Biomedical subjects
Publications and source records attributed to Charles D Stiles.
In this issue of Neuron, Jackson et al. show that adult neural stem cells (B cells) express PDGF receptors. Functional analysis of PDGF signaling in these neural progenitors resonates in provocative ways with an older body of literature on PDGF autocrine loops in malignant glioma and with more recent observations on the bHLH transcription factor Olig2.
In the adult central nervous system, two distinct populations of glial cells expressing the chondroitin sulfate proteoglycan NG2 have been described: bipolar progenitor cells and more differentiated "synantocytes." These cells have diverse neurological functions, including critical roles in synaptic transmission, repair, and regeneration. Despite their potential importance, the genetic factors that regulate NG2 cell development are poorly understood, and the relationship of synantocytes to the oligodendroglial lineage, in particular, remains controversial. Here, we show that >90% of embryonic and adult NG2 cells express Olig2, a basic helix-loop-helix transcription factor required for oligodendrocyte lineage specification. Analysis of mice lacking Olig function demonstrates a failure of NG2 cell development at embryonic and perinatal stages that can be rescued by addition of a transgene containing the human OLIG2 locus. These findings show a general requirement for Olig function in NG2 cell development and highlight further roles for Olig transcription factors in neural progenitor cells.
Within the motoneuron precursor (pMN) domain of the developing spinal cord, the bHLH transcription factor, Olig2, plays critical roles in pattern formation and the generation of motor neuron and oligodendrocyte precursors. How are the multiple functions of Olig2 regulated? We have isolated a large BAC clone encompassing the human OLIG2 locus that rescues motor neuron and oligodendrocyte development but not normal pattern formation in Olig2(-/-) embryos. Within the BAC clone, we identified a conserved 3.6 kb enhancer sub-region that directs reporter expression specifically in the motor neuron lineage but not oligodendrocyte lineage in vivo. Our findings indicate complex regulation of Olig2 by stage- and lineage-specific regulatory elements. They further suggest that transcriptional regulation of Olig2 is involved in segregation of pMN neuroblasts.
Malignant gliomas are the most common form of primary brain tumors in adults. Despite advances in diagnosis and standard therapies such as surgery, radiation, and chemotherapy, the prognosis remains poor. Recent scientific advances have enhanced our understanding of the biology of gliomas and the role of tyrosine kinase receptors and signal transduction pathways in tumor initiation and maintenance, such as the epidermal growth factor receptors, platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and the Ras/Raf/mitogen-activated protein (MAP)-kinase and phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways. Novel targeted drugs such as small molecular inhibitors of these receptors and signaling pathways are showing some activity in initial studies. As we learn more about these drugs and how to optimize their use as single agents and in combination with radiation, chemotherapy, and other targeted molecular agents, they will likely play an increasing role in the management of this devastating disease. This review summarizes the current results with targeted molecular agents in malignant gliomas and strategies under evaluation to increase their effectiveness.
Pheochromocytomas are catecholamine-secreting tumors that result from mutations of at least six different genes as components of distinct autosomal dominant disorders. However, there remain familial occurrences of pheochromocytoma without a known genetic defect. We describe here a familial pheochromocytoma syndrome consistent with digenic inheritance identified through a combination of global genomics strategies. Multipoint parametric linkage analysis revealed identical LOD scores of 2.97 for chromosome 2cen and 16p13 loci. A two-locus parametric linkage analysis produced maximum LOD score of 5.16 under a double recessive multiplicative model, suggesting that both loci are required to develop the disease. Allele-specific loss of heterozygosity (LOH) was detected only at the chromosome 2 locus in all tumors from this family, consistent with a tumor suppressor gene. Four additional pheochromocytomas with a similar genetic pattern were identified through transcription profiling and helped refine the chromosome 2 locus. High-density LOH mapping with single nucleotide polymorphism-based array identified a total of 18 of 62 pheochromocytomas with LOH within the chromosome 2 region, which further narrowed down the locus to <2 cM. This finding provides evidence for two novel susceptibility loci for pheochromocytoma and adds a recessive digenic trait to the increasingly broad genetic heterogeneity of these tumors. Similarly, complex traits may also be involved in other familial cancer syndromes.
Pheochromocytomas are neural crest-derived tumors that arise from inherited or sporadic mutations in at least six independent genes. The proteins encoded by these multiple genes regulate distinct functions. We show here a functional link between tumors with VHL mutations and those with disruption of the genes encoding for succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD). A transcription profile of reduced oxidoreductase is detected in all three of these tumor types, together with an angiogenesis/hypoxia profile typical of VHL dysfunction. The oxidoreductase defect, not previously detected in VHL-null tumors, is explained by suppression of the SDHB protein, a component of mitochondrial complex II. The decrease in SDHB is also noted in tumors with SDHD mutations. Gain-of-function and loss-of-function analyses show that the link between hypoxia signals (via VHL) and mitochondrial signals (via SDH) is mediated by HIF1alpha. These findings explain the shared features of pheochromocytomas with VHL and SDH mutations and suggest an additional mechanism for increased HIF1alpha activity in tumors.
BACKGROUND: In eukaryotic cells, RNA-binding proteins (RBPs) contribute to gene expression by regulating the form, abundance, and stability of both coding and non-coding RNA. In the vertebrate brain, RBPs account for many distinctive features of RNA processing such as activity-dependent transcript localization and localized protein synthesis. Several RBPs with activities that are important for the proper function of adult brain have been identified, but how many RBPs exist and where these genes are expressed in the developing brain is uncharacterized. RESULTS: Here we describe a comprehensive catalogue of the unique RBPs encoded in the mouse genome and provide an online database of RBP expression in developing brain. We identified 380 putative RBPs in the mouse genome. Using in situ hybridization, we visualized the expression of 323 of these RBP genes in the brains of developing mice at embryonic day 13.5, when critical fate choice decisions are made and at P0, when major structural components of the adult brain are apparent. We demonstrate i) that 16 of the 323 RBPs examined show neural-specific expression at the stages we examined, and ii) that a far larger subset (221) shows regionally restricted expression in the brain. Of the regionally restricted RBPs, we describe one group that is preferentially expressed in the E13.5 ventricular areas and a second group that shows spatially restricted expression in post-mitotic regions of the embryonic brain. Additionally, we find a subset of RBPs that share the same complex pattern of expression, in proliferating regions of the embryonic and postnatal NS and peripheral tissues. CONCLUSION: Our data show that, in contrast to their proposed ubiquitous involvement in gene regulation, most RBPs are not uniformly expressed. Here we demonstrate the region-specific expression of RBPs in proliferating vs. post-mitotic brain regions as well as cell-type-specific RBP expression. We identify uncharacterized RBPs that exhibit neural-specific expression as well as novel RBPs that show expression in non-neural tissues. The data presented here and in an online database provide a visual filter for the functional analysis of individual RBPs.
Cortical progenitor cells from rat embryos give rise to neurons or glia following exposure to platelet derived growth factor (PDGF) or ciliary neurotrophic factor (CNTF), respectively. Both growth factors impart their developmental cues quickly through a transcription-dependent mechanism. Do the alternate developmental responses to PDGF and CNTF reflect induction of qualitatively distinct genes? Alternatively, do the same genes respond to each growth factor, but with quantitatively distinct kinetics? Using differential library screening and custom cDNA microarrays we show that a common set of genes responds to either growth factor. However, quantitative differences in the onset and duration of gene induction equate to the expression of factor-specific gene signatures. Multitissue cluster analysis also reveals tissue-specific gene signatures that may play important roles in the developing brain.
Malignant gliomas are the most common form of primary brain tumors in adults. Despite advances in diagnosis and standard therapies such as surgery, radiation, and chemotherapy, the prognosis remains poor. Recent scientific advances have enhanced our understanding of the biology of gliomas and the role of tyrosine kinase receptors and signal transduction pathways in tumor initiation and maintenance, such as the epidermal growth factor receptors, platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and the Ras/Raf/mitogen-activated protein (MAP)-kinase and phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways. Novel targeted drugs such as small molecular inhibitors of these receptors and signaling pathways are showing some activity in initial studies. As we learn more about these drugs and how to optimize their use as single agents and in combination with radiation, chemotherapy, and other targeted molecular agents, they will likely play an increasing role in the management of this devastating disease. This review summarizes the current results with targeted molecular agents in malignant gliomas and strategies under evaluation to increase their effectiveness.
In the developing brain, transcription factors (TFs) direct the formation of a diverse array of neurons and glia. We identifed 1445 putative TFs in the mouse genome. We used in situ hybridization to map the expression of over 1000 of these TFs and TF-coregulator genes in the brains of developing mice. We found that 349 of these genes showed restricted expression patterns that were adequate to describe the anatomical organization of the brain. We provide a comprehensive inventory of murine TFs and their expression patterns in a searchable brain atlas database.
Olig1 and Olig2 are closely related basic helix-loop-helix (bHLH) transcription factors that are expressed in myelinating oligodendrocytes and their progenitor cells in the developing central nervous system (CNS). Olig2 is necessary for the specification of oligodendrocytes, but the biological functions of Olig1 during oligodendrocyte lineage development are poorly understood. We show here that Olig1 function in mice is required not to develop the brain but to repair it. Specifically, we demonstrate a genetic requirement for Olig1 in repairing the types of lesions that occur in patients with multiple sclerosis.
Astrocytomas, oligodendrogliomas, and oligoastrocytomas, collectively referred to as diffuse gliomas, are the most common primary brain tumors. These tumors are classified by histologic similarity to differentiated astrocytes and oligodendrocytes, but this approach has major limitations in guiding modern treatment and research. Lineage markers represent a potentially useful adjunct to morphologic classification. The murine bHLH transcription factors Olig1 and Olig2 are expressed in neural progenitors and oligodendroglia and are essential for oligodendrocyte development. High OLIG expression alone has been proposed to distinguish oligodendrogliomas from astrocytomas, so we critically evaluated OLIG2 as a marker by immunohistochemical and oligonucleotide microarray analysis. OLIG2 protein is faithfully restricted to normal oligodendroglia and their progenitors in human brain. Immunohistochemical analysis of 180 primary, metastatic, and non-neural human tumors shows OLIG2 is highly expressed in all diffuse gliomas. Immunohistochemistry and microarray analyses demonstrate higher OLIG2 in anaplastic oligodendrogliomas versus glioblastomas, which are heterogeneous with respect to OLIG2 levels. OLIG2 protein expression is present but inconsistent and generally lower in most other brain tumors and is absent in non-neuroectodermal tumors. Overall, OLIG2 is a useful marker of diffuse gliomas as a class. However, expression heterogeneity of OLIG2 in astrocytomas precludes immunohistochemical classification of individual gliomas by OLIG2 alone.
We have performed a screen combining subtractive hybridization with PCR to isolate genes that are regulated when neuroepithelial (NE) cells differentiate into neurons. From this screen, we have isolated a number of known genes that have not previously been associated with neurogenesis, together with several novel genes. Here we report that one of these genes, encoding a guanine nucleotide exchange factor (GEF), is regulated during the differentiation of distinct neuronal populations. We have cloned both rat and mouse GEF genes and shown that they are orthologs of the human gene, MR-GEF, which encodes a GEF that specifically activates the small GTPase, Rap1. We have therefore named the rat gene rat mr-gef (rmr-gef) and the mouse gene mouse mr-gef (mmr-gef). Here, we will collectively refer to these two rodent genes as mr-gef. Expression studies show that mr-gef is expressed by young neurons of the developing rodent CNS but not by progenitor cells in the ventricular zone (VZ). The expression pattern of mr-gef during early telencephalic neurogenesis is strikingly similar to that of GABA and the LIM homeobox gene Lhx6, a transcription factor expressed by GABAergic interneurons generated in the ventral telencephalon, some of which migrate into the cortex during development. These observations suggest that mr-gef encodes a protein that is part of a signaling pathway involved in telencephalic neurogenesis; particularly in the development of GABAergic interneurons.
In a widely held view of vertebrate CNS development, neurons, astrocytes, and oligodendrocytes arise from a common tripotent progenitor cell. However, tripotent progenitors have never been detected in developing embryos. In this issue of Neuron, Gabay et al. show that tripotent progenitors can be created in vitro by deregulation of normal dorsoventral positional cues.
In developing neural tube, the basic helix-loop-helix (bHLH) transcription factor Olig2 interacts with the homeodomain transcription factor Nkx2.2 at two distinct stages. During neuronogenesis, a cross-repressive interaction appears to establish the precise boundary between the p3 and pMN domains. At later times, a cooperative interaction is noted because Nkx2.2 promotes maturation of oligodendrocyte progenitor cells specified by expression of Olig2. We show here that the Olig2 protein can form a physical complex with Nkx2.2 protein in mammalian cells and yeast two-hybrid trap assay. This interaction is specific because Olig2 does not bind to a biologically irrelevant homeodomain transcription factor (Nkx6.1), and Nkx2.2 does not interact with a biologically irrelevant bHLH protein (NeuroD). Deletion mapping analysis suggests that formation of an Olig2-Nkx2.2 physical complex is insufficient for the induction of oligodendrocyte progenitors in developing spine; however, the protein-protein interaction observed might be important for the cross-repressive interaction between Olig2 and Nkx2.2 that helps to establish the pMN-p3 boundary in the developing spinal cord.
Transcription factors with bHLH motifs modulate critical events in the development of the mammalian neocortex. Multipotent cortical progenitors are maintained in a proliferative state by bHLH factors from the Id and Hes families. The transition from proliferation to neurogenesis involves a coordinate increase in the activity of proneural bHLH factors (Mash1, Neurogenin1, and Neurogenin2) and a decrease in the activity of Hes and Id factors. As development proceeds, inhibition of proneural bHLH factors in cortical progenitors promotes the formation of astrocytes. Finally, the formation of oligodendrocytes is triggered by an increase in the activity of bHLH factors Olig1 and Olig2 that may be coupled with a decrease in Id activity. Thus, bHLH factors have key roles in corticogenesis, affecting the timing of differentiation and the specification of cell fate.
Target-derived neurotrophins signal from nerve endings to the cell body to influence cellular and nuclear responses. The retrograde signal is conveyed by neurotrophin receptors (Trks) themselves. To accomplish this, activated Trks may physically relocalize from nerve endings to the cell bodies. However, alternative signaling mechanisms may also be used. To identify the vehicle wherein the activated Trks are located and transported, and to identify associated motor proteins that would facilitate transport, we use activation-state specific antibodies in concert with immunoelectron microscopy and deconvolution microscopy. We show that the'activated Trks within rat sciatic nerve axons are preferentially localized to coated and uncoated vesicles. These vesicles are moving in a retrograde direction and so accumulate distal to a ligation site. The P-Trk containing vesicles, in turn, colocalize with dynein components, and not with kinesins. Collectively, these results indicate activated Trk within axons travel in vesicles and dynein is the motor that drives these vesicles towards the cell bodies.
The oligodendrocyte lineage genes Olig1 and Olig2 encode related bHLH proteins that are coexpressed in neural progenitors. Targeted disruption of these two genes sheds light on the ontogeny of oligodendroglia and genetic requirements for their development from multipotent CNS progenitors. Olig2 is required for oligodendrocyte and motor neuron specification in the spinal cord. Olig1 has roles in development and maturation of oligodendrocytes, evident especially within the brain. Both Olig genes contribute to neural pattern formation. Neither Olig gene is required for astrocytes. These findings, together with fate mapping analysis of Olig-expressing cells, indicate that oligodendrocytes are derived from Olig-specified progenitors that give rise also to neurons.