Facilitation of embryo handling and development
One of the first challenges of developing this method was to establish in vitro conditions that permit drug delivery and support embryo viability subsequent to treatments that affect the eggshell. Conventional methods call for immersion in halocarbon oil to prevent desiccation. However, oil immersion is not compatible with delivery of aqueous solutes. Our initial attempts of incubating embryos in drops of culture medium or buffered saline solutions demonstrated a negative impact of the depth of the fluid on development, presumably due to limitations on oxygen delivery. We therefore devised a chamber that was optimal for immersion of the embryo in surrounding buffer while maximizing oxygen delivery. We modified a conventional home-made “nitex basket” used for the 50% bleach treatment in the dechorionation step (). These baskets are typically fabricated from the cut off top and screw cap of a 50 mL conical culture tube. The center portion of the cap is excised such that a patch of nitex screen can be screwed in place between the cap and the tube. When inverted, a “basket” is formed that can effectively retain embryos when dipped in a reservoir or washed from above. We modified the basket by forming four notches in the rim of the cap such that when the basket is placed in a reservoir solutes can exchange freely across the nitex (). When placed in a reservoir of 6mL of buffer in a 50mm plastic dish the buffer is retained from entering the basket due to surface tension at the nitex pores, while the reservoir level on the outside of the basket raises above the level of the nitex. Embryos placed on the nitex in the basket are seen to become two-thirds immersed in the buffer due to capillary action. When basket and reservoir are capped with an inverted beaker the reservoir evaporation is minimized and the embryo orientation in the fluid can be maintained for >48 hours without desiccation. The notches in the rim also facilitate removal of air bubbles from the underside of the nitex. We have previously shown dose-dependent delivery of the toxin methylmercury to embryos using this device (Rand et al. 2009
Alternatively, dechorionated and permeablized embryos can be arranged on slide chamber previously described by Kiehart ((Kiehart et al. 1994
), ). This slide allows for retention of embryos in a 0.3mm layer of medium between a glass coverslip and a DO membrane (Fondriest Environmental, YSI #5793) that facilitates oxygen delivery. This set up is optimal for time lapse imaging of live embryos using a microscope with confocal capabilities.
Limonene based solvents remove the waxy layer over the vitelline membrane
The Drosophila eggshell is comprised of three chorion layers, a waxy layer and the vitelline membrane (see ). Chorion layers are easily removed with sodium hypochlorite treatment with minimal adverse effects on the embryo (Hill 1945
; Strecker et al. 1994
). Permeability is almost entirely restricted by the waxy layer and thus we sought to optimize methods to remove this layer. D-limonene (hereafter referred to as limonene) is a monoterpene oil that is a major constituent of the oil in orange peels. Limonene has proven to be an exceptionally effective substitute for petroleum based solvents for removal of oily and waxy substances and has found numerous applications in industrial processes and in common household “citrus” cleaners. Limonene is a FDA registered GRAS (generally regarded as safe) substance approved for use as a food additive (Opdyke 1975
). Limonene exhibits minimal toxicity in a number of biological systems; however, it does elicit toxic responses at very high doses (IPCS 2009
; NTP 1990
). We rationalized that a limonene-based solvent would be a desirable substitute for the conventional heptane or octane applications used to remove the waxy layer. Furthermore, in the presence of suitable surfactants, limonene is miscible with water, which we predicted would eliminate the need to transition embryos in and out an organic phase to achieve waxy layer removal.
As a first approach, we evaluated the ability of the common household product Citrasolv® (Citra-Solv, Danbury, Connecticut) to render dechorionated embryos permeable. Citrasolv® consists of limonene, essential oils and biodegradable cleaning agents derived from coconut. To assess permeability, we incubated embryos treated with Citrasolv® in histological dyes of various molecular weights. In initial experiments, dechorionated embryos were immersed in a 1:10 dilution of Citrasolv® in water for five minutes, rinsed in phosphate buffer (PBS) and immersed in 0.1% solution of dye for 30 minutes. Citrasolv® treatment was highly effective in rendering the embryos permeable to cresyl violet (MW=321.3) Rhodamine B (MW=479.0) and Fast Green (MW=765.9, ). All of these dyes are excluded from untreated dechorionated embryos ( and data not shown).
Permeabilized embryos take up various dyes
Citrasolv® is composed of predominantly limonene and a proprietary mixture of surfactants and oils of which the composition is not certain. We therefore formulated a limonene/surfactant mixture of known composition. We surveyed several surfactants for their ability to render limonene miscible with water and the subsequent ability to permeablize dechorionated embryos. One optimal mixture consisted of 90% D-limonene, 5% nonionic ethoxylated alcohol and 5% coconut diethanolamide. We termed this composition EPS (embryo permeablization solvent). EPS proved to be equally effective as Citrasolv® in rendering embryos permeable to dyes (data not shown).
We then assessed the ability of EPS to remove the waxy layer of dechorionated embryos directly using scanning electron microscopy (EM). The waxy layer is a very thin (5 μm) coating over the vitelline membrane (Papassideri et al. 1991
). The waxy layer is formed by secretions from the follicle cells in the ovary and results in interleaved layers of flattened plaques of wax (, (Papassideri et al. 1993
)). This layer is removed through conventional fixation protocols for EM preparations. We therefore made preparations of dechorionated embryos, untreated and treated with EPS, omitting exposure to organic solvents and directly fixing in glutaraldehyde/paraformaldehyde and post fixing in osmium tetroxide. Scanning EM images of the control treated embryos revealed a roughened plaque-like coating of the surface consistent with the waxy plaques previously described (, (Papassideri et al. 1993
)). In contrast, EPS treated embryos showed a remarkably smooth and homogeneous surface, indicating removal of the waxy plaque layer ().
We further assessed the permeability of EPS treated embryos using a panel of fluorescent dyes. Rhodamine B is a brilliant red fluorescent dye that was seen to accumulate in the cytosol of EPS-permeabilized pre-blastoderm embryos as well as in the first cells formed at the blastoderm stage (). This distribution is consistent with the reported endosomal uptake of Rhodamine B (Vult von Steyern et al. 1996
). Remarkably, we found that Rhodamine B was excluded from the nucleus, and could be used to monitor progression of mitosis through time-lapse confocal microscopy (See and ). Calcein AM (MW=994.9) is a cell permeable dye that becomes fluorescent once cleaved by esterases in the cytosol of viable cells. Calcein is seen to label the cells in the periphery of a EPS-permeabilized blastoderm embryo (). Notably, pole cells in the posterior of the embryo are strongly labeled (). Syto® 11 is a cell permeable nucleic acid binding dye that is rendered fluorescent upon binding nucleic acids. Syto® 11 permeation is exemplified by the strong labeling of condensed chromosomes of pre-mitotic figures of an early embryo (undergoing nuclear division 13) (). In addition, Syto11 uptake can be seen in nuclei of cells at the periphery of an embryo undergoing gastrulation (). Together, these data demonstrate the efficacy of EPS to render the embryo shell permeable to a variety of dyes of various molecular weight and subcellular distribution properties.
Subcellular labeling in embryos with fluorescent dyes
Dorsal-GFP as a reporter for disruption of early embryogenesis
Manipulations to achieve various levels of eggshell permeability
With the objective of establishing optimal conditions of permeabilization and embryo viability, we investigated the effects of various conditions of EPS treatment on permeability. First, we observed that using fluorescence detection of Rhodamine B uptake could detect eggshell permeability at a much lower threshold than with light microscopy (). We initially observed that EPS treated embryos showed heterogeneous uptake of Rhodamine B (, ), presumably reflecting variability in permeabilization. We therefore assessed the effects of embryo age on the ability to permeabilize with EPS. We observed an age-dependent decrease in permeability whereby 0-2 hour embryos were readily permeabilized and 6-8 hour embryos were refractory to EPS permeabilization under the same time and concentration of treatments with EPS and Rhodamine B (). Intermediate levels of permeabilization were seen with embryos 2-4 hours and 4-6 hours old (). These data indicate a change in the properties of the vitelline membrane occurs over the first six to eight hours of development. In addition, a wide range of dye uptake was seen within each age group of embryos (e.g. ), suggesting individual variability in the permeability of eggshells of embryos of a similar age. To evaluate the effectiveness of EPS in these treatments we assessed Rhodamine B uptake in dechorionated embryos not treated with EPS. Unexpectedly, we found a substantial, albeit much lower, level of Rhodamine B uptake in 0-2 hour old embryos (). This “basal” level of Rhodamine B uptake was considerably reduced in 2-4 hour and 4-6 hour embryos () again indicating a developmental change in the vitelline membrane that excludes dye uptake.
Age dependent permeabilization of early embryos
The variable permeability among embryos of different ages calls for an ability to adjust EPS exposures to give more or less permeability. We therefore examined the effects of varying concentration and time of exposure of EPS. For these experiments, we chose to use 2-4 hour embryos. We see that a 1:10 dilution of EPS and exposure for one minute renders a majority of embryos permeable as judged by Rhodamine B fluorescence (). Increased EPS concentration (1:5 dilution) gives slightly more Rhodamine B uptake (). EPS dilution at 1:20 yields significantly less Rhodmaine B uptake than treatment with 1:10 dilution (). Increasing the time of EPS exposure yields greater permeability as seen by a high level of Rhodamine B uptake subsequent to EPS treatment for four minutes at 1:10 dilution (). A significant drop-off in permeability is seen with 1:20 and 1:40 dilutions of EPS with four minute exposures compared to 1:10 dilution (). These data demonstrate an ability to achieve a desired level of permeability though systematic adjustments to the time and concentration of EPS exposure.
Concentration and time dependent permeabilization with EPS
We next varied the time of incubation with Rhodamine B with the prediction that longer incubations would reveal a lower threshold of permeability. We used 0-2 hour embryos exposed to EPS at 1:10 dilution for 30 seconds. We observed a time dependent increase in Rhodamine B uptake with substantially higher levels of uptake seen at 15 minutes compared to five minutes or less exposure (). Again, we observed a considerable, yet lower level, uptake of Rhodamine B at 15 minutes in dechorionated embryos not treated with EPS () indicating a basal permeability of 0-2 hour embryos to this dye. The permeability revealed with extended Rhodamine B exposure occurs well below the level of detection of Rhodamine B with light microscopy ().
Rhodamine B staining, permeability levels and embryo development
Embryo viability and EPS permeabilization
We next determined the viability of permeabilized embryos under various conditions of ageing and EPS treatment. We focused initially on 0-2 hour embryo treated with EPS (1:10 dilution, 30 seconds). As described above, we were able to discern that this treatment permeabilized a majority of embryos at substantially higher levels than non-EPS treated embryos (). Embryos were incubated in nitex baskets in a modified basic incubation medium (MBIM, see methods) for 22 hours at 25°C and subsequently scored for development to Stage 17 (). This endpoint is easily discernable under light microscopy by appearance of fully-formed mid- and hindgut, or by completion of hatching as seen by empty vitelline membrane shells. EPS treated 0-2 hr embryos showed development between 47-56% as compared to non-EPS treated embryos that developed at 66% under these incubation conditions (). These data indicate that permeabilization of very early embryos results in a slight decrease in development. In addition, it is apparent from these data that Rhodamine B has no adverse effect on development (e.g. compare rates of development and Rhodamine B levels in ). It is likely the somewhat low development rate of control (dechorionated) 0-2 hr embryos (e.g. 66%, also see ) is due to the permeability we observe at this stage. Culturing 0-2 hr dechorionated embryos under halocarbon oil, common in other protocols, may compensate for this permeability and account for higher rates of development seen in other applications.
To test the effect of embryo age on viability we examined 4-6 hour embryos treated with 1:10 dilution of EPS for two minutes to achieve a similar level of permeability as seen with 0-2 hour embryos treated for 30 seconds. Monitoring the Rhodamine B fluorescence in this group we observed that permeability levels could be effectively judged by the presence of an anterior to posterior gradient of fluorescence intensity the 10-20 minutes immediately following dye exposure (). We found this “gradient” staining to be an indicator of optimal permeabilization and useful for manually sorting nearly equivalent permeabilized embryos. We sorted embryos of similar permeability into two groups: “low” and “high” permeability (). After 22 hours at 25°C all of the low permeability embryos developed to Stage 17 and three of seven embryos successfully hatched (). Six of the seven high permeable embryos developed to Stage 17 (). None of this latter group hatched, despite showing normal movements and contractions of the larva within the confines of the vitelline membrane (data not shown).
An unexpected finding was that Rhodamine B treated embryos exhibited bright green fluorescence after development, which corresponded with a reduction in the initial red fluorescence (). We predict that this results from metabolism of the Rhodamine B dye causing a spectral shift in its fluorescence. Rhodamine B (N,N,N’,N’-tetraethylrhodamine) is known to undergo de-ethylation which causes a shift in its excitation/emission properties (Qu et al. 1998
). Notably, bright green fluorescence is seen to concentrate in the mid- and hindgut in developed (Stage17) embryos ().
These unique properties of Rhodamine B dye permitted us to accurately assess viability and development of permeabilized embryos. In a batch preparation, we could score for both permeability and viability via the accumulation of green fluorescence in fully formed guts of developed embryos (). Using this endpoint, we assayed the development of embryos treated with EPS at two early developmental windows and subsequently incubated in nitex baskets at 25°C for 18-20 hours. Development subsequent to EPS treatment was compared to untreated, dechorionated embryos. Approximately 65-85% of untreated embryos 1.5-4 hours old develop to Stage 17 (). EPS treatment causes a slight reduction in the rate of development (compared to untreated embryos) with approximately 75% of 2.5-4 hour old embryos successfully developing to Stage 17 ().
Together, these data confirm that embryo permeability can be achieved that is compatible with embryo viability and development. These data also indicate that the level of permeability can be judged by the initial intensity and “gradient” profile of Rhodamine B uptake and subsequently by the elaboration of green fluorescence due to spectral conversion of Rhodamine B in living embryos.
Developmental endpoints for scoring drug effects in permeabilized embryos
By monitoring development under time-lapse imaging, we observed the highly dynamic pattern of blue auto-fluorescence of yolk proteins in the embryo. At preblastodem stages, a relatively uniformly dispersed blue fluorescence is seen across the embryo (, 0 hrs). Between 3-6 hours of development, this pattern changes drastically as the yolk proteins are pushed aside during germband elongation (, 3 and 6 hrs). At ~10 hours of development, the germband retracts and anterior and posterior extensions of the presumptive midgut fuse to complete the intestinal tract by 12 hours (, 10 and 12 hrs). Blue fluorescence is seen to consolidate in the lumen of the gut initially as one large mass in the newly fused midgut (, 12 hrs). The midgut undergoes three circular constrictions at Stage 16 (, 14hrs). The compartmentalized strong blue fluorescence in the midgut makes normally developed Stage 16/17 embryos easily discernable (, 14hrs).
An intrinsic fluorescent indicator of embryo development
The utility of the blue-autofluorescence endpoint in examining small molecule toxicity became clear with application of cycloheximide (CHX), a potent protein synthesis inhibitor and teratogen. EPS-treated 2-4 hour embryos were incubated in the presence or absence of 10μM CHX at 18°C for 20 hours (equivalent to 10 hours at 25°C) and examined under blue fluorescence. Control embryos consistently exhibited the characteristic 3-4 midgut compartments denoting development to Stage 16/17 (). In contrast, CHX treatment induced malformation as seen by the blue fluorescence centrally localized in a “ball” of tissue or dispersed anteriorly and posteriorly (). These results indicate the pattern of blue yolk auto-fluorescence can be used as an easily distinguishable endpoint to evaluate small molecule toxicity in embryonic development.
We next sought to determine the feasibility of using an early embryogenesis endpoint as an indicator of teratogenic effects. In separate experiments using flies carrying the GFP reporter for the Dorsal protein, we determined that CHX had profound effects on the morphogenetic movements of the embryo in the first 3-5 hours of development (data not shown). During these stages, formation of the cephalic furrow and germband elongation serve as landmarks that are easily identifiable under differential interference contrast (DIC) microscopy. We found that exposure of 2-3 hour permeabilized embryos to 10μM CHX for 1.5 hours results in disruption of cephalic furrow formation (). We took advantage of this easily score-able CHX phenotype to assess the efficacy of EPS in enabling drug delivery. Treatment with EPS resulted in ~70% of the embryos displaying permeabilization as determined by presence of Rhodamine B fluorescence (). This is in contrast to only 24% showing dye uptake without EPS treatment (). With CHX (10μM) treatment more than 80% of EPS treated embryos displayed an abnormal formation of the cephalic furrow. With omission of CHX roughly 17% of permeabilized embryos were abnormal (i.e. >80% were normal). The same degree of normal development was seen in non-permeabilized embryos. Furthermore, the same degree of normal development was observed in non-permeabilized embryos in the presence of CHX, indicating the EPS treatment is required for entry of CHX under these conditions (). A somewhat lower rate (65.3%) of normal development was seen in embryos that were not apparently permeablized in the preparation of EPS treatment and CHX exposure (). This likely reflects the lower levels of permeability not detected with the five-minute Rhodamine B staining used in this preparation. Together the data demonstrate that EPS treatment is an enabling step for delivery of CHX to the embryo.
EPS treatment enables drug delivery
Genetically encoded reporters for teratogenic effects
We have previously used green fluorescent protein (GFP) expression in the nervous system in transgenic flies as a reporter of abnormal neural development (Rand et al. 2009
). We sought to examine earlier markers of development. We turned to the Dorsal-GFP transgenic fusion construct reporter to examine the effects of the teratogen CHX. Signals acting through the Dorsal protein (the Drosophila NF-κB homolog) help establish the dorsal-ventral axis of the embryo (Moussian and Roth 2005
). Delotto, et al. (DeLotto et al. 2007
) demonstrated dynamic shuttling of Dorsal-GFP in and out of the nucleus with each mitotic division in the preblastoderm embryo. Using this Dorsal-GFP construct we see dynamic distribution of Dorsal-GFP in and out of the ventral nuclei of an EPS and Rhodamine B treated blastoderm embryos (). Exposure to CHX causes gross deformities in the pattern of nuclear divisions and Dorsal distribution in preblastoderm embryos (). In many cases, these early embryos are seen to stall in development. Occasionally, Dorsal accumulation in nuclei occurs at high levels in very early embryos when the syncytial nuclei first appear at the embryo surface (). Embryos affected at slightly later stages show irregular nuclear Dorsal accumulation () as well as varied sizes in the nuclei present (). Thus, the Dorsal-GFP construct serves as a robust readout for disruption of very early embryognesis events.
We next sought to examine a reporter for later stage embryogenesis and turned to the engrailed-GFP construct. The engrailed gene encodes a homeodomain transcription factor that is highly conserved in all metazoans (Gibert 2002
). Engrailed is a segment polarity gene whose expression marks the anterior boundary of each segment in the embryo (Hidalgo 1996
). Engrailed also functions in neural development to define developing brain regions and regulate growth of axons (Morgan 2006
). Expression of GFP in embryonic engrailed cells in a late stage embryo can be seen in . Exposure of EPS-permeabilized embryos to the teratogens cytochalasin D, methylmercury and cyclophosphamide results in grossly abnormal patterns of engrailed expression. Cytochalasin D is a potent inhibitor of actin polymerization and inhibitor of protein synthesis. Its teratogenic effects include exencephaly, hypognathia and neural tube defects (Shepard 1998
). We see that cytochalasin D induces a stalling of embryo development at Stage 11 with gaps in the engrailed band of cells in several segments (). In addition, we occasionally see a medial split in the engrailed bands in the anterior ventral portion of the embryo indicative of interruption of signaling events controlling ventral furrow and head formation (, lower panel). Methylmercury (MeHg) is an environmental toxin that preferentially targets embryonic neural development (Sanfeliu et al. 2003
). We see that MeHg similarly stalls the embryo at Stage 11 and disrupts the regularity of the engrailed banded pattern (). This is consistent with the known activity of MeHg in inhibiting protein synthesis, as well as being an inhibitor of cell migration. Cyclophosphamide has teratogenic effects giving rise to cleft palate and limb defects in mice and rats (Shepard 1998
). We see that cyclophosphamide results in an overall shortening of the embryo and generates breaks in the engrailed expressing bands of cells (). Thus, the engrailed GFP reporter demonstrates a robust readout of teratogenic activity in permeabilized embryos.
Engrailed-GFP as a reporter for teratogenesis