Base Columnar Cells Research Articles

UNICYSTIC VARIANT OF ADENOID AMELOBLASTOMA WITH DENTINOID

A 34-year-old male patient presented with asymptomatic swelling in the left posterior maxilla. The intraoral exam showed an expansive lesion, soft to palpation, covered by a normal-colored mucosa. Computed tomography scans revealed a well-defined unilocular hypodense tumor involving the left maxillary sinus. A plentiful yellowish fluid was collected by aspiration puncture. The lesion was enucleated, and the histopathologic examination showed a cystic tumor lined by an ameloblastic epithelium containing columnar basal cells with hyperchromatic and polarized nuclei. In some capsule areas, the tumor showed a mural infiltration by sheets of cells containing central whirling structures. Dentinoid material in association with ameloblastic-like cells was also observed. Collectively, these findings were consistent with the diagnosis of a cystic variant of adenoid ameloblastoma with dentinoid. There was no sign of recurrence after 3 years of follow-up. A 34-year-old male patient presented with asymptomatic swelling in the left posterior maxilla. The intraoral exam showed an expansive lesion, soft to palpation, covered by a normal-colored mucosa. Computed tomography scans revealed a well-defined unilocular hypodense tumor involving the left maxillary sinus. A plentiful yellowish fluid was collected by aspiration puncture. The lesion was enucleated, and the histopathologic examination showed a cystic tumor lined by an ameloblastic epithelium containing columnar basal cells with hyperchromatic and polarized nuclei. In some capsule areas, the tumor showed a mural infiltration by sheets of cells containing central whirling structures. Dentinoid material in association with ameloblastic-like cells was also observed. Collectively, these findings were consistent with the diagnosis of a cystic variant of adenoid ameloblastoma with dentinoid. There was no sign of recurrence after 3 years of follow-up.

"Basal Cell Migration" in Regeneration of the Corneal Wound-Bed.

In this issue of Stem Cell Reports, Park et al. (2019) describe real-time in vivo visual monitoring of keratin-14+, Confetti-labeled limbal epithelial stem cells and their progeny as they contribute to central corneal wound-healing. The authors show that corneal wounds initially heal by “basal epithelial cell migration” into the wound-bed.

Abstract 5022: Keratin15 (Krt15) + are radio resistant and tumor-initiating cells in the mouse small intestine

Abstract Background: Intestinal stem cells regulate the emergence of different cell lineages across the crypt-villus axis and orchestrate cellular renewal and replenishment following injury (e.g. radiation). Two major ISC have been identified to date: a rapidly cycling crypt base columnar (CBC) stem cell and a more quiescent reserve stem cell located at the +4 position above the crypt base. CBCs marked by the Wnt target gene Lgr5 are dispensable for homeostasis and are quantitatively ablated by radiation injury. In contrast, quiescent ISCs are indispensable for homeostasis, regenerate CBCs, and are radioresistant. Keratins (Krt) are intermediate filaments required for architectural and mechanical support, but also contribute to cell migration and intracellular signaling. Interestingly, Krt15 has been characterized as a stem cell marker in the hair follicle. Here we described a radio-resistant Krt15+ stem cell population in the small intestine and demonstrate that cells within this population are capable of tumor initiation. Methods: Krt15-CrePR1 mice were crossed with Rosa26 reporter mice to perform lineage-tracing experiments in the mouse small intestine. High-dose radiation was used to investigate the role of Krt15 in tissue regeneration. Finally, Krt15-CrePR1 mice were crossed with Apcfl/fl mice to characterize the tumor-initiating properties of Krt15+ cells. Results: We observed that Krt15 marks a subpopulation of long-lived small intestinal crypt cells that are able to give rise to all differentiated lineages supporting their self-renewing and multipotent capacities. Interestingly, we observed that Krt15+ cells are able to form 3D organoids when seeded in Matrigel as a single cell suspension. Irradiation of Krt15-CrePR1;R26mTomato/mGFP mice with 12 Gy revealed that Krt15+ cells are radio-resistant and participate in tissue regeneration. Interestingly, Krt15 deficiency impairs tissue regeneration following high-dose radiation. Indeed, irradiated Krt15-/- mice display reduced crypt regeneration, crypt length, microcolonies and BrdU incorporation when compared to irradiated Krt15+/+ mice. Finally, we induced Apc loss in Krt15+ cells and observed formation of adenomas as well as adenocarcinomas in all Krt15-CrePR1;Apcfl/fl mice. Metastases were detected in some Krt15-CrePR1;Apcfl/fl mice. Conclusion: We have identified a long-lived Krt15+ subpopulation of intestinal crypt cells displaying stem cell-like properties that are radio-resistant and tumor initiating. This new stem cell population may open new avenues for therapeutic targeting in colon cancer. Citation Format: Veronique Giroux, Julien Stephan, Kathryn E. Hamilton, Priya Chatterjee, Sarah Andres, Andres J. Klein-Szanto, Christopher J. Lengner, Anil K. Rustgi. Keratin15 (Krt15) + are radio resistant and tumor-initiating cells in the mouse small intestine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5022. doi:10.1158/1538-7445.AM2017-5022

Role of Krüppel-like factor 5 in the maintenance of the stem cell niche in the intestinal crypt

The intestinal epithelium is a tissue that undergoes continuous self-renewal initiated at the bottom of the crypts, which harbor the intestinal stem cell (ISC) pool. The ISC pool is sub-divided into crypt base columnar (CBC) cells at the crypt bottom and label retention cells (LRC) at position +4 from the crypt bottom. CBC cells are marked by Leucine-rich repeat-containing G-protein coupled receptor (Lgr5) while LRC cells are identified by several markers including Bmi1, mTert, Hopx, Lrig1, and Sox9. Krüppel-like factors (KLFs) belong to a family of transcription factors that exert important physiological function in various tissues. In the intestine, KLF4 is predominantly expressed in the terminally differentiated, non-proliferating cells lining the villus. Its deletion in the adult mouse intestine results in perturbed homeostasis. In contrast, KLF5 is expressed in actively proliferating cells of the intestinal crypt, including CBC cells and transit amplifying (TA) cells. We recently investigated the effect of Klf5 deletion specifically from the Lgr5-expressing CBC cells in adult mouse intestine using an inducible Cre recombinase system. Shortly (3-5 days) after Cre induction, proliferation of both CBC and TA cells ceased, which was accompanied by an increase in apoptosis in the crypt. Beginning at two weeks following Cre induction, both Klf5 expression and proliferation re-appeared but without the re-emergence of Lgr5-positive CBC cells, which were eventually depleted by four months following induction. These findings indicate that KLF5 plays an important role in regulating proliferation and survival of CBC stem cells in the intestine.

Keratocyst of the buccal mucosa: case report and immunohistochemical comparative study with sporadic intraosseous keratocystic odontogenic tumor

Describe a new case of keratocyst of the buccal mucosa and compare its immunohistochemical features with 13 sporadic intraosseous keratocystic odontogenic tumors (KOT). A male complaining about an enlargement on the left buccal mucosa was referred to the Stomatology Clinic. Clinical examination revealed a solitary nodule posterior to the parotid papilla. An excisional biopsy was performed following clinical diagnosis of epidermoid cyst. Microscopically, the lesion was characterized by a lining of five cell layers, with columnar basal cells and a corrugated parakeratinized surface. Immunohistochemical reactions for PTCH-1, Smo, Shh, mTOR, bcl-2, Ck17, and Ck19 were performed. PTCH-1 was not expressed in the keratocyst of the buccal mucosa, but was observed in suprabasal layers of eight (61.5%) cases of sporadic intraosseous KOT. Shh, mTOR, bcl-2, Ck17, and Ck19 expression was observed in all the cases investigated. The morphology and immunoprofile of this lesion are similar to sporadic intraosseous KOT.

Expression of semaphorin 3A in the rat corneal epithelium during wound healing

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1day after debridement, but it had largely returned to the normal pattern at 3days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of Sema3A is increased markedly in basal cells of the newly healed corneal epithelium, and that this up-regulation of Sema3A is not associated with cell proliferation. They further suggest that Sema3A might play a role in the regulation of corneal epithelial wound healing.

New skin-equivalent model from de-epithelialized amnion membrane

The presence of pre-existing basement membrane (BM) components improves the morphogenesis of epidermis and BM in constructing a human living skin-equivalent (LSE). De-epithelialized amniotic membrane (AM) retains key BM components. We have therefore investigated the usefulness of AM for constructing LSE. De-epithelialized AM was overlaid on type I collagen gel embedded with fibroblasts. Normal human keratinocytes (NHKs) were then seeded onto the epithelial side of the AM to construct an AM-LSE. A conventional LSE was constructed by seeding NHKs on a fibroblast-populated type I collagen gel. When the keratinocytes reached confluence, the LSE was lifted to the air-liquid interface and cultured for up to 3 weeks. Samples were harvested at various times and investigated morphologically, immunohistochemically, and ultrastructurally. In AM-LSE, the epidermis was better stratified, with more compact, polarized, columnar basal cells, and the expression of differentiation and proliferation markers was more similar to that of normal human skin than was that of LSE without AM. A more continuous BM and better-developed hemidesmosomes were found in AM-LSE. The epidermis of AM-LSE outgrew much faster than that of LSE without AM. When transplanted onto nude mice, both LSEs took well; however, the AM-LSE graft showed better morphogenesis of the epidermis, BM, and hemidesmosomes. The better epidermal morphology and better-developed BM in AM-LSE in vitro and in vivo indicates its superiority over LSE without AM for clinical applications.

Expression of p73 in normal skin and proliferative skin lesions

The p73 gene is a member of the p53 gene family and the structure and functions of p73 protein are similar to those of p53. However, these two proteins have different roles. In the present study, p73 protein was found immunohistochemically to be distributed in the basal cells of the epidermis, columnar basal cells in the hair follicle and peripheral cells without lipid droplets in the sebaceous and meibomian glands; it was expressed strongly in tumor cells in basal cell carcinomas and in the basal cell-like cells in seborrheic keratosis, and weakly or negatively in the squamous cell-like cells in seborrheic keratosis and in the tumor cells in squamous cell carcinomas. No relationship was detected between p73 and p53 protein distribution and between p73 protein expression and the proliferative potential, as shown by the Ki-67 immunopositive cell ratio. The present study shows that p73 protein is likely to play important roles in skin differentiation rather than proliferation or carcinogenesis of the skin.

Characteristics of calcifying odontogenic cyst in Taiwanese.

Calcifying odontogenic cyst (COC) is a rare type of odontogenic cyst. This retrospective study analyzed the clinical, radiographic, and histopathologic features of COC in Taiwanese. Ten cases of COC in 2 male and 8 female patients with a mean age of 29 years (range, 11 to 48 years) treated from January 1985 to December 2002 were included. Microscopic slides, clinical histories, and radiographic features of these 10 COC cases were reviewed and analyzed. COCs occurred in the maxilla in 3 cases and in the mandible in 7 cases. COCs were associated with impacted teeth in 6 cases and with odontomas in 3 cases. All COCs appeared as either unilocular (9 cases) or multilocular (1 case) radiolucencies. In 7 cases, spotty radiopaque materials were scattered throughout the radiolucency. Histologically, all of the lesions were at least partially lined by epithelium with cuboidal to columnar basal cells and stellate reticulum-like suprabasal cells. Variable numbers of ghost cells, some of which were calcified, were observed in the lining epithelium or in the fibrous connective tissue wall of all 10 cases. Juxta-epithelial dentinoid was also found in all cases. However, proliferation of ameloblastoma-like tumor nests was observed in only 1 case. Based on the above histologic findings, 6 COC lesions were classified as simple unicystic type, 3 as unicystic odontoma-producing type, and 1 as unicystic ameloblastomatous proliferating type. COC occurs frequently in the second and third decades and is commonly associated with an impacted tooth or an odontoma. It usually appears as a mixed radiolucent and radiopaque lesion radiographically. Simple unicystic type is the most common type of COC. No recurrences were found after conservative surgical removal in this series.

Ultrastructure of the basal lamina of bovine ovarian follicles and its relationship to the membrana granulosa

Different morphological phenotypes of follicular basal lamina and of membrana granulosa have been observed. Ten preantral follicles (< 0. 1 mm), and 17 healthy and six atretic antral follicles (0.5-12 mm in diameter) were processed for light and electron microscopy to investigate the relationship the between follicular basal lamina and membrana granulosa. Within each antral follicle, the shape of the basal cells of the membrana granulosa was uniform, and either rounded or columnar. There were equal proportions of follicles </= 4 mm in diameter with columnar basal cells and with rounded basal cells. Larger follicles had only rounded basal cells. Conventional basal laminae of a single layer adjacent to the basal granulosa cells were observed in healthy follicles at the preantral and antral stages. However, at the preantral stage, the conventional types of basal lamina were enlarged or even partially laminated. A second type of basal lamina, described as 'loopy', occurred in about half the preantral follicles and in half the antral follicles </= 4 mm diameter. 'Loopy' basal laminae were not observed in larger follicles. 'Loopy' basal laminae were composed of basal laminae aligning the basal surface of basal granulosa cells, but with additional layers or loops often branching from the innermost layer. Each loop was usually < 1 microm long and had vesicles (20-30 nm) attached to the inner aspect. Basal cellular processes were also common, and vesicles could be seen budding off from these processes. In antral follicles, conventional basal laminae occurred in follicles with rounded basal granulosa cells. Other follicles with columnar cells, and atretic follicles, had the 'loopy' basal lamina phenotype. Thus, follicles have different basal laminae that relate to the morphology of the membrana granulosa.

Development of the membrana granulosa of bovine antral follicles: structure, location of mitosis and pyknosis, and immunolocalization of involucrin and vimentin.

The membrana granulosa of the ovarian follicle is termed the 'follicular epithelium', yet there have been no studies considering its epithelial nature and how it changes during follicular development. Therefore, these issues were investigated using histology (n = 45 ovaries), considering its structure and the location of proliferating and dying cells, and drawing analogies with other epithelia. Additionally, differences between the layers of granulosa cells were demonstrated by immunohistochemistry (n = 7 ovaries). The structure of the membrana granulosa differed between follicles. Six arbitrary classifications were designed based on these structures, 80 follicles were allocated (n = 13 ovaries) to these classes and the follicular diameters were then measured. For the first time, differences in membrana granulosa structure were shown to correspond to follicle size. Follicles in classes 1-3, where basal granulosa cells were columnar with nuclei positioned basally in the cell, were all < or = 3 mm in diameter. All follicles larger than 3 mm had either columnar basal cells with nuclei positioned centrally (class 4), or had rounded basal cells (class 5), and all follicles > 5 mm had only rounded basal cells. In all these classes, cells in the middle zone were rounded; cells aligning the antrum were often flattened. Irrespective of follicle class, cell proliferation and cell death were shown to be predominantly in the middle portions, rather than the most antral or most basal portions, of the membrana granulosa of healthy and atretic follicles. Involucrin, a marker of keratinocyte differentiation, was localized to the suprabasal region of the membrana granulosa of healthy follicles, particularly in the second and third cellular layers in from the follicular basal lamina. Conversely, the staining intensity for the intermediate filament protein vimentin was lowest in this region, and greatest in the more antral and basal regions. In atretic follicles, there was widespread staining for involucrin and vimentin throughout the membrana granulosa. In conclusion, the membrana granulosa is highly structured, and alters with follicular development. Layers in the membrana granulosa can differ in terms of cell shape, and differ in proliferation and gene expression. In the light of the current work, and an associated study, it is proposed that proliferation occurs in the middle layers, and that granulosa cells, then progress basally or antrally, the latter undergoing terminal differentiation.

Comparative ultrastructure of the cellular components of the unconstricted notochord in the sturgeon and the lungfish.

Unconstricted notochords are found in only a few extant vertebrates. Within the bony fishes the notochord is present throughout life in the sturgeons and the lungfishes. To better understand the function of this organ, the cellular ultrastructure of the notochord was examined in the shortnose sturgeon, Acipenser brevirostratus, and in the African lungfish, Protopterus annectens. In both species, the medulla of the notochord consists of vacuolated cells connected by desmosomes. The vacuoles are surrounded by a dense cytoplasmic network of intermediate filaments. In Acipenser, the cuboidal to columnar basal cells are avacuolate and contain an extensive network of rough endoplasmic reticulum. In Protopterus, the cytoplasm of the basal cells lacks rough endoplasmic reticulum and often contains a large central vacuole surrounded by a network of intermediate filaments. Cells resembling fibroblasts are found in the fibrous sheath of the notochord of the lungfish, but the fibrous sheath of the sturgeon is acellular. On the basis of the structures observed in the two species, the medulla of the notochord has many of the characteristics of a keratinized epithelium, and the fibrous sheath may be derived from different cellular sources. J. Morphol. 236:75-104, 1998. © 1998 Wiley-Liss, Inc.

Comparative ultrastructure of the cellular components of the unconstricted notochord in the sturgeon and the lungfish

Unconstricted notochords are found in only a few extant vertebrates. Within the bony fishes the notochord is present throughout life in the sturgeons and the lungfishes. To better understand the function of this organ, the cellular ultrastructure of the notochord was examined in the shortnose sturgeon, Acipenser brevirostratus, and in the African lungfish, Protopterus annectens. In both species, the medulla of the notochord consists of vacuolated cells connected by desmosomes. The vacuoles are surrounded by a dense cytoplasmic network of intermediate filaments. In Acipenser, the cuboidal to columnar basal cells are avacuolate and contain an extensive network of rough endoplasmic reticulum. In Protopterus, the cytoplasm of the basal cells lacks rough endoplasmic reticulum and often contains a large central vacuole surrounded by a network of intermediate filaments. Cells resembling fibroblasts are found in the fibrous sheath of the notochord of the lungfish, but the fibrous sheath of the sturgeon is acellular. On the basis of the structures observed in the two species, the medulla of the notochord has many of the characteristics of a keratinized epithelium, and the fibrous sheath may be derived from different cellular sources. J. Morphol. 236:75–104, 1998. © 1998 Wiley-Liss, Inc.

The decay of autoradiographic grain number over crypt base columnar cells in murine ileum as a measure of their generation time

The decay in the number of grains over [3H]-thymidine labelled crypt base columnar cells (BCC) in autoradiographs of the ileum of BDF1 mice has been studied. The results revealed that using the conventional grain count halving (GCH) method it is possible to obtain an estimation of the generation time (Tc) of the proliferative BCC cells in the Paneth cell zone (PC-zone) of 18.8 +/- 0.74 h. This lies within the range obtained by the percent labelled mitoses (PLM) method, but is shorter than most values obtained by stathmokinetic methods. The present data show no evidence for a shortening of the cell cycle 3 days after irradiation (8 Gy) which is contrary to some earlier observations. Some reasons for this discrepancy are discussed. The comparatively high labelling index of the BCC allows a larger amount of data to be easily collected, compared with the PLM technique, and correction factors which take into account the complicated shape of the bottom of the crypt are not required.

The histology and calcification of regenerating scales in the blackspotted topminnow, Fundulus olivaceus (Storer)

The regenerating scale and tissues comprising the scale pocket of Fundulus olivaceus were examined microscopically at specific intervals. Scale removal resulted in a thickening of the epidermis which persisted through the early stages of regeneration. This thickening was due in part to the appearance of columnar basal cells which divided producing cells that became mucous cells and squamous cells. The scale regenerated as a relatively large plate of bone which first appeared between layers of scleroblasts on the floor of the scale pocket and then grew producing circuli and radii. By the fourth day of regeneration, calcium was observed in the cytoplasm of the scleroblasts and at randomly distributed foci in the osseous portion of the scale. The osseous layer was completely calcified by 15 days of regeneration.

Prevention by vitamin A of the occurrence of permanent vaginal changes in neonatally estrogen-treated mice. An electron microscopic study.

Ovary-indepedent (estrogen-independent) irreversible proliferation and cornification on the vaginal epithelium in ovariectomized mice caused by neonatal injections of 20 microng estradiol-17 beta (E2) was suppressed by injections of 200 IU vitamin A acetate (VA) given simultaneously with E2. More than 2 months after the combined treatment were required for the suppression. A new type of cell of low electron density (Light cells) appeared at 40-120 days of age on the basal lamina of the vaginal epithelium in neonatally E2 plus VA-treated ovariectomized mice only at 50 days. Light cells contained small amounts of organelles. Columnar basal cells resembling those in normal estrous mice were also found in the E2 plus VA-treatedmice at 70-120 days. In one of the 120-day-old, E2 plus VA-treated mice, the epithelium consisted of only 2-3 layers of cells. Neither Light cells nor columnar cells were found in the atrophic epithelium.

An electron microscopic study of developing chick skin

Changes that take place in the skin of the chick embryo from day 6 to day 18 of development have been studied by histochemical techniques and electron microscopy. On day 6, the embryo is covered by an epithelium that consists of one layer of columnar basal cells and one layer of flattened superficial cells. As the embryo develops, the superficial cells give rise to a bilayered periderm, the cells of which contain characteristic mesh-like granules and large accumulations of glycogen. The granules, which may be several microns in diameter, seem to be formed of interlaced strands composed of particles with a diameter of 60–80 A. By day 17 or 18, the periderm disintegrates and is sloughed off. The columnar basal cells of the covering epithelium give rise to the epidermis. By day 17 of development multigranular bodies and keratohyalin granules are present in the epidermal cells. The multigranular bodies (diameter 1000–5000 A), which contain small laminated granules, are characteristic components of chick epidermis. The keratohyalin granules are small (diameter μ ) and are composed of filaments and an amorphous matrix. By day 17, the first layers of the stratum corneum are formed and the epidermis has acquired its adult structure. A lamella that consists of 20 or more orthogonally arranged plies of collagenous fibers is formed in the dermis by day 17 of development.

The fine structure of the transitional epithelium of rat ureter.

The fine structure of the transitional epithelium of rat ureter has been studied in thin sections with the electron microscope, including some stained cytochemically to show nucleoside triphosphatase activity. The epithelium is three to four cells deep with cuboidal or columnar basal cells, intermediate cells, and superficial squamous cells. The basal cells are attached by half desmosomes, or attachment plates, on their basal membranes to a basement membrane which separates the epithelium from the lamina propria. Fine extracellular fibres, ca. 100 A in diameter, are to be found in the connective tissue layer immediately below the basement membrane of this epithelium. The plasma membranes of the basal and intermediate cells and the lateral and basal membranes of the squamous cells are deeply interdigitated, and nucleoside triphosphatase activity is associated with them. All the cells have a dense feltwork of tonofilaments which ramify throughout the cytoplasm. The existence of junctional complexes, comprising a zonula occludens, zonula adhaerens, and macula adhaerens or desmosome, between the lateral borders of the squamous cells is reported. It is suggested that this complex is the major obstacle to the free flow of water from the extracellular spaces into the hypertonic urine. The free luminal surface of the squamous cells and many cytoplasmic vesicles in these cells are bounded by an unusually thick plasma membrane. The three leaflets of this unit membrane are asymmetric, with the outer one about twice as thick as the innermost one. The vesicles and the plasma membrane maintain angular conformations which suggest the membrane to be unusually rigid. No nucleoside triphosphatase activity is associated with this membrane. Arguments are presented to support a suggestion that this thick plasma membrane is the morphological site of a passive permeability barrier to water flow across the cells, and that keratin may be included in the membrane structure. The possible origin of the thick plasma membrane in the Golgi complex is discussed. Bodies with heterogeneous contents, including characteristic hexagonally packed stacks of thick membranes, are described. It is suggested that these are "disposal units" for old or surplus thick membrane. A cell type is described, which forms only 0.1 to 0.5 per cent of the total cell population and contains bundles of tubular fibres or crystallites. Their origin and function are not known.

On the nature of tissue interactions in embryonic skin

s TUDIES of the interactions between epithelia and mesenchyme in developing embryonic organs have shown that mesenchyme has an important influence on epithelial growth and differentiation in, for example, salivary gland [5], pancreas [3], feather germs [ill, and preen gland [4]. In skin, McLaughlin [8, 91 and more recently (since this investigation began) Wessells [13] have found that the proximity of mesenchyme is necessary for the continued survival of epidermis, and for its normal differentiation. The mechanism of this dependence is less clear, but the participation of the intercellular material in such interactions has frequently been suggested (e.g. [6, 7, 91). In order fully to understand the relationship between tissues, it is necessary to know which components of one tissue contribute to the continued growth, differentiation, and functioning of the other. The work briefly reported here is an attempt to analyse the relationship between embryonic epidermis and dermis, in order to determine which mesenchymal constituents affect epidermal survival, differentiation and keratinization. The experiments were made on the scaly skin of the anterior tarsometatarsal region of the 12-day embryonic chick. After the skin had been treated with 0.04 per cent Versene (ethylene diamine tetra-acetic acid, di-sodium salt) for 20 min at 20°C the epidermis could be easily and cleanly peeled from the dermis. The dermis was subjected to various experimental treatments, and the two tissues were then recombined and cultured in vitro on cellulose acetate rafts [IO] on clots made of plasma and embryo extract [2]. The explants were fixed in acetic-Zenker for histological examination. The epidermis at 12 days consists of a layer of columnar basal cells and a single stratum of intermediate cells, above which is a two layered periderm. After separation and recombination with epidermal basal cells next to the dermis, both dermis and epidermis grew well, the periderm differentiated rapidly then degenerated, and layers of birefringent keratin were produced (Fig. 1). Isolated epidermis, however, did not survive under these conditions; the basal cells lost their columnar orientation within 3 hr, the epidermal sheet contracted and thickened, and the organization of cell layers was lost. Mitosis ceased after 18-24 hr, and most of the lower cells became necrotic, only a few cells showing traces of keratin, although the periderm underwent some differentiation (Fig. 2). No basement membrane formed. This behaviour occurred when isolated epidermis was placed on rafts, on millipore filters, or directly on the clot. Dermis was treated with trypsin to obtain a suspension of cells almost free of dermal intercellular material. When this suspension was placed on the basal surface of the epidermis, the orientation of the basal cells and the organization of cell layers were maintained, and a basement membrane stainable with the periodic acid-Schiff

Tissue interactions during skin histodifferentiation

Skin tissues from 11-day chick embryos have been cultured in protein-free nutrients. Under these conditions, epidermis displays columnar basal cells and mitoses only in regions where an intimate junction is established with either dermis or muscle. Epidermis that fails to form a junction, or that is cultured alone, develops abnormally and becomes necrotic by 4 days. Epidermal differentiation to the tonofibril stage occurs only in epidermis-dermis recombinants in regions of a junction. The dermal-epidermal interaction can occur when Millipore filters are inserted between the tissues, or in the absence of visualized periodic acid-Schiff staining at the junction. Intercellular materials form and are randomly distributed in dermal explants. In recombinants, the materials usually appear first in regions distant from the epidermis. Other properties of interacting skin tissues are described, including some aspects of scale morphogenesis.