Departments of Physiology and Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A1-162, PO Box 19024, Seattle, WA 98109-1024, USA
Authors for correspondence (e-mail: paul.martin{at}bristol.ac.uk and susanp{at}fhcrc.org)
SUMMARY
Wound healing involves a coordinated series of tissue movements that bears a striking resemblance to various embryonic morphogenetic episodes. There are several ways in which repair recapitulates morphogenesis. We describe how almost identical cytoskeletal machinery is used to repair an embryonic epithelial wound as is involved during the morphogenetic episodes of dorsal closure in Drosophila and eyelid fusion in the mouse foetus. For both naturally occurring and wound-activated tissue movements, JNK signalling appears to be crucial, as does the tight regulation of associated cell divisions and adhesions. In the embryo, both morphogenesis and repair are achieved with a perfect end result, whereas repair of adult tissues leads to scarring. We discuss whether this may be due to the adult inflammatory response, which is absent in the embryo.
Introduction
Building tissues and organs during embryogenesis involves a series of exquisite morphogenetic episodes that are driven by the marriage of regulated proliferative events with a series of precisely orchestrated tissue contractions, foldings and migrations. Slowly, a miniature model of the adult form resolves and subsequent foetal development is largely a matter of growth and remodelling phases. Once established, adult tissues are homeostatically maintained by a balance of cell death and replacement, and most tissues remain in this dynamic but fairly dormant state for the entire life of the organism. But a dramatic reawakening of the tissue building machinery is required if the organism is wounded, in order to replace missing tissues and repair the wound. Recent studies have revealed significant parallels between how tissues are built during development and how they are rebuilt during tissue repair episodes. This review outlines these parallels, focusing in particular on shared signalling cassettes and cytoskeletal machineries that drive epithelial migrations. It also discusses how studies of morphogenesis have shed light on the ways that cell:cell adhesions and cell division might be regulated as tissues move and knit together in a wound situation. In the embryo, wound healing is not accompanied by an inflammatory response and the final repair is perfect without a scar, unlike in the adult. We discuss the link between inflammation and scarring, and how studies of embryo healing might guide us in designing new wound healing therapies.
The biological basis of wound healing
Whenever an organism sustains an injury, especially to its outer protective skin layer, it must act rapidly to repair the wound to prevent further blood and tissue loss and infection. Damage to adult mammalian skin and other tissues generally leads to the rapid plugging of the defect with a fibrin-rich clot.
Subsequently, after a delay period of several hours, the epidermal layer is
repaired by the migration of keratinocytes from the cut edges and from the
amputated remains of any cut appendages, including hairs or sweat glands
(Fig. 1). From these free
edges, a sheet of keratinocytes sweeps forward across a provisional matrix of
fibronectin, vitronectin and other matrix molecules at the interface between
the wound dermis and the fibrin clot. Cells within the front few rows extend
lamellipodia and alter their integrin expression; specifically, they
upregulate fibronectin/tenascin- and vitronectin-binding integrins, and
relocalise their collagen/laminin-binding integrins so that the epidermal
sheet can attach down and drag itself forwards over the wound substratum
(reviewed by Grinnell, 1992;
Martin, 1997
;
Werner and Grose, 2003
). The
deeper connective tissue is replaced by activated fibroblasts at the wound
edge that proliferate and then migrate into the wound bed to form a
granulation tissue (so named because of its granular appearance due to massive
invasion by capillary networks), which contracts to aid in closing the wound
margins.
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Tissue repair in the mouse embryo involves largely the same tissue
movements as in the adult, although on a much smaller scale, but only at late
foetal stages is healing accompanied by an inflammatory response
(Hopkinson-Woolley et al.,
1994; Cowin et al.,
1998
). Prior to these stages, inflammation is absent and the
embryo is capable of essentially perfect, near regenerative repair, with no
resulting scar. Wound healing, even in the embryo
(Fig. 2), is a complex process
involving the coordination of several cell behaviours from several different
cell types, and for each stage of wound repair there are fundamental cell
biology issues that still need resolving (see
Box 1).
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A number of naturally occurring morphogenetic events involve tissue movements similar to those required for wound healing. Two of the clearest of these, both of which involve closure of epithelial holes, are dorsal closure in the Drosophila embryo and C. elegans ventral enclosure.
Dorsal closure in Drosophila
Near the end of the complex and intricately orchestrated cell and tissue
movements of Drosophila gastrulation, including the extension of the
germband over the dorsal surface and its subsequent retraction, a large hole
is left behind on the dorsal surface of the embryo. An extra-embryonic
membrane consisting of large flat cells the amnioserosa covers
this dorsal hole (Fig. 3A-D).
The process of bringing together the two epithelial edges over the amnioserosa
to close the hole and form a seamless dorsal midline is known as dorsal
closure. The dorsal hole is elliptical or eye shaped, and closure proceeds
from the anterior and posterior ends (or canthi) of the opening towards the
middle. The integrated efforts of three groups of cells are required for
proper closure: the dorsalmost row of ectodermal cells defining the perimeter
of the epithelial sheet, termed the leading edge (LE) cells; the more ventral
epithelial (VE) cells; and the exposed amnioserosa (AS). Dorsal closure has
been described as taking place in four phases (for detailed descriptions, see
Harden, 2002;
Jacinto et al., 2002b
). The
first phase, initiation (Fig.
3A), begins just prior to the completion of germband retraction,
with the two opposing epithelial sheets moving slowly towards one another as a
consequence of amnioserosal cell contraction. The trigger(s) required to start
the dorsal closure process are not known, but probably include a combination
of chemical and mechanical cues, including dorsoventral patterning information
and mechanical stresses generated by germband retraction.
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|
Box 1. Some of the key unanswered questions of wound repair
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As the epithelial sheets come into close proximity at the anterior and posterior ends of the opening, the third phase, zippering (Fig. 3C), begins. Filopodia from cells on the opposing epithelia meet and begin to interdigitate. Along with continued contraction of the LE cell actin cable and of the amnioserosal cells, interactions between opposing filopodial and lamellipodial protrusions appear to aid in drawing the two epithelial sheets towards one another and zipping them together.
The final phase, termination (Fig. 3D), produces the seamless midline. During this phase, filopodia regress and their transient adhesions are converted into permanent adhesions with the formation of adherens junctions (see Box S1 at http://dev.biologists.org/supplemental). As with the signal(s) required to start the dorsal closure process, those signal(s) necessary to stop the forward movement of the epithelial sheets and prevent overgrowth are currently unknown.
Ventral enclosure in C. elegans
Gastrulation in C. elegans involves a complex interplay of cell
shape changes and cell migrations within the 60 cells comprising the
dorsoposteriorly located hypodermis (the epidermis). At the start of
gastrulation, the hypodermis is arranged in three rows of 10 hypodermal cells
towards the left of the dorsal midline, and a mirror image three rows on the
right. Radiating from the dorsal midline, these three cell rows are referred
to, respectively, as dorsal hypodermis, lateral seam hypodermis (LSH) and
ventral hypodermis (VH). In a process similar to Drosophila germband
extension, the two dorsalmost rows of cells, the dorsal hypodermis,
intercalate to form a single row of cells (dorsal intercalation;
Fig. 3E). The stretching of the
hypodermis over the ventral surface of the embryo to form a seamless ventral
midline is known as ventral enclosure, and closely resembles
Drosophila dorsal closure. The integrated efforts of three
morphologically distinct cell types are required for proper ventral enclosure:
the VH, the LSH and the neuronal cells that form the ventral pocket (VP) over
which the ventral hypodermal cells migrate. Ventral enclosure has been
described as taking place in three steps
(Fig. 3F-H) (Williams-Mason et
al., 1997; Chin-Sang and Chisholm,
2000; Simske and Hardin,
2001
). The first step, leading cell migration
(Fig. 3F), begins just prior to
the completion of dorsal intercalation with the two anteriormost ventral
hypodermal cells (leading cells) elongating along the
dorsoventral axis. These cells produce filopodial extensions at their medial
tips that help to draw the hypodermis circumferentially, extending down past
the equator of the embryo.
During the second step, leading cell junction formation and fusion (Fig. 3G), the anterior pair of leading cells meet at the ventral midline followed by the rapid formation of adherens junctions for the anteriormost pair and cell fusion for the posterior pair. With the fusion of these leading cells, the remaining posterior ventral hypodermal cells become wedge shaped and elongate along the dorsoventral axis, closing a ventral gap that is called the ventral pocket. F-actin becomes concentrated in the leading edges of these migrating cells, forming an actin cable.
In the third step, ventral pocket enclosure (Fig. 3H), the ventral hypodermal cells lining the ventral pocket contract and migrate over the underlying neuronal cells to close the ventral hole. This contraction is believed to result from actin cable contraction at the leading edges of these ventral hypodermal cells using a purse-string mechanism. As the opposing ventral hypodermal cells meet, adherens junctions assemble and a seamless ventral midline forms.
Model organism paradigms
Genetic screening, genetic epistasis, cell biology, live imaging, molecular
and biochemical approaches in these two model organisms have together revealed
several of the structural and signalling molecules involved in these
morphogenetic episodes. The genetic tractability of both flies and worms has
allowed genetic screens to identify mutants that fail to undergo proper dorsal
closure or ventral enclosure (Table
1).
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Although dorsal closure and ventral enclosure do both superficially resemble re-epithelialisation of a wound hole, there are several key differences, including the fact that wound repair is initiated by tissue damage, whereas morphogenetic episodes are not. Clearly, these epithelial movements do not model all aspects of the repair process, such as inflammation or connective-tissue contraction and fibrosis, although the epithelial amnioserosa does contract during dorsal closure and so may mirror wound contraction to a certain extent.
The wound repair tool kit in embryos
Wounding skin triggers a cascade of events that leads to
re-epithelialisation of the defect and contraction of underlying wound
connective tissues. Early studies in the chick embryo showed that
re-epithelialisation occurs not by lamellipodial crawling of cells as in adult
skin healing; rather, migrating epithelial fronts sweep forward over a
mesenchymal substrata in a purse-string-like manner
(Fig. 2, Fig. 4A)
(Martin and Lewis, 1992), just
as discussed above for dorsal closure in flies and ventral enclosure in worms.
Transmission electron microscopy indicates that leading edge cells remain
adherent to the underlying basal lamina, which is drawn along with the
epithelial sheet as it moves forward, in contrast to the adult situation where
leading edge cells leave the old basal lamina behind and deposit new matrix
after they migrate forwards (McCluskey et
al., 1993
). A thick cable of actin is apparent in the leading edge
of basal marginal cells encompassing the wound, and contraction of this cable
almost certainly provides the force that draws the epidermal wound edges
together (Martin and Lewis,
1992
). Indeed, when new assembly of filamentous actin is blocked
by cytochalasin D or by loading cells with the Rho GTPase blocker, C3
transferase, wounds fail to re-epithelialise
(McCluskey and Martin, 1995
;
Brock et al., 1996
). As well
as a filamentous actin cable, other components of the contractile machinery,
including myosin II, are also assembled. These include proteins for
example E-cadherin that enable the intracellular cable to link to
neighbouring cells via adherens junctions
(Brock et al., 1996
).
In chick and mouse embryos, assembly of the actin cable is so rapid
(visible in leading edge cells within just two minutes of wounding)
(Martin and Lewis, 1992;
McCluskey and Martin, 1995
)
that it would seem that at least the early stages of cable formation must be
due to re-deployment of existing actin, myosin and junctional proteins. RHO
activity is essential for assembly of the wound-induced actin purse-string,
whereas analogous RAC-blocking experiments fail to interfere with the wound
response. Together, these results indicate that RHO is indeed the master
switch that mediates purse-string assembly at the embryonic wound margin
(Brock et al., 1996
).
Actin networks are used repeatedly to mould embryonic tissues during
organogenesis (see Box 2), and
wound purse-strings are not simply restricted to embryonic epithelia. Studies
in the adult rabbit eye suggest that small corneal lesions are drawn closed by
an analogous actin purse-string, but when this is disrupted by -catenin
blocking antibodies, epithelial migration defaults to a more
adult-like lamellipodial crawling mode
(Danjo and Gipson, 1998
).
Similarly, in vitro studies in the gut epithelial cell line
Caco2BBE show that wounds can be closed by purse-string motility
(Fig. 4B)
(Bement et al., 1993
). In all
likelihood, most adult simple epithelia use the purse-string mechanism for
closing small wounds, and size probably does really matter here, because in
the Caco2BBE studies, smaller wounds close by purse-string
contraction, larger wounds close by crawling and some middle size wounds use a
combination of both these strategies (Fig.
4B) (Bement et al.,
1993
). How cells read the mechanical cues that
direct which of these modes of motility to adopt is still unclear.
Nevertheless, they are probably able to detect the different forces exerted
upon them at the various angles of curvature around a wound, through
differential tensions on cell adhesions and on the actin stress fibres within
them.
More recent studies using transgenic ActinGFP-expressing
Drosophila embryos, wounded by a laser beam, have allowed live
confocal imaging of the actin machinery as it assembles and draws the wound
epithelium closed (Wood et al.,
2002). In one regard at least, this wound hole closure process
differs from dorsal closure because cells can be observed withdrawing from the
epithelial margin and shuffling back into submarginal rows
(Fig. 2;
Fig. 5A); this loss of leading
edge cells appears not to occur during dorsal closure except at the zipper
fronts. Concomitant with the assembly of an actin cable at the wound margin,
dynamic filopodial protrusions are also seen extending from leading edge
epithelial cells, just as during Drosophila dorsal closure
(Fig. 5A-D). These protrusions
occasionally make transient contacts with the substratum ahead of them, but
show no sign of actively adhering and tugging the epithelium forwards. Genetic
approaches using either small GTPase loss- of-function mutants or
dominant-negative transgenes for Rho1 and Cdc42 have
provided a means to analyse in real time the functions of actin cable and
filopodia respectively. In Rho1 mutants, a cable fails to assemble
but, after a lag phase of several hours, cells compensate for the absence of a
wound purse-string by tugging on their immediate neighbours using the
exuberant filopodia and lamellae that they assemble in place of the cable
(Wood et al., 2002
). These
actin-rich protrusions enable a wound to close even in the absence of a cable
by means of numerous foci where cells zipper together, but when a cable is
present these filopodia are not necessary, at least during the early phase of
healing. Rather, the key role of filopodia in this context appears to be for
epithelial fusion (Fig.
5B,D,E). Blocking the activity of CDC42, and consequently the
assembly of filopodia, using a dominant-negative transgene, does not hinder
the rate of epithelial wound closure, but it does dramatically block the final
knitting together of the wound edges as they meet one another, so that these
wounds never completely close (Wood et
al., 2002
).
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Wounding of adult flies also triggers AP1 activation, as revealed by
expression of PUC and flies mutant in kayak show retarded healing
(Ramet et al., 2002). Rather
intriguingly, eyelid closure, which occurs late in mammalian embryogenesis and
looks, at least superficially, remarkably similar to Drosophila
dorsal closure, is also absolutely dependent on JNK signalling. Two recent
studies show that tissue specific knockout of JUN in the epithelium of foetal
mice leads to a failure of eyelid closure, and these mice are born with open
eyelids, whereas their siblings have closed eyes until 10 days after birth
(Zenz et al., 2003
;
Li et al., 2003
). One of these
lines of mice also exhibits subtle defects in wound healing
(Li et al., 2003
).
Although AP1 activation during morphogenetic episodes appears to be
entirely JNK dependent, tissue repair is a response to a traumatic
intervention and so it would not be strange if the primary signals for AP1
activation were somewhat different here. Indeed, studies of in vitro scrape
wounds indicate that AP1 activation after wounding is triggered, at least
partially, not by JNK signalling, but by sublethal mechanical damage to the
very front row cells. This mechanical damage leads to a Ca2+
influx, and a subsequent wave of purine signalling that, in turn, leads to
further AP1 activity in undamaged neighbouring cells several rows back from
the leading edge (Klepeis et al.,
2001). Clearly, this AP1 activity has the potential to coordinate
and prime the leading rows of cells for some activity necessary for immediate
migration or later filling in of the gap and, if the signal is blocked, then
there is a clear slowing down of the in vitro migration/repair process. Recent
studies in fish keratocytes and rat epithelial cells suggest that the role of
JNK during migration may be at least partially mediated via phosphorylation of
the focal adhesion adaptor, paxillin
(Huang et al., 2003
), but in
vivo the signals may operate in a paracrine way also on adjacent tissues.
Indeed, wounds in mouse and rat embryos show a similarly rapid, but transient,
activation of AP1 in the front few rows of epithelial cells
(Martin and Nobes, 1992
).
These immediate-early signals may operate as kick-start activators by
triggering TGFß1 expression in wound epithelial cells, which subsequently
release this growth factor into the adjacent wound mesenchyme, directing this
tissue to contract (Martin et al.,
1993
), just as TGFß directs fibroblast contraction of
collagen gels in vitro (Montesano and
Orci, 1988
). Although the precise role of the JNK signal in these
various complex processes remains unclear, many epithelial migrations, be they
naturally occurring morphogenetic episodes or artificially activated events,
seem to be influenced by JNK signalling.
Cell movements and adhesions in wound repair
Currently, it is still not possible to observe directly the migration of
epithelial cells as they heal an adult skin wound. Nevertheless, tracking the
movements of virally tagged cells in a skin culture model that has been
wounded to simulate in vivo repair indicates that the simplest model, whereby
a coherent sheet of cells is dragged forward by its motile front row, is
probably far from true. Rather, there seems to be much shuffling of cell
positions, and a general free-for-all in the leading edge
(Garlick and Taichman, 1994;
Danjo and Gipson, 2002
).
Indeed, the expression of integrins that enable traction for crawling is
upregulated in at least the front
10 rows of cells, as well as upwards
into cell layers above the basal layer
(Hertle et al., 1992
). During
this period, there is tight regulation of cell division in the front rows of
the leading epidermis (see Box
3). In vitro studies of epithelial monolayers, in which
lamellipodial crawling is the primary mode of motility, show that closure is
not only achieved by activities restricted to front row cells, as the blocking
of RAC signalling, and thus crawling, in only these cells does not prevent
repair. To halt closure, the front four or five rows of cells must all be
prevented from crawling by disabling their RAC activity
(Fenteany et al., 2000
).
Time-lapse studies of wounds repairing in vitro and in vivo in the
Drosophila embryo have revealed how cells move relative to one
another, changing their neighbour:neighbour relationships
(Bement et al., 1993
;
Wood et al., 2002
) (see
http://www.nature.com/ncb/journal/v4/n11/suppinfo/ncb875_S1.html).
Similar cell shufflings occur during vertebrate gastrulation, and signals
that regulate and enable this fluidity in the gastrulating epithelium have now
been identified. It seems that a key role for activin during Xenopus
gastrulation may be to soften cadherin-based adhesions between epithelial
cells (Brieher and Gumbiner,
1994). If these signals are countered by exposure to
cadherin-activating antibodies, then high adhesivity is restored, the cell
rearrangements of convergent extension fail and consequently gastrulation is
blocked (Zhong et al., 1999
;
Gumbiner, 2000
). Cell matrix
interactions may also feed into this regulation of cell:cell adhesion.
Blocking integrin:fibronectin signalling appears to block the rearrangement of
cells during convergent extension, and this may define zones, perhaps domains
rich in fibronectin, in which cell:cell shuffling within an epithelium is
permitted (Marsden and DeSimone,
2003
).
Several further ways in which adhesion competence may be modulated within
an epithelial sheet are now becoming clear from cell biology studies. For
example, one consequence of scatter-factor/MET signalling, which directs cells
to break free from an epithelial sheet, is activation of a novel
E-cadherin-binding protein, Hakai, which ubiquinates the E-cadherin complex,
leading to its endocytosis (Fujita et al.,
2002). Phosphorylation of adherens junction components is clearly
key to their function and it seems that some of this regulation will turn out
to be via the Pez phosphatase, which is localised to adherens junctions in
sheets of coherent epithelial cells. Cells transfected with dominant-negative
Pez constructs show increased ß-catenin phosphorylation and loosening of
junctions leading to enhanced epithelial sheet spreading in vitro
(Wadham et al., 2003
).
Adherens junctions may not be the only junctions that are labile in migrating
epithelia; studies of desmosomes, which are anchor points for intermediate
filaments, rather than for actin, show that their adhesivity rapidly flips
from being Ca2+ independent to being Ca2+ dependent in a
wounded epithelial monolayer. This change in desmosome state spreads in a wave
mediated by protein kinase C
from wound margin cells to cells many rows
back from the wound edge (Wallis et al.,
2000
).
The biomechanics of wound repair
There is no doubt that many of the cues directing the various cell
behaviours that contribute to wound closure, or to any morphogenetic episode,
will be chemical signals, released by one cell population and operating on
another. For example, some of the growth factors released by degranulating
platelets at the wound site are known to have potent effects on the native
epidermal and fibroblast wound tissue cell lineages, and to assist in
directing cell migrations, contractions and so forth. However, it is now
becoming clear that mechanical signals are also likely to provide crucial
cues. Simply disrupting the natural tissue tensions by wounding might provide
an activating trigger. Cells stretched along the free epithelial edge as a
wound initially gapes may be mechanically stimulated to organise their actin
in alignment with the force of stress, thus setting up the purse-string that
subsequently drives epithelial closure. In vitro studies in fish keratocytes
have shown that physical tugging on cells can result in the rapid
reorganisation of actin filaments along the direction of force
(Kolega, 1986). Presumably, a
small GTPase switch transduces this mechanical signal, and, indeed, RHO can be
activated mechanically in endothelial cells in the absence of the standard
growth factor signals (Li et al.,
1999
).
In adult tissue repair, there is some evidence to indicate that mechanical
forces are, in part, responsible for the conversion of normal wound
fibroblasts into myofibroblasts at a wound site. These myofibroblasts closely
resemble smooth muscle cells with their expression of -smooth muscle
actin and the capacity for generating strong contractile forces. The signals
triggering this transformation from fibroblast to myofibroblast are believed
to be a combination of growth factors, including TGFß1, as well as
mechanical cues that are related to the forces resisting contraction (reviewed
by Grinnell, 1994
).
There is now good evidence to indicate that during morphogenesis of the fly
embryo, mechanical, stretching and pushing cues can direct transcriptional
events in cells. If gastrula-stage fly embryos are squashed in their DV axis,
within minutes they upregulate Twist throughout their epithelium, rather than,
as normal, in only a thin ventral strip
(Farge, 2003). Moreover, if
the tissues linking the posterior mesoderm to those cells destined to
invaginate and form the stomodeum are cut (thus denying them the compression
forces they would normally experience), these cells now fail to switch on
Twist and no longer invaginate.
Although it is becoming clearer that mechanical forces may be key players
during both repair and morphogenesis, we know very little about the various
tensions and forces operating in each of these scenarios. It is possible to
directly measure forces exerted by individual cells in vitro
(Wang et al., 2002), but it
is clearly much more technically challenging to do likewise with tissues in
vivo. One way of visualising the play of tensions within interacting tissues
is to release the tension in one location by cutting, and to measure the
consequential gape and movement of nearby tissues. This approach has been
undertaken for Drosophila dorsal closure using a laser beam
to make fine cuts within the amnioserosa and along the leading edge
epithelium. These studies show that the contractile amnioserosa and the
force-generating mechanisms in the adjacent epithelium make comparable
contributions to the advancement of the epithelial leading edge
(Kiehart et al., 2000
;
Hutson et al., 2003
). Similar
tissue tension geography data needs to be gathered for other
more complex vertebrate morphogenetic episodes and also for wound healing.
In this regard, labelling small groups of exposed mesenchymal cells at the
margin of an embryonic wound allows one to trace mesenchymal movements during
the wound closure process. This shows that this tissue contracts to about half
its original area by the time the wound has closed, indicating that
re-epithelialisation and connective-tissue contraction contribute equally to
the wound closure effort
(McCluskey and Martin, 1995).
A similar ratio of tissue contributions was previously described for repair of
adult skin wounds (Abercrombie et al.,
1954
). In a rather crude mirror of the elegant Drosophila
tension-cutting experiments already described, it was shown that almost all of
the contractile force generated by adult wound connective tissues is delivered
by a band of fibroblasts lying within 1-2 mm of the epidermal wound margin, as
cutting and removal of the central wound granulation tissue did not alter the
rate of wound healing (Gross et al.,
1995
).
Knitting epithelial edges together
Epithelial fusion is the climax of many morphogenetic episodes and of wound
healing. During dorsal closure in flies, the leading edge epithelial cells
extend filopodial protrusions that appear to play a key role in bonding the
two epithelial sheets together (Fig.
5A,B,E). Filopodia from confronting epithelial cells interdigitate
at the zipper front and in the fusion seam, several cell diameters back from
the zipper front, these interdigitations resolve to leave mature adherens
junctions linking opposing cells (Jacinto
et al., 2000). In vitro studies of keratinocytes adhering to one
another to form confluent sheets show that these cells too use filopodial
interdigitation to prime the formation of adherens junctions
(Vasioukhin et al., 2000
).
Live studies of equivalent filopodial interactions between opposing epithelial
leader cells during C. elegans ventral enclosure show that
-catenin is pre-localised to filopodial tips, which may aid in assembly
of rapid, transient adhesions between filopodia that go on to nucleate the
formation of mature junctions (Raich et
al., 1999
). Evidence that filopodia are pivotal for epithelial
fusion during dorsal closure comes from experiments where their assembly is
blocked by expressing a dominant-negative mutant form of CDC42 in Engrailed
stripes of the embryonic fly epithelium. In such cases, fusion of the opposing
epithelial sheets in these regions fails
(Jacinto et al., 2000
).
Moreover, these experiments reveal one further role for the filopodia in
closure of the dorsal hole. Live imaging of the leading edge in the minutes
preceding fusion shows how filopodia scan the opposing leading edge, rather
like filopodia from a growth cone sensing for axon guidance cues. If assembly
of filopodia is blocked, then the opposing epithelial fronts fail to align
properly at the midline, much like a poorly buttoned up waistcoat, indicating
that filopodia are, in part, responsible for the cell:cell matching needed to
align segments across the midline seam
(Jacinto et al., 2000
).
It seems unlikely that precise alignment of positional values would occur
during repair of an epithelial wound, but as we have already discussed,
filopodia play an integral role in finally knitting the wound hole closed
(Fig. 5C,D)
(Wood et al., 2002). Rather
strikingly, it appears that filopodial-mediated fusion probably plays a role
in all of the vertebrate developmental fusion events that have been carefully
studied to date and may be a universal phenomenon. For example, as the eyelids
transiently fuse in late mammalian gestation, filopodial interdigitation can
be observed where the opposing lid epithelial cells confront one another
(Fig. 6)
(Zenz et al., 2003
). Indeed,
there are several earlier fusion events that occur as the vertebrate face is
built that appear to use an almost identical bonding strategy (reviewed by
Cox, 2004
). Classic studies of
the fusions between the medial nasal prominence and the right and left
maxillary prominences (primary palate fusion) provide clear evidence of
filopodia from both nasal and maxillary epithelial faces; and in the classic
CPP (cleft primary palate) chick mutant, they are absent
(Yee and Abbott, 1978
).
Similarly, as the two secondary palatal shelves flip up and over the tongue to
make contact with one another, they also express exuberant filopodia that
appear to bond them together. Possible clues as to the signals regulating
assembly of palatal filopodia come from studies of TGFß3 knockout mice
that lack filopodia at the crucial time of contact, thereby failing in palatal
fusion such that mice are born with cleft palate
(Taya et al., 1999
).
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Inflammation and chemotactic factors
Beyond a late transition period in foetal development, any tissue damage always results in activation of a robust inflammatory response, whereby largely neutrophils and then macrophages are drawn to the wound site. These cells are attracted by a diverse mix of chemotactic cues, ranging from peptides cleaved from bacterial proteins, to bone fida chemokines released by degranulating platelets, damaged cells and the first patrolling leukocytes arriving at the wound site.
Once at the wound site, the prime role of neutrophils appears to be to kill
microbes, while macrophages clear the wound of cellular and matrix debris
(including spent neutrophils). It is also the case that both of these
leukocyte cell lineages release a battery of growth factors and cytokines that
themselves may supply key tissue repair signals
(Rappolee et al., 1988;
Hubner et al., 1996
). Indeed,
a classic series of experiments in the 1970s showed that although antisera
depletion of neutrophils from guinea-pig wounds did not significantly perturb
tissue repair in sterile conditions, depletion of macrophages with antisera
and steroids prevented healing of skin wounds
(Simpson and Ross, 1972
;
Leibovich and Ross, 1975
).
More recent depletion studies using knockout mouse and other approaches have
allowed more direct tests of function for each of the invading cell lineages.
Neutrophil knockdown experiments in mice result in repair that is even more
rapid than in wild-type healing as long as conditions are sterile, indicating
that these cells release signals that are inhibitory to some aspect of the
repair process (Dovi et al.,
2003
). Mice null for Kit W (Kit Mouse Genome
Informatics) are deficient in Mast cells and show reduced numbers of
neutrophils at a wound site, but otherwise normal repair
(Egozi et al., 2003
), whereas
the PU.1 (Sfpi1 Mouse Genome Informatics) knockout mouse that
lacks both neutrophils and macrophages shows slightly enhanced rates of
re-epithelialisation, again indicating that inflammatory cells release signals
that are somewhat inhibitory to repair, but are not themselves essential for
healing (Martin et al.,
2003
).
Inflammation and scarring
The inflammatory response at a wound site has clearly evolved to prevent
invasion of microbes whenever the skin barrier is broken. However, as embryos
can repair wounds perfectly in the absence of an inflammatory response, it is
tempting to consider that inflammation may cause some of the unwanted side
effects of repair in adult tissues, in particular fibrosis or scarring. This
proposal is strengthened by the observation that the transition stage of
foetal life when an inflammatory response kicks in, coincides with the
earliest stage at which scarring is a consequence of foetal surgery (about E15
in mice and the end of second trimester in human foetuses)
(Adzick et al., 1985;
Hopkinson-Woolley et al.,
1994
; Cowin et al.,
1998
). Beyond this transition period, late foetal and neonatal
tissues scar after wounding, but macrophageless PU.1 null
neonatal mice appear to repair wounds without a fibrotic response
(Martin et al., 2003
),
indicating that it is not the size of the wound that directs whether it will
scar or not, but rather whether it triggers a sustained inflammatory response.
One direct consequence of a reduced or absent inflammatory response, whether
in the embryo or in a macrophageless neonatal mouse, is a
significantly dampened profile of cytokines and growth factors at the wound
site, and one of the key growth factors in this regard appears to be
TGFß1. Indeed, there is a growing body of evidence that TGFß1, and
its downstream effector connective tissue growth factor (CTGF)
(Igarashi et al., 1996
), may
be partially responsible for inflammation-mediated fibrosis. When TGFß1
is mopped up by antibody application or its activity negated in other ways at
wound sites in adult rats, repair with reduced scarring occurs
(Shah et al., 1992
;
Shah et al., 1994
;
Shah et al., 1995
). Further
evidence that TGFß signalling may be key in mediating the link between
inflammation and fibrosis comes from studies in Smad3 mutant mice. In
these mice, wound keratinocytes, fibroblasts and inflammatory cells all have
an impaired capacity for transducing TGFß signals. One consequence of
this is a reduced number of inflammatory cells recruited to the wound, and
healing, as in the embryo, is again almost scar free
(Ashcroft et al., 1999
). All
these data reveal clear links between TGFß levels at the wound site and
subsequent scarring, and indicate that this might be a good therapeutic target
for improving tissue repair. What is much less clear is how this growth factor
signal is responsible for directing the fibroblast behaviour that leads to an
excess of collagen synthesis and its arrangement in bundles, rather than in a
basket-weave network as normally found in unwounded skin.
Summary and future directions
There are a number of lessons for better wound healing that can be learned
from the embryo. Undoubtedly, there are many parallels between those tissue
movements that shape embryos during development and those that are activated
upon tissue damage leading to repair of the wound. Indeed, in the embryo it is
very likely that tissue damage merely leads to activation of standard
morphogenetic machinery so that repair becomes a recapitulation of
morphogenesis. Perhaps the embryo reads the artificially
generated free epithelial edge and the resulting changes in epithelial
tensions that arise at a wound site, just as it does any other morphogenetic
activation cue, and acts accordingly to close the epithelial hole. The extra
complexities of adult wound healing may simply be due to additional processes,
such as inflammation, that have evolved to counter infection and cope with the
greater size of adult wounds, and of course some of these processes may be
more important than the similarities in terms of potential clinical
strategies. Equally, there are likely to be aspects of morphogenesis that are
not replicated during repair because they are activated or required only in
the unique environment of the embryo. It is unlikely, for example, that
precise cell:cell matching will occur as wound edges are stitched together, as
is the case during Drosophila dorsal closure. Furthermore, some
morphogenetic episodes that look like wound healing may be misleading. Epiboly
in the fish embryo, is a good case in point while it involves the
sweeping forward of a sheet of cells to close a hole, this movement is driven
by unique microtubule-based pulling forces generated within the underlying
yolk cell (Strahle and Jesuthasan,
1993), in ways that cannot be mirrored in a wound
re-epithelialisation scenario.
So far, the parallels we have discussed for morphogenesis and repair have
been largely the most obvious ones that occur at the level of epithelial
movements. However, there is also likely to be crossover between the signals
that guide the directed migrations and subsequent behaviours of inflammatory
cells at a wound site, and those signals that guide various migrating cell
lineages during normal development. For example, signals whose prime role was
believed to be in directing leukocytes to sites of inflammation have now also
been shown to guide germ cells from their sites of origin to the primitive
genital sites in fish and mice (Doitsidou
et al., 2002; Knaut et al.,
2003
; Molyneaux et al.,
2003
). In a reciprocal fashion, it seems that white cell
migrations might also be governed by cues previously thought to be the domain
of developmental biology, as SLIT, which repels growth cones from crossing the
midline during Drosophila neural patterning, is repulsive to
leukocytes as they attempt to respond to chemotactic cues
(Wu et al., 2001
). If one
wanted to design novel medicines for dispersing inflammatory cells from the
wound site, SLIT might be a good candidate for testing.
We suspect that there will also turn out to be parallels at the level of
wound fibroblasts and of mesenchymal cells in the developing embryo,
particularly during episodes of mesenchymal condensation (which
precedes cartilage formation and wherever dermal mesenchymal cells aggregate
beneath epidermal placodes to form appendages and glandular structures)
(Bard, 1990). These mesenchymal
condensations resemble the aggregations of previously dormant fibroblasts
recruited to wound granulation tissue and many are associated with TGFß
and BMP signals, just as both these growth factor cues are believed to be
crucial activators of wound fibroblast migrations and contractions. It will be
interesting to discover how far such parallels can take us, particularly in
understanding how connective tissues are able to undergo physiological
contractions without the inevitability of fibrosis.
In hindsight, it is not surprising that many of the tools used to repair and rebuild tissues turn out to be old tools that the embryo used to build those tissues in the first place. For the future, we need to glean which aspects of our detailed understanding of how an embryo is built will be useful in guiding us to better control the cell behaviours of repair in a clinical scenario. As more is learned about the genetics of morphogenesis, not just in flies and worms, but also in some of the more complex vertebrate episodes (as highlighted in Box 2), there will be more clues for repair aficionados.
ACKNOWLEDGMENTS
We thank Tim Cox, Jim Priess, Phil Soriano, Valera Vasioukhin and Sarah Woolner for discussions and for critically reading this manuscript. Thanks also to Jane Brock, Craig Magie, Jim Priess, Will Wood, Katie Woolley and Sarah Woolner for providing images.
Footnotes
Supplemental data available online
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