AUTHOR=Zhang Jiankang , Xia Xueping , Zhu Ying , Lian Zitong , Tian Haifeng , Xiao Hanbing , Hu Qiaomu 

TITLE=Potential antagonistic relationship of fgf9 and rspo1 genes in WNT4 pathway to regulate the sex differentiation in Chinese giant salamander (Andrias davidianus)

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2022.974348

DOI=10.3389/fmolb.2022.974348

ISSN=2296-889X

ABSTRACT=<p>Farmed chinese giant salamander (<italic>Andrias davidianus</italic>) was an important distinctive economically amphibian that exhibited male-biased sexual size dimorphism. <italic>Fgf9</italic> and <italic>rspo1</italic> genes antagonize each other in Wnt4 signal pathway to regulate mammalian gonadal differentiation has been demonstrated. However, their expression profile and function in <italic>A. davidianus</italic> are unclear<italic>.</italic> In this study, we firstly characterized <italic>fgf9</italic> and <italic>rspo1</italic> genes expression in developing gonad. Results showed that <italic>fgf9</italic> expression level was higher in testes than in ovaries and increased from 1 to 6 years while <italic>rspo1</italic> expression was higher in ovaries than in testes. <italic>In situ</italic> hybridization assay showed that both <italic>fgf9</italic> and <italic>rspo1</italic> genes expressed at 62 dpf in undifferentiated gonad, and <italic>fgf9</italic> gene was mainly expressed in spermatogonia and sertoli cells in testis while strong positive signal of <italic>rspo1</italic> was detected in granular cell in ovary. During sex-reversal, <italic>fgf9</italic> expression was significantly higher in reversed testes and normal testes than in ovaries, and opposite expression pattern was detected for <italic>rspo1.</italic> When FH535 was used to inhibit Wnt/β-catenin pathway, expression of <italic>rspo1</italic>, <italic>wnt4 and β-catenin</italic> was down-regulated. Conversely, expression of <italic>fgf9, dmrt1, ftz-f1</italic> and <italic>cyp17</italic> were up-regulated. Furthermore, when <italic>rspo1 and fgf9</italic> were knocked down using RNAi technology, respectively. We observed that female biased genes were down regulated in ovary primordial cells after <italic>rspo1</italic> was knocked down, while the opposite expression profile was observed in testis primordial cells after <italic>fgf9</italic> was knocked down. These results suggested that <italic>fgf9</italic> and <italic>rspo1</italic> played an antagonistic role to regulate sex differentiation in the process of the gonadal development and provided a foundation for further functional characterizations. The data also provided basic information for genome editing breeding to improve the Chinese giant salamander farming industry.</p>