No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Purification of neuronal precursors from the adult mouse brain: Comprehensive gene expression analysis provides new insights into the control of cell migration, differentiation, and homeostasis
Abstract
The progeny of neural stem cells in the subventricular zone (SVZ) of the adult mammalian brain consists in polysialylated NCAM-expressing immature neurons (PSA(+) cells), which migrate to the olfactory bulb (OB) to differentiate into GABAergic interneurons. We purified murine PSA(+) cells directly from the adult brain by FACS and analyzed their gene expression profile by SAGE. Comparative analyses led to the identification of precursor-enriched genes, including Survivin, Sox-4, Meis2, Dishevelled-2, C3aR1 and Riken 3110003A17, and many so far uncharacterized transcripts. Cluster analysis showed that groups of genes involved in axon guidance and gene clusters implicated in chemotaxis are strongly upregulated, indicating a role of both cues in the control of cell migration in the adult brain. Furthermore, genes involved in apoptosis and cell proliferation are co-expressed, suggesting that the amount of precursors that is present in the adult brain is a result of an equilibrium of these processes.
... PSA-NCAM is a marker of both the mitotically active neuronal progenitor cells and the early post-mitotic neurons in embryonic neural tube and in chain-migrating neuronal precursors in the subventricular zone (SVZ) of the adult brain [21,22]. PSA-NCAM was used previously to purify neuronal cells from embryonic rat neural tubes [21], adult mouse (SVZ) [22], embryonic stem cell derived neuronal cells [23] and immature neuronal cells from dNSC progeny [10]. ...
... PSA-NCAM is a marker of both the mitotically active neuronal progenitor cells and the early post-mitotic neurons in embryonic neural tube and in chain-migrating neuronal precursors in the subventricular zone (SVZ) of the adult brain [21,22]. PSA-NCAM was used previously to purify neuronal cells from embryonic rat neural tubes [21], adult mouse (SVZ) [22], embryonic stem cell derived neuronal cells [23] and immature neuronal cells from dNSC progeny [10]. Using PE-conjugated PSA-NCAM antibody and EasySep PE Selection MACS kit; we isolated PSA-NCAM IR cells after dissociating the NBA culture into single cell suspension. ...
... The purity of the isolated neurons using the shaking method was comparable to the purity level (96%) achieved by Sandra Pennartz et al. [22] using the FACS method to sort PSA-NCAM positive cells from digested tissue harvested from the subventicular zone of the adult mouse brain. However, using the FACS method and sorting neuronal cells from dNSC progeny based on cell size and internal complexity, we achieved a neuronal cell population with a purity of 75% [10]. ...
Objective: Enriched neuronal cell populations are valuable tools both for laboratory investigations and cell therapy applications. However, available cell purifying approaches demand costly equipment such as FACS or MACS that limits their universal accessibility. In this study, we developed an efficient method for purifying immature neuronal cells from differentiating neural stem cell (dNSC) progeny based on their differential substrate attachment properties without using any expensive cell separating tools.
Methods: Neural stem cells were harvested from the ganglionic eminence of embryonic day 14 mice brains using the neurosphere assay. Neurospheres were then dissociated into single cells and differentiated employing the neuroblast assay method. Following a brief trypsinization, the dNSC culture was gently shaken at 150 rpm for 30 min to detach the top neuronal cell clusters from the underlying astrocytic cell monolayer. The neuronal purification yield, astrocyte contamination, and presence of dividing cells were compared to a MACS purification method using PSA-NCAM antibody.
Results: While a neuronal yield of 97.1 ± 0.45% was achieved using MACS; it reached 97.9 ± 0.6% using the shaking method that was not significantly different.On the other hand, the percentage of astrocytes in the MACS approach was 1.18 ± 0.15%, but it significantly decreased to 0.6 ± 0.15% using the shaking method. Moreover, 4.41 ± 0.23% and 5.3 ± 0.4% of the isolated cells in the MACS and shaking methods, respectively, were Ki-67 immunoreactive dividing cells, of which 97.34 ± 1.6% and 97.9 ± 0.7% were co-expressing β III-tubulin, confirming their neuronal identity. Additionally, based on the neural-colony forming cell assay, the shaking method resulted in the generation of a homogenous neuronal cell population without any bona fide NSC contamination.
Conclusions: The shaking purification method allows easy, low cost, efficient and large-scale separation of immature neurons from dNSC progeny, potentially benefiting both basic and clinical applications.
... Currently, little is known about the molecular signals that regulate the synaptic integration of neurons into existing neuronal networks. In an attempt to identify such signals, we isolated migratory neuronal precursors from the subventricular zone (SVZ) and the rostral migratory stream (RMS) and analyzed their gene expression by Serial Analysis of Gene Expression (SAGE) (Pennartz et al., 2004) and microarray analysis (Boutin et al., 2010). One of the genes that showed strong and dynamic expression in this cell population was agrin. ...
... In a SAGE-based study (Pennartz et al., 2004) we found a strong over-representation of agrin transcripts in purified adult olfactory interneuron precursors (Pennartz et al., 2004) in comparison to total brain tissue. In agreement, in situ hybridization and immunohistochemistry showed strong signals in the RMS (Fig. 1a) as well as in the granule cell layer (GCL), the mitral cell layer (MCL), and the glomerular layer (GL) (compare Fig. 1a, arrow, http://mouse.brain-map.org/gene/show/11390). ...
... In a SAGE-based study (Pennartz et al., 2004) we found a strong over-representation of agrin transcripts in purified adult olfactory interneuron precursors (Pennartz et al., 2004) in comparison to total brain tissue. In agreement, in situ hybridization and immunohistochemistry showed strong signals in the RMS (Fig. 1a) as well as in the granule cell layer (GCL), the mitral cell layer (MCL), and the glomerular layer (GL) (compare Fig. 1a, arrow, http://mouse.brain-map.org/gene/show/11390). ...
In the adult forebrain, new interneurons are continuously generated and integrated into the existing circuitry of the olfactory bulb (OB). In an attempt to identify signals that regulate this synaptic integration process, we found strong expression of agrin in adult generated neuronal precursors that arrive in the olfactory bulb after their generation in the subventricular zone. While the agrin receptor components MuSK and Lrp4 were below detection level in neuron populations that represent synaptic targets for the new interneurons, the alternative receptor α3-Na(+)K(+)-ATPase was strongly expressed in mitral cells. Using a transplantation approach, we demonstrate that agrin-deficient interneuron precursors migrate correctly into the OB. However, in contrast to wild-type neurons, which form synapses and survive for prolonged periods, mutant neurons do not mature and are rapidly eliminated. Using in vivo brain electroporation of the olfactory system, we show that the transmembrane form of agrin alone is sufficient to mediate integration and demonstrate that excess transmembrane agrin increases the number of dendritic spines. Last, we provide in vivo evidence that an interaction between agrin and α3-Na(+)K(+)-ATPase is of functional importance in this system.
... Neuroblasts migrate through rostral migratory stream of the olfactory bulb where they differentiate into interneurons. Lower panel: the schematic summary of the findings regarding expression of SOX2 (Ferri et al., 2004), SOX3 (Rogers et al., 2013), SOX4 (Pennartz et al., 2004), SOX11 (Haslinger et al., 2009), and SOX9 (Cheng et al., 2009). NSC, neural stem cell; IN, immature neuron; MN, mature neuron; N/A, data not available. ...
... In the adult mammalian brain, the expression of SOX4 and SOX11 proteins is detected prominently in the both main neurogenic niches, SGZ ( Figure 4A) and SVZ ( Figure 4B) (Pennartz et al., 2004;Haslinger et al., 2009;Mu et al., 2012). Similar to the expression pattern in embryonic brain, onset of SOX4 and SOX11 expression in cells coincides with the downregulation of SOX2 and up-regulation of DCX (Doublecortin) expression, and remains throughout the period when newborn neurons migrate to their final destination (Figure 4). ...
The SOX proteins belong to the superfamily of transcription factors (TFs) that display properties of both classical TFs and architectural components of chromatin. Since the cloning of the Sox / SOX genes, remarkable progress has been made in illuminating their roles as key players in the regulation of multiple developmental and physiological processes. SOX TFs govern diverse cellular processes during development, such as maintaining the pluripotency of stem cells, cell proliferation, cell fate decisions/germ layer formation as well as terminal cell differentiation into tissues and organs. However, their roles are not limited to development since SOX proteins influence survival, regeneration, cell death and control homeostasis in adult tissues. This review summarized current knowledge of the roles of SOX proteins in control of central nervous system development. Some SOX TFs suspend neural progenitors in proliferative, stem-like state and prevent their differentiation. SOX proteins function as pioneer factors that occupy silenced target genes and keep them in a poised state for activation at subsequent stages of differentiation. At appropriate stage of development, SOX members that maintain stemness are down-regulated in cells that are competent to differentiate, while other SOX members take over their functions and govern the process of differentiation. Distinct SOX members determine down-stream processes of neuronal and glial differentiation. Thus, sequentially acting SOX TFs orchestrate neural lineage development defining neuronal and glial phenotypes. In line with their crucial roles in the nervous system development, deregulation of specific SOX proteins activities is associated with neurodevelopmental disorders (NDDs). The overview of the current knowledge about the link between SOX gene variants and NDDs is presented. We outline the roles of SOX TFs in adult neurogenesis and brain homeostasis and discuss whether impaired adult neurogenesis, detected in neurodegenerative diseases, could be associated with deregulation of SOX proteins activities. We present the current data regarding the interaction between SOX proteins and signaling pathways and microRNAs that play roles in nervous system development. Finally, future research directions that will improve the knowledge about distinct and various roles of SOX TFs in health and diseases are presented and discussed.
... In mammals, four Robo receptors were identified, but only two of them, Robo1 and Robo2, bind Slits Koch et al., 2011;Zelina et al., 2014). Interestingly, SVZ/RMS NPCs express Robo2 (Marillat et al., 2002;Kaneko et al., 2010) and type A and C cells express Slit1 (Nguyen-Ba-Charvet et al., 2004;Pennartz et al., 2004). Slit/Robo proteins have multiple functions in non-neural and neural tissues (Blockus and Chédotal, 2016). ...
... However, neurospheres are a mixture of stem/progenitor cells, which render the data interpretation complex with respect to cell autonomy. Since Slit1 expression is prominent in PSA-NCAM + progenitors (Pennartz et al., 2004), and in order to use an homogeneous cell population, we re-addressed the function of Slit1 in NPC migration using purified preparations of immuno-selected PSA-NCAM + NPCs. We first examined the antigenic profile of the purified PSA-NCAM + NPCs after short-term culture, using the NPC markers, Nestin, and PSA-NCAM, the combined OPC markers NG2/A2B5, the astrocyte marker GFAP, and neuronal marker NeuN. ...
Slit1 is a secreted axon guidance molecule, also involved in adult neurogenesis. In physiological conditions, Slit1 loss promotes ectopic dispersal of SVZ-derived neural precursors (SVZ-NPCs) into periventricular structures such as the corpus callosum. Demyelination of the corpus callosum triggers SVZ-NPC migration to ectopic locations and their recruitment by the lesion, suggesting a possible role for Slit1 in SVZ-NPCs ectopic dispersal regulation in pathological conditions. Here, we have investigated the function of Slit1 protein in the recruitment of SVZ-NPCs after CNS demyelination. We find that the dynamics of oligodendrogenesis and temporal profile of developmental myelination in Slit1–/– mice are similar to Slit1+/− controls. SVZ micro-dissection and RT-PCR from wild-type mice, show that Slits and Robos are physiologically regulated at the transcriptional level in response to corpus callosum demyelination suggesting their role in the process of SVZ-NPC ectopic migration in demyelinating conditions. Moreover, we find that the number of SVZ-NPCs recruited by the lesion increases in Sli1–/– mice compared to Slit1+/− mice, leading to higher numbers of Olig2+ cells within the lesion. Time-lapse video-microscopy of immuno-purified NPCs shows that Slit1-deficient cells migrate faster and make more frequent directional changes than control NPCs, supporting a cell-autonomous mechanism of action of Slit1 in NPC migration. In conclusion, while Slit1 does not affect the normal developmental process of oligodendrogenesis and myelination, it regulates adult SVZ-NPC ectopic migration in response to demyelination, and consequently oligodendrocyte renewal within the lesion.
... GLAST expression also persists in the neural progenitor stage (Pastrana et al., 2009), during which it is co-expressed with PSA-NCAM in the DG (Jungblut et al., 2012). PSA-NCAM is expressed in other migrating immature cells such as neuroblasts and immature neurons besides its expression in neural progenitor cells, owing to the fact that PSA-NCAM + cells show enriched expression of chemokines, Nestin, Notch1, and Doublecortin (Pennartz et al., 2004). ...
... Fmr1 expression is upregulated in PSA-NCAM + cells, which encompass neuroblasts and immature neurons (Pennartz et al., 2004). This suggests that FMRP plays an important role in regulating neuroblasts and immature neurons. ...
The regulation of neural precursor cells (NPCs) is indispensable for a properly functioning brain. Abnormalities in NPC proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome. Yet, no studies have examined NPCs from the early postnatal Fragile X mouse hippocampus despite the importance of this developmental time point, which marks the highest expression level of FMRP, the protein missing in Fragile X, in the rodent hippocampus and is when hippocampal NPCs have migrated to the dentate gyrus (DG) to give rise to lifelong neurogenesis. In this study, we examined NPCs from the early postnatal hippocampus and DG of Fragile X mice (Fmr1-KO). Immunocytochemistry on neurospheres showed increased Nestin expression and decreased Ki67 expression, which collectively indicated aberrant NPC biology. Intriguingly, flow cytometric analysis of the expression of the antigens CD15, CD24, CD133, GLAST, and PSA-NCAM showed a decreased proportion of neural stem cells (GLAST⁺CD15⁺CD133⁺) and an increased proportion of neuroblasts (PSA-NCAM⁺CD15⁺) in the DG of P7 Fmr1-KO mice. This was mirrored by lower expression levels of Nestin and the mitotic marker phospho-histone H3 in vivo in the P9 hippocampus, as well as a decreased proportion of cells in the G2/M phases of the P7 DG. Thus, the absence of FMRP leads to fewer actively cycling NPCs, coinciding with a decrease in neural stem cells and an increase in neuroblasts. Together, these results show the importance of FMRP in the developing hippocampal formation and suggest abnormalities in cell cycle regulation in Fragile X.
... Based on the previous finding that BIRC5 (encoding survivin) is highly expressed in hPSCs compared with somatic cells (40), we hypothesized that chemical inhibition of survivin would selectively eliminate remaining undifferentiated hiPSCs. However, since survivin is known to be important for neuronal precursors (41,42), it is important to test whether this strategy interferes with mDAP generation. Among survivin inhibitors (40), we chose the flavonoid quercetin (3,3′,4′,5,7-pentahydroxyflavone) because this natural compound is present at high concentrations in commonly consumed vegetables and fruits (43). ...
Parkinson's disease (PD) is a neurodegenerative disorder associated with loss of striatal dopamine, secondary to degeneration of midbrain dopamine (mDA) neurons in the substantia nigra, rendering cell transplantation a promising therapeutic strategy. To establish human induced pluripotent stem cell (hiPSC)-based autologous cell therapy, we report a platform of core techniques for the production of mDA progenitors as a safe and effective therapeutic product. First, by combining metabolism-regulating microRNAs with reprogramming factors, we developed a method to more efficiently generate clinical grade iPSCs, as evidenced by genomic integrity and unbiased pluripotent potential. Second, we established a "spotting"-based in vitro differentiation methodology to generate functional and healthy mDA cells in a scalable manner. Third, we developed a chemical method that safely eliminates undifferentiated cells from the final product. Dopaminergic cells thus produced express high levels of characteristic mDA markers, produce and secrete dopamine, and exhibit electrophysiological features typical of mDA cells. Transplantation of these cells into rodent models of PD robustly restores motor dysfunction and reinnervates host brain, while showing no evidence of tumor formation or redistribution of the implanted cells. We propose that this platform is suitable for the successful implementation of human personalized autologous cell therapy for PD.
... The characterization of gene expression profiles that define a particular cell state and may determine cell identity in adult NSC is technically challenging because it requires the purification of specific cell types from a complex mixture of cells present in brain tissue. To tackle this problem, different experimental strategies have been used successfully but most of them rely on the identification of cell type-specific marker(s) with specific antibodies, followed by fluorescence-activated cell sorting to purify the positive population [26][27][28][29]. These studies have provided invaluable information about gene expression profiles characteristic to discrete cell populations of adult NSC and their immature neuronal progeny. ...
Adult neural stem and progenitor cells (NSPCs) offer a unique opportunity for neural regeneration and niche modification in physiopathological conditions, harnessing the capability to modify from neuronal circuits to glial scar. Findings exposing the vast plasticity and potential of NSPCs have accumulated over the past years and we currently know that adult NSPCs can naturally give rise not only to neurons but also to astrocytes and reactive astrocytes, and eventually to oligodendrocytes through genetic manipulation. We can consider NSPCs as endogenous flexible tools to fight against neurodegenerative and neurological disorders and aging. In addition, NSPCs can be considered as active agents contributing to chronic brain alterations and as relevant cell populations to be preserved, so that their main function, neurogenesis, is not lost in damage or disease. Altogether we believe that learning to manipulate NSPC is essential to prevent, ameliorate or restore some of the cognitive deficits associated with brain disease and injury, and therefore should be considered as target for future therapeutic strategies. The first step to accomplish this goal is to target them specifically, by unveiling and understanding their unique markers and signaling pathways.
... Our transcriptional profile of the CD24+ cells provides a gene expression database for ependymal cells and should serve as an important resource for further molecular analysis of these cells (see Supplementary text). The gene expression profile of isolated type A cells has also been studied (Pennartz et al., 2004); therefore, to date, transcriptional profiles of type B, ependymal, and type A cells are available, and together they should assist investigators in the formation of hypotheses about gene function in the SVZ. ...
... There is a possible role for members of the OTX gene family in MB tumorigenesis (de Haas, et al., 2006) and in driving and defining especially the group 3 and group 4 subclasses (Marshall, et al., 2014). Specific patterns of OTX1 and OTX2 have been found delineating borders and layers between and within hindbrain, midbrain and forebrain and regulated expression of these transcription factors has been assumed important for controlling neurogenesis, neural progenitor identity and fate as well as local brain regionalization (Pennartz, et al., 2004, Rajendran, et al., 2008, Su, et al., 2009, Sun, et al., 2005a, Zupanc, et al., 2005. Importantly, OTX2 has been shown to induce proliferation and impede differentiation of cancer cells in MB (Adamson, et al., 2010, Lachyankar, et al., 2000, Ross, et al., 2001, and since it is often found amplified in group 3 and group 4 MBs (Adamson, et al., 2010, it might be argued to be a driver of those subgroups. ...
Neurogenesis, the generation of new neurons, is deregulated in neural stem cell (NSC)- and progenitor-derived murine models of malignant medulloblastoma and glioma, the most common brain tumors of children and adults, respectively. Molecular characterization of human malignant brain tumors, and in particular brain tumor stem cells (BTSCs), has identified neurodevelopmental transcription factors, microRNAs, and epigenetic factors known to inhibit neuronal and glial differentiation. We are starting to understand how these factors are regulated by the major oncogenic drivers in malignant brain tumors. In this review, we will focus on the molecular switches that block normal neuronal differentiation and induce brain tumor formation. Genetic or pharmacological manipulation of these switches in BTSCs has been shown to restore the ability of tumor cells to differentiate. We will discuss potential brain tumor therapies that will promote differentiation in order to reduce treatment resistance, suppress tumor growth, and prevent recurrence in patients.
... As polysialylated NCAM (polySia-NCAM) is considered to be a neural stem cell marker [13], and diffusely infiltrating astrocytomas are often referred as stem cell cancers [1], we studied for the first time whether the characteristics and clinical course of astrocytomas with and without polysialylated NCAM differ from each other. In addition, given that the isocitrate dehydrogenase 1 (IDH1) mutation has been found to be an essential genetic aberration in grade II-III diffusely infiltrating astrocytomas and especially in secondary GBMs [14,15], we wanted to study whether this stem cell marker, polySia-NCAM, can be of additional help in identifying the prognostic characteristics if grade II-IV astrocytomas and secondary GBMs with and without IDH1 mutations. ...
Background
The aim of the study was to assess the localization of Polysialic acid (polySia) and Neural cell adhesion molecule (NCAM) in grade I–IV astrocytomas by confocal microscopy, and also to clarify and compare their relationship to conventional clinicopathological features in these tumors.
Methods
Study material was stained immunohistochemically for polySia, NCAM and IDH1-R132H point mutation. Confocal microscopy of polySia and NCAM staining was performed on tissue micro-array samples (TMA) of 242 diffusely infiltrating astrocytomas (grade II: 28; grade III: 33; grade IV: 181) and 82 pilocytic astrocytomas. The results were statistically correlated to clinicopathological factors and survival data.
Results
PolySia was observed in 45 cases (19%) and NCAM positivity in 92 cases (38%). All 45 tumors with polySia positivity were also positive for NCAM whereas there were 47 tumors which contained positive staining for NCAM but not for polySia. The simultaneous expression was concomitant and colocalized suggesting polysialyated NCAM (polySia-NCAM). PolySia expression was significantly stronger in IDH1 mutated tumors than in IDH1 non-mutated (p = 0.001, chi-square test). There were no significant differences in polySia-NCAM between primary tumors or recurrences (p = n.s., chi-square test). PolySia positivity was associated with longer patient survival in relation to total tumor material (p = 0.020, log-rank test). Furthermore, when only glioblastomas were assessed, patients with positive polySia had significantly better prognosis (p = 0.006, log-rank test). In multivariate survival analysis, polySia was found to be an independent prognostic factor. PolySia was nearly absent in grade I pilocytic astrocytomas (1 immunopositive tumor of 82).
Conclusions
Expression of polySia is common in adult grade II–IV astrocytomas, whereas it is nearly absent in pediatric grade I pilocytic astrocytomas. PolySia positivity is associated with longer survival rates in patients with a grade II–IV astrocytomas and also grade IV glioblastomas assessed separately. The results of this study suggest that IDH1 mutation may be associated with polySia expression pathways in malignant gliomas.
... Overexpression of survivin is a hallmark of virtually every human tumor, and is prognostic for poor outcomes and disease recurrence (Altieri, 2013). Survivin expression is nearly absent from normal differentiated cells, but is high in proliferative areas of normal tissues (Ambrosini et al., 1997;Conway et al., 2003;Fukuda and Pelus, 2002;Pennartz et al., 2004). ...
Understanding the mechanisms that sustain pluripotency in human embryonic stem cells (hESCs) is an active area of research that may prove useful in regenerative medicine and will provide fundamental information relevant to development and cancer. hESCs and cancer cells share the unique ability to proliferate indefinitely and rapidly. Because the protein survivin is uniquely overexpressed in virtually all human cancers and in hESCs, we sought to investigate its role in supporting the distinctive capabilities of these cell types. Results presented here suggest that survivin contributes to the maintenance of pluripotency and that post-transcriptional control of survivin isoform expression is selectively regulated by microRNAs. miR-203 has been extensively studied in human tumors, but has not been characterized in hESCs. We show that miR-203 expression and activity is consistent with the expression and subcellular localization of survivin isoforms that in turn modulate expression of the Oct4 and Nanog transcription factors to sustain pluripotency. This study contributes to understanding of the complex regulatory mechanisms that govern whether hESCs proliferate or commit to lineages. © 2014 Wiley Periodicals, Inc.
... Furthermore, Meis1 dysregulation is a frequent occurrence in MLL-induced leukemia, implicating Meis family proteins in hematopoietic stem and progenitor cell activity in the adult under physiological and pathophysiological conditions (Calvo et al., 2001;Nakamura et al., 1996;Wong et al., 2007). Expression of Meis2 in the adult SVZ, OB and in SVZ-derived neuroblasts has been observed, but if and how Meis2 contributes to adult SVZ neurogenesis is still unknown (Allen et al., 2007;Marei et al., 2012;Parmar et al., 2003;Pennartz et al., 2004). Here, we provide experimental evidence that Meis2 functions as an essential Pax6 co-factor in adult SVZ neurogenesis and identify Dcx and tyrosine hydroxylase as direct Meis2 targets in this system. ...
Meis homeodomain transcription factors control cell proliferation, cell fate specification and differentiation in development and disease. Previous studies have largely focused on Meis contribution to the development of non-neuronal tissues. By contrast, Meis function in the brain is not well understood. Here, we provide evidence for a dual role of the Meis family protein Meis2 in adult olfactory bulb (OB) neurogenesis. Meis2 is strongly expressed in neuroblasts of the subventricular zone (SVZ) and rostral migratory stream (RMS) and in some of the OB interneurons that are continuously replaced during adult life. Targeted manipulations with retroviral vectors expressing function-blocking forms or with small interfering RNAs demonstrated that Meis activity is cell-autonomously required for the acquisition of a general neuronal fate by SVZ-derived progenitors in vivo and in vitro. Additionally, Meis2 activity in the RMS is important for the generation of dopaminergic periglomerular neurons in the OB. Chromatin immunoprecipitation identified doublecortin and tyrosine hydroxylase as direct Meis targets in newly generated neurons and the OB, respectively. Furthermore, biochemical analyses revealed a previously unrecognized complex of Meis2 with Pax6 and Dlx2, two transcription factors involved in OB neurogenesis. The full pro-neurogenic activity of Pax6 in SVZ derived neural stem and progenitor cells requires the presence of Meis. Collectively, these results show that Meis2 cooperates with Pax6 in generic neurogenesis and dopaminergic fate specification in the adult SVZ-OB system.
... Markers for neural and glial (astrocytes and oligodendrocytes) precursor cells like PSA-NCAM and A2B5 have also been identified and used in many studies [135,136] . ...
Adult Stem and Progenitor Cells.- Isolation and Enrichment of Stem Cells.- Transdifferentiation of Stem Cells: A Critical View.- Stem Cells for Cardiac Regeneration by Cell Therapy and Myocardial Tissue Engineering.- Stem Cells and Scaffolds for Vascularizing Engineered Tissue Constructs.- Embryonic Stem Cells: Isolation, Characterization and Culture.- Totipotency, Pluripotency and Nuclear Reprogramming.- Large Scale Production of Stem Cells and Their Derivatives.
... Since we did not have an appropriate mouse line to isolate neuroblasts as comparable to the cell type-specific mouse lines used in this study for other niche cells, we decided to focus our gene expression profiling using NSCs, TAPs, astrocytes, ependymal cells, and vascular endothelial cells as the SVZ niche cells. Pennartz et al. used PSA-NCAM+ cells for the serial analysis of gene expression (SAGE) to identify genes enriched in the neuroblasts [64]. Therefore, we have extracted the information embedded in this study to expand our repertoire of signaling molecules in the SVZ neurogenic niche (Table S3). ...
Neural stem cells (NSCs) reside in a unique microenvironment called the neurogenic niche and generate functional new neurons. The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the subventricular zone (SVZ) of the lateral ventricle. While several molecules produced by the niche cells have been identified to regulate adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate the SVZ niche cells including NSCs, transit-amplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and microdissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using cDNA microarray technology. Through the combination of multiple screening approaches, we identified a number of candidate genes with a potential relevance for regulating the NSC behaviors, which provide new insight into the nature of neurogenic niche signals.
... We are also beginning to identify molecules responsible for the directionality of A cell migration (Fig. 3.3). Much progress is expected in elucidating the mechanisms underlying these processes as novel techniques such as microarray analysis begin to provide a plethora of candidate genes (Pennartz et al., 2004). ...
The identification of adult germinal zones that continue generating neurons long after the end of development has generated much excitement and stimulated many questions. In direct opposition to traditional views that germinal potentials are limited to the embryonic developing brain, an increasing number of studies have shown that neurogenesis continues throughout life, albeit in restricted domains. Adult neural stem cell research has led to discoveries that challenge old views on the mechanism of nerve cell generation. Unexpected properties of the neuronal progenitors and new mechanisms for neuronal migration have emerged from these studies. Here, we shall discuss some recent findings that investigate the origin, migration, and function of new neurons derived from the adult SVZ as well as the molecular components that maintain this germinal zone and highlight areas that offer exciting opportunities for future research. © 2006 Springer Science-Business Media, Inc. All rights reserved.
... Regarding MEIS2 expression, this gene has been found in immature neuronal precursor cells, lens proliferative cells, ovarian cancer, and other tumor cell types, which underlies its possible role in sustaining proliferation [30]. We observed strong expression in leukemia-derived cell lines compared with control cells, which is in agreement with the findings of Smith et al. [31]; however, when we analyzed its expression in patients, we found no variation in the expression of this gene (Figure 3). ...
The Three-amino acid-loop-extension (TALE) superfamily of homeodomain-containing transcription factors have been implicated in normal hematopoiesis and in leukemogenesis and are important survival, differentiation, and apoptosis pathway modulators. In this work, we determined the expression levels of TALE genes in leukemic-derived cell lines, in blood samples of patients with Acute lymphoblastic leukemia (ALL), and in the blood samples of healthy donors.
Here we show increased expression of MEIS1, MEIS2, and PREP1 genes in leukemia-derived cell lines compared with blood normal cells. High levels of MEIS1 and PREP1, and low levels of PBX4 expression were also founded in samples of patients with ALL. Importantly, silencing of MEIS1 decreases the proliferation of leukemia-derived cells but increases their survival after etoposide treatment. Etoposide-induced apoptosis induces down-regulation of MEIS1 expression or PREP1 up-regulation in chemotherapy-resistant cells.
Our results indicate that up-regulation of MEIS1 is important for sustaining proliferation of leukemic cells and that down-regulation of MEIS1 or up-regulation of PREP1 and PBX genes could be implicated in the modulation of the cellular response to chemotherapeutic-induced apoptosis.
... The SOXE (Sox8, Sox9, and Sox10) group is known to function in Schwann cell development (47), oligodendrocyte development (88), and regulation of nicotinic ACh receptors (52). SOXC genes (Sox 4 and Sox11) are expressed in the immature neurons of neural stem cells in mice (66). Because SRY shares a HMG box, and thus similar DNA binding sites, with all other SOX proteins, SRY, when expressed could participate in similar roles in nervous system development and function in the adult male nervous system. ...
The Sry locus on the mammalian Y chromosome is the developmental switch responsible for testis determination. Inconsistent with this important function, the Sry locus is transcribed in adult males at times and in tissues not involved with testis determination. Sry is expressed in multiple tissues of the peripheral and central nervous system. Sry is derived from Sox3 and is similar to other SOXB family loci. The SOXB loci are responsible for nervous system development. Sry has been demonstrated to modulate the catecholamine pathway, so it should have functional consequences in the central and peripheral nervous system. The nervous system expression and potential function are consistent with Sry as a SOXB family member. In mammals, Sox3 is X-linked and undergoes dosage compensation in females. The expression of Sry in adult males allows for a type of sexual differentiation independent of circulating gonadal hormones. A quantitative difference in Sox3 plus Sry expression in males vs. females could drive changes in the transcriptome of these cells, differentiating male and female cells. Sry expression and its transcriptional effects should be considered when investigating sexual dimorphic phenotypes.
... Recent studies of the central nervous system (CNS) of many vertebrates have demonstrated that Meis genes are possibly involved in the regionalization of the developing telencephalon (Cecconi et al., 1997;Toresson et al., 2000;Takahashi et al., 2008), pretectum (Ferran et al., 2007), mesencephalon (Ferran et al., 2007;Shim et al., 2007;Agoston et al., 2009), and hindbrain (Cecconi et al., 1997;Jacobs et al., 1999;Salzberg et al., 1999;Ferretti et al., 2000;Zerucha and Prince, 2000;Biemar et al., 2001;Dibner et al., 2001Dibner et al., , 2004Vlachakis et al., 2001;Waskiewicz et al., 2001;Choe et al., 2002;Aamar and Frank, 2004;Dibner et al., 2004;Choe and Sagerström, 2004). In the forebrain, Meis2 was found to be strongly expressed in the lateral ganglionic eminence and developing striatum, being a good marker of the striatal precursors/neurons (Cecconi et al., 1997;Oulad-Abdelghani et al., 1997;Toresson et al., 1999;Takahashi et al., 2008; see also Pennartz et al., 2004). Larsen et al. (2010) recently described Meis2 expression in the proliferative zone of the human fetal telencephalon, in the ganglionic eminence, cortical plate, and putamen. ...
We are interested in stable gene network activities operating sequentially during inner ear specification. The implementation of this patterning process is a key event in the generation of functional subdivisions of the otic vesicle during early embryonic development. The vertebrate inner ear is a complex sensory structure that is a good model system for characterization of developmental mechanisms controlling patterning and specification. Meis genes, belonging to the TALE family, encode homodomain-containing transcription factors remarkably conserved during evolution, which play a role in normal and neoplastic development. To gain understanding of the possible role of homeobox Meis genes in the developing chick inner ear, we comprehensively analyzed their spatiotemporal expression patterns from early otic specification stages onwards. In the invaginating otic placode, Meis1/2 transcripts were observed in the borders of the otic cup, being absent in the portion of otic epithelium closest to the hindbrain. As development proceeds, Meis1 and Meis2 expressions became restricted to the dorsomedial otic epithelium. Both genes were strongly expressed in the entire presumptive domain of the semicircular canals, and more weakly in all associated cristae. The endolymphatic apparatus was labeled in part by Meis1/2. Meis1 was also expressed in the lateral wall of the growing cochlear duct, while Meis2 expression was detected in a few cells of the developing acoustic-vestibular ganglion. Our results suggest a possible role of Meis assigning regional identity in the morphogenesis, patterning, and specification of the developing inner ear.
... Using Serial Analysis of Gene Expression (SAGE) on purified migratory interneuron precursors, we found that CXCR7 was 5 times overexpressed compared to whole brain while CXCR4 was not detected (Pennartz et al., 2004). These data corroborate the data presented in the Gene Expression Nervous System Atlas Project (GENESAT Project; Gong et al., 2003) in which transgenic mice carrying GFP under the control of the CXCR7 locus (CXCR7-GFP mice) show GFP expression in the SVZ and RMS whereas CXCR4-GFP mice did not (www.gensat.org). ...
The chemokine CXCL12/CXCR4 signaling system is important for the regulation of neuron migration in the developing forebrain. In particular it is crucial for correct distribution of Cajal-Retzius cells and migration of cortical interneurons. Here we investigated the expression of CXCR7, the second receptor for CXCL12, in comparison to CXCR4. We found that shifts in the expression of both receptors in the above cited cell populations coincide with major changes in their migratory behavior. Furthermore, we demonstrated that postnatally generated olfactory interneuron precursors express CXCR7 but not CXCR4 and that their distribution in the rostral migratory stream is affected by CXCR7 downregulation. This suggests an involvement of CXCR7 in neuronal cell migration and indicates a possible action of CXCR7 independently of CXCR4 as a mediator of CXCL12 signaling.
... This is also reported from studies on the developing murine telencephalon [Toresson et al., 1999[Toresson et al., , 2000]. In the adult mouse, Meis2 is also expressed in migrating precursors from the subventricular zone to the olfactory bulb [Pennartz et al., 2004]. ...
The development of the human neocortex is a complex and highly regulated process involving a time-related expression of many transcription factors including the homeobox genes Pax6 and Meis2. During early development, Pax6 is expressed in nuclei of radial glia cells in the neocortical proliferative zones and controls the differentiation and neurogenetic fate of these cells in the dorsal telencephalon in rodents. Animal studies on the Meis2 gene have revealed expression in the developing telencephalon and Meis2 is known to regulate the expression of Pax6 in the eye and pancreas. Because of this functional relation between Pax6 and Meis2, we studied the spatial and temporal expression of PAX6, and MEIS2 using a developmental series of human fetal brains at 7-19 postconceptional weeks with emphasis on the forebrain to investigate whether the two genes are expressed in the same regions and zones in the same time window. We demonstrate by in situ hybridization and immunohistochemistry that the two homeobox genes are expressed during early fetal brain development in humans. PAX6 mRNA and protein were located in the proliferative zones of the neocortex and in single cells in the cortical preplate at 7 fetal weeks and in the developing cortical plate from 8 or 9 to 19 fetal weeks. The expression of PAX6 expanded into the ganglionic eminence just prior to the stage at which a stereological estimation showed an exponential rise in total cell number in this area. The MEIS2 gene was also present in the proliferative zones of the human fetal neocortex and a higher expression of MEIS2 than PAX6 was observed in these areas at 9 fetal weeks. Further, MEIS2 was expressed at a very high level in the developing ganglionic eminence and at a more moderate level in the cortical plate.
... Postnatally, survivin expression was restricted largely to rapidly proliferating cells [21] and migrating NPCs [27]. At P7, survivin mRNA was detected in the SVZ, the RMS, the OB, and the dentate gyrus (DG) ( Figure 2A, C, D, E). ...
Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family in that it exhibits antiapoptotic properties and also promotes the cell cycle and mediates mitosis as a chromosome passenger protein. Survivin is highly expressed in neural precursor cells in the brain, yet its function there has not been elucidated.
To examine the role of neural precursor cell survivin, we first showed that survivin is normally expressed in periventricular neurogenic regions in the embryo, becoming restricted postnatally to proliferating and migrating NPCs in the key neurogenic sites, the subventricular zone (SVZ) and the subgranular zone (SGZ). We then used a conditional gene inactivation strategy to delete the survivin gene prenatally in those neurogenic regions. Lack of embryonic NPC survivin results in viable, fertile mice (SurvivinCamcre) with reduced numbers of SVZ NPCs, absent rostral migratory stream, and olfactory bulb hypoplasia. The phenotype can be partially rescued, as intracerebroventricular gene delivery of survivin during embryonic development increases olfactory bulb neurogenesis, detected postnatally. SurvivinCamcre brains have fewer cortical inhibitory interneurons, contributing to enhanced sensitivity to seizures, and profound deficits in memory and learning.
The findings highlight the critical role that survivin plays during neural development, deficiencies of which dramatically impact on postnatal neural function.
... More recently cell selection methodologies have significantly improved. For example both immunopanning (Barres and Raff, 1993;Gard and Pfeiffer, 1993) and fluorescence-activated cell sorting (FACS) represent systems that quantify and separates cells from a mixed population, for example due to-fluorescence labeling of cell-surface-specific antigens (Behar et al., 1988;Pennartz et al., 2004). More recently, immunomagnetic cell separation using a specific antibody coupled to a magnetic particle has become one of the most commonly used . ...
The embryonic, neonatal, as well as adult rat spinal cords harbor a pool of neural stem cells (NSCs), which may be easily isolated and used to replace neuronal cell loss or remyelinate damaged axons following various neurodegenerative disorders. In the present study we have used magnetic cell sorting (MACs) technology to generate enriched oligodendroglial cell populations from the embryonic (E16) rat spinal cord. Target cells were separated by positive selection, using specific A2B5 antibody-labeled MicroBeads achieving optimal recovery and high purity of pro-oligodendroglial cells. Based on immunocytochemical analyses for oligodendroglial developmental markers (A2B5, NG2, RIP and MBP) we were able to characterize and quantify oligodendroglial progenitors (OPCs) and mature oligodendroglial cells in: (i) unseparated heterogeneous population of NSCs, or in (ii) antigen-antibody separated NSCs. Our results showed that MACs technology enable us to gain enriched OPCs from heterogeneous population of spinal NSCs, resulting in a 58-61% of mature oligodendrocytes content (MBP+, RIP+) in comparison to 6-12% of oligodendroglial cells acquired from unseparated population. In addition, the enriched OPCs could be cultured in vitro for several >8 passages, giving rise to a high number of newly formed spheres, as well as high expansion potential. These experiments indicate that MACs technology provide a feasible approach for experimental cell enrichment of desired oligodendroglial progeny, which may be used in future trials for cell-based therapies to treat spinal cord injury.
Background:
Predicting metastasis in melanoma patients is important for disease management and could help to identify those who might benefit from adjuvant treatment. The aim of this study was to investigate whether the tumor microenvironment-derived protein S100A8/A9 qualifies as prognostic marker for melanoma patients, also in the setting of immunotherapy.
Methods:
S100A8/A9 gene and protein expression were analyzed on melanocytic nevi, primary melanomas and metastases using a cDNA library and three independent tissue-microarrays (TMA). Serum levels of S100A8/A9 were measured using a specific ELISA in two independent cohorts of 354 stage III and stage IV melanoma patients as well as in two independent cohorts of patients treated with the PD-1 antibody pembrolizumab.
Results:
cDNA analysis revealed an upregulation of S100A8 and S100A9 gene expression in melanoma metastases compared to primary melanomas. Significantly higher numbers of infiltrating S100A8/A9 positive cells were found in tissue samples of metastasizing primary melanomas compared to non-metastasizing melanomas (P < .0001) and in melanomas of short-term survivors compared to long-term survivors (P < .0001). Serum S100A8/A9 levels > 5.5 mg/l were associated with impaired overall survival in two independent cohorts (both P < .0001). Importantly, patients with serum elevated S100A8/A9 treated with pembrolizumab showed significantly impaired survival compared to patients with lower S100A8/A9 levels (cohort 1: P = .0051; cohort 2: P < .0001).
Conclusions:
The tumor microenvironment-associated protein S100A8/A9 serves as a novel prognostic marker for metastasis and survival of metastatic melanoma patients and predicts response to immunotherapy with pembrolizumab. These data underscore the significance of tumor microenvironment-derived factors as suitable biomarkers for melanoma.
Starting from a zygote, an organism is made up of thousands, highly organized stem cells, progenitor cells and postmitotic cells which are generated in spatio-temporally coordinated proliferation and differentiation steps. The ongoing advancements in cell culture, isolation techniques, and molecular analyses have driven our basic understanding of different cell types and led to a broad classification of stem cells. This chapter outlines the most prominent techniques used for the characterization and classification of stem cells and provides an overview of many different stem cells, their function and their mRNA, miRNA and protein content.
In the postnatal forebrain regionalized neural stem cells along the ventricular walls produce olfactory bulb (OB) interneurons with varying neurotransmitter phenotypes and positions. To understand the molecular basis of this region-specific variability we analyzed gene expression in the postnatal dorsal and lateral lineages in mice of both sexes from stem cells to neurons. We show that both lineages maintain transcription factor signatures of their embryonic site of origin, the pallium and subpallium. However, additional factors, including Zic1 and Zic2, are postnatally expressed in the dorsal stem cell compartment and maintained in the lineage that generates calretinin-positive GABAergic neurons for the OB. Functionally, we show that Zic1 and Zic2 induce the generation of calretinin-positive neurons while suppressing dopaminergic fate in the postnatal dorsal lineage. We investigated the evolutionary conservation of the dopaminergic repressor function of Zic proteins and show that it is already present in C. elegans .
SIGNIFICANCE STATEMENT The vertebrate brain generates thousands of different neuron types. In this work we investigate the molecular mechanisms underlying this variability. Using a genomics approach we identify the transcription factor signatures of defined neural stem cells and neuron populations. Based thereon we show that two related transcription factors, Zic1 and Zic2, are essential to control the balance between two defined neuron types in the postnatal brain. We show that this mechanism is conserved in evolutionary very distant species.
Physiological studies indicate that the piriform or primary olfactory cortex of adult mammals exhibits a high degree of synaptic plasticity. Interestingly, a subpopulation of cells in the layer II of the adult piriform cortex expresses neurodevelopmental markers, such as the polysialylated form of neural cell adhesion molecule (PSA-NCAM) or doublecortin (DCX). The present study analyzes the nature, origin and potential function of these poorly understood cells in mice. As previously described in rats, most of the PSA-NCAM expressing cells in layer II could be morphologically classified as tangled cells and only a small proportion of larger cells could be considered semilunar-pyramidal transitional neurons. Most were also immunoreactive for DCX, confirming their immature nature. In agreement with this, detection of PSA-NCAM combined with that of different cell lineage-specific antigens revealed that most PSA-NCAM positive cells did not co-express markers of glial cells or mature neurons. Their time of origin was evaluated by birthdating experiments with halogenated nucleosides performed at different developmental stages and in adulthood. We found that virtually all cells in this paleocortical region, including PSA-NCAM positive cells, are born during fetal development. In addition, proliferation analyses in adult mice revealed that very few cells were cycling in layer II of the piriform cortex and that none of them was PSA-NCAM-positive. Moreover, we have established conditions to isolate and culture these immature neurons in the adult piriform cortex layer II. We find that, although they can survive under certain conditions, they do not proliferate in vitro either. This article is protected by copyright. All rights reserved.
The procedure of neurogenesis has made numerous achievements in the past decades, during which various molecular biomarkers have been emerging and have been broadly utilized for the investigation of embryonic and adult neural stem cell (NSC). Nevertheless, there is not a consistent and systematic illustration to depict the functional characteristics of the specific markers expressed in distinct cell types during the different stages of neurogenesis. Here we gathered and generalized a series of NSC biomarkers emerging during the procedures of embryonic and adult neural stem cell, which may be used to identify the subpopulation cells with distinguishing characters in different timeframes of neurogenesis. The identifications of cell patterns will provide applications to the detailed investigations of diverse developmental cell stages and the extents of cell differentiation, which will facilitate the tracing of cell time-course and fate determination of specific cell types and promote the further and literal discoveries of embryonic and adult neurogenesis. Meanwhile, via the utilization of comprehensive applications under the aiding of the systematic knowledge framework, researchers may broaden their insights into the derivation and establishment of novel technologies to analyze the more detailed process of embryogenesis and adult neurogenesis.
In the postnatal mammalian brain, the periventricular region lining the cerebral ventricles represents one of the main stem cells niches of the brain, generating new neurons for the olfactory bulb throughout life. Electroporation of this periventricular stem cell compartment allows the efficient and relatively simple manipulation of gene expression in vivo. This approach enables functional analysis of the molecular regulation of neurogenic processes at high resolution, thus permitting insight into the cell biology of neurogenesis in the living animal. Here, we present in detail the different steps of this approach from experimental procedure to sample analysis and discuss possible applications and perspectives.
Neural stem cells (NSCs) continually produce differentiated progeny within restricted areas of the adult mammalian brain. Not only do newly-generated neurons in the adult brain contribute to plasticity and remodeling of neuronal networks but also the neuronal network activity feeds back onto the NSCs and their progeny in order to modulate neurogenic output. A growing body of evidence indicates that neurotransmitters are fundamental niche signals that coordinate function of the neuronal network and the pace of adult neurogenesis. Among the neurotransmitters, gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain, modulates progression of multiple stages of the adult neurogenic lineage via activation of GABAA receptor ion channels. Recent data implicate metabotropic GABAB receptors, in addition to GABAA receptors, in the regulation of NSC proliferation and differentiation, adding a new level of complexity to the regulation of adult neurogenesis by GABA.
The oncogenic microRNA 17-92 cluster plays a causative role in the lymphomagenesis of nodal B-cell lymphomas, by activating proliferation and inhibiting apoptosis. Here we analyzed primary cutaneous B-cell lymphomas for the miR-17-92 cluster and its paralogs miR-106a-363 and miR-106b-25. In 22 primary cutaneous diffuse large B-cell lymphomas - leg type (PCLBCL-LT) compared to 22 primary cutaneous follicle center lymphomas (PCFCL), we found that miR-20a and miR-106a were overexpressed. Multivariate Cox analysis showed that higher miR-20a and miR-20b expression levels were associated with shorter disease-free and overall survival, independently from histological type. Gene expression profiling also showed a down-regulation of 8 candidate target genes of miR-20a, miR-20b and miR-106a in PCLBCL-LT compared to PCFCL. Among the candidate target genes, PTEN, NCOA3 and CAPRIN2 were confirmed to be underexpressed in PCLBCL-LT using quantitative RT-PCR on CD20-positive laser-microdissected tumor cells. In multivariate Cox analysis, lower PTEN mRNA expression level was associated with shorter disease-free survival, independently from the histological type. Altogether, this molecular and bioinformatics study of 44 patients skin biopsy samples showed that the oncogenic miR-17-92 cluster and its paralogs were involved in cutaneous B-cell lymphoma progression, and that the down-regulation of the target gene PTEN was associated with shorter disease-free survival.Journal of Investigative Dermatology accepted article preview online, 29 January 2015. doi:10.1038/jid.2015.27.
Starting from a zygote, an organism is made up of thousands, highly organised stem cells, progenitor cells and postmitotic cells which are generated in spatio-temporally coordinated proliferation and differentiation steps. The ongoing advancements in cell culture, isolation techniques, and molecular analyses has driven our basic understanding of different cell types and led to a broad classification of stem cells. This chapter outlines the most prominent techniques used for the characterization and classification of stem cells and provides an overview of many different stem cells, their function and their mRNA, miRNA and protein content.
Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.European Journal of Human Genetics advance online publication, 11 June 2014; doi:10.1038/ejhg.2014.81.
Cancer stem cells (CSCs) play crucial roles in tumor progression, chemo- and radiotherapy resistance, and recurrence. Recent studies on CSCs have advanced understanding of molecular oncology and development of novel therapeutic strategies. This review article updates the hypothesis and paradigm of CSCs with a focus on major signaling pathways and effectors that regulate CSC apoptosis. Selective CSC apoptotic inducers are introduced and their therapeutic potentials are discussed. These include synthetic and natural compounds, antibodies and recombinant proteins, and oligonucleotides.
Abstract In the current aging society, cognitive dysfunction is one of the most serious issues that should be urgently resolved. It also affects a wide range of age groups harboring neurological and psychiatric disorders, such as Alzheimer's disease and schizophrenia. Although the molecular mechanism of memory impairment still remains to be determined, neuronal loss and dysfunction has been revealed to mainly attribute to its pathology. The discovery of neural stem cells in the adult brain that are proliferating and able to generate functional neurons has given rise to the idea that neuronal loss could be rescued by manipulating endogenous neural progenitor and stem cells. To this end, we must characterize them in detail and their developmental programming must be better understood. A growing body of evidence has indicated that insulin-like peptides are involved in learning and memory and maintenance of neural progenitor and stem cells, and clinical trials of insulin as a memory enhancer have begun. In contrast to the expectation of insulin and IGF1, the roles of IGF2 in cognitive ability have been poorly understood. However, recent evidence demonstrated in rodents suggests that IGF2 may play a pivotal role in adult neurogenesis and cognitive function. Here, we would like to review the rapidly growing world of IGF2 in cognitive neuroscience and introduce the evidence that its deficit is indeed involved in the impairment of the hippocampal neurogenesis and cognitive dysfunction in the model mouse of 22q11.2 deletion syndrome, which deletes Dgcr8, a critical gene for microRNA processing.
Neurogenesis is the process by which cells divide, migrate, and subsequently differentiate into a neuronal phenotype. Significant rates of neurogenesis persist into adulthood in two brain regions, the subgranular zone of the dentate gyrus and the subventricular zone of the lateral ventricles. Cells of the subventricular zone divide and migrate via the rostral migratory stream to the olfactory bulb where they differentiate into granule and periglomerular cells. With the discovery of large-scale neurogenesis in the adult brain, there have been significant efforts to identify the mechanisms that control this process as well as the role of these cells in neuronal functioning. Although many questions remain unanswered, new insights appear daily about adult neurogenesis, regulatory mechanisms, and the fates of the progeny. In this review we highlight the main studies investigating factors that regulate neurogenesis in the subventricular zone, neuronal migration to the olfactory bulb, neuronal integration into the existing bulbar network and shortly discuss the functional meaning of this process.
Current prognostic clinical and morphological parameters are insufficient to accurately predict metastasis in individual melanoma patients. Several studies have described gene expression signatures to predict survival or metastasis of primary melanoma patients, however the reproducibility among these studies is disappointingly low.
We followed extended REMARK/Gould Rothberg criteria to identify gene sets predictive for metastasis in patients with primary cutaneous melanoma. For class comparison, gene expression data from 116 patients with clinical stage I/II (no metastasis) and 72 with III/IV primary melanoma (with metastasis) at time of first diagnosis were used. Significance analysis of microarrays identified the top 50 differentially expressed genes. In an independent data set from a second cohort of 28 primary melanoma patients, these genes were analyzed by multivariate Cox regression analysis and leave-one-out cross validation for association with development of metastatic disease. In a multivariate Cox regression analysis, expression of the genes Ena/vasodilator-stimulated phosphoprotein-like (EVL) and CD24 antigen gave the best predictive value (p = 0.001; p = 0.017, respectively). A multivariate Cox proportional hazards model revealed these genes as a potential independent predictor, which may possibly add (both p = 0.01) to the predictive value of the most important morphological indicator, Breslow depth.
Combination of molecular with morphological information may potentially enable an improved prediction of metastasis in primary melanoma patients. A strength of the gene expression set is the small number of genes, which should allow easy reevaluation in independent data sets and adequately designed clinical trials.
Starting from a zygote, an organism is made up of thousands, highly organised stem cells, progenitor cells and postmitotic
cells which are generated in spatio-temporally coordinated proliferation and differentiation steps. The ongoing advancements
in cell culture, isolation techniques, and molecular analyses has driven our basic understanding of different cell types and
led to a broad classification of stem cells. This chapter outlines the most prominent techniques used for the characterization
and classification of stem cells and provides an overview of many different stem cells, their function and their mRNA, miRNA
and protein content.
The mammalian adult central nervous system (CNS) has an inherent ability to generate neurons and glia. The dogma that the adult CNS remains incapable of regeneration came from the observation that most neurons and glia are post-mitotic cells. However, this idea is now widely challenged by the demonstration that indeed mitotic cells are detected in restricted areas of the adult CNS. Among these regions, the subventricular zone (SVZ) of the forebrain is the largest germinative zone of the adult brain. It is also one of the most characterized germinative area at the cellular and molecular level. While most studies highlight the neurogenic potential of the SVZ, we will review its gliogenic potential in normal and demyelinating conditions. © 2006 Springer Science-Business Media, Inc. All rights reserved.
Survivin is a structurally unique member of the inhibitor of apoptosis protein family that in addition to acting as a suppressor
of programmed cell death also plays a central role in cell division. Owing to its massive up-regulation in human tumors and
its involvement in cancer progression and treatment resistance, survivin is currently undergoing extensive investigation as
a promising target for new anticancer interventions. Several preclinical studies have demonstrated that down-regulation of
survivin expression or function, accomplished by means of various strategies (including the use of antisense oligonucleotides,
small interfering RNAs, ribozymes, dominant negative mutants and small molecule antagonists), reduced tumor growth potential,
increased the apoptotic rate, and sensitized tumor cells to chemotherapeutic drugs and ionizing radiation in different human
tumor preclinical models. Moreover, the first survivin inhibitors have already reached the clinic with some promise. However,
due to its documented role in some normal tissues, the possibility that survivin disruption could affect normal cell function,
mainly the hematopoietic and immune systems, cannot be excluded. In this context, a better understanding of the effects exerted
by survivin on normal versus malignant cells will be important for the design of optimal strategies to selectively disrupt
survivin in cancer.
KeywordsApoptosis-cell cycle-human cancers-inhibitors of apoptosis proteins-survivin
Fish, unlike mammals, can regenerate axons in the optic nerve following optic nerve injury. We hypothesized that using microarray analysis to compare gene expression in fish which had experienced optic nerve lesion to fish which had undergone a similar operation but without optic nerve injury would reveal genes specifically involved in responding to optic nerve injury (including repair), reducing detection of genes involved in the general stress and inflammatory responses. We discovered 120 genes were significantly (minimally two-fold with a P-value ≤ 0.05) differentially expressed (up or down) at one or more time point. Among these was ATF3, a member of the cAMP-response element binding protein family. Work by others has indicated that elevated cAMP could be important in axon regeneration. We investigated ATF3 expression further by qRT-PCR, in situ hybridization and immunohistochemistry and found ATF3 expression is significantly upregulated in the ganglion cell layer of the retina, the nerve fiber layer and the optic nerve of the injured eye. The upregulation in retina is detectable by qRT-PCR by 24 h after injury, at which time ATF-3 mRNA levels are 8-fold higher than in retinas from sham-operated fish. We conclude ATF3 may be an important mediator of optic nerve regeneration-promoting gene expression in fish, a role which merits further investigation.
Gamma-aminobutyric acid (GABA)ergic neurons are a diverse group of inhibitory neurons playing crucial roles in information processing. We analyzed the gene expression of regionally defined GABAergic neurons from the cortex, olfactory bulb, striatum, and cerebellum of glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mice. We introduce a generally applicable method for singularization of brain cells, flow cytometric enrichment, and global mRNA amplification for sensitive gene expression profiling. Systematic quantification elicited a high dynamic range of GABAergic cell numbers in different brain regions. Clustering of our gene expression results revealed major differences between hind and forebrain GABAergic neurons indicating that the development of GABAergic neurons depends on their regional location. While GABAergic neurons of the forebrain are characterized by three main groups of transcription factors of the Distal-less-family, the POU-family, and ETS/FOX family, specific members of the ZIC- and LHX-family of transcription factors appear to define hindbrain inhibitory neurons.
Cancer stem cell are considered to represent a population within the bulk tumor that share many similarities to normal stem cells as far as their capacities to self-renew, differentiate, proliferate and to reconstitute the entire tumor upon serial transplantation are concerned. Since cancer stem cells have been shown to be critical for maintaining tumor growth and have been implicated in treatment resistance and tumor progression, they constitute relevant targets for therapeutic intervention. Indeed, it has been postulated that eradication of cancer stem cells will be pivotal in order to achieve long-term relapse-free survival. However, one of the hallmarks of cancer stem cells is their high resistance to undergo cell death including apoptosis in response to environmental cues or cytotoxic stimuli. Since activation of apoptosis programs in tumor cells underlies the antitumor activity of most currently used cancer therapeutics, it will be critical to develop strategies to overcome the intrinsic resistance to apoptosis of cancer stem cells. Thus, a better understanding of the molecular mechanisms that are responsible for the ability of cancer stem cells to evade apoptosis will likely open new avenues to target this critical pool of cells within the tumor in order to develop more efficient treatment options for patients suffering from cancer.
Neural stem cells (NSCs) generate neurons throughout life in the hippocampal dentate gyrus (DG). How gene expression signatures differ among NSCs and immature neurons remains largely unknown. We isolated NSCs and their progeny in the adult DG using transgenic mice expressing a GFP reporter under the control of the Sox2 promoter (labeling NSCs) and transgenic mice expressing a DsRed reporter under the control of the doublecortin (DCX) promoter (labeling immature neurons). Transcriptome analyses revealed distinct gene expression profiles between NSCs and immature neurons. Among the genes that were expressed at significantly higher levels in DG NSCs than in immature neurons was the growth factor insulin-like growth factor 2 (IGF2). We show that IGF2 selectively controls proliferation of DG NSCs in vitro and in vivo through AKT-dependent signaling. Thus, by gene expression profiling of NSCs and their progeny, we have identified IGF2 as a novel regulator of adult neurogenesis.
Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system.
The isolation of neural stem cells (NSCs) has been hampered by the lack of valid cell-surface antigens on NSCs, and novel valuable markers have been proposed. Glycan (oligosaccharide chain) is a potential candidate as a marker to isolate NSCs, because the species and the combination order of saccharides in glycan generate remarkable structural diversity and specificity. At present, the expression of hundreds of glycoconjugates with glycans have been found in the NSCs; however, just a few glycan-epitopes have been identified as valuable cell-surface markers. This review focused on the isolation of NSC using glycoprotein, especially complex type N-glycans. The cell-surface N-glycan-mediated isolation of NSCs is therefore expected to provide a comprehensive understanding of the biologic characteristics of NSCs in the brain, and thereby help to develop novel strategies in the field of regenerative medicine.
Prenatal ethanol (ETOH) exposure can lead to fetal alcohol spectrum disorder (FASD). We previously showed that ETOH alters cell adhesion molecule gene expression and increases neurosphere size in fetal brain-derived neural stem cells (NSC). Here, our aim was to determine the effect of ETOH on the cell fate of NSC, premature glial-committed precursor cells (GCP), and premature neuron-committed progenitor cells (NCP).
NSC, GCP, and NCP were isolated from normal second-trimester fetal human brains (n = 3) by positive selection using magnetic microbeads labeled with antibodies to CD133 (NSC), A2B5 (GCP), or PSA-NCAM (NCP). As a result of the small percentage in each brain, NSC were cultured in mitogenic media for 72 hours to produce neurospheres. The neurospheres from NSC and primary isolates of GCP and NCP were used for all experiments. Equal numbers of the 3 cell types were treated either with mitogenic media or with differentiating media, each containing 0 or 100 mM ETOH, for 120 hours. Expression of Map2a, GFAP, and O4 was determined by immunoflourescence microscopy and western blot analysis. Fluorescence intensities were quantified using Metamorph software by Molecular Devices, and the bands of western blots were quantified using densitometry.
ETOH in mitogenic media promoted formation of neurospheres by NSC, GCP, and NCP. Under control conditions, GCP attached and differentiated, NSC and NCP formed neurospheres that were significantly smaller in size than those in ETOH. Under differentiating conditions, Map2a expression increased significantly in NSC and GCP and reduced significantly in NCP, and GFAP expression reduced significantly in GCP and NCP, and Gal-C expression reduced significantly in all 3 cell types in the presence of ETOH compared to controls.
This study shows that ETOH alters the cell fate of neuronal stem and progenitor cells. These alterations could contribute to the mechanism for the abnormal brain development in FASD.
After their generation and specification in periventricular regions, neuronal precursors maintain an immature and migratory state until their arrival in the respective target structures. Only here are terminal differentiation and synaptic integration induced. Although the molecular control of neuronal specification has started to be elucidated, little is known about the factors that control the latest maturation steps. We aimed at identifying factors that induce terminal differentiation during postnatal and adult neurogenesis, thereby focusing on the generation of periglomerular interneurons in the olfactory bulb. We isolated neuronal precursors and mature neurons from the periglomerular neuron lineage and analyzed their gene expression by microarray. We found that expression of the bHLH transcription factor NeuroD1 strikingly coincides with terminal differentiation. Using brain electroporation, we show that overexpression of NeuroD1 in the periventricular region in vivo leads to the rapid appearance of cells with morphological and molecular characteristics of mature neurons in the subventricular zone and rostral migratory stream. Conversely, shRNA-induced knockdown of NeuroD1 inhibits terminal neuronal differentiation. Thus, expression of a single transcription factor is sufficient to induce neuronal differentiation of neural progenitors in regions that normally do not show addition of new neurons. These results suggest a considerable potential of NeuroD1 for use in cell-therapeutic approaches in the nervous system.
Synaptogenesis is a decisive process for the development of the brain, its plasticity during adulthood and its regeneration after injury and disease. Despite tremendous progress during the last decades, it remains unclear, whether neurons can form synapses autonomously. In this review, I will summarize recent evidence that this is probably not the case and that distinct phases of synapse development depend on help from glial cells. The results supporting this view come from studies on the central and peripheral nervous system and on different experimental models including cultured cells as well as living flies, worms and mice. Our understanding of synapse-glia interactions in the developing, adult and diseased brain is likely to advance more rapidly as new experimental approaches to identify, visualize and manipulate glial cells in vivo become available.
Since the discovery of specific populations of cells with stem-like characteristics in human leukemias, phenotypically and/or functionally similar tumor-promoting cells have been identified in a variety of human cancers. By dint of the similarities to normal human stem cells in terms of self-renewal, differentiation, long life span, and proliferative capacity, these defined populations of cells within the bulk tumor are referred to as "cancer stem cells (CSCs)". The presence of CSCs has challenged the age-old dogma of carcinogenesis, which posits that all cells within a tissue retain the capacity to generate tumors. With respect to the frequency of CSCs, there is still a lack of consensus as in some recent models the notion that these cells constitute a very small proportion within the tumor has been challenged. Another issue that remains unresolved is the existence of a "global" marker, although reference has been made to the CD133(+), CD34(+)CD38(-), and CD44(+)CD24(-) populations as the functional stem-like cells in different cancers. Nevertheless, the identification of this sub-set within the bulk tumor and its contribution to chemotherapy resistance suggest that the CSCs could be the Achilles heel in terms of chemosensitization. Therefore, a paradigm is emerging that an effective therapeutic approach against cancers is to target this critical pool of cells that have the capacity to self-renew and proliferate as well as evade death signals. Here we provide a brief review of the literature vis a vis the various mechanisms of defective apoptotic signaling in CSCs with potential for therapeutic intervention.
It has been established that the adult mouse forebrain contains multipotential (neuronal/glial) progenitor cells that can be induced to proliferate in vitro when epidermal growth factor is provided. These cells are found within the subventricular zone of the lateral ventricles, together with other progenitor cell populations, whose requirements for proliferation remain undefined. Using basic fibroblast growth factor (bFGF), we have isolated multipotential progenitors from adult mouse striatum. These progenitors proliferate and can differentiate into cells displaying the antigenic properties of astrocytes, oligodendrocytes, and neurons. The neuron-like cells possess neuronal features, exhibit neuronal electrophysiological properties, and are immunoreactive for GABA, substance P, choline acetyl-transferase, and glutamate. Clonal analysis confirmed the multipotency of these bFGF-dependent cells. Most significantly, subcloning experiments demonstrated that they were capable of self-renewal, which led to a progressive increase in population size over serial passaging. These results demonstrate that bFGF is mitogenic for multipotential cells from adult mammalian forebrain that possess stem cell properties.
The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.
Functional properties of the neural cell adhesion molecule (NCAM) are strongly influenced by polysialylation. We used gene- targeting to generate mice lacking ST8SiaIV/PST-1, one of the polysialyltransferases responsible for addition of polysialic acid (PSA) to NCAM. Mice homozygous for the null mutation reveal normal development of gross anatomical features. In contrast to NCAM-deficient mice, olfactory precursor cells in the rostral migratory stream express PSA and follow their normal pathway. Furthermore, delamination of mossy fibers in the hippocampal CA3 region, as found in NCAM-deficient mice, does not occur in ST8SiaIV mutants. However, during postnatal development these animals show a decrease of PSA in most brain regions compared to wild-type animals. Loss of PSA in the presence of NCAM protein but in the absence of obvious histological changes al- lowed us to directly address the role of PSA in synaptic plasticity. Schaffer collateral-CA1 synapses, which express PSA in wild types, showed impaired long-term potentiation (LTP) and long- term depression (LTD) in adult mutants. This impairment was age-dependent, following the time course of developmental dis- appearance of PSA. Contrary to NCAM mutant mice, LTP in ST8SiaIV mutants was undisturbed at mossy fiber-CA3 syn- apses, which do not express PSA in wild-type mice. The results demonstrate an essential role for ST8SiaIV in synaptic plasticity in hippocampal CA1 synapses, whereas PSA produced by dif- ferent polysialyltransferase or polysialyltransferases at early stages of differentiation regulates migration of neural precursor cells and correct lamination of mossy fibers. We suggest that NCAM but not PSA is likely to be important for LTP in the hippocampal CA3 region.
The subventricular zone (SVZ) of the lateral ventricles, the largest remaining germinal zone of the adult mammalian brain, contains an extensive network of neuroblasts migrating rostrally to the olfactory bulb. Little is known about the endogenous proliferation signals for SVZ neural stem cells or guidance cues along the migration pathway. Here we show that the receptor tyrosine kinases EphB1−3 and EphA4 and their transmembrane ligands, ephrins-B2/3, are expressed by cells of the SVZ. Electron microscopy revealed ephrin-B ligands associated with SVZ astrocytes, which function as stem cells in this germinal zone. A three-day infusion of the ectodomain of either EphB2 or ephrin-B2 into the lateral ventricle disrupted migration of neuroblasts and increased cell proliferation. These results suggest that Eph/ephrin signaling is involved in the migration of neuroblasts in the adult SVZ and in either direct or indirect regulation of cell proliferation.
Little is known about the expression of the receptor for complement anaphylatoxin C3a (C3aR) in the central nervous system (CNS). In this study, we provide the first evidence that neurons are the predominant cell type expressing C3aR in the normal CNS. By using in situ hybridization (ISH) and immunohistochemistry, we found that C3aR is constitutively expressed at high levels in cortical and hippocampal neurons as well as in Purkinje cells. Moreover, we showed that primary culture of human astrocytes and microglia express the C3aR mRNA as assessed by RT-PCR. In situ hybridization performed on rat primary astrocytes confirmed the RT-PCR result demonstrating C3aR expression by astrocytes. In experimental allergic encephalitis (EAE), C3aR expression was elevated on microglia, infiltrating monocyte-macrophage cells and a few astrocytes, whereas neuronal expression remained unchanged during the course of the disease. These data demonstrate that the C3aR is expressed primarily by neurons in the normal CNS and that its neuronal expression is not dramatically upregulated under inflammation. This is in contrast to the increased neuronal expression of the C5aR in several inflammatory CNS conditions. The high constitutive expression of the C3aR by neurons suggests this receptor may play an important role in normal physiological conditions in the CNS. GLIA 26:201–211, 1999. © 1999 Wiley-Liss, Inc.
The transcription factor-encoding gene, Sox4, is expressed in a wide range of tissues and has been shown to be functionally involved in heart, B-cell and reproductive system development. Sox4 shows a high degree of sequence homology with another group C Sox gene, Sox11, which is predominantly expressed in the CNS. Since the expression of Sox4 in the CNS has not been described we have carried out such a study. Sox4 and Sox11 expression increased simultaneously in the same early differentiating cells of the developing CNS except in the external granule layer of the cerebellum where Sox11 expression preceded that of Sox4. As development proceeded, their expression always appeared to relate to the maturational stage of the cell population, with Sox11 expression more transient than Sox4, except in the spinal cord where the reverse was true. Sox4 knock-out mice have been shown to die of a heart defect half way through gestation with no observable CNS phenotype. Our more detailed analysis showed no abnormality in the spatial restriction of expression of Sox2, Sox11, Mash1, neurogenin1 or neurogenin2, although the level of expression of Sox11 and Mash1 appeared a little different from the wild-type, implying that Sox4 might indeed have a functional role in CNS development. However, since Sox4 and Sox11 expression is so similar, we propose that Sox11 might compensate for the loss of Sox4 function in the CNS such that the phenotype is extremely mild in the Sox4 null mutant.
The heterogeneity of neural gene expression and the spatially limited expression of many low-abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult. To generate amounts of nucleic acids sufficient for use in standard cloning strategies, we have devised a method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA. Whole cerebellar RNA was primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence 5' to a polythymidylate region. After second-strand cDNA synthesis, T7 RNA polymerase was used to generate amplified antisense RNA (aRNA). Up to 80-fold molar amplification has been achieved from nanogram quantities of cDNA. The amplified material is similar in size distribution to the parent cDNA and shows sequence heterogeneity as assessed by Southern and Northern blot analysis. Specific messages for moderate-abundance mRNAs for actin and guanine nucleotide-binding protein (G-protein) alpha subunits have been detected in the amplified material. By using in situ transcription to generate cDNA, sequences for cyclophilin have been detected in aRNA derived from single cerebellar tissue sections. cDNA derived from a single cerebellar Purkinje cell also has been amplified and yields material that hybridizes to cognate whole RNA and mRNA but not to Escherichia coli RNA.
The early development of the mammalian forebrain involves the massive proliferation of the ventricular zone cells lining the lateral ventricles. A remnant of this highly proliferative region persists into adult life, where it is known as the subependymal layer. We examined the proliferation kinetics and fates of the mitotically active cells in the subependyma of the adult mouse. The medial edge, the lateral edge, and the dorsolateral corner of the subependymal layer of the rostral portion of the lateral ventricle each contained mitotically active cells, but the dorsolateral region had the highest percentage of bromodeoxyuridine (BrdU)-labeled cells per unit area. Repeated injections of BrdU over 14 hr revealed a proliferation curve for the dorsolateral population with a growth fraction of 33%, indicating that 33% of the cells in this subependymal region make up the proliferating population. The total cell cycle time in this population was approximately 12.7 hr, with an S-phase of 4.2 hr. To examine the fate of these proliferating cells, we injected low concentrations of a replication-deficient, recombinant retrovirus directly into the lateral ventricles of adult mice for uptake by mitotically active subependymal cells. Regardless of the survival time postinjection (10 hr, 1 d, 2 d, or 8 d), the number of retrovirally labeled cells per clone remained the same (1 or 2 cells/clone). This suggests that one of the progeny from each cell division dies. Moreover, the clones remained confined to the subependyma and labeled cells were not seen in the surrounding brain tissue. Thus, while 33% of the dorsolateral subependymal cells continue to proliferate in adult life, the fate of the postmitotic progeny is death.
The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.
The characteristics of an organism are determined by the genes expressed within it. A method was developed, called serial
analysis of gene expression (SAGE), that allows the quantitative and simultaneous analysis of a large number of transcripts.
To demonstrate this strategy, short diagnostic sequence tags were isolated from pancreas, concatenated, and cloned. Manual
sequencing of 1000 tags revealed a gene expression pattern characteristic of pancreatic function. New pancreatic transcripts
corresponding to novel tags were identified. SAGE should provide a broadly applicable means for the quantitative cataloging
and comparison of expressed genes in a variety of normal, developmental, and disease states.
The expression patterns of four genes that are potential regulators of development were examined in the CNS of the embryonic day 12.5 mouse embryo. Three of the genes, Dlx-1, Dlx-2 (Tes-1), and Gbx-2, encode homeodomain-containing proteins, and one gene, Wnt-3, encodes a putative secreted differentiation factor. These genes are expressed in spatially restricted transverse and longitudinal domains in the embryonic neural tube, and are also differentially expressed within the wall of the neural tube. Dlx-1 and Dlx-2 are expressed in two separate regions of the forebrain in an identical pattern. The Gbx-2 gene is expressed in four domains, two of which share sharp boundaries with the domains of the Dlx genes. One boundary is in the basal telecephalon between deep and superficial strata of the medial ganglionic eminence; the other boundary is in the diencephalon at the zona limitans intrathalamica. The Wnt-3 gene is expressed in a dorsal longitudinal zone extending from the hindbrain into the diencephalon, where its expression terminates at the zona limitans intrathalamica. Reciprocal patterns of expression are found within the dorsal thalamus for the Gbx-2 and Wnt-3 genes. These findings are consistent with neuromeric theories of forebrain development, and based upon them we suggest a model for forebrain segmentation.
A number of vertebrate genes of the Dlx gene family have been cloned in mouse, frog, and zebrafish. These genes contain a homeobox related to that of Distalless, a gene expressed in the developing head and limbs of Drosophila embryos. We cloned and studied the expression of two members of this family, which we named Dlx5 and Dlx6, in human and mouse. The two human genes, DLX5 and DLX6, are closely linked in an inverted convergent configuration in a region of chromosome 7, at 7q22. Similarly, the two human genes DLX1 and DLX2 are closely linked in a convergent configuration at 2q32, near the HOXD (previously HOX4) locus. In situ hybridization experiments in mouse embryos revealed expression of Dlx5 and Dlx6 mRNA in restricted regions of ventral diencephalon and basal telencephalon, with a distribution very similar to that reported for Dlx1 and Dlx2 mRNA. A surprising feature of Dlx5 and Dlx6 is that they are also expressed in all skeletal structures of midgestation embryos after the first cartilage formation. The expression pattern of these genes, together with their chromosome localization, may provide useful cues for the study of congenital disorders in which there is a combination of craniofacial and limb defects.
During the development of the mammalian brain, neuronal precursors migrate to their final destination from their site of birth
in the ventricular and subventricular zones (VZ and SVZ, respectively). SVZ cells in the walls of the lateral ventricle continue
to proliferate in the brain of adult mice and can generate neurons in vitro, but their fate in vivo is unknown. Here SVZ cells
from adult mice that carry a neuronal-specific transgene were grafted into the brain of adult recipients. In addition, the
fate of endogenous SVZ cells was examined by microinjection of tritiated thymidine or a vital dye that labeled a discrete
population of SVZ cells. Grafted and endogenous SVZ cells in the lateral ventricle of adult mice migrate long distances and
differentiate into neurons in the olfactory bulb.
In the brain of adult mice, cells that divide in the subventricular zone of the lateral ventricle migrate up to 5 millimeters
to the olfactory bulb where they differentiate into neurons. These migrating cells were found to move as chains through a
well-defined pathway, the rostral migratory stream. Electron microscopic analysis of serial sections showed that these chains
contained only closely apposed, elongated neuroblasts connected by membrane specializations. A second cell type, which contained
glial fibrillary acidic protein, ensheathed the chains of migrating neuroblasts. Thus, during chain migration, neural precursors
moved associated with each other and were not guided by radial glial or axonal fibers.
Progression of the cell cycle and control of apoptosis (programmed cell death) are thought to be intimately linked processes, acting to preserve homeostasis and developmental morphogenesis. Although proteins that regulate apoptosis have been implicated in restraining cell-cycle entry and controlling ploidy (chromosome number), the effector molecules at the interface between cell proliferation and cell survival have remained elusive. Here we show that a new inhibitor of apoptosis (IAP) protein, survivin, is expressed in the G2/M phase of the cell cycle in a cycle-regulated manner. At the beginning of mitosis, survivin associates with microtubules of the mitotic spindle in a specific and saturable reaction that is regulated by microtubule dynamics. Disruption of survivin-microtubule interactions results in loss of survivin's anti-apoptosis function and increased caspase-3 activity, a mechanism involved in cell death, during mitosis. These results indicate that survivin may counteract a default induction of apoptosis in G2/M phase. The overexpression of survivin in cancer may overcome this apoptotic checkpoint and favour aberrant progression of transformed cells through mitosis.
Little is known about the expression of the receptor for complement anaphylatoxin C3a (C3aR) in the central nervous system (CNS). In this study, we provide the first evidence that neurons are the predominant cell type expressing C3aR in the normal CNS. By using in situ hybridization (ISH) and immunohistochemistry, we found that C3aR is constitutively expressed at high levels in cortical and hippocampal neurons as well as in Purkinje cells. Moreover, we showed that primary culture of human astrocytes and microglia express the C3aR mRNA as assessed by RT-PCR. In situ hybridization performed on rat primary astrocytes confirmed the RT-PCR result demonstrating C3aR expression by astrocytes. In experimental allergic encephalitis (EAE), C3aR expression was elevated on microglia, infiltrating monocyte-macrophage cells and a few astrocytes, whereas neuronal expression remained unchanged during the course of the disease. These data demonstrate that the C3aR is expressed primarily by neurons in the normal CNS and that its neuronal expression is not dramatically upregulated under inflammation. This is in contrast to the increased neuronal expression of the C5aR in several inflammatory CNS conditions. The high constitutive expression of the C3aR by neurons suggests this receptor may play an important role in normal physiological conditions in the CNS.
Although cell migration is crucial for neural development, molecular mechanisms guiding neuronal migration have remained unclear. Here we report that the secreted protein Slit repels neuronal precursors migrating from the anterior subventricular zone in the telencephalon to the olfactory bulb. Our results provide a direct demonstration of a molecular cue whose concentration gradient guides the direction of migrating neurons. They also support a common guidance mechanism for axon projection and neuronal migration and suggest that Slit may provide a molecular tool with potential therapeutic applications in controlling and directing cell migration.
Migration of neuronal precursor cells from the external germinal layer (EGL) to the internal granular layer (IGL) is a crucial process in the development of the mammalian cerebellar cortex. These cells make up the only precursor population known to migrate away from the surface of the brain. We studied the role of the chemokine stromal-derived factor 1 (SDF-1) in the cerebellar tissue of rats and knockout mice and found (i) that it functions as an attractive guidance cue for neuronal migration and (ii) that its secretion from non-neuronal meningeal tissue is important for controlling the migration of embryonic EGL cells
Previous studies from different laboratories have demonstrated that cigarette smoke (CS) harbours a strong oxidative stress potential, which broadly impacts exposed cells. Many of these studies have been devoted to identifying differentially expressed genes in exposed cells. Emerging DNA microarray techniques provide a sophisticated tool to characterize gene expression on a more comprehensive basis. Here, we report on kinetic studies performed to characterize gene expression profiles in Swiss 3T3 cells exposed to aqueous extracts of CS (‘smoke-bubbled phosphate-buffered saline’) up to 24 h through glass chips containing 513 different cDNA probes. The results obtained display a distinct expression pattern of up regulated and repressed genes, which was most evident after 4‐8 h of exposure. The CS-related stress response involves mainly antioxidant response genes coding for, e.g. haem oxygenase1 (HO-1), metallothionein 1/2 (MT1/2) and heat shock proteins (HSPs); genes coding for transcription factors, e.g. JunB and CAAT/enhancer binding protein (C/EBP); cell cycle-related genes, e.g. gadd34 and gadd45; and notably, genes described as mediators of an inflammatory/ immune-regulatory response, e.g. st2, kc and id3 .F rom a kinetic perspective, the stress response is characterized by the synchronized up regulation of antioxidant pathways, e.g. as reflected by the co-ordinated expression of ho-1 and ferritin. This expression pattern is obviously orchestrated by stress-responsive transcription factors, as exemplified by the early and strong expression of junB and c/ebp. Interestingly, among the 10 most up regulated genes are five which are known to counteract stress brought about by peroxynitrite. Altogether, these results demonstrate that CS induces a distinct signature of differential gene expression in exposed cells.
To obtain essential genes for neuronal development, we have performed a molecular indexing method using a human teratocarcinoma
cell line, NTera-2. We isolated a cDNA fragment, designated B18, as an upregulated gene during the neural differentiation.
From the complete cDNA sequence of B18 it was revealed that this cDNA was the human SOX11 gene. While a previous report has determined only a ∼ 2 kb of the SOX11 cDNA including the entire open reading frame, our full length cDNA was 8743 bp possessing a long 3′ untranslated region.
Human SOX11 cDNA was mapped to chromosome region 2p25.3 between markers AFMA070WC9 and WI-1412 by radiation hybrid mapping.
To gain a molecular understanding of tumor angiogenesis, we compared gene expression patterns of endothelial cells derived
from blood vessels of normal and malignant colorectal tissues. Of over 170 transcripts predominantly expressed in the endothelium,
79 were differentially expressed, including 46 that were specifically elevated in tumor-associated endothelium. Several of
these genes encode extracellular matrix proteins, but most are of unknown function. Most of these tumor endothelial markers
were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum
formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that
may have significant implications for the development of anti-angiogenic therapies.
Pfam is a large collection of protein multiple sequence alignments and profile hidden Markov models. Pfam is available on
the World Wide Web in the UK at http://www.sanger.ac.uk/Software/Pfam/, in Sweden at http://www.cgb.ki.se/Pfam/, in France at http://pfam.jouy.inra.fr/ and in the US at http://pfam.wustl.edu/. The latest version (6.6) of Pfam contains 3071 families, which match 69% of proteins in SWISS-PROT 39 and TrEMBL 14. Structural
data, where available, have been utilised to ensure that Pfam families correspond with structural domains, and to improve
domain-based annotation. Predictions of non-domain regions are now also included. In addition to secondary structure, Pfam
multiple sequence alignments now contain active site residue mark-up. New search tools, including taxonomy search and domain
query, greatly add to the functionality and usability of the Pfam resource.
Methyl tert -butyl ether (MTBE) is an octane-enhancer and oxygenate compound that was authorized as a gasoline additive by the U.S. Environmental Protection Agency (USEPA) in late 1979. MTBE has many chemical and physical properties that make it a desirable compound for these purposes. However, the aqueous solubility of MTBE, which is in the 50,000 ppm range, allows it to dissolve into groundwater where it is transported virtually without retardation. MTBE also is resistant to microbial degradation and does not air-strip from water efficiently. These characteristics have caused wells to become contaminated with MTBE that in the absence would not have become contaminated with hydrocarbons from gasoline releases. Research on innovative technologies to treat water contaminated with MTBE is underway. The final regulatory determination of allowable concentrations and whether or not future use of MTBE will be allowed has yet to be made.
The homeobox genes in the Dlx family are required for differentiation of basal forebrain neurons and craniofacial morphogenesis. Herein, we studied the expression of Dlx-1, Dlx-2, and Dlx-5 RNA and protein in the mouse forebrain from embryonic day 10.5 (E10.5) to E12.5. We provide evidence that Dlx-2 is expressed before Dlx-1, which is expressed before Dlx-5. We also demonstrate that these genes are expressed in the same cells, which may explain why single mutants of the Dlx genes have mild phenotypes. The DLX proteins are localized primarily to the nucleus, although DLX-5 also can be found in the cytoplasm. During development, the fraction of Dlx-positive cells increases in the ventricular zone. Analysis of the distribution of DLX-1 and DLX-2 in M-phase cells suggests that these proteins are distributed symmetrically to daughter cells during mitosis. We propose that DLX-negative cells in the ventricular zone are specified progressively to become DLX-2-expressing cells during neurogenesis; as these cells differentiate, they go on to express DLX-1, DLX-5, and DLX-6. This process appears to be largely the same in all regions of the forebrain that express the Dlx genes. In the basal telencephalon, these DLX-positive cells differentiate into projection neurons of the striatum and pallidum as well as interneurons, some of which migrate to the cerebral cortex and the olfactory bulb. J. Comp. Neurol. 414:217–237, 1999. © 1999 Wiley-Liss, Inc.
The properties and fate of the cells of the subependymal layer of the anterior lateral ventricle and its rostral extension into the olfactory bulb were examined. In one experiment, histological analysis was made of this structure in a large group of rats, ranging in age from newborn to adults. It was established that the ventricular subependymal layer and its rostral extension are present as proliferative and migratory matrices throughout the period studied, with relatively little reduction in size from birth to adulthood. In another, autoradiographic study, the proliferation and migration of cells of this system, and their destination and mode of differentiation, were studied in rats that were injected with thymidine-H3 at 30 days of age and killed at intervals ranging from 1 hour to 180 days. There was a declining gradient in cell proliferation in a caudorostral direction from a high level near the lateral ventricle to the absence of cell proliferation in the olfactory bulb. The labeled cells that were present in high proportion near the lateral ventricle in the rats killed 1–24 hours after injection had further multiplied and moved to the middle portion of the “rostral migratory stream” by the third day, and were located in the subependymal layer of the olfactory bulb by the sixth day after injection. By the twentieth day the labeled cells disappeared from the subependymal layer of the olfactory bulb and were distributed throughout the internal granular layer. The differentiated cells were tentatively identified as granular nerve cells and neuroglia cells. These results established that the major target structure of cell production in the subependymal layer of the lateral ventricle in young-adult rats is the olfactory bulb, with only moderate contribution made to the anterior neocortex and basal ganglia. It was postulated that the function of cell migration to the olfactory bulb is the renewal of its cell population.
l-Arginine is a substrate for the synthesis of proteins, nitric oxide (NO), creatine, urea, proline, glutamate, polyamines and agmatine. In the central nervous system (CNS), arginine is extracted from the blood and exchanged by cells through carriers called cationic amino acid transporters (CAT) and belonging to the so-called system y+. In order to better understand the arginine transport in the CNS, we studied in detail the regional distribution of the cells expressing the CAT2(B) transcript in the adult rat brain by non-radioisotopic in situ hybridization. We show that CAT2(B) is expressed in neurons and oligodendrocytes throughout the brain, but is not detected in astrocytes. The pattern of localization of CAT2(B) in the normal adult rat brain fits closely that of CRT1, a specific creatine transporter. Our study demonstrates that the in vivo expression of CAT2(B) differs from that reported in vitro, implying that local cellular interactions should be taken into account in studies of gene regulation of the CAT2(B) gene. Our work suggests that CAT2(B) may play a role in case of increased NO production as well as arginine or creatine deficiency in the brain.
The Meis and Pbx genes encode for homeodomain proteins of the TALE class and have been shown to act as co-factors for other homeodomain transcription factors (Mann and Affolter, 1998. Curr. Opin. Genet. Dev. 8, 423–429). We have studied the expression of these genes in the mouse telencephalon and found that Meis1 and Meis2 display region-specific patterns of expression from embryonic day (E)10.5 until birth, defining distinct subterritories in the developing telencephalon. The expression of the Meis genes and their proteins is highest in the subventricular zone (SVZ) and mantle regions of the ventral telencephalon. Compared to the Meis genes, Pbx genes show a broader expression within the telencephalon. However, as is the case in Drosophila (Rieckhof et al., 1997. Cell 91, 171–183; Kurrant et al., 1998. Development 125, 1037–1048; Pai et al., 1998. Genes Dev. 12, 435–446), nuclear localized PBX proteins were found to correlate highly with Meis expression. In addition, DLX proteins co-localize with nuclear PBX in distinct regions of the ventral telencephalon.
Microarray (gene chip) technology is giving biologists the ability to examine gene expression with an unprecedented scope and ease. A recently launched initiative promises to yield gene sequences valuable for using microarrays to study gene expression in the mouse brain, and new technologies have recently been developed that enhance the ability of neurobiologists to perform microarray-based experiments.
Golgi and electronmicroscopic methods were used to define the shapes and intercellular relationships of cells migrating from their sites of origin near the ventricular surface across the intermediate zone to the superficial neocortical layers of the parietooccipital region in the brains of 75- to 97-day monkey fetuses. After mitotic division in either ventricular or subventricular zones, the cells enter the intermediate zone and assume an elongated bipolar form oriented toward the cortical plate. The leading processes, 50 to 70 μ long, are irregular cytoplasmic cylinders containing prominent Golgi apparatus, mitochondria, microtubules, ribosomal rosettes, immature endoplasmic reticulum and occasional centrioles. They usually terminate in several attenuated expansions, the longest one oriented toward the cortical plate. The trailing processes are more slender, relatively uniform in caliber and display few organelles.
The possible co-existence of calretinin with other calcium binding proteins, parvalbumin and calbindin D28k, and with GABA, was studied in non-pyramidal cells of the rat dorsal hippocampal formation, using the mirror technique. The majority of the calretinin-containing neurons (83%) were found to be immunoreactive for GABA (79% in the dentate gyrus, 84% in the CA2–3, and 88% in the CA1 subfield). Most of the GABA-negative calretinin-immunoreactive neurons were located in the hilus of the dentate gyrus and in stratum lucidum of the CA3 subfield. Detailed analysis of the calretinin-immunoreactive cells of these subfields revealed that the two morphologically distinct types of calretinin neurons, i.e. the spiny and the spine-free cells, differ in their immunoreactivity for GABA. The overwhelming majority (92%) of the spine-free neurons were GABA-positive, whereas the immuno-reactivity of spiny cells was ambiguous. At the sensitivity threshold of the immunocytochemical techniques used in the present study, most of the spiny cells (89%) had to be considered as GABA-negative, although the staining intensity in their cell bodies was somewhat above background level. Colchicine treatment resulted in a degeneration of calretinin-immunoreactive neurons; therefore, its effect on the GABA content of spiny neurons could not be evaluated. Nevertheless, the observations suggest that calretinin-containing neurons are heterogeneous both morphologically and neurochemically.
Multipotential CNS stem cells receive and implement instructions governing differentiation to diverse neuronal and glial fates. Exploration of the mechanisms generating the many cell types of the brain depends crucially on markers identifying the stem cell state. We describe a gene whose expression distinguishes the stem cells from the more differentiated cells in the neural tube. This gene was named nestin because it is specifically expressed in neuroepithelial stem cells. The predicted amino acid sequence of the nestin gene product shows that nestin defines a distinct sixth class of intermediate filament protein. These observations extend a model in which transitions in intermediate filament gene expression reflect major steps in the pathway of neural differentiation.
We used retrovirus-mediated gene transfer to study the migration of clonally related cells in the developing chicken optic tectum. Clonal cohorts initially form radial arrays in the ventricular zone (approximately E5), but eventually divide into three separate migratory streams. In the first migration, a minor population of cells migrates tangentially along axon fascicles in medio-laterally directed files (approximately E6-E7); these eventually differentiate into multipolar efferent cells. After E7, the majority of cells in each clone migrate radially along fascicles of radial glia to form the tectal plate, wherein they differentiate into neurons and astrocytes. Around E9, a set of small cells leaves the radial arrays in superficial layers to form a second tangential migration; at least some of these differentiate into astrocytes. Thus, as the tectum develops, cells derived from a single multipotential precursor migrate along three separate pathways, follow separate guidance cues, and adopt distinct phenotypes.
Unilateral naris occlusion in rats on postnatal Day 1 results in dramatic decreases in the size of specific olfactory bulb cell populations when pups are examined 30 days later (Frazier and Brunjes: J. Comp. Neurol. 269:355–370, '88). The observed reductions must result from alterations in cell proliferation and/or survival, alternatives examined in the present study.
During early postnatal development, most cells destined for the bulb are produced in regions caudal to the structure and migrate to the bulb in the massive rostral migratory stream. The dynamics of the stream were examined in both normal rats and pups with a single naris closed on Day 1. 3H-thymidine injections were made on postnatal Days 2, 5, 10, 20, and 30. Groups of pups were killed 2 hours later to assess patterns of proliferation and 24 hours later to gauge initial stages of migration. A gradient of labeled cells was observed in the stream, with higher levels occurring at more caudal locations. The supply of cells to the bulb peaked on Day 5 and was still substantial as late as Day 30. The deprivation procedure did not affect patterns of cell labeling at any stage tested, indicating the procedure does not affect early cellular proliferation.
A third group of pups was examined 30 days after thymidine injection to assess both time of cell origin and survival rates. Dark granule cells and glia in the granule cell layer were produced at a consistent rate until Day 20 with cells added during the period evenly spread throughout the layer. Light granule and periglomerular cell production decreased dramatically after P5. Thirty days after injections on P2, fewer labeled dark granule cells and their associated glia were found in deprived bulbs, indicating that enhanced cell death plays a major role in the deprivation-induced decrease in cell number.
Three chicken Sox (SRY-like box) genes have been identified that show an interactive pattern of expression in the developing embryonic nervous system. cSox2 and cSox3 code for related proteins and both are predominantly expressed in the immature neural epithelium of the entire CNS of HH stage 10 to 34 embryos. cSox11 is related to cSox2 and cSox3 only by virtue of containing an SRY-like HMG-box sequence but shows extensive homology with Sox-4 at its C-terminus. cSox11 is expressed in the neural epithelium but is transiently upregulated in maturing neurons after they leave the neural epithelium. These patterns of expression suggest that Sox genes play a role in neural development and that the developmental programme from immature to mature neurons may involve switching of Sox gene expression. cSox11 also exhibits a lineage restricted pattern of expression in the peripheral nervous system.
We report the isolation of a cDNA encoding the mouse class A bHLH transcription factor ME2 and the analysis of its expression. ME2 is expressed in the cerebral cortex, Purkinje and granule cell layers of the cerebellum, olfactory neuroepithelium, pyramidal cells of hippocampal layers CA1-CA4, and in the granular cells of the dentate gyrus. The specific expression of ME2 during development and in the regions of neuronal plasticity in the adult brain suggest that ME2 may have a regulatory function in developmental processes as well as during neuronal plasticity.
Neural-cell adhesion molecules (N-CAMs) are members of the immunoglobulin superfamily mediating homo- and heterophilic cell-cell interactions. N-CAM exists in various isoforms which are generated by alternative splicing. During embryonic development, N-CAMs are expressed in derivatives of all three germ layers, whereas in the adult animal they are predominantly present in neural tissue. Processes like neurulation, axonal outgrowth, histogenesis of the retina and development of the olfactory system are correlated with the regulated expression of N-CAMs. We show here that N-CAM-deficient mice generated by gene targeting appear healthy and fertile, but adult mutants show a 10% reduction in overall brain weight and a 36% decline in size of the olfactory bulb. N-CAM deficiency coincides with almost total loss of protein-bound alpha-(2,8)-linked polysialic acid, a carbohydrate structure thought to be correlated with neural development and plasticity. The animals showed deficits in spatial learning when tested in the Morris water maze, whereas activity and motor abilities appeared normal.
N-CAM is abundantly expressed in the nervous system in the form of numerous structural variants with characteristic distribution patterns and functional properties. N-CAM-180, the variant having the largest cytoplasmic domain, is expressed by all neurons. The N-CAM-180-specific exon 18 has been deleted to generate homozygous mice unable to express this N-CAM form. The most conspicuous mutant phenotype was in the olfactory bulb, where granule cells were both reduced in number and disorganized. In addition, precursors of these cells were found to be accumulated at their origin in the subependymal zone at the lateral ventricle. Analysis of the mutant in this region suggests that the mutant phenotype involves a defect in cell migration, possibly through specific loss of the polysialylated form of N-CAM-180, which is expressed in the migration pathway. Subtle but distinct abnormalities also were observed in other regions of the brain.
The subventricular zone of the postnatal forebrain produces mainly glia, although it supports limited neurogenesis. To determine whether the subventricular zone is positionally specified, the phenotype and destination of the progeny of subventricular zone cells along the anterior-posterior axis of the lateral ventricles were analyzed. A retroviral lineage tracer containing the E. coli reporter gene lacZ was injected into different parts of the subventricular zone of neonatal rat pups, and at various times thereafter, the expression of beta-galactosidase was detected histochemically or immunohistochemically in the descendants of infected cells. A discrete region of the anterior part of the subventricular zone (SVZa) generated an immense number of neurons that differentiated into granule cells and periglomerular cells of the olfactory bulb-the two major types of interneurons. Thus, the SVZa appears to constitute a specialized source of neuronal progenitor cells. To reach the olfactory bulb, neurons arising in the SVZa migrate several millimeters along a highly restricted route. Guidance cues must be involved to prohibit widespread dispersion of these migrating neurons.
The MHC class II gene Aa was disrupted by targeted mutation in embryonic stem (ES) cells derived from C57BL/6 mice to prevent expression of MHC class
II molecules. Contrary to previous reports, the effect of the null-mutation on T cell development was investigated in C57BL/6
mice, which provide a defined genetic background. The complete lack of cell surface expression of MHC class II molecules in
B6-Aa0/Aa0 homozygous mutant mice was directly demonstrated by cytofiuorometric analysis using anti-Ab and anti-la specific mAbs. Development of CD4+CD8− T cells in the thymus was largely absent except for a small population
of thymocytes expressing high levels of CD4 together with low amounts of CD8. The majority of these cells express the TCR
at high density. Although mature CD4+CD8− T cells were undetectable in the thymus, some T cells with a CD4+CD8−TCRhigh phenotype were found in lymph nodes and spleen. Peripheral T cells from themutant mice can be polyclonally activated in vitro with the mitogen concanavalin A. However, they could not be stimulated with staphylococcal enterotoxin B in autologous lymphocyte
reactions, thereby demonstrating the absence of MHC class II expression in these mice. Peripheral B cells in B6-Aa0/Aa0 mutants were functional and responded to the T cell independent antigen levan by the production of antigenspecific IgM antibodies
similar to wild-type cells. The B6-Aa0/Aa0 mutant mice described in this study represent an important tool to investigate the involvement of MHC class II molecules
in lymphocyte maturation and the immune response.
Apoptosis is an evolutionarily conserved 'suicide' programme present in all metazoan cells. Despite its highly conserved nature, it is only recently that any of the molecular mechanisms underlying apoptosis have been identified. Several lines of reasoning indicate that apoptosis and cell proliferation coincide to some degree: many oncogenes that promote cell cycle progression also induce apoptosis; damage to the cell cycle or to DNA integrity is a potent trigger of apoptosis; and the key tumour suppressor proteins, p105rb and p53, exert direct effects both on cell viability and on cell cycle progression. There is less evidence, however, to indicate that apoptosis and the cell cycle share common molecular mechanisms. Moreover, the interleukin-1 beta converting enzyme (ICE) family of cysteine proteases is now known to play a key role in apoptosis but has no discernible role in the cell cycle, arguing that the two processes are discrete.
In the mammalian forebrain most neurons originate from proliferating cells in the ventricular zone lining the lateral ventricles. These neurons become postmitotic before they undergo migration to their final destinations. In this study we examined the proliferative and migratory properties of cells destined for the olfactory bulb that arise postnatally from progenitor cells situated at the anterior extent of the subventricular zone (SVZa). The SVZa-derived cells migrate along a stereotypical pathway to the olfactory bulb where they become interneurons. Using lineage tracers and the cell proliferation marker BrdU, we have demonstrated that SVZa-derived cells in the rat retain the capacity for division after migrating away from their initial site of generation. These cells also express a neuron-specific tubulin, recognized by the antibody TuJ1. These results suggest that, unlike other immature neurons, these SVZa-derived cells have made a commitment to become neurons before becoming postmitotic.
To gain insight into cellular and molecular mechanisms subserving neuronal cell migration in the adult mouse forebrain, we have first investigated the cellular composition of the subventricular zone-olfactory bulb pathway (SVZ-OB). The pathway was essentially composed of cells with neuronal and astrocytic identities, neuronal cells being four times more numerous than astrocytes. Neuronal cells (precursors and some young postmitotic neurons) formed continuous cellular strands of migratory cells from the anterior horn of the lateral ventricle to the olfactory bulb. These chains of migrating cells moved within channels formed by the processes of a special subpopulation of astrocytes. The neuronal cells expressed the embryonic form of polysialic acid neural cell adhesion molecule, and the astrocytes were tenascin-C positive, thus preserving an embryonic cellular environment.
A traumatic injury to the adult mammalian central nervous system (CNS) results in reactive astrogliosis and the migration of hematogenous cells into the damaged neural tissue. Chemokines, a novel class of chemoattractant cytokines, are now being recognized as mediators of the inflammatory changes that occur following injury. The expression of MCP-1 (macrophage chemotactic peptide-1), a member of the beta family of chemokines, has recently been demonstrated in trauma in the rat brain (Berman et al.: J Immunol 156:3017-3023, 1996). Using a stab wound model for mechanical injury, we studied the expression of two other beta chemokines: RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and MIP-1 beta (macrophage inflammatory protein-1 beta) in the rat brain. The stab wound injury was characterized by widespread gliosis and infiltration of hematogenous cells. Immunohistochemical staining revealed the presence of RANTES and MIP-1 beta in the injured brain. RANTES and MIP-1 beta were both diffusely expressed in the necrotic tissue and were detected as early as 1 day post-injury (dpi). Double-labeling studies showed that MIP-1 beta, but not RANTES, was expressed by reactive astrocytes near the lesion site. In addition, MIP-1 beta staining was also detected on macrophages at the site of injury. The initial expression of the chemokines closely correlated with the appearance of inflammatory cells in the injured CNS, suggesting that RANTES and MIP-1 beta may play a role in the inflammatory events of traumatic brain injury. This study also demonstrates for the first time MIP-1 beta expression in reactive astrocytes following trauma to the rat CNS.
We thank P. Golstein, R. Horvitz, A. Mudge, and R. Parnaik for helpful comments on the manuscript; J. Scholes for providing the drawings for Figure 2Figure 2; and J.-C. Ameisen, J. Burne, P. Golstein, R. Horvitz, K. Plasleitt, K. Roberts, and G. Stanfield for providing electron micrographs. Because of the limited number of references allowed, we were unable to cite many important papers; we apologize to their authors.
A comparative study was undertaken to correlate the immunohistochemical localization of polysialic acid (PSA) and the in situ localization of ST8Sia II mRNA. In situ hybridization of postnatal day 3 mouse brain showed high levels of ST8Sia II mRNA expression in the cerebral neocortex, striatum, hippocampus, subiculum, medial habenular nucleus, thalamus, pontine nuclei, and inferior colliculus; intermediate-level expression in the olfactory bulb, hypothalamus, superior colliculus, and cerebellum; and low-level expression in other regions. The distribution of ST8Sia II mRNA in the neocortex and cerebellum coincided with the immunohistochemical localization of PSA. During brain development, ST8Sia II mRNA started decreasing and had almost disappeared by postnatal day 14. Comparison between ST8Sia II and IV mRNA expression was also undertaken by northern blot analysis and competitive PCR analysis. During the late embryonic to early postnatal stages of the mouse CNS, the ST8Sia II mRNA showed abundant mRNA expression compared with the ST8Sia IV mRNA. Competitive PCR analysis of the adult mouse CNS showed weak expression of the two genes in the olfactory bulb, thalamus, hippocampus, and eyes. The regional and transient expression of ST8Sia II mRNA coincides with that of PSA, suggesting that ST8Sia II is closely involved in the biosynthesis and expression of PSA in the developing mouse CNS.
Genes differentially expressed in different tissues, during development, or during specific pathologies are of foremost interest to both basic and pharmaceutical research. "Transcript profiles" or "digital Northerns" are generated routinely by partially sequencing thousands of randomly selected clones from relevant cDNA libraries. Differentially expressed genes can then be detected from variations in the counts of their cognate sequence tags. Here we present the first systematic study on the influence of random fluctuations and sampling size on the reliability of this kind of data. We establish a rigorous significance test and demonstrate its use on publicly available transcript profiles. The theory links the threshold of selection of putatively regulated genes (e.g., the number of pharmaceutical leads) to the fraction of false positive clones one is willing to risk. Our results delineate more precisely and extend the limits within which digital Northern data can be used.
Knotted (Kn) genes are expressed within restricted domains of the plant meristems and play a key role in the control of plant morphogenesis. We have isolated the Kn-related gene Meis2 in mouse, which labels the lateral somitic compartment and its derivatives during early mouse embryogenesis and later becomes a marker for the dorso-ectodermal region overlying cells of the paraxial mesoderm. Meis2 is also highly expressed in specific areas of the developing central nervous system from embryonic day 9 (e9) onward. In later developmental stages, a strong expression is detectable in differentiating nuclei and regions of the forebrain, midbrain, hindbrain, and spinal cord. This temporal and spatial expression pattern suggests that Meis2 may play an important role in the cascade of induction leading to somitic differentiation as well as in brain regionalization and histogenesis.
Limited experimental access to the central nervous system (CNS) is a key
problem in the study of human neural development, disease, and regeneration.
We have addressed this problem by generating neural chimeras composed of human
and rodent cells. Fetal human brain cells implanted into the cerebral ventricles
of embryonic rats incorporate individually into all major compartments of
the brain, generating widespread CNS chimerism. The human cells differentiate
into neurons, astrocytes, and oligodendrocytes, which populate the host fore-,
mid-, and hindbrain. These chimeras provide a unique model to study human
neural cell migration and differentiation in a functional nervous system.
Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.
Newborn cerebral cortical neurons migrate along radial glia to the cortical plate. Experiments using a collagen gel assay revealed that the choroid plexus repelled cerebral cortical neurons and olfactory interneuron precursors, which were mimicked by Neuro-2A cells. Fractionation of Neuro-2A-conditioned medium identified a protein of 190 kDa, equivalent to full-length Slit proteins. Indeed, it cross-reacted with an antibody against Slit2, suggesting that it is either Slit2 or another Slit protein. Further, Slit2, expressed in COS cells, repelled cerebral cortical neurons and olfactory interneuron precursors. Thus, Slit2, which is expressed by the choroid plexus and the septum, acts as a chemorepulsive factor for neuronal migration. These results suggest chemorepulsion as a guidance mechanism for neuronal migration in the developing forebrain.