Genomic integration and ligand-dependent activation of the human estrogen receptor α in the crustacean Daphnia magna

The freshwater crustacean Daphnia have a long history in water quality assessments and now lend themselves to detection of targeted chemicals using genetically encoded reporter gene due to recent progress in the development of genome editing tools. By introducing human genes into Daphnia, we may be able to detect chemicals that affect the human system, or even apply it to screening potentially useful chemicals. Here, we aimed to develop a transgenic line of Daphnia magna that contains the human estrogen receptor alpha (hERα) and shows a fluorescence response to exposure of estrogens. We designed plasmids to express hERα in Daphnia (EF1α1:esr1) and to report estrogenic activity via red fluorescence (ERE:mcherry) under the control of estrogen response element (ERE). After confirmation of functionality of the plasmids by microinjection into embryos, the two plasmids were joined, a TALE site was added and integrated into the D. magna genome using TALEN. When the resulting transgenic Daphnia named the ES line was exposed to Diethylstilbestrol (DES) or 17β-Estradiol (E2), the ES line could reliably expressed red fluorescence derived from mCherry in a ligand-dependent manner, indicating that an estrogen-responsive line of D. magna was established. This is the first time a human gene was expressed in Daphnia, showcasing potential for further research.


Introduction
The crustacean Daphnia, as keystone species of freshwater ecosystems, have been used in water quality assessments for many decades [1,2]. High growth rate and high fecundity combined with cheap and easy rearing make them an excellent organism for screening. Recently, the sequencing of the Daphnia pulex and Daphnia magna genomes [3,4] has been completed and different methods for genomic integration of foreign genes like CRISPR/Cas [5] and TALEN-mediated knockin [6,7] have been successfully adapted in D. magna. In order to facilitate the expression of ectopic gene, an optimal mRNA structure in D. magna was also reported PLOS ONE | https://doi.org/10.1371/journal.pone.0198023 June 8, 2018 1 / 11 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 [8]. These progress of functional genomics allowed us for detecting one of arthropod hormones, ecdysteroid using a GFP reporter gene under control of ecdysteroid responsive elements in this species [9]. In addition, a candidate of an element responsive to another arthropod hormone, juvenile hormone, was integrated together with its reporter GFP gene into the genome [10]. These reporter constructs and transgenic lines have potential as tools to detect agonists of ecdysteroids and juvenile hormones that are widely used as pesticides, which can be used as a chemical sensor Daphnia.
Based on the progress of the development of chemical sensor Daphnia, we aimed to detect chemicals that affect the human system [11][12] by making sensor Daphnia. In this study, we aimed to develop estrogen sensor Daphnia by introducing human genes, as Daphnia hormonal system is different from human and estrogen system does not exist in Daphnia. Estrogens and estrogen like chemicals bind to estrogen receptors (ER), the resulting complex interacts with Estrogen Response Elements (ERE) on the target gene of genome and activate transcription of downstream genes [13][14][15]. In this study, we designed and integrated a plasmid to express the human estrogen receptor alpha (hERα) (EF1α1:esr1) and to report estrogenic activity via red fluorescence (ERE:mcherry) into the genome of D. magna using a set of previously established TALENs [6]. The resulting estrogen sensor line (ES line) reliably indicates presence of Diethylstilbestrol (DES), a synthetic estrogen, as well as that of 17β-Estradiol (E2), a natural estrogen. To our knowledge this is the first time a human gene has been successfully expressed in Daphnia, showcasing the potential to test the response of different human genes to environmental stimuli relatively directly, and therefore determine potential health impacts in both animals and humans.

Results
The genetic sequence for genomic integration was prepared on two separate DNA plasmids. One expresses the human estrogen receptor α (hERα) (Fig 1A) ubiquitously. The other plasmid ( Fig 1B) contains 4x repeats of the Estrogen Response Element (ERE), mCherry as a visible reporter, and the EF1α1 3'UTR truncated to 60 bp plus its poly(A) signal for higher RNA stability and/or translation efficiency [8]. The sequence between the ERE repeats and the mCherry start codon is the same as that between the EcRE (Ecdysteroid response element) repeats and its reporter start codon in a previously established ecdysteroid reporter in this species [9]. The 4xEcRE reporter was used as a positive control in this experiment ( Fig 1C).
To test the functionality of these plasmids, microinjection was conducted with wild-type (NIES) Daphnia eggs. Fifty ng/μL pRC21-hERa plasmid, 50 ng/μL pRC21-ERE:mCherry plasmid and 26.8 ng/μL (100 μM) DES were injected as single solution, in combinations of two or all three together. When only 1/3rd (data not shown) or 2/3rd of the components were injected (Fig 1E-1G), no red fluorescence could be detected after 18 h, similar to uninjected control eggs ( Fig 1D). When injecting the two plasmids together with an estrogenic compound, DES, on the other hand, fluorescence could be detected (Fig 1H), as well as after injection of the pRC21-EcRE:mCherry control plasmid that responds to endogenous ecdysteroids ( Fig 1I). Thus hERα is functional and active in an estrogen-dependent manner in D. magna embryos. We also concluded that neither hERα nor ERE are activated by endogenous compounds in D. magna eggs of this stage.
For genomic integration, we targeted the dsred2 locus of a previously generated transgenic animal [6]. To make the donor plasmid DNA, the hERα expression cassette and ERE reporter gene were cloned into a single plasmid with the target site of TALENs that cleave the dsRed2 gene [6]. Integration of the donor DNA into the DsRed2 locus causes the loss of red fluorescence in absence of estrogenic compounds. We injected this donor plasmid (25 ng/μL) together with in vitro synthesized mRNAs that code for DsRed2-targeting TALENs (250 ng/μL each). Of the 106 embryos surviving at 1 hpi, one (0.94%) that showed a loss of red fluorescence survived into adulthood. We named this transgenic animal "ES line". We then exposed neonate Daphnia of this ES line (under 24 h old) to 2 mg/L DES for seven days to find the optimal time-point for exposure evaluation. According to the values of total fluorescence of the thoracic appendages, day four showed the highest difference between control and exposed individuals (Fig 2) so all later exposures were conducted for four days. When testing this line for sensitivity regarding DES and E2, we exposed them to 0.1 mg/L-2.0 mg/L DES and 0.1 mg/L-4.0 mg/L E2. In these experiments, the detection threshold for DES was 0.5 mg/L and 4 mg/L for E2 (see Fig 3, Fig 4).

Discussion
The new estrogen responsive line of transgenic ES D. magna could successfully detect both DES and E2 at different concentrations and with different sensitivity, in accordance with their different binding affinities to hERα [16]. There was no fluorescence response without any exposure of estrogenic compounds even though the control EcRE-reporter gene [9] could detect endogenous ecdysteroids (Fig 1). The general background fluorescence in the transgenic Daphnia was relatively low so it is sensitive enough to detect estrogenic chemicals four days after exposure (Fig 2). This result is consistent with previous finding that Daphnia do not have any endogenous estrogens nor any ERs [17]. This study is the pioneering demonstration that hERα can function in Daphnia in vivo.
However it was notable that sensitivity of the transgenic Daphnia to estrogen was ranging in the low mg/L. Compared to existing biosensors for estrogen like zebrafish and medaka [18,19] or yeast, bacteria, and mammalian cell cultures [20][21][22], the sensitivity of this line was quite low. One of the future perspective of the use of the estrogen sensing Daphna may be an application to environment assessment or monitoring. In order to detect low concentration of estrogens in the ng/L ranges as a biosensor, which is where serious environmental effects can already occur [23][24][25], further improvement is necessary. This sensitivity could be improved by improving several points. One of the major problem may be a cofactor which bridges estrogen receptor and transcriptional machinery. In order to monitor Daphnia nuclear receptor (ecdysone receptor) function in mammalian cells, we have introduced Taiman as a cofactor, which suggested that direct interaction between daphnia nuclear receptor and mammalian transcriptional machinery is weak. Similarly interaction between human nuclear receptor and daphnia transcriptional machinery may be weak and introduction of a cofactor may improve its sensitivity. It may be also useful for testing further iterations of the sequence between ERE and the mCherry start codon [26], by optimizing codon usage for Daphnia [27][28][29], or by integration into a different genetic location.
Red fluorescence was expected and found all over the body of the exposed waterfleas, with stronger fluorescence in digestive tissues from feeding and the hepatopancreas which is involved in sequestering hormones taken up by feeding [30]. Stronger fluorescence was also detected in the "joints" of secondary antennae which are responsible for locomotion.
To express hERα we used the promoter of D. magna EF1a1 which produces highly abundant mRNA in this species [31], which could easily lead to over-expression of hERα. And indeed reproduction was slowed down in the ES line compared to both a wild-type strain of D. magna (NIES) and the dsred2 line. Therefore particularly the EF1α1:esr1 sequence might need to be adjusted to lower expression levels by truncating the promoter sequence or by using promoters of the other ubiquitously expressed genes such as ribosomal protein L32 and β-actin, which show moderate expression compare to EF1.
Nevertheless, both hERα and ERE could be shown to be functional in Daphnia for the first time, suggesting a huge potential for use of Daphnia to study the interaction of human genes with environmental factors like the effect of EDCs in this study. This could be applied to improved biomonitoring of water quality, or even to screen potentially useful chemicals.
Daphnia cells can conduct the post-translational modifications necessary for formation of active hERα [32], as shown by the estrogen-dependent fluorescence response of ES line Daphnia from both active hERα and its successful activation of the ERE. These results open up the possibilities to test not only single receptors or other genes in Daphnia, but partial or even whole pathways for environmental effects on them, especially with still improving methods for genome editing in this species that allow for larger and larger sizes of inserted DNA [10,33].
It would be interesting to cross this line with other transgenic lines of Daphnia and visualize more than one stressor at a time. And to increase ease of use, it would be beneficial to implement a reporter that is not based in fluorescence but that can be detected by the naked eye or a simple light microscope. Over-expression of hemoglobin and the subsequent redder color of D. magna [34,35] is a promising approach, as is black color from expressing melanin or other darker pigments which are usually only found in Daphnia species exposed to higher UV radiation like Daphnia melanica [36,37].
In conclusion, for the first time, a human gene (esr1) was successfully and stably expressed in the crustacean D. magna. The resulting new transgenic line of D. magna could indicate presence of both DES and E2 after exposure in a dose-dependent manner.

Daphnia strain
A transgenic line of Daphnia magna containing a hemizygous DsRed2 gene under the control of the D. magna EF1α-1 promoter was generated previously [6] from a D. magna NIES clone (obtained from the National Institute for Environmental Studies, NIES; Tsukuba, Japan). This dsred2 line has been maintained for more than 50 generations. It exhibits ubiquitous DsRed2 expression.

Daphnia culture conditions
Eighty neonates of dsred2 Daphnia (under 24 h old) were cultured in 5 L of the Aachener Daphnien Medium (AdaM) [38] at 22-24˚C under a light/dark cycle of 16/8 h. The culture medium was renewed once a week. Daphniids were fed every day with 5 mg of Chlorella vulgaris (Nikkai Center, Tokyo, Japan) during the first week. After maturation, offspring was removed daily and adults were fed with 10 mg Chlorella per day.
After establishment of the estrogen responsive line (ES line), ES daphnia were cultured under the same conditions with the exception of feeding and juvenile removal. ES daphniids were fed every other day with 6 mg of Chlorella vulgaris (Nikkai Center, Tokyo, Japan) until maturation. Then, offspring was removed twice a week and adults were fed with 10 mg Chlorella every other day.
All methods regarding animal use were carried out in accordance with the relevant guidelines and regulations.

Microinjection
Microinjection was conducted according to established procedures [39]. In short, adult D. magna (dsred2 line) with empty brood chambers were selected and observed until ovulation started. Then D. magna were transferred to ice-chilled M4 medium [40] containing 80 mM sucrose (M4-suc) and dissected to collect the eggs within the brood chamber. The eggs were stored in ice-chilled M4-suc medium until injection to slow the hardening of the egg membrane. Microinjection was performed within 1 hour post ovulation (hpi) for the same reason. Successfully manipulated eggs were transferred to fresh medium and were cultured in a 96-well plate at 23 ± 1˚C. From each clutch of eggs, 2-3 eggs that were not injected served as control for development.
To generate the EcRE reporter plasmid, full length mCherry and the first 60 bp of EF1α-1 3'UTR [8] were joined into a pRC21 backbone containing the last 13bp of EF1α-1 3'UTR and therefore providing a poly(A) signal via InFusion (TAKARA). A 4xEcRE promoter [9] was amplified via polymerase chain reaction (PCR) with primers introducing a restriction site for MscI. Both the PCR fragment and the mCherry plasmid were digested with MscI and EcoO109I (NewEngland BioLabs, Ipswitch, MA, USA) and joined via MightyMix ligation (TAKARA); the resulting construct was termed pRC21-EcRE:mCherry. To generate the ERE reporter plasmid, the EcRE repeats were excised out of pRC21-EcRE: mCherry with EcoO109I and XmaI (NewEngland BioLabs). A 4xERE sequence [42] was amplified with primers introducing a restriction site for XmaI, it contains an EcoO109I site. This PCR fragment was also digested with EcoO109I and XmaI (NewEngland BioLabs) and joined into the backbone plasmid via MightyMix ligation (TAKARA); the resulting construct was termed pRC21-ERE:mCherry.
For genomic integration, pRC21-hERa as the backbone was digested with SalI and NdeI (NewEngland BioLabs). pRC21-ERE:mCherry was digested with BssHII and NdeI (NewEngland BioLabs). The dsred2 TALE site was amplified from genomic DNA with PCR primers introducing digestion sites for SalI and BssHII. After digestion, all three fragments were joined via MightyMix ligation (TAKARA); in the resulting construct the ERE reporter and the ER halves face opposite directions, it was termed pRC21-estrogensensor.
All plasmids were transformed into XL10-GOLD E. coli after ligation, harvested with a PureYield Miniprep kit (PROMEGA, Fitchburg, WI, USA), purified by phenol/chloroform extraction followed by ethanol precipitation and their sequence was confirmed by sequence analysis.

In vitro RNA synthesis
For TALEN mRNA synthesis, left and right TALEN expression plasmids [6] were linearized with Acc65I (NewEngland BioLabs), and purified using the QIAquick PCR purification kit (QIAGEN GmbH, Hilden, Germany). Linearized DNA fragments were used for in vitro transcription with the mMessage mMachine kit (Life Technologies, CA, USA). Poly(A) tails were attached to TALEN RNAs using a Poly(A) Tailing Kit (Life Technologies), following the manufacturer's instructions. The synthesized RNAs were column purified using the Rneasy Mini Kit (QIAGEN GmbH, Hilden, Germany), followed by phenol/chloroform extraction, ethanol precipitation, and resuspension in DNase/RNase-free ultra pure water (Life Technologies).

Exposure
Diethylstilbestrol (DES) (Sigma Aldrich, St. Louis, MO USA) and 17β-Estradiol (E2) (Sigma Aldrich) were dissolved in 100% N,N-Dimethylformamide (DMF) (Nacalai tesque, Kyoto, Japan) to final concentrations of 10 mg/mL as stock solutions. For exposure, the stock was diluted with AdaM [36] to final solvent concentrations of under 0.2%. For exposure, neonate daphniids (under 24 h old) were kept in 24 well plates (Thermo Fisher Scientific, Waltham, MA USA), one individual in 2 mL medium per well, at 23 ± 1˚C under a light/dark cycle of 16/ 8 h, with medium renewal every day.

Fluorescence microscopy
Daphnia were partially immobilized in minimal amounts of medium on micro slide glasses (Matsunami, Osaka, Japan). Red fluorescence intensity was recorded with a color digital camera (Leica DC500) mounted on a Leica M165C fluorescence microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) equipped with a 545 nm excitation and a 620 nm barrier filter. The pictures were taken under 63 × magnification with 100% aperture, 1 s exposure, 3.0 gain, 1.5 saturation, and 1.0 gamma. The red fluorescence intensity of neonates was recorded every 24 h in the first exposure experiment, at day 4 of exposure henceforward.

Fluorescence quantification
Fluorescence intensity (fluo) was quantified using ImageJ software. Previously reported methods [8,43,44] were adapted to reduce background interference. In this study, only the area of thoracic appendages (thorap) was used for calculations with the following formula.
FluoðthorapÞ ¼ total f luorescence of thoracic appendages À ðnumber of pixels of the selected area Â mean of three background fluorescence measurementsÞ This value was then normalized for exposed Daphnia by defining the value of unexposed Daphnia of the same age as 1.