Wnt/β-catenin signaling pathway activation is required for proliferation of chicken primordial germ cells in vitro.

Hyung Chul Lee,Jae Yong Han,Sumi Lim

doi:10.1038/srep34510

Abstract

Here, we investigated the role of the Wnt/β-catenin signaling pathway in chicken primordial germ cells (PGCs) in vitro. We confirmed the expression of Wnt signaling pathway-related genes and the localization of β-catenin in the nucleus, revealing that this pathway is potentially activated in chicken PGCs. Then, using the single-cell pick-up assay, we examined the proliferative capacity of cultured PGCs in response to Wnt ligands, a β-catenin-mediated Wnt signaling activator (6-bromoindirubin-3′-oxime [BIO]) or inhibitor (JW74), in the presence or absence of basic fibroblast growth factor (bFGF). WNT1, WNT3A, and BIO promoted the proliferation of chicken PGCs similarly to bFGF, whereas JW74 inhibited this proliferation. Meanwhile, such treatments in combination with bFGF did not show a synergistic effect. bFGF treatment could not rescue PGC proliferation in the presence of JW74. In addition, we confirmed the translocation of β-catenin into the nucleus by the addition of bFGF after JW74 treatment. These results indicate that there is signaling crosstalk between FGF and Wnt, and that β-catenin acts on PGC proliferation downstream of bFGF. In conclusion, our study suggests that Wnt signaling enhances the proliferation of chicken PGCs via the stabilization of β-catenin and activation of its downstream genes.

Highlights

Degradation complex, eventually causing β-catenin degradation[18]
To verify the crosstalk between bFGF and Wnt/β-catenin signaling in PGC proliferation, Western blotting and immunocytochemistry were used to examine the translocation of β-catenin into the nucleus, which was stimulated by bFGF after JW74 treatment
In light of these findings, Wnt ligands and their receptors were well expressed in both gonadal and cultured PGCs (SNUhp26), and other key factors such as β-catenin, GSK3β, and TCFs were expressed in gonadal stromal cells and chicken embryonic fibroblasts as well as gonadal PGCs and cultured PGCs, indicating that chicken PGCs might be capable of activating Wnt/β-catenin signaling (Fig. 1A)

Summary

Introduction

BIO and JW74 were used in this study to modulate the level of β-catenin in chicken PGCs in vitro. Another growth factor family involved in cell proliferation, migration, and differentiation during embryonic development is bFGF, known as FGF219,20. The expression of Wnt/β-catenin signaling-related genes was examined to determine whether PGCs are capable of activating this signaling pathway. BIO and JW74 were applied to modulate Wnt/β-catenin signaling in cultured chicken PGCs. Subsequently, an in vitro proliferation assay was performed in the presence of a Wnt ligand, BIO, JW74, or bFGF to verify the signaling mechanism underlying PGC proliferation in chickens. To verify the crosstalk between bFGF and Wnt/β-catenin signaling in PGC proliferation, Western blotting and immunocytochemistry were used to examine the translocation of β-catenin into the nucleus, which was stimulated by bFGF after JW74 treatment

Methods

Results

Conclusion