Proliferation Of Neural Precursor Cells Research Articles

Neurotransmitters as early signals for central nervous system development.

During brain ontogenesis, the temporal and spatial generation of the different types of neuronal and glial cells from precursors occurs as a sequence of successive progenitor stages whose proliferation, survival and cell-fate choice are controlled by environmental and cellular regulatory molecules. Neurotransmitters belong to the chemical microenvironment of neural cells, even at the earliest stages of brain development. It is now established that specific neurotransmitter receptors are present on progenitor cells of the developing central nervous system and could play, during neural development, a role that has remained unsuspected until recently. The present review focuses on the occurrence of neurotransmitters and their corresponding ligand-gated ion channel receptors in immature cells, including neural stem cells of specific embryonic and neonatal brain regions. We summarize in vitro and in vivo data arguing that neurotransmitters could regulate morphogenetic events such as proliferation, growth, migration, differentiation and survival of neural precursor cells. The understanding of neurotransmitter function during early neural maturation could lead to the development of pharmacological tools aimed at improving adult brain repair strategies.

Primary neural precursor cell expansion, differentiation and cytosolic Ca 2+ response in three-dimensional collagen gel

To investigate the ability to culture neural precursor cells in a three-dimensional (3D) collagen gel, neuroepithelial cells were isolated from embryonic day 13 rat cortex, dispersed within type I collagen and maintained for up to 30 days in vitro. Cultured in Neuorobasal medium supplemented with B27 containing basic fibroblast growth factor, the collagen-entrapped precursor cells actively expanded and formed clone-like clusters. Many cells in the center of the cluster were proliferating as revealed by 5-bromo-2′-deoxyuridine uptake. Some cells began to migrate away from the center at 5 days and were labeled by either neuronal marker neuron-specific β-tubulin (TuJ1) or astrocytic marker glial fibrillary acidic protein. The differentiated neurons (TuJ1 +) exhibited characteristic cytosolic Ca 2+ oscillations in response to excitatory neurotransmitter glutamate. These findings suggest the suitability of the 3D culture system for the proliferation and differentiation of neural precursor cells.

Fibroblast growth factor 2 up regulates telomerase activity in neural precursor cells.

During brain development, neuronal and glial cells are generated from neural precursors on a precise schedule involving steps of proliferation, fate commitment and differentiation. We report that telomerase activity is highly expressed during embryonic murine cortical neurogenesis and early steps of gliogenesis and progressively decreases thereafter during cortex maturation to be undetectable in the normal adult brain. We evidenced neural precursor cells (NPC) as the principal telomerase-expressing cells in primary cultures from E15 mouse embryo cortices. Their differentiation either in neurons or in glial cells lead to a down regulation of telomerase activity that was directly correlated to the decrease of telomerase core protein (mTERT) mRNA synthesis. Furthermore, we show that FGF2 (fibroblast growth factor 2), one of the main regulators of CNS development, induces a dose-dependant increase of both the proliferation of NPC and telomerase activity in primary cortical cultures without affecting the mTERT mRNA synthesis compared to that of glyceraldehyde-3-phosphate dehydrogenase (mGAPDH). Finally, we evidenced that AZT (3'-azido-2', 3'-dideoxythymidine), known to inhibit telomerase activity, blocks in a dose dependant manner the FGF2-induced proliferation of NPC. Altogether, our results are in favor of an important role of telomerase activity during brain organogenesis. Oncogene (2000).

A Subset of Fibroblast Growth Factors (Fgfs) Promote Survival, but Fgf-8b Specifically Promotes Astroglial Differentiation of Rat Cortical Precursor Cells

Fibroblast growth factor-2 (Fgf-2 or basic Fgf) is known to promote the survival, proliferation, and differentiation of neural precursor cells. We have examined and compared the effects of Fgf-2 with those of Fgf-1, -4, -6, -7, -9, and -10, as well as three isoforms of Fgf-8 (-8a, -8b, and -8c), on the fate of cultured embryonic day 15 (E15) rat cortical cells. Clonal analysis, using retroviral tagging, shows that only Fgf-2, -4, and -8b can efficiently promote the survival of cortical precursor cells, the majority of which give rise to neurons. Surprisingly, and in contrast to other Fgfs, Fgf-8b also promotes astroglial differentiation of a subpopulation of these cells, which would otherwise appear to yield neurons. We also show that E15 cortical cells initially express the IIIc isoforms of Fgf-receptors (R-1,-2, and -3 but within 16 h of culturing they down regulate FgfR2-IIIc. These studies demonstrate that cortical precursor cells respond to Fgf stimulation in different ways depending on the ligand and by inference the Fgf receptors activated.

Basic fibroblast growth factor promotes epidermal growth factor responsiveness and survival of mesencephalic neural precursor cells

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) induce proliferation of neural precursor cells from several central nervous system regions in vitro. We have previously described two neural precursor cell populations from 13.5 days postcoitium (dpc) mesencephalon, one forming colonies in response to EGF, present in the ventral mesencephalon, and other forming colonies in response to EGF + bFGF, mainly present in the dorsal mesencephalon. In the present work, we show that 13.5 dpc dorsal mesencephalic cells required bFGF only for 1 h to form colonies in response to EGF alone, indicating that these two growth factors act in sequence rather than simultaneously. Absence of bFGF at the beginning of the culture gave rise to very few colonies, even after the addition of EGF + bFGF, suggesting that cells responsive to bFGF were very labile in the primary culture condition. This result is in contrast with cells pretreated with bFGF, which could survive for up to 5 days in the absence of bFGF or EGF, and then were capable of efficiently forming colonies in response to EGF. Basic FGF was also able to support survival of EGF-responsive neural precursors from both ventral and dorsal mesencephalon. The population requiring bFGF to form colonies in response to EGF was identified at different developmental stages (11.5-15.5 dpc), with higher contribution to the total number of neural precursors cells detected (EGF-responsive plus bFGF-responsive) at early stages and in the dorsal region. We show that the differentiation effect of bFGF resulted in the appearance of the mRNA coding for the EGF receptor. Our data suggest that bFGF-responsive neural precursors are the source of EGF-responsive neural precursors.

Id1, Id2, and Id3 gene expression in neural cells during development.

Id1, Id2, and Id3 mRNA are expressed mainly in the proliferating ependymal cell zone of the mouse brain during embryogenesis. In this study, the expression pattern and cell phenotypes of the Id family mRNA were examined in postnatal and adult rat brain. The expression of Idl and Id3 mRNA in rat brain was observed in the cortex layer 1, corpus callosum, ventricular/subventricular zone (VZ/ SVZ), and the CA1-4 layers of the hippocampus at postnatal day 1 (P1) through P14, whereby it declined at 2 months. In general, the developmental pattern of Idl mRNA coincided with the pattern observed for Id3 mRNA. Similar to Id1 and Id3, Id2 mRNA was highly expressed in the corpus callosum, VZ/SVZ, and the hippocampus. Examination of Id2 mRNA revealed high levels in the cortex and caudate putamen at P1 through P14, whereas a decline was observed in its expression in the adult cortex. In P5 rat cerebellum, all Id mRNA examined were found in the internal granular cell layers; however, at this time point, only Id2 mRNA expression was detected in the differentiating zone of the external granular cell layers, preferentially localizing to adult Purkinje cells. Furthermore, only Id2 mRNA expression in brain was observed in NF+ neurons at P5. Examination of S100alpha+ and GFAP+ astrocytes, revealed the presence of all three mRNAs, whereas the expression of Id2 and Id3 mRNA was absent in 04+ immature oligodendrocytes. These data suggest that the spatial and temporal kinetic patterns during development, as well as cellular specificity, of the Id gene family may play a critical role in neural precursor cell proliferation and cell divergence.

Fibroblast Growth Factor 2 (FGF-2) Promotes Acquisition of Epidermal Growth Factor (EGF) Responsiveness in Mouse Striatal Precursor Cells: Identification of Neural Precursors Responding to both EGF and FGF-2

Epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) induce the proliferation of neural precursor cells isolated from specific regions of the embryonic and adult brain. However, the lineage relationship between the EGF- and FGF-2-responsive cells is unknown. In this study we used phosphorylation of the transcription factor cAMP response element-binding protein as a functional readout to identify cells responding to EGF and FGF-2. In primary cultures of mouse embryonic day 14 (E14) striatum, maintained in vitro for 24 hr, 12% of the cells responded to FGF-2, whereas no response to EGF could be detected. Seventy-five percent of these FGF-2-responsive cells were beta tubulin III (TuJ1)-positive neurons, and 25% expressed nestin, a marker for neuroepithelial precursors. After growth factor treatment for 6 d, a population of nestin-positive cells responding to both EGF and FGF-2 were identified. The 6-d-old cultures also contained a small number of TuJ1-positive cells that responded to FGF-2 only. Priming of striatal cells for 24 hr with FGF-2 but not with EGF was sufficient to induce the appearance of EGF- and FGF-2 responsive cells after only 2 d in vitro. Thus, neural precursor cells from the mouse E14 striatum initially responding to FGF-2 only acquire EGF responsiveness later during in vitro development. At this stage EGF and FGF-2 act on the same cells. The acquisition of EGF responsiveness is promoted by FGF-2.

Structural Modification of Fibroblast Growth Factor-binding Heparan Sulfate at a Determinative Stage of Neural Development

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity and appear to act by coupling particular forms of FGF to appropriate FGF receptors. During neural development, one particular HS proteoglycan is able to rapidly switch its potentiating activity from FGF-2, as neural precursor cell proliferation occurs, to FGF-1, as neuronal differentiation occurs. Using various analytical techniques, including chemical and enzymatic cleavage, low pressure chromatography, and strong anion-exchange high performance liquid chromatography, we have analyzed the different HSs expressed during these crucial developmental stages. There are distinct alterations in patterns of 6-O-sulfation, total chain length, and the number of sulfated domains of the HS from the more mature embryonic brain. These changes correlate with a switch in the ability of the HS to potentiate the actions of FGF-1 in triggering cell differentiation. It thus appears that each HS pool is designed to function in the modulation of an intricate interaction with a specific growth factor and its cognate receptor, and suggests tightly regulated expression of specific, bioactive disaccharide sequences. The data can be used to construct a simple model of controlled variations in HS chain structure which have functional consequences at a crucial stage of neuronal maturation.

Multiple factors control the proliferation and differentiation of rat early embryonic (day 9) neuroepithelial cells

The proliferation and differentiation of neural precursor cells is largely controlled by environmental factors. By providing the factors that favor the proliferation or suppress the differentiation of this cell type, we isolated and expanded an early neuroepithelial pre-differentiated cell type from E9 rat neural plate in serum-free medium. This has led to the establishment of a neural epithelial precursor (NEP) cell line. The NEP cell's properties are substantially different from those of cell lines previously derived from neural tissue at later stages of development. Initial selection and survival of this cell type requires a factor secreted by an embryonic Schwann (nrESC) cell line. Continued passage of these cells requires cell-cell contact for both survival and growth. Neural cell differentiation can be induced in this nestin positive precursor cell line by bFGF and forskolin. General neuronal markers, as well as cortical neuron-specific protein kinase C isozyme, and accumulation of glutamate and aspartate were induced in most cells. Choline acetyl-transferase was also induced in a small number of cells. When implanted into neonatal rat brain, the NEP cell line gave rise to several distinct neuronal and glial phenotypes in different regions of the brain including cerebellar cortex and hippocampus.

Random catecholaminergic differentiation of mesencephalic neural precursors.

We investigated how several factors influence the catecholaminergic phenotype establishment from embryonic mesencephalic neural precursors in culture. Using a semiquantitative RT-PCR procedure we found no significant effect of several growth factors or conditioned media on tyrosine hydroxylase (TH) mRNA levels. Nevertheless, neural precursor cells expanded by epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed the ability to express TH mRNA. Subcultures of EGF expanded neural precursor cells expressed TH mRNA, but not all individual secondary colonies obtained had this characteristic. Preferential dopaminergic differentiation was observed in our culture conditions. Our results suggest that EGF stimulates the proliferation of neural precursor cells that have the potential but differentiate randomly to catecholaminergic cells.

High potassium promotes differentiation of retinal neurons but does not favor rod differentiation

Neural retinal cells of newborn rats were cultured under dissociated culture conditions. Differentiation of several types of retinal cells was confirmed by immunohistochemical detection of type-specific neural phenotypes. We used Thy-1.1 antigen as a ganglion cell marker, HPC-1 or GABA as an amacrine cell marker and rhodopsin as a rod cell marker. With a high concentration of potassium (38 mM), expression of the respective neural phenotypes were differentially affected. High K+ increased the number of Thy-1.1 positive cells 6 to 8 fold, and drastically promoted their neurite extension. The same culture conditions, however, reduced considerably the number of rhodopsin positive cells, possibly due to the unique membrane properties of photoreceptors. A high K + concentration also promoted differentiation of HPC-1 positive and GABA positive cells, but to a lesser extent than the Thy-1.1 positive cells. Several possibilities were examined to understand the effect of a high K + concentration on retinal neural cells. The total cell number in cultures with a high K + concentration was approximately half of that in control cultures at day 3 and slightly smaller at day 11, suggesting that high K + did not have a positive general effect on the proliferation or survival of retinal cells. Naturally occurring neuronal death (apoptosis) is a well-known phenomenon during retinal development. A histochemical method for detecting DNA fragmentation, a step preceding apoptosis, showed that high K + had no preventive effect. BrdU (bromodeoxyuridine) immunohistochemistry showed that high K + did not seem to enhance proliferation of neural precursor cells. These results indicate that a high K + concentration promotes the expression of neuronal phenotypes but is not a favorable condition for rod differentiation. Since a high K + concentration is considered to induce depolarization of nerve cells, the present results suggest an anterograde influence from surrounding neuronal cells, through chronic depolarization by elevated K +, is essential for the differentiation and maturation of retinal cells.

FGF2 regulates proliferation of neural crest cells, with subsequent neuronal differentiation regulated by LIF or related factors

Two of the key early events in the development of the peripheral nervous system are the proliferation of neural crest precursor cells and their subsequent differentiation into different neural cell types. We present evidence that members of the fibroblast growth factor family, (FGF1 or FGF2) act directly on the neural crest cells in vitro to stimulate proliferation in the presence of serum. These findings correlate with in situ hybridisation analysis, which shows FGF2 mRNA is expressed in cells both in the neural tube and within newly formed sensory ganglia (dorsal root ganglia, DRG) at embryonic day 10 in the mouse, when neural crest precursors are proliferating within the DRG. This data infers an autocrine/paracrine loop for FGF regulation of proliferation. Evidence supporting this notion is provided by the finding that part of the endogenous proliferative activity in the NC cultures is related to FGF. It was also found, in early neural crest cultures, that exogenous FGF completely inhibited neuronal differentiation, probably as a direct consequence of its mitogenic activity. In order to stimulate neuronal differentiation significantly, it was necessary to remove the FGF and replace it with leukemia inhibitory factor (LIF) or related factors. Under these conditions, 50% of the cells differentiated into neurons, which developed a sensory neuron morphology and were immunoreactive for the sensory markers CGRP and substance P. These data support a model of neural crest development, whereby multipotential neural crest precursor cells are stimulated to divide by FGF and subsequent development into sensory neurons is regulated by LIF or other cytokines with a similar signalling mechanism.

Fibroblast growth factor stimulates the proliferation and differentiation of neural precursor cells in vitro

We have developed an in vitro culture system to study the regulation of proliferation and differentiation of neural precursor cells contained within the neuroepithelium of embryonic day 10 mice. A number of soluble growth factors have been tested for their ability to regulate these early events and, of these factors, we have found that the fibroblast growth factors [FGFs] can directly stimulate the proliferation and survival of the neuroepithelial cells. At least 50% of the neuroepithelial cells divide in the presence of FGF whereas in the absence of FGF all of the cells die within 6 days of culture. At higher concentrations of FGF, the cells change from being nonadherent round cells in tight clusters into a more flattened cell type which adheres to the substratum. This morphological change is accompanied by the expression of both neurofilament and GFAP, which are definitive markers of the two major cell types in the central nervous system: neurons and glia. In addition a neuroepithelial cell line, which does not rely on FGF for survival or proliferation, expresses both of these markers in response to FGF. These results indicate that FGF is stimulating the differentiation of the neuroepithelial cells into mature neurons and glia.

Paludrine

Genetic variations in <i>CACNA1C</i>, which encodes the Ca_v1.2 subunit of L-type calcium channels (LTCCs), are associated with multiple forms of neuropsychiatric disease that manifest high anxiety in patients. In parallel, mice harboring forebrain-specific conditional knockout of <i>cacna1c</i> (forebrain-Ca_v1.2 cKO) display unusually high anxiety-like behavior. LTCCs in general, including the Ca_v1.3 subunit, have been shown to mediate differentiation of neural precursor cells (NPCs). However, it has not previously been determined whether Ca_v1.2 affects postnatal hippocampal neurogenesis <i>in vivo</i>. Here, we show that forebrain-Ca_v1.2 cKO mice exhibit enhanced cell death of young hippocampal neurons, with no change in NPC proliferation, hippocampal size, dentate gyrus thickness, or corticosterone levels compared with wild-type littermates. These mice also exhibit deficits in brain levels of brain-derived neurotrophic factor (BDNF), and Cre recombinase-mediated knockdown of adult hippocampal Ca_v1.2 recapitulates the deficit in young hippocampal neurons survival. Treatment of forebrain-Ca_v1.2 cKO mice with the neuroprotective agent P7C3-A20 restored the net magnitude of postnatal hippocampal neurogenesis to wild-type levels without ameliorating their deficit in BDNF expression. The role of Ca_v1.2 in young hippocampal neurons survival may provide new approaches for understanding and treating neuropsychiatric disease associated with aberrations in <i>CACNA1C</i>. Visual Abstract