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Figure 5.3 Wounds heal at different rates depending on the external media. A-F. SEMS showing wound closure in epiboly embryos wounded in either 0.3 X Danieau (A, C, E) or 1 x Danieau (B, D, F) and allowed to heal for 0 mins (A and B), 10 mins (C and D) or 20 mins (E and F). Wounds made in 0.3 x Danieau are almost re-epithelialised after 10 minutes (C) and are fully closed by 20 mins (E). In comparison, wounds made in 1 x Danieau are still open after 10 mins (D) and are only just closed by 20 mins after wounding (F).

Figure 5.4 A ctive-E R K im m unostaining o f w ounds m ade to the 24 hour Z ebrafish tail.

A-F. E m bryos were w ounded and allow ed to heal for 0 m ins (A ), 5 m ins (B), 15 m ins (C and D), 30 m ins (E) and 60 m ins (F) before fixing and im m unostaining for active-E R K . A ctive kinase is detected by the brow n precipitate at the w ound site. D show s a zoom -in o f the wound in C.

Figure 5.5 A ctive-E R K expression in w ounds m ade to epiboly-stage em bryos treated w ith the ER K inhibitor, U 0 1 2 6 .

A-C. E piboly-stage em bryos were w ounded on their dorsal surface and allow ed to heal for 0 m ins (A ), 15 m ins (B) and 30 m ins (C) before pro cessin g for active- ERK im m unostaining.

D. G roup o f control em bryos show ing norm al ER K activation (brow n staining) 15 m inutes after w ounding.

E. G roups o f U 0 1 2 6 -tre a te d em bryos show no activation o f E R K at the w ound site.

F and G. SEM s show ing control (F) and U 0126-treated (G ) em bryo fixed 30 m inutes after w ounding show ing E R K -inhibited em bryos are able to close w ounds at the sam e rate as untreated em bryos.

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Figure 5.6 Anti-active-p38 and anti-active-JNK on Zebrafish embryos.

A and B. Un wounded embryos (A) and wounded embryos (B) were fixed and stained for anti-active-p38 (green). Wounded embryos were fixed after 5 mins (B).

C and D. Un wounded epiboly-stage embryos (C) and wounded embryos (D) were fixed and immunostained for anti-active-JNK (green). 15 minutes after wounding, no increase in active-JNK is seen around the wound site (embryo is counter-stained with rhodamine-phalloidin to reveal cortical cell outlines).

E-G. SEMs showing wounds made to control embryos (E ), embryos treated with SB203580 only (F) and embryos treated with a cocktail of SB203580 and U0126 to inhibit all MAPK signalling. All wounds closed at the same rate as the control, untreated embryos.

Chapter 5 Discussion

Discussion

In this chapter I describe several signalling cascades which become activated in wound edge cells and which may be involved in directing the subsequent tissue movements of wound closure. I describe a preliminary study revealing the role of calcium in wound closure, initially looking at the wave of calcium that spreads away from the wound edge immediately following wounding and then correlating the concentration of calcium in the wound medium with the rate of wound closure. I also examine the role of MAP kinase activation during wound closure and test the role of these signals in wound closure using specific inhibitors to these kinases.

• Calcium plays an important role in wound healing

My pilot data illustrates increased levels of intracellular calcium in wound edge cells and up to 4 cells back from the wound edge by approximately 5 minutes after wounding. Although this represents only one time-point, I speculate that this corresponds to a transient wave of calcium initiated immediately upon wounding by leakage of extracellular calcium into damaged front edge cells and subsequent passage back into neighbouring cells. At the time of the study, timelapse analysis of this calcium wave was not possible but this wave could now be visualised in vivo using similar imaging techniques to those described in previous chapters for following cell movements. Since calcium influx is observed in a sim ilar number of rows of wound edge cells as appear to be involved in the

Chapter 5 Discussion

reepithelialisation process, it may be that calcium directs the epithelial cell behaviours that close the hole.

In order to part test the role of calcium influx as a wound signal I have compared repair in media of difference salt concentrations. Surprisingly it appears that repair is faster in the lower calcium conditions. This data appears not to square with previously published experiments in which the absence of calcium from the media bathing the wound either delayed (Tran et al., 1999) or inhibited (Stanisstreet, 1982) wound closure. However, analysis of my epiboly-wounded embryos at 24 hours post fertilisation using morphological and molecular markers suggest that although the wounds close more quickly, the wounds made in the low salt concentrations do not appear to close normally (Saude et al., 2000). Embryos wounded and allowed to heal in 0.3 Danieau's appear not to have sealed correctly and lead to abnormalities in the embryos whereas the embryos develop normally after wounds were made in 1 x Danieau. It may be that the osmotic shock in 0.3 x Danieau causes rapid re-epithelialisation but a failure to adhere properly when wound edges confront one another. Indeed, low extracellular calcium is inhibitory to assembly of adherens junctions between epithelial cells (Vasioukhin et al., 2000).

To formally test that calcium is indeed the key ion that might influence the reepithelialisation process, the same experiment could be repeated using Ix Danieau's with lower levels of calcium as found in 0.3x Danieau's. In theory it would also be interesting to deplete calcium entirely from the external medium at the time of wounding to test whether this inhibits wound closure. However, removing calcium entirely from the external media

Chapter 5 D iscussion

is likely to have a promiscuous effect on many cellular processes and removing calcium only at the time of wounding may merely delay the wound process until calcium is added back to the media. Another key experiment might be to block gap junction communication with halothane and thus prevent transfer of the Ca signal beyond front row cells to examine the effect on repair in these conditions.

What might increased levels of calcium do at the wound site? Intracellular calcium is important for many processes involved in cell movement such as the production of actinomyosin based contractile forces (Citi and Kendrick-Jones, 1987; Rees et al., 1989; Strohmeier and Bereiter-Hahn, 1984), the regulation of the structure and dynamics of the actin-cytoskeleton (Condeelis, 1993; Hart wig and Yin, 1988), and the formation and disassembly of cell-substratum adhesions (Crowley and Horwitz, 1995; Sjaastad and Nelson, 1997). Therefore, entry of calcium into wound edge cells might be a critical trigger for all cell shape changes during wound healing, for cellicell rearrangements and for the final epithelial adhesion of the opposing wound edges. In larger wounds that take longer to heal, it is likely that the entry of calcium into front row damaged cells might also trigger transcription of immediate early genes, but in the wounds 1 make which close in only 30 minutes, it seems unlikely that transcriptional events could be operating rapidly enough to play any significant role in the repair process.

Chapter 5 Discussion

• What role do MAP kinases play in wound healing in the Zebrafish embryo?

In this chapter I show immunocytochemical evidence for a rapid and transient activation of the Ras/MAP kinase cascade in the front few rows of cells at the wound edge. The rapidity of this signal and the domain of cells that become activated suggest that it could be involved in directing epithelial shape changes during wound closure. However, I show that pre-treatment with a specific inhibitor of the ERK cascade does not inhibit repair. This is not completely surprising, since scrape wounds made to 3T3 fibroblasts show a similar activation pattern of ERK in wound edge cells and yet, just as in my experiment, cell migration into the wound space is not altered by drugs that block ERK activation (Nobes and Hall, 1999). Epithelial repair by purse-string activity and fibroblast migration are two entirely different modes of migratory behaviour and one or both of these repair processes may be dependent on redundant MAP kinase activities. It may be that ERK feeds into one of the other MAPK pathways. However, I find that the p38 and JNK cascade appear to be constitutively active with neither overtly increasing activation levels upon wounding. Moreover, cocktails of blockers which should entirely inhibit all three of these MAP kinase cascades appear to have no effect on the rapid sealing of my small epithelial wounds.

One possible caveat with this experiment, and of experiments designed to block the p38 or JNK signals individually, is that at present we have no way of assaying whether the SB 203580 is actually blocking either the p38 or JNK pathways. Since SB 203580 acts downstream of p38 and JNK and no downstream effectors are cloned in the Zebrafish we

C hapter 5 Discussion

have no way to test this properly. As a result we cannot conclusively rule out the possibility that p38 and JNK do play a role in wound closure.

Why is ERK being activated if it has no role in wound healing? In Xenopus wounds, Ras is activated as demonstrated by the activation of ERK, proposed to stimulate growth and differentiation to replace damaged tissue (Christen and Slack, 1999). Ras feeds into several different pathways, including the ERK cascade, and it is possible that all downstream effectors of Ras are activated whether they are required or not. Clearly in my small embryonic wounds where repair is rapid and re-epithelialisation occurs in the absence of proliferation and before transcriptional events have much opportunity to play a role, the full function of ERK activity during wound repair might not be needed. It would be informative to test out the various MAP kinase blocking drugs on larger and later stage wounds where repair may indeed be hindered. Alternatively, the role of the ERK signal may be subtle and missed in my studies. For example, it may play a role in modulating cell adhesion in cell rows back from the leading edge allowing a degree of cell shufflings and fluidity in the epithelium which enhances, but is not essential, for re-epithelialisation.

C hapter 6 G eneral Discussion

CHAPTER SIX