Neocortical neurogenesis in humans is restricted to development

Stem cells generate neurons in discrete regions in the postnatal mammalian brain. However, the extent of neurogenesis in the adult human brain has been difficult to establish. We have taken advantage of the integration of (14)C, generated by nuclear bomb tests during the Cold War, in DNA to establish the age of neurons in the major areas of the human cerebral neocortex. Together with the analysis of the neocortex from patients who received BrdU, which integrates in the DNA of dividing cells, our results demonstrate that, whereas nonneuronal cells turn over, neurons in the human cerebral neocortex are not generated in adulthood at detectable levels but are generated perinatally.     

Monitoring of implanted stem cell migration in vivo: a highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat

In vivo monitoring of stem cells after grafting is essential for a better understanding of their migrational dynamics and differentiation processes and of their regeneration potential. Migration of endogenous or grafted stem cells and neurons has been described in vertebrate brain, both under normal conditions from the subventricular zone along the rostral migratory stream and under pathophysiological conditions, such as degeneration or focal cerebral ischemia. Those studies, however, relied on invasive analysis of brain sections in combination with appropriate staining techniques. Here, we demonstrate the observation of cell migration under in vivo conditions, allowing the monitoring of the cell dynamics within individual animals, and for a prolonged time. Embryonic stem (ES) cells, constitutively expressing the GFP, were labeled by a lipofection procedure with a MRI contrast agent and implanted into rat brains. Focal cerebral ischemia had been induced 2 weeks before implantation of ES cells into the healthy, contralateral hemisphere. MRI at 78-microm isotropic spatial resolution permitted the observation of the implanted cells with high contrast against the host tissue, and was confirmed by GFP registration. During 3 weeks, cells migrated along the corpus callosum to the ventricular walls, and massively populated the borderzone of the damaged brain tissue on the hemisphere opposite to the implantation sites. Our results indicate that ES cells have high migrational dynamics, targeted to the cerebral lesion area. The imaging approach is ideally suited for the noninvasive observation of cell migration, engraftment, and morphological differentiation at high spatial and temporal resolution.     

Pyramidal neurons of upper cortical layers generated by NEX-positive progenitor cells in the subventricular zone

The generation of pyramidal neurons in the mammalian neocortex has been attributed to proliferating progenitor cells within the ventricular zone (VZ). Recently, the subventricular zone (SVZ) has been recognized as a possible source of migratory neurons in brain slice preparations, but the relevance of these observations for the developing neocortex in vivo remains to be defined. Here, we demonstrate that a subset of progenitor cells within the SVZ of the mouse neocortex can be molecularly defined by Cre recombinase expression under control of the NEX/Math2 locus, a neuronal basic helix-loop-helix gene that by itself is dispensable for cortical development. NEX-positive progenitors are generated by VZ cells, move into the SVZ, and undergo multiple asymmetrical and symmetrical cell divisions that produce a fraction of the neurons in the upper cortical layers. Our data suggest that NEX-positive progenitors within the SVZ are committed to a glutamatergic neuronal fate and have evolved to expand the number of cortical output neurons that is characteristic for the mammalian forebrain.     

Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F(1) genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F(1) ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F(1) ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F(1) ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F(1) ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F(1) ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.     

A large-scale,gene-driven mutagenesis approach for the functional analysis of the mouse genome

A major challenge of the postgenomic era is the functional characterization of every single gene within the mammalian genome. In an effort to address this challenge, we assembled a collection of mutations in mouse embryonic stem (ES) cells, which is the largest publicly accessible collection of such mutations to date. Using four different gene-trap vectors, we generated 5,142 sequences adjacent to the gene-trap integration sites (gene-trap sequence tags; http://genetrap.de) from >11,000 ES cell clones. Although most of the gene-trap vector insertions occurred randomly throughout the genome, we found both vector-independent and vector-specific integration "hot spots." Because >50% of the hot spots were vector-specific, we conclude that the most effective way to saturate the mouse genome with gene-trap insertions is by using a combination of gene-trap vectors. When a random sample of gene-trap integrations was passaged to the germ line, 59% (17 of 29) produced an observable phenotype in transgenic mice, a frequency similar to that achieved by conventional gene targeting. Thus, gene trapping allows a large-scale and cost-effective production of ES cell clones with mutations distributed throughout the genome, a resource likely to accelerate genome annotation and the in vivo modeling of human disease.     

Vitamin D-dependent calcium-binding protein in rat brain: biochemical and immunocytochemical characterization

Immunoreactive calcium-binding protein (CaBP) has been characterized in rat brain both biochemically and immunocytochemically. In this study antiserum to chick CaBP was used to characterize this protein and to describe its distribution in neurons and fibers of the rat fore- and midbrain. Immunostaining in neuronal elements was judged specific for this protein by the absence of staining in tissue sections after adsorption of the antiserum with either chick intestinal CaBP or the 28,000-dalton fraction from rat brain, but not with other molecular weight fractions with calcium-binding activity. Immunoreactive CaBP was found to have a widespread distribution throughout the central nervous system, and was present in most but not all major neuronal cell groups and fiber tracts. The protein was limited primarily to neuronal elements and some ependymal cells, and was absent in glia and blood vessels. The proportion of immunoreactivity in neuronal perikarya and fibers varied among nuclei and within a given structure at different rostral-caudal levels. Immunoreactivity was prominent in neocortex, hippocampal formation (primarily in CA1 and granular cells of the dentate gyrus), hypothalamus, and amygdala. These areas are responsible for the regulation of a variety of pituitary hormones, and several bind steroids. Immunoreactive CaBP was also a major constituent of nonlimbic system pathways. The widespread distribution of immunoreactive CaBP in the central nervous system suggests that CaBP and the vitamin D endocrine system may play a significant role in the regulation of mammalian brain function.     

Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat

Because neurogenesis persists in the adult mammalian brain and can be regulated by physiological and pathological events, we investigated its possible involvement in the brain's response to focal cerebral ischemia. Ischemia was induced by occlusion of the middle cerebral artery in the rat for 90 min, and proliferating cells were labeled with 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUrd) over 2-day periods before sacrificing animals 1, 2 or 3 weeks after ischemia. Ischemia increased the incorporation of BrdUrd into cells in two neuroproliferative regions-the subgranular zone of the dentate gyrus and the rostral subventricular zone. Both effects were bilateral, but that in the subgranular zone was more prominent on the ischemic side. Cells labeled with BrdUrd coexpressed the immature neuronal markers doublecortin and proliferating cell nuclear antigen but did not express the more mature cell markers NeuN and Hu, suggesting that they were nascent neurons. These results support a role for ischemia-induced neurogenesis in what may be adaptive processes that contribute to recovery after stroke.     

Localization of P-type calcium channels in the central nervous system.

The distribution of the P-type calcium channel in the mammalian central nervous system has been demonstrated immunohistochemically by using a polyclonal specific antibody. This antibody was generated after P-channel isolation via a fraction from funnel-web spider toxin (FTX) that blocks the voltage-gated P channels in cerebellar Purkinje cells. In the cerebellar cortex, immunolabeling to the antibody appeared throughout the molecular layer, while all the other regions were negative. Intensely labeled patches of reactivity were seen on Purkinje cell dendrites, especially at bifurcations; much weaker reactivity was present in the soma and stem segment. Electron microscopic localization revealed labeled patches of plasma membrane on the soma, main dendrites, spiny branchlets, and spines; portions of the smooth endoplasmic reticulum were also labeled. Strong labeling was present in the periglomerular cells of the olfactory bulb and scattered neurons in the deep layer of the entorhinal and pyriform cortices. Neurons in the brainstem, habenula, nucleus of the trapezoid body and inferior olive and along the floor of the fourth ventricle were also labeled intensely. Medium-intensity reactions were observed in layer II pyramidal cells of the frontal cortex, the CA1 cells of the hippocampus, the lateral nucleus of the substantia nigra, lateral reticular nucleus, and spinal fifth nucleus. Light labeling was seen in the neocortex, striatum, and in some brainstem neurons.     

Molecular manipulation of neural precursors in situ: induction of adult cortical neurogenesis

Over the past three decades, research exploring potential neuronal replacement therapies have focused on replacing lost neurons by transplanting cells or grafting tissue into diseased regions of the brain. Over most of the past century of modern neuroscience, it was thought that the adult brain was completely incapable of generating new neurons. However, in the last decade, the development of new techniques has resulted in an explosion of new research showing that neurogenesis, the birth of new neurons, normally occurs in two limited and specific regions of the adult mammalian brain, and that there are significant numbers of multipotent neural precursors in many parts of the adult mammalian brain. Recent findings from our lab demonstrate that it is possible to induce neurogenesis de novo in the adult mammalian brain, particularly in the neocortex where it does not normally occur, and that it may become possible to manipulate endogenous multipotent precursors in situ to replace lost or damaged neurons. Recruitment of new neurons can be induced in a region-specific, layer-specific, and neuronal type-specific manner, and newly recruited neurons can form long-distance connections to appropriate targets. Elucidation of the relevant molecular controls may both allow control over transplanted precursor cells and potentially allow the development of neuronal replacement therapies for neurodegenerative disease and other CNS injuries that do not require transplantation of exogenous cells.     

Hematopoiesis following disruption of the Pitx2 homeodomain gene

OBJECTIVE: Study the effect of loss of expression of Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, on hematopoietic stem cells (HSCs). METHODS: We examined the fetal livers of mouse embryos with homozygous disruption of the Pitx2 gene, using flow cytometry immunophenotyping analysis, as well as immunohistochemistry techniques. We further investigated the role of Pitx2 in HSCs using a chimeric mouse model system. Pitx2 null embryonic stem (ES) cell clones were generated from embryonic day 3.5 blastocysts of Pitx2 null embryos. The Pitx2 null donor ES cell contribution to the adult hematopoietic system was confirmed by identifying donor-specific glucose-phosphate isomerase isotype in the erythrocytes using cellulose acetate eletrophoresis, and by demonstrating donor-specific major histocompatibility complex antigen allotype on the granulocytes/monocytes and T and B lymphocytes of the chimeric mice using flow cytometry analysis. RESULTS: Pitx2 homozygous null fetal livers are decreased in size and overall cellularity. The erythroid cell component of these livers is further reduced as compared to that of their wild-type and heterozygous littermates. Detailed quantitative analysis of the chimeric mice revealed contribution of Pitx2 null ES cells to erythroid, myeloid, lymphoid, and megakaryocytic lineages. The quantitative level of ES cell contribution to the peripheral hematopoietic cells was proportional to the level of general chimerism as determined by coat color. CONCLUSION: Although the fetal livers of Pitx2 null embryos displayed signs of impaired erythropoiesis, Pitx2 gene disrupted HSCs can contribute to hematopoiesis under physiological conditions.     

Permissive corridor and diffusible gradients direct medial ganglionic eminence cell migration to the neocortex

Young neurons born in the medial ganglionic eminence (MGE) migrate a long distance dorsally, giving rise to several types of interneurons in neocortex. The mechanisms that facilitate selective dorsal dispersion of MGE cells while restricting their movement ventrally into neighboring regions are not known. Using microtransplantation into fetal brain slices and onto dissociated substrate cells on floating filters (spot assay), we demonstrate that ventral forebrain regions neighboring the MGE are nonpermissive for MGE cell migration, whereas the dorsal regions leading to the neocortex are increasingly permissive. Spot assay experiments using filters with different pore sizes indicate that the permissive factors are not diffusible. We also show that MGE cells respond to chemoattractive and inhibitory factors diffusing from the neocortex and ventromedial forebrain, respectively. We propose that the final extent and regional specificity of MGE cell dispersion is largely dictated by contact guidance through a selectively permissive environment, flanked by nonpermissive tissues. In addition, we propose that chemotactic guidance cues superimposed over the permissive corridor facilitate efficient dorsal migration of MGE cells.     

The long form of the leptin receptor (OB-Rb) is widely expressed in the human brain

Leptin exerts important effects on the regulation of food intake and energy expenditure by acting in the brain. Leptin action is mediated by the interaction with a receptor that is alternatively spliced, resulting in at least five different isoforms. The long form (OB-Rb) has a long intracellular domain that is essential for intracellular signal transduction. The specific aim of this study was to further investigate the role that the brain may play in the pathogenesis of obesity in humans. We studied the expression of OB-R mRNA (both short or common and long isoforms) in the brains of obese, lean and diabetic subjects, by in situ hybridization, semiquantitative RT-PCR and Northern blots analysis. We used two alternative probes: one that recognizes all known splice variants (OB-Ra) and a second that recognizes only the long form, OB-Rb. Several brain regions, including hypothalamus, cerebellum, neocortex, entorrhinal cortex, amygdala, and rostral medulla, were evaluated. In situ hybridization studies revealed that both OB-Ra and OB-Rb mRNAs are widely distributed in the human brain. The specific hybridization signal with both probes was detected exclusively in the cytoplasm of the cell body, dendrites and proximal axons of neurons. Hypothalamic nuclei, Purkinje cells and dentate nuclei of the cerebellum, inferior olivary and cranial nerves nuclei in the medulla, amygdala and neurons from both neocortex and entorrhinal cortex demonstrated positive signals. The hybridization signal obtained in ependyma was lower than that in neurons and no specific hybridization was detected in glial cells. No significant differences were identified among the regions or among the three groups studied. These results match those previously obtained by us [Couce et al.: Neuroendocrinology 1997;66:145] in which the distribution of the OB-R protein in the human brain was first described. RT-PCR indicated that the OB-Rb was highly expressed in the hypothalamus and cerebellum. No significant differences of OB-Ra or OB-Rb mRNA expression were identified in lean or obese individuals in these two cerebral regions. The levels of OB-Rb were significantly higher in cerebellum compared to hypothalamus in lean and obese individuals. The original hypothesis that OB-Rb is present only in the hypothalamus needs to be reconsidered. This OB-Rb isoform seems to be widely expressed in the human brain with highest levels in the cerebellum. Obesity and hyperleptinemia appears not to be associated with down-regulation of the OB-Rb in the human brain.     

Construction of chimeric vaccinia viruses by molecular cloning and packaging.

Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology.     

Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells.

We have constructed and characterized a polyoma virus-based plasmid that is maintained as an autonomously replicating extrachromosomal element (episome) in mouse embryonic stem (ES) cells. Plasmid pMGD20neo contains the polyoma origin of replication harboring a mutated enhancer (PyF101), a modified polyoma early region that encodes the large tumor (T) antigen only, and a gene that confers resistance to G418 (neo). After transfection, the plasmid replicates in ES cells and is maintained as an extrachromosomal element in 15% of G418-resistant clones. Integration of the plasmid DNA is undetectable for at least 28 cell generations. In one clone, the transfected DNA persists unaltered as an episome at 10-30 copies per cell for at least 74 cell generations in the presence of G418. Cells that maintain the autonomously replicating plasmid can efficiently replicate and maintain a second plasmid that carries the polyoma origin of replication. Independent vector-containing ES cell lines showed no significant alteration of the karyotype, and two cell lines yielded several chimeric animals when introduced into blastocysts, suggesting that the presence of an episomal element and expression of polyoma large T do not eliminate the ES cells' ability to populate an embryo. This system offers an efficient means for manipulating and analyzing various aspects of gene expression in ES cells.     

Autoradiographic localization of relaxin binding sites in rat brain.

Relaxin is a member of the insulin family of polypeptide hormones and exerts its best understood actions in the mammalian reproductive system. Using a biologically active 32P-labeled human relaxin, we have previously shown by in vitro autoradiography specific relaxin binding sites in rat uterus, cervix, and brain tissues. Using the same approach, we describe here a detailed localization of human relaxin binding sites in the rat brain. Displaceable relaxin binding sites are distributed in discrete regions of the olfactory system, neocortex, hypothalamus, hippocampus, thalamus, amygdala, midbrain, and medulla of the male and female rat brain. Characterization of the relaxin binding sites in the subfornical organ and neocortex reveals a single class of high-affinity sites (Kd = 1.4 nM) in both regions. The binding of relaxin to two of the circumventricular organs (subfornical organ and organum vasculosum of the lamina terminalis) and the neurosecretory magnocellular hypothalamic nuclei (i.e., paraventricular and supraoptic nuclei) provides the anatomical and biochemical basis for emerging physiological evidence suggesting a central role for relaxin in the control of blood pressure and hormone release. We conclude that specific, high-affinity relaxin binding sites are present in discrete regions of the rat brain and that the distribution of some of these sites may be consistent with a role for relaxin in control of vascular volume and blood pressure.     

Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells.

Gene disruptions and deletions of up to 20kb have been generated by homologous recombination with appropriate targeting vectors in murine embryonic stem (ES) cells. Because we could not obtain a deletion of about 200 kb in the mouse amyloid precursor protein gene by the classical technique, we employed strategies involving the insertion of loxP sites upstream and downstream of the region to be deleted by homologous recombination and elicited excision of the loxP-flanked region by introduction of a Cre expression vector into the ES cells. In the first approach, the loxP sequences were inserted in two successive steps and after each step, ES cell clones were isolated and characterized. Deletion of the loxP-flanked sequence was accomplished by introducing the cre gene in a third step. In the second approach, ES cells containing the upstream loxP cassette were electroporated simultaneously with the downstream loxP targeting vector and the Cre expression plasmid. ES cells were obtained that gave rise to chimeric mice capable of germ-line transmission of the deleted amyloid precursor protein allele.     

Immunolocalization of the thyrotropin-releasing hormone prohormone in the rat central nervous system

The distribution of immunoreactive TRH prohormone in the rat central nervous system was studied by immunocytochemistry using an antiserum raised against a synthetic decapeptide hypothesized to represent a portion of the mammalian TRH precursor protein. Reaction product was identified in several regions of the brain in a distribution typical of that previously described for the tripeptide. In contrast to TRH, however, immunoreactive pro-TRH was largely confined to neuronal perikarya and only rarely seen in axons or axon terminals. In addition, immunoreactive pro-TRH was present in portions of the telencephalon and brainstem where TRH has not previously been described in neurons by immunocytochemistry. These studies indicate that in most regions of the brain the TRH prohormone is rapidly processed within the cell soma and not during axonal transport, and raise the possibility that in certain regions of the brain processing of the prohormone may be to non-TRH peptides, which may be of biological importance.     

Aberrant lamination in the cerebral cortex of mouse embryos lacking DNA topoisomerase IIbeta

We have examined corticogenesis in mouse embryos lacking DNA topoisomerase IIbeta (IIbeta) in the brain or in all tissues. The absence of IIbeta, a type II DNA topoisomerase normally expressed in postmitotic cells in the developing cortex, severely affects cerebral stratification: no subplate is discernible, and neurons born at later stages of corticogenesis fail to migrate to the superficial layers. This abnormal pattern of neuron positioning in the cerebral cortex is reminiscent of that observed in mouse mutants defective in the reelin-signaling pathway. Significantly, the level of reelin in the neocortex is much reduced when IIbeta is absent. These results implicate a role of IIbeta in brain development. The enzyme may be required in implementing particular genetic programs in postmitotic cells, such as reelin expression in Cajal-Retzius cells, perhaps through its action on nucleoprotein structure of particular chromosomal regions.     

Mapping of brain areas expressing RNA homologous to two different acetylcholine receptor alpha-subunit cDNAs.

We have used an in situ RNA X RNA hybridization technique to determine, in the central nervous systems of the mouse and rat, the distribution of RNA homologous to cDNA clones encoding the alpha subunit of a putative neural nicotinic acetylcholine receptor and the alpha subunit of the muscle nicotinic acetylcholine receptor. Hybridization of the neural alpha-subunit probe was strongest in the medial habenula but was also detected consistently in the compact part of the substantia nigra and ventral tegmental area, in the neocortex, and in certain parts of the thalamus and hypothalamus. The in situ hybridization technique makes it possible to compile a map of brain regions containing cell bodies expressing RNA coding for a specific receptor type and subsequently to apply the techniques of molecular biology to study these brain receptors.