Glial-restricted precursors are derived from multipotent neuroepithelial stem cells

Neuroepithelial cells in the developing ventricular zone differentiate into neurons, astrocytes, and oligodendrocytes. It is not known, however, whether this differentiation occurs in a single step or is a pathway utilizing intermediate more restricted precursor cells. To characterize the generation of glial cells from multipotent stem cells we have cultured neuroepithelial (NEP) cells from E10.5 rat embryos. Cultured NEP cells do not express any glial differentiation markers when grown on fibronectin/laminin under nondifferentiation conditions. NEP cells, however, differentiate into A2B5 immunoreactive cells which can subsequently give rise to oligodendrocytes and astrocytes. Clonal analysis of NEP cells demonstrates that the A2B5 immunoreactive cells arise in clones that contain neurons and astrocytes, indicating that A2B5(+) cells arise from multipotent NEP precursor cells. A2B5(+) cells, maintained as undifferentiated cells over multiple passages, can subsequently give rise to both oligodendrocytes and astrocytes. A2B5(+) cells, however, do not generate neurons. Thus A2B5(+) cells represent a restricted progenitor cell population that differentiates from a multipotent NEP cell. Based on our results we propose that differentiation of the multipotential NEP cells to terminally differentiated glial cells occurs via intermediate restricted precursors.     

bHLH transcription factors and mammalian neuronal differentiation

The basic helix-loop-helix (bHLH) factor Mash1 is expressed in the developing nervous system. Null mutation of Mash1 results in loss of olfactory and autonomic neurons and delays differentiation of retinal neurons, indicating that Mash1 promotes neuronal differentiation. Other bHLH genes, Math/NeuroD/Neurogenin, all expressed in the developing nervous system, have also been suggested to promote neuronal differentiation. In contrast, another bHLH factor, HES1, which is expressed by neural precursor cells but not by neurons, represses Mash1 expression and antagonizes Mash1 activity in a dominant negative manner. Forced expression of HES1 in precursor cells blocks neuronal differentiation in the brain and retina, indicating that HES1 is a negative regulator of neuronal differentiation. Conversely, null mutation of HES1 up-regulates Mash1 expression, accelerates neuronal differentiation, and causes severe defects of the brain and eyes. Thus, HES1 regulates brain and eye morphogenesis by inhibiting premature neuronal differentiation, and the down-regulation of HES1 expression at the right time is required for normal development of the nervous system. Interestingly, HES1 can repress its own expression by binding to its promoter, suggesting that negative autoregulation may contribute to down-regulation of HES1 expression during neural development. Recent studies indicate that HES1 expression is also controlled by RBP-J, a mammalian homologue of Suppressor of Hairless [Su(H)], and Notch, a key membrane protein that may regulate lateral specification through RBP-J during neural development. Thus, the Notch-->RBP-J-->HES1-Mash1 pathway may play a critical role in neuronal differentiation.     

Selective induction of c-Jun and NGF in reactive astrocytes after cholinergic degenerations in rat basal forebrain

Cholinergic basal forebrain neurons are the major source of cortical cholinergic innervation. The number of these neurons is regulated by the availability of nerve growth factor (NGF) during development while in adulthood their cholinergic activity is modulated by NGF. In previous studies we have shown that cholinergic immunolesions of basal forebrain neurons increase local immediate early gene expression and NGF synthesis in the regions of degeneration. In this study we identify the cellular source of c-Jun and NGF expression using dual immunolabeling of c-Jun and NGF in combination with neuronal and glial markers. We demonstrate that both c-Jun and NGF are exclusively expressed in reactive astrocytes but not in microglia or in GABAergic basal forebrain neurons. These observations support the hypothesis that reactive astrocytes synthesize neurotrophic substances in vivo in response to neuronal degeneration in the basal forebrain.     

The role of receptor protein tyrosine phosphatase alpha in neuronal differentiation of embryonic stem cells

In the present study, we have investigated the function of the receptor protein tyrosine phosphatase alpha (RPTP alpha) in the neuronal differentiation of E14-embryonic stem (E14-ES) cells. RNAase protection and western blot analysis revealed that E14-ES cells up regulate RPTP alpha expression upon neuronal differentiation with retinoic acid. Overexpression of RPTP alpha, by stable DNA transfection, and subsequent differentiation with retinoic acid, resulted in a temporally enhanced expression of the neuronal markers GAP-43 and NF-164. Electrophysiological experiments demonstrated that RPTP alpha overexpression also enhanced the development of neurotransmitter responses during differentiation. These results indicate that RPTP alpha plays an important role in the cascade of molecular events that lead to the formation of neurons.     

Incidence of apoptotic bodies in membrana granulosa of the patients participating in an in vitro fertilization program

Vagal primary afferent neurons have their cell bodies located in the nodose (inferior) and jugular (superior) vagal ganglia and send terminals into the nucleus tractus solitarii (NTS) which lies in the dorsomedial medulla. The presence of glutamate (Glu)-containing neurons in the rat nodose ganglion was investigated using immunohistochemistry. Glu-immunoreactivity on nodose sections was found in neuronal perikarya and nerve fibers, but not in non-neuronal elements such as Schwann cells and satellite cells. Both immunoreactive and non-immunoreactive ganglion cells were observed. The immunoreactive ganglion cells amounted to about 60% of the nodose population. No specific intraganglionic localization was observed for the non-immunoreactive cells. Immunoreactive perikarya were slightly smaller than the non-immunoreactive ones, but no relationship was found between size and staining intensities of immunoreactive neurons. The present data indicate that immunodetectable Glu is present in a large population of vagal afferent neurons. They therefore add to a growing body of evidence suggesting that Glu may be the main neurotransmitter released by vagal afferent terminals within the nucleus tractus solitarii.     

Spontaneous activity: functions of calcium transients in neuronal differentiation

Calcium ions play critical roles in neuronal development. Many excitable cells promote calcium influx across their surface membrane during early stages of differentiation, which can trigger further elevation of intracellular calcium by release from stores. Several distinct types of spontaneous elevations of intracellular calcium occur during development of amphibian spinal neurons, both in culture and in the intact spinal cord. Rapid spikes and slow waves originate by different mechanisms and have separate functions. Spikes are required for neurotransmitter expression and channel modulation. Waves occurring in growth cones appear to regulate neurite extension.     

A simple neonatal mock circulation enabling pulsatility and different hemodynamical states for neonatal ECMO research: application to assess the effect of a centrifugal pump operated neonatal ECMO system on the circulation

In the developing forebrain, mounting evidence suggests that neural stem cell proliferation and differentiation is regulated by growth factors. In vitro in the presence of serum, stem cell proliferation is predominantly mediated by fibroblast growth factor-2 (FGF-2) whereas neuronal differentiation can be triggered by FGF-1 in association with a specific heparan sulphate proteoglycan. On the other hand, astrocyte differentiation in vivo and in vitro appears to be dependent on signalling through the leukaemia inhibitory factor receptor (LIFR). The evidence suggests that in the absence of LIFR signalling, the stem cell population is present at approximately the same frequency and can generate neurons but is blocked from producing astrocytes that express glial fibrillary acidic protein (GFAP) or have trophic functions. The block can be overcome by other growth factors such as BMP-2/4 or interferon-gamma, providing further evidence that the inhibition to astrocyte development does not result from loss of a precursor population. Signalling through the LIFR, in addition to stimulating astrocyte differentiation, may also inhibit neuronal differentiation, which may explain why this receptor is expressed at the earliest stages of neurogenesis. Another signalling system which also exerts its influence on neurogenesis through active inhibition is Delta-Notch. We show in vitro that at high cell densities which impede neuronal production by FGF-1, lowering the levels of expression of the receptor Notch by antisense oligonucleotide results in a significant increase in neuronal production. Thus, stem cell differentiation appears to be dependent on the outcome of interactions between a number of signalling pathways, some which promote specific lineages and some which inhibit.     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. III. Effect on acetylcholine production

The effect of nerve growth factor (NGF) on the development of cholinergic sympathetic neurons was studied in cultures grown either on monolayers of dissociated rat heart cells or in medium conditioned by them. In the presence of rat heart cells the absolute requirement of neurons for exogenous NGF was partially spared. The ability of heart cells to support neuronal survival was due at least in part to production of a diffusable NGF-like substance into the medium. Although some neurons survived on the heart cell monolayer without added NGF, increased levels of exogenous NGF increased neuronal survival until saturation was achieved at 0.5 microgram/ml 7S NGF. The ability of neurons to produce acetylcholine (ACh) from choline was also dependent on the level of exogenous NGF. In mixed neuron-heart cell cultures, NGF increased both ACh and catecholamine (CA) production per neuron to the same extent; saturation occurred at 1 microgram/ml 7S NGF. As cholinergic neurons developed in culture, they became less dependent on NGF for survival and ACh production, but even in older cultures approximately 40% of the neurons died when NGF was withdrawn. Thus, NGF is as necessary for survival, growth, and differentiation of sympathetic neurons when the neurons express cholinergic functions as when the neurons express adrenergic functions (4, 5).     

Timing and pattern of cell fate restrictions in the neural crest lineage

The trunk neural crest of vertebrate embryos is a transient collection of precursor cells present along the dorsal aspect of the neural tube. These cells migrate on two distinct pathways and give rise to specific derivatives in precise embryonic locations. One group of crest cells migrates early on a ventral pathway and generates neurons and glial cells. A later-dispersing group migrates laterally and gives rise to melanocytes in the skin. These observations raise the possibility that the appearance of distinct derivatives in different embryonic locations is a consequence of lineage restrictions specified before or soon after the onset of neural crest cell migration. To test this notion, we have assessed when and in what order distinct cell fates are specified during neural crest development. We determined the proportions of different types of precursor cells in cultured neural crest populations immediately after emergence from the neural tube and at intervals as development proceeds. We found that the initial neural crest population was a heterogeneous mixture of precursors almost half of which generated single-phenotype clones. Distinct neurogenic and melanogenic sublineages were also present in the outgrowth population almost immediately, but melanogenic precursors dispersed from the neural tube only after many neurogenic precursors had already done so. A discrete fate-restricted neuronal precursor population was distinguished before entirely separate fate-restricted melanocyte and glial precursor populations were present, and well before initial neuronal differentiation. Taken together, our results demonstrate that lineage-restricted subpopulations constitute a major portion of the initial neural crest population and that neural crest diversification occurs well before overt differentiation by the asynchronous restriction of distinct cell fates. Thus, the different morphogenetic and differentiative behavior of neural crest subsets in vivo may result from earlier cell fate-specification events that generate developmentally distinct subpopulations that respond differentially to environmental cues.     

Production of sweat gland cholinergic differentiation factor depends on innervation

Sympathetic neurons innervating sweat glands undergo a target-directed developmental switch in neurotransmitter properties. Using cultured sympathetic neurons as a bioassay for cholinergic differentiation factors, we and others found that extracts containing soluble proteins from developing and adult footpads caused the same changes in transmitter properties in sympathetic neurons in vitro that the target does in vivo. In the present studies, using footpads from Tabby mutant mice that lack sweat glands, we found that the presence of sweat glands is correlated with the presence of cholinergic differentiation activity in footpad extracts. We examined the conditions necessary for secretion of differentiation activity from primary cultures of sweat gland cells. Surprisingly, sweat gland cells cultured alone do not produce or secrete cholinergic differentiation activity. When grown in the presence of sympathetic neurons, however, gland cells induce cholinergic function, increase vasoactive intestinal peptide content, and reduce catecholamine production in the neurons. Medium conditioned by sweat gland/neuron cocultures has a similar effect on the transmitter properties of cultured sympathetic neurons, indicating that the target influence on phenotype is mediated by a secreted factor(s). The innervation-dependence of cholinergic differentiation factor production provides evidence that reciprocal interactions between neurons and sweat glands are necessary for acquisition of the mature transmitter phenotype.     

Cholinergic immunolesions by 192IgG-saporin--useful tool to simulate pathogenic aspects of Alzheimer's disease

Alzheimer's disease, the most common cause of senile dementia, is characterized by intracellular formation of neurofibrillary tangles, extracellular deposits of beta amyloid as well as cerebrovascular amyloid accumulation and a profound loss of cholinergic neurons within the nucleus basalis Meynert with alterations in cortical neurotransmitter receptor densities. The use of the cholinergic immunotoxin 192IgG-saporin allows for the first time study of the impact of cortical cholinergic deafferentation on cortical neurotransmission, learning, and memory without direct effects on other neuronal systems. This model also allows the elucidation of contributions of cholinergic mechanisms to the establishment of other pathological features of Alzheimer's disease. The findings discussed here demonstrate that cholinergic immunolesions by 192IgG-saporin induce highly specific, permanent cortical cholinergic hypoactivity and alterations in cortical neurotransmitter densities comparable to those described for Alzheimer's disease. The induced cortical cholinergic deficit also leads to cortical/hippocampal neurotrophin accumulation and reduced amyloid precursor protein (APP) secretion, possibly reflecting the lack of stimulation of postsynaptic M1/M3 muscarinic receptors coupled to protein kinase C. This immunolesion model should prove useful to test therapeutic strategies based on stimulation of cortical cholinergic neurotransmission or amelioration of pathogenic aspects of cholinergic degeneration in the basal forebrain. Application of the model to animal species that can develop beta-amyloid plaques could provide information about the contribution of cholinergic function to amyloidogenic APP processing.     

The LHRH-astroglial network of signals as a model to study neuroimmune interactions: assessment of messenger systems and transduction mechanisms at cellular and molecular levels

Neurons and astrocytes have a close anatomic and functional relationship that plays a crucial role during development and in the adult brain. Astrocytes in the central nervous system (CNS) express receptors for a variety of growth factors (GFs), neurotransmitters and/or neuromodulators; in turn, neuronal cells can respond to astrocyte-derived GFs and control astrocyte function via a common set of signaling molecules and intracellular transducing pathways. There is also increasing evidence that soluble factors from lymphoid/mononuclear cells are able to modulate the growth and function of cells found in the CNS, specifically macroglial and microglial cells. Furthermore, glial cells can secrete immunoregulatory molecules that influence immune cells as well as the glial cells themselves. As neuronal and immune cells share common signaling systems, the potential exists for bidirectional communication not only between lymphoid and glial cells, but also between neuronal cells and immune and glial cells. In the present work, interactions of luteinizing-hormone-releasing hormone (LHRH) and the astroglial cell are proposed as a prototype for the study of neuroimmune communication within the CNS in the light of (1) the commonality of signal molecules (hormones, neurotransmitters and cytokines) and transduction mechanisms shared by glia LHRH neurons and lymphoid cells; (2) the central role of glia in the developmental organization and pattern of LHRH neuronal migration during embryogenesis, and (3) the strong modulatory role played by sex steroids in mechanisms involved in synaptic and interneuronal organization, as well as in the sexual dimorphisms of neuroendocrine-immune functions. During their maturation and differentiation in vitro, astroglial cells release factors able to accelerate markedly the LHRH neuronal phenotypic differentiation as well as the acquisition of mature LHRH secretory potential, with a potency depending on both the 'age' and the specific brain localization of the astroglia, as well as the degree of LHRH neuronal differentiation in vitro. Regional differences in astroglial sensitivity to estrogens were also measured. Different experimental paradigms such as coculture and mixed-culture models between the immortalized LHRH (GT1-1) neuronal cell line and astroglial cells in primary culture, disclosed the presence of a bidirectional flow of informational molecules regulating both proliferative and secretory capacities of each cell type. The importance of adhesive mechanisms in such cross-talk is underscored by the complete abolition of GT1-1 LHRH production and cell proliferation following the counteraction of neuronal-neuronal/neuronal-glial interactions through addition of neural-cell adhesion molecule antiserum. Other information came from pharmacological experiments manipulating the astroglia-derived cytokines and/or nitric oxide, which revealed cross-talk between the neuronal and astroglial compartments. From the bulk of this information, it seems likely that interactions between astroglia and LHRH neurons play a major role in the integration of the multiplicity of brain signals converging on the LHRH neurons that govern reproduction. Another important facet of neuronal-glial interactions is that concerning neuron-guided migration, and unraveling astroglial/LHRH-neuronal networks might then constitute an additional effort in the comprehension of defective LHRH-neuronal migration in Kallman's syndrome.     

Helix-loop-helix factors in growth and differentiation of the vertebrate nervous system

Neural development involves the initial growth phase of dividing precursor cells and the subsequent differentiation phase of postmitotic cells. Recent studies indicate that the transition from the former phase to the latter is controlled antagonistically by multiple helix-loop-helix (HLH) genes. Cascades of neuronal HLH genes promote differentiation whereas anti-neuronal HLH genes repress them under the control of Notch and keep cells at the precursor stage. This antagonistic regulation may be essential for generation of the proper number of neurons and for morphogenesis of the nervous system.     

A molecular mechanism enabling continuous embryonic muscle growth - a balance between proliferation and differentiation

Embryonic muscle growth requires a fine balance between proliferation and differentiation. In this study we have investigated how this balance is achieved during chick development. Removal of ectoderm from trunk somites results in the down-regulation of Pax-3 expression and cell division of myogenic precursors is halted. This initially leads to an up-regulation of MyoD expression and to a burst in terminal differentiation but further muscle growth is arrested. Locally applied bone morphogenetic protein-4 (BMP-4) to somites mimics the effect of the ectoderm and stimulates Pax-3 expression which eventually results in excessive muscle growth in somites. Surprisingly, BMP-4 up-regulates expression of noggin which encodes a BMP-4 antagonist. This suggests that the proliferation enhancing activity of BMP-4 can be limited via up-regulation of noggin and that myogenic cells differentiate, as an intrinsic property, when deprived of BMP-4 influence. In contrast to BMP-4, Sonic hedgehog (Shh) locally applied to somites arrests muscle growth by down-regulation of Pax-3 and immediate up-regulation of MyoD expression. Such premature muscle differentiation in somites at tongue and limb levels prevents myogenic migration and thus tongue and limb muscle are not formed. Therefore, precise limitation of differentiation, executed by proliferative and Pax-3 promoting signals, is indispensable for continuous embryonic muscle growth.     

Modulation of neuronal activity by glial cells in the retina

Glial-neuronal communication was studied by monitoring the effect of intercellular glial Ca2+ waves on the electrical activity of neighboring neurons in the eyecup preparation of the rat. Calcium waves in astrocytes and Müller cells were initiated with a mechanical stimulus applied to the retinal surface. Changes in the light-evoked spike activity of neurons within the ganglion cell layer occurred when, and only when, these Ca2+ waves reached the neurons. Inhibition of activity was observed in 25 of 53 neurons (mean decrease in spike frequency, 28 +/- 2%). Excitation occurred in another five neurons (mean increase, 27 +/- 5%). Larger amplitude Ca2+ waves were associated with greater modulation of neuronal activity. Thapsigargin, which reduced the amplitude of the glial Ca2+ increases, also reduced the magnitude of neuronal modulation. Bicuculline and strychnine, inhibitory neurotransmitter antagonists, as well as 6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and D(-)-2-amino-7-phosphonoheptanoic acid (D-AP7), glutamate antagonists, reduced the inhibition of neuronal activity associated with glial Ca2+ waves, suggesting that inhibition is mediated by inhibitory interneurons stimulated by glutamate release from glial cells. The results suggest that glial cells are capable of modulating the electrical activity of neurons within the retina and thus, may directly participate in information processing in the CNS.     

Constitutive secretion of exogenous neurotransmitter by nonneuronal cells: implications for neuronal secretion

Fibroblasts in cell culture were loaded with exogenous neurotransmitter acetylcholine (ACh). ACh secretion from loaded cells was detected by whole-cell patch clamp recordings from Xenopus myocytes manipulated into contact with ACh-loaded cells. Two different approaches were used for ACh loading. In the first approach, fibroblasts were incubated in the culture medium containing ACh. Recordings from myocytes revealed fast inward currents that resemble miniature endplate currents found at neuromuscular synapses. The currents observed in recordings from myocytes were due to exocytosis of ACh-containing vesicles. Although exogenous ACh penetrated through the plasma membrane of fibroblasts during incubation and was present in the cytoplasm at detectable levels, cytoplasmic ACh did not contribute to the quantal ACh secretion. In the second approach, exogenous ACh was loaded into the cytoplasm of fibroblasts by microinjection. Under these experimental conditions, fibroblasts also exhibited spontaneous quantal ACh secretion. Analysis of the exocytotic events in fibroblasts following two different protocols of ACh loading revealed that the vesicular compartments responsible for uptake of exogenous ACh are associated with the endocytic recycling pathway. Extrapolation of our results to neuronal cells suggest that in cholinergic neurons, in addition to genuine synaptic vesicles, ACh can be secreted by the vesicles participating in endosomal membrane recycling.     

Sonic hedgehog promotes somitic chondrogenesis by altering the cellular response to BMP signaling

Previous work has indicated that signals from the floor plate and notochord promote chondrogenesis of the somitic mesoderm. These tissues, acting through the secreted signaling molecule Sonic hedgehog (Shh), appear to be critical for the formation of the sclerotome. Later steps in the differentiation of sclerotome into cartilage may be independent of the influence of these axial tissues. Although the signals involved in these later steps have not yet been pinpointed, there is substantial evidence that the analogous stages of limb bud chondrogenesis require bone morphogenetic protein (BMP) signaling. We show here that presomitic mesoderm (psm) cultured in the presence of Shh will differentiate into cartilage, and that the later stages of this differentiation process specifically depend on BMP signaling. We find that Shh not only acts in collaboration with BMPs to induce cartilage, but that it changes the competence of target cells to respond to BMPs. In the absence of Shh, BMP administration induces lateral plate gene expression in cultured psm. After exposure to Shh, BMP signaling no longer induces expression of lateral plate markers but now induces robust chondrogenesis in cultured psm. Shh signals are required only transiently for somitic chondrogenesis in vitro, and act to provide a window of competence during which time BMP signals can induce chondrogenic differentiation. Our findings suggest that chondrogenesis of somitic tissues can be divided into two separate phases: Shh-mediated generation of precursor cells, which are competent to initiate chondrogenesis in response to BMP signaling, and later exposure to BMPs, which act to trigger chondrogenic differentiation.     

Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione

Deficiency of the antioxidant glutathione in brain appears to be connected with several diseases characterized by neuronal loss. To study neuronal glutathione metabolism and metabolic interactions between neurons and astrocytes in this respect, neuron-rich primary cultures and transient cocultures of neurons and astroglial cells were used. Coincubation of neurons with astroglial cells resulted within 24 hr of incubation in a neuronal glutathione content twice that of neurons incubated in the absence of astroglial cells. In cultured neurons, the availability of cysteine limited the cellular level of glutathione. During a 4 hr incubation in a minimal medium lacking all amino acids except cysteine, the amount of neuronal glutathione was doubled. Besides cysteine, also the dipeptides CysGly and gammaGluCys were able to serve as glutathione precursors and caused a concentration-dependent increase in glutathione content. Concentrations giving half-maximal effects were 5, 5, and 200 microM for cysteine, CysGly, and gammaGluCys, respectively. In the transient cocultures, the astroglia-mediated increase in neuronal glutathione was suppressed by acivicin, an inhibitor of the astroglial ectoenzyme gamma-glutamyl transpeptidase, which generates CysGly from glutathione. These data suggest the following metabolic interaction in glutathione metabolism of brain cells: the ectoenzyme gamma-glutamyl transpeptidase uses as substrate the glutathione released by astrocytes to generate the dipeptide CysGly that is subsequently used by neurons as precursor for glutathione synthesis.     

Maternal substance abuse during pregnancy. Policy implications in the United States

C3H10T1/2 cells are an established mesenchymal stem cell line which can differentiate into muscle, fat and cartilage cells when treated with azacytidine. Bone morphogenetic protein-2 (BMP-2) caused a dose dependent differentiation of these cells into fat, cartilage and bone cells-low concentrations favoring adipocytes and high concentrations chondrocytes and osteoblasts. The differentiated phenotypes were stable in the absence of BMP-2. Furthermore, the addition of other growth factors during the differentiation process altered the frequency of the differentiated colony formation. Transfection of the C3H10T1/2 cells with a BMP-2 cDNA also induced a phenotypic change from the parental fibroblast to adipocytes and osteoblasts. Our results in this model system indicate that a single protein factor can cause differentiation of a stem cell line to multiple phenotypes, that phenotypes induced can be regulated by factor concentration, and that other factors can also influence BMP-2 induced differentiation.