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F M Watt

Publications and source records attributed to F M Watt.

At least 127 records · Page 7Linked to original sources
PubMed1989

Onset of expression of peanut lectin-binding glycoproteins is correlated with stratification of keratinocytes during human epidermal development in vivo and in vitro.

During gestation the epidermis develops from a single layer of ectoderm into a layer of keratinocytes overlaid by a layer of periderm; this is followed by a progressive increase in the number of layers of keratinocytes, until finally the distinct granular and cornified layers characteristic of mature epidermis are formed. As part of our investigation into the function of the peanut lectin-binding glycoproteins of cultured human keratinocytes, we have examined their expression at different stages of human epidermal development. We found that the onset of expression of the glycoproteins coincided with the transition from a two- to a three-layered epidermis, both in vivo and in organ culture. In adult epidermis, the patterns of binding of peanut lectin and Limax flavus lectin are complementary, with peanut binding more strongly to suprabasal keratinocytes and Limax flavus lectin binding more strongly to cells in the basal layer. We found that the complementary pattern of binding of the two lectins was established at, or shortly after, the onset of stratification and retained throughout development. In contrast, expression by keratinocytes of involucrin, a protein precursor of the cornified envelope, occurred after stratification had begun. Finally, we identified the peanut lectin-binding glycoproteins of adult epidermis by immunoblotting with an antiserum raised against the glycoproteins of cultured neonatal keratinocytes. In conclusion, expression of the peanut lectin-binding glycoproteins is an early event in epidermal development, and this would be consistent with a role for the glycoproteins in stratification.

Embryonic and Fetal Development↗
PubMed1988

Influence of cytochalasin D-induced changes in cell shape on proteoglycan synthesis by cultured articular chondrocytes.

There is growing evidence that cell shape regulates both proliferation and differentiated gene expression in a variety of cell types. We have explored the relationship between the morphology of articular chondrocytes in culture and the amount and type of proteoglycan they synthesize, using cytochalasin D to induce reversible cell rounding. When chondrocytes were prevented from spreading or when spread cells were induced to round up, 35SO4 incorporation into proteoglycan was stimulated. Incorporation into the cell layer was stimulated more than into the medium. When the cells were allowed to respread by removing cytochalasin D, proteoglycan synthesis returned to control levels. Cytochalasin D-induced stimulation of 35SO4 incorporation reflected an increase in core protein synthesis rather than lengthening of glycosaminoglycan chains, because [3H]serine incorporation into core protein was also stimulated. The observed stimulation of proteoglycan synthesis was not due to an overall stimulation of protein synthesis, to inhibition of DNA synthesis, or to accumulation of cells in one phase of the cell cycle. Cytochalasin D-treatment of cells in suspension caused no further stimulation of 35SO4 incorporation, suggesting that the observed effects were due to cell rounding rather than exposure to cytochalasin D per se; nevertheless, we cannot completely rule out other, nonspecific, effects of the drug. Fibroblasts and chondrocytes that had been passaged to stimulate dedifferentiation did not incorporate more 35SO4 when treated with cytochalasin D, suggesting that increased proteoglycan synthesis in response to rounding may itself be a differentiated property of chondrocytes.

Animals↗
PubMed1988

The peanut lectin-binding glycoproteins of human epidermal keratinocytes.

Peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes, both in intact epidermis and in culture. In order to investigate the significance of this change in cell-surface carbohydrate we have identified the PNA-binding glycoproteins of cultured human keratinocytes and raised antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [14C]galactose- or [14C]mannose- and [14C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to Pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium (0.1 mM calcium ions) were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification.

Calcium↗
PubMed1988

Cell shape controls terminal differentiation of human epidermal keratinocytes.

Cultures of human epidermal keratinocytes provide a useful experimental model with which to study the factors that regulate cell proliferation and terminal differentiation. One situation that is known to trigger premature terminal differentiation is suspension culture, when keratinocytes are deprived of substratum and intercellular contact. We have now investigated whether area of substratum contact, and hence cell shape, can regulate terminal differentiation. Keratinocytes were grown on circular adhesive islands that prevented cell-cell contact. By varying island area we could vary cell shape from fully spread to almost spherical. We found that when substratum contact was restricted, DNA synthesis was inhibited and expression of involucrin, a marker of terminal differentiation, was stimulated. Inhibition of proliferation was not a sufficient stimulus for involucrin synthesis in fully spread cells. When DNA synthesis and involucrin expression were plotted against contact area, classic dose-response curves were obtained. Thus cell shape acts as a signal for the terminal differentiation of keratinocytes in culture.

Cell Adhesion↗
PubMed1988

An unusual strain of human keratinocytes which do not stratify or undergo terminal differentiation in culture.

We have characterized an unusual cell phenotype in third passage cultures of a human keratinocyte strain derived from newborn foreskin epidermis. The cells had the same DNA fingerprint pattern as the second passage, morphologically normal, keratinocytes; they formed desmosomes and expressed the keratin profile characteristic of normal keratinocytes in culture. However, unlike normal keratinocytes, the cells did not grow as compact colonies and did not stratify or undergo terminal differentiation, even after TPA treatment or suspension culture. For these reasons we named them ndk for "nondifferentiating keratinocytes." The ndk cells also differed from normal keratinocytes in that they did not require a feeder layer and were not stimulated by cholera toxin to proliferate. The ndk cells had an absolute requirement for hydrocortisone and their growth rate was increased when epidermal growth factor was added to the medium. Although ndk failed to undergo terminal differentiation in culture, they were not transformed, since they were still sensitive to contact inhibition of growth, did not proliferate in soft agar, and had a limited lifespan in culture. The appearance of the ndk phenotype was correlated with a doubling of chromosome number and the presence of a lp marker chromosome. We suggest that these cells are a useful experimental adjunct to cultures of normal keratinocytes, in which proliferation and terminal differentiation are tightly coordinated, because in ndk cells there appears to be a block in terminal differentiation.

Blotting, Western↗
PubMed1988

A model for in vitro studies of epidermal homeostasis: proliferation and involucrin synthesis by cultured human keratinocytes during recovery after stripping off the suprabasal layers.

In normal epidermis there is a balance between the rate of cell division and the rate of terminal differentiation. This can be perturbed by injuries such as wounding or tape-stripping, but is reestablished during recovery. The aim of the studies described in this report was to assess whether cultures of human keratinocytes could be used as an experimental model for investigating the mechanism by which epidermal homeostasis is established and maintained. The suprabasal layers were stripped from confluent keratinocyte cultures by incubation in low-calcium medium (0.1 mM calcium ions). After return to normal medium (2 mM calcium ions), the basal layer regenerated a stratified culture of approximately the same thickness as controls. The kinetics of proliferation and terminal differentiation were monitored by measuring the total number of cells and proportion of involucrin-positive cells at intervals before, during, and after stripping. During recovery the proportion of cells expressing involucrin, assessed by immunofluorescence microscopy and polyacrylamide gel electrophoresis, rapidly returned to control levels, but the total number of cells per dish rose more slowly and often failed to reach control values. Thus, terminal differentiation was initially stimulated at the expense of proliferation. Our in vitro model of epidermal regeneration should provide a useful complement to intact skin and animal models for analyzing epidermal homeostasis.

Cell Differentiation↗
PubMed1988

Prolonged expression of differentiated phenotype by chondrocytes cultured at low density on a composite substrate of collagen and agarose that restricts cell spreading.

The dedifferentiation of chondrocytes in culture is frequently associated with transition from a rounded to a spread morphology. A number of culture methods which prevent cell spreading have been described; however, all have disadvantages that limit their widespread use. In this paper we describe a new technique which allows prolonged cultivation of attached chondrocytes at low density while inhibiting spreading: the cells are grown on a composite substrate of agarose and collagen. By varying the ratio of agarose to collagen in the gel, the degree of spreading can be varied. The cultures are suitable for ultrastructural and immunofluorescence analysis and for studies of the synthesis and secretion of macromolecules. In order to determine whether the differentiated phenotype was maintained on composite gels, we compared the levels of messenger RNAs for cartilage-specific proteoglycan, link protein, alpha 1 (II) and alpha 1 (I) collagens in chondrocytes grown at low density on composite gels or at high or low density on tissue culture plastic for up to 21 days. The rate of decline in the level of mRNAs encoding the cartilage-specific products and the rate of increase in the level of alpha 1 (I) collagen mRNA were slower in the composite cultures than in the cultures on plastic. This culture technique may, therefore, prolong expression of the differentiated phenotype of chondrocytes relative to cultivation on plastic and will be useful for further studies on the role of cell shape in regulating differentiated gene expression.

Animals↗
PubMed1988

Epidermal stem cells in culture.

Cultures of human epidermal keratinocytes retain many of the characteristics of the tissue from which they are derived, and are therefore useful as an experimental model for studying stem cell properties. The cultures provide evidence that the mechanisms regulating keratinocyte proliferation are complex. The dividing cell compartment is heterogeneous, containing some cells with stem cell characteristics and others that may correspond to transit amplifying cells. The overall growth rate of the cultures is influenced by a wide range of growth stimulatory and inhibitory molecules, but it is not clear whether responsiveness to these factors differs between the different subpopulations of dividing cells. Furthermore, keratinocytes are able to express molecules that regulate their own growth. The cultures are able to achieve at least a partial homeostatic balance between proliferation and terminal differentiation and can regenerate the suprabasal cell layers following injury. Finally, a range of new experimental approaches hold promise for future studies of how keratinocyte proliferation and terminal differentiation are controlled.

Cell Differentiation↗
PubMed1988

Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture.

Articular chondrocytes are known to be phenotypically unstable in culture. One condition that has been reported to suppress dedifferentiation is cultivation at high density on tissue-culture plastic. The aim of the experiments described here was to study the effect of seeding density on chondrocyte proliferation and 35SO4 incorporation, and on the types of collagen and proteoglycan synthesized. I found that cells seeded at low or high density reached the same final density at confluence, and that 35SO4 incorporation, while initially higher (per cell) in high-density cultures, fell under both conditions, reaching the same low level after 3 weeks. The proportion of cells expressing keratan sulphate fell in low- but not high-density cultures and the decline was not prevented by inhibition of cell division. In all the cultures cells expressing keratan sulphate tended to have a rounded morphology. After 21 days in culture, chondrocytes grown at high density expressed predominantly large proteoglycans that aggregated with hyaluronic acid, whereas in low-density cultures a smaller, non-aggregating form was also present. By 21 days in culture cells at both high and low density were expressing type I collagen, although the high-density cells also had an extensive extracellular matrix of type II collagen. These observations support the conclusion that high seeding density stabilizes the chondrocyte phenotype to a greater extent than low seeding density. They also suggest that enhanced dedifferentiation at low density may be due to cell spreading, rather than to selective proliferation of a phenotypically unstable subpopulation of cells.

Animals↗
PubMed1988

Lack of keratan sulphate in the human notochord.

Intervertebral discs, formed from notochord cell expansions during embryogenesis, are known to contain proteoglycans bearing keratan sulphate chains. Keratan sulphate has previously been demonstrated in Xenopus and chick notochords and in human fetal cartilage. In contrast, we have been unable to demonstrate keratan sulphate in human fetal notochord using two monoclonal antibodies, MZ15 and 5-D-4. The contribution made by notochordal-derived keratan sulphate to the glycosaminoglycan content of the mature intervertebral disc may differ in man from that of other animal species.

Animals↗
PubMed1987

Patterns of junctional communication in animal tissues.

Gap junctions provide pathways for direct communication between cells in almost all animal tissues. The junctional channels are freely permeable to small ions and molecules but not to macromolecules. A coupled cell population is as a consequence a partial syncytium, within which metabolites, cofactors, small control molecules and inorganic ions can all diffuse freely through the combined cytoplasmic compartment, while intracellular macromolecules remain relatively fixed in space, in the cells where they are synthesized. Little is known about the extent and distribution of these communication compartments in tissues. To further understand their significance, we have mapped the patterns of junctional communication in skin by intracellular injection of the tracer dye Lucifer Yellow. Cells in the dermal layer are widely coupled, with dye spreading through hundreds of cells in a few minutes. Dye spread in the epidermis is much more restricted, passing in the same time into only a few cells. Dermal-epidermal coupling is not normally detected but is occasionally seen in new-born mouse skin when hair follicles are starting to invaginate. Dermal cells are coupled to a band of follicular cells near the base of mature hair follicles, whereas sebaceous glands appear to be coupled units, isolated from surrounding cells.

Animals↗
PubMed1987

Effect of growth environment on spatial expression of involucrin by human epidermal keratinocytes.

Involucrin, the major protein precursor of the cornified envelope, is expressed during terminal differentiation of human keratinocytes, both in vivo and in vitro. In epidermis, the onset of synthesis is several layers above the basal layer, but in stratified cultures of keratinocytes on tissue culture plastic involucrin synthesis begins in the first suprabasal layer. To investigate the reason for this premature expression, the distribution of involucrin was studied in epidermis from different body sites, in organotypic cultures and in transplants of keratinocytes onto nude mice. We found that premature expression was not associated with poor morphological differentiation, because involucrin synthesis began immediately above the basal layer even when distinct basal, spinous, granular and cornified layers were formed in organotypic cultures recombined with dermis. The site of involucrin expression in culture did not depend on the number of cornified layers present. The only conditions which resulted in an upward shift in the site of synthesis were in 3-week old transplants on nude mice. We conclude that the site of onset of involucrin synthesis is not determined by the degree of morphological differentiation of the tissue, and discuss other factors which may be involved.

Adult↗
PubMed1987

Calcium-induced changes in cytoskeleton and motility of cultured human keratinocytes.

In normal epidermis keratinocytes migrate upward from the basal layer as they undergo terminal differentiation, yet they also have the capacity for lateral movement during wound healing. The purpose of our experiments was to investigate these two types of movement by manipulating the calcium ion concentration of the medium so that keratinocytes formed monolayers (0.1 mM calcium) or stratified sheets (2.0 mM calcium). Time-lapse video recording indicated that keratinocytes in low-calcium medium were laterally more motile than keratinocytes in normal medium. This was consistent with the ultrastructural appearance of the cells and the lack of desmosomal junctions, determined by scanning and transmission electron microscopy. During calcium-induced stratification keratinocytes moved upward from the basal layer by gliding over their neighbors and forming contacts with other suprabasal cells. Keratinocytes in low-calcium medium migrated into wounds made in the cultures, a process which was inhibited by monensin; however, stratified keratinocytes in normal medium did not enter wounds. Cytochalasin D caused rapid cell rounding and disruption of actin filaments in keratinocytes grown in low-calcium but not in normal medium, indicating more rapid treadmilling of actin and consistent with the greater motility of keratinocytes in low-calcium medium. Our results suggest that desmosome formation may place constraints on the movement of individual keratinocytes and that the actomyosin cytoskeleton is involved in lateral migration.

Actins↗
PubMed1987

Patterns of junctional communication in skin: studies on cultured keratinocytes.

Junctional communication has long been suggested to play a role in coordinating the development of multicellular tissues. A better understanding of the patterns of communication between cells in such tissues is important for the identification of areas where this process may have a role. We have investigated the patterns of communication in cultures of human epidermal keratinocytes by iontophoretic injection of Lucifer Yellow CH, using involucrin expression as a marker of cells undergoing terminal differentiation. Cells that lack involucrin (i.e., the basal, proliferating cells) transfer dye preferentially to other involucrin-negative cells, whereas involucrin-positive cells either are not coupled or transfer dye with similar frequency to involucrin-positive and involucrin-negative neighbors. This decrease in communication associated with terminal differentiation was observed in both the presence and the absence of assembled desmosomes. Our observations lead us to speculate that loss of junctional communication may influence the commitment of basal keratinocytes to terminal differentiation.

Cell Communication↗
PubMed1987

Measurement of the rate of epidermal terminal differentiation: expression of involucrin by S-phase keratinocytes in culture and in psoriatic plaques.

At present little is known about the control mechanisms involved in coordinating cell production and maturation in epidermis. To investigate this, we have measured the rate of transit from the proliferative to the terminally differentiating compartment in confluent low-calcium cultures of normal epidermal keratinocytes, using involucrin as a marker of terminal differentiation. We estimate a rate of transit of 3.58 cells/5000 cells/h and a differentiation probability of 0.017, indicating a bias toward self-renewal. Surprisingly, some cells in culture synthesized DNA and expressed involucrin simultaneously. In psoriatic plaques, involucrin expression begins closer to the basal layer than in normal epidermis, and here too we found S-phase involucrin-positive cells. We also observed occasional mitotic involucrin-positive cells in psoriatic epidermis, although we were unable to detect them in culture. Our experiments show that temporal separation of proliferation and terminal differentiation is not obligatory, and thus, the kinetic organization of epidermis may be less rigid than some models imply.

Cell Differentiation↗