In this study, we have used two strategies for labeling and traceability in the initial embryonic development of P. mangurus PGC, WISH, and mRNA microinjection methodologies, aiming to develop technology for future application in conservationist actions for this endangered species. The WISH technique was performed using a specific P. mangurus nanos3 RNA antisense probe to obtain essential knowledge about the PGC migration route in P. mangurus. However, this methodology cannot be used for PGC transplantation to generate germline chimera. Then, we have mRNA microinjection to describe in vivo PGC migration, targeting P. mangurus PGC for transplantation to sterile recipients. For this, we have used two specific germline markers, nanos and bucky ball 3’UTR regulating the translation of the reporter GFP gene. We have used two nanos 3’UTR, the nanos1 3’UTR from zebrafish, commonly used for PGC labeling of various fish species, and nanos3 3’UTR from P. mangurus. Only nanos3 homolog was found in the egg transcriptome of P. mangurus (data not published), corroborating with the results obtained using Larimichthys crocea, that only nanos3 transcripts signals were detected during embryogenesis, suggesting nanos3, not nanos1 and nanos2 is a germ cell marker gene in this species (Han et al., 2018).
RNA microinjection methodology has been used in different fish species to identify and describe the migration route of PGCs (Yoon et al., 1997; Shinomiya et al., 2000; Otani et al., 2002; Saito et al., 2004 and 2006; Fujimoto et al., 2006; Mishima et al., 2006; Nagasawa et al., 2013; Linhartova et al., 2014; Fernández et al., 2015; Wu et al., 2018; Coelho et al., 2019). This technique contributes to establishing genetic banks, manipulating organisms, and assisting in biotechnologies such as the transplantation of germ cells in fish.
To identify PGCs in embryonic development by WISH, we used an RNA probe synthesized from genetic material extracted from the own marbled catfish eggs. However, the use of probe from one species can be used for other species as have been reported in other related studies, such as Loach and Goldfish that was used the vasa probe from Danio rerio (Otani et al., 2002; Fujimoto et al., 2006), and for ukigori that was used a Leucopsarion petersii probe (Saito et al., 2004). These studies point to the conserved sequence of the genes specific for PGC.
The signals obtained from the P. mangurus nanos3 probes correspond topographically to the migratory patterns of the PGC reported in other fish species ( Otani et al., 2002; Saito et al., 2004; Fujimoto et al., 2006; Ricci et al., 2018). Therefore, the results suggest that the nano3 positive cells correspond to P. mangurus PGCs and the description of the migratory route is accurate.
The origin pattern of P. mangurus PGCs observed was similar to the vasa mRNA aggregations in the cleavage grooves, as in zebrafish (Yoon et al., 1997), goldfish (Otani et al., 2002), ukigori (Saito et al., 2004) e loach (Fujimoto et al., 2006), and unlike the one presented in medaka (Shinomiya et al., 2000) with vasa mRNA distribution throughout the blastodisc.
The nanos1 3'UTR from Danio rerio have been used to in vivo visualization of PGCs for different species, with different efficiency in PGC labeling. For Leucopsarion petersii, it was obtained 100%, 97.7% Oryzias latepis, 100% Carassius auratus, 100% Danio rerio, 98.5% Danio albolineatus, 100% Clupea pallasii, 99.55% Misgurnus anguillicaudatus (Saito et al., 2006), 59.3% Anguilla japônica (Saito et al., 2011), 69.3% Tinca trinca (Linhartova et al., 2014) and 74.6% Prochilodus lineatus (Coelho et al., 2019). These results suggest the conservation of the sequence and functionality of this gene among more distant taxonomic species. However, for P. mangurus it was not possible to mark the PGCs, demonstrating that the D. rerio mRNA was not adequate for the species. Conserved regions in the alignment of P. mangurus nanos3 3’UTR and zebrafish nanos1 and nanos3 3’UTRs sequences (data not shown) were not observed, as verified in the alignment of nanos3 3’UTR in Siluriformes (Figure S3).
The non-specificity of nanos1 3'UTR from D. rerio for P. mangurus PGCs led to the construction of new species-specific mRNAs using the sequence of the 3'UTR regions of bucky ball and nanos3 from P. mangurus. The GFP-Pm-bucky ball 3’UTR and GFP-Pm-nanos3 3'UTR of the P. mangurus were injected into 246 and 213 one-cell embryos, respectively. The mRNA of the 3'UTR region of bucky ball showed no evidence of positive PGC labeling for GFP during embryonic and larval development. Only three embryos injected with GFP-Pm-nanos3 3'UTR mRNA presented 3 to 5 evidence of possible PGC marking in the hatching phase.
The PGC labeling is an essential step for PGC transplantation methods, such as blastomere transplantation or single PGC transplantation, for generating germline chimeras in fish. In blastomere transplantation, sometimes it is challenging to identify PGC labeled differentially from other cells, and generally is transplanted several blastomeres from microinjected zygotes, and PGC labeled are posteriorly observed in the gonadal region (Saito et al., 2010). In zebrafish, the germplasm has been visualized at early cleavage stage embryos using the microinjection of GFP-buc mRNA (Bontems et al., 2009). Alternatively, the microinjection of RFP-Olbuc 3'UTR mRNA in Oryzias latipes (medaka) was used for PGC labeling, validating bucky ball 3’UTR as PGC marker (Song et al., 2021). In this work, we have microinjected GFP-Pm-bucky ball 3’UTR mRNA aiming the PGC labeling at blastula stage intending blastomere transplantation; however, the PGC labeling was unsuccessful. The medaka bucky ball 3’UTR (accession code: MT622506.1) has half of the size (230 nucleotides) of the Pm-bucky ball 3’UTR (582 nucleotides) and its sequences are more variables between them (alignment not shown) compared to those used in the sequence alignment of the Siluriformes species (Figure S1). The PGC labeling using GFP-nanos 3'UTR mRNA microinjection generally permits the PGC visualization in somitogenesis, and therefore, the single PGC transplantation is possible to be performed. Moreover, the PGC ability to migrate toward the gonadal region decreased after the stage of 10 to 15 somites (Saito et al., 2008 and 2010). P. mangurus PGC labeled was identified after hatching, suggesting that using these could compromise the migration in the chimera.
Several studies have appointed that GFP-positive PGCs became evident in the final stages of gastrulation (Saito et al., 2006, 2011, Linhartova et al., 2014; Fernández et al., 2015) or during the early stage of somitogenesis (Coelho et al., 2019). The PGC numbers positive for GFP in these studies ranged on average from 5.2 to 43.2 between species. The level of GFP expression decreases during embryonic development in somatic cells, but it is kept stable in germ cells, allowing the visualization of these cells in vivo (Mishima et al., 2006).
The nanos3 expression observed during embryonic development until hatching using WISH suggests that this gene is involved in the differentiation of PGCs during migration and gonadal development in P. mangurus. This fact is reinforced by nanos3 expression detected in the initial development stages and female gonad. The gonad of the female used was found in the vitellogenin stage, suggesting that maternal RNAs had already been deposited. Bucky ball and nanos3 gene expressions during embryonic development and whole larvae using conventional PCR corroborate their higher activity in the early ontogenetic stages, as their role in PGC's differentiation, organization, migration, and maintaining, which occurs crucially in these phases (Bontems et al., 2009; Yon and Akbulut, 2015; Škugor et al., 2016). P. mangurus bucky ball expression was identified in the fertilized egg until blastula phases and female gonad, like as reported for sturgeon (Ye et al., 2018). P. mangurus nanos3 expression was identical to that observed for bucky ball; however, in the Oreochromis niloticus nanos3 expression can be observed in lower expression after blastula and in the testis (Jin et al., 2019). The pattern of P. mangurus bucky ball and nanos3 expression appoint to similar expression.