In the course of an extensive comparative, structural and developmental study of the cranial and postcranial dermal skeleton (teeth and scales) in osteichthyan fishes, we have undertaken investigations on scale development in zebrafish (Danio (Brachydanio) rerio) using alizarin red staining, and light and transmission electron microscopy. The main goal was to know whether zebrafish scales can be used as a model for further research on the processes controlling the development of the dermal skeleton in general, especially epithelialmesenchymal interactions. Growth series of laboratory bred specimens were used to study in detail : (1) the relationship of scale appearance with size and age ; (2) the squamation pattern ; and (3) the events taking place in the epidermis and in the dermis, before and during scale initiation and formation, with the aim of searching for morphological indications of epithelial-mesenchymal interactions. Scales form late in ontogeny, generally when zebrafish are more than 8n0 mm in standard length. Within a population of zebrafish of the same age scale appearance is related to standard length, but when comparing populations of different age the size of the fish at scale appearance is also related to age. Scales always appear first in the posterior region of the body and the squamation then extends anteriorly. Scales develop in the dermis but closely apposed to the epidermal-dermal boundary. Cellular modifications occurring in the basal layer of the epidermis and in the dermis before scale formation clearly indicate that the basal epidermal cells differentiate first, before any evidence of differentiation of the progenitors of the scale-forming cells in the dermis. This strongly suggests that scale differentiation could be initiated by the epidermal basal layer cells which probably produce a molecular signal towards the dermis below. Subsequently dermal cells accumulate close to the epidermis, and differentiate to form scale papillae. The late formation of the scales during ontogeny is due to a late colonisation of the dermis by the progenitors of the scale-forming cells. Because of their late formation during ontogeny and of their regular pattern of development, scales in zebrafish represent a good model for further investigations on the general mechanisms of epithelial-mesenchymal interactions during dermal skeleton development, and in particular for the study of the gene expression patterns.

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Chorionic tissue is one of the major extrarenal sites of renin production, and as such, cultured chorionic cells are a potential model for in vitro studies of renin biosynthesis and regulation. Human chorionic cells were isolated from four chorions and maintained in tissue culture for a total of eight subcultures. Total renin production was considerable in the primary cultures, but fell gradually with successive passages. The cells could be frozen and thawed without losing their ability to divide or produce renin. Both the primary cultures and the subcultures contained a single type of elongated cell containing abundant rough endoplasmic reticulum and myofibrils, but no renin granules, suggesting that the cells had smooth muscle-like features. Immunocytochemistry indicated that they contained both renin and prorenin. The renin produced by the chorionic cells was not stored within the cells, but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had biochemical and immunological properties similar to those of pure human renin. The cells contained a renin mRNA that had the same size as that for renal renin (1.6 kilobases), confirming the synthesis of renin by these cells. The cells were also examined for the presence of other components of the renin-angiotensin system. Angiotensinogen and angiotensin I were not detected, but angiotensin-converting enzyme was present in extracts of primary and secondary cultured cells. beta hCG and progesterone were also found in the medium of primary culture. However, the production of beta hCG and progesterone fell after the primary culture, and beta hCG and progesterone were indetectable in secondary and tertiary cultures, respectively. These experiments suggest that these two hormones do not influence renin synthesis or vice versa. Thus, these cultures of human chorionic cells synthesized considerable quantities of prorenin and can provide a permanent source of nonrenal prorenin-producing cells.

A human juxtaglomerular cell (JGC) tumor was used for the immortalization of renin-secreting cells. The transfection of primary JGC with three different simian virus 40 (SV40) mutants resulted in the continuous production of renin-secreting cells. The most efficient renin-producing cells (producing about 400 pg of renin per 24 hr per ml of culture medium) were those transfected with the PAS SV40 mutant. The renin production was stable and the cell cultures have been maintained for >1 year. Two types of cells were cultured together and could not be separated: round and birefringent cells, which exhibited features of mast cells, and elongated cells containing myofilaments and secretory granules. Immunocytochemical staining showed the presence of renin in this latter cell type. The renin produced by the transfected cells was not stored within the cells but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had characteristics similar to those of pure human standard renin as to its enzymatic, immunologic, and biochemical properties, except that it was less glycosylated. These stable JGC tumoral cell lines provide a unique system for studying human renin biosynthesis and its regulation in vitro.In vitro models for studying renin biosynthesis and its regulation are needed because of the multiple factors that influence renin production and secretion in vivo. Such studies have been limited, however, by the difficulty in establishing juxtaglomerular cell (JGC) cultures, which is due primarily to the scarcity of these cells in the kidney. Primary cultures of renin-secreting cells have been described recently in the rat by Kurtz et al. (1) and in the human in two cases of JGC tumors (2, 3). Galen et al. (3) cultured renin-secreting tumor cells for >1 month in both primary and secondary cultures. Renin was synthesized as an inactive precursor that had all of the biochemical and immunological characteristics of pure standard renin. The sole attempt to maintain a JGC culture has been made by Rightsel et al. (4) in the rat by subculturing and cloning dissociated cortical cells from neonatal kidney.Recent studies have shown that endocrine cells can be transformed by simian virus 40 (SV40) or SV40 mutants. This allows the establishment of cell lines that maintain their differentiated state. These differentiated cell lines are able to secrete choriogonadotropin (5) and insulin (6). We were encouraged to use a similar approach to establish reninproducing cell lines by transformation of human JGC with SV40 mutants. In the present study, continuous JGC lines, producing renin, were obtained and characterized by various morphologic techniques. METHODSPrimary Culture. The renin content of the JGC tumor used was 1128 Goldblatt units (GU)/g of tissue. More than 95% of this renin was in the active form. A 5-g fragment of this tumor was dissociated by enzymatic digestion as described by Galen et al. (3). Cells were plated at a density of 106 cells per 25-cm2 flask and ...