Abstract
Primordial germ cells (PGCs) are responsible for generating all germ cells. Therefore, they are essential targets to be used as a tool for the production of germline chimeras. The labeling and route of PGCs were evaluated during the initial embryonic development of Pseudopimelodus mangurus, using whole-mount in situ hybridization (WISH) and mRNA microinjection in zygotes. A specific antisense RNA probe constituted by a partial coding region from P. mangurus nanos3 mRNA was synthesized for the WISH method. RNA microinjection was performed using the GFP gene reporter regulated by translation regulatory P. mangurus buc and nanos3 3’UTR sequences, germline-specific markers used to describe in vivo migration of PGCs. Nanos3 and buc gene expression was evaluated in tissues for male and female adults and initial development phases and larvae from the first to seventh days post-hatching. The results from the WISH technique indicated the origin of PGCs in P. mangurus from the aggregations of nanos3 mRNA in the cleavage grooves and the signals obtained from nanos3 probes corresponded topographically to the migratory patterns of the PGCs reported for other fish species. Diffuse signals were observed in all blastomeres until the 16-cell stage, which could be related to the two sequences of the nanos3 3′UTR observed in the P. mangurus unfertilized egg transcriptome. Microinjection was not successful using GFP-Dr-nanos1 3′UTR mRNA and GFP-Pm-buc 3′UTR mRNA and allowed the identification of potential PGCs with less than 2% efficiency only and after hatching using GFP-Pm-nanos3 3′UTR. Nanos3 and buc gene expression was reported in the female gonads and from fertilized eggs until the blastula phase. These results provide information about the PGC migration of P. mangurus and the possible use of PGCs for the future generation of germline chimeras to be applied in the conservation efforts of Neotropical Siluriformes species. This study can contribute to establishing genetic banks, manipulating organisms, and assisting in biotechnologies such as transplanting germ cells in fish.
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Data Availability
The datasets generated during the current study were deposited at NCBI: BioProject ID PRJNA819998. Sequences are available from the corresponding author upon reasonable request.
Code availability
Not applicable.
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Funding
The authors are grateful to São Paulo Research Foundation—FAPESP (Award Grant #2016/16386–3) and AES Tietê (Research & Development Projects #0064–1052/2014 and #0064–1062/2020) for the financial support and ICMBio/CEPTA for providing the facilities.
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GFS performed, analyzed, and wrote WISH experiments. GCZC performed, analyzed, and wrote an embryo micromanipulation experiment. LSL performed, analyzed, and wrote tissue gene expression experiments. GPP performed, analyzed, and wrote the initial developmental gene expression experiments. SCAdS designed and supervised the experiments and reviewed the manuscript. JAS collected animals in the natural environment and performed and wrote fish reproduction. PSM performed and analyzed vector construction and in vitro RNA synthesis experiments and reviewed and edited the manuscript. GSY designed and supervised the experiments and reviewed and edited the manuscript. All authors read and approved the final manuscript.
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Pseudopimelodus mangurus were collected from the Mojiguaçu River (Pirassununga–SP, Brazil) according to Brazilian law (Sisbio #60.383–1), and the experiments were conducted following the Animal Ethics Committee from the National Center for Research and Conservation of Continental Aquatic Biodiversity (CEUA/CEPTA #010/2015).
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Shiguemoto, G.F., Coelho, G.C.Z., López, L.S. et al. Primordial germ cell identification and traceability during the initial development of the Siluriformes fish Pseudopimelodus mangurus. Fish Physiol Biochem 48, 1137–1153 (2022). https://doi.org/10.1007/s10695-022-01106-z
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DOI: https://doi.org/10.1007/s10695-022-01106-z