A transgenic mouse model of neuroepithelial cell specific inducible overexpression of dopamine D1-receptor.

Dopamine and its receptors appear in the brain during early embryonic period suggesting a role for dopamine in brain development. In fact, dopamine receptor imbalance resulting from impaired physiological balance between D1- and D2-receptor activities can perturb brain development and lead to persisting changes in brain structure and function. Dopamine receptor imbalance can be produced experimentally using pharmacological or genetic methods. Pharmacological methods tend to activate or antagonize the receptors in all cell types. In the traditional gene knockout models the receptor imbalance occurs during development and also at maturity. Therefore, assaying the effects of dopamine imbalance on specific cell types (e.g. precursor versus postmitotic cells) or at specific periods of brain development (e.g. pre- or postnatal periods) is not feasible in these models. We describe a novel transgenic mouse model based on the tetracycline dependent inducible gene expression system in which dopamine D1-receptor transgene expression is induced selectively in neuroepithelial cells of the embryonic brain at experimenter-chosen intervals of brain development. In this model, doxycycline-induced expression of the transgene causes significant overexpression of the D1-receptor and significant reductions in the incorporation of the S-phase marker bromodeoxyuridine into neuroepithelial cells of the basal and dorsal telencephalon indicating marked effects on telencephalic neurogenesis. The D1-receptor overexpression occurs at higher levels in the medial ganglionic eminence (MGE) than the lateral ganglionic eminence (LGE) or cerebral wall (CW). Moreover, although the transgene is induced selectively in the neuroepithelium, D1-receptor protein overexpression appears to persist in postmitotic cells. The mouse model can be modified for neuroepithelial cell-specific inducible expression of other transgenes or induction of the D1-receptor transgene in other cells in specific brain regions by crossbreeding the mice with transgenic mouse lines available already.

Histogenetic processes leading to the laminated neocortex: migration is only a part of the story.

The principal events of neocortical histogenesis were anticipated by work published prior to the 20th century. These were neuronal proliferation and migration and the complex events of cortical pattern formation leading to a laminated architecture where each layer is dominated by a principal neuronal class. Work that has followed has extended the knowledge of the workings of the proliferative epithelium, cellular mechanisms of migration and events through which cells are winnowed and then differentiate once their postmigratory positions are established. Work yet ahead will emphasize mechanisms that coordinate the molecular events that integrate proliferation and cell class specification in relation to the final neocortical neural system map.

Overexpression of p27 Kip 1, probability of cell cycle exit, and laminar destination of neocortical neurons.

Neocortical projection neurons arise from a pseudostratified ventricular epithelium (PVE) from embryonic day 11 (E11) to E17 in mice. The sequence of neuron origin is systematically related to mechanisms that specify neuronal class properties including laminar fate destination. Thus, the neurons to be assembled into the deeper layers are the earliest generated, while those to be assembled into superficial layers are the later generated neurons. The sequence of neuron origin also correlates with the probability of cell cycle exit (Q) and the duration of G1-phase of the cell cycle (T(G1)) in the PVE. Both Q and T(G1) increase as neuronogenesis proceeds. We test the hypothesis that mechanisms regulating specification of neuronal laminar destination, Q and T(G1) are coordinately regulated. We find that overexpression of p27(Kip1) in the PVE from E12 to E14 increases Q but not T(G1) and that the increased Q is associated with a commensurate increase in the proportion of exiting cells that is directed to superficial layers. We conclude that mechanisms that govern specification of neocortical neuronal laminar destination are coordinately regulated with mechanisms that regulate Q and are independent of mechanisms regulatory to cell cycle duration. Moreover, they operate prior to postproliferative mechanisms necessary to neocortical laminar assembly.

Cell output, cell cycle duration and neuronal specification: a model of integrated mechanisms of the neocortical proliferative process.

The neurons of the neocortex are generated over a 6 day neuronogenetic interval that comprises 11 cell cycles. During these 11 cell cycles, the length of cell cycle increases and the proportion of cells that exits (Q) versus re-enters (P) the cell cycle changes systematically. At the same time, the fate of the neurons produced at each of the 11 cell cycles appears to be specified at least in terms of their laminar destination. As a first step towards determining the causal interrelationships of the proliferative process with the process of laminar specification, we present a two-pronged approach. This consists of (i) a mathematical model that integrates the output of the proliferative process with the laminar fate of the output and predicts the effects of induced changes in Q and P during the neuronogenetic interval on the developing and mature cortex and (ii) an experimental system that allows the manipulation of Q and P in vivo. Here we show that the predictions of the model and the results of the experiments agree. The results indicate that events affecting the output of the proliferative population affect both the number of neurons produced and their specification with regard to their laminar fate.

Developmental regulation of the effects of fibroblast growth factor-2 and 1-octanol on neuronogenesis: implications for a hypothesis relating to mitogen-antimitogen opposition.

Neocortical neurons arise from a pseudostratified ventricular epithelium (PVE) that lies within the ventricular zone (VZ) at the margins of the embryonic cerebral ventricles. We examined the effects of fibroblast growth factor-2 (FGF-2) and 1-octanol on cell output behavior of the PVE in explants of the embryonic mouse cerebral wall. FGF-2 is mitogenic and 1-octanol antimitogenic in the PVE. Whereas all postmitotic cells migrate out of the VZ in vivo, in the explants some postmitotic cells remain within the VZ. We refer to these cells as the indeterminate or I fraction, because they neither exit from the VZ nor reenter S phase as part of the proliferative (P) fraction. They are considered to be either in an extremely prolonged G(1) phase, unable to pass the G(1)/S transition, or in the G(0) state. The I fate choice is modulated by both FGF-2 and 1-octanol. FGF-2 decreased the I fraction and increased the P fraction. In contrast, 1-octanol increased the I fraction and nearly eliminated the P fraction. The effects of FGF-2 and 1-octanol were developmentally regulated, in that they were observed in the developmentally advanced lateral region of the cerebral wall but not in the medial region.

Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death.

After mild ischemic insults, many neurons undergo delayed neuronal death. Aberrant activation of the cell cycle machinery is thought to contribute to apoptosis in various conditions including ischemia. We demonstrate that loss of endogenous cyclin-dependent kinase (Cdk) inhibitor p16(INK4a) is an early and reliable indicator of delayed neuronal death in striatal neurons after mild cerebral ischemia in vivo. Loss of p27(Kip1), another Cdk inhibitor, precedes cell death in neocortical neurons subjected to oxygen-glucose deprivation in vitro. The loss of Cdk inhibitors is followed by upregulation of cyclin D1, activation of Cdk2, and subsequent cytoskeletal disintegration. Most neurons undergo cell death before entering S-phase, albeit a small number ( approximately 1%) do progress to the S-phase before their death. Treatment with Cdk inhibitors significantly reduces cell death in vitro. These results show that alteration of cell cycle regulatory mechanisms is a prelude to delayed neuronal death in focal cerebral ischemia and that pharmacological interventions aimed at neuroprotection may be usefully directed at cell cycle regulatory mechanisms.

Continuity with ganglionic eminence modulates interkinetic nuclear migration in the neocortical pseudostratified ventricular epithelium.

Cells of the pseudostratified ventricular epithelium (PVE) undergo interkinetic nuclear migration whereby position of cell soma with nucleus is systematically dependent upon cell cycle phase. We examined if the interkinetic nuclear migration in the neopallial PVE is influenced by tissue continuity with the ganglionic eminence (GE) of the basal forebrain in explants from embryonic day 13 mice. We found that when continuity between the neopallial wall and the GE is intact, some neopallial PVE cells discontinue interkinetic nuclear migration following S-phase and upon entry into G2-phase. The somata and nuclei of those cells shift outward from the S-phase zone toward the subventricular and the intermediate zones. The outward migration of post-S-phase cells is observed only in the lateral region of the cerebral wall, which is closely adjacent to the GE, but not in the medial region, and only when tissue continuity with GE is maintained. We suggest that the outward moving PVE cells seed the secondary proliferative population (SPP) and that exit of the SPP seeding cells occurs in G2-phase. The phenomenon recapitulates similar migratory behavior of neopallial PVE cells in vivo and appears to represent a "choice" between two opposing options available to post-S-phase cells of the PVE. The choice appears to be imposed by mechanisms dependent upon continuity with the GE. We conclude that GE, and/or other adjacent basal forebrain structures, modulates interkinetic nuclear migration in the neopallial PVE.

Overexpression of p27Kip1 lengthens the G1 phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells.

We describe a mouse model in which p27(Kip1) transgene expression is spatially restricted to the central nervous system neuroepithelium and temporally controlled with doxycycline. Transgene-specific transcripts are detectable within 6 h of doxycycline administration, and maximum nonlethal expression is approached within 12 h. After 18-26 h of transgene expression, the G(1) phase of the cell cycle is estimated to increase from 9 to 13 h in the neocortical neuroepithelium, the maximum G(1) phase length attainable in this proliferative population in normal mice. Thus our data establish a direct link between p27(Kip1) and control of G(1) phase length in the mammalian central nervous system and unveil intrinsic mechanisms that constrain the G(1) phase length to a putative physiological maximum despite ongoing p27(Kip1) transgene expression.

Early and progressive accumulation of reactive microglia in the Huntington disease brain.

Microglia may contribute to cell death in neurodegenerative diseases. We studied the activation of microglia in affected regions of Huntington disease (HD) brain by localizing thymosin beta-4 (Tbeta4), which is increased in reactive microglia. Activated microglia appeared in the neostriatum, cortex, and globus pallidus and the adjoining white matter of the HD brain, but not in control brain. In the striatum and cortex, reactive microglia occurred in all grades of pathology, accumulated with increasing grade, and grew in density in relation to degree of neuronal loss. The predominant morphology of activated microglia differed in the striatum and cortex. Processes of reactive microglia were conspicuous in low-grade HD, suggesting an early microglia response to changes in neuropil and axons and in the grade 2 and grade 3 cortex, were aligned with the apical dendrites of pyramidal neurons. Some reactive microglia contacted pyramidal neurons with huntingtin-positive nuclear inclusions. The early and proximate association of activated microglia with degenerating neurons in the HD brain implicates a role for activated microglia in HD pathogenesis.

Increased susceptibility to ischemia-induced brain damage in transgenic mice overexpressing a dominant negative form of SHP2.

Cell culture studies have established SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) as an important factor in growth factor and cytokine-activated signaling pathways. However, the significance of SHP2 in the mammalian central nervous system (CNS) is not known since early embryonic lethality occurs in shp2 null mice. To bypass this embryonic lethality, transgenic animals containing a catalytically inactive mutant of SHP2 (SHP2-CS) under the control of a nestin intron II/thymidine kinase minimal promoter were generated. In the developing CNS of these animals, although high-level transgene expression was detected in the neuroepithelium, there was no obvious abnormality in progenitor cell proliferation or migration. In the adult brain, high-level transgene expression was detected in the subventricular zone, rostral migratory stream, dentate gyrus of hippocampus, and cerebellum. Because SHP2 function is likely important in cell survival pathways, we used a focal cerebral ischemia model to examined whether SHP2 is important during CNS injury. Ischemia-induced damage and neuronal death was found to be significantly greater in nestin-SHP2-CS mice than in wild-type littermates. These findings indicate that SHP2 is a required factor in signaling pathway(s) important for neuronal survival.

Independent controls for neocortical neuron production and histogenetic cell death.

We estimated the proportion of cells eliminated by histogenetic cell death during the first 2 postnatal weeks in areas 1, 3 and 40 of the mouse parietal neocortex. For each layer and for the subcortical white matter in each neocortical area, the number of dying cells per mm(2) was calculated and the proportionate cell death for each day of the 2-week interval was estimated. The data show that cell death proceeds essentially uniformly across the neocortical areas and layers and that it does not follow either the spatiotemporal gradient of cell cycle progression in the pseudostratified ventricular epithelium of the cerebral wall, the source of neocortical neurons, or the 'inside-out' neocortical neuronogenetic sequence. Therefore, we infer that the control mechanisms of neocortical histogenetic cell death are independent of mechanisms controlling neuronogenesis or neuronal migration but may be associated with the ingrowth, expansion and a system-wide matching of neuronal connectivity.

delta-catenin is a nervous system-specific adherens junction protein which undergoes dynamic relocalization during development.

delta-catenin is a member of the Armadillo repeat family and component of the adherens junction discovered in a two-hybrid assay as a bona fide interactor with presenilin-1 (Zhou et al., [1997], NeuroReport 8:2085-2090), a protein which carries mutations that cause familial Alzheimer's disease. The expression pattern of delta-catenin was mapped between embryonic day 10 (E10) and adulthood by Northern blots, in situ hybridization and immunohistochemistry in the mouse. In development, delta-catenin is dynamically regulated with respect to its site of expression. It is first expressed within proliferating neuronal progenitor cells of the neuroepithelium, becomes down-regulated during neuronal migration, and is later reexpressed in the dendritic compartment of postmitotic neurons. In the mouse, delta-catenin mRNA is expressed by E10, increases and peaks at postnatal day (P)7, with lower levels in adulthood. In the developing neocortex, delta-catenin mRNA is strongly expressed in the proliferative ventricular zone and the developing cortical plate, yet is conspicuously less prominent in the intermediate zone, which contains migrating cortical neurons, delta-catenin protein forms a honeycomb pattern in the neuroepithelium by labeling the cell periphery in a typical adherens junction pattern. By E18, delta-catenin expression shifts primarily to nascent apical dendrites, a pattern that continues through adulthood. The dynamic relocalization of delta-catenin expression during development, taken together with previously published data which described a role for delta-catenin in cell motility (Lu et al., [1999] J. Cell. Biol. 144:519-532), suggests the hypothesis that delta-catenin regulation is closely linked to neuronal migration and may play a role in the establishment of mature dendritic relationships in the neuropil.

Intrinsic determinants of retinal axon collateralization and arborization patterns.

Permanent, novel retinal projections to the principal thalamic somatosensory (ventrobasal) or auditory (medial geniculate) nuclei can be produced in adult hamsters if the superior colliculus is ablated bilaterally and the somatosensory and auditory lemniscal axons are transected unilaterally on the day of birth. We studied the development of those novel projections by labeling retinal axons with the fluorescent tracer 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate to examine the relative roles of intrinsic factors and axon-target interactions in the specification of retinal axon connections. Our principal findings are as follows: (1) In hamsters operated on the day of birth to produce the novel retinal projections, retinal ganglion cell axons projecting to the ventrobasal or medial geniculate nuclei develop in three morphologically distinct stages, i.e., elongation, collateralization, and arborization, as do retinal axons projecting to the dorsal lateral geniculate nucleus, the principal thalamic visual nucleus, in normal hamsters. (2) In both the ventrobasal and medial geniculate nuclei of operated hamsters, as in the dorsal lateral geniculate nucleus of normal hamsters, collateral branches were initially formed by retinal ganglion cell axons in both the superficial and internal components of the optic tract and only collaterals from the superficial component formed permanent projections. (3) The retinofugal axon terminal arbors in the ventrobasal and medial geniculate nuclei of mature, operated hamsters resemble the same three morphologic classes that are observed in the lateral geniculate nucleus of normal hamsters, although their absolute size appears to be altered. These data suggest that both superficial and internal optic tract axons can produce thalamic collaterals during development but that only superficial optic tract axons can permanently retain thalamic collaterals. Furthermore, the same morphologic types of retinofugal axons appear to contribute to normal and surgically induced retinal projections.

Proliferative behavior of the murine cerebral wall in tissue culture: cell cycle kinetics and checkpoints.

Cerebral wall from embryonic day 13 mice was cultured in a three-dimensional collagen matrix in defined, serum-free medium. The cerebral wall retained its normal architecture, including the radial glial fiber system, for up to 19 h in culture. The cell cycle was initially blocked at the S/G2/M and the G1/S phase transitions, resulting in a transient synchronization of the proliferative cells. The transient blockades correspond, we suggest, to the G2 checkpoint and G1 restriction point, adaptive mechanisms of normal proliferative cells. The blocks were relieved within a few hours of explantation with restoration of the interkinetic nuclear migration and flow of cells through the cycle phases. The duration of the reestablished cell cycle and those of G1, S, and combined G2-M phases were estimated to be 19.2, 6.3-8.3, 8.8, and 2.0-4.0 h, respectively. The leaving (Q) fraction of the cycle (0.64) was twice the in vivo value. Two-thirds of the Q fraction cells remained in the ventricular epithelium, resulting in a substantially low growth fraction of 0.73 compared with 1.0 in vivo. The embryonic murine cerebral explant, cultured in minimum essential medium, should be favorable for studies of cycle modulatory actions of cell external influences such as growth factors or neurotransmitters.

Analysis of Huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons.

Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin-associated protein 1 (HAP-1), a brain-enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP-1 is unknown, but recent evidence supports a role in microtubule-dependent organelle transport. We examined the subcellular localization of HAP-1 with an antibody made against the NH2-terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP-1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP-1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP-1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP-1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP-1 localized to membrane-bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP-1 was also strongly associated with an unusual large "dense" organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP-1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP-1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons.

The sequence of formation and development of corticostriate connections in mice.

We examined the development of the corticostriate pathway in mice by labeling corticofugal axons with the carbocyanine dye 1, 1'-dioctadecyl-3,3,3'-3'-tetramethylindocarbocyanine perchlorate (DiI). Growth cones of corticofugal axons enter the developing striatum on embryonic day 12 (E12; conception is on E0). By E15 corticofugal axons pass through the developing striatum in the internal capsule but do not produce striatal collaterals. Corticostriate collaterals are seen for the first time on E18, 6 days after the earliest arriving axons enter the striatum. At that time, presumptive synaptic contacts form between cortical axons and striatal neurons. Corticostriate collaterals arise from corticofugal axon trunks at or near axonal varicosities. Primitive corticostriate arbors form by postnatal day 2 (P2; day of birth is P0) and develop further by P7. Thus, corticostriate connections develop in three morphologically defined stages: first cortical axons elongate through the striatum to other subcortical targets, next they produce striatal collaterals, and finally they elaborate terminal arbors. The transition from elongation to collateralization stage may be triggered, among other factors, by signals from striatal neurons relayed via the synaptic contacts.

Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues.

Spinal muscular atrophy is caused by defects in the survival motor neuron (SMN) gene. To better understand the patterns of expression of SMN in neuronal cells and tissues, we raised a polyclonal antibody (abSMN) against a synthetic oligopeptide from SMN exon 2. AbSMN immunostaining in neuroblastoma cells and mouse and human central nervous system (CNS) showed intense labeling of nuclear "gems," along with prominent nucleolar immunoreactivity in mouse and human CNS tissues. Strong cytoplasmic labeling was observed in the perikarya and proximal dendrites of human spinal motor neurons but not in their axons. Immunoblot analysis revealed a 34-kDa species in the insoluble protein fractions from human SY5Y neuroblastoma cells, embryonic mouse spinal cord cultures, and human CNS tissue. By contrast, a 38-kDa species was detected in the cytosolic fraction of SY5Y cells. We conclude that SMN protein is expressed prominently in both the cytoplasm and nucleus in multiple types of neurons in brain and spinal cord, a finding consistent with a role for SMN as a determinant of neuronal viability.

The protein tyrosine phosphatase SHP-2 is expressed in glial and neuronal progenitor cells, postmitotic neurons and reactive astrocytes.

In this study we examined the distribution and developmental profile of the src homology 2 (SH2) domain-containing protein tyrosine phosphatase SHP-2 in the mouse brain. We found that SHP-2 is present in both mitotically active and postmitotic cells in the forebrains of embryonic day 12 (E12) mice. In a developmental study extending from embryonic day 12 to adulthood, Western blotting analysis demonstrated equivalent levels of SHP-2 protein at all of the ages examined. Expression of SHP-2 paralleled the level of enzymatic activity at the different developmental periods. In the adult brain SHP-2 was restricted to diverse classes of neurons, while the majority of glial cells did not express detectable levels of protein. However, reactive astrocytes in response to an ischemic brain injury showed SHP-2 immunolabelling. Our data suggest that SHP-2 may play a role in pathways of neuronal and glial progenitor cells, in a broad spectrum of neuronal responses in the adult brain and in the gliotic response to the injury.

Huntingtin localization in brains of normal and Huntington's disease patients.

The immunohistochemical localization of huntingtin was examined in the Huntington's disease (HD) brain with an antibody that recognizes the wild-type and mutant proteins. Neuronal staining was reduced in areas of the HD striatum depleted of medium-sized neurons; large striatal neurons, which are spared in HD, retained normal levels of huntingtin expression. Neuronal labeling was markedly reduced in both segments of the globus pallidus including in brains with minimal loss of pallidal neurons. In some HD cortical and striatal neurons with normal looking morphology, huntingtin was associated with punctate cytoplasmic granules that at the ultrastructural level resembled the multivesicular body, an organelle involved in retrograde transport and protein degradation. Some immunoreactive processes showed blebbing and segmentation similar to that induced experimentally by hypoxic-ischemic or excitotoxic injury. Huntingtin staining was more concentrated in the perinuclear cytoplasm and reduced or absent in processes of atrophic cortical neurons. Nuclear staining was also evident. Fibers in the subcortical white matter of HD patients had significantly increased huntingtin immunoreactivity compared with those of controls. Results suggest that there may be changes in the neuronal expression and transport of wild-type and/or mutant huntingtin at early and late stages of neuronal degeneration in affected areas of the HD brain.

Concurrent cellular output from two proliferative populations in the early embryonic mouse corpus striatum.

In mice, the striatal compartment of the forebrain is established by embryonic day 11 (E11, E0 = day of conception) when a lateral ganglionic eminence emerges surrounding the lateral and ventral margins of the forebrain ventricles. The inception of the striatal compartment is evidence of altered cell cycle kinetics, especially a rapid production of postmitotic cells, within a discrete portion of the telencephalic neuroepithelium. As a step toward understanding the mechanisms which contribute to the development of a cytokinetically distinct striatal compartment, we characterized the rate and pattern of cellular output in the lateral ganglionic neuroepithelium of mice on E11. The data show that the striatal compartment is distinguished by concurrent and equivalent levels of cell output from two proliferative populations: a dominant secondary proliferative population and a smaller, pseudostratified ventricular epithelium. In addition, although the ganglionic neuroepithelium is expanding on E11, 30-35% of the daughter cells produced leave the cell cycle and become postmitotic. These cytogenetic events, occurring in the lateral ganglionic progenitor population, may contribute to the development of a distinct striatal compartment within the telencephalon.