Differential expression of CD44s and CD4v10 proteins and syndecan in normal and irradiated mouse epidermis

 The role of the CD44s adhesion molecule, its epithelial isoforms and its relationship to epidermal proteoglycans such as syndecan was studied in normal and irradiated mouse skin. In normal mouse skin, only 10% of basal cells are strongly CD44s-immunopositive, with a cytoplasmic expression pattern. Double-label experiments with the basal cell marker keratin 14 confirmed the epithelial nature of the strongly CD44s-positive cell type in the basal layer. Some spinous keratinocytes and the majority of the remaining basal cells exhibited a weak membranous staining pattern. In contrast, the epithelial isoform, CD44v10, was strongly present in all basal and suprabasal epithelial cells of the epidermis, with a membranous staining pattern. Syndecan was found in the granular layer of the normal epidermis only. After 1 week of daily irradiation, the entire basal cell layer of the epidermis expressed CD44s in the membrane, but with a varying degree of staining intensity. This reactivity spread to the upper spinous layer after 3 weeks of treatment. In hyperproliferative epidermis, there was no difference in the staining patterns between CD44s and CD44v10. The expression of syndecan switched from the granular layer to the basal and lower spinous layers after 2 weeks of daily irradiation. Immunoreactivity for syndecan was also strongly enhanced in the dermis of irradiated samples. The results suggest an important role for syndecan and CD44 in proliferative processes during radiation-induced accelerated repopulation. Accepted: 30 September 1996     

Distribution of hyaluronan and its CD44 receptor in the epithelia of human skin appendages

Summary Biotinylated hyaluronan (HA) binding complex (HABC) from bovine articular cartilage proteoglycan was used as a histological probe to study the localization of HA in human skin. The distribution of HA was compared with its presumptive cell surface receptor, CD44, using monoclonal antibodies. In epidermis both HA and CD44 were found in the basal and spinous cell layers, but neither was present in the stratum granulosum and stratum corneum. In the keratinizing parts of hair follicles, i.e. in the outer and inner epidermal root sheath, pilosebaceous duct and the actual hair, HA and CD44 were found between the vital but not the terminally differentiated cells. In the sebaceous glands a small amount of HA was found around all cells, whereas CD44 was restricted to the basal cell layer. The secretory acini of the sweat glands stained intensively with anti-CD44 antibodies but only weakly with HABC. In the sweat gland, CD44 was localized on the basal and lateral surfaces of the clear cells, whereas the dark cells and the myoepithelial cells were negative. Both the lower and upper layers of the sweat gland ducts showed a faint but constant staining for CD44 and only minor amounts of HA. While in the keratinizing skin epithelia both HA and its CD44 receptor showed an intense staining with a close co-distribution, in the sweat and sebaceous glands their distribution patterns were not similar. It is suggested that in epithelia with divergent differentiation programs the functions of CD44 and HA may be different.     

Distribution of hyaluronan and its CD44 receptor in the epithelia of human skin appendages.

Biotinylated hyaluronan (HA) binding complex (HABC) from bovine articular cartilage proteoglycan was used as a histological probe to study the localization of HA in human skin. The distribution of HA was compared with its presumptive cell surface receptor, CD44, using monoclonal antibodies. In epidermis both HA and CD44 were found in the basal and spinous cell layers, but neither was present in the stratum granulosum and stratum corneum. In the keratinizing parts of hair follicles, i.e. in the outer and inner epidermal root sheath, pilosebaceous duct and the actual hair, HA and CD44 were found between the vital but not the terminally differentiated cells. In the sebaceous glands a small amount of HA was found around all cells, whereas CD44 was restricted to the basal cell layer. The secretory acini of the sweat glands stained intensively with anti-CD44 antibodies but only weakly with HABC. In the sweat gland, CD44 was localized on the basal and lateral surfaces of the clear cells, whereas the dark cells and the myoepithelial cells were negative. Both the lower and upper layers of the sweat gland ducts showed a faint but constant staining for CD44 and only minor amounts of HA. While in the keratinizing skin epithelia both HA and its CD44 receptor showed an intense staining with a close co-distribution, in the sweat and sebaceous glands their distribution patterns were not similar. It is suggested that in epithelia with divergent differentiation programs the functions of CD44 and HA may be different.     

Transforming growth factor-beta 1 localization in normal and psoriatic epidermal keratinocytes in situ.

Transforming growth factor-beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGF beta 1-specific polyclonal antibodies (anti-LC and anti-CC) to determine the presence of TGF beta 1 peptide in keratinocytes in sections of normal human skin in situ and in both plaque and nonplaque skin from individuals with psoriasis. In contrast to the differentiation phenotype expressed by keratinocytes in normal epidermis, keratinocytes in the psoriatic plaque exhibit a hyperproliferative/regenerative differentiation phenotype. Anti-TGF beta 1 staining was observed primarily in the epidermis. Anti-LC TGF beta 1 antibody stained nonproliferating, differentiated suprabasal keratinocytes intracellularly in normal skin but did not stain psoriatic plaques from five of seven patients. In contrast, anti-CC TGF beta 1 antibody stained suprabasal keratinocytes extracellularly in psoriatic plaques, but did not stain normal skin. Both anti-LC and anti-CC stained suprabasal keratinocytes intracellularly in nonplaque psoriatic skin. Thus, the conformation or structure of TGF beta 1 and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGF beta 1 is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGF beta 1 with nonproliferating keratinocytes in the suprabasal layers of the epidermis and its exclusion from the proliferating keratinocytes in the basal layer suggest that it may be a physiological regulator of keratinocyte proliferation. In addition, the intracellular localization of TGF beta 1 peptide in both normal and psoriatic keratinocytes suggests that it is constitutively synthesized by epidermal keratinocytes in vivo.     

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.     

Synthesis and surface expression of hyaluronan by dendritic cells and its potential role in antigen presentation

Hyaluronan (HA) is a large glycosaminoglycan consisting of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is known to act as a filling material of extracellular matrices and as an adhesive substrate for cellular migration. Here we report that dendritic cells (DC) express mRNAs for HA synthases and hyaluronidases, actively synthesize HA, and display HA on their surfaces. Interestingly, HA expression levels on DC were not significantly altered by their maturation states. With respect to physiological function, three specific HA inhibitors, i.e., bovine proteoglycan, a 12-mer HA-binding peptide (GAHWQFNALTVR) termed Pep-1, and an oligomeric Pep-1 formulation, all interfered with DC-induced activation of CD4(+) T cells isolated from DO11.10 TCR transgenic mice. For example, Pep-1 oligomer efficiently inhibited DC-dependent cluster formation, IL-2 and IFN-gamma production, and proliferation by DO11.10 T cells in vitro without affecting the viabilities of DC or T cells, DC function to uptake exogenous proteins, or DC-T cell conjugate formation at earlier time points. These observations suggest a paracrine mechanism by which DC-associated HA facilitates some of the late changes in T cell activation. Although T cells constitutively expressed mRNAs for HA synthases and hyaluronidases, their surface HA expression became detectable only after activation. Oligomeric Pep-1 and bovine proteoglycan both inhibited mitogen-triggered T cell activation in the absence of DC, suggesting an autocrine mechanism by which HA expressed by T cells assists their own activation processes. Finally, adoptively transferred DO11.10 T cells showed progressive mitosis when stimulated with Ag-pulsed DC in living animals, and this clonal expansion was inhibited significantly by administration of Pep-1 oligomer. Our findings may introduce a new concept that relatively simple carbohydrate moieties expressed on DC and perhaps T cells play an important immunomodulatory role during Ag presentation.     

Hyaluronic acid of human skin and post-burn scar: heterogeneity in primary structure and molecular weight.

Hyaluronic acid (HA) was isolated from the dermis and epidermis of normal human skin and from normal and hypertrophic scar tissue, and the molecular properties of this polysaccharide were studied by circular dichroism (CD) and high performance liquid chromatography. The molecular weights of HA of normal skin and post-burn scar tissue range from 62,000 to 180,000. Hexosamine analysis showed no galactosamine contamination and 0.37 to 2.2 w/w% of protein in the HA sample. Uronic acid analysis suggests a heterogeneous distribution of glucuronic and iduronic acids. The CD profiles of these samples are similar, indicating no significant conformational variations among them. These data suggest that the variation in the molecular properties of HA between skin and scar tissue may be due to diversity of embryonic origin between epidermis HA and dermis HA, and to the diversity of the wound-healing process between normal scar HA and hypertrophic scar HA.     

Role of hyaluronan and hyaluronan-binding proteins in lung pathobiology

Hyaluronan (HA) has diverse functions in normal lung homeostasis and pulmonary disease. HA constitutes the major glycosaminoglycan in lung tissue, with HA degradation products, produced by hyaluronidase enzymes and reactive oxygen species, being implicated in several lung diseases, including acute lung injury, asthma, chronic obstructive pulmonary disease, and pulmonary hypertension. The differential activities of HA and its degradation products are due, in part, to regulation of multiple HA-binding proteins, including cluster of differentiation 44 (CD44), Toll-like receptor 4 (TLR4), HA-binding protein 2 (HABP2), and receptor for HA-mediated motility (RHAMM). Recent research indicates that exogenous administration of high-molecular-weight HA can serve as a novel therapeutic intervention for lung diseases, including lipopolysaccharide (LPS)-induced acute lung injury, sepsis/ventilator-induced lung injury, and airway hyperreactivity. This review focuses on the regulatory role of HA and HA-binding proteins in lung pathology and discusses the capacity of HA to augment and inhibit various lung diseases.     

Correlations between hyaluronan and epidermal proliferation as studied by [3H]glucosamine and [3H]thymidine incorporations and staining of hyaluronan on mitotic keratinocytes.

The rates of keratinocyte proliferation and synthesis of Hyaluronan (HA) were studied in human whole-skin organ culture by labeling with [6-3H]glucosamine and [3H]thymidine, respectively, to reveal possible correlations between the two functions of the cell. HA distribution in epidermis was examined by staining with a specific probe prepared from cartilage proteoglycan. The keratinocyte proliferation rate was low on the first 2 culture days, but showed a tenfold increase on the third and fourth days while the synthesis of HA proceeded at a relatively stable level throughout the same period. The most intensive staining of HA occurred in the uppermost spinous cell layer, whereas mitotic cells resided in the basal and suprabasal layers. The keratinocytes under various stages of mitosis were surrounded by a HA staining not more intense than that around nondividing basal cells, but a thick pad of HA appeared rapidly between the daughter cells. These findings suggest that newly synthesized HA is associated with the separation of keratinocytes following mitosis but the majority of the synthesis and content of HA in epidermis is involved in other keratinocyte activities such as maintenance of the extracellular space and cell--cell interactions during migration and differentiation.     

Pigmented Human Skin Equivalent: New Method of Reconstitution by Grafting an Epithelial Sheet Onto a Non-Contractile Dermal Equivalent

We have established a new protocol for reconstituting a pigmented human skin equivalent (PSE) and have evaluated its functional responses to environmental stimulus, UVB. The PSE is reconstituted by grafting an epithelial sheet consisting of keratinocytes and melanocytes onto a porous non-contractile dermal equivalent populated with mitotically and metabolically active fibroblasts. i) The PSE has a multilayered, well-differentiated epidermis with cuboidal basal cells and highly organised dermis with newly synthesised extracellular matrix components. ii) Ki67-positive proliferating keratinocytes (18.1 ± 7.4%) were detected on the basal layer of the epidermis. iii) Melanocytes located exclusively within the basal layer were detected by monoclonal antibody against tyrosinase-related protein (TRP-1). iv) After exposure to UVB (100 mJ/cm² per day) for 7 consecutive days, the intensity of TRP-1 staining was increased in the PSE, showing their functional state, whereas the number of melanocytes was not changed. This non-contractile and functioning new PSE is potentially useful as a model for studying the role of melanocyte-keratinocyte-fibroblast interactions in photoprotection of the skin in more complex cutaneous microenvironment than monolayer culture, and for developing in vitro disease models and therapeutic protocols with genetically altered cells both in epidermis and dermis.     

Correlations between hyaluronan and epidermal proliferation as studied by [H]glucosamine and [H]thymidine incorporations and staining of hyaluronan on mitotic keratinocytes

The rates of keratinocyte proliferation and synthesis of Hyaluronan (HA) were studied in human whole-skin organ culture by labeling with [6-³H]glucosamine and [³H]thymidine, respectively, to reveal possible correlations between the two functions of the cell. HA distribution in epidermis was examined by staining with a specific probe prepared front cartilage proteoglycan. The keratinocyte proliferation rate was low on the first 2 culture days, but showed a tenfold increase on the third and fourth days while the synthesis of HA proceeded at a relatively stable level throughout the same period. The most intensive staining of HA occurred in the uppermost spinous cell layer, whereas mitotic cells resided in the basal and suprabasal layers. The keratinocytes under various stages of mitosis were surrounded by a HA staining not more intense than that around nondividing basal cells, but a thick pad of HA appeared rapidly between the daughter cells. These findings suggest that newly synthesized HA is associated with the separation of keratinocytes following mitosis but the majority of the synthesis and content of HA in epidermis is involved in other keratinocyte activities such as maintenance of the extracellular space and cell-cell interactions during migration and differentiation.     

Possible role of the 34-kilodalton hyaluronic acid-binding protein in visceral Leishmaniasis.

Leishmania donovani, the causative organism of human visceral leishmaniasis, invades host macrophages through its interaction with the cell surface molecules of target cells. The presence of a cell surface protein (Mr 34 kDa) having specific affinity toward hyaluronan (HA), a major extracellular matrix component, has been previously reported in macrophage cell lines. In order to identify the possible role of this HA-binding protein (HABP) in leishmaniasis, initially we demonstrated its overexpression in spleen, liver, macrophages, and serum of hamsters infected with L. donovani. We further observed higher levels of HABP in the macrophage cell line J774.G8 upon infection with L. donovani. Finally, we observed a significant increase in the level of HABP in the serum of patients with kala-azar. In order to understand its functional role in leishmaniasis, we report here a significant inhibition of cellular phosphorylation of HABP in hamster macrophages infected with L. donovani. Interestingly, the 34-kDa HABP was shown to bind with 2 proteins of promastigotes as well as amastigotes of L. donovani (with molecular masses of 55 kDa and 30 kDa respectively), suggesting a possible role for HABP in adhesion during the interaction of promastigotes and macrophages.     

Ultrastructural and histochemical study on gills and skin of the Senegal sole, Solea senegalensis

This study was undertaken to identify the normal ultrastructural features of gills and skin of the Senegal sole, Solea senegalensis, for a comparative measure to morphological alterations caused by environmental stressors such as reduced water quality and diseases. In the Senegal sole skin, four morphologically distinct layers were identified: cuticle, epidermis, dermis and hypodermis. The epidermis was composed of stratified epithelium containing three cellular layers: the outermost or mucosa layer, the middle or fusiform layer and the stratum germinativum or the basal layer. In the mucosa, two mucous cell types were differentiated: type A cells containing several round vesicles of different electron density and type B cells containing mucosomes of uniform electron density. Senegal sole have five pairs of gill arches, each containing two rows of well‐developed and compactly organized primary filaments and secondary lamellae. Fingerprint‐like microridges were observed on the surface of epithelial cells. The branchial lamellae epithelium consisted of different cell types: pavement, mucous and chloride. Between the chloride cells and the larger pavement cells, accessory cells were observed. Complexes of tight junctions and desmosomes were frequently observed between adjacent chloride and epithelial cells. Neutral mucosubstances and/or glycoconjugates were observed in the epidermis, dermis and hypodermis of S. senegalensis skin. Proteins rich in different amino acids, such as arginine and cysteine, reacted negatively or weakly positive in the epidermis, dermis and hypodermis. In gills, some mucous cells responded weakly positive to periodic acid‐Schiff (PAS) reaction but were strongly stained with Alcian Blue at pH 0.5, 1 and 2.5. When Alcian Blue pH 2.5–PAS reaction was performed, most mucous cells were stained blue (carboxylated mucins) and some mucocytes stained purple, indicating a combination of neutral and acid mucins. Proteins rich in cysteine‐bound sulphydryl (‐SH‐) and cystine disulphide (‐S‐S‐) groups were strongly detected in branchial and epidermal mucous cells, whereas lysine, tyrosine and arginine containing proteins showed very weak staining in both epidermal and branchial mucous cells. Protein reactions were strongly positive in the pillar cells, except for those rich in tryptophan, whereas the branchial cartilaginous tissue did not show an important reaction. The performed lipid reactions were negative in goblet and chloride cells. It is concluded from this study that ultrastructural and cytohistochemical features of the Senegal sole skin and gills may serve as control structures in both natural and aquaculture systems to monitor or detect environmental stress responses at the histological level.     

NG2 proteoglycan expression in mouse skin: altered postnatal skin development in the NG2 null mouse.

In early postnatal mouse skin, the NG2 proteoglycan is expressed in the subcutis, the dermis, the outer root sheath of hair follicles, and the basal keratinocyte layer of the epidermis. With further development, NG2 is most prominently expressed by stem cells in the hair follicle bulge region, as also observed in adult human skin. During telogen and anagen phases of the adult hair cycle, NG2 is also found in stem cell populations that reside in dermal papillae and the outer root sheaths of hair follicles. Ablation of NG2 produces alterations in both the epidermis and subcutis layers of neonatal skin. Compared with wild type, the NG2 null epidermis does not achieve its full thickness due to reduced proliferation of basal keratinocytes that serve as the stem cell population in this layer. Thickening of the subcutis is also delayed in NG2 null skin due to deficiencies in the adipocyte population.     

Estradiol protects dermal hyaluronan/versican matrix during photoaging by release of epidermal growth factor from keratinocytes

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

Immunolocalization of hyaluronic acid and inter-alpha-trypsin inhibitor in mice

The direct interaction of hyaluronic acid (HA) and heavy chain (HC) of the inter-alpha-trypsin inhibitor (IalphaI) family plays a critical role in the organization and stabilization of the extracellular matrix. The aim of the present investigation was to elucidate the distribution of the IalphaI HC and HA in adult mouse tissues. An immunohistochemical method using a rabbit polyclonal antibody raised against mouse IalphaI heavy-chain peptide and a specific probe for HA (biotinylated HA-binding protein) was used to demonstrate an immunolocalization of IalphaI HC and HA. Distribution and localization of HA was of three types, namely, colocalization with IalphaI HC itself (cartilaginous tissue and ovary), localization around IalphaI HC immunostaining (lung, intestine and skeletal muscle), and localization at a small distance from IalphaI HC or a different distribution pattern (brain, liver, skin and kidney). These results indicate that IalphaI HC could function as an HA-rich matrix stabilizer on the cells of cartilage and maturing ovary, in which IalphaI HC shows colocalization with its predominant ligand, HA.     

Presence and localization of proteins immunologically related to erythrocyte protein 4.1 in human skin

Analogues of human erythrocyte protein 4.1 have been examined in the human skin by immunochemical techniques using anti-human erythrocyte protein 4.1 antibodies. Immunoblot analysis revealed that human epidermis contains 4.1-like proteins of 80 kDa and 78 kDa that cross react with anti-protein 4.1 antibodies. Analysis with immunofluorescence microscopy revealed that the plasma membrane of the human epidermal keratinocyte was stained intensively in the basal cells, whereas spinous cells were moderately stained. It is noted that eccrine sweat gland cells and ductal cells were also stained in the peripheral cytoplasma. Taken together, these results demonstrate that 4.1-like proteins are present in human epidermal keratinocytes, eccrine sweat gland cells and ductal cells. The present findings enable us to suggest that a membrane skeletal protein lattice might exist in these cells.     

Changes in expression of two endogenous β-galactoside-binding isolectins in the dermis of chick embryonic skin during development in ovo and in vitro

In order to elucidate the roles of metal-independent animal lectins, we systematically investigated changes in expression of 2 kinds of -galactoside-binding isolectins (MW 14 and 16 kDa) in the dermis of chick embryonic tarsometatarsal skin during the course of development. These lectins were immunohistochemically located at different stages of development both in ovo and in vitro by light and electron microscopy. Light-microscopic observation showed that while positive staining for the 14-kDa lectin was weak at days 8 and 10 it became intense after day 13. In contrast, staining for the 16-kDa lectin was intense at days 8, 10, and 13, but it became weak after day 17 when keratinization of the epidermis was completed. Immuno-electron-microscopic observation revealed that both the 14 and 16-kDa lectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts. Distribution of the 2 isolectins was also examined in cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by the addition of vitamin A, the distribution of the 14-kDa lectin in the epidermis was significantly affected. These results indicate that (1) the expression of the 2 isolectins is differently regulated in both the dermis and epidermis, (2) the 16-kDa lectin is involved in the early stage of the formation of the dermis and the basement membrane and is replaced by the 14-kDa lectin as keratinization of the epidermis occurs, and (3) the expression of the 2 isolectins in the dermis is not significantly affected by the induction of mucous metaplasia, in contrast to their drastic changes in the epidermis.