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. 2022 Aug 12;12(8):1226.
doi: 10.3390/life12081226.

Spinyhead Croaker Germ Cells Gene dnd Visualizes Primordial Germ Cells in Medaka

Cong Xu  1   2 , Yu Li  1   2   3 , Zhengshun Wen  4 , Muhammad Jawad  1   2 , Lang Gui  1   2 , Mingyou Li  1   2
Affiliations

Spinyhead Croaker Germ Cells Gene dnd Visualizes Primordial Germ Cells in Medaka

Cong Xu et al. Life (Basel). .

Abstract

Spinyhead croaker (Collichthys lucidus) is an economically important fish suffering from population decline caused by overfishing and habitat destruction. Researches on the development of primordial germ cell (PGC) and reproduction biology were an emergency for the long-term conservation of the involved species. Dead end (dnd) gene plays an indispensable role in PGC specification, maintenance, and development. In the current study, we report the cloning and expression patterns of dnd in C. lucidus (Cldnd). RT-PCR analysis revealed that Cldnd was specifically expressed in both sexual gonads. In the ovary, Cldnd RNA was uniformly distributed in the oocytes and abundant in oogonia, and gradually decreased with oogenesis. A similar expression pattern was also detected in testis. Dual fluorescent in situ hybridization of Cldnd and Clvasa demonstrated that they almost had the same distribution except in oocytes at stage I, in which the vasa RNA aggregated into some particles. Furthermore, Cldnd 3' UTR was sufficient to guide the Green Fluorescent Protein (GFP) specifically and stably expressed in the PGCs of medaka. These findings offer insight into that Cldnd is an evolutionarily conserved germline-specific gene and even a potential candidate for PGC manipulation in C. lucidus.

Keywords: Collichthys lucidus; PGC; dnd.

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Conflict of interest statement

The authors declare no conflict of interest. The company had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Figures

Figure 1
Figure 1
Multiple alignment and phylogenetic tree of Dnd proteins. (A) Multiple alignment of Dnd proteins. Species and overall sequence identity values compared to the ClDnd protein were at the end of the alignment. RNA recognition motif (RRM) and five conserved regions were indicated in the frame (black). (B) Phylogenetic tree of Dnd proteins. Bootstrap values were given. Accession numbers followed organism.
Figure 2
Figure 2
Cldnd RNA expression. (A) RT-PCR analysis of Cldnd in adult tissues. (B,C) Ovarian and testicular cryosections using antisense Cldnd probe and the signals were visualized by chromogenic staining. sc1 and sc2, primary and secondary spermatocytes; st, spermatid; I–III, stages of oocytes. Scale bar, 200 μm.
Figure 3
Figure 3
Co-localization of Cldnd and Clvasa mRNA in testis. Dual color FISH with Cldnd and Clvasa antisense RNA probes on testis and the signals were visualized by fluorescence staining. Nuclei were stained with DAPI (blue). (A) The signals were stained for the dnd RNA (green) by FISH. (B) The vasa signals were stained red. (C) The merges of vasa with dnd and DAPI. (D–F) Larger Magnification of panels C (white frame), highlighting the different cells. sg, spermatogonia; sc1 and sc2, primary and secondary spermatocytes; st, spermatid. Scale bar, 200 μm.
Figure 4
Figure 4
Co-localization of Cldnd and Clvasa mRNA in ovary. Dual color FISH with Cldnd and Clvasa antisense RNA probes on ovary and the signals were visualized by fluorescence staining. Nuclei were stained with DAPI (blue). (A) The signals were stained for the dnd RNA (green) by FISH. (B) The vasa signals were stained red. (C) The merges of vasa with dnd and DAPI. og, oogonia; I–II, stages of oocytes. Scale bar, 200 μm.
Figure 5
Figure 5
Visualization of PGCs by Cldnd 3′ UTR. (A–P) Medaka PGCs were visualized by co-injection of gfp-Cldnd 3′ UTR mRNA and rfp-Drnos 3′ UTR mRNA during embryogenesis. The merged images are shown on the right (D,H,L,P). (M’–P’) An isolated gonad was squashed and visualized at high magnification. Concentrations of injected mRNA are all 100 ng/μL. Scale bar, 200 μm.
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