Influence of continuous light and darkness on the secretory pinealocytes of<Emphasis Type="Italic">Heteropneustes fossilis</Emphasis>

In an earlier study onHeteropneustes fossilis, evidence of secretory activity in the pinealocytes had been demonstrated at the electron microscopic (EM) level and it was found to exist in two phases: a secretory phase (light cells) and a storage phase (dark cells). In the present investigation,H. fossilis was subjected to artificial photoperiods of continuous illumination and continuous darkness for a period of ten days and the effect on the secretory pinealocytes was studied at the EM level. Marked results were observed within the short period of ten days emphasizing the role of environmental photoperiod on the secretory activity of the pinealocytes. During continuous illuminated phase, both light and dark cells were observed: the light cells showed intense secretory activity and dark cells a storage one. During the dark phase both types of cells were present but in different metabolic states and neither of the cells demonstrated synthetic nor storage activity. Light cells were metabolically active but not secretory active and dark cells showed a necrotic condition. Phagocytotic activity of the dark cells was also seen. Intense neural activity was also observed during exposure to both the artificial photoperiods. The results highlight the role of light on the secretory activities of the pinealocytes of the catfish pineal organ.     

Morphometric analysis of photoreceptive,neuronal and endocrinal cell differentiation of avian pineal cells: an in vitro immunohistochemical study on the developmental transition from neuronal to photo-endocrinal property

Little is known about the developmental origin, determination and differentiation of different pineal immunoreactive cells in the avian group, and an experimental establishment is then required to explain the differentiation of cell types (i.e. photosensory, neural and secretory types). The present in vitro study suggests that the avian pineal organ is made up of multiple types of cells with different immunoreactivity at the ontogenic state (from embryonic day 9 to day 14), before it acquires the final photoendocrinal nature of the mature state. The morphometric analysis suggests that the developmental changes in the morphology of the quail pinealocytes appear to represent a condensed expression of the phylogenic development in the ontogeny. Several types of immunoreactive cells from a neuronal line were suppressed with maturation of developing pineal glands, while other cell types such as photoreceptive and endocrinal lines became more prominent. The melatonin level in the culture medium presented a high value up to 72 hr of culture, followed by a decrease as well as dampening of the level at the end of the culture possibly because the cultures were maintained in dark. The results of the present study, a combined analysis of morphometry and RIA, open a new line for research into the pineal development and cell differentiation.     

Morphologic evidence of photoreceptor differentiation of pinealocytes in the neonatal rat

The pineal body and the retina of the neonatal Sprague-Dawley rat were studied by light and electron microscopy, and the morphologic differentiation of the parenchymal cells of the pineal body was compared with that of the developing photoreceptor cells of the retina. Between the ages of 4 and 12 days after birth, some of the developing pinealocytes were observed to become elongated and polarized, with their nuclei located at one pole. "Synaptic" ribbons were observed within the cell body. At the opposite pole the cells developed elongated cell processes that initially contained microtubules and ribosomes. These cell processes projected into luminal spaces and were attached by structures resembling zonulae adherentes to the adjacent cells. Extending from the tips of the cell processes, cilia with a 9 + 0 arrangement were observed. Lamellated and vesicular membranes were noted at the tips of the cilia. Such morphologic differentiation, however, could be observed only in rats younger than 17 days. Comparison of the morphologic features of the neonatal pinealocytes with those of the developing retinal photoreceptor cells showed much similarity. It is suggested that the pinealocytes of the neonatal rat undergo "photoreceptor-like" differentiation during a transient neonatal period. Such morphologic differentiation may provide an explanation for light-induced biochemical changes described in neonatal rats whose eyes had been enucleated.     

Twenty-four-hour changes in pinealocytes,capillary endothelial cells and pericapillary and intercellular spaces in the pineal gland of the mouse

Summary Semiquantitative electron-microscopic observations on the pineal gland of dd-mice were carried out to determine whether 24-h rhythms exist in pinealocytes, pericapillary and intercellular spaces and capillary endothelial cells. Nuclear and cytoplasmic areas of pinealocytes and the area of condensed chromatin in pinealocytes showed inversely related circadian rhythms; the former two increased, whereas the latter decreased, during the light period. The extent of pericapillary and wide intercellular spaces exhibited 24-h changes, with an increase and decrease occurring during the light period and the dark period, respectively. The cross-sectional area of endothelial cells decreased and the number of fenestrae increased during the light period; this was reversed during the dark period. The results suggest that the increase in the nuclear and cytoplasmic areas of pinealocytes, the area of pericapillary and wide intercellular spaces and the number of fenestrae, and the decrease in the area of condensed chromatin and endothelial cells during the light period may be related to an increase in synthetic activity of pinealocytes in the mouse.     

Postnatal maturation of the parenchymal cell types in the rabbit pineal gland.

An ultrastructural study on the maturation of the parenchymal rabbit pineal cell types from the first postnatal day up to 120 days is presented. Two main cell types are distinguished from the first 24h of postnatal life. Pinealocytes of the types I and II display different developmental degrees. Both immature cell types are arranged in groups. In addition, type II pinealocytes form rosette-like structures. Both cell types progressively become isolated and display cell processes. The nucleus and the cytoplasm of type I pinealocytes are barely electrondense. During the postnatal period, the number of cytoplasmic organelles, cell processes and terminal clubs increase progressively. Terminal clubs are frequently seen near blood vessels. After 30 days, type I pinealocytes show characteristics of adult pinealocytes. However, the maturation of most type I pinealocytes does not complete until the 90th postnatal day. Type II pinealocytes present a fairly electrondense nucleus and cytoplasm. Mature forms can be seen after the 5th postnatal day. During the postnatal period, a close relationship is determined among type II pinealocytes and cell processes and terminal clubs of type I pinealocytes.     

Ultrastructural analysis of malformations of the embryonic neural axis induced by in vitro hyperglycemic conditions

Neural tube defects are the most common malformations associated with diabetic pregnancies. Although the teratogenic effects of excess glucose have been investigated in in vivo and in vivo studies, a cellular basis for neural tube defects has not been elucidated. We used rat embryo culture to study the organogenesis period of development, with excess d-glucose added to the serum medium to induce neural tube anomalies. Light and electron microscopic examination of control 12-day-old embryos grown 48 hours in culture revealed blastlike cells with few organelles or cellular processes. Twelve-day-old embryos cultured in excess d-glucose had advanced cellular maturation with differentiation, including the presence of free polysomes and copious cell processes, regardless of whether they had an open neural tube. Cytoarchitectural changes such as decreased numbers of mitotic figures with mitotic cells in the mantle layer were focally distributed throughout the neural epithelium but with predominance at the site of failed closure. In vivo studies failed to demonstrate neural processes in day 12 normal embryos. Fourteen-day-old embryos grown in utero also had foci of cell processes in the neural tube but to a much lesser degree than that observed in the in vitro day 12 glucose-exposed embryos. The cellular aberrations in the excess d-glucose-treated embryos are characteristic of a premature maturational change. Since they are present in excess d-glucose-exposed embryos with or without failure of neural tube closure, these maturational and cytoarchitectural changes may contribute to the cellular basis for neural tube defects.     

Phenotypic expression of photoreceptor and endocrine cell properties by cultured pineal cells of the newborn rat

Pineal glands of newborn rats were dissociated and maintained under cell culture conditions. The phenotypic expression of both photoreceptor and endocrine cell properties was investigated using immunohistochemical techniques (specific antibodies against opsin or serotonin). After one week in culture, a number of small round cells appeared on top of a sheet of flat epithelium. Among those cells, opsin-like immunoreactive cells were observed. These cells showed a neuron-like morphology with neuritic processes and often formed rosettes. Immunoreactivity was found on the plasma membrane of both the soma and cell processes. Serotonin-like immunoreactive cells were also differentiated in culture with two different morphological types of cells being found. One type resembled cultured serotonin-containing amacrine cells of the retina, and the other type had a flat, polygonal shape similar to that of pinealocytes. Both types of immunoreactive cells possessed fine neuritic processes. These results indicated that cell culture of rat pineal gland cells allowed expression of some properties, such as opsin synthesis and neuron-like morphology with long neuritic processes, that were not expressed in the intact rat pineal gland.     

Expression of Schwann cell markers by mammalian neural crest cells in vitro

During embryonic development, neural crest cells differentiate into a wide variety of cell types including Schwann cells of the peripheral nervous system. In order to establish when neural crest cells first start to express a Schwann cell phenotype immunocytochemical techniques were used to examine rat premigratory neural crest cell cultures for the presence of Schwann cell markers. Cultures were fixed for immunocytochemistry after culture periods ranging from 1 to 24 days. Neural crest cells were identified by their morphology and any neural tube cells remaining in the cultures were identified by their epithelial morphology and immunocytochemically. As early as 1 to 2 days in culture, approximately one third of the neural crest cells stained with m217c, a monoclonal antibody that appears to recognize the same antigen as rat neural antigen-1 (RAN-1). A similar proportion of cells were immunoreactive in cultures stained with 192-IgG, a monoclonal antibody that recognizes the rat nerve growth factor receptor. The number of immunoreactive cells increased with time in culture. After 16 days in culture, nests of cells, many of which had a bipolar morphology, were present in the area previously occupied by neural crest cells. The cells in the nests were often associated with neurons and were immunoreactive for m217c, 192-IgG and antibody to S-100 protein and laminin, indicating that the cells were Schwann cells. At all culture periods examined, neural crest cells did not express glial fibrillary acidic protein. These results demonstrate that cultured premigratory neural crest cells express early Schwann cell markers and that some of these cells differentiate into Schwann cells. These observations suggest that some neural crest cells in vivo may be committed to forming Schwann cells and will do so provided that they then proceed to encounter the correct environmental cues during embryonic development.     

Neural crest stem cell property of apical pulp cells derived from human developing tooth

Recent reports have described that NCSCs (neural crest-derived stem cells) are not only present in the embryonic neural crest but also in the adult tissues. Dental pulp is one of mesenchymal soft tissues origin from cranial neural crest cells, and thought to be a source of adult stem cells. Here, we investigated the existence of NCSC-like cells in apical pulp of human developing tooth. Human impacted third molars with immature apex freshly extracted were obtained. The cells derived from the apical pulp tissue not framed by dentin or the coronal pulp tissues were cultured by primary explant culture. APDCs (apical pulp-derived cells) and CPCs (coronal pulp cells) formed spheres under neurosphere culture condition. The number of spheres from APDCs was larger than that from CPCs. The sphere-forming cells derived from APDCs had self-renewal capacity, and expressed neural crest-associated markers (p75, Snail and Slug) and NSC (neural stem cell) markers (Nestin and Musashi1). The expression pattern of mesenchymal stem cell markers, CD105 and CD166, on the surface of sphere-forming cells derived APDCs was different from that of APDCs. These sphere-forming cells could differentiate into multiple mesenchymal lineages (osteoblasts, adipocytes, chondrocytes and smooth muscle cells) and neural lineage (neurons) in vitro, and generated ectopic bone tissues on the border of HA (hydroxyapatite) scaffold in vivo. The results of this study suggest that APDCs contain cells with characteristics of NCSCs reported previously in mice. Humans developing tooth with immature apex is an effective source of cells for neural crest lineage tissue regeneration.     

Diffusible rod-promoting signals in the developing rat retina.

We previously developed a reaggregate cell culture system in which embryonic rat retinal neuroepithelial cells proliferate and give rise to opsin-expressing rod photoreceptor cells (rods) on the same schedule in vitro as they do in vivo. We showed that the proportion of neuroepithelial cells in the embryonic day 15 (E15) retina that differentiated into opsin+ rods after 5-6 days in such cultures increased by approximately 40-fold when the E15 cells were cultured in the presence of an excess of postnatal day 1 (P1) neural retinal cells. In the present study, we have further analyzed this rod-promoting activity of neonatal neural retinal cells. We show that the activity is mediated by a diffusible signal(s) that seems to act over a relatively short distance. Whereas neonatal (P1-P3) neural retina has rod-promoting activity, E15 and adult neural retina, neonatal thymus, cerebrum and cerebellum do not. Finally, we show that neonatal neural retina promotes rod but not amacrine cell development.     

Effects of fetal bovine serum and serum-free conditions on white and dark axolotl neural crest explants

Summary Neural crest cells from both white mutant and dark (wildtype) axolotls (Ambystoma mexicanum) were cultured in increasing concentrations of fetal bovine serum (FBS; 2 to 20%). For each explant, the total number of cells that migrated and the percent of differentiated melanophores were recorded. At concentrations of FBS above 2% melanophore differentiation was essentially equivalent (32 to 59%) for both the white and dark neural crest cultures, but subtle differences in cell behavior and differentiation were found between the two phenotypes. By contrast there was a significant difference in the percent melanization of cells in serum-free control cultures, wherein melanophore differentiation in dark neural crest cultures was, on average, 18% compared to 5% in white cultures. Thus, contrary to all previously published work, white and dark neural crest cells are not intrinsically equivalent. Our culture results are discussed with regard to the probable in vivo conditions that cause the white phenotype. This research was supported by grant AR 34478 from the National Institutes of Health, Bethesda, MD, and a University of Kansas Biomedical Science support grant.     

Ultrastructural evidence for the presence of secretory cells in the pineal parenchyma ofHeteropneustes fossilis

There is an extensive literature dealing with the study of indoles, especially serotonin and melatonin, but with considerably less emphasis on the cells and cell types involved in the synthetic process. In the present electron microscopical investigation of the pineal end vesicle ofHeteropneustes fossilis, pinealocytes have been revealed in the pineal parenchyma characterized with extensive synthetic apparatus viz., rough endoplasmic reticulum, free ribosomes, lipid droplets, mitochondria and Golgi bodies. Two sub-populations of the pinealocytes are easily distinguishable on the basis of electron opacity and the preponderance of one or other morphological profile: light cells and dark cells. Light cells represent the active phase of secretion while dark cells represent the storage and release phase of secretion. A neuroendocrine role for the pineal body inHeteropneustes fossilis is suggested which may be significant in view of the nocturnal habit of the fish. Dedicated to my father, Prof. C B L Srivastava     

Prospective identification and culture of rat enteric neural stem cells (ENSCs)

Hirschprung’s disease (HD), a very common congenital abnormality in children, occurs mainly due to the congenital developmental defect of the enteric nervous system. The absence of enteric ganglia from the distal gut due to deletion in gut colonization by neural crest progenitor cells may lead to HD. The capacity to identify and isolate the enteric neuronal precursor cells from developing and mature tissues would enable the development of cell replacement therapies for HD. However, a mature method to culture these cells is a challenge. The present study aimed to propose a method to culture enteric neural stem cells (ENSCs) from the DsRed transgenic fetal rat gut. The culture medium used contained 15 % chicken embryo extract, basic fibroblast growth factor, and epidermal growth factor. ENSCs were cultured from embryonic day 18 in DsRed transgenic rat. Under inverted microscope and fluorescence staining, ENSCs proliferated to form small cell clusters on the second day of culture. The neurospheres-like structure were suspended in the medium, and there were some filaments between the adherent cells from day 3 to day 6 of the culture. The neurospheres were formed by ENSCs on day 8 of the culture. Network-like connections were formed between the adherent cells and differentiated cells after adding 10 % FBS. The differentiated cells were positive for neurofilament and glial fibrillary acidic protein antibodies. The present study established a method to isolate and culture ENSCs from E18 DsRed transgenic rats in the terminal stage of embryonic development. This study would offer a way to obtain plenty of cells for the future research on the transplantation of HD.     

Characteristics of a cell line obtained from a human hypernephroma

A stable tumorigenic cell line has been obtained from the human hypernephrome. The culture grew as monolayers of large polygonal cells with foamy cytoplasm and 1-2 nucleoli. The cells formed monolayer after the third day after planting into flask and then formed multilayered growth. The electron microscopic studies of hypernephrome sections revealed the dark and light cells. The hypernephrome cells have characteristics of tumor cells. On the 200th passage the cells are aneuploid with a model number of 62 chromosomes; an acrocentric chromosome in D group is noted as a marker one. The hypernephrome cells are free of Mycoplasma contamination, able to support the several virus replication.     

Chloroplast ultrastructure,photosynthetic apparatus activities and production of steviol glycosides in <Emphasis Type="Italic">Stevia rebaudiana in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

The accumulation of steviol glycosides (SGs) in cells of Stevia rebaudiana Bertoni both in vivo and in vitro was related to the extent of the development of the membrane system of chloroplasts and the content of photosynthetic pigments. Chloroplasts of the in vitro plants, unlike those of the intact plants, had poorly developed membrane system. The callus cells grown in the light contained proplastids of almost round shape and their thylakoid system was represented by short thylakoids sometimes forming a little number of grana consisting of 2–3 thylakoids. In cells of the etiolated in vitro regenerants and the callus culture grown in the dark, only proplastids practically lacking the membrane system were observed. All the chloroplasts having developed thylakoids and forming at least a little number of grana were equipped with photochemically active reaction centers of photosystems 1 and 2. Leaves of in vivo plants accumulated greater amount of the pigments than leaves of the in vitro plants. In both the callus culture grown in the light and the etiolated in vitro regenerants, the content of the pigments was one order of magnitude lower than that in leaves of the intact plants. The callus tissue grown in the dark contained merely trace amounts of the pigments. Leaves of the intact and the in vitro plants did not exhibit any significant differences in photosynthetic O₂ evolution rate. However, photosynthetic O₂ evolution rate in the callus cells was much lower than that in the differentiated plant cells. The in vitro cell cultures containing merely proplastids did not practically produce SGs. However, after transferring these cultures in the light, both the formation of chloroplasts and the production of SGs in them were detected.     

Immunocytochemical study on photoreceptor cell differentiation in the cultured retina of the chick

Photoreceptor cell differentiation was investigated in a dissociated monolayer culture of chick embryonic retinas with electron microscopic immunohistochemistry. The antibody was raised against bovine rhodopsin purified on SDS-polyacrylamide gel electrophoresis. In the developing retina, immunoreactivity was first recognized on the 14th day of incubation and was localized on the plasma membrane of the growing inner segment. On the 16th day, immunoreactivity was observed on some differentiating outer segments but not on inner segments. In the culture from 6 1/2-day-old embryonic retinas, immunoreactivity was found on the 7th day of culturing on the plasma membrane of large-sized neurons. Electron microscopic observations confirmed that such stained cells showed reaction product on the plasma membrane, and that they displayed fine structures characteristic of intact photoreceptor cells. They had a number of microvillous processes and often one thick process, both of which were intensely stained. Outer segment formation, however, was not observed in the present monolayer culture. These results indicate that opsin synthesis and its transport to the plasma membrane begins prior to and probably independently of outer segment formation.     

Neural crest cell behavior in white and dark larvae of Ambystoma mexicanum: time-lapse cinemicrographic analysis of pigment cell movement in vivo and in culture

The pattern of migration and motile activity of developing pigment cells of the Mexican axolotl, Ambystoma mexicanum, were analyzed by time-lapse cinemicrography in vivo and in culture. In vivo, melanocytes of dark (D/-) larvae migrate from dorsal to ventral in a highly directional manner. They are elongated and aligned parallel to the direction of migration. Nearly all protrusive activity occurs at their ventral, leading edges. Translocation occurs at a mean rate of 0.7 micron/min and involves alternate or simultaneous advance of the leading and trailing edges of the cell. Indirect evidence suggests that cytoplasmic flow is common. Directional migration occurs in apparent absence of contact between melanocytes. In white (d/d) larvae, protrusive activity is infrequent and the melanocytes move slowly or not at all. Explanted neural crest cells of dark and white larvae attach, spread, and differentiate into melanophores and xanthophores in culture. Individual cultured cells are unbiased in direction of protrusive activity and path of migration. Centrifugal spreading occurs by contacting inhibition of movement. Distribution of protrusive activity, polarity, and contact behavior changes with developmental age in vivo and in culture in ways that may be important in establishing the pigment pattern.     

Effects of light on somatic embryo development and abscisic levels in carrot suspension cultures

Carrot cells were cultured under various light spectra and intensities at different times following the initiation of suspension cultures from callus. The highest intensity white and blue light treatments were inhibitory to growth and somatic embryogenesis. Red and green light were not different from dark treatments which produced the highest total number of embryoids. After extended time in culture, carrot cells in blue light produced secondary embryoids and anthocyanin. Cultures in red light had multiple cotyledons and orange-pigmented radicles. Leafy cotyledons occurred in all light treatments. Abscisic acid production peaked at the heart stage of embryogenesis and synthesis was most pronounced in blue light. Red light enhanced development to the heart stage. Both the red and blue light spectra may be used to manipulate carrot cell cultures to optimize growth.     

Identified taste bud cell proliferation in the perihatching chick [published erratum appears in Chem Senses 1998 Aug;23(4):459]

Developing taste buds in the anterior mandibular floor of perihatching chicks were studied by high voltage electron microscopic autoradiography in order to identify proliferating gemmal cell types. Montaged profiles of 29 taste buds in five cases euthanized between embryonic day 21 and posthatching day 2 were analyzed after a single [3H]thymidine injection administered on embryonic day 16, 17 or 18. Results showed that dark cells comprised 55% of identified (n = 900 cells) and 62% of labeled (n = 568 cells) gemmal cells as compared with light, intermediate, basal or perigemmal bud cells. Dark cells had both a greater (P < 0.05) number of labeled cells and a greater amount of label (grains/nucleus) than the other four bud cell types, irrespective of injection day. The nuclear area (micron 2) of dark cells was not significantly larger (P > 0.05) than that of the other gemmal cell types and therefore cannot account for the greater amount for label in the dark cells. Interestingly, only dark cells showed a positive correlation (P < 0.003) between amount of label and nuclear area. Results suggest that, during the perihatching period of robust cell proliferation, dividing dark cells may give rise primarily, but not exclusively, to dark cell progeny.