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The cellular localization of a neuronal and a glial cell specific protein (14-3-2 and S-100, respectively) has been explored in mouse hypothalamus in order to trace cell lineages. This study was performed on fixed slices, at the light microscope level, by using either the indirect peroxidase-labeled immunoglobulin technique or immunofluorescence. In the adult, only S-100 immunoreactivity was found in the ependymal layer. In contrast, the magnocellular neurons of the preoptic area displayed strong 14-3-2 immunoreactivity. At neonatal stages (fetal day 17-postnatal day 3), both 14-3-2 and S-100 immunoreactivities developed simultaneously in the same cells lining the ventral part of the third ventricle. Transient detachment of some of these ventricular cells could be visualized before migration in the hypothalamus where they remained as bipotential cells up to postnatal day 10. Later in the development, they differentiated into separate cells, one type containing 14-3-2 and the other S-100, like neurons and glial cells. These results argue for a developmental stage during which cells lining the ventricle are bipotential and may thus be candidates for the role of stem cells for both neuronal and glial lineages.

Antibodies against a lysosomal membrane antigen (A-Ly-M) have recently been obtained and characterized (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526. They recognize a 100,000-mol-wt antigen immunologically related to a purified [H+,K+]ATPase from pig gastric mucosa. In the present study, we have localized this antigen during adsorptive endocytosis in rat prolactin cells in culture using cationized ferritin (CF) as a tracer. CF was rapidly internalized (after 5 min) in coated pits and vesicles that were labeled by antibodies against clathrin. The tracer was then delivered (after 15 rain) to vacuoles and multivesicular bodies. These structures were labeled with A-Ly-M. These organelles were devoid of acid phosphatase activity. At later stages (after 30 min) CF was observed within larger structures that were strongly stained by A-Ly-M and displayed a strong acid phosphatase activity. These findings clearly indicate that A-Ly-M react with prelysosomal and lysosomal compartments involved in the endocytic pathway in cultured prolactin cells. The membrane of these structures therefore contains antigenic determinant(s) related to the 100,000-mol-wt polypeptide. Our results suggest that the prelysosomal structure stained by A-Ly-M may represent in GH3 cells the acidic prelysosomal compartment recently described in the early steps of endocytosis in other cell types (Tycko, B., and F. R. Maxfield, 1982, Cell, 28:643-651).Acidification of endocytic vesicles involved as an early step in adsorptive or receptor-mediated endocytosis has recently been demonstrated using pH-sensitive fluorescent probes (4,12,19,26). This rapid acidification occurs before the fusion of endocytic vesicles with lysosomes and in the absence of lysosomal hydrolases.To explain the molecular mechanisms responsible for the acidification of both the prelysosomal and lysosomal compartments, the existence of an ATP-dependent proton pump has been postulated (for review, see reference 16). Such an activity has recently been demonstrated pharmacologically using isolated brain clathrin-coated vesicles (3), partially isolated endosomes, and lysosomes taken from fibroblasts and macrophages (4), or intact permeabilized fibroblasts (29).A new approach for studying membrane components that are possibly involved in the acidification of the endocytic pathway is provided by the recently characterized antibodies against a lysosomal membrane fraction that recognize a 100,000-mol-wt antigen immunologically related to a purified [H+,K+]ATPase from pig gastric mucosa (17, 18). Immunoelectron microscope localization of this antigen in normal rat kidney cells, rat macrophages, and hepatocytes revealed its presence not only in the membrane of secondary lysosomes but also in the membranes of several intracellular organelles known to be involved in receptor-mediated endocytosis (4,12,19,26). Moreover, some patches on the plasma membrane and some Golgi cisternae were also labeled (18).This prompted us to localize this...

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Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.

Dissociated fetal hypothalamic cells mainly taken from 14 day-old mouse fetuses were grown in vitro for increasing time (9 to 60 days). Soon after inoculation the cells partly reaggregated and attached. The small reaggregates were then interconnected by fibers bundles. After the first week the cultures were composed of a continuous basal monolayer of flat and transparent cells, over which various types of refractile cells were lying in discontinuous areas. The ultrastructural study enabled us to identify these cell types, to describe their spatial relationships, and to follow their evolution with time in culture. The basal cell formed several superimposed layers. With increasing age, they displayed typical features of astrocytes and of ependymal cells. The latter exhibited rhythmic ciliary movements in culture. The overlying cells corresponded to three types which were associated in small clumps: primitive neuro-epithelial cells, maturing as well as mature neurons and typical neurosecretory cells. The latter cells were found as early as 9 days of culture of 14 day-old fetal hypothalamic cells and retained their typical features up to two months. Neuronal processes formed very dense networks at the surface of the cultures and terminated within the basal layers. Axon and dendrites were precociously found and were still present after two months. Within axon terminals dense-core vesicles appeared at the same time as neurosecretory cells. Synaptic vesicles and synaptic junctions were found later on.

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