Cell-intrinsic Fgf signaling contributes to primordial germ cell homing in zebrafish
Introduction
To most animals, germ cells are an essential cell type for the continuity of species. During development, primordial germ cells (PGCs) are specified early independent from the somatic cells, usually before gastrulation [1,2]. Accordingly, PGCs have to migrate across the embryos toward where the gonads develop [3]. In vertebrates, although the specifications of PGCs can be segregated into induction and preformation models [1,2,4], the CXCR4/CXCL12 signaling gradients contribute to the migratory guidance and survival during the migration of all studied PGCs [[5], [6], [7], [8]].
During the trek of PGCs, specific extracellular signaling molecules are requisite for their migration, proliferation, and survival. One of the signals thought to be instrumental in PGC development is fibroblast growth factors (FGFs). Many studies indicate that FGF signaling is conducive to maintaining PGC numbers. In cell culture, supplementation with FGF2 promoted proliferation of mouse and chicken PGCs [[9], [10], [11]]. Besides, knockout of FGFR2-IIIb, an FGF receptor (FGFR) gene, culminated in apoptosis of mouse PGCs without hampering their proliferation in vivo [12]. Interestingly, in chickens, either up- or down-regulating FGF8 increased the PGC number and the expression of the PGC marker DDX4 in vivo [13]. Hence, how Fgf signaling maintains the vertebrate PGC number was worth revisiting.
Moreover, FGF signaling might participate in the migration of PGCs. In mice, FGF signaling is crucial to the velocity of PGCs, but not their directionality during the exit from hindgut to mesenchyme [12]. Nevertheless, in Drosophila, FGF signaling regulates the cell-cell adhesion of the midgut epithelia, which collapse and trap PGCs in fgf mutants [14,15]. The phenomenon observed in Drosophila is reminiscent of the roles of FGF signaling in mice. Considering the ubiquitous roles of FGF signaling in embryogenesis [[16], [17], [18], [19]], the cell-intrinsic manipulations of the signaling are necessary to delineate the interactions between PGCs and its microenvironment. Here in this report, using zebrafish as the animal model, we aimed to validate the hypothesis that cell-intrinsic Fgf signaling plays a critical role in the PGC development in an in vivo system. To this end, the competence of zebrafish PGCs to receive Fgfs was determined and the Fgf signaling was manipulated specifically in PGCs.
Section snippets
Zebrafish
AB wild-type (WT) and Tg(kop:EGFP-F-nanos3-3′UTR) zebrafish [20] were kept in a recirculation aquaculture system at 28 °C with a light/dark cycle of 14/10 h [21]. The embryos were kept at 28.5 °C and staged depending on the developing time (hpf) and their morphogenic features [22]. All animal handling procedures in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University (NTU-105-EL-00146).
Transcriptomic analysis
Analyses of next-generation
Zebrafish PGCs express fgfrs
To determine the competence of zebrafish PGCs to perceive Fgfs, we reanalyzed published RNA-seq datasets derived from zebrafish PGCs and somatic cells [24,25]. According to transcript abundances, transcriptions of fgfrs at 7 hpf in PGCs are within about the same magnitude of a known PGC-expressing receptor, cxcr4b [5] (Fig. 1A). Among five fgfrs in the zebrafish genome, fgfr1b and fgfr4 were more abundant, whereas fgfr3 was the least (Fig. 1A). To validate the RNA-seq data, we sorted out PGCs
Discussion
Previous studies suggest the expressions of Fgfr1-IIIc and Fgfr2-IIIb in mouse PGCs [12] and FGFR1, FGFR2-IIIc, and FGFR4 in chicken PGCs [50]. To determine the competence of zebrafish PGCs to receive Fgfs, we performed the PCR-based identification, and the outcome concurred with the previous RNA-seq data (Fig. 1) [24,25]. In zebrafish PGCs, fgfr1a, fgfr1b, fgfr2, and fgfr4 were detected more than fgfr3, similar to zebrafish soma and chicken PGCs [50]. Furthermore, zebrafish PGCs are specified
Conclusion
Taken together, we explored the cell-intrinsic role of Fgf signaling in zebrafish PGC development. In zebrafish, approximately a quarter of PGC population expresses fgfrs. While we did not find any evidence suggesting roles of Fgf signaling in PGC proliferation or survival, we demonstrated that Fgf signals contribute to PGC migration in a cell-intrinsic fashion.
CRediT authorship contribution statement
Chia-Teng Chang: Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization. Yen-Hua Lee: Investigation, Resources. Wei-Chun HuangFu: Conceptualization, Supervision. I-Hsuan Liu: Conceptualization, Methodology, Resources, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition.
Acknowledgment
The authors would like to thank Dr. Harry J. Mersmann for proof-reading and helping grammatically revise this manuscript. The authors would also like to thank the Instrumentation Center at National Taiwan University for the support of the cell sorter. We thank Dr. Erez Raz for generously providing the Tg(kop:EGFP-F-nanos1-3′UTR) zebrafish and Dr. Kyo Yamasu for the plasmids containing dominant-negative as well as constitutively active fgfr genes. This work was financially supported by Ministry
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