Ian C. Mackenzie

Connective tissue influences on patterns of epithelial architecture and keratinization in skin and oral mucosa of the adult mouse

SummaryEpithelial-mesenchymal interactions play an important role during embryogenesis but it is uncertain whether such interactions influence the maintenance of epithelial structure in the adult. To examine this problem, separated epithelial and connective tissue components of skin and mucosae from various regions of adult mice were homoor heterotypically recombined and transplanted to histocompatible hosts. The patterns of tissue architecture and keratinization of the resultant epithelia were examined for changes indicative of mesenchymal influences on the epithelial phenotype. Each type of epithelium, in some recombinations, fully conserved its normal pattern of phenotypic expression indicating that subepithelial connective tissue from all regions is permissive and that regionally-specific connective tissue influences are not necessary for conservation of epithelial specificity. In other recombinations, however, the epithelium acquired features of tissue architecture or keratinization typical of the epithelium normally associated with the connective tissue component, indicating directive influ ences from the connective tissue. The patterns of epithelial response observed suggest that there may be separate connective tissue influences on epithelial architecture and cytodifferentiation and that there is a regionally-related variation in the competence of epithelia to respond to these influences.

Epithelial-mesenchymal interactions control basement membrane production and differentiation in cultured and transplanted mouse keratinocytes

SummaryThe effects of mesenchyme and substratum on epidermal differentiation and formation of a basement membrane (BM) were analyzed in vitro and in vivo. Primary epidermal cell cultures (PEC) from neonatal mice were grown: (1) on plastic culture dishes; (2) on lifted collagen gels, either alone or (3) in recombination with mesenchyme; (4) after reimplantation in vivo either directly on mesenchyme or (5) on collagen interposed between keratinocytes and mesenchyme. Differentiation of the epithelium and formation of a BM were examined by electron microscopy, and expression of BM constituents (type IV collagen, laminin, fibronectin, bullous pemphigoid antigen, and heparan sulfate proteoglycan) by indirect immunofluorescence. PEC on plastic or on collagen gels showed poor differentiation, a structured BM was not visible, and the expression and deposition of BM constituents was incomplete. Upon reimplantation in vivo, differentiation was normalized, expression of BM components complete and a structured BM reformed. This effect does not depend on immediate contact of epidermal cells with mesenchyme. When PEC on collagen gel were similarly associated with dermal mesenchyme in vitro, epidermal differentiation and expression of BM components were almost normalized, but a structured BM was absent. These findings demonstrate that formation of the BM in epidermis is a function of keratinocytes and, like differentiation is subject to mesenchymal control. A structural BM is not a prerequisite but rather an additional criterion of normal epidermal differentiation.

Label-retaining keratinocytes and Langerhans cells in mouse epithelia

SummarySlowly cycling cells in murine epithelia can be marked by their retention of a tritiated-thymidine nuclear label. The position and identity of such label-retaining cells in palatal and lingual epithelia and ear epidermis was examined using autoradiography and histochemistry. They were found to be either (a) basally positioned keratinocytes preferentially occupying sites within units of epithelial structure that correspond to those expected for epithelial stem cells, or (b) nonkeratinocytes of the Langerhans cell type which lie suprabasally except in the epidermis where they are present in low numbers and occupy a similar position to label-retaining keratinocytes.

Effects of histological processing on lectin binding patterns in oral mucosa and skin

SummaryThe effects of fixation and wax processing on lectin binding to C3H mouse palate and tail skin were evaluated using eight FITC-conjugated lectins. Sections and blocks of tissue were fixed in acetone, ethanol, methanol, formalin, glutaraldehyde or Bouins picric-acetic-formalin fixative. Tissue blocks were then processed to paraffin wax.Compared to unfixed cryostat sections, there was a weaker but similar pattern of surface binding to sections of fixed and wax-processed tissues with acetone, alcohols and Bouins fixative. Basement membrane binding was much weaker with acetone-wax and absent when alcohols or Bouins fixatives were used. Tissues fixed in formalin showed very weak general binding, while glutaraldehyde fixation resulted in considerable non-specific cytoplasmic binding. Acetone, alcohols or Bouins fixative followed by processing to paraffin wax provided convenient acceptable alternatives to the use of unfixed cryostat sections.Lipid extraction prior to lectin incubation resulted in complete elimination of detectable binding to epithelium suggesting that lectin-binding sites in the cell surface are associated with glycolipid or lipid.

Maintenance of regionally specific patterns of cell proliferation and differentiation in transplanted skin and oral mucosa

SummarySpecimens of mouse ear, footpad and tail skin and palatal, buccal and lingual mucosae were transplanted to protected sites prepared in histocompatible hosts either as intact tissues or recombined after separation of epithelial and connective tissue components using EDTA. Despite maintenance in a protected ectopic site for up to 9 weeks, transplants maintained regionally specific differences in histological appearance and rates of mitotic activity. A diurnal variation in mitotic activity comparable to host control tissues was reestablished.

The influence of differing connective tissue substrates on the maintenance of adult stratified squamous epithelia

SummaryThe interaction between adult stratified squamous epithelium and its supporting connective tissue possibly involves both permissive and directive influences. To examine the effect of vitality and specificity of connective tissue on the maintenance of epithelial structure and histo-differentiation, specimens of skin and oral mucosa from various regions of adult mice were separated using either EDTA or trypsin. Prior to transplantation, the epithelium was recombined with either inverted homologous connective tissue or with connective tissue that had been killed either by heating or repeated freeze-thawing. Epithelial sheets were also transplanted onto the graft bed alone or in combination with striated muscle or tendon.Normal patterns of cytodifferentiation were maintained when the epithelium was recombined with inverted or frozen-thawed subepithelial connective tissue but there was a loss of spatial organization on the frozen-thawed connective tissue. In contrast, heat-killed or trypsin-treated frozen-thawed subepithelial connective tissue and non-dermal connective tissue failed to maintain a viable epithelium. These observations suggest that subepithelial connective tissues (dermis, lamina propria) but not deep connective tissues facilitate epithelial proliferation and histodifferentiation.

Lectin binding to murine oral mucosa and skin

Carbohydrates on epithelial cell surfaces of oral mucosa and skin from various anatomical regions of C3H mice were demonstrated with fluoresceinated lectins. With an individual lectin, all tissues showed a similar pattern of binding: most lectins showed binding to the cell surfaces of all nucleated cell layers although that to basal cells was often weaker and was occasionally absent. The corneocytes did not typically bind lectins except that the follicular keratin of the tail showed a uniform and intense fluorescence with several lectins. Basement membrane bound all lectins. The results indicate that detectable changes occur in the cell-surface carbohydrate composition as cells differentiate but that cell-surface carbohydrates do not differ markedly from one region to the next. Lectin binding may provide a convenient method of detecting functional changes in normal cells and in cells which have undergone experimental or pathological changes.

The distribution of blood group antigens in rodent epithelia

SummaryThe pattern of distribution of antigens cross-reacting with antibodies to human blood group antigens A and B and two precursor molecules was examined by immunofluorescence in the epidermis, oral mucosa and forestomach of rats and mice. Staining for blood group antigen A was negative. In all epithelia examined, blood group antigen B was present at the surface of basal and parabasal cells, and the H antigen at the surface of spinous cells. N-acetyllactosamine was present on the cell membranes in the upper spinous and granular cell layers of epidermis and forestomach epithelium and was not expressed in the oral epithelia except for a limited area in the dorsal tongue epithelium.Thus, the expression of antigen varies both regionally and, as earlier shown in human epithelium, with the stage of maturation of cells within a given epithelium. The observed sequence of expression of these antigens during maturation differs from that of human epithelia, but the present study provides a basis for further experimental studies of the role of cell surface antigens in epithelial homeostasis and maturation.

The effect of chronic frictional stimulation on hamster cheek pouch epithelium

Abstract A controlled frictional stimulus, produced by a rotating brush, was applied daily to hamster cheek pouch for periods of up to 14 days. Pouch epithelium was examined for changes in histological appearance, and for alteration in the rate of cell proliferation as studied by injection of Vinblastine or tritiated thymidine. Specimens which had received seven or more daily applications of friction showed a marked increase in the thickness of the stratum corneum and an increase in the size and number of cells in the stratum Malpighii. There was a significant increase in the number of metaphase figures and labelled cells per unit length of rubbed epithelium. No significant difference in the length of the DNA synthesis phase was detected. The epithelial response to friction appears to be essentially similar to the epithelial response to other forms of physical or chemical trauma.

Connective tissue influences on the expression of epithelial cell-surface antigens

SummaryAdult mice were found to show regional variation in the epithelial expression of some molecules of the blood-group antigen series. To investigate connective tissue influences on such differences, heterotypic recombinants of epithelia and connective tissues from various regions were prepared and examined using monoclonal antibodies directed against bloodgroup antigens H and Ley. The results indicate that epithelia may maintain a preexisting regionally specific pattern following recombination but that, in some recombinant matches, the connective tissue is capable of signalling redirection of the pattern of expression towards that typical of the epithelium with which it is normally associated.