Elsevier

Theriogenology

Volume 158, December 2020, Pages 424-431
Theriogenology

Cell-intrinsic Fgf signaling contributes to primordial germ cell homing in zebrafish

https://doi.org/10.1016/j.theriogenology.2020.09.037Get rights and content

Highlights

  • A quarter of primordial germ cells are competent to Fgf signals.

  • Fgf signaling in primordial germ cells does not affect germ cell numbers.

  • Proper homing of primordial germ cells requires Fgf signaling revised .

Abstract

Primordial germ cells (PGCs) are specified before gastrulation and migrate toward the developing gonads. Previous in vitro studies have demonstrated a cell-intrinsic requirement of fibroblast growth factors (FGFs) by PGCs; however, no evidence suggests FGFs signal directly to PGCs in vivo. Here, using zebrafish as the animal model, we identified the mRNA expressions of Fgf receptors (Fgfrs) and determined the roles of Fgf signaling in migrating PGCs. To clarify the functions of Fgf signaling, we manipulated Fgf signaling specifically in PGCs using dominant-negative (dn) and constitutively-active (ca) Fgfrs and revealed a requirement of a basal Fgf signaling level for the robust arrival of PGCs. Repression of Fgf signaling in PGCs swayed the marginal positioning of PGCs as early as 6 h post-fertilization (6 hpf) and disrupted their arrival at the gonadal ridge at 24 hpf. On the other hand, the ectopic PGC phenotypes caused by the dn-Fgfrs could be alleviated by constitutive activation of Fgf signaling. In addition, we carefully ruled out the somatic effects in mosaic embryos by injecting RNA materials into one blastomere of the four- or eight-cell stage embryos. Injection of dn-Fgfrs into one of eight blastomeres hampered the arrival of only the treated PGCs, while the other PGCs remained unaffected. Furthermore, mosaic treatment of ca-Fgfrs rescued the ectopic rates of dn-Fgfr treated PGCs, while the other PGCs remained more ectopic within the same embryos. Interestingly, PGC-specific repression of Fgf signaling did not compromise the PGC number. To our knowledge, this is the first in vivo evidence to show that Fgf signaling plays a cell-intrinsic role in the migration of vertebrate PGCs.

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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