Primordial germ cells identification and traceability during the initial development of the Siluriformes fish Pseudopimelodus mangurus.

Abstract Primordial germ cells (PGCs) are responsible for the generation of all germ cells, and therefore, they are essential targets to be used as a tool for the production of germline chimera. 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. The WISH method was synthesized a specific RNA antisense probe using part of the coding region from P. mangurus nanos3 cDNA. The RNA microinjection was performed using the GFP gene reporter regulated by translation regulatory P. mangurus bucky ball and nanos3 3’UTR sequences, which are germline-specific markers used to describe in vivo migration of PGCs. The nanos3 and bucky ball gene expressions were evaluated in tissues for male and female adults and initial development phases and larvae from first to seventh days post-hatching. The results from the WISH technique appointed to the origin of PGCs in P. mangurus from the aggregations of nanos3 mRNA in the cleavage grooves and the signals obtained of nanos3 probes corresponded topographically to the migratory patterns of the PGC reported for other fish species. The microinjection of GFP-nanos3 3'UTR mRNA in the zygotes of P. mangurus was not successful for PGC labeling, but it was possible to identify three embryos injected, presenting 3 to 5 evidence of possible PGC labeled in the hatching phase. The nanos3 and bucky ball 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 chimera to be applied in the conservation efforts of Neotropical Siluriformes species, contributing to the establishment of genetic banks, manipulation of organisms, and assisting in biotechnologies such as the transplantation of germ cells in fish.