Mohamad Ishak Nur Syafiqah, Nong Quang Dang, Matsuura Tomoaki, Kato Yasuhiko, Watanabe Hajime
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.
Biotechnology Global Human Resource Development Program, Division of Advanced Science and Biotechnology, Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.
PLoS Genet. 2017 Nov 2;13(11):e1006953. doi: 10.1371/journal.pgen.1006953. eCollection 2017 Nov.
Divergence of upstream regulatory pathways of the transcription factor Doublesex (Dsx) serves as a basis for evolution of sex-determining mechanisms in animals. However, little is known about the regulation of Dsx in environmental sex determination. In the crustacean Daphnia magna, environmental sex determination is implemented by male-specific expression of the Dsx ortholog, Dsx1. Transcriptional regulation of Dsx1 comprises at least three phases during embryogenesis: non-sex-specific initiation, male-specific up-regulation, and its maintenance. Herein, we demonstrate that the male-specific up-regulation is controlled by the bZIP transcription factor, Vrille (Vri), an ortholog of the circadian clock genes-Drosophila Vri and mammalian E4BP4/NFIL3. Sequence analysis of the Dsx1 promoter/enhancer revealed a conserved element among two Daphnia species (D. magna and D. pulex), which contains a potential enhancer harboring a consensus Vri binding site overlapped with a consensus Dsx binding site. Besides non-sex-specific expression of Vri in late embryos, we found male-specific expression in early gastrula before the Dsx1 up-regulation phase begins. Knockdown of Vri in male embryos showed reduction of Dsx1 expression. In addition, transient overexpression of Vri in early female embryos up-regulated the expression of Dsx1 and induced male-specific trait. Targeted mutagenesis using CRISPR/Cas9 disrupted the enhancer on genome in males, which led to the reduction of Dsx1 expression. These results indicate that Vri was co-opted as a transcriptional activator of Dsx1 in environmental sex determination of D. magna. The data suggests the remarkably plastic nature of gene regulatory network in sex determination.
转录因子双性基因（Dsx）上游调控途径的分歧是动物性别决定机制进化的基础。然而，关于环境性别决定中Dsx的调控知之甚少。在甲壳动物大型溞中，环境性别决定是通过Dsx直系同源基因Dsx1的雄性特异性表达来实现的。Dsx1的转录调控在胚胎发育过程中至少包括三个阶段：非性别特异性起始、雄性特异性上调及其维持。在此，我们证明雄性特异性上调受bZIP转录因子Vrille（Vri）控制，Vri是昼夜节律基因——果蝇Vri和哺乳动物E4BP4/NFIL3的直系同源基因。对Dsx1启动子/增强子的序列分析揭示了两种溞属物种（大型溞和蚤状溞）之间的一个保守元件，其中包含一个潜在的增强子，该增强子具有一个与Dsx结合位点一致序列重叠的Vri结合位点一致序列。除了Vri在晚期胚胎中的非性别特异性表达外，我们还发现在Dsx1上调阶段开始之前，原肠胚早期存在雄性特异性表达。在雄性胚胎中敲低Vri显示Dsx1表达降低。此外，在早期雌性胚胎中瞬时过表达Vri上调了Dsx1的表达并诱导了雄性特异性特征。使用CRISPR/Cas9进行靶向诱变破坏了雄性基因组上的增强子，导致Dsx1表达降低。这些结果表明，在大型溞的环境性别决定中，Vri被选为Dsx1的转录激活因子。数据表明性别决定中基因调控网络具有显著的可塑性。
