CONFIGURACIÓN DEL MÓDULO BLUETOOTH

Introduction

• Embryonic epithelial wound front cells assemble an actin cable

As discussed in my Introductory chapter, embryonic wounds close by a combination of re- epithelialisation and contraction of the underlying mesenchymal wound bed. Re- epithelialisation is driven by an actin purse-string which forms in the leading edge of basal epidermal wound edge cells (reviewed in Woolley and Martin, 2000). This wound-induced purse-string was first observed in the chick embryo (Martin and Lewis, 1992) and in the mid-gestational mouse embryo (McCluskey and Martin, 1995), and most recently in

Xenopus tadpoles (Bement et al., 1999). In all these embryonic repair models, the cells around the wound site have a rounded morphology immediately after wounding, but several hours later the same cells appear smoothly stretched around the wound margin. No lamellipodia or filopodia, characteristic of a crawling epithelium, are visible by SEM. Generally, embryonic wounds re-epithelialise very rapidly, without a lag phase as is normal at the outset of adult skin healing, and they leave only a pimple of cell debris, where the epithelial edges have met and closed, rather than the connective-tissue scar left as a product of tissue repair in the adult.

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In the chick embryo, an actin cable begins to assemble in leading edge basal epithelial cells within minutes of wounding and, although discontinous within the first minutes, has become continuous by 5 minutes. The cable reaches its full thickness after 30 minutes and it persists until the wound is closed. After the advancing epithelial edges have made contact, the cable disassembles, transiently leaving disorganised actin in the cells which it has just zippered closed (Brock et al., 1996).

When cytochalasin D, which blocks actin polymerisation, is administered to the wound, assembly of the cable fails and wounds completely fail to epithelialise, further supporting the model that the actin cable is responsible for driving embryonic wound repair (McCluskey and Martin, 1995).

In the chick embryo, immunohistochemistry has revealed that large clusters of cadherins, the transmembrane adhesion components of adherens junctions, localise to the leading edge of basal wound edge cells co-incident with actin cable formation and presumably reflect where the cable is linked from cell to cell by adherens junctions. These junctions are seen within 5 minutes of wounding, and persist throughout wound closure.

• Actin cables are also observed in tissue culture and repair of some adult tissues

The transition from an embryonic purse-string mode of epithelial repair to the dramatically different, adult-like crawling mechanism appears to occur sometime around the late

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embryonic stage of development, coincident with epithelial stratification of the developing skin (J.Brock, thesis London 1997). In addition to embryonic wounds, some epithelial cell- lines also use contractile purse-string machinery to close a wound. Caco-Z^^^ cells, an intestinal epithelial cell line, are able to repair small wounds in confluent monolayers using a multicellular actinomyosin purse-string continuous from cell to cell, just as seen in embryonic skin wounds (Bement et al., 1993). However, in wounds that are larger than ten or so cell diameters across, they resort to lamellipodial crawling to repair, in a way more like adult skin healing. In the smaller wounds, several cytoskeletal proteins were found to co-localise with the actin filaments. Myosin-II was found in a similar pattern to actin, at the edge of the wound, tightly associated with the actin cable, along with the actin filament binding protein, tropomyosin (Bement et al., 1993).

Actin purse-strings have also been observed at the leading edge of repairing comeal wounds in adult mice, suggesting that the wound purse-string is not solely an embryonic or tissue culture phenomenon (Danjo and Gipson, 1998). Disruption of the tight cellxell junctions linking intracellular segments of this adult wound actin purse-string, using E- cadherin function-blocking antibodies, results in cable disruption and lamellipodial protrusions from leading edge cells into the denuded area. It is clearly important to understand precisely what dictates the mode of motility adopted by an epithelial cell at a free wound edge. Is the decision to purse-string or to crawl determined by the size of the wound or the angle of curvature at the wound edge and, if so, how do cells read such cues? Perhaps cells that are tightly adherent to one another can purse-string, whilst cells with less

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Strong cellx ell junctions instead utilise adhesions to their underlying matrix substrate to

drag themselves forward.

• Formation of the purse-string is dependent on the small GTPase Rho

Multicellular purse-string assembly in the wounded embryonic epithelium is dependent on firing of small GTPases. Studies in 3T3 fibroblasts have demonstrated that the small GTPase Rho mediates stress fibre assembly in response to extracellular cues (Ridley and Hall, 1992), whilst Cdc42 and Rac, mediate assembly of filopodia and lamellae respectively (Nobes and Hall, 1995; Ridley et al., 1992). Since the wound actin-cable resembles oriented, bundled stress-fibres it seemed likely that Rho might be the controlling switch. Indeed loading of wound edge cells with the Rho blocker, C3 transferase, does prevent actin-cable assembly and subsequent re-epithelialisation of the wound, whilst blocking Rac from firing does not inhibit re-epithelialisation (Brock et al., 1996).

Rho has also been shown to be important during in vitro wound healing. A selection of Rho-inhibitors, including C3 transferase, toxins A and B, and dominant negative form of Rho A, prevented wound repair in two epithelial cell lines, (IEC-6 cells and Caco-2 cells), by inhibiting stress fibre formation and subsequent cell migration (Santos et al., 1997).

Although several systems have now shown that formation of an actin cable is Rho- dependent, it is unknown what signalling cascade is activated downstream of Rho in order to trigger actin reorganisation. Rho is known to activate several signalling pathways, and

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just as initial clues as to the action of Rho came from tissue culture, similarly we can guess from the downstream events in tissue culture cells, which pathways are most likely to be important during wound healing. One of Rho’s downstream targets is Rho-kinase. Rho- kinase family members include ROCK-I (also known as ROKp and p i 60^°^^) and ROCK- II (also known as RO K -a or Rho-kinase). ROCK-I and ROCK-II induce stress fibre assembly and assembly of focal contacts, but are not involved in other downstream functions of Rho, such as SRF-regulated transcription, cell transform ation or phosphoinositide metabolism. During cytokinesis, actinomyosin contraction is thought to be controlled by another Rho-effector, citron kinase, which co-localises with Rho at the cleavage furrow in Swiss3T3 cells (Madaule et al., 1998). However a dominant-negative ROCK construct inhibits cytokinesis when injected into Xenopus embryos and mammalian cells implicating a role for Rho-kinase too (Yasui et al., 1998). Therefore, Rho-kinase is an ideal candidate for mediating the signal as it directs actin purse-string assembly at the wound site.

The substrates of Rho-kinase appear to be largely components of the cytoskeleton or proteins that directly interact with actin/myosin and all could conceivably play a role in wound healing. The myosin-binding subunit of myosin phosphatase, (MBS), and the myosin light chain (MLC), are both direct targets and are involved in regulating myosin-II binding to actin and consequently the assembly of stress fibres (Hall, 1998). Other targets include several actin binding proteins for example, adducin, which has an FH domain important in binding profilin, which in turn sequesters actin monomers but can also promote elongation of actin filaments. Several intermediate filament proteins and ERM

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proteins may also be targets and are also known to be involved in stress fibre assembly (reviewed in Aspenstrom, 1999). To test the role of Rho-kinase during wound healing, an inhibitor to this protein is now available. This inhibitor, Y-27632, is a high-affinity inhibitor of Rho-kinase and inhibits the formation of stress fibres in HeLa cells, but not any of the other activities of RhoA, such as gene transcription (Uehata et al., 1997).

• Actin purse-strings are found in single cell wounds

Repair of cell wounds in giant Xenopus oocytes has recently been shown to also involve contraction of an actinomyosin purse-string that draws the cytoplasmic defect closed (Bement et al., 1999). Whilst the plasma membrane hole is repaired within seconds by vesicle fusion (Terasaki et al., 1997; Woolley and Martin, 2000), the cytoplasmic defect takes up to 15 minutes to repair. Both actin and myosin are recruited within 30 seconds or so to the wound site. The oocyte study suggests that myosin is recruited first and may act as the purse-string template since it forms a cable at the wound margin even in the presence of cytochalasin D which blocks actin polymerisation and actin-cable assembly (Bement et al., 1999). This is somewhat surprising as earlier chick embryo studies suggested that these motor proteins are recruited to the already assembled actin-cable (Brock et al., 1996). However, it could be that undetectable levels of myosin are recruited sooner, even preceding actin localisation, in a way which might parallel Bement’s observations in single cell wounds (Bement et al., 1999). Alternatively, this discrepancy may represent a real difference between single-cell and multi-cellular wounds. Since epithelial cells already have a circumferential ring of cortical F-actin prior to wounding this presumably might act

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as a nucléation site to which more actin and myosin could be recruited. Single cells, in contrast, have to assemble the purse-string de novo. The single cell oocyte purse-string, like a multicellular embryonic wound, is also dependent on Rho activation and treatment of wounds with C3 transferase prevents assembly and subsequent wound closure (Bement et al., 1999).

• Assembly of the actin cable in X en o p u s oocytes correlates with a microtubule network

Oocyte wounding also leads to a dramatic reorganisation of the microtubule cytoskeleton - a dense microtubule network rapidly localises to the region of cortical cytoplasm adjacent to the wound site (Bement et al., 1999). Inhibition of microtubule polymerisation at the time of wounding results in a failure of actinomyosin purse-string formation and blocks cytoplasmic repair, suggesting that microtubules are in some way required for delivery of at least some of the components of the actinomyosin purse-string to the wound site. However, as soon as the cable is assembled, microtubules seem no longer to be required, and may even be a hindrance to repair, since microtubule inhibitors, delivered minutes after wounding, lead to faster repair of the cortex than in untreated oocytes.

In this chapter I report my studies of re-epithelialisation of Zebrafish embryonic wounds. I describe the cell shape changes, first in scanning electron micrographs and then in live embryos using lectin-staining of the enveloping layer to visualise these dynamic cell shape

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changes in 4-D. In addition I report a number of blocking experiments to test the roles of these elements, and the signals directing them, during wound closure.

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Results

I wounded embryos at two different developmental timepoints during Zebrafish embryogenesis, allowing me to address two different sets of questions. Wounds made to the epithelial sheet overlying a 50% epiboly embryo allow me to directly observe assembly of the cytoskeletal machinery that drives re-epithelialisation and to analyse the cell shape changes and shufflings that contribute to closure of the epithelial hole. Also, importantly, this early stage gives an opportunity to test the genetics of epithelial repair by wounding mutants that are defective in the cell movements of morphogenetic movements like gastrulation and epiboly. In addition to the early stages of development I also wounded the 24 hour embryo, which has well developed skin with mature epidermis and underlying mesenchyme, and which is more similar to the developmental stages of chick and mouse embryo wound healing studies previously performed in this lab.

• Wounds made to the 24 hour fish heal rapidly by epithelial cell shape changes.

Using a tungsten needle I made stab wounds to either the tail of the 24 hour zebrafish (approximately one third of the way between the yolk extension and the tail tip), or to the dorsal side of the embryo between the otic vesicles (see Fig 3.1 A, B)

Scanning electron microscopy reveals that wounds made by this method are fairly irregular in size, although all wounds appear to subsequently undergo a very similar set of cell rearrangements and cell shape changes (Fig 3.1 C-F). Within a couple of minutes of wounding, cells at the wound edge have become arranged radially (Fig 3.1 C). The shape change and oriented organisation is probably as a result of the release of tension in the taut epithelium (Fig 3.1 C). All wounds examined showed no trace of epithelium on the

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denuded surface but often scraps of cell debris. By 5 minutes after wounding the leading edge epithelial cells slightly extended over the exposed wound site (Fig 3.1 D). By 20 minutes, the leading edge cells have now become polarised with their long axis perpendicular to the wound margin and their apices narrow as though actively constricting (Fig 3.1 E). Cell shape changes are not restricted to the leading edge cells; cells as far back as 2-3 cell diameters from the wound edge also appear to be stretched forwards in the direction of epithelial movement. 30 minutes after wounding most epithelial holes have just re-epithelialised with the cells still stretched and with constricted apices (Fig 3.1 F). After one hour, SEMs reveal no sign of the wound, except in some cases a small pimple of cell debris remaining as the only sign that the embryo had been wounded. After closure, normal epithelial sheet tensions are re-established and cells resume normal shapes, leaving no sign of a scar.

• Wounds made to the 50% epiboly embryo heal in a standardised fashion

Wounds are made to the 50% epiboly embryo by sucking a group of cells off the animal pole of the embryo, using a pipette with 100 pm diameter tip (Fig 3.2 A-C). The resulting wound is circular and standardly about 100 pm in diameter. Because of the uniformity of wounds made at this stage, such a model is amenable to comparisons of wound closure in the presence of various blocking drugs and genetic backgrounds.

Just as in the 24 hour embryo, the cell shape changes and extent of repair in these early embryos can be observed at various timepoints post-wounding by scanning electron microscopy (Fig 3.2 D-F). Cell shape changes at the wound edge appear similar to the older embryos but occur at a slightly faster rate. Cell constriction and elongation is apparent from 5 minutes post wounding (Fig 3.2 D, E) and most 50% epiboly wounds are

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later stage wounds is the extent of cell debris at the epithelial closure site. In the rapidly closing 50% epiboly epithelial wound there is more accumulated extruded cell debris which might be deep cells of the blastoderm which have been pushed out of the wound site as the EVL closes (Fig 3.2 F).

• Timelapse studies reveal the dynamic cell shape changes during wound closure

Time-lapse analysis of wounds made to lectin-stained embryos reveals the real-time movements made by cells in relation to one another and allows more precise description of the timing of various cell behaviours during wound closure.

As seen previously by SEM studies, cells at the leading edge of a wound undergo constriction at their leading edge and elongation in the direction of the wound defect. In Fig 3.3 (and supplementary video 1) the cell marked with a white asterisk shows this occurring in real-time. When the wound is first made, this cell's leading edge is approximately 20 pm long (as part of the wound edge) (Fig 3.3 A). After 5 minutes this cell has constricted its leading edge (3-fold) (3.3 B) and started to elongate towards the wound site, elongating to as much as twice its original length (Fig 3.3 C-F). Not all cells at the wound site undergo constriction and elongation concurrently. The cells neighbouring and immediately adjacent to the white marked cell do not constrict their leading edge at the same time as the marked cell, and retain their longer edge to the wound edge for several minutes, before constricting their leading edge and migrating over the wound site.

Cells further back from the wound edge can also be seen to change shape but this seems to be restricted to the second, and occasionally cells in the third row. Some third row cells and cells further back appear not to participate at all in the re-epithelialisation process. For

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example, the cell marked with a red asterisk is three cells back from the wound edge and retains the same shape throughout the initial wound closure process (Fig 3.3 A-F).

Time-lapse videos of wounds towards the end of wound healing show how the taut epithelium appears to relax back after opposing epithelial fronts have met one another (Fig 3.4 A-F). Cells that have constricted their apices and elongated into the wound site to close the wound now shuffle to resume a normal epithelial cytoarchitecture, with characteristic 'crazy paving' cell shapes as opposed to the 'rosette' of cells seen immediately after wounding. After wound closure, cells which have stretched forward to close the wound defect are elongated with narrow apices (Fig 3.4 A). Within minutes of wound closure, these highly polarised cells gradually relax to resume a normal architecture, apparently moving away from the site of wound closure. For example, the cell marked with a white asterisk has a highly constricted leading edge at the point of epithelial closure (Fig 3.4 A). This cell appears to move back away from the wound site, gradually altering its shape until it regains its original polygonal appearance (Fig 3.4 F). The original wound gap is partially obscured by cell debris and cell outlines are difficult to discern, presumably because of the high rate of membrane turnover at the wound site leaving parts of the membrane with no florescent marker, as discussed in the following section.

• Membrane remodelling appears to occur during and following wound closure

In the addition to revealing cell shape changes in the apical membrane of EVL cells, fluorescent lectins can also reveal membrane dynamics over a period of time. Time-lapse videos of wounds both during and after wound closure reveal a higher level of membrane turnover in comparison to EVL four cells away from the wound site. At the beginning of wound closure the EVL is uniformly stained with clear and definitive cortical membrane

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fade and are partly devoid of fluorescent probe molecules (arrows in Fig 3.3 C) as if the original stained membrane has been internalised or moved to a different part of the membrane. After 17 minutes several small green particles have appeared on the apical membrane of stretched wound edge cells and these may represent membrane particles