Chronic hypoxia up-regulates fibroblast growth factor ligands in the perinatal brain and induces fibroblast growth factor-responsive radial glial cells in the sub-ependymal zone.

A number of signaling molecules have been implicated in the acute response to hypoxia/ischemia in the adult brain. In contrast, the reaction to chronic hypoxemia is largely unexplored. We used a protocol of chronic hypoxia in rat pups during the first three postnatal weeks, encompassing the period of cellular plasticity in the cerebral cortex. We find that the levels of fibroblast growth factor 1 (FGF1) and FGF2, two members of the FGF family, increase after 2 weeks of chronic hypoxia. In contrast, members of the neurotrophin family are unaffected. FGF2 is normally expressed in the nucleus of mature, glial fibrillary acidic protein (GFAP)-containing astrocytes. Under hypoxia, most FGF2-containing cells do not express detectable levels of GFAP, suggesting that chronic low O(2) induces their transformation into more immature glial phenotypes. Remarkably, hypoxia promotes the appearance of radial glia throughout the sub-ventricular and ependymal zones. Most of these cells express vimentin and brain lipid binding protein. A subset of these radial glial cells expresses FGF receptor 1, and are in close contact with FGF2-positive cells in the sub-ventricular zone. Thus, FGF receptor signaling in radial glia may foster cell genesis after chronic hypoxic damage. From the results of this study we suggest that after the chronic exposure to low levels of oxygen during development, the expression of radial glia increases in the forebrain periventricular region. We envision that astroglia, which are the direct descendants of radial glia, are reverting back to immature glial cells. Alternatively, hypoxia hinders the normal maturation of radial glia into GFAP-expressing astrocytes. Interestingly, hypoxia increases the levels of expression of FGF2, a factor that is essential for neuronal development. Furthermore, chronic hypoxia up-regulated FGF2's major receptor in the periventricular region. Because radial glia have been suggested to play a key role in neurogenesis and cell migration, our data suggests that hypoxia-induced FGF signaling in radial glia may represent part of a conserved program capable of regenerating neurons in the brain after injury.

Stem cells in neurodevelopment and plasticity.

The processes of stem cell proliferation and differentiation during embryogenesis are governed by transcription factors that regulate the regional differentiation of the central nervous system (CNS). Do neural "stem" cells persisting in the postnatal CNS disobey this sequence of events? The division of neural progenitor cells is promoted by basic Fibroblast Growth Factor Fgf2 or Epidermal Growth Factor Egf. However, while the intraventricular administration of FgF2 during embryogenesis increases the generation of cortical pyramidal neurons, the same treatment in the adult CNS produces interneurons of the olfactory bulb. The competence of neural progenitor cells to respond to Fgf is dictated by nuclear transcription factors that constrain neuronal fates through time. Developmentally regulated transcriptional programs are regulated by cell interactions, as dividing cells check their molecular signature against that of their environment. Thus, cell surface interactions account for competitive phenomena among pools of cells, including the inhibitory effect of neurons on the division of their progenitors, and may also explain the "permissive" effects of non-CNS environments. The challenge remains to understand the genetic programs that control the fate of progenitor cells within the postnatal CNS and their regulation by stress, apoptosis and environmental perturbations. These programs are likely to be similar to gene cascades that control proliferation, differentiation and migration of progenitor cells at earlier stages of development.

The subcellular localization of Otx2 is cell-type specific and developmentally regulated in the mouse retina.

Recent evidence implicates homeodomain-containing proteins in the specification of cell fates in the central nervous system. Here we report that in the embryonic mouse eye Otx2, a paired homeodomain transcription factor, was found in retinal pigment epithelial cells and a restricted subset of retinal neurons, including ganglion cells. In the postnatal and adult eye, however, both the cellular and subcellular distribution of the Otx2 protein were cell type-specific. Otx2 was detected only in the nuclei of retinal pigment epithelial and bipolar cells, but was present in the cytoplasm of rod photoreceptors. Immunohistochemical studies of retinal explants and transfected cell lines both suggested that the retention of Otx2 in the cytoplasm of immature rods is a developmentally regulated process. The differential distribution of Otx2 in the cytoplasm of rods and the nucleus of other cell types, suggests that subcellular localization of this transcription factor may participate cell fate determination during specific phases of retinal development.

Basic fibroblast growth factor (Fgf2) is necessary for cell proliferation and neurogenesis in the developing cerebral cortex.

Little is known about regionally specific signals that control the number of neuronal progenitor cells in vivo. We have previously shown that the germline mutation of the basic fibroblast growth factor (Fgf2) gene results in a reduction in the number of cortical neurons in the adult. We show here that Fgf2 is expressed in the pseudostratified ventricular epithelium (PVE) in a dorsoventral gradient and that Fgf2 and its receptor, Fgfr-1, are downregulated by mid to late stages of neurogenesis. In Fgf2 knockout mice, the volume and cell number of the dorsal PVE (the cerebral cortical anlage) are substantially smaller, whereas the volume of the basal PVE is unchanged. The dorsal PVE of Fgf2 knockout mice has a 50% decrease in founder cells and a reduced expansion of the progenitor pool over the first portion of neurogenesis. Despite this reduction, the degree of apoptosis within the PVE is not changed in the Fgf2 knockouts. Cortical neuron number was decreased by 45% in Fgf2 knockout mice by the end of neurogenesis, whereas the number of neurons in the basal ganglia was unaffected. Microscopically, the frontal cerebral cortex of neonatal Fgf2 null mutant mice lacked large neurons in deep cortical layers. We suggest that Fgf2 is required for the generation of a specific class of cortical neurons arising from the dorsal PVE.

Progressive impairment of developing neuroendocrine cell lineages in the hypothalamus of mice lacking the Orthopedia gene.

Development of the neuroendocrine hypothalamus is characterized by a precise series of morphogenetic milestones culminating in terminal differentiation of neurosecretory cell lineages. The homeobox-containing gene Orthopedia (Otp) is expressed in neurons giving rise to the paraventricular (PVN), supraoptic (SON), anterior periventricular (aPV), and arcuate (ARN) nuclei throughout their development. Homozygous Otp(-/-) mice die soon after birth and display progressive impairment of crucial neuroendocrine developmental events such as reduced cell proliferation, abnormal cell migration, and failure in terminal differentiation of the parvocellular and magnocellular neurons of the aPV, PVN, SON, and ARN. Moreover, our data provide evidence that Otp and Sim1, a bHLH-PAS transcription factor that directs terminal differentiation of the PVN, SON, and aPV, act in parallel and are both required to maintain Brn2 expression which, in turn, is required for neuronal cell lineages secreting oxytocin (OT), arginine vasopressin (AVP), and corticotropin-releasing hormone (CRH).

Identification, chromosomal assignment, and expression analysis of the human homeodomain-containing gene Orthopedia (OTP).

Homeodomain (HD) genes are helix-turn-helix transcription factors that play key roles in the specification of cell fates. In the central nervous system (CNS), HD genes not only position cells along an axis, but also specify cell migration patterns and may influence axonal connectivity. In an effort to identify novel HD genes involved in the development of the human CNS, we have cloned, characterized, and mapped the human homologue of the murine HD gene Orthopedia (Otp), whose product is found in multiple cell groups within the mouse hypothalamus, amygdala, and brain stem. Human cDNA and genomic libraries were screened with probes derived from mouse Otp sequences to find the human homologue, OTP. The deduced amino acid sequence of the open reading frame of the human cDNA is 99% homologous to mouse Otp and demonstrates a high degree of conservation when compared to sea urchin and Drosophila. OTP was mapped to human chromosome 5q13.3 using radiation hybrid panel mapping and fluorescence in situ hybridization. Flanking markers were identified from YAC clones containing OTP. A single putative OTP gene product was found in 17-week human fetal brain tissue by Western blot analysis using a novel polyclonal antibody raised against a conserved 13-amino-acid sequence at the C-terminus of the OTP protein. Expression in the developing human hypothalamus was confirmed by immunohistochemistry.

Fibroblast growth factor signaling regulates growth and morphogenesis at multiple steps during brain development.

The fibroblast growth factor (FGF) family comprises several members with distinct patterns of expression in the developing central nervous system. FGFs regulate the early specification and the subsequent growth of central nervous system regions. These different actions require the coordinated activation of distinct sets of target genes by FGFs at the appropriate stage of development. The role of FGF2 in the growth and morphogenesis of the cerebral cortex is reviewed in detail. The cellular and molecular mechanisms that underlie the action of FGF2 on cortical development are discussed.

Changes in cerebral cortex size are governed by fibroblast growth factor during embryogenesis.

We show that fibroblast growth factor 2 (FGF2) and FGF receptors are transiently expressed by cells of the pseudostratified ventricular epithelium (PVE) during early neurogenesis. A single microinjection of FGF2 into cerebral ventricles of rat embryos at E15.5 increased the volume and total number of neurons in the adult cerebral cortex by 18% and 87%, respectively. Microinjection of FGF2 by the end of neurogenesis, at E20.5, selectively increased the number of glia. Mice lacking the FGF2 gene had fewer cortical neurons and glia at maturity. BrdU studies in FGF2-microinjected and FGF2-null animals suggested that FGF2 increases the proportion of dividing cells in the PVE without affecting the cell-cycle length. Thus, FGF2 increases the number of rounds of division of cortical progenitors.

Dlx-2 homeobox gene controls neuronal differentiation in primary cultures of developing basal ganglia.

Homeodomain-containing genes of the Dlx family are expressed in the developing basal ganglia. To investigate the role of Dlx genes during development, we studied their cellular localization in primary cultures of embryonic basal telencephalon, and examined the changes in cellular phenotypes resulting from blockade of Dlx-2 expression. Cells containing Dlx-1, Dlx-2, and Dlx-5 mRNAs are immature cells of the neuronal lineage expressing the microtubule-associated proteins (MAPs) MAP1B and MAP2, but not glial fibrillary acidic protein (GFAP). Treatment of these cells with antisense oligonucleotides targeted to Dlx-2 caused a specific decrease of Dlx-2 mRNA and protein. This decrease in the Dlx-2 gene product was associated with a decrease in the expression of MAP2, a protein localized in neuronal dendrites, along with a smaller decrease in the 200-kDa neurofilament subunit (NF-H). Proteins expressed preferentially in axons were unchanged. This reduction in MAP2 expression was associated with a decrease in dendrite outgrowth and an increased level of cell proliferation. None of these changes were elicited by antisense oligonucleotides targeted to Dlx-1. We suggest that the Dlx-2 gene product regulates two interrelated aspects of neuronal differentiation: the exit from the mitotic cycle and the capability to grow MAP2-positive dendrites. As such, this gene product may be important for the establishment of neuronal polarity, setting the stage for afferent synaptic connectivity.

Characterization and sequence analysis of the human homeobox-containing gene GBX2.

Polymerase chain reaction (PCR) was used to amplify portion of homeobox genes present in a human 11-week fetal brain cDNA library. One of these PCR products was determined by sequencing to be the Gastrulation and brain specific-2 gene (GBX2). Screening this human fetal brain cDNA library with probes specific for GBX2 led to the identification of a 2151-bp cDNA clone. The nucleotide sequence of the cDNA clone encodes for a protein of 347 amino acid residues. The amino acid sequence of the GBX2 homeodomain is identical (100%) to the that of homologous gene, Gbx2, expressed in the developing mouse embryo and virtually identical (97%) to a gene expressed in the developing chicken embryo, CHox7. The 5' end of the GBX2 gene contains a CpG island in the untranslated region and a trinucleotide (CCG)8 repeat in the coding region. The amino-terminal end of the GBX2 protein is proline-rich, with 30 proline residues in one stretch of 120 amino acids. A single 2.2-kb transcript was detected by Northern analysis in the developing human CNS as well as in other tissues. The human genomic clone for GBX2 was also isolated, characterized, and mapped to 2q36(d)-q37 by somatic cell hybrid analysis and fluorescence in situ hybridization. These studies provide a framework for designing future experiments that are needed to determine the functional significance of this gene in CNS development.

Fibroblast growth factor 2 increases Otx2 expression in precursor cells from mammalian telencephalon.

Dissociated primary cultures from rat telencephalon at different developmental stages were used to study the effect of basic fibroblast growth factor (FGF2) on Otx2, Dlx1, and Emx1, three homeobox genes expressed in different regions of the developing mammalian forebrain. At embryonic day (E)13.5. the regional pattern of expression of Otx1, Otx2, Dlx1, Dlx2, Dlx5, and Emx1 is maintained in primary culture, suggesting that cells are already committed to a regional identity at this stage. In these cultures, Otx2 is expressed by precursor cells, whereas Dlx1 and Emx1 are predominantly expressed by postmitotic cells. We found that FGF2 increased Otx2 expression within precursor cells and the total number of Otx2-expressing cells. This effect was gene-specific, dose-dependent, and temporally regulated, with larger effects at earlier stages of development (E11.5). At E13.5, the effect of FGF2 on Otx2 expression was restricted to the basal telencephalon. Our results suggest that a restricted population of neuroblasts respond to FGF2 in a temporally regulated fashion by proliferating and increasing Otx2 expression. This interaction between FGF2 and Otx2 may be important for the regulation of neurogenesis in the forebrain.

Basic fibroblast growth factor increases the number of excitatory neurons containing glutamate in the cerebral cortex.

Stem cells isolated from the ventricular zone of embryonic day 12.5 rat telencephalon progressively proliferate and differentiate in vitro into three major classes of amino acid-containing neurons, glutamate, aspartate, and GABA. We quantitatively examined the effect of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on amino acid-containing neurons. bFGF caused a threefold increase in glutamate-containing neurons, while the number of GABA- and aspartate-containing neurons was not significantly changed. In contrast, NGF did not alter the number of amino acid-containing neurons. The ratio of glutamate- to GABA-containing neurons in untreated or NGF-treated cultures was 0.6:1. In the bFGF-treated cultures, this ratio was 1.4:1, which closely approximates the ratio in the cerebral cortex in vivo. Treatment with antisense oligonucleotides targeted to bFGF mRNA provoked a 50% decrease in the number of glutamate-containing neurons but had no significant effect on the GABA-containing neurons. Thus, diffusible factors such as bFGF may play an important role in determining the relative proportion of excitatory versus inhibitory neurons in the cerebral cortex by selectively regulating the proliferation of stem cells committed to different neurotransmitter phenotypes.

Excitatory amino acid receptors in glial progenitor cells: molecular and functional properties.

We have analyzed the molecular and biophysical properties of glutamate-gated channels in cells of the oligodendrocyte lineage, using both the CG-4 primary cell line (Louis et al: J. Neurosci. Res. 31:193-204, 1992a) and oligodendrocyte progenitors purified from the rat cerebral cortex. CG-4 progenitor cells, as well as primary progenitors, were stained with a specific anti-GABA antibody. In whole-cell patch-clamp recordings, rapid perfusion of the agonists L-glutamate, kainate, and AMPA produced rapidly desensitizing currents in CG-4 cells. NMDA was ineffective. Both rapidly desensitizing and steady-state components of responses to kainate were inhibited by the kainate/AMPA receptor antagonist CNQX. Northern blot analysis of total mRNA isolated from CG-4 cells revealed co-expression of both AMPA- and kainate-preferring glutamate receptor subunits. The activation of glutamate receptors in CG-4 cells caused a rapid and transient elevation of mRNAs for the immediate early gene NGFI-A.

Induction of immediate early genes by cyclic AMP in primary cultures of neurons from rat cerebral cortex.

In this paper, we tested whether physiological activators of the cAMP second messenger pathway in primary cultures of neurons from rat cerebral cortex directly induce c-fos and other immediate early gene (IEG) transcription factors. We have found that brief (30 s to 2 min) stimulation of neurons with vasoactive intestinal peptide (VIP) and SKF-38393, a D1-dopaminergic receptor agonist, potently increased mRNA levels for the IEGs c-fos, jun-B, and NGFI-A, with weaker increases for c-jun. This action was mimicked by forskolin and dibutyryl cAMP. IEG induction by VIP and dibutyryl cAMP was not blocked by excitatory amino acid receptor antagonists or by blockers of dihydropyridine-sensitive calcium channels. Moreover, calcium-free medium did not modify IEG induction by dibutyryl cAMP, suggesting that cAMP can directly regulate IEG expression in differentiated neurons independently of calcium.

Differential induction of immediate early genes by excitatory amino acid receptor types in primary cultures of cortical and striatal neurons.

In primary cultures of neurons from cerebral cortex and striatum, 30 s stimulation with the excitatory amino acid glutamate elicited a 5 to 9-fold increase in immediate early gene (IEG) mRNAs. Glutamate increased c-fos, c-jun, jun-B, and NGFI-A (zif/268) mRNAs by binding to both alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor types, and increased c-fos, jun-B, and NGFI-A mRNAs by binding to the metabotropic receptor. NMDA receptor activation elicited IEG expression by a transmembrane calcium influx; AMPA receptor-induced depolarization played a permissive role for the opening of the NMDA receptor channel. The protein kinase C (PKC) inhibitor H-7 (but not inhibitors of cyclic nucleotide-dependent and calcium/calmodulin-dependent protein kinases) partially blocked IEG expression induced by glutamate.

Modulation of protein kinase C translocation by excitatory and inhibitory amino acids in primary cultures of neurons.

In primary cultures of neurons from rat cerebral cortex and neostriatum, excitatory amino acids stimulate the translocation of protein kinase C (PKC) from the cytoplasm to the membrane. In the presence of a physiological concentration of Mg2+ in the extracellular medium, glutamate induces PKC translocation by binding to both N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) excitatory amino acid receptors. Quisqualate translocates the enzyme by stimulating primarily AMPA receptors and possibly metabotropic receptors. NMDA receptor-induced PKC translocation is sodium independent, whereas quisqualate receptor-induced PKC translocation is sodium dependent; none of the agonists is active in the absence of calcium from the extracellular medium. Muscimol does not modify excitatory amino acid stimulation; however, blockade of gamma-aminobutyric acid(A) receptors by bicuculline greatly enhances glutamate-induced PKC translocation. This enhancement is blocked by the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) and by tetrodotoxin.

Primary cultures of corticostriatal cells from newborn rats: a model to study muscarinic receptor subtypes regulation and function.

In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes increasing amounts of acetylcholine (ACh) from the third day in vitro (DIV), which reaches a plateau around the 10 day of culture. Muscarinic cholinergic receptors (mAChR), measured by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB), are detectable from the fifth DIV and increase linearly during the time of culture. At the twelfth DIV, the density of mAChRs (approximately 600 fmol/mg protein) is comparable to the density of mAChR in adult rat cortex. These receptors are coupled to second messenger systems, since muscarinic agonists inhibit adenylate cyclase activity and stimulate phosphoinositide breakdown with efficacies and potencies similar to those found in adult rat cortex. Moreover, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, we were able to demonstrate the presence of the m1, m3, and m4 mAChR subtype mRNAs in this neuronal culture at 12 DIV. Our data suggest that corticostriatal neuronal cultures develop in vitro ACh-synthesizing neurons and functionally active cholinergic receptors. This therefore makes them ideally suited to study the development and properties of brain mAChR subtypes.

Coexistence of GABA receptors and GABA-modulin in primary cultures of rat cerebellar granule cells.

GABA-modulin (GM), a basic polypeptide purified from rat brain synaptosomes, which is an allosteric inhibitor of GABA recognition sites, has been detected in primary cultures of cerebellar interneurons enriched in granule cells by immunohistochemistry, using a specific antibody raised in rabbit injected with GM purified from rat brain synaptosomes. In these cultures, GM is expressed by the granule cells, which are postsynaptic to GABAergic interneurons, but not by glial cells. In rat cerebellar sections anti-GM antiserum intensely strains the granular cell layer and Purkinje cell dendrites and cell bodies. GM has been purified from the cerebellar granule cell cultures and appears to be identical under biochemical, immunological, and functional criteria to authentic GM purified from rat brain synaptosomes. Granule cell cultures devoid of GABAergic neurons contain the GABA/BZ/Cl- receptor complex; in fact, intact cell monolayers, incubated in physiological buffer at 25 degrees C, express 3H-muscimol and 3H-flunitrazepam binding sites, which are comparable to the sites detected in cell membrane preparations and which modulate each other reciprocally. It is concluded that GM might participate in the supramolecular organization of the GABA receptor complex, perhaps functioning as a modulator of this receptor protein.