The cells of cajal-retzius: still a mystery one century after

Cajal-Retzius (CR) cells are an enigmatic class of neurons located at the surface of the cerebral cortex, playing a major role in cortical development. In this review, we discuss several distinct features of these neurons and the mechanisms by which they regulate cortical development. Many CR cells likely have extracortical origin and undergo cell death during development. Recent genetic studies report unique patterns of gene expression in CR cells, which may help to explain the developmental processes in which they participate. Moreover, a number of studies indicate that CR cells, and their secreted gene product, reelin, are involved in neuronal migration by acting on two key partners, migrating neurons and radial glial cells. Emerging data show that these neurons are a critical part of an early and complex network of neural activity in layer I, supporting the notion that CR cells modulate cortical maturation. Given these key and complex developmental properties, it is therefore conceivable for CR cells to be implicated in the pathogenesis of a variety of neurological disorders.     

VEGF expression is developmentally regulated during human brain angiogenesis

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor working as an endothelial cell-specific mitogen and exerting a trophic effect on neurons and glial cells, both these activities being essential during central nervous system vascularisation, development and repair. The vascularisation of human telencephalon takes place by means of an angiogenic mechanism, which starts at the beginning of corticogenesis and actively proceeds up to the last neuronal migration, when the basic scheme of the vascular network has been drawn. Our study focused on VEGF during this critical developmental period with the aim of identifying the cells that express VEGF and of correlating the events of angiogenesis with the main events of cerebral cortex formation. The results show that in fetal human brain VEGF protein is located on multiple cell types, cells proper to the nervous tissue, neuroepithelial cells, neuroblasts and radial glia cells, and non-neuronal cells, endothelial and periendothelial cells. In these cells VEGF expression appears developmentally regulated and is correlated with angiogenesis, which in turn responds to the high metabolic demands of the differentiating neocortex.     

Characterization of cortical neuronal and glial alterations during culture of organotypic whole brain slices from neonatal and mature mice

Background

Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented.

Methods

In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4–6), adolescent animals (P25–28) and mature adults (P50+). Cultures were prepared using the membrane interface method.

Results

Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (p<0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers.

Conclusion

In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.     

Experimental manipulation of cerebral cortical areas in primates.

The developmental mechanisms underlying the subdivision of the neocortex into structurally and functionally distinct areas is central to our understanding of the development of human cognitive capacity and the pathogenesis of congenital disorders of higher brain functions. The protomap hypothesis suggests how the cytoarchitectonic pattern of the cerebral cortex may be generated by a combination of intrinsic and extrinsic influences during embryonic development. Although little is known about the genetic and molecular mechanisms underlying this individual and species-specific diversity of cellular and synaptic architecture, experimental manipulation of development in the primate embryo provides a glimpse into the cascade of cellular events involved in the control of cell numbers, specification of neuronal phenotypes, their apportions into cytoarchitectonic areas, and establishment of area-specific synaptic circuitry.     

Aspects of biochemical differentiation in the central nervous system.

A demonstration of cell-specific patterns of development in the immature CNS is provided by examples of characteristic, cell-specific time-courses of enzyme development in different classes of brain cells isolated in highly purified form by bulk-separation from the cerebral and cerebellar cortex of the growing rat. The enzymatic analysis was carried out at the level of the nerve and glial cell lysosomes and mitochondria, two subcellular organelles crucial to the economy of all cells. The findings reveal rather similar developmental patterns for the lysosomal hydrolase N-acetyl-beta-D-glucosaminidase in neurons and glial cells of the cerebral cortex as well as in two different cerebellar nerve cell types, the Purkinje and the granule cell. However, significant differences in the post-natal chronology of development of the mitochondrial enzyme alpha-glycerophosphate dehydrogenase were noted between cortical nerve and glial cells, the glial enzyme exhibiting 6-fold higher levels of activity than the neuronal one throughout the first month of postnatal life. The findings emphasize the feasibility as well as the necessity of studies aimed at the elucidation of the cell-specific aspects of the biochemistry of developing nerve and glial cells.     

Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line

We characterized phenotype and function of a fetal human mesencephalic cell line (LUHMES, Lund human mesencephalic) as neuronal model system. Neurodevelopmental profiling of the proliferation stage (d0, day 0) of these conditionally-immortalized cells revealed neuronal features, expressed simultaneously with some early neuroblast and stem cell markers. An optimized 2-step differentiation procedure, triggered by shut-down of the myc transgene, resulted in uniformly post-mitotic neurons within 5 days (d5). This was associated with down-regulation of some precursor markers and further up-regulation of neuronal genes. Neurite network formation involved the outgrowth of 1-2, often > 500 μm long projections. They showed dynamic growth cone behavior, as evidenced by time-lapse imaging of stably GFP-over-expressing cells. Voltage-dependent sodium channels and spontaneous electrical activity of LUHMES continuously increased from d0 to d11, while levels of synaptic markers reached their maximum on d5. The developmental expression patterns of most genes and of the dopamine uptake- and release-machinery appeared to be intrinsically predetermined, as the differentiation proceeded similarly when external factors such as dibutyryl-cAMP and glial cell derived neurotrophic factor were omitted. Only tyrosine hydroxylase required the continuous presence of cAMP. In conclusion, LUHMES are a robust neuronal model with adaptable phenotype and high value for neurodevelopmental studies, disease modeling and neuropharmacology.     

Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

Expression of alpha2A adrenoceptors during rat neocortical development

Norepinephrine has been suggested to play a neurotrophic role during development and is present in the brain as early as embryonic day (E) 12. We have recently demonstrated that the alpha2A adrenoceptor subtype is widely expressed during times of neuronal migration and differentiation throughout the developing brain. Here, we report the temporal and spatial expression pattern of alpha2A adrenoceptors in neocortex during late embryonic and early postnatal development using in situ hybridization and receptor autoradiography. Functional alpha2 receptors in embryonic rat cortex were also detected using agonist stimulated [35S]GTPgammaS autoradiography. Both alpha2A mRNA and protein expression were strongly increased by E19 and E20, respectively. The increased expression was in the cortical plate and intermediate and subventricular zones, corresponding to tiers of migrating and differentiating neurons. This transient up-regulation of alpha2A adrenoceptors was restricted to the lateral neocortex. At E20, functional alpha2 adrenoceptors were also detected in deep layers of lateral neocortex. During the first week of postnatal development, the expression of alpha2A mRNA and protein changed markedly, giving rise to a more mature pattern of anatomical distribution. The temporal and spatial distribution of alpha2A adrenoceptors in developing neocortex is consistent with expression of functional proteins on migrating and differentiating layer IV to II neurons. These findings suggest that alpha2A receptors may mediate a neurotrophic effect of norepinephrine during fetal cortical development. The early delineation of the lateral neocortex, which will develop into somatosensory and auditory cortices, suggests an intrinsic regulation of alpha2A mRNA expression.     

Immunohistochemical Markers of Neural Progenitor Cells in the Early Embryonic Human Cerebral Cortex

The development of the human central nervous system represents a delicate moment of embryogenesis. The purpose of this study was to analyze the expression of multiple immunohistochemical markers in the stem/progenitor cells in the human cerebral cortex during the early phases of development. To this end, samples from cerebral cortex were obtained from 4 human embryos of 11 weeks of gestation. Each sample was formalin-fixed, paraffin embedded and immunostained with several markers including GFAP, WT1, Nestin, Vimentin, CD117, S100B, Sox2, PAX2, PAX5, Tβ4, Neurofilament, CD44, CD133, Synaptophysin and Cyclin D1. Our study shows the ability of the different immunohistochemical markers to evidence different zones of the developing human cerebral cortex, allowing the identification of the multiple stages of differentiation of neuronal and glial precursors. Three important markers of radial glial cells are evidenced in this early gestational age: Vimentin, Nestin and WT1. Sox2 was expressed by the stem/progenitor cells of the ventricular zone, whereas the postmitotic neurons of the cortical plate were immunostained by PAX2 and NSE. Future studies are needed to test other important stem/progenitor cells markers and to better analyze differences in the immunohistochemical expression of these markers during gestation.Key words: Cerebral cortex, human embryo, human development, immunohistochemistry, fetal stem cells     

Principles of neural stem cell lineage progression: Insights from developing cerebral cortex

How to generate a brain of correct size and with appropriate cell-type diversity during development is a major question in Neuroscience. In the developing neocortex, radial glial progenitor (RGP) cells are the main neural stem cells that produce cortical excitatory projection neurons, glial cells, and establish the prospective postnatal stem cell niche in the lateral ventricles. RGPs follow a tightly orchestrated developmental program that when disrupted can result in severe cortical malformations such as microcephaly and megalencephaly. The precise cellular and molecular mechanisms instructing faithful RGP lineage progression are however not well understood. This review will summarize recent conceptual advances that contribute to our understanding of the general principles of RGP lineage progression.     

C3G regulates cortical neuron migration, preplate splitting and radial glial cell attachment

Neuronal migration is integral to the development of the cerebral cortex and higher brain function. Cortical neuron migration defects lead to mental disorders such as lissencephaly and epilepsy. Interaction of neurons with their extracellular environment regulates cortical neuron migration through cell surface receptors. However, it is unclear how the signals from extracellular matrix proteins are transduced intracellularly. We report here that mouse embryos lacking the Ras family guanine nucleotide exchange factor, C3G (Rapgef1, Grf2), exhibit a cortical neuron migration defect resulting in a failure to split the preplate into marginal zone and subplate and a failure to form a cortical plate. C3G-deficient cortical neurons fail to migrate. Instead, they arrest in a multipolar state and accumulate below the preplate. The basement membrane is disrupted and radial glial processes are disorganised and lack attachment in C3G-deficient brains. C3G is activated in response to reelin in cortical neurons, which, in turn, leads to activation of the small GTPase Rap1. In C3G-deficient cells, Rap1 GTP loading in response to reelin stimulation is reduced. In conclusion, the Ras family regulator C3G is essential for two aspects of cortex development, namely radial glial attachment and neuronal migration.     

Cortical radial glial cells in human fetuses: depth-correlated transformation into astrocytes

In the human brain, the transformation of radial glial cells (RGC) into astrocytes has been studied only rarely. In this work, we were interested in studying the morphologic aspects underlying this transformation during the fetal/perinatal period, particularly emphasizing the region-specific glial fiber anatomy in the medial cortex. We have used carbocyanine dyes (DiI/DiA) to identify the RGC transitional forms and glial fiber morphology. Immunocytochemical markers such as vimentin and glial fibrillary acidic protein (GFAP) were also employed to label the radial cells of glial lineage and to reveal the early pattern of astrocyte distribution. Neuronal markers such as neuronal-specific nuclear protein (NeuN) and microtubule-associated protein (MAP-2) were employed to discern whether or not these radial cells could, in fact, be neurons or neuronal precursors. The main findings concern the beginning of RGC transformation showing loss of the ventricular fixation in most cases, followed by transitional figures and the appearance of mature astrocytes. In addition, diverse fiber morphology related to depth within the cortical mantle was clearly demonstrated. We concluded that during the fetal/perinatal period the cerebral cortex is undergoing the final stages of radial neuronal migration, followed by involution of RGC ventricular processes and transformation into astrocytes. None of the transitional or other radial glia were positive for neuronal markers. Furthermore, the differential morphology of RGC fibers according to depth suggests that factors may act locally in the subplate and could have a role in the process of cortical RGC transformation and astrocyte localization. The early pattern of astrocyte distribution is bilaminar, sparing the cortical plate. Few astrocytes (GFAP+) in the upper band could be found with radial processes at anytime. This suggests that astrocytes in the marginal zone could be derived from different precursors than those that differentiate from RGCs during this period.     

Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex

The majority of neurons in the adult neocortex are produced embryonically during a brief but intense period of neuronal proliferation. The radial glial cell, a transient embryonic cell type known for its crucial role in neuronal migration, has recently been shown to function as a neuronal progenitor cell and appears to produce most cortical pyramidal neurons. Radial glial cell modulation could thus affect neuron production, neuronal migration, and overall cortical architecture; however, signaling mechanisms among radial glia have not been studied directly. We demonstrate here that calcium waves propagate through radial glial cells in the proliferative cortical ventricular zone (VZ). Radial glial calcium waves occur spontaneously and require connexin hemichannels, P2Y1 ATP receptors, and intracellular IP3-mediated calcium release. Furthermore, we show that wave disruption decreases VZ proliferation during the peak of embryonic neurogenesis. Taken together, these results demonstrate a radial glial signaling mechanism that may regulate cortical neuronal production.     

Cortical radial glial cells in human fetuses: Depth‐correlated transformation into astrocytes

In the human brain, the transformation of radial glial cells (RGC) into astrocytes has been studied only rarely. In this work, we were interested in studying the morphologic aspects underlying this transformation during the fetal/perinatal period, particularly emphasizing the region‐specific glial fiber anatomy in the medial cortex. We have used carbocyanine dyes (DiI/DiA) to identify the RGC transitional forms and glial fiber morphology. Immunocytochemical markers such as vimentin and glial fibrillary acidic protein (GFAP) were also employed to label the radial cells of glial lineage and to reveal the early pattern of astrocyte distribution. Neuronal markers such as neuronal‐specific nuclear protein (NeuN) and microtubule‐associated protein (MAP‐2) were employed to discern whether or not these radial cells could, in fact, be neurons or neuronal precursors. The main findings concern the beginning of RGC transformation showing loss of the ventricular fixation in most cases, followed by transitional figures and the appearance of mature astrocytes. In addition, diverse fiber morphology related to depth within the cortical mantle was clearly demonstrated. We concluded that during the fetal/perinatal period the cerebral cortex is undergoing the final stages of radial neuronal migration, followed by involution of RGC ventricular processes and transformation into astrocytes. None of the transitional or other radial glia were positive for neuronal markers. Furthermore, the differential morphology of RGC fibers according to depth suggests that factors may act locally in the subplate and could have a role in the process of cortical RGC transformation and astrocyte localization. The early pattern of astrocyte distribution is bilaminar, sparing the cortical plate. Few astrocytes (GFAP+) in the upper band could be found with radial processes at anytime. This suggests that astrocytes in the marginal zone could be derived from different precursors than those that differentiate from RGCs during this period. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 288–298, 2003     

Regulation of neural progenitor cell development in the nervous system

The mammalian telencephalon, which comprises the cerebral cortex, olfactory bulb, hippocampus, basal ganglia, and amygdala, is the most complex and intricate region of the CNS. It is the seat of all higher brain functions including the storage and retrieval of memories, the integration and processing of sensory and motor information, and the regulation of emotion and drive states. In higher mammals such as humans, the telencephalon also governs our creative impulses, ability to make rational decisions, and plan for the future. Despite its massive complexity, exciting work from a number of groups has begun to unravel the developmental mechanisms for the generation of the diverse neural cell types that form the circuitry of the mature telencephalon. Here, we review our current understanding of four aspects of neural development. We first begin by providing a general overview of the broad developmental mechanisms underlying the generation of neuronal and glial cell diversity in the telencephalon during embryonic development. We then focus on development of the cerebral cortex, the most complex and evolved region of the brain. We review the current state of understanding of progenitor cell diversity within the cortical ventricular zone and then describe how lateral signaling via the Notch-Delta pathway generates specific aspects of neural cell diversity in cortical progenitor pools. Finally, we review the signaling mechanisms required for development, and response to injury, of a specialized group of cortical stem cells, the radial glia, which act both as precursors and as migratory scaffolds for newly generated neurons.     

Gene expression profiling of primate neocortex: molecular neuroanatomy of cortical areas

One hundred years have passed since Brodmann's mapping of the mammalian neocortex. Solely on the basis of morphological observations, he envisaged the conservation and differentiation of cortical areal structures across various species. We now know that neurochemical, connectional and functional heterogeneity of the neocortex accompanies the morphological divergence observed in such cytoarchitectonic studies. Nevertheless, we are yet far from fully understanding the biological significance of this cortical heterogeneity. In this article, we review our past works on the gene expression profiling of the postnatal primate cortical areas, by quantitative real-time polymerase chain reaction (PCR), DNA array, differential display PCR and in situ hybridization analyses. These studies revealed both the overall homogeneity of gene expression across different cortical areas and the presence of a small number of genes that show markedly area-specific expression patterns. In situ hybridization showed that, among such genes, occ1 and retinol-binding protein (RBP) mRNAs are selectively expressed in the neuronal populations that seem to be involved in distinct neural processing such as sensory reception (for occ1 ) and associative function (for RBP). Such a molecular neuroanatomical approach has the promise to provide an important link between structure and function of the cerebral cortex.