Anti-fibronectin serum inhibits the disorganization of the dermal-epidermal junction in cultured wounded skin

During wound-healing in cultured frog skin fragments, fibronectin (FN) was detected in the dermal-epidermal junction. Intracellular fibronectin was stained using permeabilization and DAB immunoperoxidase. With electron microscopy intracytoplasmic FN granules were localized in the epidermal processes of the stratum germinativum cells protruding towards the dermis and in their marginal regions (membrane-associated plaques). Faint staining was visible at the level of the lamina densa and inside some parts of the lamina lucida. In comparison, contrasted ultrathin sections revealed classical disorganization of the dermal-epidermal junction. In the presence of anti-fibronectin serum during the whole time of culture, fibronectin-antifibronectin binding was visualized in the form of sparse cytoplasmic granules in the epidermal processes of the stratum germinativum cells. Contrasted ultrathin sections emphasized the continuity between the tonofilaments, the anchoring filaments and the anchoring fibrils. Briefly, anti-fibronectin serum inhibits the disorganization of the dermal-epidermal junction in cultured wounded skin.     

The structure of the integument of the sea cucumber,Thyone briareus

The integument and podia of the sea cucumber Thyone briareus were examined by bright field and electron microscopy. The epidermal surface was found to be covered by an acellular, PAS positive cuticle which appeared to be secreted by the underlying epidermal cells. Although the superficial portion of the cuticle contains numerous fine filaments, their ultrastructure bears no resemblance to collagen fibers. The epidermal cells are widely spaced and have long apical processes that extend along the under surface of the cuticle forming a contiguous epithelium. The apical expansions of the epidermal cells are attached to one another by means of septate desmosomes which may run continuously around all epidermal cells. Special attachment structures within these apical expansions appear to bind the cuticle to the dermis. The epidermal cells and their apical expansions are separated from the dermis by an 800 Å thick basement membrane. Granule containing cells in the upper dermis send processes up to the cuticle where they are bound to the typical epidermal cells by septate desmosomes. The abundant membrane bound granules of the cells enter villous-like processes which pass through the cuticle. The function of these cells may be to produce an adhesive material on the podia or they may be pigment cells. The thick dermis consists of a superficial zone, containing largely ground substance; a middle or laminated zone containing laminae of collagen fibers arranged in an orthogonal fashion; and a hypodermis consisting largely of ground substance and reticular fibers. Fibroblasts are abundant in the superficial dermis and between the collagen laminae. Wandering coelomocytes, or morula cells, accumulate between the collagen laminae and in the hypodermis. They may also become an integral part of the epidermis by forming septate desmosomes with epidermal cells. Morula cells contain highly specialized spherules whose tinctorial properties and electron microscopic appearance suggest that they contain protein and mucopolysaccharide.     

Ultrastructure of human dermal blood vessels with special reference to the endothelial filaments.

The ultrastructure of endothelial cytoplasmic filaments of small blood vessels from the human dermis has been described. The material consisted of biopsies from normal abdominal and thoracic skin and also from the skin of patients with urticaria pigmentosa. Most vessels were surrounded by multiple layers of basal lamina and corresponded to the small venules of the subpapillary dermis. The wall of many vessels was composed by endothelial cells with clear cytoplasm which was rich in filaments and by endothelial cells with a dense cytoplasm which was poor in filaments. Some vessels had walls composed of clear endothelial cells only. The filaments varied in diameter between 80-120 A. Curling, recoiling and whorling of cytoplasmic filaments were obvious in endothelial cells of contracted vessels. Bulging of endothelial nuclei and nuclear indentations were seen in the skin lesion of urticaria pigmentosa. The possibility that the clear endothelial cells which are rich in filaments may be more actively involved in contraction than the dense cells, is discussed.     

松江鲈鱼皮肤的显微和亚显微结构

采用光学显微镜、扫描电镜和透射电镜,对松江鲈鱼(Trachidermus fasciatus)成体皮肤的显微和亚显微结构进行了观察。结果表明,松江鲈鱼体表不同部位皮肤的厚薄不一,但基本结构相似。皮肤由表皮和真皮层构成。松江鲈鱼的皮肤裸露无鳞,表皮层较薄,由约4～8层细胞构成,主要由复层上皮细胞和黏液细胞及基底细胞组成。表层细胞呈扁平、多边形,细胞之间主要靠桥粒紧密连接,连接处形成增厚的边缘嵴状突起。表皮细胞游离面向内凹陷,表面形成指纹状微嵴。黏液细胞呈圆形或卵圆形,散布在上皮细胞之间。黏液细胞内的黏原颗粒具有椭圆颗粒状、均匀致密的块状和疏松丝状3种不同形态。真皮通过基膜与表皮相连,由稀疏层和致密层构成。真皮结缔组织在腹部较厚而在其他部位较薄。表皮与真皮连接处有色素层,头部、背部、尾柄和体侧皮肤色素细胞分布多,色素层明显,而腹部和颏部皮肤缺少色素。松江鲈鱼黄河群体真皮层中有角质棘状突起,而滦河群体则无。头部、体侧和尾柄处皮肤上还分布有侧线孔和表面神经丘等感觉器官。     

Freeze-fracture study of the epidermal cells of a teleost with particular reference to intercellular junctions and permeability to tracer

The plasmatic membranes, the intercellular junctions and the intercellular spaces of the epidermis of the fish Pimelodus maculatus were studied by freeze-fracture and by lanthanum methods. The observations has confirmed the presence of desmosomes. Gap junctions were not found and the tight junctions can be seen very rarely, arranged to form small discrete maculae. The finger-print pattern due to the microridges of the apical plasma membrane of the superficial cells was studied by direct replicas. The tracer penetrates all the intercellular epidermal spaces but failed to penetrate the dermis, suggesting the presence of a barrier at the dermo-epidermal level.     

Collagen type IV of the basal membrane of the epithelium of the skin in certain forms of pathology

Using immunofluorescence, attenuation or complete disappearance of type IV collagen antiserum reaction were shown in the epidermal basal membrane zone of SLE patients. Some positive material was revealed in the intercellular spaces of the epidermis. There is an inverse correlation between the amount of immune complexes in the dermo-epidermal region and the intensity of anticollagen serum reaction in the basal membrane zone. This can be explained by the toxic effect of immune complexes on collagen-synthesizing cells and the disturbance of this protein fixation in the basal membrane followed by its sequestration from the affected regions. Complex therapy including hemoperfusion restores normal collagen location in the epidermal basal membrane.     

HUMAN WOUND REPAIR : I. Epidermal Regeneration

A series of linearly incised superficial skin wounds was made on the forearms of young adult male volunteers. Wounds were sampled at several intervals between 3 hr and 21 days after wounding, for study by light and electron microscopy. The light microscopic observations show that regeneration of epidermis in human wounds conforms chronologically to that reported for the epidermis in superficial wound repair in laboratory animals. It is further shown that "ruffling" of cell membranes characterizes the cells of the migrating epidermis in early wound healing. This study reveals that the basement lamina and hemidesmosomes are established by epidermis in contact with the fibrin net at the base of early wounds. Epidermal cells in the wound environment are shown to be phagocytic. Analysis of the submicroscopic cytology of differentiating and maturing regenerated epidermis reveals that, in the sequence of events, the formation of filaments, basal lamina, and desmosomes is followed chronologically by evolution of keratohyalin granules and, subsequently, by keratinization of the surface epidermal elements. The entire sequence of migration, differentiation, and ultimate keratinization in the superficial wounds studied requires 3–5 days for completion.     

ELECTRON MICROSCOPE STUDIES OF EPIDERMAL MELANOCYTES, AND THE FINE STRUCTURE OF MELANIN GRANULES

Melanocytes and melanin granules have been studied by electron microscopy in normal human and cat skin, and in hyperplastic human skin lesions. The melanocytes have always been found as free cells within the epidermis,i.e., on the epidermal side of the dermal membrane. Melanocytes frequently rest on the dermal membrane or bulge towards the dermis. In such cases the uninterrupted dermal membrane is uniformly thin and smooth in appearance, in contrast with the regions alongside Malpighian cells, where it appears appreciably thicker and seemingly anchored to the basal cell layer. Two types of melanin granules have been distinguished according to their location in the melanocytes and to morphological characteristics which may only express different stages in the maturation of the granules: (a) light melanin granules in which a structure resembling a fine network is apparent; (b) dense melanin granules which, in osmium-fixed preparations, appear as uniformly dense masses surrounded by a coarsely granular, intensely osmiophilic shell. Treatment of sections of osmium-fixed tissues with potassium permanganate has revealed within the dense granules the existence of an organized framework in the form of a regular, crystalline-like lattice. It is suggested that this basic structure is protein in nature and may include the enzymatic system capable of producing melanin. The existence is reported of fine filaments located in the cytoplasm of melanocytes and morphologically distinct from the tonofilaments found in Malpighian cells.     

An ultrastructural study of connective tissue in mollusc integument III. Cephalopoda

We studied structure and ultrastructure of the subepidermal connective tissue (SEC) of the integument of three cephalopods (Sepia officinalis, Octopus vulgaris and Loligo pealii). In all species, three distinct regions of the SEC were recognised: (a) an outer zone (OZ) that included the dermal-epidermal junction, and consisted of a thin layer of connective tissue containing muscles, (b) an extensive middle zone (MZ) containing a compact network of collagen fibres and numerous cells, (c) an inner zone (IZ) of loose connective tissue that merged with muscular fascia. This arrangement differs from that in bivalves and gastropods and recalls vertebrate integument. The dermal-epidermal junction of cephalopods differed from that of bivalves, gastropods and mammals in that the epidermal cells did not possess hemidesmosomes, and their intermediate filaments terminated directly in the plasmamembrane. The thick (120-500 nm) basal membrane (BM) had a superficial zone containing a regular array of granules; a lamina densa composed of a compact network of small filaments and granules; and an IZ distinguished by expansions of granular material protruding into underlying structures. Collagen fibres contained fibroblast-derived cytoplasmic thread, running through their centres and were surrounded by granular material that joins them to adjacent fibres. The collagen fibrils were of medium diameter (30-80 nm) had the typical ultrastructure of fibrillar collagens, and were surrounded by abundant interfibrillar material. The hypodermis was loose, with a network of small bundles of collagen fibrils. Cephalopod integument appears to represent a major evolutionary step distinguishing this class of molluscs.     

The terminal web. A reevaluation of its structure and function

The apical cytoplasm of epithelial cells of the small and large intestines has been examined by freeze-etch techniques as well as conventional and high voltage electron microscopy of sectioned material to gain a better understanding of the fine structural organization of the terminal web region. In the small intestine the terminal web exhibits a distinct stratification caused by the association of different sets of filaments with the three members of the junctional complex. Individual filaments of this network are closely associated with the sealing elements of the tight junctions, the surface of the core microfilament bundles, and the intermicrovillar plasma membrane. This region of the terminal web is the apical zone. The adherens zone appears as a band of interwoven filaments of two different diameters extending across the cytoplasm at the level of the intermediate junction. Within this region of the terminal web, individual 60-70 A actin-like filaments separate from the bundles of core microfilaments to interact with one another and with filaments of similar diameter from the zonula adherens. 100 A tonofilaments also contribute to the adherens zone, presumably stabilizing the orientation of the actin-like filaments. The basal zone which underlies the adherens zone consists of closely interwoven bundles of tonofilaments that are anchored to and interconnect the spot desmosomes. Within the large intestine the cytoplasmic microfilaments form a looser and less clearly stratified network which nevertheless retains the same basic organization found in the small intestine. Transmembrane linkers appear to originate within the cytoplasmic plaques of the spot desmosomes, pass through the plasma membranes, and meet in a staggered configuration in the intercellular space; these linkers may thus mediate the actual mechanical coupling between the cytoskeletal networks of tonofilament bundles of adjacent cells. This integrated system of cytoplasmic filaments and intercellular junctions endows the apical cytoplasm with both the flexibility and the stability necessary for the normal functioning of the epithelium.     

Terminal differentiation of cultured human epidermal cells.

Three aspects of terminal differentiation of the epidermal keratinocyte have been studied in cell culture—the development of detergent-insoluble cytoplasmic filaments, the formation of a cornified cell envelope and the destruction of the cell nucleus.In the presence of lethally irradiated 3T3 cells, single human epidermal keratinocytes grow into stratified colonies. After the colonies become confluent, the culture enters a steady state in which the upper cells are shed from the surface of the cell layer like stratum corneum cells in vivo and are replaced by the proliferation of dividing cells in the basal layer. The cells shed into the medium are flattened and elongated squames, and are insoluble in solutions of sodium dodecylsulfate. Since the squames usually detach before their nuclei are digested, the cultures behave like some wet-surfaced, stratified squamous epithelia in that they possess little or no anucleate stratum corneum. The rates of proliferation and squame detachment in confluent cultures are increased by the presence of epidermal growth factor.Most of the squames harvested from the medium are permeable to trypan blue. The permeable squames may or may not have a visible nucleus, but squames not permeable to trypan blue nearly always possess a nucleus. When freshly detached squames containing nuclei are incubated in medium containing serum, their nuclei are digested and disappear within a few days. On the other hand, if the squames are washed and incubated in serum-free medium, their nuclei are not digested. This suggests that the permeable cell membrane permits a serum component essential for nuclear digestion to enter the cytoplasm.When growing colonies of epidermal keratinocytes are disaggregated and the cells suspended in medium containing methyl cellulose, they cannot multiply, but within a few days the cells become permeable to trypan blue and insoluble in sodium dodecylsulfate. This insolubility is due to disulfide linking of the proteins of the abundant cytoplasmic filaments, for the filaments are dissolved when β-mercaptoethanol is added as well, leaving the emptied cornified cell envelopes. Nuclear digestion follows some days later. In the absence of serum, cells become permeable and develop detergent-insoluble filaments and a cornified envelope, but, as in the case of spontaneously detached squames of surface cultures, their nuclei are not destroyed. Purified plasminogen supports nuclear destruction, whereas serum depleted of plasminogen does not.Earlier studies on intact skin have suggested that chemical gradients between epidermis and dermis might be responsible for the differentiation of the epidermal cells. In surface culture, basal cells multiply and nonbasal cells undergo terminal differentiation, even though all the cells are bathed in the same medium and the terminally differentiating cells have, if anything, better access to the medium than do the basal cells. Differentiation also begins in virtually all singly suspended cells uniformly exposed to the medium. The program of differentiation is therefore independent of the orientation of any chemical gradients in the cellular environment. Cell-cell contacts are not required for the development of detergent insolubility, the formation of the cornified envelope or the process of nuclear digestion, although they are essential for the formation of flattened squames. Unlike proliferation, which is strongly dependent upon fibroblast products, terminal differentiation proceeds in the absence of fibroblast support.     

The skin of primates. XXXVII. The skin of the pig-tail macaque (Macaca nemestrina)

The skin of the pig-tail macaque is basically similar to that of the rhesus monkey and the stump-tail macaque. The epidermis is thin and contains occasional basal melanocytes. The dermis, rich in elastic fibers, is practically free of pigment-containing cells. The upper dermis is highly vascular in the perianal region and sex skin. Cholinesterase-reactive nerve endings are plentiful beneath the friction surfaces of the pes and manus, mucous membranes, and junction of the hairy gluteus and glabrous ischial callosity. Hederiform-like endings are present in the eyelid, pinna, and frontal scalp. Apocrine and eccrine sweat glands occur throughout the hairy skin in a 2–3: 1 ratio. Both types are invested by nerves reactive for acetyl- and butyrylcholinesterase.     

Actin localization in male germ cell intercellular bridges in the rat and ground squirrel and disruption of bridges by cytochalasin D

Filaments about 6-7 nm in diameter were seen associated with germ cell intercellular bridges in detergent-permeabilized cells treated with tannic acid. Approximately 40-50 filaments were present subjacent to the bridge density. Filaments encircled the bridge channel in a manner similar to contractile ring actin filaments of dividing cells. NBD-phallacidin and myosin S-1 subfragments were employed to demonstrate that the filaments observed at intercellular bridges are actin. Intratesticular injection of a single dose of cytochalasin D, a specific inhibitor of actin filaments, caused certain intercellular bridges of spermatids to open within 3 hr after injection, leading to the production of symplasts. During bridge opening, remnants of bridge densities were gradually incorporated into the lateral aspect of the plasma membrane of the symplast. Thus actin, present in bridge structures, appeared to participate in maintaining certain intercellular bridges. A model of intercellular bridge structure is presented.     

Ultrastructure of the embryonic snake skin and putative role of histidine in the differentiation of the shedding complex.

The morphogenesis and ultrastructure of the epidermis of snake embryos were studied at progressive stages of development through hatching to determine the time and modality of differentiation of the shedding complex. Scales form as symmetric epidermal bumps that become slanted and eventually very overlapped. During the asymmetrization of the bumps, the basal cells of the forming outer surface of the scale become columnar, as in an epidermal placode, and accumulate glycogen. Small dermal condensations are sometimes seen and probably represent primordia of the axial dense dermis of the growing tip of scales. Deep, dense, and superficial loose dermal regions are formed when the epidermis is bilayered (periderm and basal epidermis) and undifferentiated. Glycogen and lipids decrease from basal cells to differentiating suprabasal cells. On the outer scale surface, beneath the peridermis, a layer containing dense granules and sparse 25-30-nm thick coarse filaments is formed. The underlying clear layer does not contain keratohyalin-like granules but has a rich cytoskeleton of intermediate filaments. Small denticles are formed and they interdigitate with the oberhautchen spinulae formed underneath. On the inner scale surface the clear layer contains dense granules, coarse filaments, and does not form denticles with the aspinulated oberhautchen. On the inner side surface the oberhautchen only forms occasional spinulae. The sloughing of the periderm and embryonic epidermis takes place in ovo 5-6 days before hatching. There follow beta-, mesos-, and alpha-layers, not yet mature before hatching. No resting period is present but a new generation is immediately produced so that at 6-10 h posthatching an inner generation and a new shedding complex are forming beneath the outer generation. The first shedding complex differentiates 10-11 days before hatching. In hatchlings 6-10 h old, tritiated histidine is taken up in the epidermis 4 h after injection and is found mainly in the shedding complex, especially in the apposed membranes of the clear layer and oberhautchen cells. This indicates that a histidine-rich protein is produced in preparation for shedding, as previously seen in lizard epidermis. The second shedding (first posthatching) takes place at 7-9 days posthatching. It is suggested that the shedding complex in lepidosaurian reptiles has evolved after the production of a histidine-rich protein and of a beta-keratin layer beneath the former alpha-layer.     

FORMATION AND ORIGIN OF BASAL LAMINA AND ANCHORING FIBRILS IN ADULT HUMAN SKIN

The purpose of this investigation was to study the formation and origin of basal lamina and anchoring fibrils in adult human skin. Epidermis and dermis were separated by "cold trypsinization." Viable epidermis and viable, inverted dermis were recombined and grafted to the chorioallantoic membrane of embryonated chicken eggs for varying periods up to 10 days. Basal lamina and anchoring fibrils were absent from the freshly trypsinized epidermis before grafting although hemidesmosomes and tonofilaments of the basal cells remained intact. Basal lamina and anchoring fibrils were absent from freshly cut, inverted surface of the dermis. Beginning 3 days after grafting, basal lamina was noted to form immediately subjacent to hemidesmosomes of epidermal basal cells at the epidermal-dermal interface. From the fifth to the seventh day after grafting, basal lamina became progressively more dense and extended to become continuous in many areas at the epidermal-dermal interface. Anchoring fibrils appeared first in grafts consisting of epidermis and viable dermis at five day cultivation and became progressively more numerous thereafter. In order to determine the epidermal versus dermal origin of basal lamina and anchoring fibrils, dermis was rendered nonviable by repeated freezing and thawing 10 times followed by recombination with viable epidermis. Formation of basal lamina occurred as readily in these recombinants of epidermis with freeze-thawed, nonviable dermis as with viable dermis, indicating that dermal viability was not essential for synthesis of basal lamina. This observation supports the concept of epidermal origin for basal lamina. Anchoring fibrils did not form in recombinants containing freeze-thawed dermis, indicating that dermal viability was required for anchoring fibrils formation. This observation supports the concept of dermal origin of anchoring fibrils.     

Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation

Fixation of HeLa cells with a mixture of 100 mM glutaraldehyde, 2 mg/ml tannic acid and 0.5 mg/ml saponin allows the tannic acid to penetrate intact cells without disruption of membranes or extraction of the cytoplasmic matrix. After subsequent treatment with OsO4 cytoplasmic structures are stained so densely that fine details are visible even in very thin (dark gray) sections. Actin filaments are protected from disruption by OsO4 so that straight, densely stained filaments are seen in the cell cortex, filopodia, ruffling membranes, and stress fibers. Stress fibers also have 15-18-nm densities similar in appearance to myosin filaments. Tannic acid staining reveals that the coats of coated vesicles, pits, and plaques have a 12-nm layer of amorphous material between the membrane and the clathrin basketwork. HeLa cells have very large clathrin-coated membrane plaques on the basal surface. These coated membrane plaques appear to be a previously unrecognized site of cell-substrate adhesion.     

Collagen XVI is expressed by human dermal fibroblasts and keratinocytes and is associated with the microfibrillar apparatus in the upper papillary dermis.

Indirect immunofluorescence staining of normal skin with affinity-purified antibodies revealed a conspicuous presence of collagen XVI at the dermo-epidermal interface where it occurs in close vicinity to collagen VII. In addition, the protein co-localizes with fibrillin 1 at the cutaneous basement membrane zone and the adjacent papillary dermis, but not in deeper layers of the dermis. Both fibronectin and collagen XVI are distributed throughout smooth muscles of hair follicles but do not co-localize. These data suggest, therefore, that collagen XVI contributes to the structural integrity of the dermo-epidermal junction zone by interacting with components of the anchoring complexes and the microfibrillar apparatus. A strong immunofluorescence signal associated with the extracellular matrix of individual cells was observed for keratinocytes or fibroblasts in monolayer cultures. Therefore, both cell types are likely sources of the protein also in situ. The rate of expression of collagen XVI mRNA in keratinocytes is about half of that in normal human skin fibroblasts. In both cell types, TGF-beta2 treatment results in an up-regulation of the collagen XVI-mRNA by approximately 50%. In keratinocytes, synthesis of collagen XVI protein and deposition to the cell layer and the extracellular matrix is stimulated fivefold and twofold, respectively. Since TGF-beta2 also upregulates the biosynthesis of other matrix macromolecules in the subepidermal zone the factor is likely to contribute to the stabilization of matrix zones near basement membranes in healing wounds.     

Expression of vimentin and prekeratins in solid and ascites variants of Zajdela hepatoma

Using indirect immunofluorescence with monoclonal antibodies against prekeratins and vimentin, the contents and intracellular distribution of these proteins have been investigated in Seidel hepatoma cells. In ascitic tumour, cells were organized in multicellular unilayer spheric or ellipsoid complexes with an inner cavity. Such complexes have been found to express intracellular vimentin and chaotically distributed prekeratin filaments. One of the constituents of the normal epithelial basal membrane--laminin was not found on the basal surface of cellular complexes but was localized in their inner lumens only. The expression of vimentin and prekeratin filaments was preserved in metastatic tumour cells found in paratracheal lymph nodes and in the majority of solid tumour cells induced by subcutaneous cell injections. In both cases tumour cells did not form regular morphological structures and laminin was visualized as extracellular granules and short fibrils. In several cases subcutaneous injections of Seidel hepatoma cells gave rise to adenocarcinomas. Prekeratin filaments in these tumours were localized predominantly under cellular membranes. Laminin "membranes" outlined the basal surface of adenomatous structures. Vimentin in these cellular structures was completely absent. It is suggested that vimentin expression in Seidel hepatoma cells was suppressed with morphological normalization of tumour structures manifested in the regular distribution of intercellular contacts and in basal membrane reconstitution.     

Ultrastructural studies of regenerating spines of the sea urchin Strongylocentrotus purpuratus I. Cell types without spherules

The fine structure of regenerating tips of spines of the sea urchin Strongylocentrotus purpuratus was investigated. Each conical tip consisted of an inner dermis, which deposits and contains the calcite skeleton, and an external layer of epidermis. Although cell types termed spherulecytes containing large, intracellular membrane bound spherules were also present in spine tissues, only epidermal and dermal cell types lacking such spherules are described in this paper. The epidermis was composed largely of free cells representing several functional types. Over the apical portion of the tip these cells occurred in groups, while proximally they were distributed within longitudinal grooves present along the periphery of the spine from the base to the tip. The terminal portions of apical processes extending from some of the epidermal cells formed a thin, contiguous outer layer consisting of small individual islands of cytoplasm bearing microvilli. Adjacent islands were connected around the periphery by a junctional complex extending roughly 200 Å in depth in which the opposing plasma membranes were separated by a narrow gap about 145 Å in width bridged by amorphous material. Other epidermal cells were closely associated with the basal lamina, which was 900 Å in thickness and delineated the dermoepidermal junction; some of these cells appeared to synthesize the lamina, while others may be sensory nerve cells. The dermis at the spine tip also consisted of several functional types of free cells; the most interesting of these was the calcoblast, which deposits the skeleton. Calcoblasts extended a thin, cytoplasmic skeletal sheath which surrounded the tips and adjacent proximal portions of each of the longitudinally oriented microspines comprising the regenerating skeleton, and distally, formed a conical extracellular channel ahead of the mineralizing tip. The intimate relationship between calcoblasts and the growing mineral surface strongly suggests that these cells directly control both the kinetics of mineral deposition and morphogenesis of the skeleton. Other cell types in the dermis were precalcoblasts and phagocytes. Precalcoblasts may function as fibroblasts and are possible precursors of calcoblasts. Closely associated with the basal lamina at the dermoepidermal junction were extracellular unbanded anchoring fibrils 150 Å to 200 Å in diameter. Scattered proximally among dermal cells were other extracellular fibrils, presumably collagenous, about 300 Å in diameter with a banding periodicity of 210 Å.