Identification of gene isoforms and their switching events between male and female embryos of the parthenogenetic crustacean Daphnia magna.

The cladoceran crustacean Daphnia exhibits phenotypic plasticity, a phenomenon that leads to diverse phenotypes from one genome. Alternative usage of gene isoforms has been considered a key gene regulation mechanism for controlling different phenotypes. However, to understand the phenotypic plasticity of Daphnia, gene isoforms have not been comprehensively analyzed. Here we identified 25,654 transcripts derived from the 9710 genes expressed during environmental sex determination of Daphnia magna using the long-read RNA-Seq with PacBio Iso-Seq. We found that 14,924 transcripts were previously unidentified and 5713 genes produced two or more isoforms. By a combination of Illumina short-read RNA-Seq, we detected 824 genes that implemented switching of the highest expressed isoform between females and males. Among the 824 genes, we found isoform switching of an ortholog of CREB-regulated transcription coactivator, a major regulator of carbohydrate metabolism in animals, and a correlation of this switching event with the sexually dimorphic expression of carbohydrate metabolic genes. These results suggest that a comprehensive catalog of isoforms may lead to understanding the molecular basis for environmental sex determination of Daphnia. We also infer the applicability of the full-length isoform analyses to the elucidation of phenotypic plasticity in Daphnia.

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.

Development of transgenic Daphnia magna for visualizing homology-directed repair of DNA.

In the crustacean Daphnia magna, studying homology-directed repair (HDR) is important to understand genome maintenance during parthenogenesis, effects of environmental toxicants on the genome, and improvement of HDR-mediated genome editing. Here we developed a transgenic D. magna that expresses green fluorescence protein (GFP) upon HDR occurrence. We utilized the previously established reporter plasmid named DR-GFP that has a mutated eGFP gene (SceGFP) and the tandemly located donor GFP gene fragment (iGFP). Upon double-strand break (DSB) introduction on SceGFP, the iGFP gene fragment acts as the HDR template and restores functional eGFP expression. We customized this reporter plasmid to allow bicistronic expression of the mCherry gene under the control of the D. magna EF1α-1 promoter/enhancer. By CRISPR/Cas-mediated knock-in of this plasmid via non-homologous joining, we generated the transgenic D. magna that expresses mCherry ubiquitously, suggesting that the DR-GFP reporter gene is expressed in most cells. Introducing DSB on the SceGFP resulted in eGFP expression and this HDR event could be detected by fluorescence, genomic PCR, and quantitative reverse-transcription PCR, suggesting this line could be used for evaluating HDR. The established reporter line might expand our understanding of the HDR mechanism and also improve the HDR-based gene-editing system in this species.

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.

Roles of and cross-talk between ecdysteroid and sesquiterpenoid pathways in embryogenesis of branchiopod crustacean Daphnia magna.

The ecdysteroid and sesquiterpenoid pathways control growth, developmental transition, and embryogenesis in insects. However, the function of orthologous genes and the cross-talk between both pathways remain largely uncharacterized in non-insect arthropods. Spook (Spo) and Juvenile hormone acid o-methyltransferase (Jhamt) have been suggested to function as rate-limiting factors in ecdysteroid and sesquiterpenoid biosynthesis, respectively, in insects. In this study, we report on the functions of Spo and Jhamt and the cross-talk between them in embryos of the branchiopod crustacean Daphnia magna. Spo expression was activated at the onset of gastrulation, with the depletion of Spo transcript by RNAi resulting in developmental arrest at this stage. This phenotype could be partially rescued by supplementation with 20-hydroxyecdysone, indicating that Spo may play the same role in ecdysteroid biosynthesis in early embryos, as reported in insects. After hatching, Spo expression was repressed, while Jhamt expression was activated transiently, despite its silencing during other embryonic stages. Jhamt RNAi showed little effect on survival, but shortened the embryonic period. Exposure to the sesquiterpenoid analog Fenoxycarb extended the embryonic period and rescued the Jhamt RNAi phenotype, demonstrating a previously unidentified role of sesquiterpenoid in the repression of precocious embryogenesis. Interestingly, the knockdown of Jhamt resulted in the derepression of ecdysteroid biosynthesis genes, including Spo, similar to regulation during insect hormonal biosynthesis. Sesquiterpenoid signaling via the Methoprene-tolerant gene was found to be responsible for the repression of ecdysteroid biosynthesis genes. It upregulated an ortholog of CYP18a1 that degrades ecdysteroid in insects. These results illuminate the conserved and specific functions of the ecdysteroid and sesquiterpenoid pathways in Daphnia embryos. We also infer that the common ancestor of branchiopod crustaceans and insects exhibited antagonism between the two endocrine hormones before their divergence 400 million years ago.

Monitoring ecdysteroid activities using genetically encoded reporter gene in Daphnia magna.

Ecdysteroid is an important hormone that regulates growth, reproduction, and embryogenesis in arthropods. However, little is known about its role and action mechanism in crustaceans, despite their pivotal role in aquatic ecosystem. Daphnia magna, a freshwater crustacean, is used as a classic model organism in ecology and ecotoxicology. Its ecdysteroid activity has been partially characterized previously. However, the spatio-temporal behavior of ecdysteroid, especially during early embryogenesis, when it is thought to have pivotal roles, is still unclear. Thus, we proposed a genetic modification approach by integrating a reporter gene exhibiting ecdysteroid activity in vivo. We used the clustered regularly interspaced palindromic repeats (CRISPR) genome editing technique, followed by non-homologous end-joining (NHEJ) pathway as the transgenesis method to generate the ecdysteroid reporter transgenic Daphnia. One transgenic Daphnia containing one copy of the ecdysone response element (EcRE)-controlled reporter gene mCherry was successfully obtained and was designated EcRE-mCh. The expression of mCherry was observed during early embryogenesis starting from 12 h after ovulation (hao). The time-lapse imaging during 12-24 hao showed the growing expression of mCherry signal originating from the posterior section of embryo and then migrating toward the anterior section. From 18 hao, the signal was detected around the developing thoracic appendages and localized between the first to third thoracic segments. The establishment of this EcRE-mCh line and its ability to exhibit ecdysteroid activity spatio-temporally might serve as convenient tool to elucidate the roles of ecdysteroid during the early stage of animal development. Moreover, the expression of mCherry in response to the presence of ecdysteroid in water suggests that EcRE-mCh could be used for monitoring ecdysteroid activities in environmental water.