Name: zebrafish keratocyte explants to study collective cell migration and reepithelialization in cutaneous wound healing
Uploaded: 2015-02-25T17:00:00
Description: Watch this Scientific Journal Video about Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing at JoVE.com

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
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	<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 w...

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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 border="0" cellpadding="0" cellspacing="0" width="777">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:320px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">35 mm Glass Bottom Culture Dishes (Poly-D Lysine Coated)&#160;</td>
			<td style="width:71px;">MatTek</td>
			<td style="width:119px;">P35GC-1.5-14-C</td>
			<td style="width:320px;">1.5 mm&#160; thickness is best for coverslip removal.&#160; 14 mm diameter provides more culture area.&#160;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Swiss Jewelers Style Forceps, Integra, Miltex 17-303</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">21909-460</td>
			<td style="width:320px;">We find that narrow, fine forceps work the best for scale removal.&#160;&#160;</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 ...

Research

JoVE Journal

Developmental Biology

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...