Reelin transiently promotes N-cadherin–dependent neuronal adhesion during mouse cortical development

Abstract

Reelin is an essential glycoprotein for the establishment of the highly organized six-layered structure of neurons of the mammalian neocortex. Although the role of Reelin in the control of neuronal migration has been extensively studied at the molecular level, the mechanisms underlying Reelin-dependent neuronal layer organization are not yet fully understood. In this study, we directly showed that Reelin promotes adhesion among dissociated neocortical neurons in culture. The Reelin-mediated neuronal aggregation occurs in an N-cadherin-dependent manner, both in vivo and in vitro. Unexpectedly, however, in a rotation culture of dissociated neocortical cells that gradually reaggregated over time, we found that it was the neural progenitor cells [radial glial cells (RGCs)], rather than the neurons, that tended to form clusters in the presence of Reelin. Mathematical modeling suggested that this clustering of RGCs could be recapitulated if the Reelin-dependent promotion of neuronal adhesion were to occur only transiently. Thus, we directly measured the adhesive force between neurons and N-cadherin by atomic force microscopy, and found that Reelin indeed enhanced the adhesiveness of neurons to N-cadherin; this enhanced adhesiveness began to be observed at 30 min after Reelin stimulation, but declined by 3 h. These results suggest that Reelin transiently (and not persistently) promotes N-cadherin-mediated neuronal aggregation. When N-cadherin and stabilized β-catenin were overexpressed in the migrating neurons, the transfected neurons were abnormally distributed in the superficial region of the neocortex, suggesting that appropriate regulation of N-cadherin-mediated adhesion is important for correct positioning of the neurons during neocortical development.T he mammalian neocortex is highly organized into six neuronal layers. This laminar structure is responsible for the complex motor, sensory, and cognitive functions of the mammalian brain (1). Neuronal migration plays an important role in the establishment of this layered structure. Cortical neurons are generated within the ventricular zone (VZ) or subventricular zone (SVZ), and migrate along radial fibers toward the pial surface. Newly born excitatory neurons migrate radially into the cortical plate (CP) past the neurons born earlier, resulting in a birth date-dependent "inside-out" alignment of the neurons in the CP (2-4).Reelin is a glycoprotein that is secreted by the Cajal-Retzius cells in the marginal zone (MZ) of the cortex during neocortical development (5-7). This glycoprotein is essential for establishment of the aforementioned birth date-dependent layered structure of the neocortex, because the CP neurons show an almost inverted alignment in the neocortex of the Reelindeficient, reeler mice. In addition, neurons born at the same time tend to be distributed broadly and not to form clear layers in the reeler cortex (8, 9). Although extensive studies, including at the molecular level, have been conducted to determine the role of Reelin in neuronal migration i...

Both excitatory and inhibitory neurons transiently form clusters at the outermost region of the developing mammalian cerebral neocortex

“…Although Reelin controls multiple aspects of neocortical development, the mechanisms of Reelin function have not been completely understood. One of the important functions of Reelin is that it can cause neuronal aggregation both in vitro and in vivo (Hirota, Kubo, Fujino, Yamamoto, & Nakajima, 2018;Ishii et al, 2015;Kohno et al, 2015;Kubo et al, 2010;Matsunaga et al, 2017). Notably, the ectopic overexpression of Reelin in the developing neocortex causes neuronal aggregation in a birth-datedependent manner (Kubo et al, 2010), suggesting that this aggregation is a properly controlled phenomenon rather than simple aggregation.…”

How Do Cortical Excitatory Neurons Terminate Their Migration at the Right Place? Critical Roles of Environmental Elements

“…Interestingly, Reelin appears to play a direct role in the increase of neuron-neuron interaction. Matsunaga et al (2017) have found that application of Reelin to dissociated cortical neurons transiently enhances neuronal adhesion. This is consistent with the finding that forced Reelin expression in migrating neurons induces neuronal aggregation (Kubo et al, 2010).…”

“…Although a low level of Reelin expressed in the lower IZ (Uchida et al, 2009;Hirota et al, 2015) is reported to have a different role for migrating multipolar neurons in the IZ (initiation of the multipolar-bipolar transition/control of neuron-RG cell interaction, Jossin, 2011;Jossin and Cooper, 2011;Kon et al, 2019), accumulation of these neurons in the lower IZ (Tabata et al, 2009) may also support this view. The increase of neuron-neuron interaction seems to be N-cadherindependent (Matsunaga et al, 2017). In this context, a Reelinreceptor-Dab1 pathway likely functions, because the aggregation fails to occur when binding of Reelin to the receptor is prevented by 2A-Reelin, or when Dab1 is removed from the system.…”

“…The analysis also involves technical limitations in gene manipulation: in utero electroporation or viral infection to introduce a gene of interest usually targets neural stem cells, which can sometimes prevent us from examining the gene's role in migration or migration termination when the transgene severely impairs neuronal differentiation and/or neuronal migration in the early phase. Nevertheless, recent studies using conditional knockout mice, or temporally and/or spatially controlled gene manipulation, are increasingly uncovering the process of migration termination, with particular attention being directed to sequential changes in adhesiveness between a migrating neuron and the extracellular components, including neighboring neurons, in its environment (Franco et al, 2011;Sekine et al, 2011Sekine et al, , 2012Gil-Sanz et al, 2013;Kohno et al, 2015;Ha et al, 2017;Matsunaga et al, 2017;Hirota et al, 2018;Hatanaka et al, 2019;Hirota and Nakajima, 2020).…”

N-Cadherin Orchestrates Self-Organization of Neurons within a Columnar Unit in the Drosophila Medulla

“…Numerous studies have shown that Ncad has a wide range of functions in the developing nervous systems of fly and mammals (Redies et al, 1992;Redies and Takeichi, 1996;Hayashi and Carthew, 2004;Nern et al, 2008). Ncad is essential for the cortical organization of the mouse brain (Kadowaki et al, 2007;Matsunaga et al, 2017). Therefore, Ncad-dependent differential adhesion and interlayer interaction may be the essential mechanism underlying the 3D organization of column formation that is evolutionarily conserved from the fly optic lobe to mammalian brains.…”