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Differential expression of F-actin in in utero fetal wounds

European Journal of Dermatology. Volume 15, Number 3, 133-9, May-June 2005, Investigative report

Summary

Author(s) : Allison J Cowin, Child Health Research Institute, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, SA, 5006, Australia.

Summary : Fetal skin possesses the capacity to heal a wound by a process of regeneration rather than repair, resulting in the absence of scar formation. The actin cytoskeleton may be involved in this process of scar-free wound healing. The effect of wounding on the expression of contractile filamentous actin (F-actin) was investigated in utero in mice between embryonic day 16 (E16) and embryonic day 18 (E18). Increased F-actin staining in the epidermis of the E16 fetal wounds was observed as early as 3 hours post-wounding, peaking in intensity after 24 hours. By 48 hours the intensity of staining had returned to background levels. In marked contrast, E18 fetal wounds did not show increased epidermal F-actin fluorescence, instead increased staining was observed in cells lying perpendicular to the wound margin within the dermis. This developmental switch from epidermal actin expression in “scar-free” fetal wounds to dermal actin expression in late “scar-forming” gestation wounds may be important in fetal wound contraction and scar-free wound repair.

Keywords : actin, cytoskeleton, fetal, repair, skin, wound

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ARTICLE

Auteur(s) :, Allison J Cowin^*

Child Health Research Institute, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, SA, 5006, Australia

accepté le 19 Janvier 2005

Fetal dermal wounds heal without scar formation by a process resembling regeneration rather than repair [1-4]. A number of major differences between the adult and fetal wound have been elucidated, including the ability to heal rapidly with reduced inflammation [5, 6]. The extracellular matrix deposited in fetal wounds contains essentially the same structural components as that in the adult wound and the organisation of collagen in the healed fetal wound is indistinguishable from the normal surrounding tissue [2]. Additionally, fetal wounds are rich in the extracellular matrix molecule hyaluronic acid, which may be important in influencing cellular and matrix events within the wound [7, 8]. Growth factors play a major role in adult wound healing and their role in fetal wound repair has been extensively studied [9, 10]. A particular example of this is the absence of detectable TGFß in fetal wounds compared to that detected in adult wounds by immunocytochemistry [11, 12]. There are obvious differences in the surrounding environment of the fetal in utero wounds and the adult wounds, e.g. amniotic fluid, constant temperature, and moisture [13]. In addition, there are less obvious differences in the nature of the fetal cells. Fetal fibroblasts are continuously proliferating, dividing and differentiating and may be able to respond more quickly to wounding whereas adult cells must undergo proliferation and phenotypic change. Fetal fibroblasts may also lack the necessary signals, possibly from growth factors, to change to a myofibroblast phenotype and hence wound closure may proceed differently from that of an adult wound [14].The migration of fibroblasts and keratinocytes into the wound site is dependent upon cell motility. This requires the remodelling of the cytoskeleton, an internal filamentous structure that governs cell shape and motility [15, 16]. Several studies have indicated that cytoskeletal proteins may mediate this wound healing process [3, 17]. The mechanism of fetal wound contraction may also be different to that of the adult. Adult wound closure involves active movements of both connective tissue and epidermis. The granulation tissue contracts to bring the wound edges together and, as this happens, the epidermis migrates to cover the exposed tissue. It is well accepted that the process of epithelialisation in the adult wound proceeds by cells crawling across an adhesive substrate [18] using lammellipodial like-fingers to attach and drag themselves over the wound substratum [19]. In contrast, studies in embryos suggest that lamellipodial crawling is not the migratory mechanism used to cover embryonic wounds. In vitro, excisional fetal wounds on the embryonic day 4 fetal chick or E12.5 rodent heal by both mesenchymal contraction and active movement of the epithelium over the dermal wound margins of the wound [20]. The leading epithelial edge of a chick limb bud wound in in vitro culture does not extend lamellipodia that would facilitate a “crawling” mechanism of epithelialisation, instead fetal re-epithelialisation appears to involve the formation of an actin cable in the basal marginal epidermal cells that may act like a purse-string to draw the wound margins together [20]. Localisation of cadherins into clusters at the wound margins of fetal in vitro wounds suggests that this cable may join adjacent cells via adherens junctions enabling the leading cells in a fetal wound to be drawn forward by concerted tugging on their epithelial cell neighbours. Additional studies have shown that these actin cables also contain myosin and can act in a zipper-like manner to close incisional wounds in fetal skin [21].Although these studies clearly show a role of actin in fetal wound healing in vitro, the mechanism behind fetal wound closure in utero still remains to be elucidated and debate remains about whether it depends on the purse string closure suggested by Martin and Lewis (1992) or whether there is more of an adult mechanism of wound closure, where regeneration of the extracellular matrix components facilitate the closure of the wound space. In these studies we have utilised an in utero fetal mouse model of wound repair to assess the effect of wounding on the expression of contractile filamentous actin (F-actin) in fetal wounds at the time of the developmental switch from scar-free to scar-forming wound repair.

Methods

Fetal surgical technique

Time mated female MF1 mice were anaesthetised on day 16 and day 18 of gestation. A midline laparotomy was performed to access the pregnant uterus. Using an operating microscope one horn of the pregnant uterus was exposed to reveal the left flank of a fetus. A purse string suture of 9/0 nylon was placed through all layers of the uterine wall on the anti-mesenteric surface. An incision was made through the myometrium and membranes to expose the left flank of the fetus. A 2 mm linear full thickness incision was made on the left flank of the fetus with microsurgical scissors. The wound was left unsutured. The purse string was closed by gently easing the flank of the embryo back into the uterus. The maternal abdominal wound was repaired using a 6/0 nylon suture. Two fetuses were wounded in each animal. The animals were allowed to recover from anaesthesia and the pregnancies continued until the fetuses were harvested or birth occurred on day 19-20 of gestation.

Wound harvest and sectioning

Unborn fetuses were killed by decapitation, pups were killed by overdose of chloroform followed by cervical dislocation. Fetal wounds were harvested at 0, 1, 3, 6, 12, 18, 24, 36, 48, 72 hours and 28 days post-wounding. The 28 days wounds were harvested in the neonatal period and were recognised by a thin line absent of hair growth at the site of the wound approximately 5 days after birth, when hair growth had begun. This site was then marked regularly with an indelible marker pen to aid identification when the fur had thickened. Fetal limbs and tails were amputated and the remaining body was fixed in 4% paraformaldehyde for visualising F-actin staining.

Localisation of filamentous actin

Whole wounded E16 or E18 fetuses were fixed in buffered formalin (4% paraformaldehyde in phosphate buffered saline) for 24 hours and then rinsed in tris-buffered saline (TBS: 25 mM Tris, 140 mM NaCl 3 mM KCl, pH 7.4). The skins were carefully dissected free of the fetuses and incubated at room temperature in the actin-binding alkaloid phalloidin linked to the fluorochrome FITC (2.5 μg/mL in TBS) [20]. The skins were then rinsed in two 20 minute washes of TBS at room temperature before being carefully mounted on microscope slides using Immu-mount (Shandon, Pittsburg, PA) and viewed using confocal microscopy.

Confocal imaging of filamentous actin

FITC-phalloidin labelled specimens were viewed using Bio-Rad MRC-1000 Laser Scanning Confocal Microscope System in combination with a Nikon Diaphot 300 inverted microscope in fluorescence mode with excitation at 488/510 nm and emission at 522/32 nm. Objective lenses × 20 NA 0.40 and × 40 water immersion NA 1.15 were used to produce horizontal and vertical optical slices from the specimens. The images were captured as digital computer files and quantitative examination of the fluorescence was performed using CoMOS (Bio-Rad) computer image analysis software.

Results

Staining of filamentous actin in in utero E16 and E18 fetal wounds

To create a wound on the flank of a fetus, the flank was exposed and using microdissecting scissors a snip was made in the skin. This created a “V” shaped wound that could be clearly seen in E16 and E18 mice (figure 1A, 2A-D and 3A respectively). In the E16 skin, the wounds clearly gaped open at 3 hours post-wounding ( (figure 2A) ), but by 6 hours the wound edges were opposed ( (figure 2C) ) and by 24 hours the wound edges were less distinct ( (figure 2E) ). By 48 hours post-wounding it was almost impossible to detect where the original wounds were created ( (figure 2G) ). Following incubation of the fetal wounds with FITC-phalloidin to stain F-actin, increased F-actin was observed as early as 3 hours post wounding in the fetal wounds particularly at the tip of the v shaped incision ( (figure 2B) ). This expression increased markedly and was strongly fluorescing 6 hours post wounding ( (figure 2D) ), Maximum fluorescence was observed 24 hours post-wounding ( (figure 2F) ) but by 48 hours the fluorescence had almost disappeared with only remnants of staining remaining ( (figure 2H) ).

Wounds created in E18 fetal skin still gaped open up to 24 hours post-wounding ( (figure 3A) ) and were still clearly visible 48 hours post-wounding. In marked contrast to the E16 wounds, no epidermal actin staining was observed at the wound margins at any of the timepoints post injury. However, actin positive filaments were observed surrounding the wound margins within the dermis adjacent to the wound edges ( (figure 3B,C and D) ). These actin positive bundles, which may be developing muscle fibres, were arranged perpendicular to the wound margins. The filaments were assembled in units between 1-2 μm in diameter, extending perpendicular to the wound for some 15 to 20 μm. These dermal actin positive filaments were observed as early as 3 hours post wounding ( (figure 3B) ) and were present surrounding the wound margins over the following 24-48 hours. A higher magnification view of the dermal filaments staining positive for F-actin is shown in ( figure 3D ).

Position of actin in E16 and E18 wounds

Using the confocal scanning microscope transverse cross-sections were created through the E16 and E18 wound margins ( (figure 4) ). These cross-sections confirmed that the actin was localised within the epidermis at E16 ( (figure 4A-B) ) and in the dermis at E18 ( (figure 4C-D) ). Fluorescence could be seen at the wound margins in epidermal cells up until 48 hours post-wounding and can be seen at 6 and 24 hours post wounding in ( figure 4A and B ) respectively. In contrast to the epidermal location of the actin cable in the E16 wounds, vertical sections through the E18 wounds confirmed that the actin filaments surrounding these wounds were localised in the deep dermal layer of the skin at the wound edges ( (figure 4C-D) ). These large filaments fluoresced strongly and can be seen at 3 and 24 hours post-wounding ( (figure 4C and D ) respectively). The position of these large filaments at E18, in the deep dermis, indicates that they are most likely to be within developing panniculus carnosus muscle fibres which may have become aligned around the edges of the E18 wounds in response to the change in tension that has occurred following wounding.

Discussion

The process of re-epithelialization is an important component of wound repair as it serves to restore the barrier function of skin and aids in reducing scar formation. Cells participating in the re-epithelialization of wounds include basal and suprabasal keratinocytes lining the wound edge, and keratinocytes derived from hair follicles and sweat ducts. Keratinocyte motility is driven by rearrangements of the actin cytoskeleton to produce lamellipodia and filopodia, two protrusive structures that adhere to the surrounding tissue via integrins, and serve to drag the cell forward [22]. The mechanism of keratinocyte motility is different in adult vs. fetal wounds. Adult wound closure involves active movements of both connective tissue and epidermis. The granulation tissue contracts to tug the wound edges together and the epidermis migrates to cover over the exposed connective tissue [19]. In contrast, early embryonic in vitro excisional wounds do not extend lamellipodial type epidermal cells as seen at the adult wound edges, which would facilitate a “crawling” mechanism of wound closure [20].

The current study was designed to identify if the cytoskeletal protein actin was developmentally regulated in response to wound healing in the fetus. Two important developmental time points were chosen, embryonic day 16 and embryonic day 18, due to the transition that occurs between these time points from scar-free healing to scar-forming healing in mice [9]. F-actin was upregulated in response to wounding at both these developmental time points with expression peaking only hours post-injury. No actin cable was observed surrounding the cut edges of the wounds at any time point post-wounding. This was in contrast to in vitro data whereby the formation of an actin cable in the basal marginal epidermal cells has been suggested to draw epidermal edges forward simply by concerted tugging on their epithelial cell neighbours [19]. This cable of actin has been shown to run in the front row of basal epidermal cells at the margin of the embryonic wounds in vitro and possibly act like a purse-string to draw the wound margins together [20]. However, in this study using in vivo wounds, no actin cable formation was observed at the wound edge, instead more diffuse, cytoplasmic actin staining was observed in the outer layers of the epidermis and at the margin of the wounds. The absence of actin cable formation may be due to the later gestational age of the mouse used in this study. Previous studies have been performed on E12.5 mice [21] and the different observations may reflect further developmental regulation of the actin cytoskeleton and its involvement in epithelial contraction. We have previously reported actin cable formation in E17 excisional skin explants in vitro [17]. These explants were suspended in culture with no dermal matrix upon which the epidermal cells could crawl across. Even so, the wounds were still able to contract and close via the formation of an actin cable. The absence of actin cable formation observed in this study in the same gestational age skin is therefore interesting and may highlight the difference between in vitro and in vivo situations. Clearly actin levels are significantly elevated at the wound edges in vivo indicating that reorganisation of the cytoskeleton is still an important feature of fetal wound healing.

The major difference between E16 and E18 fetal wounds however was clearly the position of the actin staining within the skin. At E16, similar to our previous in vitro studies [17], actin was upregulated within the epidermal cells just back from the wound margins. In marked contrast, in the skin of E18 fetal mice, wounding did not cause up regulation of actin in the epidermis but instead increased F-actin staining was observed within the dermis in bundles of filaments arranged perpendicular to the wound edges. No cable structures were observed in the wound margins of the E18 fetal skins at any time point post-wounding; however, actin filaments assembled in units between 1 and 2 μm in diameter, extending perpendicular to the wound for some 15-20 μm. Upon full wound closure the actin staining dispersed indicating that there was no longer a requirement for actin at the wound site. In adult wounds, actin-positive myofibroblasts exert contractile forces in granulation tissue within a wound by generating stress fibres in the surrounding extracellular matrix producing localised contraction of the dermal matrix [23]. It is possible that the actin positive fibres observed in the E18 fetal wounds are exerting similar contractile forces to those exhibited by myofibroblasts resulting in the dermal contraction of the E18 wounds.

Members of the transforming growth factor beta family play critical roles both in wound healing and in stimulating morphological changes of the epidermis that require epithelial cell movement [24]. Activin and TGFβ cause the activation of RhoA but not of Rac and CDC42, leading to MEKK1-dependent phosphorylation of JNK and transcription factor c-Jun. MEKK1-mediated JNK and p38 activities are both essential for activin-stimulated and transcription-dependent keratinocyte migration. These pathways lead to the control of actin cytoskeleton reorganization and epithelial cell migration, contributing to the physiologic and pathological effects of TGFβs/activins on epithelial morphogenesis. Our previous studies have shown a differential switch in TGFβ levels in fetal vs adult wounds occurring at the same gestational time points observed in this study [11]. Neither TGFβ1 or TGFβ2 are upregulated in response to wounding in fetal wounds, however TGFβ3, which has been shown to have anti-scarring properties [25] is upregulated in E16 fetal wounds. It is therefore possible that TGFβ3 may activate RhoA expression in epithelial cells leading to increased expression and reorganisation of actin observed in this study. In support of this, studies have shown that RhoA is required for the formation of actin cables in fetal wounds [21]. In adult wounds, changes in actin expression are localised to cells within the dermis with little expression occurring in the epidermis. The TGFβs may also be involved in this process because increased expression of TGFβ1 and TGFβ2 are observed in these late gestation fetal wounds and they may be stimulating the differentiation of fibroblasts to actin positive myofibroblasts. It is unclear why TGFβ1 and TGFβ2 do not upregulate actin expression in the epidermis in late gestation and adult wounds and may reflect either different properties of these TGFβ isoforms or intrinsic developmental changes that occur in the skin and the change that occurs in the fetus to be able to regenerate rather than repair a wound.

In conclusion, the change from epidermal to dermal actin expression occurs at the same time in gestation that the transition occurs between scar-free and scar-forming wound repair. This may reflect a change from an epidermally driven mechanism of wound repair to the more mature, dermally driven wound healing observed in adults which results in the occurrence of scar formation.

Acknowledgements

The author thanks Prof Mark Ferguson for his assistance in this study. The study was supported by the Channel 7 Children’s Research Foundation of South Australia and the Women’s and Children’s Hospital Foundation. AJC is funded in part by the Jean B Reid Research Associateship, University of Adelaide SA.

References

1 Longaker MT, Adzick NS. The biology of fetal wound healing: a review. Plast Reconstr Surg 1991; 87: 788-98.

2 Adzick NS, Lorenz HP. Cells, matrix, growth factors, and the surgeon. The biology of scarless fetal wound repair. Ann Surg 1994; 220: 10-8.

3 Martin P. Wound Healing-aiming for perfect skin regeneration. Science 1997; 276: 75-81.

4 Ferguson MWJ, Whitby DJ, Shah M, Armstrong J, Siebert JW, Longaker MT. Scar formation: The spectral nature of fetal and adult wound repair. Plast Reconstr Surg 1996; 97: 854-60.

5 Hopkinson-Woolley J, Hughes D, Gordon S, Martin P. Macrophage recruitment during limb development and wound healing in the embryonic and foetal mouse. J Cell Sci 1994; 107: 1159-67.

6 Cowin AJ, Brosnan MP, Holmes TM, Ferguson MW. Endogenous inflammatory response to dermal wound healing in the fetal and adult mouse. Dev Dyn 1998; 212(3): 385-93.

7 Kennedy CI, Diegelmann RF, Haynes JH, Yager DR. Proinflammatory cytokines differentially regulate hyaluronan synthase isoforms in fetal and adult fibroblasts. J Pediatr Surg 2000; 35(6): 874-9; Jun.

8 Mast BA, Diegelmann RF, Krummel TM, Cohen IK. Scarless wound healing in the mammalian fetus. Surg Gynecol Obstet 1992; 174: 441-51.

9 Whitby DJ, Ferguson MW. Immunohistochemical localization of growth factors in fetal wound healing. Dev Biol 1991; 147: 207-15.

10 Dang CM, Beanes SR, Soo C, Ting K, Benhaim P, Hedrick MH, Lorenz HP. Decreased expression of fibroblast and keratinocyte growth factor isoforms and receptors during scarless repair. Plast Reconstr Surg 2003; 111(6): 1969-79.

11 Cowin AJ, Holmes TM, Brosnan MP, Ferguson MWJ. Expression of TGF-β and its receptors in murine fetal and adult dermal wounds. Eur J Dermatol 2001; 11: 424-31.

12 Soo C, Beanes SR, Hu FY, Zhang X, Dang C, Chang G, et al. Ontogenetic transition in fetal wound transforming growth factor-beta regulation correlates with collagen organization. Am J Pathol 2003; 163(6): 2459-76.

13 Longaker MT, Whitby DJ, Ferguson MWJ, Lorenz HP, Harrison MR, Adzick NS. Adult skin wounds in the fetal environment heal with scar formation. Ann Surg 1994; 219: 65-72.

14 McCallion RL, Ferguson MWJ. Fetal wound healing and the development of antiscarring therapies for adult wounding. In: Clark RAF, ed. The molecular and cellular biology of wound repair 2^nd ed. New York and London: Plenum Press, 1996: 561-600.

15 Pollard TD, Blanchoin L, Mullins RD. Molecular mechanisms controlling actin filament dynamics in non-muscle cells. Annu Rev Biophys Biomol Struct 2000; 29: 545-76.

16 dos Remedios CG, Chhabra D, Kekic M, Dedova IV, Tsubakihara M, Berry DA, Nosworthy NJ. Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol Rev 2003; 83(2): 433-73.

17 Cowin AJ, Hatzirodos N, Teusner JT, Belford DA. Differential effect of wounding on actin and its associated proteins, paxillin and gelsolin, in fetal skin explants. J Invest Dermatol 2003; 120(6): 1118-29.

18 Stenn KS, Malhotra R. Epithelialization. In: Cohen IK, Diegelmann RF, Lindblad WJ, eds. Wound Healing Biochemical and Clinical Aspects. Philadelphia: W.B.Sauders Company., 1992: 115-27.

19 Nodder S, Martin P. Wound healing in embryos: a review. Anat Embryol (Berl) 1997; 195(3): 215-28; Mar.

20 Martin P, Lewis J. Actin cables and epidermal movement in embryonic wound healing. Nature 1992; 360: 179-83.

21 Brock J, Midwinter K, Lewis J, Martin P. Healing of incisional wounds in the embryonic chick wing bud: characterization of the actin purse-string and demonstration of a requirement for Rho activation. J Cell Biol 1996; 135(4): 1097-107.

22 Lambrechts A, Van Troys M, Ampe C. The actin cytoskeleton in normal and pathological cell motility. Int J Biochem Cell Biol 2004; 36(10): 1890-909.

23 Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases. J Pathol 2003; 200(4): 500-3.

24 Zhang L, Wang W, Hayashi Y, Jester JV, Birk DE, Gao M, Liu CY, Kao WW, Karin M, Xia Y. A role for MEK kinase 1 in TGF-beta/activin-induced epithelium movement and embryonic eyelid closure. EMBO J 2003; 22(17): 4443-54.

25 Shah M, Foreman DM, Ferguson MW. Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 1995; 108(Pt 3): 985-1002.