Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.
Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.
Bioelectricity refers to endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane1. Ionic exchanges across the cellular membrane, and the coupled electrical potential and current changes, are essential for signaling processes of excitable neuronal and muscular cells. In addition, bioelectricity and ion gradients have a variety of other important biological functions including energy storage, biosynthesis, and metabolite transportation. Bioelectrical signaling was also discovered as a regulator of embryonic pattern formation, such as body axes, the cell cycle, and cell differentiation1. Thus, it is critical for understanding many human congenital diseases that result from the mis-regulation of this type of signaling. Although patch clamp has been widely used for recording single cells, it is still far from ideal for the simultaneous monitoring of multiple cells during embryonic development in vivo. Furthermore, voltage sensitive small molecules are also not ideal for in vivo applications due to their specificities, sensitivities, and toxicities.
The creation of a variety of genetically encoded fluorescent voltage indicators (GEVIs) offers a new mechanism to overcome this issue, and allows for easy application to study embryonic development, even though they were originally intended for monitoring neural cells2,3. One of the currently available GEVIs is the Accelerated Sensor of Action Potentials 1 (ASAP1)4. It is composed of an extracellular loop of a voltage-sensing domain of voltage sensitive phosphatase and a circularly permuted green fluorescent protein. Therefore, ASAP1 allows visualization of cellular electric potential changes (polarization: bright green; depolarization: dark green). ASAP1 has 2 ms on-and-off kinetics, and can track subthreshold potential change4. Thus, this genetic tool allows for a new level of efficacy in real-time bioelectric monitoring in live cells. Further understanding of the roles of bioelectricity in embryonic development and many human diseases, such as cancer, will shed new light on the underlying mechanisms, which is critical for disease treatment and prevention.
Zebrafish have been proven a powerful animal model to study developmental biology and human diseases including cancer5,6. They share 70% orthologous genes with humans, and they have similar vertebrate biology7. Zebrafish provide relatively easy care, a large clutch size of eggs, tractable genetics, easy transgenesis, and transparent external embryonic development, which make them a superior system for in vivo imaging5,6. With a large source of mutant fish lines already present and a fully sequenced genome, zebrafish will provide a relatively unlimited range of scientific discovery.
To investigate the in vivo real-time electrical activity of cells, we take advantage of the zebrafish model system and ASAP1. In this paper, we describe how to incorporate the fluorescent voltage biosensor ASAP1 into the zebrafish genome using Tol2 transposon transgenesis, and visualize cellular electrical activity during embryonic development, fish larval movement, and in live tumor.
The zebrafish are housed in an AAALAC-approved animal facility, and all experiments were carried out according to the protocols approved by the Purdue Animal Care and Use Committee (PACUC).
1. Tol2 Transposon Plasmid Construct Preparation
NOTE: Tol2, a transposon that was discovered in medaka fish, has widely been used in the zebrafish research community8,9. It has been successfully adopted to the Gateway site-specific recombination-based cloning system and known as the Tol2 kit10. The Tol2 kit allows for a more convenient way of creating customized expression constructs, while also increasing the efficiency of transgenesis. Thus, it was an easy decision to take advantage of this system, and create a ubiquitous ASAP1 expression zebrafish line using a validated ubiquitin promoter to drive ASAP111.
2. Prepare Tol2 Transposase mRNA and Injection Solution
3. Microinjection
4. Establish Transgenic ASAP1 fish, Tg(ubi:ASAP1)
5. Imaging
In a successful injection, more than 50% injected fish embryos will display some degree of green fluorescence in the somatic cells, and most of them will be positive by Tol2 transposon excise assay (Figure 2). After 2-4 generations of out-cross with wildtype fish (until the fluorescent fish reach 50%, the expected Mendelian ratio), the transgenic fish were used for the imaging experiment to track cell membrane potentials during embryonic development. First, membrane potential changes were examined throughout the cell cycle during zebrafish early embryonic developmental stages. It was observed that the cells hyperpolarized before the cleavage furrow formation (Figure 3A-3C, and Supplementary Video 1). Moreover, different tissues showed a variety of membrane potentials in 1-3 day old fish embryos. (Figure 3D-3G). For example, the somites and notochord are generally hyperpolarized, compared to the adjacent tissues/organs. Once the zebrafish embryos were able to move, we were also able to detect the neuromuscular electrical activities (Figure 4, Supplementary Video 2). As bioelectric properties of cancer cells could be altered, we took advantage of this ASAP1 reporter fish, and crossed it with an rpL35 gene mutant, which is prone to spontaneous malignant peripheral nerve sheath tumors21,24,25. Although only a few fish tumors were examined, due to the long potential growth period for the fish tumor mutant, it was noticeable that there were voltage differences between tumors and surrounding tissues in live tumor-bearing zebrafish (Figure 5). Thus, these representative results demonstrated the successful generation of a cellular electric reporter fish line, and its potential application to developmental and cellular biology.
Figure 1: Illustration of the Tol2 transposon-based plasmid construction.
(A) BP recombination was used for ASAP1 sub-cloning into the pDONR221 middle entry vector. attB sequences were added to the 5-end of the primers for ASAP1. (B) Diagram for Tol2 transposon-based construct assembling based on LR recombination. Purple oval shape shows Tol2 inverted repeats. The dashed lines indicate homologous recombination. Please click here to view a larger version of this figure.
Figure 2: Typical results of injected embryos by epifluorescence and Tol2-excise assay.
(A) Non-positive 1dpf fish embryo. (B) Successfully injected 1dpf fish embryo. GFP spots are evident in the trunk. (C) Non-positive 2dpf fish embryo. (D) Successfully injected 2dpf fish embryo. GFP spots are evident in the trunk. (E) A representative result of Tol2 excise assay. Lane 1-7 PCR were amplified from 7 randomly selected fish embryos 8 hours after injection. The last one is a negative control (NC) without any genomic DNA. Scale bar = 250 μm. Please click here to view a larger version of this figure.
Figure 3: Dynamic voltage changes during zebrafish embryo development.
(A-C) Differential cellular voltage polarity during mitosis in the fish embryos. (A) 2-cell stage zebrafish embryo. (B) 4-cell stage embryo. (C) 8-cell stage embryo. The red arrow heads indicate the positions of the cleavage furrows in the panels (A–C). The changes are also evident in the corresponding movie (Supplementary Video 1). The region around the cleavage furrow is more polarized compared the rest of the cell. (D-G) Dynamic electric voltage changes in the different early stages of zebrafish embryos. (D) 12-somite stage. (E) 22-somite stage. (F) 48 hours post fertilization. (G) 72 hours post fertilization. e, eye; ht, heart; nt, notochord; op, optic vesicle; ov, otic vesicle; pf, pectoral fin; so, somite; yk, yolk. Scale bar = 250 μm. Please click here to view a larger version of this figure.
Figure 4: Electrical voltage changes of the fish body during fish embryo movement.
2-day old fish embryos show the neuro-muscular electric activities during movement. (A) – (F) Sequential imaging of the same fish embryo. Color density changes are corresponding to the electric signaling transduction. The interval time between two consecutive images is about 12.4 milliseconds. The red arrows indicate the positions that voltage changed during the imaging period. The changes are also evident in the corresponding movie (Supplementary Video 2). All the panels are in the same scale. Scale bar = 250 μm. Please click here to view a larger version of this figure.
Figure 5: Tumor cells tend to be more polarized.
A 10-month old fish (rpL35hi258/wt; Tg(ubi:ASAP1) developed a malignant peripheral nerve sheath tumor in the abdomen. (A) & (C) Bright field image. (B) & (D) Image with GFP channel. (A) & (B) Intact fish. (C) & (D) Abdomen tumor was dissected out. Tumor cells are more polarized (brighter green) compared to surrounding tissues (dark green). Arrow heads show the tumors. All the panels are in the same scale. Scale bar = 25 mm. Please click here to view a larger version of this figure.
Supplementary Video 1. Epifluorescent imaging of electrical signaling during cleavage stages in Tg(ubi: ASAP1) fish embryo. This movie was recorded from the view of animal pole. The ASAP1 fluorescence is associated with the formation of cell cleavage furrow, a temporary structure during cell division. Please click here to download this file.
Supplementary Video 2. Epifluorescent imaging of electrical signaling during 2-day old Tg(ubi: ASAP1) fish embryo movement. The move was recorded from the lateral view of the 2-day old fish embryo after anesthetization. The ASAP1 fluorescence alterations are evident in neuromuscular tissue during the moving process. Please click here to download this file.
Although the cellular and tissue level electrical activities during embryonic development and human disease were discovered a long time ago, the in vivo dynamic electrical changes and their biological roles still remain largely unknown. One of the major challenges is to visualize and quantify the electrical changes. Patch clamp technology is a break-through for tracking single cells, but its application to vertebrate embryos is limited because they are composed of many cells. The current chemical voltage dyes are also not ideal due to their sensitivities, specificities, and toxicities. The recent efforts on the invention of GEVIs provide us a new path to visualize cellular electric activities in vivo and in real time. Here, we showed the process of creating a zebrafish electric reporter line, Tg(ubi:ASAP1).
Using this reporter fish line, we show cellular electrical activities that can be monitored in zebrafish embryos. The electric voltage change is highly related to the cell cycle during early embryonic development. We have observed that hyperpolarization happens before the formation of the cleavage furrow/cell division (Figure 3). This is in contrast to the current knowledge that depolarization happens before cell division26. Thus, more details of cell membrane voltage changes during the cell cycle of other animal and human cells, and whether this is related to tissue context, require further studies. Related studies are currently underway in our laboratory. Moreover, we have verified that ASAP1 is able to track physiological voltage changes in the neural-muscular system (Figure 4), in which the alteration is relatively fast compared to the changes during cell cycles.
It was also demonstrated that this reporter can also be used to visualize zebrafish tumors (Figure 5). It was interesting to find tumor cells were generally more polarized compared to the surrounding normal tissues. However, whether this is a general phenomenon for all malignant tissues requires further investigation, due to the limitation of tumor samples and fish tumor types in this study. Future investigations on cell membrane polarization and voltage quantification on other types of tumors and human cancer cells will be informative for better understanding its roles during tumorigenesis.
In this protocol, we chose a ubiquitous promoter to drive ASAP1 expression to track all the cells in fish embryos. Tissue or organ specific promoters could be another option if only a certain cell/tissue type is preferred. The ASAP1 voltage sensor is a relatively well characterized biosensor, and it is composed of a voltage sensitive domain of sea squirt voltage-sensitive phosphatase (S3-S4 loop) and a circular permutation of GFP (default is low fluorescence). It was reported to be expressed on the outside cellular membrane in human neuron cells and mouse brain slices4,27,28. The brightness of the sensor is dominantly determined by the conformational positions of the S3-S4 loop and GFP. The rapid green fluorescence change was unlikely caused by protein concentration, due to the speed of the brightness changes and protein synthesis. However, the transgene, ASAP1, may have altered expression in tumor cells, due to the nature of genomic instability. In addition to ASAP1, other GEVIs, such as archaerhodopsin-based voltage indicators (QuasAr1 and QuasAr2), may also be a good complementary option, since they use a completely different mechanism and they also have a high sensitivity and speed 29. In addition, their emission is in the red color range. This makes them particularly complimentary to the green ASAP1, if there is already another florescent protein in the same cell. For example, ASAP1 and QuasAr can be combined with Fucci zebrafish30 for studying the relationship between cell cycle and electric potential changes.
The authors have nothing to disclose.
The research work reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under the Award Number R35GM124913, Purdue University PI4D incentive program, and PVM Internal Competitive Basic Research Funds Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agents. We thank Koichi Kawakami for the Tol2 construct, Michael Lin for the ASAP1 construct, and Leonard Zon for the ubi promoter construct through Addgene.
14mL cell culture tubes | VWR | 60818-725 | E.Coli culture |
Agarose electrophoresis tank | Thermo Scientific | Owl B2 | DNA eletrophoresis |
Agarose RA | Amresco | N605-500G | For making the injection gels |
Attb1-ASAP1-F primer | IDT DNA | GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGAGACGACTGTGAGGTATGAACA | ASAP1 coding region amplification for subcloning |
Attb2-ASAP1-R primer | IDT DNA | GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAGGTTACCACTTCAAGTTGTTTCTTCTGTGAAGCCA | ASAP1 coding region amplification for subcloning |
Bright field dissection scope | Nikon | SMZ 745 | Dechorionation, microinjection, mounting |
Color camera | Zeiss | AxioCam MRc | Fish embryo image recording |
Concave slide | VWR | 48336-001 | For holding fish embryos during imaging process |
Disposable transfer pipette 3.4 ml | Thermo Scientific | 13-711-9AM | Fish embryos and water transfer |
Endonuclease enzyme, Not I | NEB | R0189L | For linearizing plasmid DNA |
Epifuorescent compound scope | Zeiss | Axio Imager.A2 | Fish embryo imaging |
Epifuorescent stereo dissection scope | Zeiss | Stereo Discovery.V12 | Fish embryo imaging |
Fluorescent light source | Lumen dynamics | X-cite seris 120 | Light source for fluorescence microscopes |
Forceps #5 | WPI | 500342 | Dechorionation and needle breaking |
Gateway BP Clonase II Enzyme mix | Thermo Scientific | 11789020 | Gateway BP recombination cloning |
Gateway LR Clonase II Plus enzyme | Thermo Scientific | 12538120 | Gateway LR recombination cloning |
Gel DNA Recovery Kit | Zymo Research | D4002 | DNA gel purification |
Loading tip | Eppendorf | 930001007 | For loading injection solution into capilary needles |
Methylcellulose (1600cPs) | Alfa Aesar | 43146 | Fish embryo mounting |
Methylene blue | Sigma-Aldrich | M9140 | Suppresses fungal outbreaks in Petri dishes |
Microinjection mold | Adaptive Science Tools | TU-1 | To prepare agaorse mold tray for holding fish embryos during injection |
Microinjector | WPI | Pneumatic Picopump PV820 | Microinjection injector |
Micro-manipulator | WPI | Microinjector mm33 rechts | Microinjection operation |
Micropipette puller | Sutter instrument | P-1000 | For preparing capillary needle |
Mineral oil | Amresco | J217-500ml | For calibrating injection volume |
mMESSAGE mMACHINE SP6 Transcription Kit | Thermo Scientific | AM1340 | mRNA in vitro transcription |
Monocolor camera | Zeiss | AxioCam MRm | Fish embryo image recording |
Plasmid Miniprep Kit | Zymo Research | D4020 | Prepare small amount of plasmid DNA |
Plastic Petri dishes | VWR | 25384-088 | For holding fish or fish embryos during imaging process |
RNA Clean & Concentrator-5 | Zymo Research | R1015 | mRNA cleaning after in vitro transcription |
Spectrophotometer | Thermo Scientific | NanoDrop 2000 | For measuring DNA and RNA concentrations |
Stage Micrometer | Am Scope | MR100 | Microinjection volume calibration |
Thermocycler | Bio-Rad | T100 | DNA amplification for gene cloning |
Thin wall glass capillaries | WPI | TW100F-4 | Raw glass for making cappilary needle |
Tol2-exL1 primer | IDT DNA | GCACAACACCAGAAATGCCCTC | Tol2 excise assay |
Tol2-exR primer | IDT DNA | ACCCTCACTAAAGGGAACAAAAG | Tol2 excise assay |
TOP10 Chemically Competent E. coli | Thermo Scientific | C404006 | Used for transformation during gene cloning |
Tricaine mesylate | Sigma-Aldrich | A5040 | For anesthetizing fish or fish embryos |
UV trans-illuminator 302nm | UVP | M-20V | DNA visualization |
Water bath | Thermo Scientific | 2853 | For transformation process of gene cloning |