Identification of Merkel cells in human skin by specific cytokeratin antibodies:

Merkel cells are special neurosecretory cells which, in adult human skin, are usually very scarce. By immunofluorescence microscopy using antibodies to human cytokeratin polypeptide no. 18, we localized distinct non-keratinocyte cells in the glandular ridges of human fetal and adult plantar epidermis. Using electron and immunofluorescence microscopy, these cells were identified as Merkel cells containing typical neurosecretory granules as well as bundles of intermediate-sized filaments and desmosomes. Two-dimensional gel electrophoresis of the cytoskeletal fractions of microdissected epidermal preparations highly enriched in Merkel cells indicated the presence of cytokeratin polypeptides nos. 8, 18 and 19 which are typical of diverse simple epithelia of the human body. Double immunofluorescence microscopy showed that these human Merkel cells contain neither neurofilaments nor vimentin filaments. In human fetuses of 18-24 weeks of age, conspicuously high concentrations of Merkel cells, reaching a density of approximately 1,700 Merkel cells/mm2 skin, were found in the glandular ridges of plantar skin. The concentration decreased considerably at newborn and adult stages. Thin cell processes (up to 20 microns long) were observed in many fetal epidermal Merkel cells. In addition, we detected isolated Merkel cells deeper in the dermis (i.e. at distances of, at most, 100 microns from the epidermis) in fetal and newborn plantar skin. Our results show that Merkel cells are true epithelial cells which, however, differ profoundly from epidermal keratinocytes in their cytokeratin expression. The findings are discussed in relation to the much disputed question of the origin of Merkel cells. The present data speak against the immigration of Merkel cells from the neural crest, but rather suggest that they originate from epithelial cells of the skin, although most probably not from differentiated keratinocytes.     

Resident and circulating mast cells in propulsative organs of frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

A histochemical and ultrastructural examination of mast cells (MCs) in “blood” and lymph hearts of the adult frog Rana temporaria showed that they are represented by two populations, i.e., resident and circulating MCs. Resident cardiac MCs have an oval or elongated shape and are located in the atrial and ventricular myocardium, as well as in connective tissue of the epicardium. Circulating MCs were identified in heart lumen lacunas and in narrow clefts produced by ventricular trabecular myocardium, as well as in blood-filled atrial and ventricular central cavities. The small round shape circulating MCs resemble lymphocytes, but their cytoplasm is filled with granules that are ultrastucturally similar to granules of cardiac resident MCs. In the lymph heart, elongated resident MCs are located in the interstitial space between the cross-striated muscle fibers and smooth muscle cells of afferent and efferent heart valves. Round circulating MCs are occasionally visible in the cavity of the lymph heart. More commonly, circulating MCs are found in the lymphatic sinus cavity located adjacent to lymph hearts. In certain parts of the lymphatic sinus walls, MCs form clusters that are in tight contact with the mesothelial cells that line the lymphatic cavity. Our histochemical investigation revealed that both resident and circulating MCs of the propulsative organs are strongly alcian blue positive, but are weakly red after safranin staining and weakly metachromatic with toluidine blue. The presence of populations of circulating MCs in adult frogs suggests that there are differences in MC biology in lower and higher vertebrates.     

Intermediate filaments in muscle and epithelial cells of nematodes

Current concepts of the developmentally controlled multigene family of intermediate filament (IF) proteins expect the origin of their complexity in evolutionary precursors preceding all vertebrate classes. Among invertebrates, however, firm ultrastructural as well as molecular documentation of IFs is restricted to some giant axons and to epithelia of a few molluscs and annelids. As Ascaris lumbricoides is easily dissected into clean tissues, IF expression in this large nematode was analyzed by electron microscopic and biochemical procedures and a monoclonal antibody reacting with all mammalian IF proteins. We document for the first time the presence of IFs in muscle cells of an invertebrate. They occur in three muscle types (irregular striated pharynx muscle, obliquely striated body muscle, uterus smooth muscle). IFs are also found in the epithelia studied (syncytial epidermis, intestine, ovary, testis). Immunoblots on muscles, pharynx, intestine, uterus, and epidermis identify a pair of polypeptides (with apparent molecular masses of 71 and 63 kD) as IF constituents. In vitro reconstitution of filaments was obtained with the proteins purified from body muscle. In the small nematode Caenorhabditis elegans IF proteins are so far found only in the massive desmosome-anchored tonofilament bundles which traverse a special epithelial cell type, the marginal cells of the pharynx. We speculate that IFs may occur in most but perhaps not all invertebrates and that they may not occur in all cells in large amounts. As electron micrographs of the epidermis of a planarian--a member of the Platyhelminthes--reveal IFs, the evolutionary origin of this cytoplasmic structure can be expected either among the lowest metazoa or already in some unicellular eukaryotes.     

Development of Merkel cell populations with contrasting sensitivities to neonatal deafferentation in the rat whisker pad

In this study, we used the quinacrine fluorescence technique to investigate the embryonic and early postnatal development of two distinct populations of Merkel cells in the rat whisker pad and the consequences of neonatal deafferentation on their subsequent development. Annular clusters of Merkel cells first appear in the epidermis near the caudal margin of the mystacial region between embryonic days E14 and E15 at dome sites located on horizontal ridges where the primordial vibrissal follicles develop. The development of these cells progresses in a caudorostral sequence across the whisker pad as does the development of the vibrissal follicles. Each cluster eventually forms a conical ridge or collar of about 130 Merkel cells that surrounds the vibrissal hair shaft as it penetrates the overlying pad epidermis. In the vibrissae, which develop as downgrowths from the horizontal ridges at the dome sites, Merkel cells first appear (caudally) between E16 and E17 and form a cylindrical cuff within the outer root sheath; cells are added progressively until about the end of the first postnatal week when a plateau level of about 750-800 cells is reached. Following unilateral transection of the infraorbital nerve at 24-36 hr after birth, these vibrissal Merkel cells continued to develop along a time course that was indistinguishable from normal, at least over the first 2 weeks of postnatal life. In contrast, all or most of the Merkel cells that normally develop within collars or annular clusters in the pad epidermis (around both the vibrissal and intervibrissal or pelage hairs) either disappeared within a few days or failed to develop. Other light and electron microscopic procedures supported the main findings and confirmed that the denervation was successful. Thus, the vibrissal Merkel cells, like those in the glabrous hindpaw, behaved as a distinct class which develops postnatally and is maintained (at least over a 2-week period) without the presence of sensory nerves. Since both the mystacial vibrissae and glabrous hindpaw have specialized cortical representations, a possible relationship between these findings and the organization of the somatosensory cortex during development is discussed.     

Formation of the dorsal root ganglia in the avian embryo: Segmental origin and migratory behavior of neural crest progenitor cells

The segmental origin and migratory pattern of neural crest cells at the trunk level of avian embryos was studied, with special emphasis on the formation of the dorsal root ganglia (DRG) which organize in the anterior half of each somite. Neural crest cells were visualized using the quail-chick marker and HNK-1 immunofluorescence. The migratory process turned out to be closely correlated with somitic development: when the somites are epithelial in structure few labeled cells were found in a dorsolateral position on the neural tube, uniformly distributed along the craniocaudal axis. Following somitic dissociation into dermomyotome and sclerotome labeled cells follow defined migratory pathways restricted to each anterior somitic half. In contrast, opposite the posterior half of the somites, cells remain grouped in a dorsolateral position on the neural tube. The fate of crest cells originating at the level of the posterior somitic half was investigated by grafting into chick hosts short segments of quail neural primordium, which ended at mid-somitic or at intersomitic levels. It was found that neural crest cells arising opposite the posterior somitic half participate in the formation of the DRG and Schwann cells lining the dorsal and ventral root fibers of the same somitic level as well as of the subsequent one, whereas those cells originating from levels facing the anterior half of a somite participate in the formation of the corresponding DRG. Moreover, crest cells from both segmental halves segregate within each ganglion in a distinct topographical arrangement which reflects their segmental origin on the neural primordium. Labeled cells which relocate from posterior into anterior somitic regions migrate longitudinally along the neural tube. Longitudinal migration of neural crest cells was first observed when the somites are epithelial in structure and is completed after the disappearance of the last cells from the posterior somitic region at a stage corresponding to the organogenesis of the DRG.     

Mallory bodies: Lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group

Summary Mallory's alcoholic hyalin in hepatocytes was found also in other diseases and is now referred to as Mallory bodies. Data concerning their histochemical, immuno and electron microscopic properties are partly contradictory. In this study, early stages of Mallory bodies reacted strongly with configurational technics for myosins; affinity tended to decrease when material with the properties of keratohyalin and the matrix of stratum corneum was formed. Thus, many Mallory bodies contained histochemically distinct myoid and keratin-like proteins. Electron microscopists demonstrated thick and thin filaments resembling contractile systems in Mallory bodies; the failure of immunologists to visualize actomyosin may be due to the heterogeneity of these proteins. The currently popular term prekeratin has been applied to a variety of substances extracted from epidermis, hoof and hair under different conditions. The prekeratin of recent immunofluorescence studies seems to contain mainly epidermin and low molecular matrix proteins; both were studied extensively by chemists. Epithelial filaments, including tonofibrils and contractile fibrils regarded as a subgroup of myofibrils, were well known half a century ago, but were banished by electron microscopy. Observations in this study and data on epidermal actomyosin indicate that different proteins of the k-m-e-f group can indeed coexist in epithelial cells. The formation and resolution of Mallory bodies can be regarded as an example of the well known shifts of epithelial cells between secretory and keratinizing states.     

Mallory bodies: lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group

Mallory's alcoholic hyalin in hepatocytes was found also in other diseases and is now referred to as Mallory bodies. Data concerning their histochemical, immuno and electron microscopic properties are partly contradictory. In this study, early stages of Mallory bodies reacted strongly with configurational technics for myosins; affinity tended to decrease when material with the properties of keratohyalin and the matrix of stratum corneum was formed. Thus, many Mallory bodies contained histochemically distinct myoid and keratin-like proteins. Electron microscopists demonstrated thick and thin filaments resembling contractile systems in Mallory bodies; the failure of immunologists to visualize actomyosin may be due to the heterogeneity of these proteins. The currently popular term prekeratin has been applied to a variety of substances extracted from epidermis, hoof and hair under different conditions. The prekeratin of recent immunofluorescence studies seems to contain mainly epidermin and low molecular matrix proteins; both were studied extensively by chemists. Epithelial filaments, including tonofibrils and contractile fibrils regarded as a subgroup of myofibrils, were well known half a century ago, but were banished by electron microscopy. Observations in this study and data on epidermal actomyosin indicate that different proteins of the k-m-e-f group can indeed coexist in epithelial cells. The formation and resolution of Mallory bodies can be regarded as an example of the well known shifts of epithelial cells between secretory and keratinizing states.     

Melanocyte destruction and repigmentation in vitiligo: A model for nerve cell damage and regrowth

Melanocytes (MCs) are melanin-producing cells of the skin that are derived from neural crest cells. Vitiligo vulgaris is a common depigmentation disorder resulting from the destruction of functional MCs in the affected skin. The three prevailing pathomechanisms of vitiligo are the immune hypothesis, the neural hypothesis and the autocytotoxic hypothesis. None of these mechanisms has been conclusively proven. Melanoblasts (MBs) in the outer root sheath of the hair follicles are the reservoir cells for repigmentation. Recovery from vitiligo is initiated by activation and proliferation of these MBs, followed by upward migration to the nearby epidermis that forms perifollicular pigmentation islands. Migration, proliferation and differentiation of MCs and MBs are regulated by keratinocyte-derived factors and some coat color genes. Any therapy for vitiligo must explain not only the repopulation of MCs but also their functional development. In patients with vitiligo, MCs are destroyed in the skin, the eyes, and possibly the ears. However, the concept of vitiligo as a systemic disease will be clearly established only when the mechanisms involved in vitiligo are identified. Recent advances in the fields of neural crest cell culture and molecular genetics have opened new perspectives in the understanding of vitiligo. Not only will this result in better treatments for vitiligo patients, but possibly will also provide a key to triggering nerve cell regrowth in other nervous diseases.     

Ultrastructure and complex carbohydrate ultracytochemistry of the cat parotid gland

The structure and glycoconjugate content of the cat parotid gland were analyzed at electron microscopic level by applying morphological techniques and three ultrastructural histochemical methods - HID-TCH-SP, LID-TCH-SP and PA-TCH-SP. This gland appeared as a typical salivary gland composed of acinar secretory cells, intercalated ducts, striated ducts and excretory ducts. The most common configuration of secretory granules consisted of a dense core surrounded by a variable electron-lucent halo. All ductal segments were characterized by the presence of different cell populations and small apical granules greatly different from those localized in the acinar cells. By using HID-TCH-SP we were able to demonstrate that in a few acinar cells there are sulphated sites, whereas PA-TCH-SP staining revealed the presence of vic-glycol radicals in all acinar cells preferentially located on the halo of secretory granules.     

Temporospatial cell interactions regulating mandibular and maxillary arch patterning

The cellular origin of the instructive information for hard tissue patterning of the jaws has been the subject of a long-standing controversy. Are the cranial neural crest cells prepatterned or does the epithelium pattern a developmentally uncommitted population of ectomesenchymal cells? In order to understand more about how orofacial patterning is controlled we have investigated the temporal signalling interactions and responses between epithelium and mesenchymal cells in the mandibular and maxillary primordia. We show that within the mandibular arch, homeobox genes that are expressed in different proximodistal spatial domains corresponding to presumptive molar and incisor ectomesenchymal cells are induced by signals from the oral epithelium. In mouse, prior to E10, all ectomesenchyme cells in the mandibular arch are equally responsive to epithelial signals such as Fgf8, indicating that there is no pre-specification of these cells into different populations and suggesting that patterning of the hard tissues of the mandible is instructed by the epithelium. By E10.5, ectomesenchymal cell gene expression domains are still dependent on epithelial signals but have become fixed and ectopic expression cannot be induced. At E11 expression becomes independent of epithelial signals such that removal of the epithelium does not affect spatial ectomesenchymal expression. Significantly, however, the response of ectomesenchyme cells to epithelial regulatory signals was found to be different in the mandibular and maxillary primordium. Thus, whereas both mandibular and maxillary arch epithelia could induce Dlx2 and Dlx5 expression in the mandible and Dlx2 expression in the maxilla, neither could induce Dlx5 expression in the maxilla. Reciprocal cell transplantations between mandibular and maxillary arch ectomesenchymal cells revealed intrinsic differences between these populations of cranial neural crest-derived cells. Research in odontogenesis has shown that the oral epithelium of the mandibular and maxillary primordia has unique instructive signaling properties required to direct odontogenesis, which are not found in other branchial arch epithelia. As a consequence, development of jaw-specific skeletal structures may require some prespecification of maxillary ectomesenchyme to restrict the instructive influence of the epithelial signals and allow development of maxillary structures distinct from mandibular structures.     

Histochemical and ultrastructural features of the epidermis of the land planarian Bipalium adventitium

The epidermis of the land planarian Bipalium adventitium was examined by light and electron microscopy. In all regions, the epidermis consists of a simple columnar ciliated epithelium associated with a prominent basement membrane. The epithelial cells, possessing abundant microvilli and poorly developed terminal webs, are conjoined laterally at their apical ends by septate junctions. The epidermis of the creeping sole is distinguished from that of adjoining regions by a “insunken” condition of the epithelial cells, a greater number of cilia per cell, and an absence of glandular secretions other than mucus. The insunken cells of the sole possess large glycogen disposits and attributes of metabolically active cells. Unusual intranuclear inclusions of unknown significance are also found in many of the epidermal cells in all regions. The basement membrane lacks distinct layering and consists of fine fibrils displaying a beaded appearance but no obvious cross-banding. Histochemical tests indicate that the fibrils are collagenous. In addition to mucus, secretory material found in nonsole regions includes lamellated granules and rhabdites, both stained intensely by acidic dyes. Rhabdites and the basement membrane also contain disulfide-enriched proteins. In scanning electron micrographs, the sole appears as a faint, longitudinally oriented band extending along the entire length of the animal. In all regions except the sensory border of the head, the microvilli are generally obscured by the densely arranged cilia. The sensory border consists of a row of toothlike papillae and grooves covered almost exclusively by microvilli, small club-shaped structures, and larger spherical protrusions.     

Intercellular adhering junctions with an asymmetric molecular composition: desmosomes connecting Merkel cells and keratinocytes

Merkel cells (MCs) are special neuroendocrine epithelial cells that occur as individual cells or as cell groups within the confinements of a major epithelium formed and dominated by other epithelial cells. In the epidermis and some of its appendages MCs are mostly located in the basal cell layer, occasionally also in suprabasal layers and generally occur in linear arrays in outer root sheath cell layers of hair follicles. As MCs are connected to the adjacent keratinocytes by a series of adhering junctions (AJs), of which the desmosomes are the most prominent, these junctions represent heterotypic cell–cell connections, i.e. a kind of structure not yet elucidated in molecular terms. Therefore, we have studied these AJs in order to examine the molecular composition of the desmosomal halves. Using light- and electron-microscopic immunolocalization and keratin 20 as the MC-specific cell type marker we show that the plaques of the MC half of the desmosomes specifically and constitutively contain plakophilin Pkp2. This protein, however, is absent in the keratinocyte half of such heterotypic desmosomes which instead contains Pkp1 and/or Pkp3. We discuss the developmental, tissue-architectonic and functional importance of such asymmetric junctions in normal physiology as well as in diseases, in particular in the formation of distant tumor cell metastasis.     

Ultrastructural observation of possible nerve endings in rat sebaceous gland

Summary Neural elements within the parenchyma of the sebaceous gland have not been reported previously. Nerve endings have been observed only in the connective tissue surrounding the gland or in close association with the undifferentiated basal cells.In this study, electron microscopy revealed the possible presence of nerve endings (or terminal portions of neural elements) in the suprabasal level of functional sebaceous glands of pinnae of white rats. Morphologically, there are two distinct types of nerve endings. Type 1 is bordered by a membrane of relatively irregular contour and contains a single mitochondrion, various-sized vesicles, numerous microtubules, fine neurofilament-like fibrils, and occasional ribosome-like granules. Type II is also bordered by a membrane, but its contour was relatively smooth and rounded. Moreover, Type II contains many mitochondria, varying in size, density, and the arrangement of cristae. While ribosome-like granules are scattered throughout the structure in relative abundance, there are scarcely any fine neurofilament-like fibrils or microtubules. Whether these two structures are sensory or autonomic fibers could not be determined by electron microscopic examination.     

Immunohistochemical localization of glucose transporters and insulin receptors in human fetal membranes at term

The localization has been investigated of the isoforms GLUT1, GLUT3 and GLUT4 of glucose transporter proteins as well as of insulin receptors. Fetal membranes (n=10) were examined by immunohistochemical methods at the light and electron microscopic levels using mono- and polyclonal antibodies. In all amnion epithelial cells, GLUT1 and GLUT3 antibodies were bound to the apical membrane. Very rarely the GLUT1 antibody also immunostained the basolateral membrane and reacted weakly with the endomembrane system and membranes of the lateral cell protrusions. Fibroblasts reacted with the antibodies against GLUT1, GLUT4 and insulin receptor, whereas they were labelled only in one case with GLUT3 antibody. Cytotrophoblast cells were only stained with antibodies against GLUT1 and GLUT3. Antibodies against GLUT4 only reacted with fibroblasts in the membranes. On amnion epithelial cells, weak immunoreactivity with insulin receptor antibodies was detected only at the electron microscopic level. The data indicate: (1) GLUT1 is located on all cells of the amnion, whereas GLUT3 is present in detectable amounts only on amnion epithelial cells and cytotrophoblast; (2) GLUT1 and GLUT3 on amnion epithelial cells are predominantly located on the apical surface; (3) GLUT4 and insulin receptors are not regularly expressed. We suggest that amnion epithelial cells cover their basal glucose requirements from the amniotic fluid and not from the maternal circulation.     

Distinct Neuronal Lineages of the Ascidian Embryo Revealed by Expression of a Sodium Channel Gene

The ascidian larva contains tubular neural tissue, one of the prominent anatomical features of the chordates. The cell-cleavage pattern and cell maps of the nervous system have been described in the ascidian larva in great detail. Cell types in the neural tube, however, have not yet been defined due to the lack of a suitable molecular marker. In the present work, we identified neuronal cells in the caudal neural tube of theHalocynthiaembryo by utilizing a voltage-gated Na⁺channel gene, TuNa I, as a molecular marker. Microinjection of a lineage tracer revealed that TuNa I-positive neurons in the brain and in the trunk epidermis are derived from the a-line of the eight-cell embryo, which includes cell fates to epidermal and neural tissue. On the other hand, TuNa I-positive cells in the more caudal part of the neural tissue were not stained by microinjection into the a-line. These neurons are derived from the A-line, which contains fates of notochord and muscle, but not of epidermis. Electron microscopic observation confirmed that A-line-derived neurons consist of motor neurons innervating the dorsal and ventral muscle cells. Isolated A-line blastomeres have active membrane excitability distinct from those of the a-line-derived neuronal cells after culture under cleavage arrest, suggesting that the A-line gives rise to a neuronal cell distinct from that of the a-lineage. TuNa I expression in the a-line requires signals from another cell lineage, whereas that in the A-line occurs without tight cell contact. Thus, there are at least two distinct neuronal lineages with distinct cellular behaviors in the ascidian larva: the a-line gives rise to numerous neuronal cells, including sensory cells, controlled by a mechanism similar to vertebrate neural induction, whereas A-line cells give rise to motor neurons and ependymal cells in the caudal neural tube that develop in close association with the notochord or muscle lineage, but not with the epidermal lineage.