Abstract
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.
Highlights
The diversity and evolution of sex-determining pathways among animals are fundamental issues in developmental and evolutionary biology
We found that early male embryos transiently express the bZIP transcription factor, Vrille (Vri), known to be a circadian regulator, before male-specific Dsx1 activation begins
We found a potential enhancer that contains a consensus sequence of the Dsx binding site and an overlapping element for binding of an ortholog of the bZIP transcription factors, Drosophila Vrille (Vri) and vertebrate E4BP4/NFIL3, which are known to be involved in various general development processes including growth [21,22], circadian clock regulation [23,24], metamorphosis [25], apoptosis [26], and human T cell function [27]
Summary
The diversity and evolution of sex-determining pathways among animals are fundamental issues in developmental and evolutionary biology. There are numerous studies about genetic sex determination (GSD) mechanisms from various model organisms, including the mouse, nematode, and fruit fly. These studies have shown that, through interactions of several genes in a hierarchical manner, initial cues lead to sex-specific expression of the major effector of sexual differentiation, a DM-domain gene that encodes a transcription factor containing a DNA binding domain called DM-domain [4]. Pioneering studies using model organisms have demonstrated that sex-determining genes differ among species upstream of the hierarchies [4,5]. Little is known about the mechanisms of environmental sex determination (ESD) because organisms with ESD systems are poor genetic models
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