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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↗
PubMed2005

A novel bovine virus efficiently transduces inner ear neuroepithelial cells.

Disruption of the cellular composition or arrangement of the sensory epithelia due to hair cell or supporting cell damage leads to hearing loss and vestibular dysfunctions. These peripheral hearing disorders make good targets for gene therapy; however, development requires efficient gene transfer methods for the inner ear. Here we characterized the cellular tropism of a novel adeno-associated bovine virus vector (BAAV) in cultured rat inner ear epithelia. To help identify transduced cells, we used beta-actin-GFP as a reporter gene. We found that BAAV efficiently transduced auditory and vestibular hair cells as well as all types of supporting cells with no apparent pathological effects. The number of transduced hair cells significantly increased in both a dose- and a time-dependent manner. Transduction was independent of the cells' maturation state and was observed in both P2 and P10 cultures. Interestingly, even after several days of incubation with BAAV, hair cells demonstrated varying progression of beta-actin-GFP incorporation into the stereocilia. This suggests that the onset of viral transduction can occur throughout the course of the experiment. Of the other tested AAVs, AAV2 and AAV5 transduced only a small percentage of inner and vestibular hair cells, respectively, whereas no transduction was detected with AAV4.

Actins↗
PubMed2006

Asymmetric localization of Numb:EGFP in dividing neuroepithelial cells during neurulation in Danio rerio.

In the neural plate and tube of the zebrafish embryo, cells divide with their mitotic spindles oriented parallel to the plane of the neuroepithelium, whilst in the neural keel and rod, the spindle is oriented perpendicular to it. This change is achieved by a 90 degrees rotation of the mitotic spindle. We cloned zebrafish homologues of the gene for the Drosophila cell fate determinant Numb, and analyzed the localization of EGFP fusion proteins in vivo in dividing neuroepithelial cells during neurulation. Whereas Numb isoform 3 and the related protein Numblike are localized in the cytoplasm, Numb isoform 1 is localized to the cell membrane. Time-lapse analyses showed that Numb 1 is distributed uniformly around the cell cortex in dividing cells during plate and keel stages, but becomes localized at the basolateral membrane of some dividing cells during the transition from neural rod to tube. Using in vitro mutagenesis and Numb:EGFP deletion constructs, we showed that the first 196 amino acids of Numb are sufficient for this localization. Furthermore, we found that an 11-amino acid insertion in the PTB domain is essential for localization to the cortex, whereas amino acids 2-12 mediate the basolateral localization in the neural tube stage.

Amino Acid Sequence↗
PubMed2004

Vigorous neuronal differentiation of amplified and grafted basic fibroblast growth factor-responsive neurospheres derived from neuroepithelial stem cells.

Neuroepithelial stem cells (NESCs) have emerged as a possible donor material aimed at neural transplantation for the repair of damaged neural circuitry, particularly because of their propensity to differentiate into neurons. We previously ascertained in vitro that NESCs derived from rat early embryos could be amplified in culture containing basic fibroblast growth factors (bFGF), and that neurospheres grown for 7 days in the culture had a strong tendency to differentiate into neurons. In this report, we analyze immunohistochemically the biological nature of bFGF-responsive neurospheres derived from NESCs. We first succeeded in amplifying the number of NESCs from the mesencephalic neural plate of embryonic day 10 Wistar rats with the addition of bFGF. Grown neurospheres were labeled with bromodeoxyuridine (BrdU) in vitro and were stereotactically transplanted into the right striatum of the normal adult Wistar rat. Two weeks after transplantation, a viable graft in the host brain was observed. While many BrdU/Hu double positive cells were seen in the graft, and a few BrdU/nestin double positive cells were also seen, no BrdU/GFAP double positive cells could be identified. These results suggested that bFGF-responsive neurospheres derived from NESCs demonstrated a propensity to differentiate into neurons in the adult brain environment. Furthermore, following in vitro amplification of the original stem cell number with bFGF, the grown neurospheres preserved their propensity to differentiate vigorously into neurons. NESCs are thus suggested as a feasible candidate for intracerebral grafting donor materials aimed at reconstruction of damaged neural circuits.

Animals↗
PubMed2005

Comparative study of gill neuroepithelial cells and their innervation in teleosts and Xenopus tadpoles.

Peripheral O(2) chemoreceptors initiate adaptive cardiorespiratory responses to hypoxia in vertebrates. Morphological and physiological evidence suggests that, in fish, neuroepithelial cells (NECs) of the gill perform this role. We conducted a comparative examination in three species of teleosts (zebrafish, goldfish and trout) and larvae of the amphibian Xenopus laevis, using whole-mount gill preparations and confocal immunofluorescence, to elucidate the distribution, morphology and innervation of gill NECs. Nerve fibres were immunolabelled with the neuronal marker zn-12 and were associated with serotonin-immunoreactive NECs in the gills of all species tested. With the exception of trout, innervated NECs were present on all gill arches in the filaments and respiratory lamellae in fish and on homologous structures in Xenopus (i.e. gill "tufts", including respiratory terminal branches). Thus, the distribution and innervation of NECs of the internal gills of amphibians and teleosts are relatively well conserved, suggesting an important role for gill NECs as O(2) chemoreceptors in aquatic vertebrates. Furthermore, the size and density of gill NECs is variable among teleosts and developmental stages of Xenopus larvae and may be dependent on general gill dimensions or environmental conditions. This report constitutes the first comparative study of gill NECs in fish and amphibians and highlights the significance of gill NECs as an evolutionary model for studying O(2) sensing in vertebrates.

Animals↗
PubMed1986

Positioning and stability of mitotic spindle orientation in the neuroepithelial cell.

To verify non-random positioning and to define the stability of the mitotic spindle orientation in neuroepithelial cells of mouse foetuses, computer - assisted morphometric analysis at the light microscopy level was performed. It was confirmed that the mitotic spindle axis is positioned non-randomly in relation to the cell polarity axis and could be displaced only within a narrow range. This orientation was found to be attained at metaphase and it does not change until telophase is completed. However, in relation to the long axis of the neural tube the mitotic spindle axis was found to be positioned randomly. In the light of these findings centrosome movement and positioning are discussed.

Animals↗
PubMed1994

The engraftment of transplanted primary neuroepithelial cells within the postnatal mouse brain.

Primary neuroepithelial precursor cells carrying the reporter gene lacZ were transplanted into postnatal murine brain and assessed for their engraftment capacity. Freshly dissected precursors, derived from lacZ transgenic embryonic day 10 mouse brain, predominantly engrafted as discrete clusters, whereas the same precursors cultured in vitro with fibroblast growth factor-2, engrafted as single cells within the parenchyma of the hippocampus. Approximately 0.5% of the transplanted cells survived in the host brain for up to 3 months. Many of these cells displayed neuronal and astrocyte morphologies. These observations suggest that transplanted primary precursors derived from the embryonic brain can engraft and commit in situ to a variety of developmental fates.

Animals↗
PubMed2006

Apical localization of ASIP/PAR-3:EGFP in zebrafish neuroepithelial cells involves the oligomerization domain CR1, the PDZ domains, and the C-terminal portion of the protein.

Neurulation in zebrafish (Danio rerio) embryos is characterized by oriented cell divisions and the progressive establishment of cellular polarity. Mitoses in the neural plate and neural tube are planar, but in the neural keel/rod stage, the mitotic spindle rotates by 90 degrees, causing cell divisions to occur perpendicular to the plane of the neuroepithelium. The mechanisms and molecules that establish cellular polarity and cause the stereotypic orientation of the mitotic spindle during neurulation are largely unknown. In Caenorhabditis elegans and Drosophila, the PAR/aPKC complex has been shown to be involved in both establishment of cellular polarity and spindle orientation. Here, we show that the conserved N-terminal oligomerization domain (CR1) and the PDZ domains of ASIP/PAR-3:EGFP are involved in its localization to the apical membrane in zebrafish neuroepithelial cells. We further show that the C-terminal part of ASIP/PAR-3 contributes to proper localization and that the apical localization signals in ASIP/PAR-3 prevent the basolateral localization of a Numb:PAR-3 fusion protein. The parallel orientation of the mitotic spindle in the neural tube, however, is only weakly impaired upon overexpression of various ASIP/PAR-3:EGFP constructs.

Amino Acid Sequence↗
PubMed1996

Human olfactory neuroepithelial cells: tyrosine phosphorylation and process extension are increased by the combination of IL-1beta, IL-6, NGF, and bFGF.

Olfactory neuroepithelial cells (ONC) grown from biopsies of human donors are a novel cell culture system that may facilitate studies into normal and disease-related human neurobiology. We further characterized the expression of cell surface markers and intermediate filaments, and responses to neurotrophic factors by ONC. ONC are positive for cell surface markers N-CAM, PSA-N-CAM, neutral endopeptidase, N-aminopeptidase, NGF low-affinity receptor homologue (CD40), and transferrin receptor by flow cytometry for the intermediate filament proteins peripherin, vimentin, and NF-H by immunocytochemistry. Responses to neurotrophic factors measured were process outgrowth, cytoskeletal protein expression, and protein phosphorylation. Process outgrowth was increased by interleukin-beta 164-171 (IL-1beta) or by the combination of IL-1beta, interleukin-6 (IL-6), nerve growth factor (NGF), and basic fibroblast growth factor (bFGF). This combination of IL-1beta, IL-6, NGF, and bFGF (16NF) increased expression of two cytoskeletal proteins, NF-H protein and microtubule-associated protein tau. Application of the individual neurotrophic factors IL-1beta, IL-6, NGF, and bFGF increased protein phosphorylation, while 16NF produced an immediate increase in tyrosine phosphorylation of several proteins (MW of 40-80, 120, 150, and 190 kDa). The 16NF combination appears to act through a tyrosine-kinase-mediated pathway to induce process extension and increase NF-H expression. The ONC culture has the potential to be further explored to examine the relationship among process outgrowth, protein phosphorylation, and synergy between neurotrophin and cytokine receptor systems.

Antigens, CD↗
PubMed1985

The effect of N-nitrosobis(2-hydroxypropyl)amine on pulmonary neuroepithelial cells in Syrian golden hamsters.

The present study was carried out for determination of the effect of N-nitrosobis(2-hydroxyproyl)amine (BHP) on pulmonary neuroepithelial cells (NECs) in Syrian golden hamsters. This study was also carried out for elucidation of the histologic, histochemical, and ultrastructural features of NECs in the normal lungs of the hamsters. In the normal hamster lungs, NECs occurred exclusively in bronchioles and in bronchi lacking cartilage, appearing in groups as neuroepithelial bodies (NEBs). The number of NEBs was dependent on the age of the animals. Neonatal lungs contained large numbers of NEBs, but these seemed to disappear or remain as inconspicuous cell nests in adult lungs. Chronic BHP exposure (21 weeks) induced subepithelial spherical or fungating nodules composed of proliferating NECs, which projected into the bronchial lumens. The nodules appeared to arise from inconspicuous cell nests, which were rudiments of neonatal NEBs.

Aging↗
PubMed2000

Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepithelial cells.

Ionotropic glutamate receptors mediate fast forms of excitatory synaptic transmission in mature neurons and may play critical roles in neuronal development. However, the developmental stage at which neuronal cells begin to express functional receptors and their roles in lineage progression remain unclear. In the present study, neural precursor cells were isolated from the cortical neuroepithelium of embryonic day 13 rats, and rapidly expanded in serum-free medium in response to basic fibroblast growth factor. RT-PCR revealed the presence of mRNAs encoding AMPA(A), AMPA(C), KA(1), KA(2), NMDA(1), and NMDA(2D) subunits after 3 days in culture. The functional expression of AMPA/kainate and NMDA receptors was investigated using Ca(2+) imaging and whole-cell patch-clamp recording techniques in cells pulse-labeled with bromodeoxyuridine (BrdU) for 1-4 hr. The recorded cells were then double-immunostained for BrdU incorporation and neuron-specific beta-tubulin (TuJ1). The results show that AMPA/kainate and NMDA induced increases in cytosolic Ca(2+) and inward currents only in differentiating neurons. In contrast, proliferating (BrdU(+)TuJ1(-)) cells failed to respond to any ionotropic glutamate receptor agonists. Interestingly, Ca(2+) imaging revealed that a subpopulation of BrdU(+)TuJ1(+) cells also responded to AMPA, indicating the emergence of functional ionotropic AMPA/kainate receptors during terminal cell division and the earliest commitment to neuronal cell lineage. These in vitro results were supported by flow cytometric sorting of AMPA-responsive cells pulse-labeled with BrdU for 1 hr in vivo, which revealed that functional AMPA receptors appear in BrdU(+)TuJ1(+) cells under physiological conditions and may play a role in terminal cell division.

Animals↗
PubMed1995

Regulation of neurite outgrowth from differentiated human neuroepithelial cells: a comparison of the activities of prothrombin and thrombin.

The mechanism by which thrombin and prothrombin control neurite retraction was studied in Ad12E1HER10 human neuroepithelial cells. Morphological changes in differentiated cells were apparent within minutes of the addition of very low concentrations of thrombin (3 pM). Higher concentrations (2 nM) of prothrombin were required to elicit a similar response. Doses of thrombin and prothrombin sufficient to cause neurite retraction stimulated protein tyrosine kinase activity. Protein tyrosine kinase activation also correlated positively with thrombin- and prothrombin-induced phosphoinositide 3-kinase activation and InsP6 dephosphorylation. However, thrombin-stimulated Ins(1,4,5)P3 generation and intracellular Ca2+ mobilization only occurred at concentrations in excess of those needed to induce retraction. No fluctuations in Ins(1,4,5)P3 were detected after stimulation with prothrombin, and no rapid synchronized release of Ca2+ was observed, even at very high concentrations. Prothrombin did, however, cause small oscillations in the intracellular Ca2+ concentration, similar to those produced by low concentrations of thrombin, after approximately 30 min. We conclude that prothrombin- and thrombin-induced neurite retractions are not dependent on PtdIns(4,5)P2 and Ca2+ mobilization, but are more probably mediated through an effector mechanism involving protein tyrosine kinase activation. No intracellular Ca2+ mobilization, protein tyrosine kinase activity or neurite retraction was observed after treatment of cells with proteolytically inactive mutant thrombin (S205-->A). Prothrombin-mediated intracellular Ca2+ mobilization and neurite retraction were inhibited by hirudin, which was shown to interact with thrombin but not prothrombin. It is concluded that cleavage of prothrombin to thrombin is a necessary prerequisite for biological activity on differentiated Ad12E1HER10 cells and that differences in agonist concentration are capable of coupling the thrombin receptor to different pathways within the cell.

Blotting, Western↗
PubMed1998

Intrinsic polarity of mammalian neuroepithelial cells.

Progenitor cells in the mammalian forebrain can undergo either symmetric or asymmetric cell divisions by varying their cleavage orientation. In asymmetric divisions, cells distribute apically and basally localized proteins differentially to their daughters. Here we explore the intrinsic polarity of neuroepithelial cells in the developing telencephalon. Actin microfilaments are concentrated apically, forming beltlike structures that encircle spots of gamma-tubulin immunoreactivity. Staining for N-cadherin, beta-catenin, and the tyrosine kinase substrates pp120 and paxillin is also enriched at the lumenal surface, presumably due to the localization of these proteins at adherens junctions. Phosphotyrosine immunoreactivity is concentrated apically in rings, suggesting that adherens junctions are enriched for signaling molecules. In mitotic cells it appears that adherens junction proteins and phosphotyrosine immunoreactivity may be inherited either symmetrically or asymmetrically, depending on the cell's cleavage orientation during mitosis. The differential inheritance of junctional proteins may determine whether a daughter cell can respond to extrinsic signals after mitosis.

Actin Cytoskeleton↗
PubMed1994

Immunocharacterization of H-2Kb-tsA58 transgenic mouse hippocampal neuroepithelial cells.

From dissected fragments of embryonic H-2Kb-tsA58 transgenic mouse hippocampal neuroepithelium, we have derived a population of rapidly proliferating, nestin-positive conditionally immortal hippocampal neuroepithelial cells. Treatment with dibutyryl cAMP in non-permissive culture conditions resulted in cessation of cell division and differentiation of the precursor cells into neuronal or glial phenotypes.

Animals↗
PubMed1996

Determination of neuroepithelial cell fate: induction of the oligodendrocyte lineage by ventral midline cells and sonic hedgehog.

Near the floor plate of the embryonic neural tube there is a group of neuroepithelial precursor cells that are specialized for production of the oligodendrocyte lineage. We performed experiments to test whether specification of these neuroepithelial oligodendrocyte precursors, like other ventral neural cell types, depends on signals from the notochord and/or floor plate. We analyzed heterozygous Danforth's short tail (Sd/+) mutant mice, which lack a notochord and floor plate in caudal regions of the neural tube, and found that oligodendrocyte precursors did not appear at the ventricular surface where there was no floor plate. Moreover, oligodendrocytes did not develop in explant cultures of Sd/+ spinal cord in the absence of a floor plate. When a second notochord was grafted into an ectopic position dorsolateral to the endogenous notochord of a chicken embryo, an additional floor plate was induced along with an ectopic focus of oligodendrocyte precursors at the ventricular surface. Oligodendrocytes developed in explants of intermediate neural tube only when they were cocultured with fragments of notochord or in the presence of purified Sonic hedgehog (Shh) protein. Thus, signals from the notochord/floor plate, possibly involving Shh, are necessary and sufficient to induce the development of ventrally derived oligodendroglia. These signals appear to act by specifying the future fate(s) of neuroepithelial cells at the ventricular surface rather than by influencing the proliferation or differentiation of prespecified progenitor cells in the parenchyma of the cord.

Animals↗
PubMed1985

Ultrastructural analysis of basal neuroepithelial cells in dysraphic mice.

Ultrastructural aspects of the cellular pathology in the basal neuroepithelium of the hindbrain and spinal cord were analyzed in dysraphic loop-tail mice at nine days of gestation. Whereas the basal cytoplasm of the neuroepithelium in normal littermates showed a consistent electron density, the neuroepithelium in abnormal embryos was characterized by "light" and "dark" cells scattered randomly along the basal aspect of the hindbrain and spinal cord. In the abnormals, gaps occurred in the neural basal lamina, and the neuroepithelial cells often were in direct contact with cytoplasmic processes from mesenchymal cells and from notochordal cells; in normal littermates, contact was observed only between the intact and continuous neural basal lamina and mesenchymal cells and notochordal cells. Thus, it is possible that the pathological features observed ultrastructurally in the basal neuroepithelium in dysraphic embryos may represent faulty tissue interaction with adjacent notochordal and mesenchymal cells.

Animals↗
PubMed1997

Recovery of spatial learning by grafts of a conditionally immortalized hippocampal neuroepithelial cell line into the ischaemia-lesioned hippocampus.

Transient global cerebral ischaemia in rats causes relatively circumscribed and specific damage to the CA1 pyramidal cells of the dorsal hippocampus, along with a cognitive deficit manifest as difficulties in the performance of a range of spatial learning and memory tasks. Our previous studies have shown that restoration of behavioural performance in ischaemic rats by neural grafts taken relatively late in fetal development occurs only after local replacement of cells homotypic to those lost through the ischaemic insult. This lesion-plus-behaviour model therefore offers a powerful means for establishing whether multipotent embryonic neuroepithelial cells will engraft the damaged CA1, develop into appropriate neuronal phenotypes and produce behavioural recovery. Here we report that, in rats subjected to 15 min of global cerebral ischaemia, intrahippocampal implants of a conditionally immortal, multipotent cell line, directly derived from the embryonic day 14 hippocampal neuroepithelium of the H-2Kb-tsA58 transgenic mouse, selectively repopulated the lesioned CA1 pyramidal layer and restored ischaemia-induced deficits in acquisition of a hidden platform location in the Morris water maze.

Animals↗