• A historical perspective
To date there have been very few wound healing studies in fish with early studies of wound healing in fish focused mainly on the development and regeneration of the scales, (Yamada,
Chapter 1 Introduction
(1935), (reviewed in Mittal et al., 1977). Fin regeneration has been studied ever since and recently several groups have begun to dissect out the molecular mechanism underlying the repair process using the Zebrafish as a model..
During the 1970’s several authors contributed to describing the sequential histopathology of skin wounds in teleosts, culminating in work by Mittal et al (1977), who described the pattern of wound healing and overall structural changes in the skin and underlying muscles as a result of a skin wound in the adult Cat-Fish Heteropneustes fossilis (Bloch). Repair of the comeal epithelium has also been studied in marine and freshwater fish (Ubels and Edelhauser, 1982). A more recent paper (Quilhac and Sire, 1999) described healing of superficial skin wounds made to the adult cichlid fish, Hemichromis bimaculatus, detailing the spreading, proliferation and differentiation of the epidermis and the interactions between the epidermal cells and the underlying substrate.
It was not until 1988 that embryonic wound healing in fish first began to be studied using the killifish Fundulus heteroclitus (Fink and Trinkaus, 1988). Since this time no further work has been published in the field of embryonic fish wounds.
• Even relatively large skin lesions in adult fish close within minutes
In the past, work on adult fish wounds has focused on either the inflammatory responses invoked (Mittal et al., 1977) or the underlying mesenchymal substrate on which the epidermal cells migrate (Quilhac and Sire, 1999). Immediately after injury to the adult Cat- Fish Heteropneustes fossilis (Bloch) (Mittal et al., 1977), a plug consisting of inflammatory cells and exudate fills the wound area, just as in an adult mammalian wound. Following injury the wound edge initially retracts and then, within fifteen minutes of injury, the epidermis begins to migrate towards the wound gap. The wound area is very rapidly re-
Chapter 1 Introduction
epithelialised within 4 hours, at a rate of 1.25mm / hour but the underlying tissues are much slower at recovering. Granulation tissue forms from the blood capillaries and fibroblasts, and gradually resumes normal synthesis of collagen. It takes 35 days for the epidermis to resume its normal structure and appearance, and interestingly, there is no trace of a scar left at the wound site (Mittal et al., 1977).
A more recent study focused on the healing of superficial skin wounds in adult cichlids,
Hemichromis biaculatus (Quilhac and Sire, 1999). Again, re-epithelialisation was fast, with a 1cm square wound surface completely covered by the healing epidermis 9 hours after surgery. Although it takes longer for the epithelium to resume its normal structure there is no mention of whether a scar forms. Re-epithelialisation does not require proliferation of surrounding epithelium, as revealed by incorporation of tritiated thymidine, but there does appear to be a burst of proliferation following completion of re- epithelialisation. This lack of proliferation required for epithelialisation has been noted previously (Misof and Wagner, 1992). Therefore re-epithelialisation occurs by the spreading and migration of pre-existing cells from around the wound area. This spurt of new division seen in areas around the wound, especially in intermediate layers, lasts for a few days, indicating that new cell division is required in the restructuring of the tissue around the wound.
Ubels and Edelhauser (1982) noted that the comeal healing mechanism was essentially the same in fish and mammals. Histology of wounds made to the cornea of various fish species shows that the wound initially becomes covered with a single layer of epithelial cells which migrate from the wound edge across the basement membrane.
C hapter 1 Introduction
None of the studies above describe the mode of motility utilised by the rapidly repairing epidermis in adult fish wounds so it is unknown whether a crawling or a purse-string mechanism may be involved.
• Studies in Fundulus embryos show that both EVL movements and migration of deep cells occur during repair
Although wound healing in adult fish allows a comparison with the mammalian process, the fish embryo provides a far better tool for studying directional cell movements in vivo for the reasons mentioned in the previous two sections. Largely because of their transparency, Fink and Trinkaus (1988) used the killifish Fundulus heteroclitus to study directional cell movements during development of the embryo and used wound healing as a model of these movements. As with Zebrafish the embryo is large and transparent allowing the study of these movements in vivo, using DIC optics. An obvious advantage of studying repair rather than natural morphogenetic movements is that the process can be followed from the very beginning because the wound can be induced at any time. Fink and Trinkaus made small wounds, of less than 100 fxm to the embryo and movements of the EVL and underlying deep cells were observed by Nomarsky optics (Fink and Trinkaus, 1988). Within the first minute the EVL margins contracted and by 2 minutes deep cells began to move towards the site of injury and continued to migrate towards the wound area over the next two hours. The wound was completely closed within 150 minutes. The focus of this study was the movement of deep cells and chemotactic cues for deep cell blebbing with no mention of how EVL epithelial cells might be migrating forward, but their puckered leading edge suggests that like embryo repair in other organisms studied in our lab, it is probably driven by epithelial shape changes which in turn are driven by purse-string contraction. Despite some literature in the field it is clear that studies in both adult and embryonic fish wounds have largely ignored the molecular and cellular machinery
Chapter 1 Introduction
controlling wound closure. To address these issues we have looked for clues from other systems.
• Molecular clues from fin regeneration studies
Teleosts have the capacity to regenerate fins following their amputation. Fin regeneration in zebrafish leads to re-establishment of full fin pattern and is preceded by upregulation of a battery of genes that may be important in the repair process. Fin regeneration proceeds through several stages similar to those which occur during urodele limb regeneration. Within 24 hours of fin amputation, a wound epithelium has migrated over the fin stump. It is in direct contact with the underlying mesenchyme and is thought to play a role similar to the epithelium of developing fin buds. This epithelium is believed to operate in a similar way to the apical ectodermal ridge (AER) of developing limbs in higher vertebrates because it is positioned at the exact boundary of ventrally located expression of engrailedl
gene (Ekker et al., 1992; Hatta et al., 1991). Also expression of sonic hedgehog (shh)
corresponds to the ZPA of vertebrate limbs (Krauss et al., 1993). Mesenchymal cells are subsequently recruited to form a blastema underneath the wound epithelium, probably by de-differentiation. Finally, differentiation and outgrowth of the regenerate proceeds until the fin reaches its normal size (Johnson and Weston, 1995). Treatment with retinoic acid induces apoptosis specifically in the wound epithelium (Ferretti and Geraudie, 1995). This appears not to impair the process of wound healing but does block the formation of a blastema, suggesting that signals from the wound epithelium may be important for blastema formation. In newts, the wound epidermis is itself a source of retinoids (Viviano et al., 1995) and so the regulation of endogenous retinoic acid may be an important factor in regenerative processes.
Chapter 1 Introduction
In a temperature-sensitive screen for mutations affecting growth and regeneration of the zebrafish caudal fin several mutant lines were identified in which fin regeneration fails, (Johnson and Weston, 1995). Characterisation of these mutants will provide more clues about the downstream signals involved and the genes required for patterning the regenerate, but is unlikely to shed light on the basic repair processes that are the topic of my thesis. No screens have yet been undertaken that address re-epithelialisation or closure of a wound in Zebrafish.
In my thesis I aim to analyse the process of wound repair in the Zebrafish embryo. My main focus will be to study how cytoskeletal reorganisations and contractions control re- epithelialisation of a wound. I will attempt to elucidate which signalling cascades direct these events and to compare them with the signals controlling the natural morphogenetic movement of epiboly in the Zebrafish embryo.
C hapter 2 G eneral M aterials and M ethods