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Chlorpromazine and trifluoperazine HCl, antipsychotic drugs known to bind to calmodulin, reversibly inhibited elevation of neural folds by interfering with the contractile activity of apical microfilament bundles in developing chick neuroepithelial cells.
Peripheral chemoreceptors responsive to hypoxia have been well characterized in air-breathing vertebrates, but poorly in water-breathers. The present study examined the distribution of five populations of neuroepithelial cells (NECs), putative O(2) chemoreceptors, and innervation patterns in the zebrafish gill using whole-mounts and confocal immunofluorescence. Nerve bundles and fibers of the gill were labeled with zn-12 (a zebrafish-specific neuronal marker) and SV2 antisera and NECs were characterized by serotonin (5-HT) immunoreactivity (IR), SV2-IR and the purinoceptor P2X(3)-IR. A zn-12-IR nerve bundle extended the length of the gill filament and gave rise to a nerve plexus surrounding the efferent filament artery (eFA) and a rich network of fibers that innervated both serotonergic and nonserotonergic NECs of the filament and lamellar epithelium. Three populations of serotonergic, SV2-IR neurons intrinsic to the gill filaments are described, one of which provided innervation to NECs of the filament epithelium. Degeneration of nerve fibers in gill arches maintained in explant culture for 2 days revealed the extrinsic origin of nerve fibers of the plexus and lamellae and the innervation of filament NECs by both intrinsic and extrinsic fibers. Intrinsic innervation surrounding the eFA survived in explant cultures, suggesting a mechanism of local vascular control within the gill. In addition, NECs survived in explants after degeneration of extrinsic nerve fibers. Thus, NECs of the zebrafish gill are organized in a manner reminiscent of O(2) chemoreceptors of mammalian vertebrates, suggesting a role in respiratory regulation.
Shaping of the neural plate, one of the most striking events of neurulation, involves rapid craniocaudal extension. In this study, we evaluated the roles of two processes in neural plate extension: neuroepithelial cell rearrangement and cell division. Quail epiblast plugs of constant size were grafted either just rostral to Hensen's node or paranodally and the resulting chimeras were examined at selected times postgrafting. By comparing the size of the original plug, the number of cells it contained and the distribution of cells within it to those same features of the grafts in chimeras, we were able to ascertain that, during transformation of the flat neural plate into the closed neural tube (a period requiring 24 h), the graft undergoes a maximum of 3 rounds of craniocaudal extension (each round of craniocaudal extension was defined as a doubling of graft length, so 3 rounds equaled an 8-fold increase in length). Such extension is accompanied by 2 rounds of cell rearrangement and 2-3 rounds of cell division (cell rearrangement occurred mediolaterally, so each round was defined as a halving of the number of cells in the width of the graft and a doubling of the number of cells in its length; each round of cell division was defined as a doubling of graft cell number). Modeling studies demonstrate that these amounts of cell rearrangement and division are sufficient to approximate the shaping of the neural plate that normally ensues during neurulation, provided that some of the cell division occurs within the longitudinal plane of the neural plate and some within its transverse plane: longitudinal cell division results in craniocaudal extension of the neural plate, whereas transverse cell division results in lateral expansion of the neural plate such as that occurring at its cranial end; cell rearrangement results in craniocaudal extension of the neural plate as well as in its narrowing. In conclusion, our results provide evidence that shaping of the neural plate involves mediolateral cell rearrangement and cell division, with the latter occurring within both the longitudinal and transverse planes of the neural plate.
Cardiotrophin-1 (CT-1) belongs to the interleukin (IL-)6 family of cytokines that share membrane glycoprotein 130 (gp130) as a receptor component critical for signal transduction. We here observed that CT-1 was expressed in mouse fetal neuroepithelial cells, and was capable of inducing astrocyte differentiation from these cells in a synergistic manner with bone morphogenetic protein (BMP)-2, whose expression was also found in the fetal brain. CT-1-induced astrocyte differentiation was solely gp130-dependent. CT-1-stimulation led to promoter activation of the gene for an astrocyte marker, glial fibrillary acidic protein (GFAP), which was clearly inhibited by expression of a dominant negative form of a gp130-downstream transcription factor, signal transducer and activator of transcription 3(STAT3), or by introduction of a mutation in a single STAT3-binding site in the promoter, suggesting a critical role of STAT3 in the CT-1-induced GFAP transcription. These results suggest that astrocyte differentiation in the developing brain involves CT-1-signaling which cooperates with BMP2.
The distribution of cell death in the ventral pycnotic zone of the chick embryo retina was studied in Hamburger-Hamilton's stages 16 to 25 (2 1/2 to 4 1/2 days of incubation). The number of fragments appearing in the retina increases notably from stage 20 at which stage they are limited almost exclusively to the optic disc region. At the same time optic fibers are seen in this area for the first time. In stage 24 cell death phenomena are numerous in the ventral retina, and become even more extensive in the following stage. Stage 25 meanwhile sees a drop in cell death in the dorsal retina. The overall picture presented by cell remains and young ganglion cells indicates that in stages 19-23 cell death occurs mainly in the zone between the ganglion cells of the posterior pole and the optic stalk. In the stage 25 retina most of the cell fragments of the ventral retina are found on either side of the fissure, while ganglion cells in the process of sending out axons toward the fissure appear laterally (nasally and temporally) to these zones of degeneration. Hence a spatial and temporal correlation is established between fiber growth and neuroepithelial cell degeneration, allowing us to construct a hypothesis with regard to the role that cell death might play in setting up an initial pattern of optic fiber growth.
The interleukin (IL)-6 family of cytokines share gp130 as a signal-transducing receptor component. We here show that oncostatin M (OSM), a member of this family, and its receptor components were expressed in embryonic mouse brain and that OSM induced astrocytes in cultured fetal mouse neuroepithelial cells. OSM induced promoter activation of the gene for an astrocyte marker, glial fibrillary acidic protein. The activation was completely blocked by expression of a dominant negative form of STAT3, a transcription factor activated downstream of gp130, or by introduction of a mutation in the STAT3 binding motif in the promoter. Taken together with its expression, we suggest that OSM contributes to the induction of astrocyte differentiation in a fetal developing brain via STAT3 activation.
Cardiotrophin-like cytokine (CLC), also known as novel neurotrophin-1/B cell stimulating factor-3 (NNT-1/BSF-3), is a recently identified member of the interleukin (IL)-6 family of cytokines that share gp130 as a signal-transducing receptor component. In this study, we demonstrate that CLC is expressed in fetal mouse neuroepithelial cells and has a potential to induce their astrocyte differentiation in a synergistic manner with bone-morphogenetic protein (BMP)-2, which is also expressed in the fetal mouse brain. CLC-stimulation led to promoter activation of the gene for an astrocyte marker, glial fibrillary acidic protein (GFAP), which was clearly inhibited by expression of a dominant negative form of a transcription factor, STAT3, or by introduction of a mutation in a single STAT3-binding site in the promoter, suggesting a critical role of STAT3 in the CLC-induced GFAP transcription. These results suggest that CLC plays a role in astrocyte differentiation via STAT3 activation within the developing brain.
The importance of lateral inhibition mediated by NOTCH signaling is well demonstrated to control neurogenesis both in invertebrates and vertebrates. We have identified the chicken homolog of Drosophila numb, which suppresses NOTCH signaling. We show that chicken NUMB (c-NUMB) protein is localized to the basal cortex of mitotic neuroepithelial cells, suggesting that c-NUMB regulates neurogenesis by the modification of NOTCH signaling through asymmetrical cell division. Consistent with this suggestion, we show (1) that c-NUMB interferes with the nuclear translocation of activated c-NOTCH-1 through direct binding to the PEST sequence in the cytoplasmic domain of c-NOTCH-1 and (2) that c-NUMB interferes with c-NOTCH-1-mediated inhibition of neuronal differentiation.
Peptide growth factors have been shown to have diverse effects on cells of the CNS, such as promoting neuronal survival, neurite outgrowth, and several other aspects of neuronal differentiation. In addition, some of these factors have been shown to be mitogenic for particular classes of glial cells within the brain and optic nerve, and recently two peptide growth factors, fibroblast growth factor and nerve growth factor, have been shown to have mitogenic activity on the CNS neuronal progenitors. We now report that two members of another peptide growth factor, epidermal growth factor and transforming growth factor-alpha, are mitogenic for retinal neuroepithelial cells in primary cultures and provide evidence for the presence of both of these factors in normal developing rat retina.
Fibroblast growth factors have been increasingly assigned mitogenic and trophic roles in embryonic and postnatal development of the nervous system. Little is known, however, of their functional roles in early embryonic neural development at the neural tube stage. We have examined the effect of basic fibroblast growth factor (bFGF) on the adhesive behavior in culture of dissociated brachio-thoracic neural tube cells from 26- to 30-somite stage chick embryos. Cells plated on collagen-coated substratum at a low density attach to the substratum but show poor cell spreading. Addition of bFGF markedly promotes cell spreading, yielding an epithelial morphology. This effect becomes discernible 6-8 hours after cell plating with bFGF and is completed by 24 hours, with half-maximal and maximal effects attained at around 0.4 and 10 ng/ml, respectively. The number of cells remain largely constant up to 24 hours, and then cell survival and/or mitogenic effects of bFGF become apparent. The cell spreading effect is abolished by cycloheximide treatment, inhibited by the anti-beta 1-integrin antibody CSAT, and accompanied by about twofold increases in the expression of beta 1-integrin and vinculin, components of focal adhesion complexes. Cells cultured with bFGF for 24 hours exhibit enhanced cell attachment and cell spreading with little time lag following cell plating. In earlier embryonic stages, developmentally less mature cells depend much more on bFGF for their cell spreading and survival, while in later stages the cell spreading response to bFGF becomes undetectable as neural tube develops to spinal cord. The cell spreading effect of bFGF is realized on specific extracellular matrix proteins including laminin, fibronectin and collagen, but not on vitronectin, arg-gly-asp peptide (PepTite-2000), poly-L-ornithine or others. These results suggest that, in an early stage of neural tube development, bFGF is involved in the developmental regulation of adhesive interactions between neuroepithelial cells and the extracellular matrix, thereby controlling their proliferation, migration and differentiation.
XLalphas ('extra large' alpha) is a 78 kDa splice variant of the alpha-subunit of the heterotrimeric G protein, Gs (Nature 372 (1994) 804). Prompted by its neuroendocrine-specific tissue distribution in the adult (J. Biol. Chem. 275 (2000) 33622) and its ability to activate adenylyl cyclase (J. Biol. Chem. 275 (2000) 33633), we investigated the expression of XLalphas in the developing rat nervous system using immunofluorescence. Remarkably, XLalphas expression in the neural tube was found to begin at the onset of neurogenesis, being observed in a subset of mitotic neuroepithelial cells as well as in young neurons. At later developmental stages, XLalphas was associated with a subset of neurons in certain regions of the nervous system such as diencephalon, midbrain, hindbrain, spinal cord and sympathetic trunk. These results suggest a role of XLalphas in neuronal differentiation.
alpha-Adrenoreceptors are present in the ventricular (VZ) and subventricular (SVZ) zones of the mammalian embryonic forebrain, and contribute towards cell cycle controls in the germinal neuroepithelium [Pabbathi et al., Brain Res. 760 (1997) 22-33.]. Since noradrenaline-containing fibres from the locus coeruleus (LC) and other brainstem nuclei innervate many parts of the CNS from an early stage of embryogenesis, we have used heterochronic cocultures to investigate whether noradrenergic innervation may be the source of trophic support. Dorsal neopallium from E13 rat embryo was cultured in proximity to E15 rostral pons (RP), enabling rapid innervation of the neuroepithelium by differentiated noradrenergic neurons of the LC. The projection of interconnecting fibres into germinal areas of the cortex was established in vitro by retrograde tracing and dopamine beta-hydroxylase (DBH) immunocytochemistry. When functional innervation was prevented by transection of axons connecting the explants, the number of apoptotic (TUNEL-positive) cells in the neopallium was increased. Bromodeoxyuridine (BrdU) double-labelling confirmed that germinal cells were amongst those which underwent apoptosis. When cortical explants were innervated by a source of non-monoaminergic (cholinergic) axons from the E18 basal forebrain diagonal band/septum complex (DS), numbers of apoptotic cells were comparable to those in non-innervated cultures. alpha1 and alpha2 adrenoreceptor antagonists included in the medium of cortical cocultures innervated by noradrenergic axons reversed the survival-promoting effect of innervation. Blockade of alpha1 but not of alpha2 receptors also reduced the numbers of S-phase nuclei in the cortex. The results provide evidence that local delivery of neurotransmitter from embryonic noradrenergic axons terminating in the neocortex can regulate survival and proliferation of neuroepithelial cells.
Angiotensin-converting enzyme (CE) occurs in three types of cell: endothelial, epithelial, and neuroepithelial. In all three, it appears to be bound to plasma membrane. With antisera to the human enzyme, CE is demonstrated in paraffin sections on the apical surface of epithelial cells in the proximal tubule of the kidney, the mucosa of the small intestine, the syncytial trophoblast of the placenta, and the choroid plexus. Epithelial CE is characteristically found on microvillous surfaces in contact with an effluent, well placed to act on substrate in flux. In the brain, CE occurs in nerve fibers and terminals, mainly mesiobasally and in basal ganglia. Mesiobasal CE coincides with other components of the renin-angiotensin system (RAS) in the choroid/ventricular fluid, the subfornical organ, and the magnocellular neurosecretory system of the hypothalamus. Extrapyramidal CE, however, may not be related to the RAS. In the substantia nigra and the globus pallidus, the enzyme has the same cellular distribution as two putative neuromodulators, substance P and enkephalin, the latter a known substrate of CE.
To determine the initial growth pattern of pioneering axons and investigate the factors that may influence their guidance, the lateral margin of a stage 16+ chick brachial spinal cord was examined in serial thin sections. The specimen was prepared with hypertonic fixative which partially shrank the tissue and increased extracellular space. The retention of surface contact after shrinkage was used as an index of the relative adhesiveness between cells in situ. Six axons and growth cones were found within the reconstructed tissue; five were oriented dorsoventrally and one apparent motor neuron growth cone was oriented radially. The five circumferential axons originated from presumptive interneurons distributed in a dispersed pattern along the neural tube lateral wall. Four terminated with growth cones, and each extended a short distance (less than 30 microns) ventrally along the outer margin. No contact was found between these nonfasciculating axons or growth cones. Thus, the earliest intracentral axons appear to grow dorsoventrally from the outset with no appreciable wandering. Morphological features that may indicate their mechanism of guidance, including preformed cellular guides, extracellular channels, contact with basal lamina, and intercellular junctions were not found. The preferential retention of surface contact between adjacent endfeet, as well as between pioneering circumferential axons and neuroepithelial cells, suggests that these particular surfaces are mutually adherent. These findings are consistent with a proposed dorsal-to-ventral adhesive gradient mechanism of circumferential axonal guidance.
Neuronal microtubules have unique stability properties achieved through developmental regulation at the expression and posttranslational levels on tubulins and microtubule associated proteins. One of the most specialized tubulins specific for neurons is class-III beta-tubulin (also known as beta6-tubulin). Both the upregulation and the post-translational processing of class-III beta-tubulin are believed to be essential throughout neuronal differentiation. The present investigation documents the temporal and spatial patterns of class-III beta-tubulin expression throughout neurogenesis. For this study a novel polyclonal antiserum named U-beta6, specific to unphosphorylated class-III beta-tubulin has been developed, characterized and compared with its commercial homologue TuJ-1. Our experiments indicate that the two antibodies recognize different forms of class-III beta-tubulin both in vitro and in vivo. Biochemical data revealed that U-beta6 bound unphosphorylated soluble class-III beta-tubulin specifically, while TuJ-1 recognized both the phosphorylated and unphosphorylated forms of the denatured protein. In vivo U-beta6 was associated with neurogenesis and labelled newly committed CNS and PNS neuroblasts expressing neuroepithelial cytoskeletal (nestin and vimentin) and surface markers (the anti-ganglioside supernatant, A2B5 and the polysialic acid neural adhesion molecule, PSA-NCAM), as well as differentiating neurons. These studies with U-beta6 illustrate three main developmental steps in the neuronal lineage: the commitment of neuroepithelial cells to the lineage (U-beta6 +ve/TuJ-1-ve cells); a differentiation stage (U-beta6 +ve/TuJ-1 +ve cells); and, finally, neuronal maturation correlating with a drop in unphosphorylated class-III beta-tubulin immunostaining levels. These investigations also conclude that U-beta6 is an earlier marker than TuJ-1 for the neuronal lineage in vivo, and it is thus the earliest neuronal lineage marker known so far.
Neuronal microtubules have unique stability properties achieved through developmental regulation at the expression and post-translational levels on tubulins and microtubule associated proteins. One of the most specialized tubulins specific for neurons is class-III beta-tubulin (also known as beta6-tubulin). Both the upregulation and the post-translational processing of class-III beta-tubulin are believed to be essential throughout neuronal differentiation. The present investigation documents the temporal and spatial patterns of class-III beta-tubulin expression throughout neurogenesis. For this study a novel polyclonal antiserum named U-beta6, specific to unphosphorylated class-III beta-tubulin has been developed, characterized and compared with its commercial homologue TuJ-1. Our experiments indicate that the two antibodies recognize different forms of class-III beta-tubulin both in vitro and in vivo. Biochemical data revealed that U-beta6 bound unphosphorylated soluble class-III beta-tubulin specifically, while TuJ-1 recognized both the phosphorylated and unphosphorylated forms of the denatured protein. In vivo U-beta6 was associated with neurogenesis and labelled newly committed CNS and PNS neuroblasts expressing neuroepithelial cytoskeletal (nestin and vimentin) and surface markers (the anti-ganglioside supernatant, A2B5 and the polysialic acid neural adhesion molecule, PSA-NCAM), as well as differentiating neurons. These studies with U-beta6 illustrate three main developmental steps in the neuronal lineage: the commitment of neuroepithelial cells to the lineage (U-beta6 +ve/TuJ-1 -ve cells); a differentiation stage (U-beta6 +ve/TuJ-1 +ve cells); and, finally, neuronal maturation correlating with a drop in unphosphorylated class-III beta-tubulin immunostaining levels. These investigations also conclude that U-beta6 is an earlier marker than TuJ-1 for the neuronal lineage in vivo, and it is thus the earliest neuronal lineage marker known so far.
Neuroepithelial b2T cells were derived from the hindbrain and the spinal cord of mouse transgenic embryos, which expressed SV40 T antigen under the control of a Hoxb2 enhancer. Strikingly, b2T cell lines of either origin exhibit a very similar gene expression pattern, including markers of the hindbrain and the spinal cord, such as Hox genes, but not of more anterior cephalic regions. In addition, the broad expression pattern of b2T cells, probably linked to culture conditions, appeared to be appropriately modulated when the cells were reimplanted at different longitudinal levels into chick host embryos, suggesting that these cells are responsive to exogenous signalling mechanisms. Further support for these allegations was obtained by culturing b2T cells in defined medium and by assessing the expression of Krox20, an odd-numbered rhombomere marker, which appeared to be modulated by a complex interplay between FGF, retinoic acid (RA), and noggin. With respect to these as yet unique properties, b2T cells constitute an original alternative tool to in vivo models for the analysis of molecular pathways involved in the patterning of the neural tube.
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