Functional differentiation of two lhx8 paralogs and possible regulatory role of lhx8a in Japanese flounder (Paralichthys olivaceus)

Abstract

Lhx8, belonging to the LIM-Homebox family, is involved in the tooth, nervous system, and primordial follicles development in mammals. However, little is known about the regulatory roles of lhx8 in teleosts. In this study, two lhx8 duplicates were identified in Paralichthys olivaceus, termed Polhx8a and Polhx8b, respectively. Bioinformatic analysis showed that Polhx8a was more likely to be a teleost-specific paralog. According to expression analysis, Polhx8a transcripts were almost exclusively concentrated in the oocytes, while Polhx8b was weakly expressed in the spleen, gill, and some facial organs, indicating sub-functionalization of this gene pair during evolution. Furthermore, Polhx8a mRNA level elevated from perinucleolar oocyte (PNO) stage to vitellogenic oocyte (VO) stage transition and changed after exogenous hormone stimulation, proving that Polhx8a was involved in the oocyte development and could be regulated by sex hormones. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) experiments captured the positive protein interactions between PoLhx8a and the other two oocyte-specific transcription factors: PoFigla and PoNobox. After knocking down lhx8a in embryos or adult ovaries in vivo, the expression of oocyte-associated genes was significantly down-regulated (P < 0.05). Our findings suggest the evolution and functional differentiation of lhx8 genes, and shed light on the potential role of lhx8a in protein interactions and gene regulation in teleosts.