Developmental malformations in Huntington disease: neuropathologic evidence of focal neuronal migration defects in a subset of adult brains.

Neuropathologic hallmarks of Huntington Disease (HD) include the progressive neurodegeneration of the striatum and the presence of Huntingtin (HTT) aggregates that result from abnormal polyQ expansion of the HTT gene. Whether the pathogenic trinucleotide repeat expansion of the HTT gene causes neurodevelopmental abnormalities has garnered attention in both murine and human studies; however, documentation of discrete malformations in autopsy brains of HD individuals has yet to be described. We retrospectively searched the New York Brain Bank (discovery cohort) and an independent cohort (validation cohort) to determine whether developmental malformations are more frequently detected in HD versus non-HD brains and to document their neuropathologic features. One-hundred and thirty HD and 1600 non-HD whole brains were included in the discovery cohort and 720 HD and 1989 non-HD half brains were assessed in the validation cohort. Cases with developmental malformations were found at 6.4-8.2 times greater frequency in HD than in non-HD brains (discovery cohort: OR 8.68, 95% CI 3.48-21.63, P=4.8 × 10-5; validation cohort: OR 6.50, 95% CI 1.83-23.17, P=0.0050). Periventricular nodular heterotopias (PNH) were the most frequent malformations and contained HTT and p62 aggregates analogous to the cortex, whereas cortical malformations with immature neuronal populations did not harbor such inclusions. HD individuals with malformations had heterozygous HTT CAG expansions between 40 and 52 repeats, were more frequently women, and all were asymmetric and focal, aside from one midline hypothalamic hamartoma. Using two independent brain bank cohorts, this large neuropathologic series demonstrates an increased occurrence of developmental malformations in HD brains. Since pathogenic HTT gene expansion is associated with genomic instability, one possible explanation is that neuronal precursors are more susceptible to somatic mutation of genes involved in cortical migration. Our findings further support emerging evidence that pathogenic trinucleotide repeat expansions of the HTT gene may impact neurodevelopment.

Basic and clinical neuroscience applications of embryonic stem cells.

There have been recent dramatic advances in our understanding of the molecular mechanisms governing the elaboration of mature tissue-specific cellular subpopulations from embryonic stem (ES) cells. These investigations have generated a range of new biological and potential therapeutic reagents to allow us to dissect specific stages of mammalian development that were previously experimentally inaccessible. Ultimately, we will be able to reconstitute seminal signaling pathways to promote regeneration of the nervous system. Totipotent ES cells possess an unlimited proliferative capacity that make them attractive candidates for use in a series of innovative transplantation paradigms. Elucidation of the molecular and physiologic properties of ES cells also has important implications for our understanding of the integrative cellular processes underlying neural induction, patterning of the neural tube, neural lineage restriction and commitment, neuronal differentiation, regional neuronal subtype specification, and the specific pathological consequences of alterations in discrete components of these fundamental neurodevelopmental pathways. In addition, recent experimental observations suggest that neurodegenerative disease pathology may involve alterations in a range of progressive neural inductive and neurodevelopmental events through novel biological mechanisms that result in sublethal impairments in cellular homeostasis within evolving regional neuronal precursor populations containing the mutant proteins, culminating in increased vulnerability of their differentiated neuronal progeny to late-onset apoptosis. Future discoveries in ES cell research will offer unique conceptual and therapeutic perspectives that representing an alternative to neural stem cell therapeutic strategies for ameliorating the pathologic consequences of a broad range of genetic and acquired insults to the developing, adult, and aging brain. Evolving regenerative strategies for both neurodevelopmental and neurodegenerative diseases will likely involve the targeting of vulnerable regional neural precursor populations during "presymptomatic" clinicopathological stages prior to the occurrence of irrevocable neural cell injury and cell death.

Developmental mechanisms in the pathogenesis of neurodegenerative diseases.

Cellular genes that are mutated in neurodegenerative diseases code for proteins that are expressed throughout neural development. Genetic analysis suggests that these genes are essential for a broad range of normal neurodevelopmental processes. The proteins they code for interact with numerous other cellular proteins that are components of signaling pathways involved in patterning of the neural tube and in regional specification of neuronal subtypes. Further, pathogenetic mutations of these genes can cause progressive, sublethal alterations in the cellular homeostasis of evolving regional neuronal subpopulations, culminating in late-onset cell death. Therefore, as a consequence of the disease mutations, targeted cell populations may retain molecular traces of abnormal interactions with disease-associated proteins by exhibiting changes in a spectrum of normal cellular functions and enhanced vulnerability to a host of environmental stressors. These observations suggest that the normal functions of these disease-associated proteins are to ensure the fidelity and integration of developmental events associated with the progressive elaboration of neuronal subtypes as well as the maintenance of mature neuronal populations during adult life. The ability to identify alterations within vulnerable neuronal precursors present in pre-symptomatic individuals prior to the onset of irrevocable cellular injury may help foster the development of effective therapeutic interventions using evolving pharmacologic, gene and stem cell technologies.

Temporal expression of neuronal connexins during hippocampal ontogeny.

Communication through gap junction channels provides a major signaling mechanism during early brain histogenesis, a developmental time during which neural progenitor cells are inexcitable and do not express ligand-gated channel responses to the major CNS neurotransmitters. Expression of different gap junction types during neurogenesis may therefore define intercellular pathways for transmission of developmentally relevant molecules. To better understand the molecular mechanism(s) by which growth and differentiation of neurons are modulated by gap junction channels, we have been examining the developmental effects of a specific set of cytokines on differentiation and gap junction expression in a conditionally immortalized mouse embryonic hippocampal neuronal progenitor cell line (MK31). When multipotent MK31 cells are in an uncommitted state, they uniformly express the neuroepithelial intermediate filament class VI marker, nestin, are strongly coupled by gap junctions composed of connexin43 (Cx43) and express connexin45 (Cx45) at the mRNA level. As these cells undergo neuronal lineage commitment and exit from cell cycle, they begin to express the early neurofilament marker, NF66, and coupling strength and expression of Cx43 begin to decline with concurrent expression of other connexin proteins, including Cx26, Cx33, Cx36, Cx40 and Cx45. Terminal neuronal differentiation is heralded by the expression of more advanced neurofilament proteins, increased morphologic maturation, the elaboration of inward currents and action potentials that possess mature physiological properties, and changing profiles of expression of connexin subtypes, including upregulation of Cx36 expression. These important developmental transitions are regulated by a complex network of cell cycle checkpoints. To begin to examine the precise roles of gap junction proteins in traversing these developmental checkpoints and in thus regulating neurogenesis, we have focused on individual members of two classes of genes involved in these seminal events: ID (inhibitor of differentiation)-1 and GAS (growth arrest-specific gene)5. When MK31 cells were maintained in an uncommitted state, levels of ID-1 mRNA were high and GAS5 transcripts were essentially undetectable. Application of cytokines that promote neuronal lineage commitment and cell cycle exit resulted in down-regulation of ID-1 and upregulation of GAS5 transcripts, whereas additional cytokine paradigms that promoted terminal neuronal differentiation resulted in the delayed down-regulation of GAS5 mRNA. Stable MK31 transfectants were generated for ID-1 and GAS5. In basal conditions, cellular proliferation was enhanced in the ID-1 transfectants and inhibited in the GAS5 transfectants when compared with control MK31 cells. When cytokine-mediated neurogenesis was examined in these transfected cell lines, constitutive expression of ID-1 inhibited and constitutive expression of GAS5 enhanced initial and terminal stages of neuronal differentiation, with evidence that terminal neuronal maturation in both transfectant lines was associated with decreased cellular viability, possibly due to the presence of conflicting cell cycle-associated developmental signals. These experimental reagents will prove to be valuable experimental tools to help define the functional interrelationships between changing profiles of connexin protein expression and cell cycle regulation during neuronal ontogeny in the mammalian brain. The present review summarizes the current state of research involving the temporal expression of such connexin types in differentiating hippocampal neurons and speculates on the possible role of these intercellular channels in the development and plasticity of the nervous system. In addition, we describe the functional properties and expression pattern of the newly discovered neuronal-specific gap junctional protein, Cx36, in the developing mouse fetal hippocampus and in the rat retina and brain.

Brain dystrophin, neurogenetics and mental retardation.

Duchenne muscular dystrophy (DMD) and the allelic disorder Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders that are associated with a spectrum of genetically based developmental cognitive and behavioral disabilities. Seven promoters scattered throughout the huge DMD/BMD gene locus normally code for distinct isoforms of the gene product, dystrophin, that exhibit nervous system developmental, regional and cell-type specificity. Dystrophin is a complex plasmalemmal-cytoskeletal linker protein that possesses multiple functional domains, autosomal and X-linked homologs and associated binding proteins that form multiunit signaling complexes whose composition is unique to each cellular and developmental context. Through additional interactions with a variety of proteins of the extracellular matrix, plasma membrane, cytoskeleton and distinct intracellular compartments, brain dystrophin acquires the capability to participate in the modulatory actions of a large number of cellular signaling pathways. During neural development, dystrophin is expressed within the neural tube and selected areas of the embryonic and postnatal neuraxis, and may regulate distinct aspects of neurogenesis, neuronal migration and cellular differentiation. By contrast, in the mature brain, dystrophin is preferentially expressed by specific regional neuronal subpopulations within proximal somadendritic microdomains associated with synaptic terminal membranes. Increasing experimental evidence suggests that in adult life, dystrophin normally modulates synaptic terminal integrity, distinct forms of synaptic plasticity and regional cellular signal integration. At a systems level, dystrophin may regulate essential components of an integrated sensorimotor attentional network. Dystrophin deficiency in DMD/BMD patients and in the mdx mouse model appears to impair intracellular calcium homeostasis and to disrupt multiple protein-protein interactions that normally promote information transfer and signal integration from the extracellular environment to the nucleus within regulated microdomains. In DMD/BMD, the individual profiles of cognitive and behavioral deficits, mental retardation and other phenotypic variations appear to depend on complex profiles of transcriptional regulation associated with individual dystrophin mutations that result in the corresponding presence or absence of individual brain dystrophin isoforms that normally exhibit developmental, regional and cell-type-specific expression and functional regulation. This composite experimental model will allow fine-level mapping of cognitive-neurogenetic associations that encompass the interrelationships between molecular, cellular and systems levels of signal integration, and will further our understanding of complex gene-environmental interactions and the pathogenetic basis of developmental disorders associated with mental retardation.

Developmental changes in neural progenitor cell lineage commitment do not depend on epidermal growth factor receptor signaling.

Multipotent neural progenitor cells become progressively more biased towards a glial fate during development coincident with an increase in expression of the epidermal growth factor receptor (EGFR). To determine whether differences in lineage commitment of neural progenitor cells from different stages are causally related to expression of the EGFR and whether generation of glia is EGFR-dependent, we used an EGFR-specific tyrosine kinase inhibitor, PD158780, to block the activation of EGFR in progenitor cells. Treatment of cultured neonatal progenitor cells with PD158780 completely blocked EGF-induced proliferation of the cells but did not affect bFGF-induced proliferation. Nevertheless, treatment with the inhibitor failed to inhibit the generation of astroglia in the presence of either EGF or bFGF. Treatment with bone morphogenetic protein-2 (BMP2) enhanced astroglial differentiation and suppressed oligodendroglial (OL) differentiation. PD158780 treatment had no effect on the BMP2-induced astroglial differentiation or OL suppression. These observations suggest that the generation of astroglia is not dependent on EGFR activation. Because it was still possible that the progenitor cell responses reflected a prior history of EGFR signaling, rat forebrain cells were cultured in the presence of PD158780 from a time (E12.5) preceding expression of the EGFR. After time in culture, the E12.5 cells expressed EGFR by Western analysis both in the presence and in the absence of PD158780, but activation of EGFR kinase (receptor autophosphorylation) was undetectable in the presence of PD158780 and the cells did not proliferate in response to EGF. Nevertheless, astroglial differentiation was normal in PD158780-treated cells both in the absence and in the presence of BMPs or CNTF. Furthermore, the propensity towards glial differentiation increased with time in culture even in the absence of EGFR signaling. This suggests that the increased bias towards glial differentiation during development does not depend on EGFR signaling.

Developmental changes in progenitor cell responsiveness to bone morphogenetic proteins differentially modulate progressive CNS lineage fate.

Although multipotent progenitor cells capable of generating neurons, astrocytes and oligodendrocytes are present within the germinal zones of the brain throughout embryonic, postnatal and adult life, the different neural cell types are generated within discrete temporospatial developmental windows. This might suggest that multipotent progenitor cells encounter different signals during each developmental stage, thus accounting for separate waves of lineage commitment and cellular differentiation. This study demonstrates, however, that progenitor cell responses to the same class of signals, the bone morphogenetic proteins (BMPs), change during ontogeny, and that these same signals may thus initiate progenitor cell elaboration of several different lineages. BMPs promote cell death and inhibit the proliferation of early (embryonic day 13, E13) ventricular zone progenitor cells. At later embryonic (E16) stages of cerebral cortical development, BMPs exhibit a concentration-dependent dissociation of cellular actions, with either enhancement of neuronal and astroglial elaboration (at 1-10 ng/ml) or potentiation of cell death (at 100 ng/ml). Finally, during the period of perinatal cortical gliogenesis, BMPs enhance astroglial lineage elaboration. By contrast, oligodendroglial lineage elaboration is inhibited by the BMPs at all stages. Further, application of the BMP antagonist noggin to cultured progenitors promotes the generation of oligodendrocytes, indicating that endogenous BMP signaling can actively suppress oligodendrogliogenesis. These observations suggest that developmental changes in neural progenitor cell responsiveness to the BMPs may represent a novel mechanism for orchestrating context-specific cellular events such as lineage elaboration and cellular viability.

Mechanisms underlying neural cell death in neurodegenerative diseases: alterations of a developmentally-mediated cellular rheostat.

Genes associated with neurodegenerative diseases are normally expressed throughout neural development and are essential for the elaboration and maintenance of neuronal subpopulations. Disease-causing mutations can compromise defined subsets of these neural specification events in subtle ways that initially lead to impairments in the cellular homeostasis of evolving regional neuronal subpopulations, and adult-onset cell death from normally non-lethal environmental stressors. Neurodegenerative diseases may, therefore, represent an emerging class of developmental disorders characterized by novel biological responses to subthreshold neurodevelopmental abnormalities that impair targeted neuronal biosynthetic pathways without causing obvious developmental deficits. This developmental model of pathogenesis predicts that it will soon be possible to identify these dysfunctional pathways prior to the occurrence of irreversible cellular injury, and to successfully intervene using innovative neuroprotective and neural regenerative strategies.

Sonic hedgehog and BMP2 exert opposing actions on proliferation and differentiation of embryonic neural progenitor cells.

Although Sonic Hedgehog (Shh) plays a critical role in brain development, its actions on neural progenitor cell proliferation and differentiation have not been clearly defined. Transcripts for the putative Shh-receptor genes patched (Ptc) and smoothened (Smo) are expressed by embryonic, postnatal, and adult progenitor cells, suggesting that Shh can act directly on these cells. The recombinant human amino-terminal fragment of Shh protein (Shh-N) alone did not support the survival of cultured progenitor cells, but treatment with Shh-N in the presence of bFGF increased progenitor cell proliferation. Furthermore, treatment of embryonic rat progenitor cells propagated either in primary culture or after mitogen expansion significantly increased the proportions of both beta-tubulin- (neuronal marker) and O4- (oligodendroglial marker) immunoreactive cells and reduced the proportion of nestin- (uncommitted neural progenitor cell marker) immunoreactive cells. By contrast Shh-N had no effect on the elaboration of GFAP- (astroglial marker) immunoreactive cells. Cotreatment with Shh-N and bone morphogenetic protein-2 (BMP2) inhibited the anti-proliferative, astroglial-inductive, and oligodendroglial-suppressive effects of BMP2. Our observations suggest that Shh-N selectively promotes the elaboration of both neuronal and oligodendroglial lineage species and inhibits the effects of BMP2 on progenitor cell proliferation and astroglial differentiation.

Developmental changes in progenitor cell responsiveness to cytokines.

Multipotent progenitor cells have been identified within periventricular generative zones of the developing and adult brain. To determine whether the environmental responsiveness of these cells changes during development, progenitor cells were cultured from embryonic, postnatal, and adult rat brain in the presence of either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Embryonic cells cultured as intact progenitor neurospheres proliferated more robustly in response to bFGF than to EGF, whereas proliferation of postnatal and adult progenitor cells was enhanced more by EGF than bFGF. Progenitor cells generated in the presence of either bFGF or EGF had the capacity to generate neurons, astrocytes, and oligodendrocytes at all developmental stages. Most embryonic and neonatal bFGF-generated cells differentiated predominantly into neurons, whereas late stage embryonic and neonatal EGF-generated progenitors largely remained in an undifferentiated state. However, later postnatal and adult progenitor species, irrespective of whether they were generated in the presence of bFGF or EGF, gave rise preferentially to astrocytes. Treatment with bone morphogenetic protein (BMP)2 or BMP7 enhanced astroglial differentiation and suppressed oligodendroglial differentiation of both EGF- and bFGF-generated progenitor species, suggesting that the effects of the BMPs are not dependent on EGF receptor activation. Thus, while central nervous system (CNS) progenitor cells retain multipotent capacity and responsiveness to the BMPs throughout development, they exhibit significant changes in other cellular response properties, perhaps reflecting differences in the requirements for specific generative versus regenerative events.

Multiple roles of bone morphogenetic protein signaling in the regulation of cortical cell number and phenotype.

Members of the bone morphogenetic protein (BMP) family have been implicated in multiple aspects of neural development in both the CNS and peripheral nervous system. BMP ligands and receptors, as well as the BMP antagonist noggin, are expressed in the developing cerebral cortex, making the BMPs likely candidates for regulating cortical development. To define the role of these factors in the developing cerebral cortex, we examined the effects of BMP2 and BMP4 on cortical cells in vitro. Cells were cultured from embryonic day 13 (E13) and E16 rat cerebral cortex in the absence or presence of different concentrations of fibroblast growth factor 2, a known regulator of cortical cell proliferation and differentiation. At E13, the BMPs promoted cell death and inhibited proliferation of cortical ventricular zone cells, resulting in the generation of fewer neurons and no glia. At E16, the effects of the BMPs were more complex. Concentrations of BMP2 in the range of 1-10 ng/ml promoted neuronal and astroglial differentiation and inhibited oligodendroglial differentiation, whereas 100 ng/ml BMP2 promoted cell death and inhibited proliferation. Addition of the BMP antagonist noggin promoted oligodendrogliogenesis in vitro, demonstrating that endogenous BMP signaling influences the differentiation of cortical cells in vitro. The distribution of BMP2 and noggin within the developing cortex suggests that local concentrations of ligands and antagonists define gradients of BMP signaling during corticogenesis. Together, these results support the hypothesis that the BMPs and their antagonist noggin co-regulate cortical cell fate and morphogenesis.

Progenitor cell biology: implications for neural regeneration.

A few brief years ago, damage to the central nervous system was generally perceived to be irreparable, and loss of neurons was largely viewed as an irreversible process. However, major advances in the study of neural progenitor cells have altered these perceptions, and rational approaches to the repair of the damaged nervous system using transplanted progenitor cells now seem feasible. This review will discuss the basic biology of neural progenitor cells, the mechanisms regulating the generation of neurons and glia from these cells, and the techniques that are available for preparing such cells for transplantation into the nervous system. The potential uses for these cells in treating neurologic disease will then be reviewed, and the theoretical and technical problems that may be encountered will be discussed.

Postnatal cerebral cortical multipotent progenitors: regulatory mechanisms and potential role in the development of novel neural regenerative strategies.

In the developing postnatal cerebral cortex, protracted generation of glia and neurons occurs and precise matching of local cell types is needed for the functional organization of regional microdomains characteristic of complex CNS tissues. Recent studies have suggested that multipotent progenitors play an important role in neural lineage elaboration during neurogenesis and gliogenesis after migration from paramedian generative zones. The presence of a separate reservoir of cerebral cortical multipotent cells under strict local environmental regulation would provide an appropriate mechanism for terminal developmental sculpting and for reconstitution of regional cellular pools after injury. We have isolated distinct pools of EGF- and bFGF-responsive multipotent progenitors from the postnatal mammalian cerebral cortex independent of the subventricular zone. These progenitor populations are under tight environmental regulation by specific hierarchies of cytokine subclasses that program the progressive elaboration of intermediate lineage-restricted progenitors and differentiated type I and II astrocytes, myelinating oligodendrocytes and neuronal subtypes that express specific neuromodulatory proteins. Neural lineage development from these cortical multipotent progenitors is a graded developmental process involving sequential induction of specific cytokine receptors, acquisition of factor responsiveness and complex lineage interdependence. The cortical multipotent progenitor pathways program the elaboration of neural lineage species with distinct cellular response properties when compared with analogous species derived from subventricular zone progenitors, indicating that the cortical multipotent cells contribute to the establishment of lineage diversity within the developing cortical cortex. In addition, the cortical multipotent cells generate dynamic intermediate progenitor pools that utilize temporally-coded environmental cues to alter neural fate decisions. These cumulative observations suggest that postnatal cerebral cortical multipotent cells represent a novel set of progenitor pathways necessary for normal mammalian cortical maturation, and may have important implications for our understanding of a wide variety of neuropathological conditions and for the development of more effective regenerative strategies to combat these pervasive neurological disorders.

Differentiation of oligodendroglial progenitors derived from cortical multipotent cells requires extrinsic signals including activation of gp130/LIFbeta receptors.

We have previously isolated epidermal growth factor (EGF)-responsive multipotent progenitor cells from the early postnatal rodent cerebral cortex independent of generative zones. In this study we have examined the mechanisms regulating the generation of differentiated oligodendrocytes (OLs) from these multipotent cells. Although cultures of primary cortical OL progenitor cells propagated at clonal density spontaneously gave rise to differentiated OLs in defined medium, cultures of multipotent progenitors isolated from identical regions supported the elaboration of OL progenitors but not differentiated OLs. These observations indicate that the terminal maturation of OL progenitors derived from multipotent cells is dependent on signals present within the cellular environment. Application of cytokines such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), or neurotrophin 3 (NT3) to clonal density cultures of cortical multipotent progenitors increased the proportion of OL progenitors but failed to support the generation of differentiated OLs. By contrast, application of factors that activate gp130/leukemia inhibitory factor beta (LIFbeta) heterodimeric receptors, such as ciliary neurotrophic factor (CNTF), activated signal transducers and activators of transcription-3 in these OL progenitor cells and promoted the generation of differentiated OLs. Clonal analysis also demonstrated that CNTF directly targets OL progenitors derived from the multipotent cells. These observations suggest that two distinct progenitor cell pathways contribute to the generation of differentiated OLs during postnatal cortical gliogenesis. Although oligodendroglial maturation of classical OL progenitor cells is driven by cell autonomous mechanisms, our findings demonstrate that the generation of differentiated OLs from cortical multipotent progenitor cells is dependent on environmental cues, including activation of gp130/LIFbeta receptors.

Bone morphogenetic proteins induce apoptosis and growth factor dependence of cultured sympathoadrenal progenitor cells.

Neuron numbers in developing vertebrate organisms are regulated by the availability of growth factors which promote their survival. However, neuron survival may also be regulated by growth factors which promote rather than prevent cell death. This study examined the effects of bone morphogenetic proteins (BMPs) in inducing apoptosis of MAH cells, an immortalized sympathoadrenal progenitor cell line. Treatment of MAH cells with BMP2 or BMP4 killed the cells in a dose-dependent manner. By contrast, treatment with BMP7 or TGFbeta1 failed to affect survival, suggesting that induction of apoptosis is specific to the dpp subgroup of BMPs. Survival after treatment with BMP2 or BMP4 required addition of fibroblast growth factor (FGF) and nerve growth factor (NGF), indicating that BMP treatment made the neurons dependent upon an exogenous factor for survival. Several experimental observations suggested an apoptotic mechanism for BMP-induced death. After BMP2 treatment, the cells progressively shrank and became pyknotic. Further, there was prominent endonucleosomic cleavage of DNA (laddering) as well as TUNEL staining. Moreover, BMP-induced death was inhibited by the caspase inhibitor z-VAD and was partially prevented by the endonuclease inhibitor aurintricarboxylic acid. These observations suggest that neuron numbers may be regulated by factors which promote death and that exposure to such factors may be a signal for the development of dependence upon other growth factors for survival.

Development of bone morphogenetic protein receptors in the nervous system and possible roles in regulating trkC expression.

Characterization of bone morphogenetic protein receptor (BMPR) expression during development is necessary for understanding the role of these factors during neural maturation. In this study, in situ hybridization analyses demonstrate that BMP-specific type I (BMPR-IA and BMPR-IB) and type II (BMPR-II) receptor mRNAs are expressed at significant levels in multiple regions of the CNS, cranial ganglia, and peripheral sensory and autonomic ganglia during the embryonic and neonatal periods. All three BMP receptor subunits are expressed within periventricular generative zones. BMPR-IA is more abundant than the other receptor subtypes, with widespread expression in the brain, cranial ganglia, and peripheral ganglia. By contrast, BMPR-IB mRNA displays significant expression within more restricted regions, including the anterior olfactory nuclei. BMPR-II mRNA exhibits peak expression within the cerebellar Purkinje cell layer and the hippocampus, as well as within cranial ganglia. The distribution of BMP receptors within large neurons in adult dorsal root ganglia suggested a possible role in regulating expression of the neurotrophin receptor trkC. This hypothesis was tested in explant cultures of embryonic day 15 (E15) and postnatal day 1 (P1) sympathetic superior cervical ganglia (SCG). Treatment of the E15 or the P1 SCG with BMP-2 induced expression of trkC mRNA and responsiveness of sympathetic neurons to NT3 as measured by neurite outgrowth. The pattern of expression of BMP receptors in embryonic brain suggests several potentially novel areas for further developmental analysis and supports numerous recent studies that indicate that BMPs have a broad range of cellular functions during neural development and in adult life.

Changes in the properties of gap junctions during neuronal differentiation of hippocampal progenitor cells.

The cellular mechanisms that regulate progenitor cell lineage elaboration and maturation during embryonic development of the mammalian brain are poorly understood. Conditionally immortalized mouse hippocampal multipotent progenitor cells (MK31 cells) were found to be strongly coupled by gap junctions comprising connexin 43 (Cx43) during early neuronal ontogeny; the presence of this Cx type was confirmed by electrophysiological, molecular biological, and immunocytochemical assays. However, as progenitor cells underwent intermediate stages of neuronal differentiation under the influence of interleukin 7 (IL-7) alone or terminal differentiation after composite exposure to basic fibroblast growth factor, IL-7, and transforming growth factor alpha, coupling strength and the level of Cx43 expression declined. An additional population of junctional channels with distinct properties was detected at an intermediate stage of neuronal differentiation. Reverse transcription-PCR assays detected mRNA encoding Cx40 in IL-7-treated cells and Cx33 after both treatment conditions. Because functional channels in exogenous expression systems are not formed by pairing Cx40 with Cx43 or by pairing Cx33 with itself or additional connexins, these experimental observations raise the possibility that the progressive loss of coupling during differentiation of neural progenitor cells may involve downregulation of Cx43 coupled with potentiation of expression of Cx33 and Cx40. Furthermore, continued expression of Cx43 in differentiating neuroblasts could mediate intercellular communication between neuronal precursor cells and astrocytes by direct signaling via homotypic gap junction channels.

Isolation and developmental characterization of cerebral cortical multipotent progenitors.

Multipotent neural progenitor species present within developing and adult periventricular generative zones can give rise to all of the major cellular elements of the brain. Although lineage specification during development has been thought to be restricted to these generative zones, we have utilized quantitative immunoselection techniques to isolate an enriched population of multipotent neural progenitor cells that express polysialylated neural cell adhesion molecule (PSA-NCAM) from postnatal day 2 cerebral cortex independent of generative zones. This population of cerebral cortical progenitor cells exhibited robust proliferation in response to epidermal growth factor and subsequently gave rise to clonally derived neurons, astrocytes, and oligodendrocytes. Quantitative regional analysis further demonstrated that while the multipotent cells derived from the cerebral cortex uniformly expressed PSA-NCAM, multipotent cells derived from generative zones contained equal proportions of PSA-NCAM-positive and -negative multipotent progenitor cells. The generation of individual cellular lineages from cortical multipotent progenitors could be enhanced by specific cytokines that are expressed within the cerebral cortex. Further, while oligodendroglial progenitor cells derived from cortical multipotent progenitors exhibited responsiveness to platelet-derived growth factor (PDGF) and neurotrophin-3 (NT-3), primary cultures of cortical oligodendroglial progenitors were responsive to PDGF but not to NT-3. These observations suggest that in addition to glial progenitors that commit to a specific lineage prior to migration from generative zones, there is within the cerebral cortex a separate pool of multipotent cells that are capable of generating mature glial progeny in response to specific environmental cues. Therapeutic interventions aimed at differentiation of endogenous cerebral pools of multipotent progenitors may provide a novel strategy for amelioration of the sequelae of environmental and genetic insults to the postnatal cerebrum.

Generation and regulation of developing immortalized neural cell lines.

The genetic and environmental signals that regulate progressive lineage elaboration in the mammalian brain are poorly understood. In addition, characterization of the developmental profiles of early central nervous system (CNS) stem/ progenitor cells and analysis of the mechanisms involved in their clonal expansion, lineage restriction, and cellular maturation have been fragmentary and elusive. These seminal neurodevelopmental issues have been examined using a series of clonally derived neural stem/progenitor cell lines established by retroviral transduction of embryonic (E16.5-E17.5) murine hippocampal and cerebellar cells using temperature-sensitive alleles (A58/U19) of the simian virus (SV) 40 large tumor (T) antigen. Under conditions permissive for T-antigen expression (33 degrees C), single neural stem cells exhibited self-renewal, clonal expansion, and both symmetric and asymmetric modes of cell division. By contrast, at the nonpermissive temperature for T-antigen expression (39 degrees C), specific sets of cytokines potentiated the progressive elaboration of neuronal, oligodendroglial, and astroglial lineage species. These observations demonstrate that a spectrum of genetic and epigenetic signals and distinct cellular processes are involved in orchestrating the evolution of individual neural lineages from regional CNS stem/progenitor species. Further, the availability of conditionally immortalized neural cell lines that can be transplanted back into the mammalian brain may represent an important experimental resource for the detailed characterization of cellular and molecular mechanisms involved in the developmental sculpting, plasticity, and regeneration of the mammalian CNS.