LYVE-1 (also termed CRSBP-1), a 120-kDa disulfide-linked dimeric type I membrane glycoprotein, is a specific marker for lymphatic endothelial cells (LECs) and exhibits multiple ligand (hyaluronic acid and growth factors/cytokines) binding activity in mammals. Recent studies indicate that LYVE-1/CRSBP-1 ligands (VEGF-A165, PDGF-BB, oligopeptides containing the cell-surface retention sequence (CRS) motifs of VEGF-A165 and PDGF-BB) induce opening of lymphatic intercellular junctions in vitro and in vivo. To determine the function of the ortholog of mammalian LYVE-1 in zebrafish, we cloned it (zLyve-1). The cloned cDNA (zLyve1) encodes a 328-amino-acid type I membrane glycoprotein. The protein and genomic structure evidence supports the notion that the cloned zLyve-1 is the ortholog of LYVE-1 in zebrafish. zLyve-1 expressed in cultured cells by transfection exhibits hyaluronic acid binding activity but lacks the growth factor binding activity seen in mammalian homologs. Knockdown of zLyve-1 levels by embryo microinjection with a specific antisense morpholino oligonucleotide (MO2) in wild-type and Tg(fli1:EGFP)-transgenic zebrafish causes defects in thoracic duct (TD) formation. Such zebrafish injected with MO2 also exhibit impaired TD flow (as determined by intramuscular injection of FITC-dextran). The phenotypes in these zebrafish injected with MO2 are reversed by co-injection with zLyve1cDNA. In situ hybridization reveals that zLyve-1 is expressed in the posterior cardinal vein (PCV). Expression of zLyve-1 at the highest level in the PCV occurs at 3 dpf which coincides with the time for TD formation in zebrafish development. These results suggest that zLyve-1 is required for TD formation. They also suggest that zLyve-1 is distinct from mammalian LYVE-1 in its role in lymphatic function.
Reepithelialization, a part of the process of the wound healing, is essential for organisms to survive. The patterns of reepithelialization are known to differ during the developmental stages [1-6]. First, the wounds in mammalian adult skin are sealed with fibrin clot and then the wound closure is driven by epidermis crawling from the wound margin [
However, the late stages of reepithelialization after the wound closure have not yet been well investigated. In this study, we focused on the phenomenon that, once fullthickness incisional wounds made on the skin of neonatal rats were closed, the thickness of the covering epidermis increased and temporarily overran and, in turn, restored its normal level.
First, we examined the possible fluctuation of the epidermal cell number at the wound site, and calculated the rate of the cell proliferation as well as that of the cell death detected by anti-BrdU immunostaining and TUNEL, respectively. Second, we analyzed the terminal differentiation of the epidermal keratinocytes. In order to identify the differentiation stage of the wound epidermis after the wound closure, the immunohistochemistry was performed using antibodies of involucrin, profilaggrin/ filaggrin, and loricrin as the epidermal differentiation markers [9-12]. Finally, we examined the distribution of CCAAT/enhancer-binding protein-α (C/EBP-α) and caspase-14. These two proteins are known to be endogenous factors regulating the terminal differentiation of the epidermal keratinocytes at the early or late stages, respectively [12-20].
The findings in the present study are summarized as follows. The delay or derangement in the terminal differentiation of keratinocyte occurs within the areas from the upper spinous to the granular layer in the thickening epidermis at the wound region. The thinning of the wound epidermis may also be associated with the cornification probably accompanied by an enhancement of the cell death and the action of caspase-14.
Sprague-Dawley rats were anesthetized at 1 day after birth. Two incisional wounds 3 mm in length were made in the back skin on either side of the dorsal midline with a disposable scalpel (FEATHER). The wound sites were immediately covered with the cover agent Opsite (Smith & Nephew), and the wounded individuals were returned to the mother rats. The Opsite treatment had no effects on the wound healing except that it avoided unnecessary retardation of healing due to the infection that otherwise occasionally occurred (Koizumi et al., 2004).
Two hours before sampling, 10 μl/gram body-weight 10 mM 5-bromo-2’-deoxyuridine (BrdU) were injected into the abdominal cavity of rats.
The skin surrounding the wound was cut out at 0, 48, 72, 96, 120, or 144 h PW. Pieces of the skin were fixed with 4% paraformaldehyde in Ca2+, Mg2+-free phosphate-buffered saline [PBS(−)] for 1 h at room temperature, and embedded in paraffin. Sections (4 μm thickness) were stained with hematoxylin-eosin (HE) or analyzed immunohistochemically. For immunohistochemistry, deparaffinized sections were pretreated with citric acid buffer (pH 6.0) at 95˚C, and rinsed with PBS(−). Alternatively, the sections were pretreated with 2N HCl, followed by digestion with 0.05% trypsin at 37˚C. The pretreated sections were incubated with 1% normal horse serum in PBS(−) for 20 min, and then reacted with primary antibodies at 4˚C overnight. They were washed with PBS(−), then incubated with secondary antibodies for 2 h. Samples were washed with PBS(−), counterstained with 4’,6- diamino-2-phenylindole dihydrochloride (DAPI, Polysciences) at 0.1 μg/ml, and mounted with Fluoromount (Japan Tanner Corporation). The following primary antibodies were used: mouse anti-cytokeratin 14 monoclonal antibody (mAb) (Chemicon, 1:100); mouse anti-cytokeretin 10/13 mAb (Lab Vision, 1:100); mouse anticytokeratin 6 mAb (Progen, 1:50); rabbit anti-mouse involucrin poriclonal antibody (pAb) (Berkley antibody company, 1:500); rabbit anti-mouse loricrin pAb (abcam, 1:500); rabbit anti-rat profilaggrin/filaggrin pAb (466) (generously supplied by Dr. R. B. Presland, 1:250); and mouse anti-BrdU mAb (Dako Cytomation, 1:100). Alexa fluor 594 goat anti-mouse IgG1 (Molecular Probes, 1:1500); Alexa fluor 488 goat anti-mouse IgG2a (Molecular Probes, 1:500); Alexa fluor 488 goat anti-mouse IgG1 (Molecular Probes, 1:500); and Alexa fluor 488 goat anti-rabbit IgG (Molecular Probes, 1:500) were used as secondary antibodies.
The double staining of loricrin with involucrin and involucrin with filaggrin, and the staining of C/EBP-α and caspase 14 were performed using immunoenzyme technique. Deparaffinized sections were treated with citric acid buffer (pH 6.0) at 95˚C or 10% TritonX-100 in PBS(−), and endogenous peroxidase was inactivated by H2O2 before blocking process. After incubation with HRP-conjugated anti-rabbit immunogrobulins (DakoCytomation, 1:100) as the secondary antibody, the samples were reacted with DAB for color reaction. The samples for the double staining were subjected to staining with the next primary antibodies, followed by staining the alkaline phosphatase-conjugated anti-rabbit immunogrobulins (sigma-aldrich, 1:100) as the next secondary antibody. The immunostained sections were finally counterstained with hematoxylin. The following primary antibodies were used: anti-C/EBP-α rabbit pAb (Santa Cruz, 1:100); and anti-caspase 14 rabbit pAb (IMGENEX, 1:1000).
The TUNEL reaction was performed using in situ cell death detection kit and TMR red (Roche Diagnotics) according to the manufacture’s instructions.
The ratios of the numbers of BrdUor TUNEL-positive cells to those of DAPI positive cells in the wound areas or the intact areas of epidermis were calculated. The epidermal regions between both wound edges and the normal interfollicular epidermal regions more than 5 mm away from the wound edges were defined as the wound areas and the intact areas of epidermis, respectively, in this study.
The standard technique using a scalpel No. 11 (FEATHER) in this study usually generated the full-thickness incisional wounds 1 mm in depth, as shown in
ening peaked around 96 h PW (Figures 1(C)-(E)). At 144 h PW, the thickened epidermis tended to become thinner (
In addition, the immunohistochemical analyses using the epidermal differentiation markers showed that K14 keratin was localized at the basal layer, and the localization of K10 keratin was observed at the suprabasal layer, whereas K6 keratin was not detectable, at 0 h PW (Figures 1(G), (M), (S)). The localizations of these keratinspositive cells in the wound-covering and the woundsurrounding epidermis fell into disorder within 48 hours after wounding (Figures 1(H), (N), (T)), and gradually recovered the normal patterns from the distal region of wound during reepithelialization (Figures 1(I)-(L), (O)- (R), (U)-(X)).
From the above-mentioned histological observations, we noticed that the epidermis was thickened just after wound closure, and thereafter became thinner. Then, we became interested in what was the cause of the fluctuation of the epidermal thickness at the wound areas. To begin with, we speculated that the number of the epidermal cells would change at the wound. Exactly the living cells in the thickened epidermis outnumbered those in the control epidermis, as long as the total numbers of DAPI-positive nuclei were compared (
BrdU-positive cells as the proliferating cells were detected in the basal layer at 0 h PW and in the woundcovering epidermis from 48 h PW onward, too (Figures 2(A)-(F)). Here, the absolute number of BrdU-positive cells in the epidermal wound regions suggested that the cell proliferation took place during the thickening of the epidermis in the wound region. However, when the ratios of the number of the proliferating cells to the total number of living cells were estimated, the rate in the wound areas of epidermis were almost constant throughout the period tested and, moreover, there was no great difference between these rates in the wound and the intact epidermis (Figures 2(G) and (H)). Consequently, it was likely that the down-regulation of cell proliferation, if any, did not so much contribute to the thinning of the wound-covering epidermis.
Next, we did TUNEL staining to test the possibility that apoptosis is induced in the thickened epidermis as the epidermis became thinner and reduced in cell number. From 0 h to 96 h PW, the rates of TUNEL-positive cells in both the wound and intact epidermis were generally similar, though more or less variable (Figures 2(I)-(L), (O), (P)). At 120 h PW, however, the rate of TUNEL-positive cells in the epidermal wound site significantly increased, as compared with that in the intact or 0 h PW (Figures 2(M), (O), (P)). The wound region at 144 h PW had still a high rate of TUNEL-positive cells (Figures 2(N), (O), (P)). The epidermis in the wound area reached a rate equivalent to that of the normal epidermis at 192 h PW (data not shown). It should be noted that TUNEL-positive cells were observed only in the vicinity of the nucleated upper granular layer in both
wound and intact epidermis.
These results suggested that cornification accompanying cell death is significantly elevated during the thinning of the epidermis at the wound region.
The epidermal keratinocytes generate diverse differentiation marker proteins during the terminal differentiation.
We examined the localizations of these markers. First, the double immunostaining of loricrin and involucrin was executed during the process of wound healing. In both the wound and normal areas of the neonatal skin at 0 h PW, loricrin (brown) was expressed at and above the granular layer, whereas involucrin (blue) at and above the upper spinous layer (
at the wound-covering epidermis (
Next, we carried out the double immunostaining of the epidermis of the neonates using the anti-involucrin and anti-profilaggrin/filaggrin antibodies. In both the wound and normal areas at 0 h PW, the expression of profilaggrin/filaggrin (blue) was observed at the zone lower than involucrin (brown), concretely, at and above the middle spinous cell layer (
We immunohistochemically examined the expression of C/EBP-α and caspase-14, known to participate in the terminal differentiation of the epidermal keratinocytes at the early and late stages, respectively. At 0 h PW, C/EBP- α was intensely stained at both the nuclei and the cytoplasm of the cells in the suprabasal layer, while caspase- 14 at the cytoplasm in the granular layer (Figures 5(A) and (H)). The staining intensities of both molecules at the leading edge of the epidermis were obviously fallen by 24 h PW (Figures 5(B) and (I)). At 48 h PW, these immunostaining intensities were restored to the normal levels at the wound-covering epidermis. The epidermis at 72 h PW showed a similar tendency (Figures 5(C), (D), (J), (K)). By 96 h PW when the thickness of the woundcovering epidermis reached the peak, the region intensely stained with anti-caspase-14 was confined to the upper layer at the thickening epidermis, and the unstained region remarkably extended (
hardly changed (Figures 5(E) and (F)). The epidermis at 144 h PW showed that both of these two molecules tended to restore the normal level of expression (Figures 5(G) and (N)).
These results suggested that the modulation of the expression of caspase-14 might be responsible for the epidermal thickness.
HE and immunostaining experiments in this study revealed the healing process of the incisional wounds on neonatal rat skin to be outlined as follows (
We estimated the rates of cell proliferation and cell death using anti-BrdU immunostaining and TUNEL staining, respectively, to verify the possible causal relationship between the epidermal cell numbers and the fluctuation of the epidermal thickness in the wound area. The analyses of cell proliferation revealed that there is no clear correlation between the fluctuation of thickness at the wound-covering epidermis and the rate of the proliferating cells (Figures 2(A)-(H)). By contrast, the rate of TUNEL-positive cells rose shortly after peak (96 h PW) of the thickness (and the cell number) of the epidermis in the wound area, and reached the peak at 120 h PW, at which the thinning phase of epidermis was considered to start (Figures 2(M) and (O)). The peak value was 1.4 times as much as that at 96 h PW. It should be emphasized here that the rates of cell death in the wound areas at 120 and 144 h PW were significantly higher than those in the intact areas at 120 and 144 h PW, respectively (Figures 2(O) and (P)). Thus, the fluctuation of the rate of cell death and that of the thickness of epidermis seemed to coincide with each other. In other words, an elevation of the apoptosis in some cell populations in the upper epidermis may partially contribute to the reduction in the epidermal thickness.
A previous study showed that TUNEL-positive cells were often observed at the granular layer just below the horny layer of epidermis [
One of the two daughter cells that generates from an epidermal stem cell via the asymmetric cell division is known to have the developmental fate toward the terminal differentiation through which, following several cycles of proliferation, the cell differentiates into spinous, granular and horny cells in order [
Taken together, we speculated the post-wound-closure process of the epidermal wound healing, as follows (
spinous and granular layers. Next, the epidermal keratinocytes are rapidly cornified by a sudden acceleration of the differentiation to restore the steady state of epidermis. Although more evidence is necessary to establish this line of scheme, another noteworthy fact is the presence of the thick-multilayered horny layers on the thickening epidermis at the wound. This is an indication of the occurrence of transient and active cornification that may couple with the thinning of epidermis at the wound region.
As the last analysis in this study, we examined the expression of C/EBP-α and caspase-14 that are known to closely participate in the terminal differentiation of keratinocytes. C/EBP-α is a transcription factor, acting on the early phase of the terminal differentiation [14,15], whereas caspase-14 a proteolytic enzyme involved in the cornification step [12,17]. During the early stage when the epidermal sheets were migrating for the wound closure, C/EBP-α and caspase-14 were absent from the leading edges (
In conclusion, one of the most plausible causes for the fluctuation of the epidermal thickness at the wound areas is considered to be a delay and acceleration of the terminal differentiation, including cornification, in the epidermal keratinocytes. Further analyses from the other viewpoints of cellular migration, intercellular and intracellular signaling, and regeneration of some specific cells, etc. are needed for thorough elucidation of how and why the thickness of the wound-covering epidermis fluctuates.
We thank the members of our Morphogenesis Laboratories for their support and helpful discussions in weekly seminars for furthering this study.