A study of cell interactions involved in Pleurodeles waltlii epidermal differentiation

Summary A polyclonal antibody (SP-2) has been produced, which recognizes antigens expressed in epidermal cells of Pleurodeles waltlii embryos. The antigens appear first at the end of gastrulation in the external surface of the embryo and are selectively expressed in ectodermally derived epidermal structures. Ectodermal commitment was investigated using cell cultures and blastocoel graft experiments. The four animal blastomeres of the 8-cell stage as well as the animal cap explants of the early gastrula stage cultured in vitro differentiate into epidermis, and SP-2 antigens are expressed. The expression of SP-2-defined antigens is inhibited both in vivo and in vitro by the inductive interaction of chordomesoderm. Once dissociated, ectodermal cells do not react with SP-2. Conversely, the aggregation of ectodermal cells may restore the expression of SP-2 antigens. Transplantation of animal cap explants or isolated ectodermal cells into the blastocoel of a host embryo at the early gastrula stage shows that only cells integrated into the epidermis express the marker antigens. When vegetal cells were dissociated from donor embryos before the mid-blastula stage and implanted into the blastocoel of host embryos at the early gastrula stage, their progeny were found in all germ layers, cells that were found in the host epidermis were stained with SP-2, whereas those contributing to mesoderm and endoderm were not. Thus the acquisition of cell polarity in epidermal differentiation and the organization of cells into epithelial structures are essential for SP-2-defined antigen expression.     

Mesoderm induction in Xenopus laevis: responding cells must be in contact for mesoderm formation but suppression of epidermal differentiation can occur in single cells

When Xenopus embryos are cultured in calcium- and magnesium-free medium (CMFM), the blastomeres lose adhesion but continue dividing to form a loose heap of cells. If divalent cations are restored at the early gastrula stage the cells re-adhere and eventually form muscle (a mesodermal cell type) as well as epidermis. If, however, the cells are dispersed during culture in CMFM, muscle does not form following reaggregation although epidermis does. This suggests that culturing blastomeres in a heap allows the transmission of mesoderm-induction signals from cell to cell while dispersion effectively dilutes the signal. In this paper, we have attempted to substitute for cell proximity by culturing dispersed blastomeres in XTC mesoderm-inducing factor (MIF). We find that dispersed cells do not respond to XTC-MIF by forming mesodermal cell types after reaggregation, but the factor does inhibit epidermal differentiation. One interpretation of this observation is that an early stage in mesoderm induction is the suppression of epidermal differentiation and that formation of mesoderm may require contact-mediated signals that are produced in response to XTC-MIF. We have gone on to study the suppression of epidermal differentiation in more detail. We find that this is a dose-dependent phenomenon that can occur in single cells in the absence of cell division. Animal pole blastomeres become more difficult to divert from epidermal differentiation at later stages of development and by stage 12 they are 'determined' to this fate. Fibroblast growth factor (FGF) also suppresses epidermal differentiation in isolated animal pole blastomeres and transforming growth factor-beta 1 acts synergistically with FGF in doing so.     

Glycogen Distribution in Relation to Epidermal Cell Differentiation During Embryonic Scale Morphogenesis in the Lizard Anolis lineatopus

The differentiation of the epidermis during scale morphogenesis in the lizard Anolis lineatopus has been studied by autoradiographic and immunocytochemical techniques and by electron microscopy, in relation to mitotic activity and to the distribution of glycogen. The flat embryonic epidermis of the early embryo is transformed into symmetric epidermal papillae which progressively become asymmetric and eventually form scales with stratified epidermal and peridermal layers. Papilla asymmetrization and epidermal stratification derive from cell hypertrophy and multiplication in the “basal hypertrophic layer of the forming outer side of scales” (BLOS). Glycogen is scarce or absent during early stages of epidermis development. In the dermis no glycogen is found at any stage of scale morphogenesis. Glycogen particles 25–40 nm in size accumulate in hypertrophic basal cells and peridermal cells during scale development. Conversely cells in the forming inner side of scales do not accumulate glycogen, divide less frequently than in the outer side and do not form a β–keratinized layer. It is suggested that an osmotic effect related to glycogen deposition causes increased hydration of the BLOS, whose cells become swollen and contribute to the asymmetrization of the epidermal papillae. Glycogen decreases in suprabasal differentiating cells and disappears from the BLOS at the stage of complete keratinization of the scale, around the period of hatching. Terminal differentiation in the peridermis and suprabasal epidermal layers takes place by cell flattening and condensation of the nucleus and cytoplasm as typical for apoptotic cells.     

Changes in keratin gene expression during terminal differentiation of the keratinocyte

Cells of the inner layers of the epidermis contain small keratins (46-58K), whereas the cells of the outer layers contain large keratins (63-67K) in addition to small ones. The changes in keratin composition that take place within each cell during the course of its terminal differentiation result largely from changes in synthesis. Cultured epidermal cells resemble cells of the inner layers of the epidermis in synthesizing only small keratins. The cultured cells possess translatable mRNA only for small keratins, whereas mRNA extracted from whole epidermis can be translated into both large and small keratins. As no synthesis takes place in the outermost layer of the epidermis (stratum corneum), the keratins of this layer must be synthesized earlier, but in some cases they then become smaller: this presumably occurs by post-translational processing of the molecules during the final stages of differentiation. Stratified squamous epithelia of internal organs do not form a typical stratum corneum and do not make the large keratins characteristic of epidermis. Their keratins are also different from those of cultured keratinocytes, implying that they have embarked on an alternate route of terminal keratin synthesis.     

Patterns of Cell Division,Cell Differentiation and Cell Elongation in Epidermis and Cortex of Arabidopsis pedicels in the Wild Type and in erecta

Plant organ shape and size are established during growth by a predictable, controlled sequence of cell proliferation, differentiation, and elongation. To understand the regulation and coordination of these processes, we studied the temporal behavior of epidermal and cortex cells in Arabidopsis pedicels and used computational modeling to analyze cell behavior in tissues. Pedicels offer multiple advantages for such a study, as their growth is determinate, mostly one dimensional, and epidermis differentiation is uniform along the proximodistal axis. Three developmental stages were distinguished during pedicel growth: a proliferative stage, a stomata differentiation stage, and a cell elongation stage. Throughout the first two stages pedicel growth is exponential, while during the final stage growth becomes linear and depends on flower fertilization. During the first stage, the average cell cycle duration in the cortex and during symmetric divisions of epidermal cells was constant and cells divided at a fairly specific size. We also examined the mutant of ERECTA, a gene with strong influence on pedicel growth. We demonstrate that during the first two stages of pedicel development ERECTA is important for the rate of cell growth along the proximodistal axis and for cell cycle duration in epidermis and cortex. The second function of ERECTA is to prolong the proliferative phase and inhibit premature cell differentiation in the epidermis. Comparison of epidermis development in the wild type and erecta suggests that differentiation is a synchronized event in which the stomata differentiation and the transition of pavement cells from proliferation to expansion are intimately connected.     

EFFECT OF ACTINOMYCIN D ON THE DIFFERENTIATION OF HATCHING GLAND CELL AND CILIA CELL IN THE FROG EMBRYO

The forehead epidermis of the stage 18–20 R. japonica embryo includes the hatching gland cell (HGC) which contains cell-specific secretory granules. The cilia cell (CC) and common epidermal cell (CEC) constitute the epidermis of the entire body surface, in addition to the forehead region.
Culture of superficial epidermal explants from various embryonic portions at various developmental stages revealed that HGCs are derived from cells localized on the neural crest in the stage 13a (early neural plate) embryo. When explants from the presumptive HGC area were treated with 1 ug/ml actinomycin D (AMD), the formation of secretory granules in HGCs was inhibited either by continuous treatment from stage 13 or by an 8-hr treatment at stage 13b. Similarly, the ciliogenesis in CCs was inhibited. The differentiation of CECs was entirely unaffected by any of the AMD treatment. After release from AMD, mucous vesicles, characteristic of the CEC, were formed in cells whose differentiation into HGC and CC had been suppressed by the antibiotic. Thread complexes and clumps of coiled strings were found in the nuclei of AMD-affected cells.
It is concluded that the DNA-dependent RNA syntheses which direct secretory granule formation in the HGC and ciliogenesis in the CC occur during a limited period at stage 13b, viz. , 20 hr before their cytodifferentiation becomes appreciable.     

Two ways to skin a plant: The analysis of root and shoot epidermal development in Arabidopsis

The post-embryonic architecture of higher plants is derived from the activity of two meristems that are formed in the embryo: the shoot meristem and the root meristem. The epidermis of the shoot is derived from the outermost layer of cells covering the shoot meristem through repeated anticlinal divisions. By contrast, the epidermis of the root is derived from an internal ring of cells, located at the centre of the root meristem, by a precise series of both periclinal and anticlinal divisions. Each epidermis has an independent origin. In Arabidopsis the mature shoot epidermis is composed of a small number of cell types: hair cells (trichomes), stomatal guard cells and other epidermal cells. In shoots, hairs take the form of branched trichomes that are surrounded at their base by a ring of accessory cells in a sheet of epidermal cells. The root epidermis is composed of two cell types: trichoblasts that form root hair cells and atrichoblasts that form non-hair cells. Mutations affecting both the patterning and the morphogenesis of cells in both shoot and root epidermis have recently been described. Most of these mutations affect development in a single epidermis, but at least one, ttg, is involved in development in both epidermal systems.     

INDUCTION OF THE FROG HATCHING GLAND CELL FROM EXPLANTED PRESUMPTIVE ECTODERMAL TISSUE BY LICL

When cells of the superficial layer explanted from the presumptive ectoderm of a Rana japonica early gastrula embryo at stage 10 were cultured in standard salt solution for 4–7 days, they differentiated into cement gland cells (CGCs), cilia cells (CCs) and common epidermal cells (CECs). When, however, these explants were treated with LiCl and transferred to Barth's solution, hatching gland cells (HGCs) and pigment cells were induced.
The optimum condition for inducing differentiation of HGC was treatment with 70 mM LiCl for 6–8 hr at 18°C. The best ability to react to the HGC-inducing stimuli resided in the superficial layer of the dorsal presumptive epidermis of the embryo at stage 10. Upon repeated stimulation, explants from stage 8 embryos underwent differentiation into nerve and pigment cells, whereas those from stage 11 embryos differentiated into CCs and CECs. Under optimum conditions, the total volume of HGCs induced amounted to about 70% of the explanted tissue. The culture media from LiCl-induced HGCs showed an apparent jelly-digesting activity, strongly indicating that the cells were functionally identical with those differentiated in situ .     

Coordination of mitosis and morphogenesis: role of a prolonged G2 phase during chordate neurulation

Chordates undergo a characteristic morphogenetic process during neurulation to form a dorsal hollow neural tube. Neurulation begins with the formation of the neural plate and ends when the left epidermis and right epidermis overlying the neural tube fuse to close the neural fold. During these processes, mitosis and the various morphogenetic movements need to be coordinated. In this study, we investigated the epidermal cell cycle in Ciona intestinalis embryos in vivo using a fluorescent ubiquitination-based cell cycle indicator (Fucci). Epidermal cells of Ciona undergo 11 divisions as the embryos progress from fertilization to the tadpole larval stage. We detected a long G2 phase between the tenth and eleventh cell divisions, during which fusion of the left and right epidermis occurred. Characteristic cell shape change and actin filament regulation were observed during the G2 phase. CDC25 is probably a key regulator of the cell cycle progression of epidermal cells. Artificially shortening this G2 phase by overexpressing CDC25 caused precocious cell division before or during neural tube closure, thereby disrupting the characteristic morphogenetic movement. Delaying the precocious cell division by prolonging the S phase with aphidicolin ameliorated the effects of CDC25. These results suggest that the long interphase during the eleventh epidermal cell cycle is required for neurulation.     

Tornado1 and tornado2 are required for the specification of radial and circumferential pattern in the Arabidopsis root

The cell layers of the Arabidopsis primary root are arranged in a simple radial pattern. The outermost layer is the lateral root cap and lies outside the epidermis that surrounds the ground tissue. The files of epidermal and lateral root cap cells converge on a ring of initials (lateral root cap/epidermis initial) from which the epidermal and lateral root cap tissues of the seedling are derived, once root growth is initiated after germination. Each initial gives rise to a clone of epidermal cells and a clone of lateral root cap cells. These initial divisions in the epidermal/lateral root cap initial are defective in tornado1 (trn1) and trn2 plants indicating a requirement for TRN1 and TRN2 for initial cell function. Furthermore, lateral root cap cells develop in the epidermal position in trn1 and trn2 roots indicating that TRN1 and TRN2 are required for the maintenance of the radial pattern of cell specification in the root. The death of these ectopic lateral root cap cells in the elongation zone (where lateral root cap cells normally die) results in the development of gaps in the epidermis. These observations indicate that TRN1 and TRN2 are required to maintain the distinction between the lateral root cap and epidermis and suggest that lateral root cap fate is the default state. It also suggests that TRN1 and TRN2 repress lateral root cap fate in cells in the epidermal location. Furthermore, the position-dependent pattern of root hair and non-root hair cell differentiation in the epidermis is defective in trn1 and trn2 mutants. Together these results indicate that TRN1 and TRN2 are required for the maintenance of both the radial pattern of tissue differentiation in the root and for the subsequent circumferential pattern within the epidermis.     

The embryonic development of the temnocephalid flatworms Craspedella pedum and Diceratocephala boschmai

We have analyzed the embryonic development of the temnocephalid flatworms Craspedella pedum and Diceratocephala boschmai, using a combination of fuchsin-labeled whole-mount preparation, histology, and transmission electron microscopy. Following the staging system recently introduced for another flatworm species (Mesostoma lingua), we can distinguish eight morphologically defined stages. Temnocephalids produce eggs of the neoophoran type in which a small oocyte is surrounded by a layer of yolk cells. Cleavage takes place in the center of the yolk mass (stages 1-2) and results in an irregular, multilayered disc of mesenchymal cells that moves to the future ventral egg pole (stage 3). Organ primordia, including those of the brain, pharynx, male genital apparatus, sucker, and epidermis "crystallize" within this disc without undergoing gastrulation movements (stage 4). An invagination of the epidermal primordium pushes the embryo back into the center of the yolk ("embryonic invagination"). As a result, organogenesis begins while the embryo is invaginated (stage 5). The brain differentiates into an outer cortex of cell bodies that surround a central neuropile. Precursor cells of the epidermis, pharynx, and protonephridia become organized into epithelia. During stage 6, the embryonic primordium everts back to the surface, where organogenesis and cell differentiation continues. Epidermal cells fuse into a syncytium that expands around the yolk. Myoblasts initially do not spread out in the way epidermal cells do; they remain concentrated in two narrow, longitudinal bands that extend along the sides of the embryo. Three pairs of axon tracts extending posteriorly from the brain follow the bands of myoblasts. Stages 7 and 8 are characterized by the appearance of eye pigmentation, brain condensation, and the formation of tentacles and a sucker that bud out from the epidermis of the anterior and posterior end, respectively. Comparison of morphogenesis in temnocephalids with observations in other flatworm taxa suggests a phylotypic stage for this phylum of invertebrates.     

Emigration of bilayered epidermal cell sheets from tadpole tails (Xenopus laevis)

Summary Migration of bilayered epidermal cell sheets out of explants of tadpole tails (Xenopus laevis) were investigated with time-lapse cinemicrography using reflection-contrast optics. Cell-sheet formation begins beneath the explant in a region where it is closely attached to the coverslip. A single basal cell extends a lamellipodium through the outer (surface) epidermal layer and starts moving in a direction free of attached cells. This cell remains connected to the following basal cell, which the also extends a lamellipodium onto the glass. The cell sheet develops as increasingly more adjacent basal cells start to migrate. Surface cells do not actively locomote but they remain attached to the basal cells and to adjacent surface cells. Thus, they are transported as an intact cell layer, and consequently the in situ arrangement of the tadpole epidermis is largely preserved in the cell sheet, i.e., basal cells adhere to the substratum and are covered by outer cells (surface cells) which face the culture medium. Basal cells extend lamellae beneath the rear end of the preceding cell, which is slightly fifted off the substratum. The direction of locomotion is determined by the frontal cells. Cell-sheet enlargement and locomotion cease when all the epidermal cells facing the coverslip have left the explant, and the cell sheet and epidermis covering the explant form a continuous layer.     

Temporal and spatial sequence expression of cytokeratin K19 in cultured human keratinocyte

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.     

Reconstitution of the epidermal basement membrane after enzymatic dermal-epidermal separation of embryonic mouse skin

Pieces of trypsin-isolated 14-day embryonic mouse epidermis were recombined with various living or non-living dermal or non-dermal substrates, in order to analyse the reconstruction of the dermal-epidermal junction. The constitution and ultrastructure of the epidermal basement membrane were characterized by immunolabelling of laminin, type IV collagen and bullous pemphigoid antigen, and by transmission electron microscopy. Trypsin treatment of dorsal skin followed by dermal-epidermal separation does not visibly damage the epidermal basement membrane, which remains attached to the lower face of epidermis. When freshly isolated epidermis is reassociated with dermis, the basement membrane is first degraded during the first 4 h of culture, then reconstituted within 24 h. When epidermis is cultured in isolation the basement membrane disappears within 4 h and is not reconstructed. Epidermis, precultured for 4 h and thus deprived of its basement membrane prior to reassociation, is able to reconstruct an antigenically and ultrastructurally normal basement membrane, when recombined with living or frozen-killed (-20 degrees C) dermis, with muscle tissue, or with a film of fibrous type I collagen. No basement membrane is reconstituted when the epidermis is recombined with heat (100 degrees C) killed dermis. It is concluded that, in the reconstituted epidermal basement membrane, laminin, type IV collagen, bullous pemphigoid antigen, and lamina densa are of exclusive epidermal origin.     

Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters

BACKGROUND: Human epidermis is renewed throughout life from stem cells in the basal layer of the epidermis. Signals from the surrounding keratinocytes influence the differentiation of the stem cells, but the nature of the signals is unknown. In many developing tissues, signalling mediated by the transmembrane protein Delta1 and its receptor Notch1 inhibits differentiation. Here, we investigated the role of Delta-Notch signalling in postnatal human epidermis. RESULTS: Notch1 expression was found in all living epidermal layers, but Delta1 expression was confined to the basal layer of the epidermis, with highest expression in those regions where stem cells reside. By overexpressing Delta1 or Delta(T), a truncated form of Delta1, in primary human keratinocytes and reconstituting epidermal sheets containing mixtures of Delta-overexpressing cells and wild-type cells, we found that cells expressing high levels of Delta1 or Delta(T) failed to respond to Delta signals from their neighbours. In contrast, wild-type keratinocytes that were in contact with neighbouring cells expressing Delta1 were stimulated to leave the stem-cell compartment and initiate terminal differentiation after a few rounds of division. Delta1 promoted keratinocyte cohesiveness, whereas Delta(T) did not. CONCLUSIONS: We propose that high Delta1 expression by epidermal stem cells has three effects: a protective effect on stem cells by blocking Notch signalling; enhanced cohesiveness of stem-cell clusters, which may discourage intermingling with neighbouring cells; and signalling to cells at the edges of the clusters to differentiate. Notch signalling in epidermal stem cells thus differs from other progenitor cell populations in promoting, rather than suppressing, differentiation.     

Differentiation of the epidermis of scutes in embryos and juveniles of the tortoise Testudo hermanni with emphasis on beta-keratinization

The sequence of differentiation of the epidermis of scutes during embryogenesis in the tortoise Testudo hermanni was studied using autoradiography, electron microscopy and immunocytochemistry. The study was mainly conducted on the epidermis of the carapace, plastron and nail. Epidermal differentiation resembles that described for other reptiles, and the embryonic epidermis is composed of numerous cell layers. In the early stages of differentiation of the carapacial ridge, cytoplasmic blebs of epidermal cells are in direct contact with the extracellular matrix and mesenchymal cells. The influence of the dermis on the formation of the beta‐layer is discussed. The dermis becomes rich in collagen bundles at later stages of development. The embryonic epidermis is formed by a flat periderm and four to six layers of subperidermal cells, storing 40–70‐nm‐thick coarse filaments that may represent interkeratin or matrix material. Beta‐keratin is associated with the coarse filaments, suggesting that the protein may be polymerized on their surface. The presence of beta‐keratin in embryonic epidermis suggests that this keratin might have been produced at the beginning of chelonian evolution. The embryonic epidermis of the scutes is lost around hatching and leaves underneath the definitive corneous beta‐layer. Beneath the embryonic epidermis, cells that accumulate typical large bundles of beta‐keratin appear at stage 23 and at hatching a compact beta‐layer is present. The differentiation of these cells shows the progressive replacement of alpha‐keratin bundles with bundles immunolabelled for beta‐keratin. The nucleus is degraded and electron‐dense nuclear material mixes with beta‐keratin. In general, changes in tortoise skin when approaching terrestrial life resemble those of other reptiles. Lepidosaurian reptiles form an embryonic shedding layer and crocodilians have a thin embryonic epidermis that is rapidly lost near hacthing. Chelonians have a thicker embryonic epidermis that accumulates beta‐keratin, a protein later used to make a thick corneous layer.     

Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis

Human interfollicular epidermis is renewed by stem cells that are clustered in the basal layer in a patterned, non-random distribution. Stem cells can be distinguished from other keratinocytes by high expression of beta1 integrins and lack of expression of terminal differentiation markers; they divide infrequently in vivo but form actively growing colonies in culture. In a search for additional stem cell markers, we observed heterogeneous epidermal expression of melanoma chondroitin sulphate proteoglycan (MCSP). MCSP was expressed by those keratinocytes with the highest beta1 integrin levels. In interfollicular epidermis, expression was confined to non-cycling cells and, in culture, to self-renewing clones. However, fluorescence-activated cell sorting on the basis of MCSP and beta1 integrin expression gave no more enrichment for clonogenic keratinocytes than sorting for beta1 integrins alone. To interfere with endogenous MCSP, we retrovirally infected keratinocytes with a chimera of the CD8 extracellular domain and the MCSP cytoplasmic domain. CD8/MCSP did not affect keratinocyte proliferation or differentiation but the cohesiveness of keratinocytes in isolated clones or reconstituted epidermal sheets was greatly reduced. CD8/MCSP caused stem cell progeny to scatter without differentiating. CD8/MCSP did not alter keratinocyte motility but disturbed cadherin-mediated cell-cell adhesion and the cortical actin cytoskeleton, effects that could be mimicked by inhibiting Rho. We conclude that MCSP is a novel marker for epidermal stem cells that contributes to their patterned distribution by promoting stem cell clustering.     

Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia

Different stratified squamous epithelia, whether they bear a stratum corneum or not, are shown by immunofluorescence to possess the precursor protein of the cross-linked envelope that is characteristic of epidermal s. corneum. This protein, involucrin, is not present in the deepest epithelial cells but appears in the course of their outward migration. The boundary at which involucrin first appears can sometimes by correlated with a visible boundary between zones of large and small cells. Cultured keratinocytes, derived from all stratified squamous epithelia (epidermal, corneal, conjuctival, esophageal, lingual, and vaginal), form colonies that grow together to form a stratified epithelium. The cells of the basal layer are nearly always free of detectable involucrin, but, in contrast to the natural epithelium, this protein usually makes its appearance in the cells immediately above the basal layer. When a cultured epithelium derived from epidermal keratinocytes is detached and applied as a graft to animals, the cells flatten and the distinctness of the basal layer is at first reduced; but with time the organization of the epithelium becomes more characteristic of epidermis. Cell size and shape become more orderly along the cell migration pathway, and involucrin first appears at some distance from the basal layer, instead of in immediately suprabasal cells, as in the cultured epithelium. The progeny of dissociated and cultured keratinocytes are therefore able, when grafted, to reassemble an epidermis in which the timing of specific gene expression is restored to that of the original tissue.