1Department of Biological Sciences, Center for Zebrafish Research, University of Notre Dame, 2Department of Microbiology, Immunology, and Pathology, Colorado State University, 3Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University School of Medicine
Hyde, D. R., Godwin, A. R., Thummel, R. In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin. J. Vis. Exp. (61), e3632, doi:10.3791/3632 (2012).
Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.
1. Resuspend Morpholino
2. Fin Amputation
3. Morpholino Injection
4. Electroporation of the Morpholino
6. Representative Results
Figure 1. Schematic of the various events that occur during fin regeneration. The underlying times for each event are given in hours post-amputation (hpa) and correspond to a tank temperature of 33 °C.
Figure 2. A. Schematic of the injection plate, which is made from agarose and contains a small well to hold the fish during microinjection of the morpholino.
Figure 3. Schematic of morpholino microinjection. A. Place the fish in the dish with the head of the fish in the notch cut out of the well, which will help the fish stay stable. B. At low magnification, arrange the needle so that it is close to the regenerating tissue of the fin. C. At higher magnification, inject the morpholino distal to each bony fin ray (i.e. in each blastema). The needle should enter the tissue just distal to the bony ray (1), and then continue to the location of the blastema (2). Note: the green circles in the schematic are only meant to show the location of the injection. The morpholino can briefly be visualized as a green/yellow "puff" following each injection; however, this does not persist as shown in the schematic.
Figure 4. Schematic of fin electroporation. A. Following microinjection, place the fish in a Petri dish full of anesthesia and electroporate both the dorsal and ventral halves. B. Make sure to not touch the fin tissue. Electrodes should be placed ~ 1 mm from the tissue.
Figure 5. Schematic of the methods used to calculate fin outgrowth inhibition. A. Take a picture of the fin of each fish at 2 dpa, either immediately before or after morpholino injection and electroporation. Trace the regenerative tissue of both the dorsal (green) and ventral (blue) halves of the fin using NIH Image, (black dashed lines). B. At 3 dpa, take another picture of each fin and again trace the dorsal and ventral areas of regrowth using NIH Image. C. Subtract the area of regrowth at 2 dpa from the total regrowth at 3 dpa for both the dorsal and ventral haves. The percent area of dorsal versus ventral re-growth can be calculated using the formula: ((D3dpa - D2 dpa)/(V3dpa - V2dpa)) X 100. Percent inhibition = 100 - Percent Area.
Figure 6. Examples of expected outcomes. A. Fluorescent image showing a fluorescein-tagged control morpholino in the dorsal half of the fin, 24 hours post-electroporation (hpe). B. Brightfield image of a fin that was injected and electroporated with a control morpholino in the dorsal half. The image shows equal regrowth of both the dorsal and ventral halves of a fin, 24 hpe. C. Brightfield image of a fin that was injected and electroporated with an experimental morpholino in the dorsal half. The image shows inhibition of regrowth on the dorsal/injected side. The line shows the amount of regrowth at 2 dpa, immediately prior to morpholino injection and electroporation.
Figure 7. Schematic of an alternate injection and electroporation procedure to target proteins involved in wound healing and blastema formation. A. Inject the morpholino between each bony fin ray on the dorsal half of the fin. B. Electroporate the morpholino as per normal. C. Amputate the fin immediately proximal (~ 1 bony segment) to the injection site. D. The fluorescein-tagged morpholino can be observed in wound epithelium and blastema at 24 hpe.
Figure 8. Using the technique to target wound epithelium and blastema formation. A. Brightfield image of a fin at 24 hpa that was injected and electroporated with a control morpholino immediately prior to amputation. B. Fluorescent image of fin shown in panel A. Note that the injected dorsal half of the fin shows good uptake of the morpholino in the regenerative tissue. C - C". Higher magnification of the dorsal half of the fin shown in panels A and B. The injection sites are often still visible (arrowheads), but many targeted cells have migrated to participate in wound epithelium and blastema formation (arrow).
Figure 9. Using the technique to target cells that migrate to form the blastema. A. Fluorescent and brightfield inset images showing a fin injected and electroporated with morpholino on both the dorsal and ventral halves. The fin was then amputated at two planes. The dorsal half was cut immediately distal to the injection sites, where as the ventral half was cut 9-10 bony segments distal to the injection sites. B. At 24 hpa, the fluorescent and brightfield inset images show that morpholino has migrated to the dorsal regenerate (1), but not the ventral regenerate (2), indicating that only the cell immediately proximal to the cut site participate in blastema formation. The two sets of white arrowheads show the level of each amputation plane. Panels marked 1 and 2 on the far right of the image show a higher magnification view of the dorsal and ventral halves of the fin, respectively.
Supplemental Figure 1. A video of a confocal z-stack of a region corresponding to the location of a blastema in a fin injected and electroporated with a control morpholino. The image was taken at 24 hpe. Since a single fluorescein molecule cannot be visualized, not all of the morpholino can be visualized or quantified. However, these images give some idea of the varying degrees of uptake that can be visualized in cells, from individual punctate dots, to entire cells full of fluorescent morpholino. On the still image, the orientation is shown. Scale bar: 25 microns.
Here, we describe a powerful loss-of-function approach to conditionally knockdown proteins of interest during fin regeneration in adult zebrafish. This technique has been used to study gap junction genes, signaling receptors, transcription factors, and microRNAs during regenerative fin outgrowth16, 20-22.
We anticipate that this technique could also be used to study genes required for wound healing and blastema formation by adapting the technique. For example, we injected and electroporated a control morpholino in the space between the bony fin rays on the dorsal half of the fin prior to amputation (Figure 7). We then amputated the fin immediately distal to the injection plane. 24 hpa, we observed that the morpholino-targeted cells had migrated distally to form both the wound epithelium and blastema (Figure 8), indicating that cells during these early stages of regeneration can also be targeted.
The technique has a few notable limitations. For example, fluorescence from the fluorescein tag does not persist following fixation and processing for immunohistochemistry, which makes it impossible to correlate a particular cellular phenotype (i.e. cell proliferation) with the amount of morpholino present in a cell. In addition, we have been unable to consistently achieve the electroporation of plasmids into the regeneration tail fin, although a previous group did report the successful electroporation of DNA into fin tissue23. Finally, we have noted that the morpholino is only effective for ~48 hours post electroporation21, which prohibits using this technique in its current form for testing genes involved in the differentiation of new cell types. Additional testing and modification of the procedure may overcome these current limitations.
In addition, it is possible that this technique could be used to test proteins involved in cell migration from the underlying tissue to the blastema. For example, we injected and electroporated a control morpholino into both sides of the fin (as described in Figure 7) prior to amputation. We then amputated the dorsal half of the fin immediately distal to the injection plane and we amputated the ventral fin much more distally. At 24 hpa, the morpholino-positive cells on the dorsal side had migrated from the injection site to the overlying wound epithelium and blastema. However, that was not the case on the ventral side (Figure 9). This supports the idea that only the cells that underlie the amputation plane participate in the regenerative response. These data also suggest that proteins hypothesized to be required for cell migration could be tested using this technique.
We have nothing to disclose.
The authors would like to thank the Freimann Life Science Center and Center for Zebrafish Research staff for their care and maintenance of the zebrafish.
|CUY21-EDIT or CUY21-SC Square wave electroporator||Protech International, Inc.||CUY21EDIT or CUY21SC||Both units work for this protocol|
|3-mm diameter paddle electrodes||Protech International, Inc.||CUY 650-P3|
|Morpholino||GeneTools, LLC||Morpholino should be custom designed to your protein of interest|
|2-Phenoxyethanol||Sigma-Aldrich||77861-1L||Anesthesia; dilute 1:1000 in fish system water for procedure, 1:500 for euthanasia|
|Micro-injection pump||World Precision Instruments, Inc.||PV830 Pneumatic PicoPump||Many different microinjection systems could be used|
|Micro-manipulator||World Precision Instruments, Inc.||MMJR||Right-handed (MMJL for left-handed)|
|Micro-injection needles, 1.0 mm outside diameter||World Precision Instruments, Inc.||1B100F-4||These are borosilicate glass capillaries, pulled into a needle|
|Needle holder||World Precision Instruments, Inc.||5430-ALL||Pico Nozzle Kit; make sure to inset the 1.0 mm pipette gasket|
|Needle puller||Sutter Instrument Co.||P-97||Other micropipette/needle pullers should also work|
|Microscope||Leica, Nikon, Zeiss||Number varies depending on the manufacturer||Any stereomicroscope with 20X optics and the ability to work with micromanipulators|