CELF1 represses Doublesex1 expression via its 5' UTR in the crustacean Daphnia magna.

In sex determination of the crustacean Daphnia magna, male-specific expression of DM-domain transcription factor Doublesex1 (Dsx1) orchestrates the male developmental program triggered by environmental stimuli. We previously identified the CELF1 ortholog as a candidate of proteins associated with the 5' UTR of the Dsx1α isoform. Here we report the CELF1-dependent suppression of Dsx1 expression in D. magna. During embryogenesis, CELF1 expression was not sexually dimorphic. Silencing of CELF1 led to the activation of Dsx1 expression both in female and male embryos. Overexpression of CELF1 in male embryos resulted in a reduction of Dsx1 expression. By these manipulations of CELF1 expression, the Dsx1 transcript level was not significantly changed. To investigate whether the CELF1 controls Dsx1 expression via its 5' UTR, we injected the GFP reporter mRNA having intact Dsx1α 5' UTR or mutated one lacking the GU-rich element (GRE) that is known as a binding site of the CELF1 ortholog. We found that deletion of the GRE significantly increased the reporter gene expression. These results indicate that CELF1 suppresses Dsx1 expression both in females and males, possibly at the post-transcriptional level. We speculate that CELF1 may avoid unintended Dsx1 expression and generation of sexual ambiguity by setting a threshold of Dsx1 expression.

Sense-overlapping lncRNA as a decoy of translational repressor protein for dimorphic gene expression.

Long noncoding RNAs (lncRNAs) are vastly transcribed and extensively studied but lncRNAs overlapping with the sense orientation of mRNA have been poorly studied. We analyzed the lncRNA DAPALR overlapping with the 5´ UTR of the Doublesex1 (Dsx1), the male determining gene in Daphnia magna. By affinity purification, we identified an RNA binding protein, Shep as a DAPALR binding protein. Shep also binds to Dsx1 5´ UTR by recognizing the overlapping sequence and suppresses translation of the mRNA. In vitro and in vivo analyses indicated that DAPALR increased Dsx1 translation efficiency by sequestration of Shep. This regulation was impaired when the Shep binding site in DAPALR was deleted. These results suggest that Shep suppresses the unintentional translation of Dsx1 by setting a threshold; and when the sense lncRNA DAPALR is expressed, DAPALR cancels the suppression caused by Shep. This mechanism may be important to show dimorphic gene expressions such as sex determination and it may account for the binary expression in various developmental processes.

Mutation of the Cytochrome P450 CYP360A8 Gene Increases Sensitivity to Paraquat in Daphnia magna.

The freshwater crustacean Daphnia magna has traditionally been a model for ecotoxicological studies owing to its sensitivity to many xenobiotics. Because it is used in many toxicity assessments, its detoxification mechanism for xenobiotics is important and requires further study. However, studies related to detoxification genes are limited to transcriptomic profiling, and there are no D. magna mutants for use in the understanding of xenobiotic metabolism in vivo. We report the generation of a D. magna CYP360A8 mutant-the gene is a cytochrome P450 (CYP) clan 3 gene. Based on RNA sequencing of adult D. magna, we found that CYP360A8 has the highest expression level among all CYP genes. At ovarian maturation, its expression level is up-regulated 6-fold compared to the juvenile stages and is maintained thereafter. Using the CRISPR/CRISPR-associated 9 (Cas9) system, we disrupted CYP360A8 by coinjecting CYP360A8-targeting guide RNA and Cas9 proteins into D. magna eggs and established one monoallelic CYP360A8 mutant line. This CYP360A8 mutant had a higher sensitivity to the herbicide paraquat compared to the wild type. We confirmed the up-regulation of CYP360A8 by paraquat. The results demonstrate the role of CYP360A8 in paraquat detoxification. The present study establishes a CYP mutant of D. magna, and this strategy can be a basic platform to document a range of CYP gene-xenobiotic relationships in this species. Environ Toxicol Chem 2021;40:1279-1288. © 2020 SETAC.

Production of genome-edited Daphnia for heavy metal detection by fluorescence.

Aquatic heavy metal pollution is a growing concern. To facilitate heavy metal monitoring in water, we developed transgenic Daphnia that are highly sensitive to heavy metals and respond to them rapidly. Metallothionein A, which was a metal response gene, and its promoter region was obtained from Daphnia magna. A chimeric gene fusing the promoter region with a green fluorescent protein (GFP) gene was integrated into D. magna using the TALEN technique and transgenic Daphnia named D. magna MetalloG were produced. When D. magna MetalloG was exposed to heavy metal solutions for 1 h, GFP expression was induced only in their midgut and hepatopancreas. The lowest concentrations of heavy metals that activated GFP expression were 1.2 µM Zn2+, 130 nM Cu2+, and 70 nM Cd2+. Heavy metal exposure for 24 h could lower the thresholds even further. D. magna MetalloG facilitates aqueous heavy metal detection and might enhance water quality monitoring.

Two Doublesex1 mutants revealed a tunable gene network underlying intersexuality in Daphnia magna.

In recent years, the binary definition of sex is being challenged by repetitive reports about individuals with ambiguous sexual identity from various animal groups. This has created an urge to decode the molecular mechanism underlying sexual development. However, sexual ambiguities are extremely uncommon in nature, limiting their experimental value. Here, we report the establishment of a genetically modified clone of Daphnia magna from which intersex daphniids can be readily generated. By mutating the conserved central sex determining factor Doublesex1, body-wide feminization of male daphniid could be achieved. Comparative transcriptomic analysis also revealed a genetic network correlated with Doublesex1 activity which may account for the establishment of sexual identity in D. magna. We found that Dsx1 repressed genes related to growth and promoted genes related to signaling. We infer that different intersex phenotypes are the results of fluctuation in activity of these Dsx1 downstream factors. Our results demonstrated that the D. magna genome is capable of expressing sex in a continuous array, supporting the idea that sex is actually a spectrum.

Mapping the expression of the sex determining factor Doublesex1 in Daphnia magna using a knock-in reporter.

Sexually dimorphic traits are common and widespread among animals. The expression of the Doublesex-/Mab-3-domain (DM-domain) gene family has been widely studied in model organisms and has been proven to be essential for the development and maintenance of sex-specific traits. However, little is known about the detailed expression patterns in non-model organisms. In the present study, we demonstrated the spatiotemporal expression of the DM-domain gene, doublesex1 (dsx1), in the crustacean Daphnia magna, which parthenogenetically produces males in response to environmental cues. We developed a dsx1 reporter strain to track dsx1 activity in vivo by inserting the mCherry gene into the dsx1 locus using the TALEN-mediated knock-in approach. After confirming dsx1 expression in male-specific traits in juveniles and adults, we performed time-lapse imaging of embryogenesis. Shortly after gastrulation stage, a presumptive primary organiser, named cumulus, first showed male-specific dsx1 expression. This cell mass moved to the posterior growth zone that distributes dsx1-expressing progenitor cells across the body during axial elongation, before embryos start male-specific dsx1 expression in sexually dimorphic structures. The present study demonstrated the sex-specific dsx1 expression in cell populations involved in basal body formation.

Co-option of the bZIP transcription factor Vrille as the activator of Doublesex1 in environmental sex determination of the crustacean Daphnia magna.

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.