Mary E. Hatten

Riding the glial monorail: A common mechanism for glialguided neuronal migration in different regions of the developing mammalian brain

In vitro studies from our laboratory indicate that granule neurons, purified from early postnatal mouse cerebellum, migrate on astroglial fibers by forming a migration junction with the glial fiber along the length of the neuronal soma and extending a motile leading process in the direction of migration. Similar dynamics are seen for hippocampal neurons migrating along hippocampal astroglial fibers in vitro. In heterotypic recombinations of neurons and glia from mouse cerebellum and rat hippocampus, neurons migrate on astroglial processes with a cytology and neuron-glia relationship identical to that of homotypic neuronal migration in vitro. In all four cases, the migrating neuron presents a stereotyped posture, speed and mode of movement, suggesting that glial fibers provide a generic pathway for neuronal migration in developing brain. Studies on the molecular basis of glial-guided migration suggest that astrotactin, a neuronal antigen that functions as a neuron-glia ligand, is likely to play a crucial role in the locomotion of the neuron along glial fibers. The navigation of neurons from glial fibers into cortical layers, in turn, is likely to involve neuron-neuron adhesion ligands.

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.

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.

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.

Antibodies that recognize astrotactin block granule neuron binding to astroglia

To provide a rapid, specific assay for receptor systems involved in the binding of cerebellar granule neurons to astroglia, granule cells, purified from early postnatal mice, or from E15-E16 chicks, were radiolabeled with [35S]methionine and plasma membranes were prepared. The kinetics of binding of radiolabeled material to primary mouse or chick glia or to the mouse G26-24 astrocytoma cell line was measured in the presence or absence of antibodies against astrotactin, neural cell adhesion molecules, cadherins, or integrins. Addition of Fab fragments of astrotactin antibodies reduced the amount of granule cell membrane binding to astroglia by 70%. In contrast, Fab fragments of antibodies against the neural adhesion molecules N-CAM, L1, and N-cadherin and against integrin did not reduce the level of granule cell membrane binding to astroglia. Combinations of antibodies against N-CAM, L1, N-cadherin, and integrin also did not impair neuron binding to glia.

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.

Culturing nerve cells

Although many of the principles of cell culture of nerve cells were presented by Ross Harrison early in the century, the hows and whys of neuronal cell culture remain an obstacle to many who would use in vitro approaches to examine the processes of neuronal development. The reader of this book is given a tour through a number of major laboratories that use in vitro approaches, with detailed descriptions of experimental methods combined with the biological problems that prompted such experiments. Edited by Gary Banker, a major figure in cell culture approaches to hippocampal neuronal differentiation, and his colleague Kimberly Goslin, the chapters are contributed by leading experts in each of the branches of CNS and PNS cell culture, in both vertebrate and invertebrate systems. The variety of approaches and culture systems described in the book is comprehensive, and the level of detail in any given chapter is great enough to get a novice started using the methods of that laboratory. However, unlike many other books on culture methods the chapters balance these detailed protocols with the rationale of the approach and the range of studies to which they might be applied. Therefore, the book is an invaluable resource to any who might wish to scan the field for appropriate in vitro systems. The chapters are organized into three general sections. In the first Getting started the outline of the book as well as the aim and scope of the individual chapters are presented. In this opening section, in a chapter entitled In defense of neuronal cultures, the editors address the time-worn issue of the relevance of cell culture experiments to development in vivo. In a second section entitled General principles, the editors provide an excellent review of the types of nerve cell cultures, ranging from primary cells to cell lines of neuronal cells and cultures of glial cells. The editors then review classical methods of primary dissociated cell culture, including extensive how to sections ranging from culture methods to book-keeping and the characterization of cell types in primary cell cultures. These opening chapters are especially well organized and well written, and will be useful to anyone wishing to learn the nuts and bolts of cell culture. The book then turns to the biological problems amenable to cell culture experimentation, and illustrates how specific culture systems can be designed to address particular problems. Here, prominent figures in the field, including Denis Bray, Mu-Ming Poo, Dan Goldberg, Dennis Higgins, Mary Johnson, Lloyd Greene, Robert Baughman, Ekkhart Trenkner, Ken McCarthy, Pat Wood, Richard Bunge, Beat Gahwhiler, and the editors and their colleagues, describe how problems of axon growth, neuronal differentiation, neuronal migration,