The effect of oxygen on the growth of Oncorhynchus mykiss embryos with and without a chorion

Growth was measured in dechorionated and chorionated embryos of rainbow trout Oncorhynchus mykiss exposed to three oxygen treatments: hypoxia, normoxia and hyperoxia (nominally 5, 10 and 15 mg l⁻¹). Dechorionated embryos grew faster than chorionated ones in all three oxygen treatments. Similar patterns, however, were found in both dechorionated and chorionated groups; embryos exposed to hypoxia grew less than normoxic and hyperoxic exposed embryos. Moreover, there was no significant interaction between the effect of oxygen levels and presence or absence of the chorion on growth after hatching. These results suggest that the effect of oxygen on growth was not affected by the presence of the chorion.     

Laser surgery of zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos using femtosecond laser pulses: Optimal parameters for exogenous material delivery,and the laser's effect on short- and long-term development

Background  

Femtosecond (fs) laser pulses have recently received wide interest as an alternative tool for manipulating living biological systems. In various model organisms the excision of cellular components and the intracellular delivery of foreign exogenous materials have been reported. However, the effect of the applied fs laser pulses on cell viability and development has yet to be determined. Using the zebrafish (Danio rerio) as our animal model system, we address both the short- and long-term developmental changes following laser surgery on zebrafish embryonic cells.     

Model of creation and evolution of stable electropores for DNA delivery

Electroporation, in which electric pulses create transient pores in the cell membrane, is becoming an important technique for gene therapy. To enable entry of supercoiled DNA into cells, the pores should have sufficiently large radii (>10 nm), remain open long enough for the DNA chain to enter the cell (milliseconds), and should not cause membrane rupture. This study presents a model that can predict such macropores. The distinctive features of this model are the coupling of individual pores through membrane tension and the electrical force on the pores, which is applicable to pores of any size. The model is used to explore the process of pore creation and evolution and to determine the number and size of pores as a function of the pulse magnitude and duration. Next, our electroporation model is combined with a heuristic model of DNA uptake and used to predict the dependence of DNA uptake on pulsing parameters. Finally, the model is used to examine the mechanism of a two-pulse protocol, which was proposed specifically for gene delivery. The comparison between experimental results and the model suggests that this model is well-suited for the investigation of electroporation-mediated DNA delivery.     

Photoporation of biomolecules into single cells in living vertebrate embryos induced by a femtosecond laser amplifier

Introduction of biomolecules into cells in living animals is one of the most important techniques in molecular and developmental biology research, and has potentially broad biomedical implications. Here we report that biomolecules can be introduced into single cells in living vertebrate embryos by photoporation using a femtosecond laser amplifier with a high pulse energy and a low repetition rate. First, we confirmed the efficiency of this photoporation technique by introducing dextran, morpholino oligonucleotides, or DNA plasmids into targeted single cells of zebrafish, chick, shark, and mouse embryos. Second, we demonstrated that femtosecond laser irradiation efficiently delivered DNA plasmids into single neurons of chick embryos. Finally, we successfully manipulated the fate of single neurons in zebrafish embryos by delivering mRNA. Our observations suggest that photoporation using a femtosecond laser with a high pulse energy and low repetition rate offers a novel way to manipulate the function(s) of individual cells in a wide range of vertebrate embryos by introduction of selected biomolecules.     

New method to deliver exogenous material into developing planarian embryos

The ability to report or modify the embryological processes in living embryos is pivotal for developmental biology research. Planarian embryology has experienced renewed interest as the genetic pathways that drive adult regeneration were found to be involved in the development of embryos. The major drawback to the study of planarian embryology is the absence of methods that give access to the embryos and enable their manipulation. Herein, we report on a novel method for delivering external material into developing embryos using nanosecond laser pulses. When focused on the eggshell surface under optimal parameters, laser pulses ablate the protective case and open a pathway throughout which foreign material can be delivered. In this study, we used egg capsules from Schmidtea polychroa (Schmidt, 1861) to microinject 1 microm fluorescein isothiocyanate fluorescent beads into the live embryos. We obtained viability values ranging from 15% in early egg capsules to 100% in late developmental stages. Moreover, we measured the delivery effectiveness as the number of hatchlings containing fluorescent beads per microinjected egg capsule, reaching 100% in early stages and almost 40% in late stages. This is the first time that planarian embryos have been modified without compromising normal development. We consider that this technique will be of extreme value to future work on planarian developmental biology and regeneration, enabling the application of modern functional tools to the study of this Lophotrochozoan.     

Morphological score assignment guidelines for the dechorionated zebrafish teratogenicity assay

BACKGROUND: Recently we reported the development and optimization of a zebrafish teratogenicity assay using dechorionated AB strain embryos, a promising assay that was 87% concordant in correctly identifying in vivo teratogens and non‐teratogens from a set of 31 compounds (Brannen et al., 2010: Birth Defects Res 89:66–77). METHODS: This assay utilizes a zebrafish morphological score system to characterize adverse effects and identify the no‐observed‐adverse‐effect level (NOAEL). RESULTS: This report describes in detail the morphological score system used in the dechorionated zebrafish embryo culture teratogenicity assay. The morphological assessment includes evaluation of most structures and organ systems and grades relative severity of abnormalities. CONCLUSIONS: To this end, the morphological score system provides information of tissue‐specific teratogenicity that has been found to have good concordance with structures found affected in vivo and can also be used to rank compounds based on the severity of malformations. Birth Defects Res (Part B) 89:382–395, 2010. © 2010 Wiley‐Liss, Inc.     

Differential scanning calorimetry studies of intraembryonic freezing and cryoprotectant penetration in zebrafish (Danio rerio) embryos

Nucleation temperatures of intraembryonic water and cryoprotectant penetration in zebrafish embryos were studied using differential scanning calorimetry. The effects of embryo developmental stage, dechorionation, partial removal of yolk, cooling rate, and cryoprotectant treatment on the temperatures of intraembryonic freezing were investigated. Embryo stages were found to have a significant effect on the nucleation temperatures of intact embryos. Freeze onset temperatures of -11.9 +/- 1.5, -15.6 +/- 0.3, and -20.5 +/- 0.1 degrees C were obtained for intact embryos at 6-somite, prim-6, and high-pec stages, respectively. After dechorionation, the freeze onset temperatures of intraembryonic water shifted to significantly lower temperatures, being -23.5 +/- 0.8, -18.7 +/- 0.7, -24.9 +/- 0.8 degrees C for 6-somite, prim-6, and high-pec stages, respectively. Yolk-reduced high-pec stage embryos showed significantly lower nucleation temperatures with an average onset at -27.9 +/- 0.4 degrees C. The effect of cryoprotectant treatment on the nucleation temperatures of intraembryonic water varies among different embryo stages and different cryoprotectants. Thirty-minute treatment with 2 M methanol significantly decreased the nucleation temperatures of dechorionated 6-somite embryos whilst no temperature decrease was observed for prim-6 or yolk-reduced high-pec embryos. Thirty-minute exposure to 1 M propylene glycol did not significantly affect the nucleation temperatures of dechorionated 6-somite, prim-6, or yolk-reduced high-pec embryos. In order to increase the permeability of embryos to cryoprotectants, the yolk sacs of dechorionated embryos at 6-somite or prim-6 embryos were punctured with a sharp micro-needle before exposure to cryoprotectants. The punctured prim-6 embryos showed significantly lower temperatures of intraembryonic freezing after 30 min of exposure to 2 M methanol following the multi-punctures. The nucleation temperatures of punctured 6-somite or prim-6 embryos were also decreased significantly after exposure to 1 M propylene glycol for 30 min. These results suggested that in intact embryos, intraembryonic freezing appeared to be seeded by the external ice in the perivitelline fluid and that in dechorionated embryos (in the absence of external water) intraembryonic freezing was more likely a consequence of heterogeneous nucleation. Methanol was demonstrated to show a limited degree of penetration into prim-6 stage embryos, but it did not penetrate later-stage embryos such as prim-6 and yolk-reduced high-pec. No propylene glycol permeation was observed for embryos at all stages. However, multi-punctures of yolk resulted in the permeation of both cryoprotectants into prim-6 embryos and propylene glycol permeation into 6-somite embryos. These findings may have important implications in overcoming the problem associated with the low membrane permeability of zebrafish embryos to cryoprotectants.     

One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos

We constructed a photoactivatable Drosophila histone 2 A variant green fluorescent fusion protein (H2AvD-paGFP) for tracking chromatin loci in living Drosophila embryos. Activation of paGFP was achieved by irradiation from a single-photon diode laser at 408 nm, but activated nuclei failed to divide. Photoconversion could also be achieved by two-photon fs pulses in the range of 780-840 nm. Viability in whole-mount embryos could only be maintained at 820 nm, at which we could activate, simultaneously track and quantitate the mobility of multiple fluorescent loci. This report constitutes the first demonstration of two-photon activation of paGFP and the use of a paGFP-fusion protein in investigations of whole organisms.     

Electrical currents associated with rhythmic contractions of the blastoderm of the medaka, Oryzias latipes

1. We used a vibrating probe to measure extracellular electrical currents near the surface of dechorionated Oryzias latipes eggs as contraction waves moved slowly across the blastoderm. 2. Although we found no detectable current outside dechorionated embryos, we recorded large current pulses near the edge of wounds made in the surface of the blastoderm. 3. The maximum net inward current--or in some cases, the least net outward current--correlated temporally with the contraction of cells near the edge of the wound. 4. The current pulses were superimposed on steady currents of variable magnitude and polarity. 5. We discuss possible mechanisms for the initiation and propagation of the contraction wave.     

Laser‐enhanced drug delivery of antiretroviral drugs into human immunodeficiency virus‐1 infected TZMbl cells

The introduction of highly active antiretroviral therapy (HAART) has significantly increased life expectancy and improved management of the human immunodeficiency virus‐1 (HIV‐1) disease globally. This well‐established treatment regime has shown to reduce viral capacity to undetectable limits when using traditional clinical assays. The establishment of viral reservoirs during the early stages of infection are the major contributors to failure of the current regimens to eradicate HIV‐1 infection since the reservoirs are not affected by antiretroviral drugs (ARVs). Therefore, advanced modification of the present treatment and investigation of novel antiretroviral drug delivery system are needed. The aim of this study was to use femtosecond (fs) laser pulses to deliver ARVs into HIV‐1 infected TZMbl cells. Different ARVs were translocated into TZMbl cells using fs pulsed laser (800 nm) with optimum power of 4 μW and 10 ms laser to cell exposure time. Changes in cellular processes were evaluated using cellular morphology, viability, cytotoxicity and luciferase activity assays. Cells treated with the laser in the presence of ARVs showed a significant reduction in viral infectivity, cell viability and an increase in cytotoxicity. This study demonstrated that fs laser pulses were highly effective in delivering ARVs into HIV‐1 infected TZMbl cells, causing a significant reduction in HIV‐1 infection.     

Proximal and distal sequences control UV cone pigment gene expression in transgenic zebrafish

The molecular basis of cone photoreceptor-specific gene expression is largely unknown. In this study, we define cis-acting DNA sequences that control the cell type-specific expression of the zebrafish UV cone pigment gene by transient expression of green fluorescent protein transgenes following their injection into zebrafish embryos. These experiments show that 4.8 kb of 5'-flanking sequences from the zebrafish UV pigment gene direct expression specifically to UV cones and that this activity requires both distal and proximal sequences. In addition, we demonstrate that a proximal region located between -215 and -110 bp (with respect to the initiator methionine codon) can function in the context of a zebrafish rhodopsin promotor to convert its specificity from rod-only expression to rod and UV cone expression. These experiments demonstrate the power of transient transgenesis in zebrafish to efficiently define cis-acting regulatory sequences in an intact vertebrate.     

Optoporation and Genetic Manipulation of Cells Using Femtosecond Laser Pulses

Femtosecond laser optoporation is a powerful technique to introduce membrane-impermeable molecules, such as DNA plasmids, into targeted cells in culture, yet only a narrow range of laser regimes have been explored. In addition, the dynamics of the laser-produced membrane pores and the effect of pore behavior on cell viability and transfection efficiency remain poorly elucidated. We studied optoporation in cultured cells using tightly focused femtosecond laser pulses in two irradiation regimes: millions of low-energy pulses and two higher-energy pulses. We quantified the pore radius and resealing time as a function of incident laser energy and determined cell viability and transfection efficiency for both irradiation regimes. These data showed that pore size was the governing factor in cell viability, independently of the laser irradiation regime. For viable cells, larger pores resealed more quickly than smaller pores, ruling out a passive resealing mechanism. Based on the pore size and resealing time, we predict that few DNA plasmids enter the cell via diffusion, suggesting an alternative mechanism for cell transfection. Indeed, we observed fluorescently labeled DNA plasmid adhering to the irradiated patch of the cell membrane, suggesting that plasmids may enter the cell by adhering to the membrane and then being translocated.     

Optoporation and Genetic Manipulation of Cells Using Femtosecond Laser?Pulses

Femtosecond laser optoporation is a powerful technique to introduce membrane-impermeable molecules, such as DNA plasmids, into targeted cells in culture, yet only a narrow range of laser regimes have been explored. In addition, the dynamics of the laser-produced membrane pores and the effect of pore behavior on cell viability and transfection efficiency remain poorly elucidated. We studied optoporation in cultured cells using tightly focused femtosecond laser pulses in two irradiation regimes: millions of low-energy pulses and two higher-energy pulses. We quantified the pore radius and resealing time as a function of incident laser energy and determined cell viability and transfection efficiency for both irradiation regimes. These data showed that pore size was the governing factor in cell viability, independently of the laser irradiation regime. For viable cells, larger pores resealed more quickly than smaller pores, ruling out a passive resealing mechanism. Based on the pore size and resealing time, we predict that few DNA plasmids enter the cell via diffusion, suggesting an alternative mechanism for cell transfection. Indeed, we observed fluorescently labeled DNA plasmid adhering to the irradiated patch of the cell membrane, suggesting that plasmids may enter the cell by adhering to the membrane and then being translocated.     

Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio)

Prior to hatching, the zebrafish embryo is surrounded by an acellular envelope, the chorion. Despite repeated speculations, it could not be clarified unequivocally whether the chorion represents an effective barrier and, thus, protects the embryo from exposure to distinct chemicals. Potentially, there is a risk of generating false negative results in developmental toxicity studies due to limited permeability of the chorion for some compounds. The simplest way to exclude this is to remove the chorion and expose the “naked” embryo. In the context of ecotoxicity testing, standardized protocols do not exist for fish embryo dechorionation, and survival rates of dechorionated embryos have usually not been subjected to statistical analysis. Since reproducibly high survival rates are of fundamental importance for chemical toxicity assessment, the present study was designed to develop and optimize a dechorionation procedure. With appropriate modifications of the fish embryo test protocol, embryos can be dechorionated at 24 h post-fertilization (hpf) with survival rates of ≥ 90%. However, for fish embryo tests with dechorionated embryos, the standard positive control test substance, 3,4-dichloroaniline, should be replaced by another compound, e.g., acetone, since 3,4-dichloroaniline exerts its effects during the first 24 h of development. Dechorionation of younger stages (< 24 hpf) is generally possible, however with lower survival rates. The effect of dechorionation was demonstrated with the cationic polymer Luviquat HM 552, which is blocked by the chorion non-dechorionated embryos due to its molecular weight of ~ 400,000 Dalton, but becomes strongly toxic after dechorionation.     

Transient expression of RSVCAT in transgenic zebrafish made by electroporation.

We report the fish use of an exponential decay electroporation system to introduce foreign DNA into fertilized zebrafish embryos. The plasmid RSVCAT (Rous sarcoma viral promoter (RSV) upstream from the chloramphenicol acetyltransferase gene (CAT)) was linearized and introduced into fertile zebrafish embryos by electroporation no later than the four-cell stage. Conditions for the procedure were empirically derived, and 68% of the treated animals survived through hatching to at least 6 days after fertilization and well beyond. Dot-blot analysis on DNA extracted from individual hatching fry demonstrated that 65% of the animals tested carried the foreign construct. Enzyme assays on the soluble proteins of treated animals were positive for chloramphenicol acetyltransferase activity. These data demonstrate that the foreign construct was being transiently expressed in the developing tissues of the embryo. The simplicity of this technique will greatly enhance the ability to analyze gene promoter regulation in vivo in transgenic zebrafish. The ability of the electroporated DNA to integrate into the host genome and to generate stable lines of transgenic fish is discussed.     

Imaging intercellular calcium waves during late epiboly in intact zebrafish embryos

Through the injection of f-aequorin and the use of a photon imaging microscope, we have previously reported that a rhythmic series of intercellular Ca2+ waves circumnavigate zebrafish embryos over a 10 h period during gastrulation and axial segmentation. These waves first appear at about 65% epiboly and continue to arise every 5-10 min up to at least the 16-somite stage. In response to our publication, it was suggested that the waves may be an artefact caused by dechorionation of the embryos and would not be observed during the development of intact embryos (i.e. those with chorions). Here we demonstrate (again initially by aequorin imaging) that the rhythmic intercellular Ca2+ waves that traverse the blastoderm margin can also be observed in embryos that have an intact chorion. In addition, the appearance time, propagation pathway, velocity, duration and Ca2+ rise of the waves, as well as the interwave interval and the timing of wave onset, are approximately the same in both dechorionated embryos and those with an intact chorion. Furthermore, by loading intact embryos with Ca(2+)-green dextran at the single-cell stage and then using scanning confocal microscopy to obtain high-resolution images, we confirm the presence of circumferential Ca2+ waves and show that they pass through a population of deep cells located at the blastoderm margin. The confirmation of these pan-embryonic Ca2+ waves in zebrafish further corroborates our earlier suggestion that such waves might play a fundamental role in normal embryonic patterning during the gastrula period.     

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat(2), mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene/ethanol (1 : 20, v/v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cell-penetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active beta-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat(2)-mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene/ethanol (1 : 40, v/v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat(2)-mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat(2)-GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat(2)-plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat(2)-plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent Lipofectamine 2000 to the Tat(2)-plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.