Diffusion of small molecules into medaka embryos improved by electroporation
© Jung et al.; licensee BioMed Central Ltd. 2013
Received: 14 February 2013
Accepted: 17 June 2013
Published: 1 July 2013
Diffusion of small molecules into fish embryos is essential for many experimental procedures in developmental biology and toxicology. Since we observed a weak uptake of lithium into medaka eggs we started a detailed analysis of its diffusion properties using small fluorescent molecules.
Contrary to our expectations, not the rigid outer chorion but instead membrane systems surrounding the embryo/yolk turned out to be the limiting factor for diffusion into medaka eggs. The consequence is a bi-phasic uptake of small molecules first reaching the pervitelline space with a diffusion half-time in the range of a few minutes. This is followed by a slow second phase (half-time in the range of several hours) during which accumulation in the embryo/yolk takes place. Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules. Testing electroporation we could establish conditions to overcome the diffusion barrier. Applying this method to lithium chloride we observed anterior truncations in medaka embryos in agreement with its proposed activation of Wnt signalling.
The diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion. These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos.
Japanese medaka (Oryzias latipes) are small egg-laying freshwater fish that are native to brackish waters and rice paddies in South-East Asia. Economic husbandry, high fecundity, and ex utero development make them a popular vertebrate model organism in developmental biology and molecular genetics and their transparent chorion facilitates non-invasive observation. Furthermore, they are very hardy and highly resistant to common fish diseases (reviewed in ). These properties make medaka ideal for testing of toxic substances [2–5].
Toxicity tests are conducted to evaluate the adverse effects of chemicals or biological substances on organisms and are mainly performed by animal experiments. Even if testing of cosmetic and personal care products has been reduced over the last years, there remain numerous chemicals, such as pharmaceuticals or food additives, where animal tests are necessary. On the other hand, several programs like the OECD HPV (High Production Volume) Program or the European Union REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) initiative were introduced to regulate the chemical industry globally (reviewed in ). They require extensive test regimes for most chemicals. Without further alternatives, these new regulations would therefore dramatically increase the number of animal experiments, particularly for mammals and birds.
Historically, fish toxicity testing plays an important role in ecotoxicology and aquatic toxicology. It is however a controversial question, whether adult fish can be a replacement for mammals and birds, because they may experience the same levels of pain and distress. One alternative is the use of embryos [7, 8]. In the fish embryo toxicity (FET) test, newly fertilized eggs are exposed to a chemical for 48 hours and various lethal and sub-lethal endpoints are recorded as described by Lammer and colleagues . Due to their high fecundity and the synchronous extra-uterine development of the transparent embryos, fish are particularly well suited for such a strategy. Most FET tests have been conducted with zebrafish but inter-species comparison with fathead minnow and medaka demonstrated applicability also for other species . Furthermore, fish embryos are suitable for adaption to high throughput protocols .
Contrary to zebrafish, medaka embryonic development is relatively long, thus providing extended time for testing. In combination with their hardiness, medaka embryos therefore represent an ideal system for toxicologic/pharmacologic testing. During FET tests the substances have to diffuse into the developing embryo. Medaka embryos are surrounded by an acellular envelope, the chorion, which protects the developing embryo from environmental influences. Once fertilized, the chorion turns into a rigid structure  and might therefore act as an effective barrier that protects the embryo also against chemicals.
Compared to zebrafish, we found a substantially reduced sensitivity for chemicals like lithium chloride for medaka. Closer inspection revealed a limiting diffusion rate of membranes positioned closely to the embryo. Contrary to our expectations, the rigid outer chorion is readily passed by small molecules. In an effort to overcome these problems, we tested the addition of detergents. This effectively improved diffusion, but also affected the internal distribution of the chemicals. Electroporation also enhanced the uptake of fluorescent tracer molecules. When applied to Wnt-signalling, electroporation facilitated the transfer of the GSK-3 inhibitor lithium, thereby inducing deficiencies in medaka anterior-posterior development, similar to those reported for zebrafish .
Fish stocks and maintenance
Japanese medaka from the wild-type cab strain were used for this study. Adult fish were maintained at 26°C with an artificial 14 hours light and 10 hours dark cycle. Stages were determined according to Iwamatsu .
Dechorionation using hatching enzyme
For dechorionation, the embryos were placed in a small drop of water on a plastic surface. Excess liquid was removed and hatching enzyme was added (10 μl hatching enzyme per 15 embryos). The embryos were incubated at 27°C until holes appeared in the chorion. The remaining chorion was removed using forceps and the embryos were kept in ERM (17 mM NaCl; 0.4 mM KCl; 0.27 mM CaCl2; 0.65 mM MgSO4) in dishes coated with agarose.
Hatching enzyme was prepared as follows: embryos with visible hatching glands were homogenized in pre-cooled PBS (0.75 μl PBS per embryo), incubated over night at 4°C and debris was removed by centrifugation (15.000 rpm, 4°C, 10 minutes). The hatching enzyme solution was stored in aliquots at −20°C.
Sodium fluorescein (Roth; 10 mg/ml), rhodamine B (Roth; 10 ng/ml) and acridine orange (Roth; 1 μg/ml) solutions were prepared in 1x Yamamoto’s medium (0.128 M NaCl; 0.27 mM KCl; 0.14 mM CaCl2; 0.24 mM NaHCO3). Four embryos were incubated in 50 μl staining solution for 40 minutes (if not mentioned otherwise) at 27°C protected from light. Standard post incubation washes were performed with 0.5 ml ERM at room temperature on a shaker. The embryos were washed 3 times with fresh ERM in the first seven minutes and subsequently 10, 20, 30 and 60 minutes after the incubation.
For yolk injections, sodium fluorescein (10 mg/ml) was injected into the yolk using a microinjector (Eppendorf) and borosilicate glass capillaries (Clark Electromedical Instruments).
For methylene blue diffusion, 0.001% methylene blue in 1x ERM was used. The embryos were incubated for 10 minutes at 27°C. Standard washing steps were performed as described above.
Quantification of fluorescence intensity
Embryos were examined in vivo by fluorescence microscopy using a Nikon Eclipse TS100 inverted microscope equipped with an Infinity 2 camera. Pictures with defined exposure times were taken and the fluorescence intensity was quantified from the pictures with the ImageJ software . The quantification of internal concentrations was performed by measuring the fluorescence intensity at different time points of washing and extrapolating back to time zero (start of washing).
As a reference we used empty cellulose sulphate beads with an average diameter of 730 μm , which were soaked with different concentrations of the fluorescent dyes for several days to reach equilibration. The beads were shortly washed and then transferred into mineral oil, where they were immediately quantified as described above.
Electroporation of medaka embryos was performed in 1× Yamamoto’s medium. Prior to electroporation the embryos were pre-incubated with the staining solution for 40 minutes in Gene Pulser® disposable Cuvettes with 0.4 cm gap width (Bio-Rad) at 27°C and then electroporated in the same cuvettes. For better comparison, the control experiments were also performed in cuvettes omitting the electroporation pulses. Electroporation was performed with a home-made device consisting of a function generator (sine wave with added DC-voltage and variable modulation depth), a pulse generator (generating a defined number of pulses with varying length), a switch (regulated by the pulse generator, controlling the output of the function generator to the amplifier) and an amplifier connected to the cuvette filled with 100 μl 1× Yamamoto’s medium. An oscilloscope was used to display the signals. The conditions for the experiments were: modulation frequency 1 to 105 Hz, modulation depth 1; voltage 5 to 50 V; 1 to 3 pulses with intervals of varying length.
For the experiments mimicking chorion diffusion alone, dead embryos were prepared as follows: fifty embryos at stage 17 were electroporated in 400 μl 1× Yamamoto’s medium using 3 pulses of 60 ms with 200 ms interval, 60 V, 0.5 A and 35 kHz, followed by 30 minutes incubation in 1× ERM at 27°C.
Embryos at stage 14 were incubated in 100 μl 0.4 M lithium chloride (Roth) solution for 10 minutes at 27°C, followed by electroporation using the following settings: modulation frequency 330 Hz; voltage 15 V; pulse time 100 ms; 1 pulse. After diffusion and electroporation the embryos were washed as described before. Embryos were examined in vivo by light microscopy using a Zeiss Stemi 2000-C microscope equipped with an AxioCam HRc camera.
We previously tested the effects of the Wnt signalling pathway on medaka anterior-posterior development by overexpressing wnt1. Another well-established way to activate this pathway is the application of the GSK-3 inhibitor lithium . However, when we used conditions established for zebrafish embryos  no effects could be observed. Except at very high concentrations (above 1 M) where a low number of embryos showed phenotypes. We therefore reasoned that the diffusion through the chorion into the embryos was not efficient. In order to understand this problem in more detail and to find possible solutions, we started analysing the diffusion properties of the embryo’s chorion and membranes. As small and easily quantifiable molecular tracers we selected fluorescing substances.
Diffusion into medaka embryos
We compared several fluorescing small molecules. Depending on their molecular properties, they were effective at variable concentrations and differed in their distribution within the egg (embryo/yolk). We finally selected fluorescein, which showed enrichment within the embryo (see below) and therefore appeared well suited as a model for small molecules affecting embryonic development. To quantify the uptake, we compared fluorescence spectroscopic determination of egg extracts and quantification of live embryos under a fluorescence microscope (measurement of pixel intensity of microscopic pictures, for details see Materials and Methods). Although the spectroscopic measurements were more sensitive, we selected the microscopic determination of fluorescence intensity as a standard quantification method. This allowed us to follow the same individuals during different stages of development and thereby to qualify tissue distribution, phenotypic alterations and survival of the embryos.
We also looked at the fate of fluorescein. For this, embryos at the 1-cell stage were treated as described before, but the fluorescence signal was determined after 4 days of incubation in ERM. This extensive washing removed the majority of fluorescein. However, residual fluorescence was detected in the gallbladder and the liver. Even in bright field pictures (Additional file 1) yellow staining of fluorescein can be seen in the gallbladder. Therefore, the fate of fluorescein in older medaka embryos is comparable to that of mammals .
Diffusion barriers within the medaka egg
Based on the bi-phasic out-diffusion observed for fluorescein, we questioned which structures of the medaka egg are responsible for the diffusion kinetics. The egg is shielded by a rigid acellular envelope, the chorion, which represents a prime candidate for a diffusion barrier . However, inside the chorion the embryo and the yolk are covered by additional extra-embryonic membrane systems, which could also affect diffusion. In order to differentiate between these possibilities we manipulated the eggs. In eggs with dead embryos the yolk and membranes shrink to a small ball within the chorion (Figure 2B,C). The quantified fluorescence signal in the egg therefore mainly depends on the diffusion through the chorion. In order to qualify the diffusion properties of the membrane systems covering embryo and yolk, we used hatching enzyme to dechorionate the eggs (Figure 2B,D; for experimental details see Methods).
Quite unexpectedly, diffusion of fluorescein into dead embryos resulted in strong fluorescence (Figure 2C’), indicating that the chorion is readily passed by small molecules. Quantification of the signals (Figure 2E, for details see Methods) showed that the interior concentration almost reached saturation after 40 minutes (50% after 20 minutes). On the other hand, diffusion into dechorionated embryos was extremely slow, resulting in an average concentration similar to that for intact eggs (<0.1%) after 40 minutes (some of the dechorionated embryos showed high signals, which was however due to disruption of the fragile membranes). Therefore, not the chorion, but membrane systems within the medaka egg represent the main diffusion barrier for small molecules. These data would predict that diffusion of small molecules into intact embryos should rapidly proceed through the chorion into the perivitelline space, but would then be stopped at inner membranes. To test this hypothesis also with life embryos, we looked at eggs shortly exposed to fluorescein (10 minutes), which should be sufficient to reach high signal levels in the perivitelline space, but not in the embryo. Indeed, after a short washing step (2 minutes) the expected distribution was visible (Figure 2F-F”). Based on the previous experiments (Figure 1), the cells of the embryo should however show a signal when exposed for longer times (40 minutes). After a short washing step fluorescein should therefore be detectable in both the perivitelline space and the embryo (Figure 2G-G”; white arrowhead and arrow, respectively). During extended washing fluorescein should rapidly disappear from the perivitelline space (fast out-diffusion), but a signal would be retained in the embryo (slow out-diffusion). The expected results could be observed (Figure 2H-H”). In order to verify this model also with another small molecule we tested methylene blue. As seen for fluorescein, methylene blue rapidly (after 10 minutes of incubation) accumulated in the perivitelline space (Additional file 2A). Longer washing times (20 minutes) quickly removed methylene blue again, but no signals were detectable in the cells of the embryo (Additional file 2B).
An internal diffusion barrier is also in perfect agreement with the bi-phasic out-diffusion profile (Figure 2A). The first phase represents loss of fluorescein from the perivitelline space (line a) and the second phase (line b) is caused by the considerably slower diffusion out of the embryo/yolk (resulting in a low steady state concentration in the perivitelline space). The half-time calculated for the initial out-diffusion (4 minutes) roughly fits to that of in-diffusion through the chorion (20 minutes; a factor of 5 differing the values can be explained by the larger interior volume of dead embryos compared to that of the perivitelline space). Therefore, the slow diffusion of small molecules into medaka embryos is caused by membrane systems close to the embryo and dechorionation does not improve the uptake.
Detergents affect membrane behaviour
In an attempt to overcome this diffusion barrier, we first focussed on the injection of the small molecules. Injection into single cells of the embryo is well established for early stages of development, leading to a rapid distribution in all cells of the embryo. During later development injection into large internal cavities, like the neural tube, would be an option. However this is again limited to certain stages. In order to accomplish uniform diffusion into the embryos, we tested injection into the yolk, a method that is often performed in zebrafish. Within 30 minutes the injected fluorescein evenly distributed throughout the yolk (Additional file 3A-C and F-G). However, the detection of signals in the embryo was only possible after 4 hours (Additional file 3D,K) and the obtained concentrations were low. Furthermore, yolk injection in medaka results in low reproducibility, due to rapid clogging of the injection needles by the sticky yolk.
Electroporation improves small molecule uptake into embryos
Lithium induces deficiencies in anterior-posterior development
Several studies have been performed to qualify the effect of small molecules on fish embryos [21–23]. In addition, toxicological applications like the FET test are discussed as an alternative to experiments with mammals and birds . The uptake of substances by the embryos has been recognized as a critical factor in these experiments [24, 25]. However, little is known about the diffusion properties of the chorion and other membrane systems covering fish embryos. We used fluorescing substances as a model to study the diffusion of small molecules into medaka embryos. Combined with fluorescence microscopy quantification this offers a number of advantages over conventional techniques like radioactive labelling. Firstly, individual embryos can be followed over the time and quantified repeatedly. Secondly, the internal distribution of the molecules in the embryo due to the physicochemical nature of the molecules can be examined and compared at different stages of development. Alterations in the compartment specific distribution, as for example after incubation with detergents (Figure 3), are highly important for the assessment of pharmacokinetic properties of drugs. Thirdly, metabolic processing can be followed (e.g. as for the accumulation of fluorescein and acridine orange in the liver/gallbladder). And finally, the distribution in different compartments due to variable diffusion rates can be detected, as the accumulation in the perivitelline space (Figure 2F-H”).
We used fluorescein, rhodamine B, acridine orange, and lithium chloride for our experiments. They represent small molecules which cover a large spectrum of different properties from charged (lithium chloride) to non-charged (acridine orange). The substances exhibited varying distributions in medaka eggs, indicating differences in their hydrophobic properties (rhodamine B being most strongly enriched in the yolk). Nevertheless, all substances showed low diffusion rates into medaka embryos. In contrast to zebrafish, medaka eggs contain a hard chorion. Based on suggestions from the literature , we therefore suspected the chorion to represent a diffusion barrier, shielding the embryos from their chemical environment. Following sperm entry, a calcium wave moves along the surface and the chorion matures , thereby reaching maximum hardness approximately 6 hours after fertilization . We therefore expected a dramatic reduction of the diffusion rate after chorion hardening. However, no differences appeared in the experiments (Figure 1E). As the chorion permeability is connected to calcium ions [27, 28] we incubated the embryos right after fertilization in a calcium-free medium , with different pH values and even tried incubation in distilled water. However, the uptake of fluorescein into the embryos did not improve (data not shown). Therefore, conditions affecting chorion hardening did not alter the diffusion rates into the egg, neither did dechorionation of the embryos (Figure 2D’, E). Instead we observed a rapid diffusion through the chorion into dead embryos (Figure 2C’.E). The fact that inner membrane systems and not the chorion represent the main diffusion barrier in medaka eggs was further supported by the fast accumulation of signals in the perivitelline space (early in-diffusion; Figure 2F-F”) and the rapid loss of this signal during out-diffusion (Figure 2G-H”). On the contrary, accumulation in the embryo needed extensive in-diffusion times, but once achieved stayed considerably longer than that of the perivitelline space (Figure 2F-H”). The result is a bi-phasic progression of diffusion (Figure 2A), caused by a strong diffusion barrier positioned closer to the embryo than the chorion. During blastula stage, the egg can be subdivided into three distinct cell lineages. One lineage consists of the pluripotent deep layer blastomeres (DEL), which produce the future embryo proper. The second domain consists of a syncytium of multiple nuclei, called the yolk syncytial layer (YSL). The third domain is the envelope layer (EVL), a thin cell layer which covers the DEL and will eventually form the periderm . Both the second and the third domain are extra-embryonic. According to our results, the EVL/periderm would be a candidate to block the diffusion into the egg.
These results have important implications for pharmacologic and toxicologic experiments with medaka embryos. Due to the bi-phasic diffusion process, the substance is initially enriched in the perivitelline space (diffusion half-time in the range of a few minutes). Subsequently, the slow diffusion into the embryo/yolk starts (diffusion half-time in the range of several hours). Therefore extended exposure to the test substances is necessary to evaluate pharmacologic/toxicologic consequences on embryonic development. Due to the slow accumulation in the embryo, effects on the first hours of development (e.g. early teratogenic properties) will not be detectable without enhanced transfer.
Assuming that the main diffusion barrier is a membrane system, it should be possible to increase its permeability by addition of detergents. Indeed, addition of Triton X-100 improved the uptake of fluorescein dramatically, but severely affected the membrane systems of the embryo (see Figure 3). Also injection into the yolk turned out to be inefficient. A possibility to make membranes permeable is electroporation. We applied current shifted radio frequency pulses  and could optimize the conditions to substantially increase the dye uptake compared to diffusion alone. Also other electroporation techniques like nanosecond pulsed electric fields have been tested for this purpose . Such techniques would not only be of interest for small molecules, but also for the transfer of DNA or RNA. Indeed, Hostetler and colleagues initially proposed this technique to obtain transgenic medaka fish. In our hand the method worked effectively for small molecules, however, we failed with DNA. Closer inspection revealed that not even fluorescence labelled oligonucleotides (MW 6000) could pass the chorion, suggesting a size exclusion for the pores of the chorion in that range (data not shown). Electroporation did not improve this transfer. We also used DNA expression constructs containing gfp and luciferase for more sensitive assays, but failed to detect any marker gene expression in intact electroporated embryos. Only after dechorionation we could successfully transfer DNA into the embryos by electroporation (detection of gfp expression; data not shown). However, the survival of dechorionated embryos is extremely low during electroporation, making the method inapplicable for routine experiments. Injection of DNA into the perivitelline space and subsequent electroporation would be an option. However, direct injection of DNA into the zygote represents a simple alternative.
Having established electroporation for the uptake of small molecules into the living medaka embryo, we then returned to our initial question, the application of lithium to medaka development. The effects of lithium on developmental processes have been shown in several organisms. Already more than 60 years ago it was known that amphibian embryos developed severe anterior truncations when exposed to lithium during gastrulation (reviewed in ). This dorsalization effect was explained by the finding that lithium inhibits GSK-3β, a key component of the Wnt signalling pathway [32, 33]. Indeed, diffusion and subsequent electroporation led to axis truncations in medaka embryos, similar to those observed upon ectopic expression of wnt1. The embryos either failed to develop anteriorly to the midbrain (Figure 7D,G), lacking eyes and the forebrain (strong phenotype), or they developed eyes with reduced size (Figure 7C,F; weak phenotype). Electroporation therefore effectively improves the transfer of small molecules into medaka embryos. Hence, this method can be used to enhance the uptake of chemicals. Examples of such applications are specific inducers or repressors of signalling pathways to study embryonic development. For these experiments timing represents an important parameter. Slow diffusion and consequently slow accumulation prevents an exact timing of the effective concentration in the embryo. Electroporation strongly increases the internal concentration within a single step as exemplified for lithium chloride (the timing of Wnt signalling pathway activation critically affects the observed phenotypes; ).
During our experiments we found that membrane layers surrounding the medaka embryo represent a diffusion barrier for small molecules, whereas the hard outer chorion is readily passed. The slow diffusion has to be considered when toxicologic or pharmacologic experiments are performed with medaka embryos. A possible way to overcome this problem is electroporation, which substantially improves the uptake of small molecules. We thus were able to induce medaka embryos with the GSK-3 inhibitor lithium and could show that the resulting activation of the Wnt signalling pathway causes deficiencies in anterior-posterior development.
The work was supported by the Austrian Science Fund (FWF, grant P19571-B11) and the City of Vienna (MA23 – project 10-20).
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