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R G Nagele

Publications and source records attributed to R G Nagele.

At least 37 records · Page 2Linked to original sources
PubMed1991

A morphometric and computer-assisted three-dimensional reconstruction study of neural tube formation in chick embryos.

The origin of the driving forces for neural tube formation remains uncertain but is currently thought to involve the participation of microfilament bundles situated in the apical ends of neuroepithelial cells. In the work presented here, we show how morphometric measurements that map local variations in the apical geometry of neuroepithelial cells (especially apical constriction) can provide information on the distribution of motive forces within the neuroepithelium during neural tube formation. When used in combination with computer-assisted, three-dimensional reconstruction, it becomes possible to analyze the morphometric data from a dynamic, three-dimensional perspective. As an example application of this method, we have used morphometry to evaluate the effects of ionomycin on the developing neuroepithelium. Treatment of early (stages 6-8) chick embryos with 5 microM ionomycin was found to cause rapid bending of the neuroepithelium within 1 min of exposure and a dramatic acceleration of the normal sequence of neural tube formation. Electron microscopy and morphometry revealed that this acceleration was coincident with a marked increase in the local degree of apical constriction of neuroepithelial cells, presumably a consequence of enhanced contractile activity of apical microfilament bundles. This work shows that transient elevation of free calcium levels can accelerate the usual sequential phases of NT formation. The rapidity of the response (hours of normal development reduced to minutes), increased prominence of apical microfilament bundles, and the enhanced degree of apical constriction strongly support a direct causal role for apical microfilament bundles and apical constriction of neuroepithelial cells in bending of the neuroepithelium.

Actin Cytoskeleton↗
PubMed1990

Neural tube formation in the mouse: a morphometric and computerized three-dimensional reconstruction study of the relationship between apical constriction of neuroepithelial cells and the shape of the neuroepithelium.

Morphometry and computerized three-dimensional reconstruction were used to study the relationship between apical constriction of neuroepithelial cells and the pattern of bending of the neuroepithelium in the developing neural tube of the 12-somite mouse embryo. The neuroepithelium of the mouse exhibits prominent regional variations in size and shape along the embryo axis. The complex shape of most of the cephalic neural tube (e.g., forebrain and midbrain) is due to the coexistence of concave and convex bending sites whereas more caudal regions (e.g., hindbrain and spinal cord) generally lack sites of convex bending and have a relatively simple shape. The apical morphology of neuroepithelial cells was found to be correlated more closely with the local status of bending of the neuroepithelium than with the specific region of the neural tube in which they are located. In areas of enhanced apical constriction, microfilament bundles were particularly prominent. Morphometry revealed that patterns of bending of the neuroepithelium were correlated almost exactly with those of apical constriction throughout the forming neural tube. These findings support the idea that apical constriction of neuroepithelial cells, resulting from tension generated by microfilament bundles, plays a major role in bending of the neuroepithelium during neural tube formation in the mouse.

Actin Cytoskeleton↗
PubMed1989

Intrinsic and extrinsic factors collaborate to generate driving forces for neural tube formation in the chick: a study using morphometry and computerized three-dimensional reconstruction.

The formation of the neural tube, the rudiment of the entire central nervous system, is one of the earliest morphogenetic movements. The origin of the driving forces for this process remains uncertain, but recent studies suggest the involvement of both intrinsic and extrinsic factors. In the present study, we have used morphometry, analysis of stereopair photographs of whole embryos, and computerized three-dimensional reconstruction to investigate the factors which constitute the bulk of the driving forces for neural tube formation in the developing midbrain of Hamburger and Hamilton stages 5-9 chick embryos. Results support the notion that neural tube formation is driven by a coordinated interplay of intrinsic and extrinsic forces. Initial bending of the neural plate along the midline of the embryo and uplifting of the neural folds is accomplished primarily through the combined action of intrinsic forces (resulting from apical constriction of neuroepithelial cells) and extrinsic forces (mostly a passive consequence of head-fold formation). However, once in the uplifted position, curling over of neural folds and closure of the neural tube is driven largely by apical constriction-mediated (intrinsic) forces that are generated by cells in the midlateral walls of the forming neural tube.

Animals↗
PubMed1989

Biomechanical basis of diazepam-induced neural tube defects in early chick embryos: a morphometric study.

The biomechanical basis of diazepam (Valium/Roche)-induced neural tube defects in the chick was investigated using a combination of electron microscopy and morphometry. Embryos at stage 8 (four-somite stage) of development were explanted and grown for 6 hr in nutrient medium containing 400 micrograms/ml diazepam. Nearly 80% of these embryos exhibited neural tube defects that were most pronounced in the forming midbrain region and typified by a "relaxation" or "collapse" of neural folds. The hindbrain and spinal cord regions were less affected. Electron microscopy revealed that neuroepithelial cells in diazepam-treated embryos had smoother apical surfaces and broader apical widths than did controls. Morphometric measurements supported this observation and further showed that these effects were focused at sites within the wall of the forming neural tube that typically exhibit the greatest degree of bending and apical constriction (i.e., the floor and midlateral walls). Overall results indicate that neural tube defects associated with exposure to diazepam are due largely to a general inhibition of the contractile activity of apical microfilament bundles in neuroepithelial cells. These findings 1) emphasize the important contribution of microfilament-mediated apical constriction of neuroepithelial cells in providing the driving forces for bending of the neuroepithelium during neural tube formation and 2) suggest that agents or conditions that impair their contractile activity could play a role in the pathogenesis of certain types of neural tube defects.

Animals↗
PubMed1988

Immunoelectron microscopic localization of actin in neurites of cultured embryonic chick dorsal root ganglia: actin is a component of granular, microtubule-associated crossbridges.

Axons have been shown to contain substantial quantities of actin distributed along their length. However, the general lack of information on the structure and organizational state of this protein in axons has made it difficult to assign it a functional role. In the present study, we used electron microscopic immunocytochemistry (immunogold labeling) on neurites from cultured embryonic chick dorsal root ganglia to: (1) determine the distribution of actin in neurites: (2) identify actin-containing structures; and (3) reveal whether or not actin is associated preferentially with microtubules. Results show that actin is present throughout neurites but is organized primarily into short filaments that are localized almost exclusively to granular, microtubule-associated crossbridges. We propose that these short actin filaments are part of the framework of a supramolecular 'carrier complex' for the slow component b polypeptides. In addition, actin-containing crossbridges are often closely associated with the surfaces of membrane-bound organelles. This suggests that actin and microtubule-associated crossbridges are involved somehow in fast axonal transport, although the nature of their participation in this process still needs to be resolved.

Actins↗
PubMed1988

A morphometric study of cytoskeletal reorganization in rat sciatic nerve axons following beta,beta'-iminodipropionitrile (IDPN) treatment.

Electron microscopy and morphometry were used to study changes in the organization of cytoskeletal elements (microtubules (MTs) and neurofilaments (NFs] in myelinated rat sciatic nerve axons resulting from exposure to beta,beta'-iminodipropionitrile (IDPN). Adult male rats were injected subepineurially with IDPN and sacrificed at intervals. Electron micrographs of thin sections through injected nerve segments revealed that MTs segregated from NFs in axons and formed prominent clusters. Morphometric measurements of intermicrotubular distances within these clusters showed that adjacent MTs had a fairly uniform spatial separation that appeared to be maintained by granular MT-associated cross-bridges. In addition, MT clusters underwent a progressive, but reversible, compaction during the first 12 h of IDPN treatment.

Animals↗
PubMed1988

Time-lapse photographic study of neural tube closure defects caused by xylocaine in the chick.

Sequential changes in the morphology of early chick embryos were, for the first time, photographically recorded. Embryos were explanted at stage 8 (four-somite) or 9- (six-somite) of development using New's technique and grown in nutrient medium (thin albumen) with or without a teratologic dose (200 micrograms/ml) of xylocaine. They were photographed using a Nikon Diaphot inverted microscope equipped with both phase-contrast optics and photomicrographic accessories maintained in an incubator. It was found, among other things, that a characteristic neural tube closure defect often seen in the midbrain and anterior portion of the hindbrain of xylocaine (200 micrograms/ml)-treated chick embryos was a consequence of failure of the neural tube to withstand the tension generated by the rapidly expanding cephalic region, which occurred, regardless of the stage at explanation, when corresponding control embryos had advanced to stage 10+ (11-somite) of development.

Abnormalities, Drug-Induced↗
PubMed1987

Studies on the mechanisms of neurulation in the chick: morphometric analysis of the relationship between regional variations in cell shape and sites of motive force generation.

Microfilaments, which are organized into bundles in the apical ends of neuroepithelial cells, are generally thought to play a major role in generating the driving forces for neural tube closure. Because of their proximity to the luminal surface, the contractile activity of these microfilament bundles results in conspicuous changes in the overall shape of neuroepithelial cells, most notably apical constriction and apical surface folding. In the present study, we have used morphometric methods and computer-assisted image analysis to reveal the distribution of microfilament-mediated forces in the developing midbrain during initial contact of apposing neural folds in chick embryos at Hamburger and Hamilton stage 8+ of development (Hamburger and Hamilton (1951) J. Morphol., 88:49-92). The degree of apical constriction, apical surface folding, and bending of the neuroepithelium was used as a barometer of local microfilament activity. Results indicate that cells forming the floor and midlateral walls of the developing midbrain consistently show a higher degree of apical constriction and surface folding than those at other locations. These same regions of the neuroepithelium also exhibit the greatest degree of bending. We conclude that the principal driving forces for closure of the neural tube, at the level of the midbrain, are concentrated in certain regions of the neuroepithelium (i.e., the floor and midlateral walls of the forming neural tube) rather than uniformly distributed.

Actin Cytoskeleton↗
PubMed1987

Studies on the mechanisms of neurulation in the chick: morphometric analysis of force distribution within the neuroepithelium during neural tube formation.

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197-205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V-shaped neuroepithelium), 2) the midlateral walls (during transformation of the V-shaped neuroepithelium into a C-shaped neuroepithelium), and 3) the upper walls (during the transformation of the C-shaped neuroepithelium into a closed neural tube).

Animals↗
PubMed1987

A new method for the preparation of 'double-fixed', quick-frozen, freeze-substituted cells for whole-cell transmission electron microscopy.

A method is described in which quick-frozen, freeze-substituted, cultured cells can be prepared for whole-cell transmission electron microscopy (WCTEM). This method is simple and reliable and can be carried out in most laboratories without special equipment. Cells grown of Formvar-carbon-coated nickel grids are quick-frozen in Freon 22, freeze-substituted in an ethanolic solution of glutaraldehyde, post-fixed in osmium tetroxide and critical-point-dried. The quality of ultrastructural preservation using this 'double fixation' protocol is comparable to that of conventional WCTEM. However, the combination of quick-freezing and WCTEM has the decided advantage over conventional WCTEM in that cellular activities are arrested almost instantaneously. Thus, this new method could potentially yield a more faithful representation of cytoarchitecture and is especially useful for studies on the structural basis of rapid cytoplasmic events which may remain undetected when using conventional fixation methods.

Cells, Cultured↗
PubMed1986

Toxic and teratologic effects of verapamil on early chick embryos: evidence for the involvement of calcium in neural tube closure.

Toxic and teratologic effects of verapamil, a calcium antagonist, on chick embryos explanted at stage 8 (four-somite stage) and cultured for 6-8 hours were investigated. In general, embryos responded to verapamil in a dose-related manner. Concentrations lower than 2 micrograms/ml had no apparent effect on the development of embryos. A concentration of 15 micrograms/ml significantly increased the incidence of embryos (approximately 80% of viable embryos) with neural tube closure defects and less numerous somites. Higher concentrations (e.g., 30 micrograms/ml) were embryotoxic and over 90% of the embryos were either severely malformed or dead after 8 hours of incubation. Compared to controls, verapamil-treated neuroepithelial cells had smoother apical surfaces and less conspicuous microfilament bundles. The deleterious effects of verapamil (15 micrograms/ml) could be reversed by subculturing the affected embryos, within 3 hours of treatment, on nutrient medium alone or on nutrient medium containing 25 micrograms/ml chlorotetracycline (CTC), a calcium agonist, the latter being more effective provided that treatment did not exceed 4 hours. Exposure of the developing neuroepithelium to 15 micrograms/ml verapamil for 3-4 hours resulted in a significant reduction in free Ca2+ levels, as revealed by the pyroantimonate precipitation method, throughout neuroepithelial cells. Overall results suggest that verapamil causes neural tube closure defects by reducing intracellular free Ca2+ levels, thereby relaxing apical microfilament bundles of developing neuroepithelial cells.

Animals↗
PubMed1985

Nerve growth factor induces neural differentiation in undifferentiated cell of early chick embryos.

Nerve growth factor (NGF) induced differentiation in postnodal pieces (PNPs) of stage 4 chick embryos. This induction was highly selective for neural tissue; no other structures developed in the NGF-treated PNPs. Furthermore, the number of PNPs showing neural differentiation was dependent on the concentration of NGF, but there was no correlation between the concentration of NGF (5-100 ng/ml) and extent of neuralization. The neural inducing capacity of NGF could be abolished by anti-NGF antibody. NGF-induced neural differentiation was accompanied by elevated intracellular levels of cyclic AMP. Exogenous cyclic AMP (175 micrograms/ml) was able to stimulate neural differentiation but, unlike NGF, induced other structures (e.g., notochord and pulsatile tissue). Overall results suggest that cells from chick embryos at developmental stages much earlier than previously thought are responsive to NGF and NGF or a a closely related substance may serve as a neural inducer in the chick embryo.

Animals↗
PubMed1985

Studies on the mechanisms of neurulation in the chick: interrelationship of contractile proteins, microfilaments, and the shape of neuroepithelial cells.

Electron microscopy and indirect immunofluorescence were employed to correlate the distribution patterns of major contractile proteins (actin and myosin) with 1) the organizational state of microfilaments, 2) the apical cell surface topography, 3) the shape of the neuroepithelial cells, and 4) the degree of bending of the neuroepithelium during neurulation in chick embryos at Hamburger and Hamilton stages 5-10 of development. Both actin and myosin are present at these developmental stages and colocalize in the neural plate as well as in later phases of neurulation. During elevation of neural folds, actin- and myosin-specific fluorescence is always most intense in regions where the greatest degree of bending of the neuroepithelium takes place [e.g., the midline of the V-shaped neuroepithelium (early neural fold stage) and the midlateral walls of the "C"-shaped neuroepithelium (mid-neural-fold stage)]. This intense fluorescence coincides with 1) a particularly dense packing of microfilaments and 2) highly constricted cell apices. After neural folds make contact, there is an overall reduction in both the intensity of apical fluorescence and the thickness of apical microfilament bundles, especially in the roof and floor of the neural tube. The remaining fluorescence in the contact area is apparently related to cellular movements during fusion of neural folds.

Actin Cytoskeleton↗
PubMed1985

Neural tube defects caused by local anesthetics in early chick embryos.

The effects of local anesthetics (ketamine HCl, lidocaine HCl, procaine HCl, and tetracaine HCl) on stage 8 (four-somite) chick embryos were investigated. In general, embryos responded to drug treatment in a dose-related manner during the first 6 hr of incubation. Concentrations of 500 micrograms/ml (ca. 2 mM) or higher were embryolethal, whereas 100-200 micrograms/ml (0.1-0.8 mM) preferentially inhibited elevation of neural folds. The latter effect was detectable within 3 hr of treatment and was readily reversible. Tetracaine was the most potent among the four local anesthetics tested at any given dose. Compared to controls, cells in the defective neuroepithelium were less elongated and exhibited smoother apical (luminal) surfaces, thinner microfilament bundles, and less intense actin-specific fluorescence. Furthermore, the effects of local anesthetics (100-200 micrograms/ml) on stage 8 chick embryos were not identical to those of cytochalasin D (0.05 micrograms/ml), colchicine (1 microgram/ml), or ionophore A23187 (25 micrograms/ml), although all treatments produced neural tube defects. Overall results suggest that local anesthetics inhibit closure of the neural tube through their disruptive action on the organization and function of microfilaments in developing neuroepithelial cells.

Anesthetics, Local↗
PubMed1985

A method for preparing quick-frozen, freeze-substituted cells for transmission electron microscopy and immunocytochemistry.

A quick-freeze, freeze-substitution method is described which employs glutaraldehyde as well as osmium tetroxide (OsO4) in a 'double-fixation' protocol comparable to that used for conventional transmission electron microscopy. Cultured cells are quick-frozen in Freon 22 and freeze-substituted in an ethanolic solution of glutaraldehyde. Specimens destined for TEM are postfixed in OsO4 in acetone, embedded in Epon-Araldite, and sectioned. This method yielded ultrastructural preservation which was comparable to that obtained from methods employing OsO4 alone as a freeze-substitution fixative. However, if glutaraldehyde is used alone as a freeze-substitution fixative, specimens can be processed for immunocytochemistry without additional treatment with permeabilizing agents.

Actins↗
PubMed1984

Examination of geniculate ganglion cells contributing sensory fibers to the rat facial 'motor' nerve.

Using a method to visualize HRP-containing cells in the geniculate ganglion (GG) in situ after decalcifying surrounding bone, we found that about 30% of the total (about 1000) GG cells contributed sensory fibers to the posterior auricular branch of the facial motor nerve. These cells are relatively large for GG cells in general. The remaining facial motor nerve branches, including those involved in vibrissal movement, contained few sensory afferent fibers originating from GG cells.

Animals↗