Benjamin Deneen

Astrocytes and disease: a neurodevelopmental perspective

Astrocytes are no longer seen as a homogenous population of cells. In fact, recent studies indicate that astrocytes are morphologically and functionally diverse and play critical roles in neurodevelopmental diseases such as Rett syndrome and fragile X mental retardation. This review summarizes recent advances in astrocyte development, including the role of neural tube patterning in specification and developmental functions of astrocytes during synaptogenesis. We propose here that a precise understanding of astrocyte development is critical to defining heterogeneity and could lead advances in understanding and treating a variety of neuropsychiatric diseases.

The Transcription Factor NFIA Controls the Onset of Gliogenesis in the Developing Spinal Cord

The mechanisms controlling the transition from neurogenesis to gliogenesis in the vertebrate CNS are incompletely understood. We identified a family of transcription factors, called NFI genes, which are induced throughout the spinal cord ventricular zone (VZ) concomitantly with the induction of GLAST, an early marker of gliogenesis. NFIA is both necessary and sufficient for GLAST induction in the VZ. Unexpectedly, NFIA is also essential for the continued inhibition of neurogenesis in VZ progenitors. This function is mediated by the requirement of NFIA for the expression of HES5, a Notch effector. However, Notch effectors are unable to promote glial-fate specification in the absence of NFIA. Thus, NFIA links the abrogation of neurogenesis to a generic program of gliogenesis, in both astrocyte and oligodendrocyte VZ progenitors. At later stages, NFIA promotes migration and differentiation of astrocyte precursors, a function that is antagonized in oligodendrocyte precursors by Olig2.

Committed Neuronal Precursors Confer Astrocytic Potential on Residual Neural Precursor Cells

During midgestation, mammalian neural precursor cells (NPCs) differentiate only into neurons. Generation of astrocytes is prevented at this stage, because astrocyte-specific gene promoters are methylated. How the subsequent switch from suppression to expression of astrocytic genes occurs is unknown. We show in this study that Notch ligands are expressed on committed neuronal precursors and young neurons in mid-gestational telencephalon, and that neighboring Notch-activated NPCs acquire the potential to become astrocytes. Activation of the Notch signaling pathway in midgestational NPCs induces expression of the transcription factor nuclear factor I, which binds to astrocytic gene promoters, resulting in demethylation of astrocyte-specific genes. These findings provide a mechanistic explanation for why neurons come first: committed neuronal precursors and young neurons potentiate remaining NPCs to differentiate into the next cell lineage, astrocytes.

Identification of Positionally Distinct Astrocyte Subtypes whose Identities Are Specified by a Homeodomain Code

Astrocytes constitute the most abundant cell type in the central nervous system (CNS) and play diverse functional roles, but the ontogenetic origins of this phenotypic diversity are poorly understood. We have investigated whether positional identity, a fundamental organizing principle governing the generation of neuronal subtype diversity, is also relevant to astrocyte diversification. We identified three positionally distinct subtypes of white-matter astrocytes (WMA) in the spinal cord, which can be distinguished by the combinatorial expression of Reelin and Slit1. These astrocyte subtypes derive from progenitor domains expressing the homeodomain transcription factors Pax6 and Nkx6.1, respectively. Loss- and gain-of-function experiments indicate that the positional identity of these astrocyte subtypes is controlled by Pax6 and Nkx6.1 in a combinatorial manner. Thus, positional identity is an organizing principle underlying astrocyte, as well as neuronal, subtype diversification and is controlled by a homeodomain transcriptional code whose elements are reutilized following the specification of neuronal identity earlier in development.

Sox9 and NFIA Coordinate a Transcriptional Regulatory Cascade during the Initiation of Gliogenesis

Transcriptional cascades that operate over the course of lineage development are fundamental mechanisms that control cellular differentiation. In the developing central nervous system (CNS), these mechanisms are well characterized during neurogenesis, but remain poorly defined during neural stem cell commitment to the glial lineage. NFIA is a transcription factor that plays a crucial role in the onset of gliogenesis; we found that its induction is regulated by the transcription factor Sox9 and that this relationship mediates the initiation of gliogenesis. Subsequently, Sox9 and NFIA form a complex and coregulate a set of genes induced after glial initiation. Functional studies revealed that a subset of these genes, Apcdd1 and Mmd2, perform key migratory and metabolic roles during astro-gliogenesis, respectively. In sum, these studies delineate a transcriptional regulatory cascade that operates during the initiation of gliogenesis and identifies a unique set of genes that regulate key aspects of astro-glial precursor physiology during development.

Loss of p16 pathways stabilizes EWS/FLI1 expression and complements EWS/FLI1 mediated transformation.

Ewings sarcoma and primitive neuroectodermal tumors (ES/PNET) are characterized by the fusion of the N-terminus of the EWS gene to the C-terminus of a member of the ETS family of transcription factors. While such fusion proteins are thought to play dominant oncogenic roles, it is unlikely that a single genetic alteration by itself will support cellular transformation. Given that EWS/FLI1 is only able to transform immortalized 3T3 fibroblasts and that 30% of ES/PNET tumors contain a homozygous deletion of the p16 locus, it is likely that other genetic events are required for EWS/FLI1 oncogenesis. Here we describe a complementary mechanism utilized in the establishment ES/PNET tumors. EWS/FLI1 has the capacity to induce apoptosis and growth arrest in normal MEFs. Such effects prevent the establishment of stable expression of the protein in these cells. When expressed in p16, p19ARF, or p53 deficient MEFs, the apoptotic and growth arrest effects are attenuated, creating a environment permissive for stable expression of the protein. While loss of a single tumor suppressor is sufficient to establish expression of EWS/FLI1, cellular transformation requires further genetic perturbation.

Glial Development: The Crossroads of Regeneration and Repair in the CNS

Given the complexities of the mammalian CNS, its regeneration is viewed as the holy grail of regenerative medicine. Extraordinary efforts have been made to understand developmental neurogenesis, with the hopes of clinically applying this knowledge. CNS regeneration also involves glia, which comprises at least 50% of the cellular constituency of the brain and is involved in all forms of injury and disease response, recovery, and regeneration. Recent developmental studies have given us unprecedented insight into the processes that regulate the generation of CNS glia. Because restorative processes often parallel those found in development, we will peer through the lens of developmental gliogenesis to gain a clearer understanding of the processes that underlie glial regeneration under pathological conditions. Specifically, this review will focus on key signaling pathways that regulate astrocyte and oligodendrocyte development and describe how these mechanisms are reutilized in these populations during regeneration and repair after CNS injury.

PIM3 Proto-Oncogene Kinase Is a Common Transcriptional Target of Divergent EWS/ETS Oncoproteins

ABSTRACT Despite significant structural diversity, present evidence suggests that EWS/ETS fusion proteins promote oncogenesis by transcriptionally modulating a common set of target genes. In order to identify these genes, microarray expression analyses were performed on NIH 3T3 polyclonal populations expressing one of three EWS/ETS fusion genes. The majority of these genes can be grouped into seven functional categories, including cellular metabolism and signal transduction. The biologic significance of these target genes was pursued. The effects of modulating genes involved in metabolism were assessed by flux studies and demonstrated shifts in glucose utilization and lactate production as a result of EWS/FLI1 expression. The proto-oncogene coding for serine/threonine kinase PIM3 was found to one of several genes encoding signal transduction proteins that were up-regulated by EWS/ETS fusions. PIM3 was found to be expressed in a panel of human Ewings family tumor cell lines. Forced expression of PIM3 promoted anchorage-independent growth. Coexpression of a kinase-deficient PIM3 mutant attenuated EWS/FLI1-mediated NIH 3T3 tumorigenesis in immunodeficent mice.

Olig2 neuroepithelial motoneuron progenitors are not multipotent stem cells in vivo

Neurons and glia are thought to arise from multipotent and self-renewing stem cells, which comprise the majority of neuroepithelial cells in the ventricular zone (VZ) of the early embryonic CNS. However, this idea remains to be tested rigorously, because CNS stem cells have been identified only by using in vitro assays, from which their abundance in vivo cannot be directly inferred. In the hematopoietic system, stem cells are characterized by using prospective isolation and direct in vivo transplantation. Here we have used this approach to ask whether most VZ progenitors behave as stem cells in vivo. The best-studied region of the embryonic CNS for addressing this problem is, arguably, the ventral spinal cord, within which progenitors in the motoneuron progenitor (pMN) domain sequentially generate motoneurons (MNs) and oligodendrocyte precursors (OPs). Virtually all VZ cells in pMN express the transcription factor Olig2. If most of these cells are stem cells, then they should maintain neurogenic potential, even at later, gliogenic stages. To test this hypothesis, we have prospectively isolated Olig2+ cells from murine embryonic day (E)9.5 and E13.5 spinal cord and directly transplanted them to E2 chick spinal cord. Transplanted E9.5 cells generate both neurons, including MNs and OPs, whereas E13.5 cells generate. The observation that most Olig2+ progenitors do not maintain neurogenic potential into the period of gliogenesis argues that they do not self-renew. These results do not support the commonly held view that most neuroepithelial cells in the embryonic CNS VZ are stem cells in vivo.

Identification of diverse astrocyte populations and their malignant analogs

Astrocytes are the most abundant cell type in the brain, where they perform a wide array of functions, yet the nature of their cellular heterogeneity and how it oversees these diverse roles remains shrouded in mystery. Using an intersectional fluorescence-activated cell sorting–based strategy, we identified five distinct astrocyte subpopulations present across three brain regions that show extensive molecular diversity. Application of this molecular insight toward function revealed that these populations differentially support synaptogenesis between neurons. We identified correlative populations in mouse and human glioma and found that the emergence of specific subpopulations during tumor progression corresponded with the onset of seizures and tumor invasion. In sum, we have identified subpopulations of astrocytes in the adult brain and their correlates in glioma that are endowed with diverse cellular, molecular and functional properties. These populations selectively contribute to synaptogenesis and tumor pathophysiology, providing a blueprint for understanding diverse astrocyte contributions to neurological disease.