For our wound model we chose neonatal mouse back skin which raises a robust inflammatory response to wounding that is not dissimilar to that seen at sites of tissue damage in adult skin, but which heals rapidly, such that incisional lesions are generally fully re-epithelialized by 24 hours. This compression of the repair process reduces the temporal 'noise' and thus the potential loss of gene-expression synchrony between wild-type and PU.1 null animals, which naturally will increase with time after the initial wound insult.
Wounding of neonatal back skin results in rapid healing with or without an associated inflammatory response
Resin histology of healing incisional wounds in neonatal mouse skin reveals closure of the wound commencing within 3 hours of the lesion; by 24 hours, the epidermal wound edges have generally met and fused along much, if not all, of the length of the wound. This is true for both wild-type neonates and for PU.1 null sibs, with the only obvious differences apparent in the histology being an absence of inflammatory cells in the PU.1 null wounds (Figure ). As previously described, in situ hybridization studies using a c-fms macrophage-specific probe reveal large numbers of these cells drawn to the wound connective tissue just beneath the epidermal fusion seam at 24 hours after wounding wild-type skin, but their complete absence in PU.1 null equivalent wound sections (Figure ). The same difference, although with an earlier temporal profile, is observed if wounds of wild-type and PU.1 null skin are probed with histochemical stains that reveal neutrophil influx (data not shown). These wound dynamics lead us to believe that the neonatal mouse skin wound model provides a good opportunity to analyze the transcriptional events that regulate the various tissue-repair episodes from initial activation steps through to the 'stopping' signals that occur when the tissue defect has been filled in, both in the presence and absence of an inflammatory response.
Figure 1 Wound histology. The location of skin wounds on the back of a neonatal mouse is shown. (a) For the array studies a series of criss-cross wounds were made so that all the skin cells were as close as possible to a wound edge for collection of wound RNA. (more ...)
More than 1,000 genes are differentially expressed post-wounding
For microarray comparison, a consistent series of horizontal and vertical incisional (criss-cross) wounds were made to the back skin of 2-day-old neonatal PU.1 null mice and their wild-type littermates. Each time-matched pair (PU.1 null and wild-type) were chosen from the same litter to reduce the possibility of differential expression 'noise' due to environmental differences. Wound tissues were harvested at either 30 minutes to identify immediate early genes, 3 hours for early tissue repair effector genes, and 12 or 24 hours to reveal later tissue repair effectors as well as inflammatory genes. Total RNA was then extracted and hybridized to Affymetrix GeneChips, and differentially expressed genes were identified by comparison of expression levels for each time point with unwounded skin samples that served as baseline controls. Genes were selected if transcript levels exceeded a twofold increase over either the unwounded baseline, or between time points, or between the wild-type and PU.1 null wounds. On the basis of these criteria, 1,001 genes were identified as wound-induced (see Additional data file 1 for an annotated database of all these genes together with full details of expression levels at all time points).
Cluster analysis to group these genes reveals temporal profiles that correlate with distinct physiological episodes in the repair process
Cluster analysis with Spotfire Array Explorer 3.0 software was used to organize the 1,001 wound-induced genes into groups according to the cosine coefficient similarity measurement; this includes within a group all those genes that have a similarly shaped temporal profiles, regardless of the levels of gene expression. Nine clusters were identified in this way, and of these, seven correlated with clear episodes in the repair process. The other two had profiles that, as far as we can tell, do not correspond to any currently understood step in the repair process and so were discarded for further analysis, although they appear in our supplementary data (see Additional data file 2 for median graphs of these clusters). Of the seven clusters associated with known repair episodes, five contain one or more known genes with good functional associations to that repair episode, and this encourages us to name each cluster according to that physiological episode. This does not provide definitive proof of function for any gene in that cluster, but it gives the best opportunity to predict function, particularly for expressed sequence tags (ESTs) with no further sequence information.
Four clusters have profiles that are independent of an inflammatory response
Four clusters of genes have profiles that are largely independent of inflammation. Genes in these clusters are expressed with similar profile whether wounds are in wild-type skin, where there is an influx of inflammatory cells, or in PU.1 null skin, where there is none. In both these situations there is full and complete repair, and so we propose that these four clusters represent the basic repertoire of repair genes that are activated during the repair response. Figure shows line graphs that display the temporal profile of the median expression levels at each time point to give a representation of that cluster and these have been termed the 'activation' (Figure ), 'early effector' (Figure ), 'late effector' (Figure ) and 'stop' (Figure ) clusters. The number of genes found in each cluster is displayed on each graph.
Figure 2 Median temporal profile graphs of identified repair and inflammation clusters. Line graphs displaying the median level of absolute mRNA expression (y-axis) at each time point: 0, 0.5, 3, 12 and 24 h (x-axis), for genes within each of the four repair clusters (more ...)
Three gene clusters correlate with various phases of the inflammatory response
Three further clusters of genes represent expression profiles that correlate with the onset of inflammation and thus we consider them inflammation-associated genes. In neonatal animals, the inflammatory response is generally induced by 12 hours and is well established by 24 hours post-wounding. Two of these inflammation-associated gene clusters contain genes that are not expressed in unwounded skin or at early stages of repair; rather, they are upregulated in wild-type skin directly coincident with the onset of the wound inflammatory response but are generally not upregulated in the PU.1 null wound site at any stage. We have called these two groups of genes, the 'early inflammatory' cluster (Figure ) and the 'late inflammatory' cluster (Figure ). A third cluster does not display the standard inflammatory response profile as typified by the early and late inflammatory clusters. Rather, this cluster contains genes that are expressed at early stages of repair in both PU.1 null and wild-type mice but, whereas expression appears to increase in the wild-type wound coincident with the inflammatory response, the same genes are downregulated in the PU.1 null wound, where there is no inflammatory response; we have called this group of genes the 'inflammation-maintained' cluster (Figure ).
Nearly 100 genes are expressed with an immediate early gene profile at the wound site
One of the most clear-cut clusters of genes is of those whose temporal expression profiles are suggestive of a transient, immediate early response to wounding. These genes show almost identical profiles, whether in the wild-type or PU.1 null situation, and thus are independent of an inflammatory response. We have named this group the activation cluster, as many will be kick-start activators of the various cell behaviors that together comprise the wound-healing process. This cluster is dominated by transcription factors and contains several well known immediate early genes, such as Egr1 (Krox 24), JunB, Myc, and I-Kappa-Bα (Nfkbia). We present a heatmap for the 90 genes in this cluster arranged with the most highly expressed at the top of the map (Figure ). Heatmaps provide a visual representation of temporal profiles only, and so for a small sample of these genes we also include in situ hybridization data on wounded skin sections to illustrate which cells and tissues express that particular gene. This spatial expression profile reveals expression in the in vivo setting, giving clues to the function of that gene during repair.
Figure 3 Heatmaps for activation and effector clusters. Color depiction of the temporal profiles of mRNA intensity during the 24 h repair period for genes in (a) the activation cluster, (b) the early effector cluster and (c) the late effector cluster. Higher levels (more ...) Krox24
(Figure ) has previously been shown to be transiently induced in both embryonic and adult mouse wounds [7
]. In situ
hybridization reveals Krox24
to be expressed by those epidermal cells extending back 10-12 cell diameters from the cut edge of neonatal wounds and all the associated hair follicles within this zone also (Figure ).
Figure 4 Temporal and spatial expression profiles of sample genes from the activation and effector clusters. Temporal and spatial profiles of the (a-h) activation and (i-p) effector clusters. The line graphs display temporal expression: absolute expression levels (more ...)
MKP-1 (Figure ) is a dual-specificity phosphatase with close homology to Drosophila
puckered, which has been shown genetically to be key in braking the Jun N-terminal kinase (JNK) cascade activated during morphogenetic episodes such as dorsal closure in the fly embryo [8
]. In situ
hybridization shows that the front few rows of wound epidermis express MKP-1
, although expression extends less far back from the wound edge than for Krox24
(Figure ). By analogy to Drosophila
morphogenetic episodes, it may be that MKP-1 operates as suppressor of MAP kinase (MAPK) signaling by phosphorylation of extracellular-regulated kinases 1 and 2 (ERK1 and 2), and so may actually function as a brake on the earliest tissue movements activated at the wound site.
Expression of Fos-like antigen 1 (Fosl1
), has previously been associated with epithelial migrations during tumorigenesis but has not been analyzed in a wound-repair model [9
has a classic activator temporal profile (Figure ) and a similar spatial profile to Krox24
, with high levels of expression in wound-margin epidermal cells but somewhat weaker expression in damaged hair follicles at the wound site (Figure ). Its close relative c-fos
has previously been shown to be upregulated during repair of embryonic skin wounds [11
], and in vitro
studies show that blocking wound-induced fos
induction may hinder cell migration [12
Because cluster analysis allows us to group genes together that are likely to have similar functions [13
], the temporal profiles of, as yet, uncharacterized ESTs in the activation cluster implicates them as having an immediate-early activator function during repair. A good example of such a gene is EST GenBank accession number AI853531 (Figure ), which is weakly homologous to human Mitogen-Inducible-Gene-6
, Gene 33
). The exact function of Mig-6 remains elusive but it has been shown to interact with Cdc42, a member of the Rho family of GTPases, via the activation of stress-activated protein kinases (SAPKs) [14
]. In situ
hybridization reveals clear expression of this gene in wound fibroblasts (Figure ); together with its potential Cdc42 effector status and its induction in quiescent fibroblasts upon mitogenic stimulation and expression in many human cancer cell lines [15
], this suggests that Mig-6 may mediate a fibroblast migration signal. The remaining genes in the activation cluster all have very similar temporal profiles, suggesting that they too may have important roles in activating or modulating early cell behavior at the wound edge.
A further 200 genes are also expressed independently of inflammation, but with later onset and a less transient time course
Two further clusters of genes have increased expression levels post-wounding in a manner that is also inflammation-independent but where expression occurs at a later time than with the activation genes. The profiles of these two clusters are temporally distinct from one another and so we have called them the early effector and late effector clusters. Between them they contain 184 genes that fit the expected profile of genes that might direct re-epithelialization and granulation tissue assembly events. The temporal profiles of all these genes can be seen by heatmap in Figure (early effector cluster) and Figure (late effector cluster). These two clusters contain varied types of tissue repair effectors such as tissue remodelers, genes encoding extracellular matrix (ECM) proteins, those involved in the signaling machinery and structural genes required for cell migration. Again, we provide here several examples of genes within these clusters with accompanying in situ hybridization data to provide an insight into the spatial localization of some genes in these clusters.
Map4k4, a member of the serine/threonine protein kinase family that activates the JNK and MAPK signaling pathways in response to stress signals, cytokines and growth factors [16
], is a member of the early effector cluster. The temporal profile (Figure ) and expression of Map4k4
, in both keratinocytes up to 10-12 cell diameters from the wound edge and a subset of dermal fibroblasts extending a similar distance back from the wound edge (Figure ), confirms the activation of this intracellular signaling cascade at sites of tissue repair. The JNK pathway has recently been shown to have a role in Paxillin regulation during fibroblast migrations triggered by in vitro
scratch wounds [17
], and so expression of Map4k4
is also suggestive of a cell migratory regulatory role for this signaling pathway in keratinocytes and fibroblasts during in vivo
Also in the early effector cluster, retinol binding protein-1 (Rbp1), a Fabp/p2/Crbp/Crabp family retinol transporter is expressed in wound epidermal cells approximately 15 cell diameters back from the wound site (Figure and ). This suggests a role for retinoids in re-epithelialization of the wound, and indeed, there is some evidence that these molecules can trigger epidermal proliferation via heparin-binding epidermal growth factor (HB-EGF) expression in suprabasal epidermal cells [18
Typifying the late effector profile is Keratin 6
), a classic wound-induced gene [19
] (Figure ). K6
encodes a nonconventional keratin which is thought to facilitate the packaging up of other intermediate filaments in activated keratinocytes, so that these cells can migrate forward to re-epithelialize the wound [19
]. High levels of expression of K6
by the front 10-12 rows of wound-edge keratinocytes were confirmed by in situ
hybridization (Figure ).
Interestingly, another member of the late effector cluster, the intracellular Ca2+
-binding protein MRP8 (S100A8) is expressed in a similar temporal and spatial pattern to K6
(Figure and ). MRP8 binds to keratin filaments as an MRP8/14 heterodimer in a Ca2+
dependent manner [20
] and is postulated to interact with these keratin filaments and guide cytoskeletal rearrangements during tissue repair [22
]. The temporal and spatial coexpression of K6
may highlight a relationship between them and as such reveals another advantage of cluster analysis - the ability to identify potential interactions between genes and genetic pathways within the same cluster.
Not all functionally related genes cluster together, however. The heterophilic binding partner of MRP8 is MRP14, which does not appear in the same cluster but rather is expressed within the early inflammation cluster (see later), since, in addition to keratinocyte expression, it is expressed at high levels by wound leukocytes. As both the MRP8/MRP14 heterodimer and a homodimer, MRP8 is a potent chemoattractant [22
] and, interestingly, the MRP8/14 heterodimer also has an entirely different role, operating as a wound antimicrobial factor, although the MRP14 subunit seems to be responsible for this activity [24
]. The pleiotropic activities of MRP8/MRP14 may reflect different functions of monomeric versus complexed subunits.
A final cluster of inflammation-independent genes may indicate players in the 'contact inhibition' stopping process
At the end of the repair process many of the cell behaviors that drive repair - such as migration and proliferation - clearly need to cease as tissues re-establish approximately their pre-wound state. This will be a gradual process and yet we might expect to see such genes depressed during the repair period and becoming upregulated as wound edges meet and closure is finishing. We see a cluster of genes with exactly this profile, suggesting that some of these genes are re-expressed to control the later stages of repair. We have loosely termed this the stop cluster. Because of their known biology, several genes in this cluster make ideal candidates for players in the processes of contact inhibition and epithelial fusion that occurs as cells from the two epidermal wound fronts confront one another.
The Eph receptors and their ligands, the ephrins, have features that might make them ideal for sensing and responding to stop cues. In vitro
studies show that both ligand- and receptor-bearing cells become activated upon cell-cell contact [25
], and this interaction leads to a repulsive response by receptor-expressing growth cones during the developmental wiring of the nervous system [27
]. Further evidence for ephrin-mediated control of epithelial sheet movement and fusion comes from studies in Caenorhabditis elegans
, where Eph receptor mutants display defects in the movement of epidermal cells over neuroblasts, and in Eph knockout mice, where various morphogenetic epithelial fusions fail, leading, for example, to cleft palate and hypospadius [28
]. All these results suggest that the transcriptional regulation of EphB1 revealed in the heatmaps for our stop cluster (Figure ) may reflect a functional role in the stopping or final fusion episodes of wound re-epithelialization.
Figure 5 Heatmap and in situ hybridization data for genes in the stop cluster. (a) The temporal expression profiles of genes of the cluster are represented by a heatmap. The highest levels of expression are indicated by the brightest shades of red, while lower (more ...)
Similarly, the expression levels of the receptor Notch also dip and rise during the repair period, and in situ
hybridization studies reveal that this transcriptional regulation is also occurring within leading wound-edge epidermal cells (Figure ). Notch has exceptionally complex biology with several ligands, including Delta and Serrate, and is a widely used as a signaling cassette at various stages of embryogenesis, and has been shown to be downregulated in several invasive tumors [30
]. In Drosophila
, Notch signaling has been implicated in the contact inhibition and fusion events that occur during dorsal closure at the end of embryogenesis (A. Martinez-Arias, personal communication), and during gut cell migratory episodes, which are also dependent on transcriptional activation of the short stop
], the mammalian orthologue of which, Actin crosslinking family 7 (ACF7), is another member of our wound stop cluster.
Several other genes within the stop cluster have characteristics that indicate they may be involved in sensing contact-inhibition cues or be downstream of these signals and operate to adhere epidermal fronts together. They include genes for Plexin 3 (Plxn3), a member of the plexin family of semaphorin receptors [32
], Desmocollin 3 (Dsc3), which is a cadherin component of intercellular desmosomal junctions [33
] and ACF7, a cytoskeletal linker protein [34
As with the other clusters, suggestive biology is no proof of function, and it is worth noting that several other genes with this temporal profile do not have biology suggestive of a role in these late stages of wound healing. We feel that this cluster, more than any other, can only hint at function, and definitive function testing using knockout or knockdown assays will be necessary to investigate any speculative roles in the repair process.
Expression of 200 genes at the wound site is dependent on the inflammatory response
A comparison of those genes expressed during the repair process in wild-type versus PU.1 null mice reveals most clearly genes that are dependent on the presence of an inflammatory response at the wound site. The heatmaps for early and late inflammatory gene clusters strikingly reveal robust expression in wild-type wounds, but little or no expression in the PU.1 null mice for these genes (Figure ). Together, the early and late inflammatory clusters comprise 169 genes that are not expressed in unwounded wild-type skin or at early stages of repair but appear to be upregulated in the wild-type wound directly, coincident with the onset of the inflammatory response. The early inflammatory cluster typically contains genes whose expression is upregulated rapidly in the wild-type, often reaching a peak by 12 hours (Figure ), coincident with the influx of neutrophils to the wound site. In the late inflammatory cluster, expression typically peaks a little later, at 24 hours post-wounding (Figure ), more suggestive of a link to the later influx of macrophages. A further 17 genes are initially expressed at both the wild-type and PU.1 wound site, but are maintained at high level only in the wild-type wound, where there is an influx of leukocytes. In PU.1 null wounds, where there is no such influx, these genes are only transiently expressed. We assume that expression of these inflammation-maintained genes (Figure ) is directly or indirectly dependent on signals released by inflammatory cells.
Figure 6 Heatmaps for inflammation-dependent genes. (a-c) Heatmaps of the temporal profiles of mRNA intensity during the 24 h repair period for inflammation-dependent genes in wild-type and PU.1 null wounds. (a) The early inflammatory cluster corresponds to the (more ...)
Inflammation-dependent genes may be expressed by leukocytes or by host cells as a 'response signature' to inflammatory signals
Genes that are expressed only in wild-type wounds and whose temporal expression patterns are coincident with the influx of neutrophils and/or macrophages will include those genes that are constitutively expressed by one or both of these lineages, or genes that are upregulated as part of the leukocyte activation state, or may be expressed by cells other than the invading leukocytes as a downstream consequence of host fibroblast, endothelial and muscle cells being exposed to signals from these leukocytes. We present a selection of in situ hybridization studies to illustrate each of these scenarios as revealed by very distinct classes of spatial expression pattern.
Early inflammatory cluster
L-plastin (Lcp1) is a pan-leukocyte, calcium-dependent, actin-bundling protein that has previously been implicated in macrophage activation and migration, although it is also overexpressed in many types of malignant human tumors [35
]. It is first expressed in the wild-type wound coincident with the early stages of the wound inflammatory response, with a peak of expression at 12 hours post-wounding; our temporal data indicate no expression at any stage in PU.1
null wounds (Figure ). In situ
hybridization studies reveal intense expression by leukocytes clustered within the wild-type wound site but no expression in surrounding skin (Figure ), and they confirm the absence of expression in PU.1
null wounds (Figure ). The wound-restricted expression pattern of L-plastin
suggests that expression of this gene is limited to activated leukocytes only.
Figure 7 Temporal and spatial expression profiles of sample genes from the three inflammation-dependent clusters. Temporal and spatial profiles of the (A) early inflammatory, (B) late inflammatory and (C) inflammation-maintained clusters. Line graphs display absolute (more ...)
Also expressed by leukocytes in the early inflammatory cluster are C3, a key component of the classical and alternative complement pathways, and its receptor, C3R. C3 is expressed at similar levels in unwounded PU.1 null and wild-type skin, but whereas expression is rapidly upregulated by 30 minutes post-wounding and continues until 24 hours in wild-type wounds, upregulation of C3 is delayed and much weaker in the PU.1 null wound (Figure ). This delay in C3 expression suggests that inflammation has a significant role in raising and maintaining a rapid complement response at the wound site.
also appears as a member of the early inflammatory cluster; it encodes a leukaemia-inhibitory factor-regulated protein that has previously been identified in a screen for genes controlling inflammatory dermatitis [36
]. Unwounded wild-type skin expresses Onzin
at low levels but it is completely absent in PU.1
null, unwounded skin and remains so until 12 hours post-wounding, when it is upregulated, but to a much lesser extent than in wild-type (Figure ). In situ
hybridization studies reveal a rather similar expression pattern in both wild-type and PU.1
null wounds (Figure ). This suggests that Onzin
might be expressed in wild-type skin by resident inflammatory cells and in the PU.1
null wound, either by inflammatory cells whose development is delayed, such as T cells, or that there may be an alternative or compensatory mechanism of gene regulation in non-inflammatory cells at the wound site.
As discussed previously, both the genes for MRP8 and its binding partner MRP14 are upregulated by wound-edge keratinocytes. Both are also expressed by leukocytes, and in the case of MRP14
this expression predominates and leads to cluster separation of the two genes, with MRP14
categorized as part of the early inflammatory cluster. In the wild-type wound, it is expressed from 3 hours, with expression peaking at 12 hours post-wounding, whereas in the PU.1
null, expression does not begin until 12 hours post-wounding and levels are much reduced compared with wild-type (Figure ). In situ
hybridization clearly shows MRP14
to be expressed, in addition to expression in keratinocytes, in the region of the wound populated by inflammatory cells in the wild-type only (Figure ), and indeed, previous experiments suggest that both neutrophils and macrophages express MRP8
It may be that genes expressed by host connective-tissue cells at the wound site as a consequence of inflammatory signals are detrimental to healing and lead to some of the imperfect aspects of repair seen in adult healing such as fibrosis and scarring. One candidate for such a gene is Osteopontin
), encoding a glycoprotein that can mediate cell-matrix interactions via the engagement of a number of adhesive receptors (reviewed in [37
]). Previous wound-healing studies on Spp1
null mice report differences from wild-type in that repair is characterized by abnormal macrophage debridement and abnormal maturation of collagen bundles [38
has a clear inflammation-related profile (Figure ) and in situ
hybridization reveals an unusual pattern of expression at the wild-type wound site, with some expression by a subset of leukocytes but with most positive cells located in what appears to be the deep dermal or muscle layers of the wound region (Figure ).
Both the early and late inflammatory clusters contain chemokine and growth factor receptors unique to leukocytes, and presumably used by these cells to detect various chemotactic cues that will guide them to the wound site. For example, the gene for chemokine receptor 1 (CCr1), a receptor for several chemokines including MIP-1α, CCL5 and Scya7, is expressed as early as 3 hours post-wounding, with expression levels peaking by 12 hours. There is no expression at the PU.1 null wound site (Figure ). In situ studies show CCr1 to be expressed in the wild-type wound by leukocytes recruited to the wound site (Figure ). As well as chemokine receptors, chemokines themselves are found in these clusters. CXCL10 (IP-10) encodes an α-chemokine that functions as a potent chemoattractant for macrophages and T cells, and is upregulated by 12 hours in wild-type wounds but is absent in PU.1 null wounds (Figure ). In situ studies reveal intense staining by what could be either leukocytes or host fibroblasts at the wild-type wound site (Figure ). Either this chemokine is an amplifying chemotactic signal expressed by leukocytes to draw in further leukocytes, or its expression is triggered in fibroblasts, but only if they receive signals from the first influx of neutrophils.
Late inflammatory cluster
Cathepsin S is a typical gene of the late inflammatory cluster, being highly upregulated at 24 hours post-wounding in the wild-type, but with no expression in the PU.1 null wound (Figure ). Cathepsin S is one of a large family of leukocytic proteases - this one largely macrophage-specific - that catalyze the remodelling of ECM proteins. In situ hybridization studies in the wild-type wound show Cathepsin S to be expressed by macrophages clustered around the wound site, but also by cells in the dermis at skin sites well away from the wound (data not shown), suggesting that it is constitutively expressed by cells of the monocyte lineage, rather than being part of the macrophage activation profile. No expression of Cathepsin S is seen in wounded or unwounded skin of the macrophageless PU.1 null mouse (Figure ).
is an epidermal differentiation gene and a member of the fused gene subgroup of the S100 family that encodes multifunctional epidermal matrix proteins [39
]. This temporal profile at the wound site implicates Repetin
as being responsive to inflammatory signals (Figure ), and yet in situ
hybridization studies reveal it is not expressed by inflammatory cells, but rather by leading-edge keratinocytes in both wild-type and PU.1
null wounds (Figure ). While not absolutely dependent on inflammatory signals, it appears that Repetin
expression by wound keratinocytes is significantly enhanced by inflammatory cues. As several studies have shown somewhat enhanced rates of re-epithelialization where one or more components of the inflammatory response are reduced during healing [6
], it is tempting to speculate that genes like Repetin
, which are upregulated in the wound epidermis in response to inflammatory signals, may in some way retard the re-epithelialization process.
As with the early inflammatory cluster, there are several genes in the late inflammatory cluster that may directly or indirectly, via their effects on signaling pathways, be responsible for wound fibrosis. The angiotensin II receptor has previously been implicated in mediating the fibrotic response in several tissue injury situations, such as myocardial infarction [42
]; its gene is also a member of the late inflammatory cluster but is expressed at both wild-type and PU.1
null wounds. Expression is clear in both wild-type and PU.1
null wounds but significantly higher in the wild-type (Figure ). The spatial expression pattern of Angiotensin II receptor
is reminiscent of Osteopontin
in the early inflammatory cluster, with the brightest staining in the deep dermal or muscle layer of the wild-type wound and only very faint expression seen at the PU.1
null wound site (Figure ). Presumably, a subset of genes found in these inflammatory clusters, which are upregulated by host granulation tissue lineages rather than by leukocytes, may turn out to be markers, or direct regulators, of the fibrotic response that is routinely activated in adult wound granulation tissue. Clearly, therapeutic reduction of the products of these genes at the wound site might result in the reduction of wound fibrosis.
A final cluster of genes appears to be regulated by the inflammatory response in that they are generally expressed at early stages post-repair in both PU.1
null and wild-type mice, but, whereas their expression subsequently diminishes in the PU.1
null mouse, expression is maintained, or increases, coincident with the inflammatory response in wild-type wounds. This temporal expression profile is most clearly visualized from heatmap data (Figure ). Some of the genes in this cluster implicate mast cells in the recruitment of other leukocyte lineages which then amplify the inflammatory signal. For example, Mast Cell Protease 5 (Mcpt5) is a serine chymase stored in the secretory granules of mast cells and acts as a potent chemoattractant [46
is rapidly and transiently upregulated immediately post-wounding and by 12 hours is back to near basal levels in the wild-type wound. However, it is secondarily upregulated at 24 hours. Expression is also clear at the PU.1
null wound site as an immediate response but levels remain low and there is no second peak of expression (Figure ). In situ
hybridization studies show expression by scattered cells within the wild-type wound, with low levels of expression detected at the PU.1
null wound site also (Figure ). These data suggest that Mcpt5
is initially expressed independently of signals from macrophages and neutrophils, but that leukocytes are subsequently responsible for a secondary expression, either directly by expressing Mcp5
themselves, or indirectly by triggering expression in another cell type, possibly supplying cues that reinforce expression by mast cells or prevent their dispersal from the wound site.
Chemokines are also represented in this inflammation-maintained cluster. CCL2 and CCL7 are C-C chemokines with roles in directing the cellular composition of the inflammatory response. They are upregulated at 3 hours with expression tailing off by 24 hours post-wounding in the wild-type. In the PU.1 null wound, CCL2 and CCL7 are also upregulated at 3 hours but to a lesser degree than in the wild-type, and unlike in the wild-type, expression is immediately downregulated, so that by 12 hours post-wounding there is a complete absence of expression (Figure ). This suggests that expression is enhanced and maintained in the wild-type by the presence of macrophages and neutrophils, whereas in the PU.1 null wound, initial expression is independent of these leukocytes but without them expression cannot be amplified and maintained. Our in situ studies suggest that these chemokines are expressed by host wound connective-tissue cells rather than leukocytes at both the wild-type and PU.1 null wound sites (Figure , and ).