Research reportLocalization of ApoER2, VLDLR and Dab1 in radial glia: groundwork for a new model of reelin action during cortical development
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
References (44)
- et al.
Reelin is a ligand for lipoprotein receptors
Neuron
(1999) Evidence for the occurrence of early modifications in the ‘Glia limitans’ layer of the neocortex of the reeler mutant mice
Neurosci. Lett.
(1979)Comparative localization of Cajal–Retzius cells in the neocortex of normal and reeler mutant mice fetuses
Neurosci. Lett.
(1985)- et al.
Reelin binds alpha3beta1 integrin and inhibits neuronal migration
Neuron
(2000) - et al.
Cortical development: receiving reelin
Curr. Biol.
(2000) - et al.
Characterization of CNS precursor subtypes and radial glia
Dev. Biol.
(2001) - et al.
Direct binding of reelin to VLDL receptor and ApoE receptor 2 induces tyrosine phosphorylation of disabled-1 and modulates tau phosphorylation
Neuron
(1999) - et al.
Whole-mount confocal immunofluorescence of mammalian CNS
Brain Res. Protoc.
(2001) - et al.
Rescue of ataxia and preplate splitting by ectopic expression of reelin in reeler mice
Neuron
(2002) - et al.
Asymmetric inheritance of radial glial fibers by cortical neurons
Neuron
(2001)