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The morphogenesis of hairs is initiated and maintained by reciprocal interactions between groups of epithelial and mesenchymal cells. To examine whether cell adhesion molecules play a role in this process, prenatal distribution patterns of various cell adhesion molecules were studied during hair follicle morphogenesis in the dorsal skin of C57BL mouse embryos, using monoclonal antibodies. E-cadherin was present on all epithelial cells of the skin when the ectoderm gave rise to periderm and epidermis. E-cadherin was reduced in the follicle placodes and hair plugs, then disappeared from the presumptive hair matrix of elongating follicles. P-cadherin was initially present on all cells of periderm and epidermis and was later retained at a reduced level in the basal epidermal layer. P-cadherin was prominent in all follicle placodes and hair plugs and in the presumptive hair matrix of elongating follicles. N-CAM was present on all mesenchymal cells of the presumptive dermis at the prefollicle stage, then temporarily restricted to a few cells just below the dermal-epithelial junction. Later, N-CAM reappeared in the interfollicular mesenchyme and was prominent in the mesenchymal sheath and dermal papilla of elongating follicles. In addition, N-CAM was expressed in the hair plugs, then became progressively restricted to the upper caudal part of the elongating follicles. The results suggest that the main role of cell adhesion molecules is to mould the follicle by relaxing or reinforcing cell contacts in areas of increased morphogenetic activity.

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Wild-type mice have three main types of hair in their pelage: tylotrichs, awls and zigzags. Tabby mice have a yellowish coat consisting of awls only, whereas downy mice have a sparse grayish coat consisting of unusually fine hairs. The spatial and temporal distribution of cell adhesion molecules (CAMs) during hair follicle morphogenesis was investigated in the mutants and compared with that in nonmutant mice. In Tabby embryos, awl follicles developed normally and showed normal immunostaining patterns for E-cadherin, P-cadherin and N-CAM. Prior to follicle initiation, however, some deviations from normal skin morphology and staining patterns indicated a delay in the development of the basal epidermal layer. On the other hand, the stratum corneum was formed prematurely. Therefore, the lack of tylotrich and zigzag follicles in Tabby mice might be explained by a general defect in epidermal development rather than by abnormal CAM expression. In downy embryos, tylotrich and awl follicles were initiated within the normal time periods, but elongation and differentiation of most follicles were abnormal. At birth, most follicles were small and/or severely deformed but showed normal CAM expression patterns. Extreme distortion and disorientation of follicles seemed to be associated with disintegration of the dermal papilla and abnormal mesenchymal cell condensations between the follicles. This suggests that abnormal hair development in downy mice might result from a defect in dermal rather than epidermal components of the skin.

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In order to explore the origin and significance of Merkel cells in the hairy skin of mammals, the development of Merkel cells and nerve endings in the dorsolateral skin of C57BL mouse embryos was studied in serial cryostat sections. At 13 and 14 days of gestation, application of a monoclonal antibody to mouse keratin 8 (mK8) resulted in specific immunofluorescence of all cells in the epidermis and periderm. The periderm retained specific staining until it was shed, around 18 days. At 15 days, mK8-specific staining elsewhere was restricted to scattered immature Merkel cells in the developing tylotrich follicles and the adjacent epidermis. Between 16 and 17 days, these cells assembled within the basal epidermal layer, caudal to each tylotrich follicle, to form a disc-shaped rudiment of a 'haarscheibe' or touch dome. No Merkel cells were found in association with the later developing awl and zigzag follicles. In mice homozygous or hemizygous for the Tabby mutation, in which tylotrich follicles never form, no Merkel cells were found in any part of the dorsolateral skin. In mice homozygous for the recessive downy mutation, in which all three types of hair are present but reduced in size, Merkel cell development was the same as in wild-type mice. Nerve endings were located in the upper dermal mesenchyme by a monoclonal antibody to neural cell adhesion molecule. This antibody also stained plasma membranes in specific parts of the hair follicles during their development. From 14 to 19 days, none of the nerve endings were seen in contact with the epidermis or the follicle epithelium, even in areas where Merkel cells were located. These findings support the view that both location and early differentiation of Merkel cells in the dorsolateral epidermis are independent of neural influences but linked to the development of tylotrich follicles.

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Rat pheochromocytoma PC12 cells were genetically modified in vitro to express recombinant beta-nerve growth factor (beta-NGF) using a replication-deficient retroviral vector carrying the mouse beta-NGF gene and subsequently implanted into the striatum of a mouse model of Parkinson's disease. The fate of the genetically modified PC12 cells (PC12N.8) was assessed at varying times postimplantation by studying immunoreactivity (IR) to tyrosine hydroxylase (TH) or the rat NGF receptor (NGFR). In vitro, the genetically modified PC12 cells displayed a neuronal morphology in the absence of exogenous NGF which was characterized by extensive neurite outgrowth. In addition, the genetically modified PC12 displayed a catecholaminergic phenotype in vitro as assessed by TH-IR. Following implantation into the striatum, the survival of PC12N.8 cells was limited. Surviving cells could be identified by NGFR-IR, but not by TH-IR. In addition, PC12N.8 cells with a neuronal morphology similar to that observed in vitro were only rarely observed in vivo. No tumors were observed in PC12N.8 graft recipients up to 30 days postimplantation. In contrast, intrastriatal tumors were observed in 50% of the PC12 cell recipients. These data demonstrate that PC12 cells genetically modified in vitro to synthesize beta-NGF do not revert to the mitotic phenotype of the parent PC12 cell line following implantation into the adult striatum, an observation that suggests that these cells may continue to express recombinant beta-NGF in vivo. The data further suggest that the genetically modified PC12 cells lose the catecholaminergic phenotype following implantation into the striatal parenchyma.(ABSTRACT TRUNCATED AT 250 WORDS)

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In the brain, the action of glucocorticoid steroids is mediated via two intracellular receptors, the mineralocorticoid (MR), or type I receptor, and the glucocorticoid (GR), or type II receptor. These receptors are expressed in many types of neurons and are co-expressed in some neurons such as the hippocampal pyramidal cells. Although glucocorticoids are known to affect gliogenesis and glial cell differentiation, the expression of the GR in different types of glial cells throughout the brain has not been thoroughly studied and the expression of the MR in glia not previously reported. Here we review studies suggesting that both receptors are expressed in astrocytes and oligodendrocytes.

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Glucocorticoid hormones affect gene expression directly at the level of transcription via intracellular receptors that translocate to the nucleus in the presence of steroid. In the brain, two types of high-affinity receptors bind glucocorticoids, the type I, mineralocorticoid receptor and the type II, glucocorticoid receptor (GR). Both receptor types are expressed by many types of neurons. Although binding studies have suggested that glial cells may also express receptors, the expression of these receptors in specific classes of glia has not been studied previously. This immunocytochemical study was undertaken to determine which of the different classes of glial cells express type II GR. Primary cultures of mixed glial cells from rat cerebrum and cerebellum, purified oligodendrocytes and astrocytes, as well as two glial tumor cell lines were screened for the expression of glucocorticoid receptors using a mouse monoclonal antibody directed against rat liver GR (BuGR-2). Glial cell types were identified by morphology and immunoreactivity (IR) with antibodies directed against glial fibrillary acidic protein (GFAP), cyclic nucleotide phosphodiesterase (CNP), or myelin basic protein (MBP). Double immunofluorescence microscopy revealed that all GFAP-IR cells (type 1 and type 2 astrocytes), all CNP- or MBP-IR cells (oligodendrocytes), as well as immature and intermediate cell types expressed GR, although at different levels. C6 glioma and JScl1 Schwannoma cells were observed to express moderate to high levels of GR. Furthermore, cells grown in the absence of glucocorticoids had diffuse GR staining over the cytoplasm, whereas cells grown in the presence of the synthetic glucocorticoid dexamethasone had strong nuclear staining. These results demonstrate that, in vitro, all classes of glial cells express glucocorticoid receptors that can translocate to the nucleus in the presence of hormone. These observations suggest that glial cells are major targets for glucocorticoid-directed control of gene transcription in the nervous system.

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Pluripotent P19 embryonal carcinoma cell cultures can be induced to differentiate into neurons and glial cells by the addition of 10(-6) M retinoic acid. During early neural differentiation, a bundle of colchicine-stable, acetylated microtubules is formed. This acetylated microtubule array apparently extends to form neurites during neurogenesis. In this paper, we analyze changes in vimentin and MAP 2 distributions during neural differentiation with respect to the changes in the acetylated microtubule array. During a brief period early in differentiation, indirect immunofluorescence staining shows the colocalization of colchicine-stable acetylated microtubules, vimentin, and MAP 2. Using acrylamide to disrupt the organization of vimentin intermediate filaments and estramustine to disrupt the binding of MAP 2 to microtubules, we show that acetylated microtubules, MAP 2, and vimentin intermediate filaments are arranged in an interdependent cytoskeletal array. We suggest this array may serve to stabilize processes in neural stem cells, before the final decision to differentiate into neurons or glia is made.

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In recent studies on the cytoskeletal organization of T51B rat liver cells by indirect immunofluorescence microscopy, we have been unable to achieve double-staining of microtubules and intermediate filaments within the same cell. In acetone-fixed cells, microtubules were poorly preserved, and two out of three monoclonal antibodies tested did not stain them properly. In formaldehyde-fixed cells, the monoclonal anti-cytokeratin produced an incomplete staining pattern against a diffuse background. We have now developed a fixation protocol which includes simultaneous fixation and extraction with formaldehyde and nonionic detergent in the present of microtubule stabilization buffer. Although developed for a specific purpose, it is of general application as it yields excellent preservation of all cytoskeletal components tested so far, without masking antigenic determinants. The procedure is both simple and fast and will, therefore, be valuable for efficient processing of samples from large-scale experiments, such as the screening for cytoskeletal changes during longterm treatment of cells with drugs or carcinogens.

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Two posttranslational modifications of alpha-tubulin, acetylation and detyrosination, are associated with stable microtubule (MT) populations, including those of neuronal processes. We have used a pluripotent embryonal carcinoma cell line, P19, to investigate changes in MT isotype and stability found in MT arrays during neurogenesis. This cell line has an advantage in that both commitment- and differentiation-related events can be observed. Uncommitted P19 cells have minimal arrays of acetylated and detyrosinated MTs. Following neuronal induction with retinoic acid (RA), indirect immunofluorescence microscopy shows that the first MT modifications occur during commitment and before any morphological change is observed. RA-induced cells initially polymerize a temporarily enlarged population of MTs. Included in this population is a new array of acetylated MTs arranged in a bundle of parallel MTs. This bundle is colchicine-stable, although no MT-associated proteins (MAPs) are detectable using a battery of anti-MAP antibodies. Observation of MT arrays with patterns that are intermediate between the early bundles and short neurites suggests that the acetylated MT bundle subsequently extends to form a neurite. MAP 2 is first detected at about the time of neurite extension. However, at this early stage of differentiation, MAP 2 is not yet limited to dendritic processes. This report provides the first evidence that the stable MTs of mature neurons may be initiated during neuronal commitment.

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Retinal pigmented epithelium (RPE) from 7-day-old chicken embryos (stages 29 to 31) was isolated and dissociated into single cells using different procedures. The results were assessed in two ways. (1) The yield of single RPE cells per embryo was determined, and their ability to form pigmented colonies in clonal culture was tested. The most efficient and gentle procedure included isolation of the RPE in EDTA solution, trypsinization at low temperature and low enzyme concentration in the presence of EDTA, followed by incubation in culture medium for up to 4 hr. The completely dissociated cells thus obtained had a much higher plating efficiency and more uniform pattern of colony growth and differentiation than those obtained under any other conditions tested. (2) The effects of different treatments on cell junctions and morphological integrity of the cells were determined by transmission electron microscopy. EDTA solution yielded excellent separation of the epithelial sheet from the mesenchyme by dissociating it from Bruch's membrane, but had little effect on the junctions between adjacent RPE cells. Trypsinization of the epithelium under various conditions separated the basal lateral cell borders and caused loss of gap junctions, but left many cells still joined by apical tight junctions. Final disruption of the tight junctions occurred during recovery of the trypsinized cells in culture medium and was accompanied by dedifferentiation of the RPE cells.

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Distribution and organization of the extracellular glycoproteins, fibronectin and laminin, in clonal cultures of chick retinal pigmented epithelial cells have been investigated using indirect immunofluorescence microscopy. Fibronectin is located on the apical and basal cell surfaces and between the cells in the undifferentiated regions of the colony (outer edge and stratified region). It seems to run parallel to intracellular microfilament bundles and to be associated with them across the cell membrane. In the differentiated region of the colony (center), it is located exclusively on the basal cell surface and seems to be primarily associated with the collagen bundles of the basement membrane. The locations suggest that it may be necessary to permit rapid division and movement of the undifferentiated cells in the outer region of the colony, while stabilizing the sheet of differentiated cells in the colony center. In all regions except the outer edge of the colony, laminin is associated with the basal cell surfaces where it forms a meshwork of short, fine strands. The laminin has a totally different staining pattern from the fibronectin and does not seem to be associated with collagen bundles. The location suggests that laminin may be present in the basal lamina and may be involved in adhesion of the cells to the substratum.

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We introduce inbred strains of platyfish and swordtails as well as their hybrids as laboratory animals for carcinogenicity testing. We especially propose to take advantage of the platyfish-swordtail melanoma system, which permits the breeding of animals with high susceptibility to carcinogen-induced melanoma. Based on our present view that, in melanoma formation, such fundamental processes as the determination and differentiation of a specific cell type are affected, we suggest the use of embryos and embryo-derived cell cultures. In both systems, all stages of pigment cell differentiation can be followed and screened for abnormal development. The in vitro culture of the embryos of the viviparous fish and the establishment of cell cultures are described.

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We report genetic transformation in an intact higher organism, i.e., in xiphophorine fish. The gene to be transferred (Tu) is responsible for the formation of T-melanophores in the platyfish and is involved in the formation of melanomas in platyfish-swordtail hybrids. After injection of Tu-donor DNA into the neural crest region of embryos from Tu-free fish, some of the recipients developed T-melanophores. In a few cases, one or two single T-melanophores were formed during late embryo-genesis. In most cases, many T-melanophores developed in young fish and were arranged in several colonies or in a pattern. DNase-degraded Tu-donor DNA, Tu-free fish DNA, as well as DNA from E. coli and adenovirus-2, did not induce T-melanophores. When using DNA from different strains of Tu-donor fish which differed in a regulating gene linked to Tu, the percentages of fish showing T-melanophores paralleled the degree of phenotypic expression of the Tu gene in the DNA donor. The results suggest that the Tu gene has been successfully transferred together with the linked regulating gene.

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In poeciliid fish, melanoma of different degrees of malignancy can be produced by crossing specific genotypes. For a detailed investigation of the processes leading to proliferation or differentiation of the melanoma cells, it is necessary to establish cell cultures. The aim of the present study was to find out the optimal conditions for initiating and culturing poeciliid fish cells for the purpose of establishing cell cultures of melanoma. The optimal method was developed by using small pieces of late embryos as starting material and includes: (a) dispersion of tissue by mild stepwise treatment with a trypsin-EDTA mixture at low temperature; (b) culture of cells in the complex medium 199; (c) supplementation of medium with high percentage (20%) of fetal bovine serum; and (d) stabilization of pH by buffering the medium with HEPES. Under these conditions, primary and secondary cultures of embryonic cells have been initiated. An epithelial-like cell line has been subcultured for more than 80 passages. The method developed for embryonic tissues was used to start cell cultures from melanoma of platyfish-swordtail hybrids. Until now, only cells of rapidly growing malignant albino melanoma could be maintained in primary cultures. Secondary cultures could not be initiated since the melanoma cells tended to differentiate and stopped growing before a confluent monolayer was formed.

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Embryonic skin and eyes, and melanomas of xiphophorine fish were investigated by fomaldehyde-induced fluorescence in order to test whether the pigment cells in these tissues may be identified by a specific green-yellow fluorescence. Skin of pigmented fish embryos showed no fluorescence in the black pigment cells (melanocytes and melanophores), while skin of albino embryos showed a green-yellow fluorescence in all cells which correspond to the black pigment cells of pigmented embryos. The skin of both pigmented and albino embryos showed a bright orange fluorescence in the red pigment cells (pterinophores). No fluorescence was observed in the retinal pigment epithelium of pigmented embryos, while a green-yellow fluorescence was observed in the pigment epithelium of albino embryos. Neither the melanotic melanomas of pigmented fish nor the amelanotic melanomas of albino fish showed any specific fluorescence.

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In the genetically determined pigment cell tumors of platyfish and platyfish-swordtail hybrids, the degree of malignancy of pigment cells which have been neoplastically transformed by a tumor gene (Tu) depends on the type and number of certain regulating genes (R). In the present study, the tyrosinase activities in tumors of different degrees of malignancy (black spots, premelanomas, melanomas) have been determined. The results demonstrate a close correlation between the level of tyrosinase activity and the degree of malignancy. Spot patterns consisting of completely differentiated (benign) Tu-transformed cells show no tyrosinase activity. Premelanomas containing a few incompletely differentiated (malignant) Tu-transformed cells in addition to many differentiated ones show moderate tyrosinase activities. Melanomas which contain increasing numbers of incompletely differentiated cells with increasing growth rates show high to extremely high tyrosinase activities. Thus, the tyrosinase levels present in these tumors can be used as an indicator for the degree of differentiation and, thereby, for the degree of malignancy of the neoplastically transformed pigment cells.

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In certain fish hybrids, malignant transformation of pigment cells is due to the presence of a tumor gene (Tu), the action of which is controlled by several regulatory elements. Absence of these controlling genes causes rapid proliferation of the Tu-transformed cells and ultimately results in melanoma formation. One of these genes has been identified as a differentiation gene (Diff), since it seems to control the differentiation of the transformed pigment cells. Light and electron microscopy of Tu-transformed cells of fish differing in the dosage of Diff, and the determination of tyrosinase activity in homogenates of the respective tissues revealed that the degree of cellular differentiation depends on the dosage of Diff present in the genome. It is concluded that the gene Diff promotes the differentiation of malignant melanoma cells into benign melanophores.

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