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Transient expression of a neurofilament protein by replicating neuroepithelial cells of the embryonic chick brain.

An immunohistochemical survey was carried out on frozen sections of the early embryonic chick brain between 1 and 6 days of incubation, with antisera to the three neurofilament proteins (NF-L, NF-M, NF-H). Large numbers of replicating neuroepithelial cells were found to express one of these proteins, NF-M, generations before the existence of any postmitotic neuroblasts (Days 1-2 1/2 of incubation). NF-L and NF-H could not be detected. Not all primordial brain regions contained NF-M-positive cells, but in those that did, every cell was positive. These regions included the dorsal forebrain, optic vesicles, and dorsal hindbrain, but not the dorsal midbrain. All cells in all regions of the cephalic neural tube contained vimentin, whether or not they also contained NF-M. This NF-M expression was transient in the sense that later generations of these NF-M-positive neuroepithelial cells became NF-M negative, before finally giving rise to some descendents that ultimately express all three NF proteins. This transient NF-M expression was found in certain other cells of early embryos, including cardiac myoblasts. The identity of the component in these early neural and nonneural tissues, that bound the antibody, was demonstrated to be identical to adult brain NF-M by one- and two-dimensional immunoblots. These findings demonstrate an unusual kind of biochemical heterogeneity among neuroepithelial cells, and they are relevant to considerations regarding lineage analysis and lineage "markers" in the vertebrate central nervous system.

Age Factors↗

[The reactions of neuroepithelial cells in different parts of the brain in mouse embryos to the action of ionizing radiation].

Dynamics of neuroepithelial cells reaction was studied in different areas of brain obtained from mouse embryos affected by X-rays irradiation on 10th, 12th and 13th days of pregnancy. It was shown that as differentiation starts after the irradiation regeneration processes arise along with cell death. This develops in all brain areas due to total mitotic division of unchanged neuroepithelial cells. Forming of rosettes on proliferative centers is mainly characteristic for the forebrain. After the irradiation of mice at 1-2 days pregnancy rosettes form only in rostral part of anterior encephalic vesicle. In irradiation on 13th pregnancy day a significant number forms in rostral, lateral and dorsal parts of the embryonic forebrain. Irradiation on the 14th day causes the forming of the rosettes in the forebrain ventral part.

Animals↗

Ca2+ mobilization and capacitative Ca2+ entry regulate DNA synthesis in cultured chick retinal neuroepithelial cells.

Release of Ca2+ from intracellular Ca2+ stores (Ca2+ mobilization) and capacitative Ca2+ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca2+ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and of SK&F 96365, an inhibitor of capacitative Ca2+ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [3H]-thymidine incorporation in a dose-dependent manner without affecting cell viability or morphology. The inhibition of [3H]-thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC50: approximately 4 microM) as the blockade of capacitative Ca2+ entry in the E3 retinal organ culture. At a concentration of 5 microM SK&F 96365. DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca2+ stores also inhibited [3H]-thymidine incorporation with IC50 values of 20-30 microM in the different cultures. Our results suggest the involvement of Ca2+ mobilization and capacitative Ca2+ entry in the regulation of DNA synthesis in the developing neural retina.

Animals↗

Directly linked soluble IL-6 receptor-IL-6 fusion protein induces astrocyte differentiation from neuroepithelial cells via activation of STAT3.

Signals of interleukin 6 (IL-6) are transduced by binding of IL-6 to its cell surface receptor (IL-6R) and subsequent association of the resultant IL-6/IL-6R complex with gp130, the signal transducing receptor component utilized in common by all the IL-6 family of cytokines. A soluble form of IL-6R (sIL-6R), which lacks transmembrane and cytoplasmic regions, retains the ability to bind IL-6 and signal through gp130. We show here that a fusion protein of sIL-6R and IL-6 without a polypeptide linker, termed FP6, induces differentiation of astrocytes from fetal mouse neuroepithelial cells as potently as a representative IL-6 family cytokine, leukaemia inhibitory factor (LIF). FP6 has a potential to activate a transcription factor, signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinases, ERK1 and ERK2, in these cells as does LIF. FP6 activates a promoter of the gene for an astrocytic marker, glial fibrillary acidic protein (GFAP), in neuroepithelial cells. This activation is virtually abolished by ectopic expression of a dominant-negative form of STAT3, or by introducing a point mutation into the STAT3 response element located in the GFAP promoter. These results suggest that FP6 induces astrocyte differentiation from neuroepithelial cells through STAT3 activation and that FP6 could be of use as a substitute for natural IL-6 family cytokines.

Animals↗

Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex.

Pax6 is a transcription factor with key functional roles in the developing brain. Pax6 promotes neuronal differentiation via transcriptional regulation of the Neurogenin2 (Ngn2) gene, although Pax6 expression appears in proliferating neuroepithelial cells before the onset of neurogenesis. Here, we identified Fabp7 (BLBP/B-FABP), a member of the fatty acid-binding protein (FABP) family, as a downregulated gene in the embryonic brain of Pax6 mutant rat (rSey2/rSey2) by microarray analysis. Marked reduction of Fabp7 expression was confirmed by quantitative PCR. Spatiotemporal expression patterns of Fabp7 in the wild-type rat embryos from embryonic day 10.5 (E10.5) to E14.5 were similar to those of Pax6, and expression of Fabp7 was undetectable in the rSey2/rSey2 cortex. The expression pattern of Fabp7 in the wild-type mouse embryo at E10.5 (corresponding to E12.5 rat) was different from that in the rat embryo, and no change of expression was observed in the Sey/Sey mouse embryo. Overexpression of exogenous Pax6 mainly induced ectopic expression of Fabp7, rather than of Ngn2, in the early cortical primordium. Interestingly, knocking-down FABP7 function by electroporation of Fabp7 small interfering RNA severely curtailed cell proliferation but promoted neuronal differentiation. We conclude that Fabp7 is a downstream gene of Pax6 transcription factor in the developing rat cortex and essential for maintenance of neuroepithelial cells during early cortical development.

Animals↗

[Ultrastructure of the neuroepithelial cells of the respiratory portion of the lungs in experimental inflammatory diseases of the lungs].

Neuroepithelial cells (NEC) and neuroepithelial bodies (NEB) were found in the lungs of rats among cells of the alveolar epithelium. The peculiar features of NEC structure included microvilli with microfibrils penetrating deep into the cytoplasm, and a complicated structure of the endoplasmic reticulum: reticulum membrane complex. NEC's are arrayed on the alveolar basal membrane singly, in pairs and in larger numbers forming NEB. The latter consist of cells differing in shape, size, and ultrastructure. Most of them have no microvilli with microfibrils inside. In chronic pneumonia NEC's occur more frequently, the ultrastructure of most cells being well-developed.

Animals↗

Selective in vitro blockade of neuroepithelial cells proliferation by methylazoxymethanol, a molecule capable of inducing long lasting functional impairments.

In order to characterize the antiproliferative effect of methylazoxymethanol neuroepithelial cells derived from the rat striata primordia at embryonic day 14 have been exposed to graded doses of this compound. It was found that methylazoxymethanol application to striatal neuroblasts elicits a blockade of cell proliferation at a dose which does not interfere with cell survival. By using synchronized cells and short term exposures to this compound, we found that the antiproliferative effect of methylazoxymethanol is strikingly correlated to the number of cells actively dividing in culture, thus indicating that the cells targeted by methylazoxymethanol must be in an active mitotic phase. To test for the selectivity of action of Methylazoxymethanol for dividing neuroblasts either cultures composed of mature proliferating astrocytes or muscle cells have been subjected to the same treatment. It has been observed that astrocytes proliferation was not affected by the dose of methylazoxymethanol shown to be effective on neuroepithelial cells. Finally we demonstrated that methylazoxymethanol is able only transiently to interfere with smooth muscle cell division, further supporting its selectivity of action within the developing CNS.

Animals↗

PAR3 acts as a molecular organizer to define the apical domain of chick neuroepithelial cells.

Neural progenitors in the vertebrate nervous system are fully polarized epithelial cells, with intercellular junctions at the apical region. These progenitor cells remain within the neuroepithelium throughout neurogenesis, and will ultimately give rise to all the neurons in the mature nervous system. We have addressed the role of the PAR polarity complex in vertebrate neuroepithelial polarity and show that PAR3 functions as the initial scaffold to assemble and organize the PAR complex at the apical region of neuroepithelial cells, coordinating also the recruitment of additional polarity complexes and junction-associated proteins to the same region, while restricting other polarity proteins to the basolateral membrane. We propose that PAR3 acts as a molecular organizer to connect the acquisition of apico-basal polarity with the positioning and formation of junctional structures in neuroepithelial cells, a function of upmost importance for the morphogenesis of embryonic neural tissue and the process of neurogenesis.

Animals↗

Neuropoietin induces neuroepithelial cells to differentiate into astrocytes via activation of STAT3.

Neuropoietin (NP) is a recently identified member of the interleukin (IL)-6 family of cytokines, which share glycoprotein 130 (gp130) as a signal-transducing receptor component, and is highly expressed in embryonic brain. In this study, we show that NP has the potential to induce neuroepithelial cells to differentiate into astrocytes. NP stimulation leads to promoter activation of the gene for an astrocyte marker, glial fibrillary acidic protein (GFAP), which is clearly inhibited by either expression of a dominant-negative form of a transcription factor, signal transducer and activator of transcription 3 (STAT3) or by a nucleotide-substitution in the STAT3-binding element within the gene promoter. We further show that NP induces binding of endogenous STAT3 to its cognate sequence within the gfap gene promoter in neuroepithelial cells. Moreover, like the other IL-6 cytokine family members, NP promotes astrocyte differentiation in a synergistic manner with bone-morphogenetic protein (BMP)-2. Taken together, our data indicate that NP can be considered as a new astrocyte-inducing cytokine in the developing brain.

Animals↗

Induction of differentiation of rat retinal, germinal, neuroepithelial cells by dbcAMP.

Little is known about the factors that regulate the production of neurons during the development of the vertebrate central nervous system (CNS); however, evidence from several neuronal cell lines suggests that an increase in intracellular cAMP might trigger the process of differentiation. To determine if a similar process is involved in differentiation during normal CNS neurogenesis, we raised the intracellular level of cAMP in primary cultures of mitotically active, germinal neuroepithelial cells from fetal and postnatal rat retina. This treatment induced differentiation of the CNS precursors, causing the cells to cease DNA synthesis and increase their expression of proteins normally found in differentiated retinal cells. These results indicate that germinal neuroepithelial cell differentiation can be controlled through the cAMP second messenger system, and that the regulation of this system may in part determine the numbers and ratios of the various classes of neurons during the normal development of the CNS.

Animals↗

A rod end deletion in the intermediate filament protein nestin alters its subcellular localization in neuroepithelial cells of transgenic mice.

Neuroepithelial and radial glial cells span between the ventricular and the pial surfaces of the neural tube and express two intermediate filaments (IFs), nestin and vimentin, which form a filamentous network throughout the length of the cells. In this report we study the polymerization characteristics of nestin and examine how mutations affect the assembly and localization of the nestin protein in cultured cells and in the developing CNS of transgenic mice. A wild-type rat nestin gene transfected into the IF-free SW13 cell line failed to assemble into a filamentous network but was incorporated into the existing IF network of a subclone expressing vimentin, demonstrating that nestin requires vimentin for proper assembly. In transgenic mice, rat nestin formed a network indistinguishable from that formed by endogenous nestin and vimentin, but a mutant form lacking five amino acids at the carboxy terminus of the rod domain was largely restricted to the pial endfeet. Since nestin mRNA is localized to the pial endfoot region we propose that both transgenes are translated there, but that the wild-type protein is preferentially incorporated into the IF network. These observations provide evidence for hierarchical assembly and a complex organization of the IF network along the ventricular-pial axis in the early CNS.

Amino Acid Sequence↗

Lipid droplets of neuroepithelial cells are a major calcium storage site during neural tube formation in chick and mouse embryos.

In situ precipitation of calcium (Ca2+) with fluoride and antimonate shows that Ca(2+)-specific precipitate is localized almost exclusively within lipid droplets of neuroepithelial cells during neural tube formation in chick and mouse embryos. The density of Ca2+ precipitate within lipid droplets is generally greater in the apical ends of cells situated in regions of the neuroepithelium that are actively engaged in bending. These findings suggest that lipid droplets, in addition to providing a source of metabolic fuel for developing neuroepithelial cells, also serve as Ca(2+)-storage and -releasing sites during neurulation.

Animals↗

[Vimentin and neuroepithelial cell differentiation in the spinal cord of chick embryos: an immunohistochemical study].

Using vimentin immunohistochemical staining, the differentiation processes of neuroepithelium in the neural tube were examined in chick embryos from stages 8 through 28. At an early stage of the neural groove, stage 8, no morphological differences could be found among the neuroepithelial cells, but vimentin staining allowed us to identify four different regions in the groove wall. The epithelial cells in the ventral wall exhibited moderate staining of vimentin, and vimentin was detected in the basal and middle cytoplasmic areas. A weak staining limited to the basal cytoplasm was observed in the dorsal wall. In contrast, epithelial cells in the median hinge region and in the lateral edge of the neural groove had little vimentin. On the basis of this vimentin staining, four similar regions could also be observed in the neuroepithelium of the neural tube at stage 12; the ventral wall, dorsal wall, floor plate and roof plate. Prior to the morphological changes in the neuroepithelial cells, vimentin expression showed dramatic changes, and our immunohistochemical data suggest that cell differentiation into motor areas and sensory areas starts at an early stage of the neural groove.

Animals↗

Effects of colchicine on the shape of chick neuroepithelial cells during neurulation.

We have analyzed the effects of colchicine on the cell shapes in chick neuroepithelium. Cell shapes were ascertained by the position of the nucleus in plastic serial sections. We tested three colchicine doses (5 X 10(-5) M, 5 X 10(-6) M, and 5 X 10(-7) M) by two experimental treatments (in ovo and in vitro). Colchicine treatment in vitro is always effective in depolymerizing microtubules of neuroepithelial cells and reduces the percentages of wedge-shaped cells in the median area of neuroepithelium. The same effect can be observed when the embryos are treated with 5 X 10(-5) M or 5 X 10(-6) M colchicine in ovo. A concentration of colchicine of 5 X 10(-7) M in ovo cannot disrupt microtubules in stage 8 and stage 10 embryos, and the percentage of wedge-shaped cells is the same as that of the untreated cells. In stage 6 embryos this colchicine dose effects the microtubules and the percentages of wedge-shaped cells. These facts are interpreted in respect to variations in microtubular resistance to microtubular-disrupting agents that are shown by the neuroepithelial cells from different developmental stages.

Animals↗

Aberrant differentiation of neuroepithelial cells in developing mouse brains subsequent to retinoic acid exposure in utero.

All-trans-retinoic acid induced 2 types of disorganized neuroepithelium, localized and continuous, in the exencephaly of 9-day-old mouse embryos exposed to 60 or 40 mg/kg for 27 to 30 hr in utero. The localized effect appeared as a protuberance in the wall of the telencephalon and thick neural folds in the mesencephalon with the discontinuity of the apical terminal sheet. The continuous disorganization was seen from the olfactory placode to the myelencephalon with rosettes of cells and many dense bodies in the neuroepithelium. Ultrastructurally, cells in the localized disorganizations showed swelling of Golgi complexes, coated vesicles, and rough endoplasmic reticulum resulting in degeneration. The continuous disorganizations consisted of undifferentiated homogeneous cells in which the nuclei exhibited expansion of nucleolar granular portions and coagulated heterochromatin, and cytoplasm showed monosomal dispersion. In both types of disorganized neuroepithelium, junctional complexes were seen focally at the apical side or apical processes of the rosette, with few or no microfilament bundles. A layer of microfilaments at the base of the neuroepithelial cells in controls, just above the basal lamina, was not present in the monosome dispersed cytoplasm. In the neuroepithelium of controls, one phagosome was seen in the perinuclear region in 0.8% of the cells examined, whereas in the experimental neuroepithelium 2 or more phagosomes were seen in a cell, and phagocytosis occurred by pseudopods. These findings suggest that all-trans-retinoic acid induces not only cytotoxicity but also dedifferentiation in the neuroepithelial cells leading to more cell death, which activates the phagocytosis. These lesions in the neuroepithelium may be a cause of exencephaly.

Abnormalities, Drug-Induced↗

Fate alteration of neuroepithelial cells from neurogenesis to astrocytogenesis by bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs), a class of cytokines belonging to the transforming growth factor-beta superfamily, have been shown to play a wide variety of roles during development including those in the central nervous system. We here report that BMP2, BMP4 and BMP7 have an equivalent potential to inhibit neurogenesis and concomitantly induce astrocytogenesis of mouse fetal neuroepithelial cells. We further show that these BMPs activate a promoter of the gene for negative helix-loop-helix (HLH) factor, Id1, which is known to inhibit the function of such neurogenic transcription factors as Mash1 and neurogenin. These results suggest that BMP2, BMP4 and BMP7 alternate the fate of neuroepithelial cells from neuronal type to astrocytic one via a common mechanism involving negative HLH factor.

Activin Receptors, Type I↗

Insulin and insulin-like growth factor system components gene expression in the chicken retina from early neurogenesis until late development and their effect on neuroepithelial cells.

To better understand the role of insulin-related growth factors in neural development, we have characterized by in situ hybridization in chicken embryonic retina the patterns of gene expression for insulin, insulin-like growth factor I (IGF-I), their respective receptors and the IGF binding protein 5 (IGFBP5) from early stages (E6) until late stages (E18)--an analysis not performed yet in any species. In addition, we studied the effect of insulin and IGF-I on cultured neuroepithelial cells. Insulin receptor mRNA and IGF-I receptor mRNA were both present and showed a similar, widespread pattern throughout retina development. Insulin mRNA could be detected only by reverse transcription coupled to polymerase chain reaction. IGF-I mRNA was concentrated in the ciliary processes and extraocular muscles early in development (embryonic day 6; E6) and in maturing retinal ganglion cells subsequently (E9-15). IGFBP5 mRNA was preferentially localized in the more differentiated central retinal zone and was maximally concentrated in the inner nuclear and ganglion cell layers at E9. These findings suggest a near constitutive expression of insulin receptor and IGF-I receptor genes, while IGF-I and IGFBP5 showed a highly focal spatiotemporal regulation of gene expression. Insulin and IGF-I, already at 10(-8) M, increased the proportion of PM1-positive neuroepithelial cells found in E5 retinal cultures without affecting significantly the total number of proliferating cells. Together, these data support the finding that, during early neurogenesis in chicken retina, insulin and IGF-I have a specific paracrine/autocrine action. This action, as well as possible effects elicited subsequently, may be dictated by restricted-local synthesis of the ligands and limited access to the factors contained in the vitreous humour. In the case of IGF's role, local IGFBPs expression can contribute to the fine modulation.

Animals↗