Temporal control of gene recombination in astrocytes by transgenic expression of the tamoxifen-inducible DNA recombinase variant CreERT2

Inducible gene modification using the Cre/loxP system provides a valuable tool for the analysis of gene function in the active animal. GFAP-Cre transgenic mice have been developed to achieve gene recombination in astrocytes, the most abundant cells of the central nervous system, with pivotal roles during brain function and pathology. Unfortunately, these mice displayed neuronal recombination as well, since the GFAP promoter is also active in embryonic radial glia, which possess a substantial neurogenic potential. To enable the temporal control of gene deletions in astrocytes only, we generated a transgenic mouse with expression of CreERT2, a fusion protein of the DNA recombinase Cre and a mutated ligand-binding domain of the estrogen receptor, under the control of the human GFAP promoter. In offspring originating from crossbreedings of GFAP-CreERT2-transgenic mice with various Cre-sensitive reporter mice, consecutive intraperitoneal injections of tamoxifen induced genomic recombination selectively in astrocytes of almost all brain regions. In Bergmann glia, which represent the main astroglial cell population of the cerebellum, virtually all cells showed successful gene recombination. When adult mice received cortical stab wound lesions, simultaneously given tamoxifen induced substantial recombination in reactive glia adjacent to the site of injury. These transgenic GFAP-CreERT2 mice will allow the functional analysis of loxP-modified genes in astroglia of the postnatal and adult brain.     

Failure to express GAP-43 leads to disruption of a multipotent precursor and inhibits astrocyte differentiation

The nervous system-specific protein GAP-43 is significantly upregulated in neurons and glia that are differentiating. In P19 EC cells that do not express GAP-43, neurogenesis is inhibited; many immature neurons apoptose and the survivors do not mature morphologically. Here we show that the initial defect is in an early precursor with characteristics of a neural stem cell, which failed to respond normally to retinoic acid (RA). As a consequence, its progeny had altered cell fates: In addition to the neuronal defects previously reported, RC1-labeled radial glia failed to exit the cell cycle, accumulated, and failed to acquire GFAP immunoreactivity. However, leukemia inhibitory factor (LIF) could stimulate GFAP expression suggesting that astrocytes not derived from radial glia are less affected by absence of GAP-43. Differentiation of radial glia-derived astrocytes was also inhibited in glial cultures from GAP-43 (-/-) cerebellum, and in GAP-43 (-/-) telencephalon in vivo, differentiation of astrocytes derived from both radial and nonradial glia lineages were both affected: In the glial wedge, GFAP-labeled radial glia-derived astrocytes were reduced consistent with the interpretation that they may be unable to deflect GAP-43 (-/-) commissural axons toward the midline. At the midline, both radial and nonradial glia-derived astrocytes were also decreased although it fused normally. The results demonstrate that GAP-43 expressed in multipotent precursors is required for appropriate cell fate commitment, and that its absence affects astrocyte as well as neuronal differentiation.     

Radial glia and astrocytes in developing and adult telencephalon of the lizard Gallotia galloti as revealed by immunohistochemistry with anti-GFAP and anti-vimentin antibodies

The development of radial glia and astrocytes in the telencephalon of the lizard Gallotia galloti was studied by immunohistochemistry with anti-vimentin and anti-GFAP antibodies. Vimentin appears at embryonic stage 32 (E32) in the proliferative zone of the lateral ventricle and subpial end-feet in the marginal zone. At E34-35 the staining intensity for vimentin in all radial glia is maximal. It then decreases and disappears in most structures in adult animals. GFAP appears at E35 in the end-feet in the marginal zone and its intensity increases until adulthood, particularly in radial and sinuous fibers and in fibers that originate from the sulci and invade the ventral striatum and the septum. In contrast, the reaction is weak in the cortex, in the anterior dorso-ventricular ridge, and in the amygdala nuclei. Radial glia is still present in the adult, and the composition of its intermediate filaments changes during development from vimentin to GFAP. No GFA-positive cell bodies except those of ependymal glia were detected in telencephalon.     

Radial Glia-Like Cells in the Supraoptic Nucleus of the Adult Rat

Conventional light and confocal microscopy of thick vibratome sections of the hypothalamus of adult male and female rats immunostained for the astrocytic marker glial fibrillary acidic protein (GFAP) revealed that the supraoptic nucleus (SON) contains two morphologically distinct types of astrocytes. One has a stellate form, similar to that of most astrocytes in the adult CMS. The other has a morphology reminiscent of radial glia in the developing CNS: from their cell bodies, located along the ventral glia lamina (VGL), arise one long thick process that spans the SON in the coronal plane, several horizontally-oriented processes that form a dense network in the VGL, and a short process oriented towards the pia. The latter astrocytes are immunoreactive for vimentin, an intermediate filament protein of immature glial cells and a marker for radial glia. The stellate astrocytes showed no vimentin immunoreactivity. The functional significance of each type of supraoptic astrocyte is at present unknown but the presence of radial glia-like cells in this hypothalamic region suggests that the SON retains a certain degree of immaturity during adulthood, that may be linked to its well known capacity to undergo neuronal-glial plasticity under physiological and experimental stimulation.     

Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis

Reactive glia, including astroglia and oligodendrocyte progenitors (OPCs) are at the core of the reaction to injury in the mammalian brain with initially beneficial and later partially adverse functions such as scar formation. Given the different glial composition in the adult zebrafish brain with radial ependymoglia but no parenchymal astrocytes, we examined the glial response to an invasive stab wound injury model in the adult zebrafish telencephalon. Strikingly, already a few days after injury the wound was closed without any scar tissue. Similar to mammals, microglia cells reacted first and accumulated close to the injury site, while neither GFAP+ radial ependymoglia nor adult OPCs were recruited to the injury site. Moreover, OPCs failed to increase their proliferation after this injury, while the number of proliferating GFAP+ glia was increased until 7 days after injury. Importantly, neurogenesis was also increased after injury, generating additional neurons recruited to the parenchyma which survived for several months. Thus, these data suggest that the specific glial environment in the adult zebrafish telencephalon is not only permissive for long-term neuronal survival, but avoids scar formation. Invasive injury in the adult zebrafish telencephalon may therefore provide a useful model to untangle the molecular mechanisms involved in these beneficial glial reactions.     

Radial glial cells as neuronal precursors: a new perspective on the correlation of morphology and lineage restriction in the developing cerebral cortex of mice

Radial glia is a ubiquitous cell type in the developing central nervous system (CNS) of vertebrates, characterized by radial processes extending through the wall of the neural tube which serve as guiding cables for migrating neurons. Radial glial cells were considered as glial precursor cells due to their astroglial traits and later transformation into astrocytes in the mammalian CNS. Accordingly, a hypothetical morphologically distinct type of precursor was attributed the role of neurogenesis. Recent evidence obtained in vitro and in vivo, however, revealed that a large subset of radial glia generates neurons. We further demonstrate here that the progeny of radial glial cells does not differ from the progeny of precursors labeled from the ventricular surface, implying that there is no obvious relation between precursor morphology and neuron-glia lineage decisions in the developing cerebral cortex of mice. Moreover, we show that many radial glial cells seem to maintain their process during cell division and discuss the implications of this observation for the orientation of cell division. These new data are then related to radial glial cells in other non-mammalian vertebrates persisting into adulthood and suggest that radial glia are not only neurogenic during development, but also in adulthood.     

Differentiation of radial glia from radial precursor cells and transformation into astrocytes in the developing rat spinal cord

Radial glial cell origins and functions have been studied extensively in the brain; however, questions remain relating to their origin and fate in the spinal cord. In the present study, radial glia are investigated in vivo using the neuroepithelial markers nestin and vimentin and the gliogenic markers GLAST, BLBP, 3CB2, and glial fibrillary acidic protein (GFAP). This has revealed heterogeneity among nestin/vimentin-positive precursor cells and suggests a lineage progression from neuroepithelial cell through to astrocyte in the developing spinal cord. A population of self-renewing radial cells, distinct from an earlier pseudo-stratified neuroepithelium, that resemble radial glial cells in morphology but do not express GLAST, BLBP, or 3CB2, is revealed. These radial cells arise directly from the spinal cord neuroepithelium and are probably the progenitors of neurons and the earliest appearing radial glial cells. GLAST/BLBP-positive radial glia first appear in the ventral cord at E14, and these cells gradually transform through one or more intermediate stages into differentiated astrocytes. Few if any neurons appear to be derived from radial glial cells, which are instead the major sources of astrocytes in the spinal cord. Evidence for the nonradial glial cell origins of some white matter astrocytes is also presented.     

Genetic lineage tracing defines distinct neurogenic and gliogenic stages of ventral telencephalic radial glial development

Background

Radial glia comprise a molecularly defined neural progenitor population but their role in neurogenesis has remained contested due to the lack of a single universally accepted genetic tool for tracing their progeny and the inability to distinguish functionally distinct developmental stages.

Results

By direct comparisons of Cre/loxP lineage tracing results obtained using three different radial glial promoters (Blbp, Glast, and hGFAP), we show that most neurons in the brain are derived from radial glia. Further, we show that hGFAP promoter induction occurs in ventral telencephalic radial glia only after they have largely completed neurogenesis.

Conclusion

These data establish the major neurogenic role of radial glia in the developing central nervous system and genetically distinguish an early neurogenic Blbp ⁺ Glast ⁺ hGFAP ^- stage from a later gliogenic Blbp ⁺ Glast ⁺ hGFAP ⁺ stage in the ventral telencephalon.     

Astroglia disturbances during development of the central nervous system in fetuses with Down's syndrome

Down's syndrome (DS), caused by aneuploidy of chromosome 21, is the most common chromosomal disorder. The most significant symptom of this disorder is mental retardation. Neuropathological changes found in the DS central nervous system (CNS), such as reduced number of neurons, alteration of synapses and synaptic spines or delayed myelination have been widely described. But there are only a few studies of DS-related glia disturbances. A growing number of astroglia new functions have recently been described. In our study we compared the number of astrocytes and radial glial cells in the frontal lobe of DS fetuses at 18-20 weeks of gestation with that observed in age-matched controls. We found a substantially increased number of glial fibrillary acid protein (GFAP) positive cells in all age range samples of DS brains. We also noticed that in our study astrocytes in DS brains seem to be morphologically more mature than in controls of corresponding age. The same observation was made for radial glia. Taking into consideration the role played by astroglia during CNS development we believe that any change in their number, reduced or increased, can affect CNS development and lead to disturbances of both neurogenesis and synaptogenesis. A possible correlation between the increased number of astroglia and disturbances in CNS development is discussed.     

GFAP-expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes

Neural stem cells in the adult subventricular zone (SVZ) derive from radial glia and express the astroglial marker glial fibrillary acidic protein (GFAP). Thus, they have been termed astrocytes. However, it remains unknown whether these GFAP-expressing cells express the functional features common to astrocytes. Using immunostaining and patch clamp recordings in acute slices from transgenic mice expressing green fluorescent protein (GFP) driven by the promoter of human GFAP, we show that GFAP-expressing cells in the postnatal SVZ display typical glial properties shared by astrocytes and prenatal radial glia such as lack of action potentials, hyperpolarized resting potentials, gap junction coupling, connexin 43 expression, hemichannels, a passive current profile, and functional glutamate transporters. GFAP-expressing cells express both GLAST and GLT-1 glutamate transporters but lack AMPA-type glutamate receptors as reported for dye-coupled astrocytes. However, they lack 100 microM Ba2+-sensitive inwardly rectifying K+ (K(IR)) currents expressed by astrocytes, but display delayed rectifying K+ currents and 1 mM Ba2+-sensitive K+ currents. These currents contribute to K+ transport at rest and maintain hyperpolarized resting potentials. GFAP-expressing cells stained positive for both K(IR)2.1 and K(IR)4.1 channels, two major K(IR) channels in astrocytes. Ependymal cells, which also derive from radial glia and express GFAP, display typical glial properties and K(IR) currents consistent with their postmitotic nature. Our results suggest that GFAP-expressing cells in concert with ependymal cells can perform typical astrocytic functions such as K+ and glutamate buffering in the postnatal SVZ but display a unique set of functional characteristics intermediate between astrocytes and radial glia.     

Nestin mRNA expression correlates with the central nervous system progenitor cell state in many,but not all,regions of developing central nervous system

Nestin is a recently discovered intermediate filament (IF) gene. Nestin expression has been extensively used as a marker for central nervous system (CNS) progenitor cells in different contexts, based on observations indicating a correlation between nestin expression and this cell type in vivo. To evaluate this correlation in more detail nestin mRNA expression in developing and adult mouse CNS was analysed by in situ hybridization. We find that nestin is expressed from embryonic day (E) 7.75 and that expression is detected in many proliferating CNS regions; at E10.5 nestin is expressed in cells of both the rostral and caudal neural tube, including the radial glial cells; at E15.5 and postnatal day (P) 0 expression is observed largely in the developing cerebellum and in the ventricular and subventricular areas of the developing telencephalon. Furthermore, the transition from a proliferating to a post-mitotic cell state is accompanied by a rapid decrease in nestin mRNA for motor neurons in the ventral spinal cord and for neurons in the marginal layer of developing telencephalon. In contrast to these data we observe two proliferating areas, the olfactory epithelium and the precursor cells of the hippocampal granule neurons, which do not express nestin at detectable levels. Thus, nestin mRNA expression correlates with many, but not all, regions of proliferating CNS progenitor cells. In addition to its temporal and spatial regulation nestin expression also appears to be regulated at the level of subcellular mRNA localization: in columnar neuroepithelial and radial glial cells nestin mRNA is predominantly localized to the pial endfeet.     

Cortical radial glia: identification in tissue culture and evidence for their transformation to astrocytes

Radial glia are transiently present in the developing cerebral cortex, where they are thought to guide the migration of neurons from the proliferative zone to the forming cortical plate. To provide a framework for experimental studies of radial glia, we have defined morphological and immunocytochemical criteria to identify them in primary cultures of cortical cells obtained at embryonic day 13 in the mouse. Cortical radial glia in culture for 1-2 d resemble radial glia in vivo: they have a long, thin, unbranched process extending from one or both ends of the elongated cell body and are labeled with the monoclonal antibody RC1 but not with antibodies to glial fibrillary acidic protein (abGFAP). We tested the specificity of RC1 by double-labeling with a panel of cell-type specific antibodies, and found that it labels radial glia, astrocytes, and fibroblast-like cells, but not neurons. Fibroblasts are easily distinguished from glia by morphology and by labeling with antibodies to fibronectin. To test the hypothesis that radial glia become astrocytes when their developmental role is complete, we examined their morphological and immunocytochemical development in culture. After 3-4 d in vitro radial glia develop several branched processes; in this transitional stage they are labeled by both RC1 and abGFAP. Many radial glia lose RC1 immunoreactivity as they become increasingly branched and immunoreactive to abGFAP. In areas of the cultures that have few neurons and in cultures depleted of neurons by washing, flat, nonprocess-bearing glia predominate. These cells do not lose immunoreactivity to RC1 during the 9-d period of observation even though they acquire GFAP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental potential of radial glia investigated by transplantation into the developing rat ventricular system in utero

During development there is a clear correlation between position of dividing progenitor cells, mode of division and developmental potential, suggesting that the local environment of progenitor cells may influence their cell fate [ 17 (6), 639-647]. The contribution of these conditions was investigated here by transplantation of radial glial progenitor cells into isotopic, isochronic, heterotopic and heterochronic environment conditions. Neuronal cells were removed from E14 spinal cords using negative immunoselection. The remaining radial glia were transplanted into the ventricular system of host embryos and pups. Distance of migration as well as morphological and antigenic phenotype of transplanted radial glia was examined after various survival times post transplantation. Host age clearly influenced migration and differentiation of transplant cells, with transplant cells migrating further in younger hosts and differentiating earlier in older aged host environments. Evidence is presented showing that most transplanted spinal cord radial glia give rise to astrocytes. In addition some transplanted radial glia were shown to give rise to neurons in spinal cord regions. Radial glia did not appear to generate neurons in the brains of host animals until postnatal ages, perhaps because transplanted radial glia were isolated from spinal cord and thus may not have been influenced to behave as endogenous radial glia in the brain which commonly produce neurons.     

Ethanol exposure during embryogenesis decreases the radial glial progenitorpool and affects the generation of neurons and astrocytes

Prenatal ethanol exposure induces functional abnormalities during brain development affecting neurogenesis and gliogenesis. We have previously reported that alcohol exposure during embryogenesis disrupts radial glia (RG) and gliogenesis. Taking into account the new role of RG as neural progenitors, we have investigated whether ethanol affects RG as a neural stem cell. We found that in utero ethanol exposure impairs cell proliferation and decreases neurons and astrocytes generated in cultured RG and in embryonic cerebral cortex. Telencephalic cultures obtained at E12 from ethanol-treated rats displayed a reduction in the proportion of actively dividing RG progenitors, as demonstrated by 5-bromo-2'-deoxyuridine incorporation, and in the percentage of brain lipid binding protein-positive RG. Consistently, neurosphere formation assay from E12 telencephalon showed a reduced number of multipotent progenitor cells in cultures isolated from ethanol-treated rats in comparison with pair-fed control group. Moreover, levels of activated Notch1 and fibroblast growth factor receptor 2, which regulate the maintenance of the progenitor state of RG, are decreased by prenatal ethanol exposure. These findings demonstrate that ethanol reduces the telencephalic RG progenitor pool and its transformation into neurons and astrocytes, which may contribute to an explanation of the defects in brain function often observed in fetal alcohol syndrome.     

Prenatal exposure to ethanol alters the postnatal development and transformation of radial glia to astrocytes in the cortex

Postmitotic neurons migrate from a zone(s) near the ventricles to the neocortex. During this migration, neurons associate with radial glia. After serving their role as guides for neuronal migration, the radial glia transform into astrocytes. Prenatal exposure to ethanol causes abnormal neuronal migration. We examined the effects of gestational exposure to ethanol on radial glia and astrocytes. Radial glia were stained immunohistochemically with the antibody RAT-401, and astrocytes were labeled with an antibody directed against glial-fibrillary acidic protein (GFAP). The subjects were the offspring of rats fed an ethanol-containing liquid. diet (Et), pair-fed a liquid control diet (Ct), or fed chow and water (Ch). During the first postnatal week, radial glial fibers (in Et-treated rats and controls) stretched from the ventricular surface through the developing. cerebral wall to the pial surface. In the Et-treated rats, the radial processes were less dense and more poorly fasciculated than they were in the Ch-and Ct-treated rats. Moreover, by postnatal day (P) 5, there was a significant reduction in RAT-401 immunostaining in the Et-treated rats, particularly in the superficial cortex. A similar reduction in control rats did not begin until P10. In all three treatment groups, GFAP-immunoreactive astrocytes were in the cortex throughout the period from P1 to P45. In neonates, GFAP-positive cells were distributed in the marginal zone (layer I) and the intermediate zone (the white matter). The number of GFAP-positive cells in the cortical plate increased steadily with time so that, by P26, GFAP-immunoreactive astrocytes were distributed evenly through all cortical laminae. Interestingly, between P5 and P12, the number of astrocytes was significantly greater in Et-treated rats than in controls. Thus prenatal exposure to ethanol induces the premature loss of RAT-401-positive processes and the precocious increase in GFAP immunostaining. These ethanol-induced changes in glial development indicate that ethanol accelerates the transformation of radial glia into astrocytes. Moreover, the ethanol-induced premature degradation of the network of radial glial fibers may underlie the migration of late-generated neurons to ectopic sites. © 1993 Wiley-Liss, Inc.     

Adult Nestin-expressing Subependymal Cells Differentiate to Astrocytes in Response to Brain Injury

The adult brain contains a small population of central nervous system (CNS) cells in the subependyma which, like embryonic CNS progenitor cells, express the intermediate filament nestin. In this report, the differentiation capacity in vivo of these cells was analysed following a standardized trauma. Before the trauma, the subependymal cells expressed nestin but not the astrocytic and neuronal differentiation markers glial fibrillary acidic protein (GFAP) and neurofilament respectively. In response to injury, the majority of the subependymal cells coexpressed nestin and GFAP, but never nestin and neurofilament. Furthermore, cells coexpressing nestin and GFAP were found progressively further away from the subependyma and closer to the lesion at later time points after the injury, indicating that these cells migrate towards the lesion. Nestin was in addition re-expressed in reactive astrocytes near the lesion and in non-reactive astrocytes very far from the lesion throughout the ipsilateral cortex. In conclusion, our data indicate that the nestin-positive subependymal cells are an in vivo source for the generation of new astrocytes but not neurons after injury, and that nestin re-expression in astrocytes following traumatic stimuli can be used as a sensitive marker for astroglial activation.     

Nestin expression in hippocampal astrocytes after injury depends on the age of the hippocampus

Analysis of the expression of nestin in reactive astrocytes facilitates quantification of the extent of activation of astrocytes after injury in the mature CNS. We hypothesize that the capability of astrocytes for re-expressing nestin in response to CNS injury diminishes as a function of age. We quantified astrocytes positive for S-100beta protein, glial fibrillary acidic protein (GFAP) and nestin in the hippocampus of young adult, middle-aged, and aged Fischer 344 rats after an intracerebroventricular kainic acid (KA) administration. In all age groups, KA administration induced degeneration of CA3 pyramidal neurons, which led to a significant deafferentation in the CA1 region. The KA-induced neurodegeneration and deafferentation resulted in an increased population of astrocytes positive for S-100beta and glial fibrillary acidic protein (GFAP) in all age groups. Interestingly, these increases were highly comparable across the three age groups. However, in areas of both neurodegeneration and deafferentation, the overall numerical density of nestin-positive reactive astrocytes varied depending on the age at the time of injury with noticeably decreased numerical density in the injured middle-aged and aged hippocampus. In contrast, nestin-immunoreactive radial glia framework after lesion is not impaired with aging in the ependymal lining of the CA3 region.     

Activation of MAPK/PI3K/SMAD pathways by TGF-β(1) controls differentiation of radial glia into astrocytes in vitro

The major neural stem cell population in the developing cerebral cortex is the radial glia cells, which generate neurons and glial cells. The mechanisms that modulate the maintenance of the radial glia stem cell phenotype, or its differentiation, are not completely elucidated. We previously demonstrated that transforming growth factor-β(1) (TGF-β(1)) promotes radial glia differentiation into astrocytes in vitro [Glia 2007;55:1023-1033]. Here we investigated the intracellular signaling pathways involved in the TGF-β(1)-induced radial glia fate commitment. We demonstrate that the mechanisms underlying the TGF-β(1) effect on radial glia cell differentiation or progenitor potential maintenance diverge. Whereas radial glia differentiation into astrocytes is mediated by the activation of the MAPK signaling pathway, neurogenesis is modulated by different levels of PI3K and SMAD2/3 activity. Our work demonstrates that radial glia cells are a heterogeneous population and a potential target of TGF-β(1), and suggests that its effect on radial glia fate commitment is mediated by the recruitment of a complex multipathway mechanism that controls astrocyte and neuronal generation in the developing cerebral cortex.