Elsevier

Developmental Brain Research

Volume 140, Issue 2, 16 February 2003, Pages 195-203
Developmental Brain Research

Research report
Localization of ApoER2, VLDLR and Dab1 in radial glia: groundwork for a new model of reelin action during cortical development

https://doi.org/10.1016/S0165-3806(02)00604-1Get rights and content

Abstract

The reelin signaling pathway regulates laminar positioning of radially migrating neurons during cortical development. It has been suggested that reelin secreted by Cajal–Retzius cells in the marginal zone could provide either a stop or an attractant signal for migratory neurons expressing reelin receptors, but the proposed models fail to explain recent experimental findings. Here we provide evidence that the reelin receptor machinery, including the lipoprotein receptors ApoER2 and VLDLR along with the cytoplasmic adaptor protein Dab1, is located in radial glia precursors whose processes span the entire cortical wall from the ventricular zone to the pial surface. Moreover, in reeler mice, defective in reelin, decreased levels of Dab1 in the ventricular zone correspond to an accumulation of the protein in radial end-feet beneath the pia matter. Our results support that neural stem cells receive a functional reelin signal. They are also consistent with a working model of reelin action, according to which reelin signaling on the newborn neuron-inherited radial process regulates perikaryal translocation and positioning.

Introduction

An intricate choreography of physical forces due to neural proliferation, differentiation and migration cause morphogenetic tissue growth during brain development. During the development of the cerebral cortex, young neurons originating in discrete neurogenic areas modify their positions by means of radial and tangential migratory mechanisms that eventually determine the correct cytoarchitecture of the mature cerebral cortex. Radially migrating neurons, using radial glia processes as a scaffold, transit from the ventricular zone (VZ) to the cortical plate where they become arranged in an ‘inside-out’ manner. This pattern is achieved by the migration of later born neurons past their predecessors, and for the most part determines the laminar organization of the mature cortex (for reviews see Refs. [15], [32]).

Some of the molecular mechanisms that orchestrate this migration are now appreciated. Recent studies have revealed that the secretable glycoprotein reelin, synthesized during development by Cajal–Retzius cells in the cortical marginal zone (MZ), is required for the ‘inside-out’ organization of the cortex. The apolipoprotein receptor 2 (ApoER2) and the very low-density lipoprotein receptor (VLDLR), and the cytoplasmic adaptor protein disabled 1 (Dab1), are downstream components of the reelin signaling pathway. Mutant mice for reelin, Dab1 or both ApoER2 and VLDLR show defects in the lamination of several brain regions, including the genesis of an anomalous cortical plate under the subplate with a dispersed, roughly inverted, inside-out pattern of lamination and neuronal invasion of cortical layer I (for review see Ref. [33]). It has been established that reelin binding to ApoER2 and VLDLR induces tyrosine phosphorylation of Dab1, a tyrosine kinase signal transduction cascade [3], [13], [16], [36], [43]. It has subsequently been shown that the receptors physically interact with reelin. The receptors are required for reelin-induced Dab1 tyrosine phosphorylation along with Dab1-regulated turnover [33]. In addition, lack of a reelin-evoked signal led to an accumulation of Dab1 in several neuronal populations [34].

Pioneering expression studies have detected enriched expression of ApoER2, VLDLR and Dab1 transcripts on migrating cortical neurons and Purkinje cells [3], [13], [17], [34]. Recently, it has been established that transcripts of the reelin receptor machinery are also located in the VZ, where the reelin signaling pathway is activated by phosphorylation of Dab1. It has been suggested that such an activation may occur through the apical dendrites that premigratory neurons extend from the VZ to the marginal zone [20]. However, how reelin functions to position neurons remains unclear, although several models have been suggested. It has been proposed that reelin functions as an inhibitory signal that terminates radial migration of young neurons by releasing them from their radial glia guides [6], [7], [37]. Alternatively, reelin has been postulated to act as a repellent for early neuronal populations [28], [35] or as a stop signal [29]. Reelin has also been proposed to act as a chemoattractant for migrating young Purkinje cells [9]. Highlighting the difficulties to define a coherent model of reelin action, the results from a recent analysis about the effects of ectopic reelin expression are not consistent with any of the proposals. It is unlikely that reelin functions as a stop signal or as an attractant because animals ectopically expressing reelin in the VZ show neither evidence of premature termination of neuronal migration in the intermediate or VZ, nor evidence of cortical plate heterotopia in the VZ [20].

The mechanisms of radial migration must be revisited in light of several recent reports demonstrating that radial glia are the progenitor cells of neurons [11], [21], [23], [25], [42]. Moreover, during asymmetrical cell division of the radial glia, the newly generated neurons inherit the radial glia-process, while the sibling radial glia grow new ascending ones [23], [42]. Here we provide evidence that the reelin receptor machinery, including ApoER2, VLDLR and Dab1, is located in the neuronogenic radial glia. This precise compartmentalization of reelin signaling partners along with recent findings showing migration of cortical neurons by perikaryal translocation [24] could be consistent with a working model of reelin action according to which reelin signaling on the inherited radial processes regulates perikaryal translocation and positioning of newborn cortical neurons.

Section snippets

Animals

Wistar rats along with wild type and Orleans (Balb/c background) reeler mice were used. The care and handling of the animal prior to and during the experimental procedures followed European Union regulations and were approved by the Animal Care and Use Committees of the authors’ institution. All efforts were made to reduce the number of animals used and minimize animal suffering. Animals were housed in a pathogen-free environment on a 12:12 h light–dark cycle.

Tissue collection

Timed mating was established, and

Results

To compare the localization pattern of ApoER2, VLDLR and Dab1 in the developing forebrain, immunohistochemical analysis was performed throughout corticogenesis (E12–E17). At the preplate stage of development (E12), ApoER2 and Dab1 immunoreactivities were homogeneously distributed throughout the VZ, with Dab1 having the strongest signal (Fig. 1a–c). Radiated fibers spanning the preplate were clearly decorated using polyclonal Dab1 B3 antibody (Fig. 1a). The use of a Dab1 monoclonal antibody (H3)

Radial glia as a compartment for reelin signal decoding

Here we provide evidence that during cortical development the reelin receptor machinery, including the reelin receptors ApoER2 and VLDLR along with the cytoplasmatic adaptor protein Dab1, is located in postmitotic neurons and also in radial glia precursors. Double-labelling immunofluorescence experiments using the markers nestin and vimentin confirmed the radial glia nature of some cell populations expressing reelin receptors. Moreover, in the reeler mutant, an increase of Dab1 in radial

Acknowledgements

We are grateful to B. Howell, C. Sotelo, M. Giménez-Ribotta and M. Valdeolmillos for critical reading of the manuscript. We thank A. Goffinet, J. Nimpf, W. Stockinger and B. Howell for antibodies, cDNA and mutant tissue. We also thank C. Colmenero for technical assistance and S. Ingham and E. Fajardo for help with Fig. 6. This work was supported in part by Ministerio de Ciencia y Tecnologı́a Grants PB97-0582-CO2-01, PGC2000-2756-E and BFI 2001-1504 to A.F. and Generalitat Valenciana Grant

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