ARTICLE |
Correspondence to: Raija H. Tammi, Dept. of Anatomy, University of Kuopio, PO Box 1627, 71120 Kuopio, Finland.
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Summary |
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The cell surface glycoprotein CD44 is involved in active cell movement, cancer metastasis, and morphogenesis. We studied its expression in fetal human skin using an antibody specific for CD44v3 and another that recognizes all CD44 forms. In embryonic and early fetal skin, only cells with dendritic morphology expressed CD44. The first keratinocyte expression of CD44 occurred in the basal cells on the eleventh week. Later, the suprabasal cells also turned positive, whereas periderm and the terminally differentiated cells remained negative at all stages. Maturation of the early mesenchyme towards dermis at the eleventh week was associated with an increase in the number of CD44-positive cells, and later the fibrous extracellular matrix also became CD44-positive. During hair induction, the epithelium showed a transient downregulation of CD44. Later, the follicular cells regained CD44. Cells in the primordial dermal papilla displayed a continuously strong signal. The sweat gland anlagen showed faint CD44 positivity. Exon 3 was present in the CD44 of keratinocytes and their derivatives but was absent in dermis. CD44 expression in human fetal skin is a relatively late event, associated with maturation and adult-type differentiation both in epidermal keratinocytes and in dermal fibroblastic cells. (J Histochem Cytochem 47:16171624, 1999)
Key Words: hyaluronan receptor, immunohistochemistry, development
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Introduction |
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The cell surface GLYCOPROTEIN CD44 is the major hyaluronan receptor (
Adult skin is one of the major organs that show high expression of CD44 under normal tissue homeostasis, both in the epithelial and dermal cells (
Adult epidermal keratinocytes show distinct changes in CD44 expression during their differentiation from basal cells to corneocytes (
During embryonic development, CD44 is often expressed in sites of active morphogenesis, such as the apical ectodermal ridge (
The present work was undertaken to establish a more systematic description of the temporal and spatial changes of CD44 expression in human fetal skin We also wanted to reveal the time of appearance of the heparan sulfate proteoglycan CD44v3 because of its high abundance in adult epidermis (
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Materials and Methods |
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Fetal Material
Human fetal skin was obtained after legally approved abortions in Kuopio University Hospital. The use of fetal skin for these studies was approved by the Ethics Committee of the hospital, and the authors were in no way involved in decisions to terminate the pregnancies. Fifteen fetuses at estimated gestational ages (EGA) of 620 weeks were included in the study. Immediately after abortion, samples of skin were removed from the back of the fetus at the level of the iliac crest and from the head at the site of the anterior fontanelle. The skin was immersed overnight in a fixative containing 2% paraformaldehyde, 2.5% glutaraldehyde, and 0.5% cetylpyridinium chloride in 0.1 M sodium phosphate buffer, pH 7.4 (PB) (
Immunostaining of CD44
The slides were incubated with the blocking buffer (1% fat-free milk powder in PB) for 30 min to block nonspecific binding. The generation of the anti-human CD44 antibody Hermes3 has been previously described (
The monoclonal antibody against the CD44 variant exon 3 was generated by immunizing mice with a recombinant fusion protein containing exons v3v10 (
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Results |
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Dermal Mesenchyme
Most of the primitive mesenchymal cells in the embryonic skin were negative for CD44. Only occasional strongly CD44-positive stellate or elongated cells were present when the fetus was about 6 weeks old, first in the lower part of the dermal mesenchyme (Figure 1A). Three weeks later, cells with similar morphology and CD44 signal emerged also in the upper dermis and close to the epidermaldermal junction (not shown).
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In the eleventh week, most of the dermal cells had acquired at least a low level of CD44 expression (Figure 1C). However, at 12 weeks (Figure 1D), 17 weeks (Figure 1E, Figure 2C, and Figure 2D), and 20 weeks (Figure 2E) the signal was also associated with the fibrillar matrix. This matrix-associated CD44 was less intense in the deep connective tissue below the dermis (Figure 2E) and did not contain variant exon 3 (Figure 1J).
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Nonkeratinocytes
The germinative epithelial cells and the peridermal cells in the embryonic skin did not express CD44 (Figure 1A, and Figure 1B). However, as early as 6 weeks, occasional strongly Hermes3-positive but v3-negative cells with dendritic morphology were seen above the germinative cells (not shown). Two weeks later, cells with similar morphology resided more frequently between the periderm and the germinative layer (Figure 1B) and subsequently also within the basal layer (Figure 1D and Figure 2A2C).
Epidermal Keratinocytes
The formation of the first intermediate cell layer at 1112 weeks coincided with the first signs of CD44 expression in the germinative (basal) layer (Figure 1C, inset), whereas the newly formed intermediate cell layer and the peridermal layer remained CD44-negative (Figure 1C). With increasing epidermal thickness, the CD44 signal grew stronger in the basal layer and the intermediate cells became CD44-positive (Figure 1D and Figure 2A2C). From the beginning, the stratifying epidermal cells were also positive for variant exon 3 (Figure 1I).
Maturation of epidermis was associated with the loss of CD44 expression from the upper intermediate cells at the age of 17 weeks (Figure 1E). With complete keratinization of epidermis (EGA 20 weeks), the granular cells (Figure 1F), and the corneocytes were CD44-negative (Figure 1F, inset). At this stage, the lower cell layers (basal and spinous cells) were intensely CD44-positive (Figure 1F).
Hair Anlagen and Dermal Condensation
The tightly packed cylindrical basal epithelial cells in the initial hair anlagen (pre-germ) stained less intensely for CD44 than the surrounding interfollicular basal cells or the intermediate cells (Figure 2A). This was even more evident in a late pre-germ follicle (Figure 2B). When the hair germ was formed, its outer cells showed less intense staining for CD44 than the inner cells (Figure 2C). Among the latter, occasional strongly positive cells with dendritic morphology were present (Figure 2C). The mesenchymal cell aggregation below the epithelial germ, which at first displayed faint CD44 expression (Figure 2B and Figure 2E), became strongly CD44-positive during elongation of the follicle (Figure 2C2E).
The outer epithelial cells on the deep part of the hair peg were more strongly CD44-positive than those in the more superficial end and at the base of the peg (Figure 2D and Figure 2E). Formation of bulbous pegs was associated with the compartmentalization of CD44, with strong expression in the ORS (outer root sheath) and loss of CD44 from the terminally differentiated part of IRS (inner root sheath) and the hair (Figure 2E and Figure 2F). In the matrix of the hair and IRS, the progenitor cells remained CD44-positive (Figure 2F).
Interestingly, the putative stem cells in the hair bulge exhibited staining from faint to negative, clearly different from the rest of the ORS (Figure 2E).
The mesenchymal cells in the dermal papillae were strongly CD44-positive (Figure 2F). In addition, the cells forming the connective tissue sheath around the follicle showed a strong signal (Figure 2E).
Sebaceous Cells
The undifferentiated progenitor cells in the sebaceous gland primordium were CD44-positive, whereas the maturing sebocytes were only occasionally positive and fully mature cells expressed no CD44 (Figure 2E and Figure 2G).
Sweat Gland
In the sweat gland bud, the outer keratinocytes were CD44-positive, whereas the inner cells were almost negative (Figure 2H). A strongly positive cell with dendritic morphology was often present in the bud (Figure 2H and Figure 2I). The more elongated sweat gland ducts exhibited a very low level of CD44 expression, most of the outer and inner cells being negative (Figure 2J and Figure 2L). Occasional intensely stained cells and cell processes were seen among the inner cells (Figure 2J and Figure 2L). Cells surrounding the intraepidermal sweat pore were CD44-negative, in contrast to the surrounding basal and intermediate keratinocytes (Figure 2K).
CD44v3 Expression
Early fetal skin showed no signal with the monoclonal antibody specific for v3 (Figure 1H). When epidermis started to stratify, a faint v3 signal appeared in basal and suprabasal layers (Figure 1I), similar to that of Hermes3 (Figure 1C), and increased in intensity with epidermal maturation and particularly with the onset of true cornification (Figure 1J). The signals for v3 in the epithelial cells of developing hair follicles were also virtually superimposable with those obtained with Hermes3 (not shown). In a contrast, all mesenchymal cells and cells with dendritic morphology remained negative, indicating restriction of this variant to the keratinocyte lineage in human skin (Figure 1H1J). The v3 signal was also absent from the dermal extracellular matrix, suggesting that this CD44 did not originate from epidermis.
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Discussion |
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Epidermis
The present study showed that CD44, with its variant exon v3, first appears in the basal cells of epidermis during the transition to a stratified epithelium. At the same time, the basal cells adopt columnar morphology, contain less glycogen, and express new keratin types (reviewed by
The importance of CD44 is largely due to its ability to bind and immobilize hyaluronan in the epidermis (
Interestingly, peridermal cells and early epithelial cells in human epidermis display hyaluronan (
Epidermal Nonkeratinocytes
The strongly CD44-positive but v3-negative cells with dendritic morphology in the early epithelia probably represent Langerhans cells, melanocytes, or Merkel cells (
Dermis
CD44 appeared relatively late in the mesenchyme, apparently being expressed with the gene set associated with the transition of dermis from a cellular to a fibrous tissue (
The cells in the developing dermal papilla showed strong CD44 expression, as reported earlier in murine skin (
Skin Appendages
The basal cells at the initiation site of the hair follicle (pre-germ) expressed little CD44 and hyaluronan (
The fetal hair bulge differed from the rest of the ORS by its lower CD44 signal. The bulge cells have a morphology characteristic of undifferentiated keratinocytes (
The developing sweat gland anlagen exhibited only little CD44 and hyaluronan (
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Acknowledgments |
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Supported by a grant from the Hulda Tossavainen Fund of the North Savo Cultural Foundation to A-LT.
We thank Ms Eija Rahunen, Ms Arja Venäläinen, Mr Kari Kotikumpu, Mrs Aija Parkkinen, Ms Riikka Eskelinen, and Ms Tiina Räsänen for skillful technical assistance.
Received for publication June 16, 1999; accepted July 13, 1999.
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