This work was supported by funds from Midwestern University College of Health Sciences Research Facilitation Grant awarded to EEH and Midwestern University Office of Research and Sponsored Programs Intramural Grant awarded to KJL.

This protocol conforms to the AVMA Animal Welfare Principles for the care and use of laboratory animals and has been approved by the Institutional Animal Care and Use Committee (IACUC) at Midwestern University.
1. Preparation of Anesthesia, Equipment, &#38; Media
<ol>
	<li>Dechlorinate approximately 1.5 L of tap water using either a commercial dechlorinating agent (available at pet stores) or by letting water stand for 24 hr. Obtain three 1 L beakers and fill each with 500 ml dechlorinated water. Label one for holding fish before any procedure and one for recovery. To the third beaker, add 100 &#181;g/ml of tricaine methanesulfonate; this becomes the beaker in which fish will be anesthetized.</li>
	<li>Fill a shallow tray with ice, warm the culture medium (RPMI 1640, 10% FBS (fetal bovine serum), 50 &#181;g/ml gentamycin, 100 &#181;g/ml kanamycin, 25 mM HEPES) to RT, and gather clean jeweler&#8217;s tweezers. Clean tweezers by immersing the tips in 70% ethanol and pass through a Bunsen burner, allowing the ethanol to burn off.</li>
	<li>Decide if the procedure will be performed without magnification, with a commercial, table-top lighted magnifying glass with 2 - 10X magnification, or dissecting microscope (entirely dependent on personal preference).</li>
</ol>
2. Establish Explant Cultures
<ol>
	<li>Catch the desired number of fish and place in a holding beaker. Transfer a single fish to the anesthesia beaker; it is best to anesthetize fish individually.</li>
	<li>Monitor the effects of anesthesia by observing the swimming and gill movement of the fish. As anesthesia progresses, gill movement slows and the fish will swim erratically and frequently upside down; consider fish anesthetized as soon as gill movement ceases. Do not leave the fish for longer periods in the anesthesia bath as this may result in the death of the fish.</li>
	<li>Remove fish from the anesthesia beaker and transfer to ice, placing the fish horizontally with the tail facing the hand holding the tweezers. If experienced with the technique and if multiple fish are to be used to make explants, consider placing the next fish in the anesthesia beaker at this time.</li>
	<li>To remove scales, position tweezers parallel to the fish with tip at edge of scale. Depress flat side of the tweezers slightly into the side of the fish to cause the scale to elevate from the body of the fish. Grasp scale with tweezers, pluck, and place (without inversion) in tissue culture dish. Repeat the procedure, removing a maximum of 12 scales from a large adult (6 from each side), avoiding the gill region, the lateral line, and fins.</li>
	<li>Place the fish into the recovery beaker and monitor to ensure it recovers from the effects of anesthesia. At this time, fish can be transferred back to an individual tank. Allow the fish to recover for at least 21 days to allow for complete scale regeneration.</li>
	<li>Depending on the experimental design, place up to 20 scales per plastic 35 mm tissue culture-treated dish or 4 - 6 scales per glass bottom dish. Allow scales to adhere to the dish before gently adding media so as not to detach scales from the dish. 
	NOTE: With experience, multiple fish can be processed before media is added to the first dish.</li>
	<li>Add 1.2 ml of complete media (see 1.2 above) into each 35 mm dish.</li>
	<li>Preferably incubate explant cultures at 28 &#176;C in 5% CO2 for the desired period of time with or without additional treatment. Alternatively, incubate cultures on a heated microscope stage or within a live cell culture chamber for video microscopy.</li>
</ol>
3. Brightfield and Video Microscopy
<ol>
	<li>Position dishes containing cell sheets onto the microscope stage and initially focus using the 4X objective lens. 
	NOTE: Higher magnifications may be used, depending on the experiment. Marking the location of each cell sheet and/or orientation of the dish on the stage will facilitate taking images over time with the sheets in approximately the same orientation.</li>
	<li>Remove dishes from the incubator and photograph at various time points, according to each experimental protocol and place back into the incubator if only still images are needed over the experimental time frame.</li>
	<li>While performing video microscopy, pay attention to the following:
	<ol>
		<li>Position the scale/emerging cell sheet within the field of view such that the migrating cell sheet remains in the field of view for the duration of the video. 
		NOTE: This may take some experience in observing how cells migrate from under the scale. For short videos, this is less of a concern than for longer videos.</li>
		<li>For shorter time frames, incubate at RT. For longer time frames (18+ hr), use a heated stage or an incubation chamber to maintain temperature, pH balance, and to minimize evaporation of the culture medium.</li>
	</ol>
	</li>
</ol>
4. Fixation for Immunofluorescence Microscopy
<ol>
	<li>Prepare and pre-warm all solutions to 28 &#176;C. In order to view keratocytes under fluorescence, grow explant cultures as described above using glass-bottom 35 mm tissue culture dishes. Alternatively, use glass chamber slides.</li>
	<li>Add 0.8 ml of 4% paraformaldehyde in 1.1x PBS to each dish. Do not remove the culture media first. Incubate at RT for 5 min then aspirate to remove the solution.</li>
	<li>Add 0.8 ml of 4% paraformaldehyde in 1.1x PBS to each dish. Incubate at RT for 5 min then aspirate to remove the solution.</li>
	<li>Unless using external ligands or external epitopes, permeabilize cells by adding 0.5 ml of 0.2% Triton X-100 in 1X PBS. Incubate at RT for 5 min then aspirate to remove the solution.</li>
	<li>Add 0.5 ml of 1% BSA in 1x PBS and incubate for 1 hr (at RT) or O/N (at 4 &#176;C). 
	NOTE: Depending on experimental conditions, phalloidin can be added at this step.</li>
	<li>After incubation, aspirate to remove the solution and proceed with antibody staining. Alternatively, store the culture dishes by adding at least 1 - 2 ml of 1% BSA in 1x PBS for 3 - 4 weeks at 4 &#176;C.</li>
</ol>
5. Immunofluorescence Protocol
<ol>
	<li>Prepare primary and secondary antibody solutions according to the manufacturer&#8217;s recommendations. 
	NOTE: If manufacturer recommendations are not available, a good starting dilution of primary antibodies (in 1% BSA in 1x PBS) is 1:500 for polyclonal and 1:1,000 for monoclonal antibodies; a good starting dilution for secondary antibodies is 1:1,000. Adjust these dilutions of the original stock obtained from the manufacturer higher if the signal is weak, lower if the signal is strong and background is a problem.</li>
	<li>Aspirate the 1% BSA in 1x PBS solution. Add 0.5 ml of primary antibody solution and incubate for 1 hr (at RT) or O/N (at 4 &#176;C).</li>
	<li>Remove the primary antibody solution, place into a clean and labeled 1.5 ml microcentrifuge tube, and freeze at -20 &#176;C. 
	NOTE: This allows for the reuse of the primary antibody, if needed.</li>
	<li>Wash the dish 3 - 5 times with 1x PBS, removing and discarding the PBS after each wash.</li>
	<li>Add 0.5 ml of secondary antibody solution. If desired, add fluorescently-labeled phalloidin (following any manufacturer&#8217;s recommended concentration) during this step. Incubate for 1 hr (at RT) or O/N (at 4 &#176;C). 
	NOTE: Use of fluorescently-labeled phalloidin allows for visualization of actin filaments; we have found that 488-labeled phalloidin works best in our zebrafish keratocytes, but other fluorophores can be used.</li>
	<li>Remove and discard the secondary antibody solution.</li>
	<li>Wash the dish 3 - 5 times with 1x PBS, removing and discarding the PBS after each wash. 
	NOTE: The dish can be stored, covered from light, at 4 &#176;C if unable to view immediately; make sure 1x PBS is added into the dish before storing to ensure the cells do not dry out but warm to RT (20 - 30 min), then remove the PBS just prior to proceeding to the next step.</li>
	<li>When ready to observe, add 2 - 3 drops of a mounting medium, with or without DAPI, depending on the experimental needs, into the dish and let sit for 10 min at RT. 
	NOTE: We use a commercially-available, glycerol-based mounting medium with DAPI, but other mounting media can be used. Allowing 10 min for the mounting medium to sit in the dish facilitates easy removal of the glass coverslip from the bottom of the dish.</li>
	<li>After incubation, gently squeeze on the sides of the dish to loosen and remove the coverslip. Add an additional drop of mounting media onto a glass slide and place the coverslip, cells down, onto the slide. 
	NOTE: Consider sealing the coverslip to the slide using clear fingernail polish if the slide needs to be kept for longer than 1 - 2 days.</li>
	<li>Observe the slide under a fluorescence microscope. 
	NOTE: Most of our images were taken using a 40X or 63X objective lens under oil immersion, but the specific objective lens used will depend on the experimental protocol.</li>
</ol>

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The authors have nothing to disclose.

The most critical step for success of keratocyte explant culture is allowing the scale to adhere to the culture dish for approximately 2 - 3 min before addition of the culture medium. Approximately 75% of scales will adhere and grow sheets (the number of cultures established for each experiment will need to be adjusted accordingly). Keratocyte sheets do not form around every scale removed from the fish and placed in culture for several reasons. First, DAPI staining of explants reveals that some scales have few cells atta...

Studies of cell motility have traditionally focused on individually migrating cells. Keratocytes cultured from fish and amphibian explants have proven to be a powerful model system to study the mechanisms of cell motility using both experimental and mathematical modeling approaches1-6. Experimental work on individually migrating cells is aided by the rapid, smooth gliding motion of the cells, while maintaining a uniform shape, speed, and direction. However, in vivo, cells frequently move collectively while maintaining cell-cell junctions, particularly during embryogenesis, wound healing, and metastasis. Thus, collective cell migration is a growing and increasingly prominent area of research within the field of cell migration. The collective migration of these cells has recently been described7 and it has many unique features which make it a powerful system to study collective cell migration in a wound healing model.
When scales are plucked from an anesthetized adult zebrafish, some epithelial tissue remains attached to the underside of the scale. When cell culture medium is added, these epithelial cells, or keratocytes, migrate rapidly as a collective unit from the scale. The explant culture can be viewed as an epithelial wound healing model, in which the cells migrate away from the explant as they would across the provisional matrix of a wound bed to reestablish an intact epithelium. Recent data suggests that this ex vivo culture mimics many features of in vivo wound healing in adult zebrafish. Wound closure rates8 translate to a migration speed similar to that observed in the ex vivo system7. In addition, in an in vivo wound healing model, treatment with warfarin suggests that hemostasis has no effect on rate of healing, while treatment with hydrocortisone to decrease immune response does not delay reepithelialization8. As the zebrafish does not bleed when the scale is removed from the animal, hemostasis is not a major player. Although we have found immune cells in explants, we have not studied the effects they might have on collective cell migration.
The protocol presented here describes how to establish zebrafish keratocyte explant cultures that ensure consistency and reproducibility for use in many biological assays. These explant cultures are a particularly compelling in vitro model for collective cell migration for several reasons. First, zebrafish keratocytes are primary cells used within hours of establishing explant cultures. Therefore, these cells have not undergone the morphological and gene expression changes associated with passage of primary cells9-13. Second, this system represents a model for the study of reepithelialization in response to wounding14 and, as part of the response to wounding, an epithelial to mesenchymal transition (EMT) process is initiated as evidenced by changes in gene expression and cytoskeletal rearrangements14,15. Thus, the background changes in gene expression and motility which occur in untreated cells have been characterized and provide a context to interpret changes with treatment. Furthermore, the fish keratocyte and the human keratinocyte are functionally equivalent; both are the primary epithelial cells of their respective species and play a key role in epithelial wound healing. Third, adult zebrafish are gaining recognition as a model system for a variety of human diseases16-18 including melanoma and other cancers19-21, cutaneous wound healing8,14 and tissue regeneration22-24.
Technical advantages of this model system are equally compelling. A growing number of mutant and transgenic lines are available from non-profit, centralized facilities. Specifically, the Zebrafish Mutation Project (ZMP) at the Wellcome Trust Sanger Institute aims to create a knockout allele in every protein coding gene in the zebrafish genome. Currently, they have mutated 11,892 genes, approximately 45% of the genome, with 24,088 alleles characterized. In addition, ZMP accepts proposals and will generate knock-outs free of charge. Recent methods in gene knockdown in larval and adult zebrafish25-28 provide flexibility to study the function of developmentally important genes for which transgenic approaches are problematic or impractical.
Due to their extremely rapid rates of motility of ~145 &#181;m/hr shortly after establishment of cultures29, assays can be completed rapidly (usually in 24 hr or less). As experiments can be completed rapidly and cultures grow well at RT, several of the technical hurdles associated with mammalian cell migration assays involving video microscopy are avoided. In addition, in the age of tightening research budgets, zebrafish are easily and inexpensively maintained.
The structure and behavior of the explant is complex. As the fish do not bleed when the scale is removed, it is unlikely that much more than the superficial epidermal layers are removed with the scale. Keratocytes appear to be the predominant cell type in the explant when scales are initially removed from the fish as the vast majority of cells appear to stain with an anti-E-cadherin antibody14. Furthermore, there is no evidence of fibroblasts in the initial culture as judged by the absence of vimentin staining by immunofluorescence14. However, with repeated scale removal, there appear to be numerous neutrophils in the explant (see Video 1). We have identified these cells as neutrophils based on morphological observations of their size, rapidly motility, and their migration out of the cell sheet when exposed to LPS (data not shown). Additionally, these cells stain brightly with an antibody to neutrophil cytosolic factor as well as with a variety of secondary IgG antibodies, which indicates that they have abundant FcRs on their surface (data not shown). Multiple cell layers are present within the explant. Confocal microscopy reveals the presence of a single layer near the leading edge to more than two layers of keratocytes near the scale with neutrophils visible above, below, and between the cell keratocyte sheets.
At early time points, cells on the edge of the multi-layer explant polarize, initiating collective migration of keratocytes from the explant at initial rates of ~145 &#181;m/hr. The area covered by the explant rapidly increases, leading to cell spreading, tension within the sheet, and a decreased rate of advance of the leading edge. Rapid interconversions between leader and follower cells are observed at the leading edge during the formation and closure of spontaneously formed holes within the sheet7. As the EMT process initiated with the explant continues, the sheet fragments and the keratocytes lose their specialized, rapid motility. Gene expression and morphological changes consistent with EMT, wound healing, and inflammatory responses occur within 7 days of culture with explants being considered viable for approximately 10 days14.

<table><tbody><tr><td>35 mm Glass Bottom Culture Dishes (Poly-D Lysine Coated)</td><td>MatTek</td><td>P35GC-1.5-14-C</td><td>1.5 mm thickness is best for coverslip removal. 14 mm diameter provides more culture area.</td></tr><tr><td>Swiss Jewelers Style Forceps, Integra, Miltex 17-303</td><td>VWR</td><td>21909-460</td><td>We find that narrow, fine forceps work the best for scale removal.</td></tr></tbody></table>

zebrafish keratocyte explants to study collective cell migration and reepithelialization in cutaneous wound healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

As illustrated in Figure 1, sheets of keratocytes can be observed migrating out from beneath the scale within hours of establishing the explant culture. Occasionally, an individually migrating keratocyte which has broken away from the collectively migrating sheet can be observed. In addition, as the explant was established from a fish which had been previously plucked, there are abundant neutrophils migrating through the sheet. At longer culture periods, the keratocyte sheet fragments as cells undergo EM...

Watch this Scientific Journal Video about Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing at JoVE.com

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

zebrafish-keratocyte-explants-to-study-collective-cell-migration

Research

JoVE Journal

Developmental Biology

9.9K Views.  Midwestern University.  Zebravis keratocyten migreren celvellen van explantaten en verschaffen een in vitro model voor de studie van de mechanismen van collectieve celmigratie in de context van epitheliale wondgenezing. Deze protocollen detail een effectieve manier om primaire explantkweken stellen voor gebruik in collectieve celmigratie assays. 

Video: Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Bioengineering

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Biology

Robust Zebrafish Cell Transplantation for Regeneration and Development Research

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

Author Spotlight: Mapping Cellular Connectivity in the Zebrafish Nervous System During Development and Regeneration

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

Immunology and Infection

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. Indeed, the eye injuries most frequently encountered clinically are those to the cornea. While corneal wound healing has been extensively studied in small mammals (i.e., mice, rats, and rabbits), corneal physiology studies have neglected other species, including zebrafish, despite zebrafish being a classic research model.
This report describes a method of performing a corneal abrasion on zebrafish. The wound is performed in vivo on anesthetized fish using an ocular burr. This method allows for a reproducible epithelial wound, leaving the rest of the eye intact. After abrasion, wound closure is monitored over the course of 3 h, after which the wound is reepithelialized. By using scanning electron microscopy, followed by image processing, the epithelial cell shape, and apical protrusions can be investigated to study the various steps during corneal epithelial wound closure.
The characteristics of the zebrafish model permit study of the epithelial tissue physiology and the collective behavior of the epithelial cells when the tissue is challenged. Furthermore, the use of a model deprived of the influence of the tear film can produce&#160;new answers regarding corneal response to stress. Finally, this model also allows the delineation of the cellular and molecular events involved in any epithelial tissue subjected to a physical wound. This method can be applied to the evaluation of drug effectiveness in preclinical testing.

This protocol focuses on damaging the ocular surface of zebrafish through abrasion to assess the subsequent wound closure at the cellular level. This approach exploits an ocular burr to partly remove the corneal epithelium and uses scanning electron microscopy to track changes in cell morphology during wound closure.

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. ...

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

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