Nerve Growth Factor influences potassium movements in chick embryo dorsal root ganglionic cells

Recently we have shown that Nerve Growth Factor (NGF) influences the movement of Na⁺ across the membrane of chick embryo dorsal root ganglion (DRG) cells. When cell dissociates from 8-day embryonic chick DRG, equilibrated with ⁸⁶Rb⁺ (a K⁺ analog) in the presence of NGF, were transferred to NGF-free medium a marked loss of intracellular K⁺ occurred over several hours. The time course of K⁺ loss was similar to the time course of Na⁺ accumulation which occurs in the absence of NGF. NGF-deprived, K⁺-depleted DRG cells reaccumulated K⁺ within minutes of delayed NGF presentation, just as delayed NGF administration results in the rapid extrusion of Na⁺ from Na⁺-loaded cells. Restoration of K⁺ competence was dependent upon NGF concentration. The occurrence of this K⁺ response to exogenous NGF in other ganglionic preparations correlated with traditional responses to NGF in culture and previously observed Na⁺ responses. Neither the development nor the expression of the ionic defect (K⁺ depletion, Na⁺ filling) during NGF deprivation required the presence of both cations in the medium. NGF-dependent restoration of intracellular K⁺ in NGF-deprived chick DRG cells required the presence of intracellular Na⁺, and NGF-dependent extrusion of Na⁺ required extracellular K⁺. Thus NGF appears to influence the coupled (active) movements of Na⁺ and K⁺ across the membrane of its target cells, possibly by means of the classical Na⁺, K⁺-ATPase pump.     

Effects of pulsed magnetic fields on neurite outgrowth from chick embryo dorsal root ganglia

We have previously shown that neurite outgrowth from 6-day chick embryo dorsal root ganglia (DRG) in vitro was stimulated when nerve growth factor (NGF) and pulsed magnetic fields (PMF) are used in combination. 392 DRGs were studied in a field excited by a commercial PMF generator. We have now analyzed an additional 416 DRGs exposed to very similar PMF's produced by an arbitrary wave from generator and power amplifier. We reproduced our previous findings that combination of NGF and bursts of asymmetric, 220 μs-wide, 4.0 mT-peak pulses induced significantly (p<0.05) greater outgrowth than NGF alone, that fields without NGF do not significantly alter outgrowth, and that, unlike NGF alone, 4.0 mT fields and NGF can induce asymmetric outgrowth. The asymmetry does not seem to have a preferred orientation with respect to the induced electric field. Analysis of the data for the entire 808 DRGs confirms these findings. Importantly, we find similar results for pulse bursts repeated at 15 or 25 Hz. © 1996 Wiley-Liss, Inc.     

The neural tube origin of ventral root sheath cells in the chick embryo

The embryonic origin of peripheral nerve Schwann/sheath cells is still uncertain. Although the neural crest is known to be an important source, it is not clear whether the ventral neural tube also contributes a progenitor population for motor axons. We have used the techniques of immunohistochemistry, electron microscopy and quail-chick grafting to examine this problem. Immunohistochemistry with monoclonal antibody HNK-1 identified a cluster of immunoreactive cells in the sclerotome, at the site of the future ventral root. With the electron microscope, nucleated cells could not be seen breaching the basal lamina of the neural tube, exclusively in the region of the ventral root and preceding axon outgrowth. After grafting a length of crest-ablated quail neural tube in place of host chick neural tube, a population of quail cells was found localized to the ventral root exit zone, associated with the ventral root axons. Taken together, these observations support the possibility of a neural tube origin for ventral root sheath cells, although we found no evidence for a more extensive migration of these cells. The ventral root cells share certain phenotypic traits, such as HNK-1 immunoreactivity, with neural-crest-derived Schwann cells, but are not necessarily identical to them. We argue that while they may help motor axons to exit the neural tube at the correct position, they are unlikely to guide axons beyond the immediate vicinity of the neural tube.     

Regulation of Na+,K+ pump activity by nerve growth factor in chick embryo dorsal root ganglion cells

Nerve growth factor (NGF) is required for the growth and development of sensory and sympathetic neurons. Incubation of chick dorsal root ganglionic cells without NGF resulted in a decrease of active (Na+,K+-pump-mediated) K+ influx over a period of several hours. Addition of NGF to NGF-deprived cells caused 1) a return of the active K+ influx to the values occurring in cells continuously exposed to NGF, preceded by 2) a very rapid, but transient overstimulation of the Na+,K+-pump-mediated K+ influx. Restoration of normal Na+,K+-pump activity occurred at NGF concentrations of 1 biological unit/ml or greater, whereas the NGF concentration in the 1-100 biological unit/ml range affected the rapidity with which the pump restoration took place. The transient pump behavior was only observed in NGF-deprived cells and could not be elicited in NGF-supported steady-state cells or in cells having already received delayed NGF once. This transient Na+,K+-pump behavior was exclusively displayed in conjunction with a high intracellular Na+ concentration. Decreasing the external Na+ concentration below 70 mM reduced the hyperstimulation response to NGF, until at 10 mM Na+ the delayed presentation of NGF caused no overshoot at all. The effect of NGF on the Na+,K+-pump was specific for the NGF molecule and could not be mimicked by other proteins.     

The differentiation of prolactin cells in an organ culture of chick embryo adenohypophysis

We have studied differentiation of prolactin cells in explants of cephalic and caudal parts of Rathke's pouch of 4.5 day and 5.5 day old chick embryos after their incubation in vitro lasting for 7-8 days. Indirect immunofluorescence using an antiserum against bovine prolactin was used to detect prolactin cells in the cultures. Differentiation of prolactin cells was detected regularly in explants of the cephalic lobe of the adenohypophysis anlage in 5.5 day old embryos; under certain growth conditions prolactin cells were found in explants of the same lobe in 4.5 day old embryos. Prolactin cells were either absent or found in small numbers in cultures of the caudal part of adenohypophysis of 5.5 day old embryos. Our results provide evidence for the appearance of the committed precursors of prolactin cells in the Rathke's pouch at late stages of its formation and for their regional localization in the cephalic part of the anlage. This localization is in correspondence with the distribution of differentiated cells of this type in definitive adenohypophysis.     

Phenotypic modulation of chick embryo muscle cells in a suspension culture

Three-dimensional aggregates are formed in the suspension culture by myoblasts of the chick embryo femoral muscle. Cells present in these aggregates undergo mitotic divisions just as in a monolayer; thereafter they fuse with the formation of myosimplasts. Further stages of myogenesis are impaired under the conditions of the suspension culture. This was manifested in the formation of atypical, massive spherical or vesicle-like thick-walled myosimplasts with dozens or hundreds of randomly clustered nuclei. After the replating and attachment to a glass surface myosimplasts undergo gladual elongation and differentiate into muscle fibers. Thus the attachment to the artificial solid support is a necessary prerequisite for the completion of morphogenesis in vitro.     

Localization of myosin IIB at the leading edge of growth cones from rat dorsal root ganglionic cells.

Primary cultures of rat dorsal root ganglionic (DRG) cells were stained with isoform-specific antibodies against non-muscle myosin II. Antibodies against the brain type myosin (MIIB) stained the peripheries of growth cones and non-neuronal cells. Double staining of the cells with the anti-myosin antibodies and rhodamine-phalloidin or anti-actin antibodies indicated that MIIB co-exists, with F-actin, at the leading edge. Antibodies against platelet myosin stained neither leading edges nor neurites, but stained the cell bodies of neurons and the stress fibers of non-neuronal cells. These results suggest that MIIB functions in the motility of the leading edge of DRG cells.     

Kinetics of chick embryo cell types in culture

The growth kinetics and population doubling limits of chick embryonic fibroblasts, chondroblasts, and retinal pigment cells were compared. Chondroblasts were found to have a cumulative population doubling level (37 +/- 3 PDL) similar (p = 0.05) to that of control fibroblasts (42 +/- 2 PDL), in individual and pooled clones. While both cell types have similar doubling potential, the proportion of tritium-labeled nuclei decreases, and differs significantly as doubling level increases. This age-associated decline is due to an extension in the population doubling time. Direct cell-cycle analysis shows this increase to occur in the G1 phase. Furthermore, cartilage colonies maintain their phenotypic expression (metachromasia) throughout their lifespan under conditions of subcloning at sparse density. When fibroblasts derived from 15 day chick embryos are compared with fibroblasts from 10 day embryos (41 +/- 2 PDL) there is no significant difference (p = 0.05) in cumulative PDL or percent labeled nuclei, indicating that fibroblasts of different embryonic age have similar potential. The addition of hydrocortisone and insulin to the medium significantly shortens (25 +/- 2 PDL) the lifespan of 10 day chick fibroblasts. Kinetics of retinal pigment cells show a population doubling potential (29 +/- 1 PDL) different from fibroblasts and chondroblasts, suggesting that different cell types may not have similar limits on doubling potential when first determined in embryogenesis. Hydrocortisone and insulin have no effect on the growth kinetics or lifespan of retinal pigment cells in culture.     

Primary culture of chick embryo skeletal muscle on dextran microcarrier

We report the conditions to obtain primary suspension cultures using embryonic skeletal muscle from 12-day chick breast muscle. Further, the conditions are described to obtain scanning electron micrographs of whole cells and transmission electron micrographs of sections of plastic-embedded cells on microcarriers. A positively charged hydrated dextran microcarrier, Cytodex I (Pharmacia), provided support for the cells; the myogenic stages of proliferation, myoblast alignment and fusion to form myotubes coincided temporally with replicate cultures grown on gelatin-coated plastic dishes. Microcarrier-grown cells, including non-muscle cells, had microvilli, lamellipodia, bleb, and other surface modifications but no ruffling membranes. Myoblasts and myotubes on beads had fewer microvilli compared to homologous cells grown in the static culture medium of plastic dishes. Myoblasts aligned laterally during fusion, starting at 48 h. Myotube cytodifferentiation proceeded to myofibril formation by day 4 of microcarrier culture. The sarcomeres of aligned myofibrils had normal banding with an hexagonal lattice of thick and thin myofilaments in the A-bands. Caveolae intracellulares and sarcoplasmic reticulum were evident. Scaling-up to larger volumes promises to provide a cost-effective way to obtain a large harvest of cultured skeletal muscle which may prove especially useful for studies of minor constituents.