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Biomedical subjects

M E Hatten

Publications and source records attributed to M E Hatten.

At least 37 records · Page 2Linked to original sources
PubMed1996

Embryonic precursor cells from the rhombic lip are specified to a cerebellar granule neuron identity.

The specification of diverse classes of neurons is critical to the development of the cerebellar cortex. Here, we describe the purification of early embryonic precursors of cerebellar granule neurons from the rhombic lip, the dorsal aspect of the midbrain/hindbrain region. Isolation of rhombic lip cells reveals a homogenous population of precursor cells that express general neuronal markers and the granule cell marker RU49, but fail to extend neurites or express differentiation markers. Differentiation is induced by coculture with external germinal layer (EGL) cells, or their membranes, suggesting that a local inducing factor acts after formation of the EGL. Thus, proliferating precursors within the rhombic lip are specified to be granule cells very early, with the availability of an inducing factor increasing over the course of development.

Animals↗
PubMed1995

Radial glial cell transformation to astrocytes is bidirectional: regulation by a diffusible factor in embryonic forebrain.

During development of mammalian cerebral cortex, two classes of glial cells are thought to underlie the establishment of cell patterning. In the embryonic period, migration of young neurons is supported by a system of radial glial cells spanning the thickness of the cortical wall. In the neonatal period, neuronal function is assisted by the physiological support of a second class of astroglial cell, the astrocyte. Here, we show that expression of embryonic radial glial identity requires extrinsic soluble signals present in embryonic forebrain. Moreover, astrocytes reexpress features of radial glia in vitro in the presence of the embryonic cortical signals and in vivo after transplantation into embryonic neocortex. These findings suggest that the transformation of radial glia cells into astrocytes is regulated by availability of inducing signals rather than by changes in cell potential.

Animals↗
PubMed1995

Motility and cytoskeletal organization of migrating cerebellar granule neurons.

To characterize CNS neuronal precursor migration along astroglial fibers, we examined the motility of the migratory leading process and cytoskeletal-based mechanisms of locomotion of early postnatal mouse cerebellar granule neurons in vitro. To visualize the surface motility of the leading process, granule neurons were labeled with the fluorescent lipophilic dye, PKH-26, and imaged by time lapse fluorescence microscopy. The motile behavior and cytoskeletal organization of the migrating neuron had several distinctive features. As the migrating neuron moved along the glial fiber, the leading process rapidly extended, projecting up to 40 microns, and retracted, withdrawing towards the cell soma. Broad lamellipodia were common along the entire length of the leading process, giving it a ruffled appearance. Within the cell soma, a cage-like distribution of microtubules encircled the nucleus and actin filaments formed a subcortical rim underneath the plasma membrane. Disruption of actin filaments with cytochalasin B inhibited migration, suggesting involvement of actin subunit assembly in neuronal migration. Both microtubules and actin filaments were heavily concentrated in the leading process; the leading process did not show the development of a distinct actin-rich domain at its tip.

Actins↗
PubMed1994

Cell-cell interactions influence survival and differentiation of purified Purkinje cells in vitro.

To determine the role of cell-cell interactions in Purkinje cell survival and dendritic differentiation, perinatal mouse Purkinje cells were purified, and their development was analyzed in vitro. In isolation at low density, Purkinje cell survival was poor, improved by neuronal contacts, either with purified granule neurons or with Purkinje cells themselves. Moreover, coculture with specific cell populations led to widely different degrees of Purkinje cell differentiation. Purified Purkinje cells cultured alone or with an inappropriate afferent, the mossy fibers, did not progress beyond immature forms. With astroglia, Purkinje cells had thin smooth processes. Proper Purkinje cell differentiation was driven only by coculture with granule cells, resulting in dendrites with spines receiving synapses. These results suggest that Purkinje cell differentiation is regulated by local epigenetic factors, provided in large part by the granule neuron.

Afferent Pathways↗
PubMed1994

Brain lipid-binding protein (BLBP): a novel signaling system in the developing mammalian CNS.

Using a polyclonal antibody against postnatal cerebellar cells, we have isolated a new, brain-specific member of the lipid-binding protein family (BLBP). Members of this family, such as cellular retinoic acid-binding protein, have been shown to carry small hydrophobic signaling molecules between cellular compartments. The expression of BLBP is spatially and temporally correlated with neuronal differentiation in many parts of the mouse CNS, including postnatal cerebellum, embryonic spinal cord, and cerebral cortex. In situ hybridization and immunocytochemistry show that BLBP is transiently expressed in radial glia in both the embryonic ventricular zone and the postnatal cerebellum. Subcellular localization studies by immunoelectron microscopy demonstrate that BLBP is present in the nucleus as well as the cytoplasm. Affinity-purified anti-BLBP antibodies block glial and neuronal differentiation in primary cell cultures, but have no effect on cell proliferation or adhesion. Based on these results, we propose that BLBP is required for the establishment of the radial glial fiber system in developing brain, a system that is necessary for the migration of immature neurons to establish cortical layers.

Amino Acid Sequence↗
PubMed1994

Immortalizing oncogenes subvert the establishment of granule cell identity in developing cerebellum.

After implantation into the external germinal layer of early postnatal cerebellum, primary external germinal layer progenitor cells gave rise exclusively to granule neurons. In contrast, all major classes of cerebellar cells were observed following implantation of embryonic day 13 cerebellar precursor cells into the external germinal layer. These results suggest that granule cells arise from precursors with a restricted potential. In contrast to results with the primary external germinal layer population, cell lines established from external germinal layer cells, by infection with a retrovirus containing the SV40 large T-antigen oncogene, gave rise to several cerebellar cell types upon implantation. These included granule neurons, one subclass of stellate interneurons, Golgi cells, Bergmann glia and astrocytes. From these results, we conclude that early postnatal external germinal layer progenitors are normally fated to a granule cell identity and that expression of the SV40 large T-antigen oncogene subverts mechanisms that control granule neuron fate.

Animals↗
PubMed1994

Astroglial differentiation is required for support of neurite outgrowth.

Models of astrocyte differentiation stress a lineage program that involves a progressive loss of astroglial support of neuronal differentiation. These models predict that astroglial promotion of neurite extension declines with the "age" of the astrocyte. An alternative view is that astroglial support of neurite growth is regulated by epigenetic factors that induce the cells either to differentiate and support neuronal functions or to undergo cell proliferation and fail to support neurons. To compare the contribution of astroglial cell "age" to astroglial support of neurite extension, mouse cerebellar astroglia were maintained in vitro for 3-90 d, and assayed for their ability to support neurite formation. When cultured in isolation, astroglial support of neurite extension declined with time in vitro, as assayed by quantifying outgrowth from explants of pontine nuclei, falling from a robust level just after the astroglia were harvested to negligible levels 21-90 d later. Since previous studies have shown that neurons can change the state of astroglial cells (Hatten, 1985), we tested the neurite promoting activity of astroglia that were cultured for 21-90 d in vitro and subsequently induced to differentiate by the addition of neurons. When granule neurons were added to aged astroglia and pontine explants plated 2 d later, neurite growth from the explants was exuberant, regardless of the time astroglia spent in vitro prior to the addition of neurons. The state of astroglia that were growth promoting or growth inhibiting was examined by bromodeoxyuridine staining and with antisera to glial filament protein. Aged astroglia cultured alone and thus inhibitory to axon growth, proliferated at high rates and had polygonal shapes. In contrast, aged astroglia to which neurons had been added, proliferated at low rates and developed process-bearing stellate shapes. To test further whether proliferation levels related to the growth-supporting properties of astroglia, astroglia were plated alone in medium without serum, or with the addition of transforming growth factor-beta 1, each treatment known to arrest proliferation. In both cases, promotion of neurite growth was restored in aged astroglia, but the morphology of astroglia did not correlate with the ability to support neurite growth. Finally, the growth-inhibiting properties of aged astroglia do not appear to be mediated by diffusible factors, and require close apposition with living astroglial cells. We conclude that astroglial support of neurite extension depends on the state of differentiation of astroglial cells, and that these properties can be modified by coculture with neurons or conditions that arrest of astroglial proliferation, irrespective of astroglial "age".

Animals↗
PubMed1993

Neuronal differentiation rescued by implantation of Weaver granule cell precursors into wild-type cerebellar cortex.

The migration of postmitotic neurons away from compact, germinal zones is a critical step in neuronal differentiation in the developing brain. To study the molecular signals necessary for cerebellar granule cell migration in situ, precursor cells from the neurological mutant mouse weaver, an animal with phenotypic defects in migration, were implanted into the external germinal layer (EGL) of wild-type cerebellar cortex. In this region, labeled weaver precursor cells of the EGL progressed through all stages of granule neuron differentiation, including the extension of parallel fibers, migration through the molecular and Purkinje cell layers, positioning in the internal granule cell layer, and extension of dendrites. Thus, the weaver gene acts nonautonomously in vivo, and local cell interactions may induce early steps in neuronal differentiation that are required for granule cell migration.

Animals↗
PubMed1993

Dispersion of neural progenitors within the germinal zones of the forebrain.

One of the early events in the establishment of regional diversity in brain is the subdivision of the forebrain into the cerebral cortex and underlying basal ganglia. This subdivision is of special interest, owing to the striking difference in cellular patterning in these two regions. Whereas the dorsal aspect of the telencephalon gives rise to the laminar, cortical regions of brain, the basal aspect gives rise to nuclear, subcortical regions. To examine early events in the regionalization of the forebrain, we visualized cell movement within the ventricular zones of the dorsal and basal regions of the E15 murine telencephalon. Over an 8-24-hour observation period, labelled cells moved extensively in the plane of the cortical ventricular zone. Cell dispersion was restricted, however, at the border between the cortical ventricular zone and the lateral ganglionic eminence, the basal telencephalic ventricular zone. We suggest that this restriction of cell movements establishes a regional pattern of neurogenesis in the developing brain.

Animals↗
PubMed1993

The role of migration in central nervous system neuronal development.

The past year has seen the emergence of significant new information on the control of neurogenesis and migration, and the establishment of neuronal identity in three systems: developing cerebellum, cortex, and optic tectum. These findings have important implications for the role of glial-guided migrations in central nervous system neuronal development.

Animals↗
PubMed1993

Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation.

Among CNS neuronal populations, the cerebellar granule cell provides a simple model for analysing the molecular regulation of CNS neurogenesis. In this study, polyclonal antisera raised against immature granule cell precursors, purified from early postnatal mouse cerebellum, were used to isolate 39 unique cDNA clones from a lambda gt11 cDNA expression library made from the same cell population. Northern blot analysis revealed developmental stage and tissue-specific expression of 28 of the clones. In situ localization of mRNAs encoded by these novel cDNAs, as well as those encoding the axonal glycoprotein TAG-1 and the alpha 6 subunit of the GABAA receptor, reveal four distinct stages in cerebellar granule cell differentiation. The developmentally transient and spatially restricted expression of clones GC9 and GC44 identify a previously unrecognized step in cerebellar histogenesis.

Animals↗
PubMed1993

Multiple receptor systems promote CNS neural migration.

To identify glial receptor systems in CNS migration, cerebellar granule neuron migration was assayed on glass fibers coated with polylysine, astroglial membranes (AM fibers), or the extracellular matrix proteins collagen (COLL fibers), fibronectin (FN fibers), and laminin (LAM fibers). By video microscopy, granule cells migrated along AM fibers with the cytology, neuron-fiber apposition, and dynamics seen on living glia. The demonstration that immobilized astroglial membranes support neural migration suggests that astroglial receptor systems, in combination with glial fiber geometry, promote CNS neural migration. Moreover, granule neurons migrated rapidly on LAM fibers, moved relatively slowly on FN fibers, and not at all on COLL fibers. Antibody perturbation analyses suggested that, whereas astrotactin provides the neural receptor for migration on astroglial membranes, integrin beta 1 provides the neural receptor for migration on LAM fibers. These results suggest that multiple receptor systems support CNS neural migration.

Animals↗
PubMed1992

The weaver gene encodes a nonautonomous signal for CNS neuronal differentiation.

In the neurological mutant mouse weaver, CNS precursor cells in the external germinal layer (EGL) of the cerebellar cortex proliferate normally, but fail to differentiate and die in the proliferative zone. To examine the autonomy of expression of the weaver gene, we carried out cell-mixing experiments in vitro. In homotypic, reaggregate cultures, weaver EGL precursor cells expressed the general neuronal markers N-CAM, L1, and MAP2, but failed to express the late neuronal antigens TAG-1 and astrotactin, to extend neurites or to migrate on glial fibers. After reaggregation with wild-type EGL precursor cells, weaver precursor cells extended neurites equivalent in length to wild-type cells, migrated along astroglial fibers, and expressed TAG-1 and astrotactin. Rescue of neurite production was also achieved by the addition of membranes from, but not by medium conditioned by wild-type cells. These findings suggest that the weaver gene acts non-autonomously, encoding a membrane-associated ligand that induces EGL neuronal differentiation.

Animals↗
PubMed1992

Cerebellar target neurons provide a stop signal for afferent neurite extension in vitro.

The contributions of cell-cell interactions to the establishment of specific patterns of innervation within target brain regions are not known. To provide an experimental analysis of the regulation of afferent axonal growth, we have developed an in vitro assay system, based on the developing mouse cerebellum, in which afferent axons from a brainstem source of mossy fiber afferents, the basilar pontine nuclei, were cocultured with astroglia or granule neurons purified from the cerebellum. In the absence of cells from the cerebellum, pontine explants produced axons that fasciculated and extended rapidly on a culture surface treated with poly-lysine or laminin. When pontine neurites grew onto cerebellar astroglial cells, outgrowth was more abundant than on substrates alone, suggesting that glial cells provide a positive signal for axon extension. Time-lapse video microscopy indicated that the rate of neurite extension increased from less than 50 microns/hr to more than 100 microns/hr when axonal growth cones moved from the culture substratum onto an astroglial-cell surface. Acceleration of neurite extension was also observed as pontine neurites grew onto other pontine neurites. By contrast, when pontine neurites grew on granule neurons, the appropriate targets of mossy fibers, the length of pontine neurites was greatly reduced. As growing axons terminated on granule neurons, the target cells appeared to provide a "stop-growing signal" for axon extension. The length of pontine neurites decreased with increasing granule neuron density. Two lines of evidence suggested that the stop signal was contact mediated. First, video microscopy showed that pontine growth cones stopped extending after contacting a granule neuron. Second, the length of afferent axons was not reduced when pontine neurites grew at a distance from granule neurons. Competition experiments where both astroglia and granule neurons were plated together suggested that the growth arrest signal provided by granule neurons could override the growth-promoting signal provided by astroglial cells. These results suggest that specific cell-cell interactions regulate the growth of pontine afferent axons within their cerebellar target, with axoaxonal and axoglial interactions promoting axon extension and axon-target cell interactions interrupting axon extension.

Afferent Pathways↗
PubMed1991

Astroglia in CNS injury.

The astroglial response to CNS injury is considered in the context of neuron-glial relationships. Although previous models suggested that astroglial cells present in "scars" impede axon regrowth owing to irreversible changes in the glial cell following injury, recent in vivo and in vitro studies indicate that astroglial cells exhibit considerable plasticity, elevating expression of the glial filament protein and altering expression of properties which support axons, including extracellular matrix components and cell surface adhesion systems. Both in vivo and in vitro studies on neuron-glia interactions in different brain regions suggest that glia express region-specific properties, including ion channels, neurotransmitter uptake and receptor systems, and cell surface adhesion systems. Together these findings suggest that a more detailed analysis of glial response to injury in different brain regions will lead to an appreciation of the diversity of the astroglial response to injury, and its regulation by neuron-glia relationships.

Animals↗
PubMed1991

Cerebellar granule cell neurogenesis is regulated by cell-cell interactions in vitro.

When CNS precursor cells purified from the external germinal layer of the early postnatal mouse cerebellum are cultured in cellular reaggregates, DNA synthesis increased 10-fold above that of cells dispersed in a monolayer or embedded in a collagen matrix. Dividing precursor cells gave rise to neurons immunopositive for the neural antigens N-CAM, L1, and TAG-1, but not to astroglial cells immunopositive for glial filament protein. Moreover, proliferating precursor cells did not generate other types of cerebellar neurons, as judged by the lack of expression of glutamic acid decarboxylase, the synthetic enzyme for gamma-amino-n-butyric acid. By contrast, the addition of astroglial cells, or astroglial cell membranes, to cellular reaggregates of granule cell neuroblasts arrested precursor cell DNA synthesis in a dose-dependent manner. These results suggest that homotypic contact interactions among CNS neural progenitors control precursor cell proliferation and fate in generative zones of developing brain.

Aminobutyrates↗
PubMed1991

Molecular mechanisms of glial-guided neuronal migration.

The migration of young neurons from their site of origin in proliferative zones out into neuronal layers is a hallmark of cortical development. Neuroanatomic studies show that astroglial fibers provide the primary substrate for neuronal migration. In vitro studies on living cells provide evidence that migrating neurons express distinguishing cytologic features including the formation of a specialized junction at the site of neuron-glia contact and the extension of an active leading process in the direction of migration. Our in vitro functional assays point to a critical role for astrotactin in neuron-glia binding during the developmental periods of glial-guided cell migration and assembly in brain. Other receptor systems, including neural cell adhesion systems, cadherins, and integrins are expressed by granule cells but do not appear to contribute to neuron-glia binding or to glial-guided neuronal migration. A role for astrotactin in glial-guided migration and assembly is supported by our observation that astrotactin is expressed by neurons and not glial cells and by restricted spatiotemporal expression of astrotactin in vivo, wherein astrotactin is expressed by migrating neurons and by neurons during periods of assembly into neuronal layers in developing brain. Understanding the regulation of astrotactin expression and its role in migration will provide fundamental insights into the role of glial-guided migration in the histogenesis of the brain.

Animals↗