A comparative study of the differentiation of dissociated nerve cells under different culture conditions

Summary In vitro differentiation of chick embryo brain cells was compared under several culture conditions. Morphological observations and acetylcholinesterase histochemical staining revealed that the development was similar in all conditions tested if cells have been derived from 7 days embryos. Considering the cultures from 11 days embryos, the cell dissociation by trypsin and the plastic surface proved to be the most favourable conditions in contrast to mechanical dissection and collagen surface.M. Sensenbrenner is Maitre de Recherche au CNRS.     

Development of rhythmic melatonin synthesis in cultured pineal glands and pineal cells isolated from chick embryo

The chick pineal gland exhibits circadian rhythms in melatonin synthesis under in vivo and in vitro conditions. A daily rhythm of melatonin production was first detectable in pineal glands isolated from chick embryos at embryonic day 16 and incubated under a LD cycle. All pineal glands isolated from 17-day-old and older embryos were rhythmic while no gland isolated at embryonic day 14 and 15 exhibited a daily rhythm in melatonin synthesis. Melatonin production in static cultures of embryonic pineal cells was rhythmic over 48 h if the cells were kept under a LD cycle. When embryonic pineal cells were incubated in constant darkness the rhythm in melatonin production was damped within 48 h. These results suggest that chick pineal cells from embryonic day 16 onwards are photosensitive but that the endogenous component of the melatonin rhythm is not completely developed at that age. A soluble analogue of cAMP stimulated and norepinephrine inhibited melatonin synthesis in cultured embryonic pineal cells. These findings indicate that the stimulatory and inhibitory pathways controlling melatonin synthesis in the mature pineal gland are effective in pineal cells isolated from chick embryos at least 2 days before hatching.     

Progesterone induced diminished incorporation of exogenous14C-amino acids in diapausing mouse blastocysts

Summary Under identical in vitro labeling conditions, significantly lower amounts of radioactivity were found a) in proteins of early diapausing blastocysts (days 7–9 p.c.) vs normal, late blastocysts (day 5 p.c.) and b) in proteins of days 19–23 p.c. diapausing blastocysts from ovariectomized mothers treated with progesterone vs similar embryos from untreated mothers. Thus, progesterone, which maintains the viability of embryos during prolonged diapause, causes diminished utilization of exogenous amino acids for protein synthesis in these embryos.Acknowledgment. This study was supported, in part, by NICHD grant HD-06234.     

Imprinting of the Peking duck (Anas platyrhynchos) and dependence on exposure to light during ontogenesis

Embryos of Peking ducks were either incubated in complete darkness up t o hatching or were put into light one week before hatching. Control embryos were incubated under dim light conditions which corresponded broadly to the natural conditions. Under standardized imprinting conditions the controls and both groups of the light deprived ducklings showed the 'following response'. Most of the dark-incubated embryos, however, did not distinguish between imprinting and test objects of different shapes. Since most of the embryos kept in darkness only for 21 days also failed to develop the capacity for shape discrimination, there is apparently a critical period for light influences on the development of this capacity at some time during the early prenatal period.     

Embryonic development of an insect myocardium

Summary Evidence from light and electron microscopic studies indicates that differentiation of myocardial cells in moth embryos begins at 6 days post-oviposition and is completed on eclosion. Fibrillogenesis, and development of cytoplasmic organelles and membranes are described. The heart is fully differentiated and functional at eclosion.Acknowledgments. This work was supported by the New Hampshire Heart Association and the National Science Foundation.     

Comparison of the effects of concanavalin A on cell growth and the transport of 3-O-methylglucose in chick embryo fibroblasts

The proliferative capacity of Chick embryo fibroblasts was modified by Con A treatment. Con A decreased the growth of fibroblasts from young embryos (8 days), whereas the lectin stimulated the growth of fibroblasts from older embryos (16 days). This differential effect of Con A did not result from changes in cellular permeability to thymidine, but rather from Con A induced modifications of hexose transport. Changes in hexose transport would cause, as a response, parallel modifications in glycolysis and hence energy charge, which would alter proliferative capacity.     

In vitro development of rat embryos obtained from diabetic mothers

Rat embryos of 9.5 or 10 days of gestation were removed from control or streptozotocin-diabetic mothers and cultured in normal rat serum (180 mg% glucose) or in diabetic serum (600 mg% glucose). The development of control embryos in normal serum was adequate. Embryos from normal mothers cultured in diabetic serum showed signs of developmental retardation. The development of embryos obtained from diabetic mothers was severely impaired, regardless of the gestational age or the culture medium. These results suggest that a diabetic maternal milieu produces irreversible effects in the embryo very early in gestation.     

Germ cell population of chick embryos incubated 5 1/2 days after ultraviolet irradiation of young blastoderms]

Chick blastoderms, incubated for 5 hrs., were wholly irradiated with ultraviolet light (2 537 A). With ultraviolet doses of 14,400 and 16,200 erg/mm2, the embryos, ofter morphologically abnormal, which developed from these blastoderms, were shown to contain, at 5 1/2 days of incubation, very few or no germ cells in the region of their primordia.     

Scanning electron microscopy of cells from hydroxyurea-arrested blastulae ofXenopus laevis

Summary Cells fromXenopus embryos blocked at the blastula stage by treatment with hydroxyurea have been isolated and cultured in vitro. The morphology of these cells has been compared with that of cells from normal embryos using scanning electron microscopy. Cells from such hydroxyurea-blocked embryos do not show the features, or changes in features, in culture shown by cells from normal embryos.Acknowledgments. We wish to thank Mr C. Veltkamp for his help with the electron microscopy, and Mrs J. Clumpus for technical assistance.     

Mast cells are present during angiogenesis in the chick extraembryonic vascular system

The extraembryonic vascular membranes of 3-day-18-day chick embryos were examined for the presence of mast cells. As early as 3.5 days mast cells were found on the area vasculosa. It is suggested that these cells have a role in angiogenesis of the chick extraembryonic vascular system.     

Ventral neural tube cells differentiate into hepatocytes in the chick embryo

A population of ventral neural tube cells has recently been shown to migrate out of the hind brain neural tube via the vagus nerve and contribute to the developing gastrointestinal tract. Since liver is also innervated by the vagus nerve, we sought to determine if these cells also migrate into the liver. Ventral neural tube cells in the caudal hindbrain of chick embryos were tagged with a replication-deficient retroviral vector containing the LacZ gene on embryonic day 2. Embryos were processed for detection of labeled cells on embryonic day 5 and 11. Labeled cells were seen in the liver on both days and identified as hepatocytes. Previously, it was believed that all hepatocytes develop from the gut endoderm. Results of the present study show an additional source for the formation of liver cells. Received 25 August 1998; received after revision 5 November 1998; accepted 5 November 1998     

Abortive effect of autologous anti-alpha-fetoprotein antibodies in the rat]

Female Commentry Rats 80--85 days old, were immunized by 5 intramuscular injections of Mouse alphafetoprotein (AFP) and then mated. After fertilization a supplementary injections was administered. The animals were bled at different times and killed immediately after the last bleeding on the 19 or 20th day of gestation. Titers of AFP and of autologous anti-AFP antibodies in the maternal blood were determined as well as the AFP concentration in the pooled amniotic fluids from live embryos of each litter. Compared to non-immunized control series, the total of live and dead embryos per litter in animals immunized with Mouse AFP showed no difference. However, the number of live embryos was on the average 50% lower than that in the control series. The serum titers of AFP and of antibodies to autologous AFP in immunized pregnant Rats bearing dead embryos decreased concomitantly with the number of live embryos. The results reported herein demonstrate that the presence of anti-autologous AFP antibodies in pregnant Rats correlates with the interruption of development in a significant proportions of embryos. This suggests that certain spontaneous abortions in the Rat and perhaps in other mammals can be explained by the rupture of immunological tolerance to autologous AFP.     

Imprinting of the Peking duck (anas platyrhynchos) and dependence on exposure to light during ontogenesis

Summary Embryos of Peking ducks were either incubated in complete darkness up to hatching or were put into light one week before hatching. Control embryos were incubated under dim light conditions which corresponded broadly to the natural conditions. Under standardized imprinting conditions the controls and both groups of the light deprived ducklings showed the following response. Most of the dark-incubated embryos, however, did not distinguish between imprinting and test objects of different shapes. Since most of the embryos kept in darkness only for 21 days also failed to develop the capacity for shape discrimination, there is apparently a, critical period for light influences on the development of this capacity at some time during the early prenatal period.Acknowledgments. We are grateful to Dr E. Pröve for his advice on the carrying out of our imprinting tests, to K. Weigel for help with the drawings and to Dr. J. R. Wolff for helpful discussions.     

The maternal protection reaction against a mother's rejection of her own larva in Salamandra salamandra L]

In vitro experiments have shown that maternal spleen cells from Salamandra salamandra are cytotoxic to cells from their embryos. This reaction can be inhibited by maternal serum. In this paper, we show that maternal serum protection acts through two effects: by inactivating spleen cells and by protecting embryonic cells. The more numerous the embryos are in a female, the stronger the protection is. The effect of the maternal serum does not appear to be individual specific.     

Complete replacement of serum in primary cultures of chick embryo brain cells by growth-promoting alpha-globulin

Summary Growth-promoting alpha-globulin (GPAG), a specific serum protein complex which induces mitotic activity in continually replicating metazoan cells in vitro, was shown in this study to support growth of astrocytes and mesenchymal cells as well as process formation of nerve cells isolated from cerebral hemispheres of chick embryos.     

Action du DDT sur le peuplement en gonocytes des ébauches gonadiques de l'embryon de Poulet agé de 6 jours

Summary A brief immersion of chick eggs in a DDT aqueous solution, before the incubation, provokes a strong reduction of the number of germ cells in the gonads, when the embryos are 6 days old. Some hypertrophied gonocytes are pycnotic. Hypotheses are proposed in order to explain the mechanism of DDT action.     

Complete replacement of serum in primary cultures of chick embryo brain cells by growth-promoting alpha-globulin

Growth-promoting alpha-globulin (GPAG), a specific serum protein complex which induces mitotic activity in continually replicating metazoan cells in vitro, was shown in this study to support growth of astrocytes and mesenchymal cells as well as process formation of nerve cells isolated from cerebral hemispheres of chick embryos.     

Differentiating abilities of avian somatopleural mesoderm.

Quail-to-chick grafting experiments were performed on 2-day embryos in order to test the differentiating abilities of the somatopleure. After orthotopic and heterotopic transplatations of different parts of quail somatopleural mesoderm into chick embryos it is demonstrated that avian somatopleural cells differentiate into skeletal elements, smooth muscles, tendons and connective tissues. However, skeletal muscle fibres do not originate from somatopleural cells.     

Survival of rabbit embryos after rapid freezing and thawing

Summary Frozen storage of rabbit embryos at the 16-cell stage in 2.0 M dimethylsulfoxide (DMSO) in phosphate-buffered saline (PBS) was achieved by a 2-step procedure. After storage for 10 days at–196°C they were revived by rapidly thawing at 500°C/min. On transfer of these embryos to pseudopregnant foster mothers, 50% survived to term. The difference in in vivo survival between frozen-thawed and frozen-thawed-cultured embryos was not significant.Acknowledgment. The authors thank the Director for the facilities. VHR is supported by Council of Scientific and Industrial Research (India).     

Intrinsic forces alone are sufficient to cause closure of the neural tube in the chick

Summary An isolated neural plate or a postnodal piece of early chick embryos, when cultured under appropriate experimental conditions, can undergo morphogenetic movements and form tubular structures closely resembling neural tubes of early chick embryos.This work was supported by grants from the NIH (NS 23200 and NS 21730) and the Busch Fund of Rutgers University.