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Black carp vasa identifies embryonic and gonadal germ cells

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Abstract

Identification of molecular markers is an essential step in the study of germ cells. Vasa is an RNA helicase and a well-known germ cell marker that plays a crucial role in germ cell development. Here, we identified the Vasa homolog termed Mpvasa as the first germ cell marker in black carp (Mylopharyngodon piceus). First, a 2819-bp full-length Mpvasa complementary DNA (cDNA) was cloned by PCR using degenerated primers of conserved sequences and gene-specific primers. The Mpvasa cDNA sequence encodes a 637-amino acid protein that contains eight conserved characteristic motifs of the DEAD box protein family, and shares high identity to grass carp (81%) and zebrafish (74%) vasa homologs. Second, Mpvasa expression was restricted to the gonad in adulthood by RT-PCR and Western blot analysis. The dynamic patterns of temporal-spatial expression of Mpvasa during gametogenesis were examined by in situ hybridization, and Mpvasa transcripts were strictly detected in gonadal germ cells throughout oogenesis, predominantly in immature oocytes (stage I, II, and III oocytes). Third, Mpvasa transcripts were highly detected in unfertilized eggs and early embryos, and the signal indicated a dynamic migration of the primordial germ cells during embryogenesis, suggesting that Mpvasa transcripts were maternally inherited and specifically distributed in germ cells. Taken together, these results demonstrated that Mpvasa is an applicable molecular marker for identification of gonadal and embryonic germ cells, which facilitates the isolation and utilization of germ cells in black carp.

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Abbreviations

ISH:

In situ hybridization

PGCs:

Primordial germ cells

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Acknowledgments

This work was supported by grants from the National Key Basic Research Program of China (2013CB967700 to TC) and Huazhong Agricultural University Scientific and Technological Self-innovation Foundation (2013RC014; 2662015PY049). This work contains part of the Ph.D. thesis of XT.

Contribution statement

XT contributed to the study design and the acquisition, analysis, and interpretation of the data. YM, FJ, DY, CK, PQ, LL, WY, and WQ contributed to the analysis and interpretation of data. CT contributed to the conception and design of the research, the analysis and interpretation of the data, and drafting/revising the article. All authors approved the final version to be published. CT is responsible for the integrity of the work as a whole.

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Authors and Affiliations

  1. Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture and College of Fisheries, Huazhong Agricultural University, No. 1, Shizishan Street, Hongshan District, Wuhan, Hubei, 430070, China

    Ting Xue, Miao Yu, Qihua Pan, Yizhou Wang, Jian Fang, Lingyu Li, Yu Deng, Kai Chen, Qian Wang & Tiansheng Chen

  2. College of Fishery, Engineering Technology Research Center of Henan Province for Aquatic Animal Cultivation, Engineering Lab of Henan Province for Aquatic Animal Disease Control, Henan Normal University, Xinxiang, 453007, China

    Miao Yu

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  1. Ting Xue
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  2. Miao Yu
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  3. Qihua Pan
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  4. Yizhou Wang
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  5. Jian Fang
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Correspondence to Tiansheng Chen.

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Communicated by Matthias Hammerschmidt

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Fig. S1

Detection by Mpvasa sense probe or pre-immune serum in the adult ovary. A. The negative fluorescent signal detected by Mpvasa sense probe. B. Merged micrographs of PI staining and Mpvasa sense staining. C. The negative fluorescent signal detected by pre-immune serum. D. Merged micrographs of PI staining and pre-immune serum staining. CA, Cortical alveoli; ZR, Zona Radiata; FE, Follicular Epithelium; YG, Yolk Granule. Scale bars: 100 μm. (PPTX 363 kb)

Fig. S2

The whole mount in situ hybridization in developing embryos by Mpvasa sense probe. Whole embryos at various stages were subjected to ISH with sense Mpvasa probe and observed under microscopy. A: 2-cell stage. B: blastula stage. C: neurula stage. D: tail bud stage. E: heart pulsation stage. Scale bars, 200 μm. (PPTX 1235 kb)

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Xue, T., Yu, M., Pan, Q. et al. Black carp vasa identifies embryonic and gonadal germ cells. Dev Genes Evol 227, 231–243 (2017). https://doi.org/10.1007/s00427-017-0583-6

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