Buc-GFP injected in 1-cell salmon embryos. Magnified signals are shown in the white box. c Zebrafish Buc-GFP localized to putative PGCs in salmon embryos during segmentation. White arrows indicate fluorescent signals. Immunostained salmon embryo with secondary antibody only is shown in Additional file 8: Figure S5
Fig. 5. Gene expression of salmon buc1, buc2a, buc2b, dnd and vasa. a Ovarian mRNA levels during oogenesis at the freshwater (parr) and early seawater (smolt) stages compared to those in 3-year old ovary (n = 6). b Tissue expression in 2-year old females (n = 2). All tissues were compared to liver. The transcriptional levels of target genes were normalized to eEf1-a. Data are presented as –ΔΔCt ± SE. Significant difference is marked with * (p < 0.05)
Fig. 6. Immunohistochemical staining of ovarian sections from maturing 2-year old salmon. a Buc1a restricted to granular ooplasm of immature oocytes with magnified section below showing immature oocytes positively stained for Buc1a (orange). b Buc2a in ooplasm of immature oocytes with magnified view of Buc2b positive oocytes (orange) below. c Vasa positive signals (orange) in immature oocytes with magnified view below. d Dazl signals (orange) in Bb and nucleoli of immature oocytes and granulosa cells of more mature follicles with magnified oocyte positively stained for Dazl. e Population of oocytes at different developmental stages showing Dnd signals (orange) in both immature and cortical alveolus stage oocytes. Magnified view of Dnd signals is shown below. Proteins are stained orange and nuclei are stained blue with DAPI. Ovaries used to prepare the sections were collected from 3 different females. Bb- Balbiani body, gc- granulosa cell, n- nucleolus. Negative (secondary antibody alone) and positive (collagen staining) controls are shown in Additional file 8: Figure S5
Fig. 7. Immunohistochemical staining of juvenile ovarian sections. Adjacent sections were used for staining of (a) Buc1a (orange) and (b) Vasa (orange). c Co-localization of Buc1a and Vasa in the granular ooplasm of primary growth oocytes. Ovaries used to prepare the sections were collected from 3 different females. Nuclei were stained with DAPI (blue); Bb- Balbiani body, on- oocyte nucleus
Fig. 8. Ultrastructure of Atlantic salmon oocytes. a Stage 2 oocytes with granular ooplasm/Balbiani body. Black arrows indicate accumulations of nuage close to nuclear envelope and numerous nucleoli. b An area with aggregates of nuage and numerous mitochondria. c Ooplasm of stage 2 oocytes with endoplasmic reticulum and Golgi complex near the nuclear membrane. d Stage 4 oocyte surrounded with follicular cells. BL-basal membrane; CA-cortical alveoli; CT-connective tissue; ER-endoplasmic reticulum; GC-Golgi complex; GO-granular ooplasm; GRC-granulosa cell; HO- homogeneous ooplasm; LB-lipid body; M-mitochondria; NU-nuage; N-nucleus; n-nucleolus; TC-theca cell
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