PTEN-mediated Akt activation in human neocortex during prenatal development

Akt is a crucial factor for cell survival and migration. Phosphatase and tensin (PTEN) negatively regulates cell growth and survival by inhibiting PI3K-dependent signaling. PTEN also blocks Akt phosphorylation, a main downstream molecule of PI3K cascade. So far, no studies have shown PTEN expression and Akt phosphorylation levels in the developing human neocortex. Our hypothesis is that spatial and temporal expression of PTEN is likely to modulate developing human brain cortical modeling by regulating Akt activation. Therefore, our aim is to analyze the expression pattern of PTEN and phospho-Akt levels using immunohistochemistry, Western blot, and semiquantitative analysis in the developing human neocortex (n=13 fetuses from first, second, and third trimesters). PTEN expression was decreased parallel to development, but some cells revealed strong nuclear immunoreactivity in the developing neocortex while the active Akt level was increased. Double immunohistochemistry was performed for proliferating cell nuclear antigen (PCNA)-Tuj1 (as neuronal marker) and PCNA-GFAP (Glial marker) to the subsequent sections of PTEN and Akt-stained slides. PCNA (+) cells were mostly positive for glial fibrillary acidic protein (GFAP) and correlated with active-Akt immunoreactivity. Our results suggest that Akt-mediated signaling plays an active role in cell migration, survival, and cerebral cortical modeling throughout prenatal life and that PTEN is the most likely protein to regulate this signaling.     

A monoclonal antibody against a neuron-specific 65-kDa protein with laminar expression in the developing cerebral cortex

The extracellular matrix component chondroitin sulfate supports the survival of neocortical neurons and influences their differentiation in culture. During development of the rat forebrain expression of chondroitin sulfate peaks at around birth. To elucidate functional partners of this important player of neurogenesis, a monoclonal antibody, termed MAb-9, was generated after immunization with chondroitin sulfate-binding proteins from neonatal rat brain. In western blots of neonatal tissue, MAb-9 recognized a major brain-specific 65-kDa protein band. While most of the 65-kDa protein was present in the soluble compartment, a significant fraction was membrane-associated. Prolonged extraction of brain membranes with CHAPS revealed three additional minor protein bands of approximately 48, 50, and 58 kDa. Of these, the 50-kDa protein specifically bound to chondroitin sulfate C-Sepharose. Immunocytochemical studies and western blot analyses of cultures of neocortical neurons and astrocytes demonstrated that MAb-9 recognizes a neuron-specific cytosolic protein. In the developing cerebral cortex the protein was detectable by immunohistochemistry in the preplate and ventricular zones as early as embryonic day 15. On embryonic day 18, the protein was found in the marginal zone and the subplate, but it was not present in the emerging cortical plate. At the neonatal stage the immunoreactivity was distributed throughout the cerebral cortex with prominent staining of the marginal zone. A similar pattern was observed in the adult animal. Notably, the laminar distribution of MAb-9 immunoreactivity in the cerebral cortex during the prenatal period closely resembled the expression pattern reported for chondroitin sulfate. Thus, MAb-9 recognizes a neuronal cytosolic protein which might participate in neurotrophic signaling events triggered by chondroitin sulfate.     

Ghrelin receptors in human gastrointestinal tract during prenatal and early postnatal development

The aim of our study was to investigate the appearance, density and distribution of ghrelin cells and GHS-R1a and GHS-R1b in the human stomach and duodenum during prenatal and early postnatal development. We examined chromogranin-A and ghrelin cells in duodenum, and GHS-R1a and GHS-R1b expression in stomach and duodenum by immunohistochemistry in embryos, fetuses, and infants. Chromogranin-A and ghrelin cells were identified in the duodenum at weeks 10 and 11 of gestation. Ghrelin cells were detected individually or clustered within the base of duodenal crypts and villi during the first trimester, while they were presented separately within the basal and apical parts of crypts and villi during the second and third trimesters. Ghrelin cells were the most numerous during the first (∼11%) and third (∼10%) trimesters of gestation development. GHS-R1a and GHS-R1b were detected at 11 and 16 weeks of gestation, showed the highest level of expression in Brunner's gland and in lower parts of duodenal crypts and villi during the second trimester in antrum, and during the third trimester in corpus and duodenum. Our findings demonstrated for the first time abundant duodenal expression of ghrelin cells and ghrelin receptors during human prenatal development indicating a role of ghrelin in the regulation of growth and differentiation of human gastrointestinal tract.     

Protein Expression Profiles Characterize Distinct Features of Mouse Cerebral Cortices at Different Developmental Stages

The proper development of the mammalian cerebral cortex requires precise protein synthesis and accurate regulation of protein expression levels. To reveal signatures of protein expression in developing mouse cortices, we here generate proteomic profiles of cortices at embryonic and postnatal stages using tandem mass spectrometry (MS/MS). We found that protein expression profiles are mostly consistent with biological features of the developing cortex. Gene Ontology (GO) and KEGG pathway analyses demonstrate conserved molecules that maintain cortical development such as proteins involved in metabolism. GO and KEGG pathway analyses further identify differentially expressed proteins that function at specific stages, for example proteins regulating the cell cycle in the embryonic cortex, and proteins controlling axon guidance in the postnatal cortex, suggesting that distinct protein expression profiles determine biological events in the developing cortex. Furthermore, the STRING network analysis has revealed that many proteins control a single biological event, such as the cell cycle regulation, through cohesive interactions, indicating a complex network regulation in the cortex. Our study has identified protein networks that control the cortical development and has provided a protein reference for further investigation of protein interactions in the cortex.     

Glutamate decarboxylase in developing rat neocortex: Does it correlate with the differentiation of GABAergic neurons and synapses?

Postnatal development of glutamate decarboxylase was studied in the rat cerebral cortex. Two methods were used: estimation of the enzymatic activity of glutamate decarboxylase in homogenates of developing cortical tissue and visualization of structures containing glutamate decarboxylase-like immunoreactivity. Glutamate decarboxylase-like immunoreactivity appeared first in perikarya and dendrites and only later in axons and axon varicosities. The most rapid increase in the glutamate decarboxylase activity took place during the second postnatal week and this coincided with a rapid increase in the density of axon varicosities containing glutamate decarboxylase-like immunoreactivity but preceded the most rapid phase in the formation of GABAergic synapses by several days. However, there was a change in the characteristics of glutamate decarboxylase which correlated with GABA synaptogenesis: two fractions of glutamate decarboxylase with different sensitivities to the activating effects of Triton X-100 could be distinguished as from about the time when most of the GABAergic synapses are formed.     

N-cadherin, NCAM, and integrins promote retinal neurite outgrowth on astrocytes in vitro

Retinal ganglion neurons extend axons that grow along astroglial cell surfaces in the developing optic pathway. To identify the molecules that may mediate axon extension in vivo, antibodies to neuronal cell surface proteins were tested for their effects on neurite outgrowth by embryonic chick retinal neurons cultured on astrocyte monolayers. Neurite outgrowth by retinal neurons from embryonic day 7 (E7) and E11 chick embryos depended on the function of a calcium-dependent cell adhesion molecule (N-cadherin) and beta 1-class integrin extracellular matrix receptors. The inhibitory effects of either antibody on process extension could not be accounted for by a reduction in the attachment of neurons to astrocytes. The role of a third cell adhesion molecule, NCAM, changed during development. Anti-NCAM had no detectable inhibitory effects on neurite outgrowth by E7 retinal neurons. In contrast, E11 retinal neurite outgrowth was strongly dependent on NCAM function. Thus, N-cadherin, integrins, and NCAM are likely to regulate axon extension in the optic pathway, and their relative importance varies with developmental age.     

Immunohistochemical localization of phosphoenolpyruvate carboxykinase in adult and developing mouse tissues.

We used immunohistochemical techniques to analyze the cell distribution of phosphoenolpyruvate carboxykinase (PEPCK) in adult and developing mouse tissues. PEPCK immunoreactivity was detected in many tissues, including some that had not been previously reported to contain PEPCK enzyme activity (bladder, stomach, ovary, vagina, parotid gland, submaxillary gland, and eye). In some multicellular tissues, PEPCK immunoreactivity was observed in multiple cell types. Several tissues (spleen, thyroid, and submaxillary gland) contained no detectable PEPCK immunoreactivity. During development, PEPCK immunoreactivity was associated with the developing nervous system and somites in 15-day embryos. At prenatal day 18, PEPCK immunoreactivity was detected only in the nervous system. At prenatal day 20, PEPCK immunoreactivity was observed in many of the tissues that contain PEPCK in the adult, with the exception of liver, lung, and stomach. PEPCK immunoreactivity was detected in liver at postnatal day 1, lung at postnatal day 7, and stomach after postnatal day 21. The only tissue in which PEPCK immunoreactivity decreased during development was the pancreas, where PEPCK immunoreactivity was detected at prenatal day 20 and was present until postnatal day 21. These results suggest that PEPCK expression is cell-type specific, more widespread than previously thought, and differentially expressed during development.     

Developmental aspects of galectin-3 expression in the lens

In order to investigate the temporal and spatial expression pattern of the lectin galectin-3 during lens development we performed immunohistochemical studies using monoclonal and polyclonal antibodies against galectin-3 on paraffin sections of human, mouse and rat eyes. Galectin-3 has been shown to be involved in various biological functions related to cell adhesion, proliferation, apoptosis and differentiation in other tissues. In the human lens, galectin-3 shows a selective expression pattern during lens development. It is present in all cells of the early lens vesicle and at later stages it is strongly expressed during the elongation phase in differentiating primary lens fibres. From about 7 weeks onwards the anterior lens epithelium fails to express galectin-3. Adult lenses, however, exhibit immunoreactivity in the anterior epithelial cells and in the early differentiating secondary fibres of the lens' outer cortex prior to the onset of degradation of the nuclei. In contrast to the observed expression pattern in prenatal human lenses, mouse and rat lenses exhibited immunoreactivity for galectin-3 during postnatal and adult stages only. At these stages, the expression pattern closely resembles that seen in the corresponding human lenses. The spatiotemporal pattern of galectin-3 distribution during lens development favours a role of this lectin in adhesion processes and in the regulation of programmed organelle elimination during lens cell differentiation.     

Developmental pattern of three vesicular glutamate transporters in the myenteric plexus of the human fetal small intestine

Three vesicular glutamate transporters (VGLUT1-3) have previously been identified in the central nervous system, where they define the glutamatergic phenotype, and their expression is tightly regulated during brain development. In the present study we applied immunocytochemistry to examine the distribution of the immunoreactivity of all three VGLUTs during prenatal development of the myenteric plexus in the human small intestine. We also investigated changes in their localization in the different segments of the small intestine and in the different compartments of the developing myenteric ganglia. Immunoreactivity against all three VGLUTs was found predominantly in the ganglionic neuropil, interganglionic varicose fibers and perisomatic puncta, but cytoplasmic labeling with different intensities also occurred. Each transporter displayed a characteristic spatiotemporal expression pattern, with the transient increase or decrease of immunoreactive cell bodies, varicosities or perisomatic puncta, depending on the fetal age, the gut segment or the ganglionic compartment. Throughout gestational weeks 14-23, VGLUT1 immunoreactivity always predominated over VGLUT2 immunoreactivity, though both peaked around week 20. VGLUT3 immunoreactivity was less abundant in the developing myenteric plexus than those of VGLUT1 and VGLUT2 immunoreactivity. It was mainly expressed in the ganglionic neuropil and in the perisomatic puncta throughout the examined gestational period. Neuronal perikarya immunoreactive for VGLUT3 were restricted to between weeks 18 and 20 of gestation and exclusively to the oral part of the small intestine.     

Appearance of parvalbumin-specific immunoreactivity in the cerebral cortex and hippocampus of the developing rat and gerbil brain

Summary Developmental changes in the distribution of parvalbumin-specific immunoreactivity in the brain, in particular in the cerebral cortex and hippocampus, were followed immunohistochemically in two different species, the rat and the Mongolian gerbil (Meriones unguiculatus) using an antibody raised against for rat parvalbumin. The gerbil is known to develop its auditory and visual capacity later than rat. In both the rat and gerbil, parvalbumin-specific immunoreactivity appeared after birth in both the cerebral cortex and hippocampus. The timing of the development of expression of parvalbumin varied among different parts of the cerebral cortex. The parietal cortex showed evidence of the earliest expression of parvalbumin whilst the occipital and temporal cortices expressed parvalbumin at a later stage of a development. This feature was common to both the rat and gerbil but occurred at a relatively later stage in the gerbil. The profile of the distribution of parvalbumin in the brain of the developing and adult gerbil was similar to that of the rat, but there were some differences. The frequency of bead-like structures on the dendrites of the parvalbumin-positive cells in the CA1 region of the hippocampus was markedly lower in the gerbil; instead, straight non-beaded fibers which ran vertically into the pyramidal layer were stained. Parvalbumin-positive fibers were also found in the cerebral cortex of the gerbil.     

E-Cadherin Can Replace N-Cadherin during Secretory-Stage Enamel Development

Background

N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development.

Methodology/Principal Findings

The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. μCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ.

Conclusions/Significance

The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous increase in E-cadherin expression due to N-cadherin ablation in a healthy developing tissue.     

Molecular and cellular mechanisms of human cortical connectivity

Comparative studies of the cerebral cortex have identified various human and primate-specific changes in both local and long-range connectivity, which are thought to underlie our advanced cognitive capabilities. These changes are likely mediated by the divergence of spatiotemporal regulation of gene expression, which is particularly prominent in the prenatal and early postnatal human and non-human primate cerebral cortex. In this review, we describe recent advances in characterizing human and primate genetic and cellular innovations including identification of novel species-specific, especially human-specific, genes, gene expression patterns, and cell types. Finally, we highlight three recent studies linking these molecular changes to reorganization of cortical connectivity.     

Radial Columnar Patches in the Chimeric Cerebral Cortex Visualized by Use of Mouse Embryonic Stem Cells Expressing β-Galactosidase

Presumed radial migration of neuroblasts from the ventricular to pial surface during formation of the cerebral cortex predicts radial columnar patches in chimeric brains. Lack of adequate cell marker for neurons, however, has hindered such chimera analysis. We used a mouse embryonic stem cell line expressing β-galactosidase gene to produce chimeric brains. Patches of the labeled cells were examined by whole mount staining and also by computer-assisted three-dimensional reconstruction from serial paraffin sections. Our study revealed presence of coherent radial columnar patches in the prenatal cerebral cortex, thus giving a direct evidence for the radial migration of neurons. These columnar patches were less clear in adult brains, suggesting cell mixing during later development and maturation.     

The expression of aristaless-related homeobox in neural progenitors of gyrencephalic carnivore ferrets

Aristaless-related homeobox (ARX) has important functions in the development of various organs including the brain. Mutations of the human ARX gene have been associated with malformations of the cerebral cortex such as microcephaly and lissencephaly. Although the expression patterns of ARX in the lissencephalic cerebral cortex of mice have been intensively investigated, those in expanded gyrencephalic brains remained unclear. Here, we show the expression patterns of ARX in the developing cerebral cortex of gyrencephalic carnivore ferrets. We found that ARX was expressed not only in intermediate progenitor (IP) cells but also in outer radial glial (oRG) cells, which are neural progenitors preferentially observed in the gyrencephalic cerebral cortex. We found that the majority of ARX-positive oRG cells expressed the proliferating cell marker Ki-67. These results may indicate that ARX in oRG cells mediates the expansion of the gyrencephalic cerebral cortex during development and evolution.     

Comparative analysis of the subventricular zone in rat, ferret and macaque: evidence for an outer subventricular zone in rodents

The mammalian cerebral cortex arises from precursor cells that reside in a proliferative region surrounding the lateral ventricles of the developing brain. Recent work has shown that precursor cells in the subventricular zone (SVZ) provide a major contribution to prenatal cortical neurogenesis, and that the SVZ is significantly thicker in gyrencephalic mammals such as primates than it is in lissencephalic mammals including rodents. Identifying characteristics that are shared by or that distinguish cortical precursor cells across mammalian species will shed light on factors that regulate cortical neurogenesis and may point toward mechanisms that underlie the evolutionary expansion of the neocortex in gyrencephalic mammals. We immunostained sections of the developing cerebral cortex from lissencephalic rats, and from gyrencephalic ferrets and macaques to compare the distribution of precursor cell types in each species. We also performed time-lapse imaging of precursor cells in the developing rat neocortex. We show that the distribution of Pax6+ and Tbr2+ precursor cells is similar in lissencephalic rat and gyrencephalic ferret, and different in the gyrencephalic cortex of macaque. We show that mitotic Pax6+ translocating radial glial cells (tRG) are present in the cerebral cortex of each species during and after neurogenesis, demonstrating that the function of Pax6+ tRG cells is not restricted to neurogenesis. Furthermore, we show that Olig2 expression distinguishes two distinct subtypes of Pax6+ tRG cells. Finally we present a novel method for discriminating the inner and outer SVZ across mammalian species and show that the key cytoarchitectural features and cell types that define the outer SVZ in developing primates are present in the developing rat neocortex. Our data demonstrate that the developing rat cerebral cortex possesses an outer subventricular zone during late stages of cortical neurogenesis and that the developing rodent cortex shares important features with that of primates.     

Diverse functions of N-cadherin in dendritic and axonal terminal arborization of olfactory projection neurons

The cadherin superfamily of cell adhesion molecules have been proposed to play important roles in determining synaptic specificity in developing nervous systems. We examine the function of N-cadherin in Drosophila second order olfactory projection neurons (PNs), each of which must selectively target their dendrites to one of approximately 50 glomeruli. Our results do not support an instructive role for N-cadherin in selecting dendritic targets; rather, N-cadherin is essential for PNs to restrict their dendrites to single glomeruli. Mosaic analyses suggest that N-cadherin mediates dendro-dendritic interactions between PNs and thus contributes to refinement of PN dendrites to single glomeruli. N-cadherin is also essential for the development of PN axon terminal arbors in two distinct central targets: regulating branch stability in the lateral horn and restricting high-order branching in the mushroom body. Although the N-cadherin locus potentially encodes eight alternatively spliced isoforms, transgenic expression of one isoform is sufficient to rescue all phenotypes.     

Developmental Expression and Functional Characterization of the 4C5 Antigen in the Postnatal Cerebellar Cortex

Abstract: The monoclonal antibody 4C5 recognizes a neuron-specific surface antigen (4C5 antigen) in the CNS and PNS of the rat. In the present study we investigated the expression of 4C5 antigen in the developing cerebellum of the rat and the functional role of this molecule during cerebellar morphogenesis. Immunoblotting and immunohistochemistry in sections of cerebellar cortex revealed an age-dependent decrease in the expression of the 4C5 antigen. In cerebellar primary cell cultures, 4C5 immunoreactivity was detected both on granule and on Purkinje neurons. Granule cell migration was inhibited in cerebellar explants derived from 8-day-old rats and cultured for 2 days in the presence of antibodies against the 4C5 antigen. Electron microscope immunocytochemistry revealed that in 8-day-old rat cerebellum, 4C5 immunoreactivity was localized on the cell bodies of granule neurons in the external and internal granular layers and on parallel fibers in the developing molecular layer as well as at contact sites between these cellular elements. It was not detected on Bergmann glia. These results suggest strongly that the 4C5 antigen is involved in granule cell migration during cerebellar development, possibly via neuron-neuron interactions.