Notochordal induction of cell wedging in the chick neural plate and its role in neural tube formation

Cells in the median hinge point (MHP) of the bending chick neural plate are tightly apposed to the underlying notochord. These cells differ from those in adjacent lateral neuroepithelial areas (L) in that MHP cells are short and mainly wedge-shaped and line a furrow, whereas L cells are tall and mainly spindle-shaped and do not line a furrow. Cell generation time also differs in these regions. These consistent differences are detectable only after the notochord has formed and established contact with the neural plate; it is unclear whether they result from self-differentiation or induction. Two experiments were performed to evaluate the hypothesis that MHP characteristics develop owing to inductive interactions between the notochord and overlying neuroepithelial cells. First, notochordless chick embryos were generated to determine whether midline neuroepithelial cells still developed typical MHP characteristics. In the absence of the notochord, such characteristics did not develop. Second, isolated segments of quail notochord were transplanted subjacent to L of chick hosts to ascertain whether the notochord is capable of inducing MHP characteristics in L cells. When transplanted notochordal segments established apposition with host L cells, the apposing L cells usually developed typical MHP characteristics. Collectively, these results provide strong evidence that the notochord plays an inductive role in the formation of MHP characteristics. This investigation further revealed that bending can occur in the absence of MHP characteristics, forming a neural tube with an abnormal morphology. Thus, the formation of such characteristics, particularly cell wedging, is not required for bending but plays a major role in generating the normal cross-sectional morphology of the neural tube.     

Patterns of neurepithelial cell rearrangement during avian neurulation are determined prior to notochordal inductive interactions

In the epiblast of elongating primitive-streak-stage avian embryos, MHP cells--short wedge-shaped neurepithelial cells contained within the median hinge point of the bending neural plate--arise from the midline prenodal and nodal area, whereas L cells--tall spindle-shaped neurepithelial cells constituting the lateral neural plate--arise from paired areas flanking the cranial primitive streak. These characteristic differences in neurepithelial cell shape are acquired as a result of inductive interactions with the notochord. Both MHP and L cells undergo extensive rearrangement (intercalation) during shaping and bending of the neural plate, but their pattern of rearrangement differs. MHP cells intercalate with other MHP cells and the population always spans the midline, whereas L cells intercalate with other L cells, remaining in bulk lateral to the midline. The following experiment was performed to establish whether these distinctive rearrangement patterns are determined prior to notochordal inductive interactions. Quail prospective MHP and L cells were transplanted isochronically and heterotopically to chick host blastoderms at stages prior to formation of the notochord (to wit, prospective MHP cells were transplanted into prospective L cell territory and vice versa) and the distribution, fate, and morphological characteristics of grafted cells were determined in chimeras collected 24 hr later. Our results demonstrate that heterotopic MHP and L cells do not adopt the rearrangement pattern characteristic of their new site; rather, they change their position so that grafted MHP cells intermix with MHP cells of the host and grafted L cells intermix with L cells of the host. Thus, patterns of neurepithelial cell rearrangement are determined prior to notochordal inductive interactions. When and how this determination occurs are topics for further studies.     

Fate mapping the avian neural plate with quail/chick chimeras: origin of prospective median wedge cells

The origin of prospective M cells, which are median neuroepithelial cells that become wedge-shaped during bending of the neural plate and eventually form the midline floor of the neural tube, was determined by constructing quail/chick chimeras and using the quail nucleolar marker to identify quail donor cells in chick host blastoderms. Two possible sites of prospective M-cell origin in the epiblast were examined: a single, midline rudiment located just rostral to Hensen's node and paired rudiments flanking the cranial part of the primitive streak. Our results suggest that M cells arise exclusively from the midline, prenodal rudiment. From this rudiment, M cells extend caudally throughout the entire length of the neuroepithelium. This new information on the origin of prospective M cells will aid in the analysis of their role in neurulation.     

Parasite immunity and the major histocompatibility complex

Parasite infestations offer fertile ground for investigation of the relationship between immunity, disease and the major histocompatibility complex (MHC). However, due to the complexities of parasite life cycles and the success of parasites in evading the immune response, immune reactions against the parasite often do not parallel protective immunity, and immunity does not imply lack of disease. — An additional level of complexity is introduced in some forms of parasite immunity by accessory effector cells, e. g., macrophages and eosinophils, that need to be activated for maximal effectiveness, and the activated form of these cells may partly compensate for a deficiency in specific immune responses. — It is not surprising, therefore, that polygenic effects operate in parasite immunity and reports linking non-MHC genes with parasite immunity far out number those linking MHC genes with it. From the reports that do link MHC genes with parasite immunity, two areas emerge that are interesting. First, the increased incidence of certainHLA genes in people with schistosomiasis who develop hepatosplenic disease may pinpoint individuals at risk of morbidity and direct early treatment to them. Second, mechanisms that intimately involve MHC products but are not linked to a particular MHC haplotype, may indicate newer areas in the investigation of parasite immunity.     

Identification of amino acid sequence in the hinge region of human vitamin D receptor that transfers a cytosolic protein to the nucleus

The localization of human vitamin D receptor (VDR) in the absence of its ligand 1,25-dihydroxyvitamin D(3) was investigated using chimera proteins fused to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fusion proteins were transiently or stably introduced into COS7 cells, and the subcellular distribution of the fusion proteins was examined. GFP-tagged wild-type VDRs were located predominantly in nuclei but with a significant cytoplasmic presence, while GFP alone was equally distributed throughout the cells. 10(-8) M 1,25-dihydroxyvitamin D(3) promoted the nuclear import of VDR in a few hours. To identify the NLS, we constructed several mutated VDRs fused to GFP. Mutant VDRs that did not bind to DNA were also localized predominantly in nuclei, while the deletion of the hinge region resulted in the loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to translocate to nuclei. These results indicate that 1) VDR is located predominantly in nuclei with a significant presence in cytoplasm without the ligand and 2) an NLS consisting of 20 amino acids in the hinge region facilitates the transfer of VDR to the nucleus.     

LHRH-like system in the brain of Xenopus laevis daud

Summary Rabbit antiserum to synthetic LHRH was used with the immunofluorescence technique to identify the LHRH-secreting neurons and their axonal pathways in the brain of Xenopus laevis. Three groups of immunoreactive neurons were identified: the first, in the telencephalon, is a paired group of cells scattered near the two telencephalic ventricles; the second group lies near the preoptic recess; the third group occurs in the ventral wall of the infundibulum. Two principal neuronal pathways were observed: Fibres originating from the dorsally located telencephalic neurons converge on the cephalic median plane where they form a single bundle behind the telencephalic furrow. This bundle descends towards the anterior border of the preoptic recess where it divides into two nerve bundles which pass on either side of the preoptic recess, run above the optic chiasma then cross the infundibular floor and finally terminate in the median eminence. The second pathway is more direct. The more ventrally located telencephalic LHRH cells give rise to this second pathway. Their axons converge with the other LHRH fibres near the lateral border of the preoptic recess. Most of the LHRH nerve fibres terminate in the median eminence although some terminate near the paired pars tuberalis. No reaction was observed after the use of antiserum absorbed with synthetic antigen.Equipe de Recherche associée C.N.R.S. n 492. This work was financed by the D.G.R.S.T., Contract n 7470046     

A Golgi study of the hypothalamus of Actinopterygii

Summary The posterior hypothalami of the polypteriform, Calamoichthys, and of the teleost, Anguilla, were studied by means of the Golgi technique. In Calamoichthys, the lateral lobes are not developed and the median lobe is simple. In Anguilla, the median (tuberal) lobe shows lophodendritic, CSF-contacting cells and horizontal cells in the periventricular grey and some reticular elements directed toward the cell-poor lateral areas. In the lateral lobes the periventricular grey is formed by multipolar neurons and a diffuse population of multipolar cells of uncertain identity. The nucleus diffusus lobi lateralis is formed by scarce multipolar neurons, often placed next to the external surface of the brain. The organization of the lateral lobes in Actinopterygii is reminiscent of highly developed integrative regions.Work performed under CNR Project Biology of Reproduction     

Intrathalamic afferent connections of the median center of the thalamus

Microiontophoretic local injection of horseradish peroxidase (HP) have been performed into the median center (MC). Many thalamic nuclei are sources of projections into MC, though the role of each nucleus is not equivalent. MC is predominantly connected with nonspecific formations (reticular, parafascicular, central-lateral, paracentral, ventromedial, paraventricular). Among them the reticular nucleus is distinguished, it sends its efferent fibers from the ventral, ventrolateral and lateral areas. In the anterior part of the reticular nucleus there are no HP-labelled cells. In MC little projections from specific nuclei (ventrobasal complex, ventrolateral nucleus, geniculate body) are presented, as well as simple projections from the associative nuclei. The data obtained are in keeping with electron physiological investigations.     

Localization of α1,2,3-subunit isoforms of Na,K-ATPase in cultured neonatal and adult rat myocardium: The immunofluorescence and immunocytochemical study

By indirect immunofluorescence and preembedding peroxidase-diaminobenzidine technique the localization of polyclonal and monoclonal antibodies against 1, 2 and 3 isoforms of the Na,K-ATPase were studied in rat myocardium.The 1-subunit was identified predominantly on sarcolemma of cultured myocytes, neonatal, as well as adult cardiocytes. The 2 signal was localized around nuclei of cultured cardiocytes, very weak signals were seen in neonatal and more intense signal, were dispersed throughout the adult myocytes. The 3-subunit immunoreactivity was weak and localized in cell processes connecting individual cultured cells, on sarcolemma and intercalated discs of neonatal cells and very weak in adult working myocytes. Cytochemically demonstrated ouabain resistant Na,K-ATPase localized in junctional sarcoplasmic reticulum may represent 1 isoenzyme which is directly involved in modulation of action potential fluxes.     

Tubular inclusions within polyploid nuclei ofGerris najas do not react with a monoclonal anti-β-tubulin antibody

Summary Variable aggregates, composed of tubules with a mean diameter of 19 nm, were found exclusively within polyploid nuclei of the midgut, Malpighian tubes, cyst cells, testis epithelium, and trophocytes ofGerris najas. The nuclear inclusions are always in direct contact with the nucleoplasm, and no other structures are associated with them. They appear most abundant within degenerating nuclei of the midgut surface epithelium, where they form paracrystalline bodies or spindle-shaped inclusions with tapered ends. Smaller fusiform inclusions occur in younger epithelial nuclei but not in the diploid nuclei of regenerative cells. In other tissues, mainly spindle-shaped inclusions can be observed, the longest (4.5 m) in cyst cell nuclei. The mean diameter of the tubules determined from transverse sections, resembles that of cytoplasmic microtubules and was verified statistically. The inclusions within trophocyte nuclei failed to react with monoclonal anti--tubulin antibody, although the antibodies could penetrate the nuclei after extensive lysis of the cells.     

Developmental pattern of three vesicular glutamate transporters in the rat superior olivary complex

Vesicular glutamate transporters (VGLUTs) mediate the packaging of the excitatory neurotransmitter glutamate into synaptic vesicles. Three VGLUT subtypes have been identified so far, which are differentially expressed in the brain. Here, we have investigated the spatiotemporal distribution of the three VGLUTs in the rat superior olivary complex (SOC), a prominent processing center, which receives strong glutamatergic inputs and which lies within the auditory brainstem. Immunoreactivity (ir) against all three VGLUTs was found in the SOC nuclei throughout development (postnatal days P0–P60). It was predominantly seen in axon terminals, although cytoplasmic labeling also occurred. Each transporter displayed a characteristic expression pattern. In the adult SOC, VGLUT1 labeling varied from strong in the medial nucleus of the trapezoid body, lateral superior olive, and medial superior olive (MSO) to moderate (ventral and lateral nuclei of the trapezoid body) to faint (superior paraolivary nucleus). VGLUT2-ir was moderate to strong throughout the SOC, whereas VGLUT3 was only weakly expressed. These results extend previous reports on co-localization of VGLUTs in the auditory brainstem. As in the adult, specific features were seen during development for all three transporters. Intensity increases and decreases occurred with both VGLUT1 and VGLUT3, whereas VGLUT2-ir remained moderately high throughout development. A striking result was obtained with VGLUT3, which was only transiently expressed in the different SOC nuclei between P0 and P12. A transient occurrence of VGLUT1-immunoreactive terminals on somata of MSO neurons was another striking finding. Our results imply a considerable amount of synaptic reorganization in the glutamatergic inputs to the SOC and suggest differential roles of VGLUTs during maturation and in adulthood. This work was supported by the Graduate Research School Molecular, physiological and pharmacological analysis of cellular membrane transport, DFG GRK 845/1.     

The lateral eyes of the scorpion,Androctonus australis

Summary The dioptric apparatus of the lateral eyes of the scorpion, Androctonus austrails, consists of a cuticular lens, but lacks a vitreous body. The retina is formed by (1) retinula cells displaying a contiguous network of rhabdoms; (2) arhabdomeric cells bearing a distal dendrite that contacts retinula cells via numerous projections and ends before the rhabdomere of the retinula cells; (3) pigment cells that ensheath retinula and arhabdomeric cells with the exception of the contact regions; and (4) neurosecretory fibres possibly originating in the supraesophageal ganglion. The ratio of the number of retinula to arhabdomeric cells is determined to be close to 2 1 in the three larger anterolateral eyes, in contrast to the median eyes where the ratio is 5 1.The construction of the dioptric apparatus as well as the anatomy of the retina imply that in the lateral eyes of Androctonus australis visual acuity is reduced. A certain degree of spatial discrimination, however, may be retained by the presence of a relatively high number of arhabdomeric cells. It is suggested that the lateral eyes of A. australis mainly function as light detectors, e.g., for Zeitgeber stimuli.Supported by grant no. FL 77/8-10 from the Deutsche Forschungsgemeinschaft     

Brain area-specific effect of TGF-beta signaling on Wnt-dependent neural stem cell expansion

Regulating the choice between neural stem cell maintenance versus differentiation determines growth and size of the developing brain. Here we identify TGF-beta signaling as a crucial factor controlling these processes. At early developmental stages, TGF-beta signal activity is localized close to the ventricular surface of the neuroepithelium. In the midbrain, but not in the forebrain, Tgfbr2 ablation results in ectopic expression of Wnt1/beta-catenin and FGF8, activation of Wnt target genes, and increased proliferation and horizontal expansion of neuroepithelial cells due to shortened cell-cycle length and decreased cell-cycle exit. Consistent with this phenotype, self-renewal of mutant neuroepithelial stem cells is enhanced in the presence of FGF and requires Wnt signaling. Moreover, TGF-beta signal activation counteracts Wnt-induced proliferation of midbrain neuroepithelial cells. Thus, TGF-beta signaling controls the size of a specific brain area, the dorsal midbrain, by antagonizing canonical Wnt signaling and negatively regulating self-renewal of neuroepithelial stem cells.     

Fine structure changes in the development of the nodules of Trifolium subterraneum L. and Medicago tribuloides Desr.

Summary Electron microscope observations of thin sections of nodules of subterranean clover and barrel medic, after fixation in KMnO₄ or OsO₄, show that following infection there is a marked increase in the amount of endoplasmic reticulum, in the number of ribosomes, Golgi bodies, mitochondria and proplastids in the host cells.As the infection thread approaches the nucleus, large gaps appear in the nuclear membrane. During the formation of the membrane envelopes around the rhizobia, after their release from the infection thread, the reticulum changes from a predominantly plate-like to a vesicular form. As the bacteroids develop the plastids of the host cells become filled with starch, and become aligned, with the mitochondria, against the cell walls of the host cells. Plastids in noninvaded cells also become starch-filled. Bacteroids and host cells enlarge further and finally the bacterioids occupy most of the cytoplasm of the host cell, except for the nuclear region and vacuole. With OsO₄ fixation the nucleoplasm, predominantly fibrillar before infection, with a dense staining nucleolus, becomes packed with dense ribosome-like (150 A° diameter) granules. No such changes occur in the nuclei of non-infected cells. In the proplastids and plastids many small, electron dense particles (60 A° diameter) (phytoferritin?) are observed.     

Neural tube closure defects caused by papaverine in explanted early chick embryos.

Papaverine (50 micrograms/ml) preferentially inhibited uplifting of neural folds in explanted stage 8 chick embryos. Affected neuroepithelial cells often lost their wedge-shaped and elongated appearance. Also, luminal surfaces of most affected cells were smoother than usual as evidenced by the marked decrease in the number of cytoplasmic extensions, but the integrity of other structures (including cytoskeletal components) was not noticeably affected. The observed changes in cell surface topography were due, at least in part, to the imparied ability of apical microfilaments to contract and their eventual relaxation. The "relaxing" effect of papaverine on neural folds could be reversed by subsequent treatment with ionophore A23187. Since papaverine and ionophore A23187 are known to alter the normal distribution of intracellular Ca2+ and changes in cell surface topography are correlated with contractile activities of apical microfilaments, papaverine elicits neural tube closure defects by lowering intracellular free Ca2+ levels, thereby relaxing contracted apical microfilaments in neuroepithelial cells.     

Scanning electron microscopy of rat maturation ameloblasts

Summary Post-secretory, maturation-phase ameloblasts were studied by scanning electron microscopy of freeze-fractured or dry-dissected rat incisors. These cells are in contact with the enamel which they secreted at an earlier time and which undergoes a process of continuing mineralization. The lateral intercellular compartment between maturation ameloblasts is sometimes continuous with the intercellular space of the papillary layer of the enamel organ, but often closed by basal ring contacts which correspond to terminal bars seen in transmission electron microscopy. The distal poles of the cells sometimes possess striated borders. Lateral cell surfaces may show longitudinal gutter-like depressions between ridges from which numerous intercellular connections arise; or a maze of lateral folds and ridges; or they may have mostly microvillous surface projections bordering a minimal intercellular space compartment. Preliminary correlations of groupings of basal, lateral and distal cell features indicate that basal-closed plus distal striated border cells may show every type of lateral surface. Cells without a striated border, whether open or closed basally, have ridge or maze lateral surfaces bordering a wide intercellular compartment. Basal-open plus striated border cells have microvillous or maze-like surfaces. These combinations of features are encountered a few times along the length of the maturation zone of individual incisors and suggest the existence of cyclical changes in the type of activity of maturation ameloblasts.     

Gamete Structure and Fertilization in the Barents Sea Sponge Leucosolenia complicata

The ultrastructure of male and female gametes of asconoid sponge Leucosolenia complicata(Calcispongiae, Calcaronea), a hermaphrodite species that reproduces in autumn, is described. The mature sponge's oocytes were up to 70 m in diameter, had no coatings, and contained a nucleus about 31 m in diameter with large nucleoli (up to 6.6 m). There were vacuoles with fibrillar contents typical of calcareous sponges in ooplasm. During vitellogenesis, a cluster of a great number of nurse cells developed above each oocyte from transformed choanocytes. Mature spermia of L. complicatalooked like orbicular cells about 2.5 m in diameter, with no acrosome or tail. The spermium nucleus (diameter about 2.2 m) was formed by incompletely condensed chromatin and was surrounded with a thin layer of cytoplasm of nonuniform thickness. In the thick layer of cytoplasm beyond the ribosomes, there were two or three mitochondria, dictyosomes, and electron-dense protein bodies lying freely under the nucleus. Fertilization occurred with the aid of a carrier cell. During spawning (mass release of spermia), any nurse cell complex can seize a spermium and transform into a carrier cell in situ. The transformation of a seized spermium into a spermiocyst was connected with the rapid isolation of the spermium nucleus from the protein body. Fertilization began with the penetration of the protein body into the oocyte cytoplasm. Only after this did the spermium's nucleus penetrate into the oocyte.     

Analysis of localization of cell-cycle regulators in Neurospora crassa

Most fungi are multinucleated organisms. In some fungi, they have asynchronous nuclei in the same cytoplasm. We analyzed a cell-cycle regulation mechanism using a model fungus Neurospora crassa, which can make heterokaryon cells. G1/S cyclin CLN-1 and cyclin-dependent kinase CDC-2 were tagged with different fluorescence in different strains and expressed. By forming a heterokaryon strain of these, two different fluorescence-tagged proteins were expressed in the same cytoplasm. CDC-2 was localized in all nuclei, whereas CLN-1 was not detected in most of the nuclei and was dispersed in the cytoplasm with small granular clusters. This indicates that in multinucleated fungi, cell-cycle regulators, similar to other proteins, are shared around the nuclei regardless of different cell-cycle stages. Moreover, each nucleus can select and use a special cell-cycle regulator only when it is necessary. Fungal nuclei may have a novel pickup mechanism of necessary proteins from their cytoplasm at the point of use.     

Nerve growth factor inhibits osmotic swelling of rat retinal glial (Müller) and bipolar cells by inducing glial cytokine release

Osmotic swelling of neurons and glial cells contributes to the development of retinal edema and neurodegeneration. We show that nerve growth factor (NGF) inhibits the swelling of glial (Müller) and bipolar cells in rat retinal slices induced by barium‐containing hypoosmotic solution. NGF also reduced Müller and bipolar cell swelling in the post‐ischemic retina. On the other hand, NGF prevented the swelling of freshly isolated Müller cells, but not of isolated bipolar cells, suggesting that NGF induces a release of factors from Müller cells that inhibit bipolar cell swelling in retinal slices. The inhibitory effect of NGF on Müller cell swelling was mediated by activation of TrkA (the receptor tyrosine kinase A), but not p75^NTR, and was prevented by blockers of metabotropic glutamate, P2Y₁, adenosine A₁, and fibroblast growth factor receptors. Basic fibroblast growth factor fully inhibited the swelling of freshly isolated Müller cells, but only partially the swelling of isolated bipolar cells. In addition, glial cell line‐derived neurotrophic factor and transforming growth factor‐β1, but not epidermal growth factor and platelet‐derived growth factor, reduced the swelling of bipolar cells. Both Müller and bipolar cells displayed TrkA immunoreactivity, while Müller cells were also immunostained for p75^NTR and NGF. The data suggest that the neuroprotective effect of NGF in the retina is in part mediated by prevention of the cytotoxic glial and bipolar cell swelling.

     

Shaping and bending of the avian neuroepithelium: morphometric analyses

Changes in the size and shape of the neuroepithelium were measured from serial transverse sections of 30 plastic-embedded chick embryos at stages 4-11. The neural plate folds into a neural tube during this period. Changes in volume, length, apical and basal widths, apical and basal surface areas, and thickness of the neuroepithelium were measured and correlated with the amount of folding that had occurred. These measurements were made to provide data for comparison with those available from other systems, to gain insight into the mechanisms of shaping and bending of the neuroepithelium, and to obtain normal parameters for eventual comparison with those obtained from embryos with induced neural tube defects. During stages 4-11, the volume, length, apical and basal surface areas, and lateral thickness of the neuroepithelium increase, whereas apical and basal widths and median thickness of the neuroepithelium decrease. Models are presented to demonstrate the effects of possible changes in neuroepithelial cell number, position, and size on the shaping of the neural plate.