The structural role of radial glial endfeet in confining spinal motor neuron somata is controlled by the Reelin and Notch pathways

Highlights

• We study a novel role of radial glia in confining motor neurons within the CNS.

• Radial glia scaffold provides a structural support to retain motor somata.

• Motor somata escape the ventral root in reeler mice.

• Enhanced Notch signals prevent ectopic motor neurons in reeler mice.

Abstract

Neuronal migration is a fundamental biological process that enables the precise positioning of neurons to form functional circuits. Cortical neurons migrate along glial scaffolds formed by radial glia guided by Reelin ligand. However, it is unclear whether the Reelin-directed behavior of radial glia is also critical for positioning the spinal neurons. Here we demonstrate a novel role of radial glia that confines motor neurons within the neural tube and is promoted by Reelin and Notch signaling. Spinal radial glia express the Dab1 adaptor for Reelin signaling and are surrounded by Reelin. In reeler mice, in which Reelin is absent, ectopic motor neurons are found outside the neural tube, although they appear to maintain their identity. Boundary cap (BC) cells, Schwann cell precursors and the basal lamina at motor exit points are intact, whereas the glia limitans of radial glia are disorganized and detached from the basement membrane. The sparse and irregular radial scaffold is wide enough to allow motor somata to pass. Forced activation of Notch signaling rescued the structural defects in radial glia in reeler mice and the appearance of extraspinal neurons. In the absence of Reelin, Notch intracellular domain (NICD) protein level was reduced. In addition, disrupting the radial glia scaffold by destroying its polarity induced ectopic motor neurons in chick embryos. These findings suggest that activation of the Notch pathways by Reelin is required to establish the radial glial scaffold, a structure that actively constrains motor neuron somata and specifies the CNS–PNS boundary.

Introduction

The precise positioning of neurons is critical for proper wiring and for brain function. Numerous modes of migration of neurons from their birthplace to their ultimate destination have been described, revealing diverse and dynamic migratory paths and cues optimized for individual neuronal subsets. In the cortex, newborn neurons undergo radial migration to reach their destined lamina, so building up the multi-layered cortex. This migration requires a specialized progenitor population called radial glia that exhibit characteristic radial processes, extending from the ventricular zone to the pial surface. Radial glia do not merely support migration but themselves divide to generate neurons that migrate along the radial fibers to reach the cortical plate (Noctor et al., 2001). The cues or guidance mechanisms that lead cells to move and stop at the right position have been extensively studied.

One of the best characterized cues that controls neuronal migration is Reelin, a ligand secreted from Cajal Retzius cells in the cortex that guide radial migration along the radial glial scaffold (D'Arcangelo et al., 1995, Rice and Curran, 2001). In reeler mice where Reelin is absent, the laminar organization of the cortex is disturbed due to defective radial migration. As a result, reeler mice display severe behavioral defects including a reeling gait, postural imbalance and so on. The role of Reelin in neuronal migration is not limited to the cortex, since Reelin and its signaling components are widely expressed in the CNS (Rice and Curran, 2001, Yip et al., 2004). Some neurons in other areas of the CNS such as the cerebellum, hippocampus, brainstem and spinal cord are also mis-positioned in reeler mice, pointing to a role of Reelin throughout the CNS (Forster et al., 2002, Hartfuss et al., 2003, Pinto-Lord et al., 1982). Interestingly, in reeler mice, the radial glial cells are also disorganized or reduced in number in the brain, suggesting a link between Reelin signaling and radial glia in neuronal migration (Benjelloun-Touimi et al., 1985, Forster et al., 2002, Fukaya et al., 1999, Hartfuss et al., 2003, Pinto-Lord et al., 1982). However, our understanding of the role of Reelin and radial glia-guided migration in other regions of the CNS is limited.

Unlike neuronal movement in the cortex, the migration of spinal neurons is rather simple: some neurons require radial glia for their migration and others do not (Barry et al., 2012, Yip et al., 2000). For instance, pregangionic column (PGC) motor neurons that innervate the peripheral autonomic nervous system and LMC neurons that connect to the limb are mispositioned within the neural tube when either Reelin or its adaptor, Dab1, is absent (Palmesino et al., 2010, Phelps et al., 2002, Yip et al., 2000). The existence of radial glia and the prominent expression of major genes that deliver Reelin signaling in the spinal cord raise the possibility that Reelin may serve an additional role in the developing spinal cord (Barry and McDermott, 2005, Luque et al., 2003, Yip et al., 2004).

The spinal cord is a unique structure in that it builds boundaries between the CNS and PNS: the dorsal root entry zone (DREZ) and ventral motor exit points (MEPs). At the DREZ, sensory axons enter from the dorsal root ganglia to deliver sensory information to the CNS. Motor neurons whose cell bodies are located within the ventral neural tube extend their axons to pass the ventral motor exit points and send out motor functions to the periphery. Thus, the barrier that defines and maintains the CNS–PNS transition zone must be formed during development. Boundary cap (BC) cells are well-characterized structures that separate the CNS and PNS and are of neural crest origin. Removal of either BC cells themselves or repulsive cues within them results in an emigration of motor somata out of the neural tube, indicating that they form a physical barrier at the transition zone (Chauvet and Rougon, 2008, Mauti et al., 2007, Vermeren et al., 2003). Alternatively, other peripheral glia such as perineurial glia or Schwann cells play similar roles in restricting the motor neurons, which otherwise are inclined to escape from the neural tube (Coulpier et al., 2010, Kucenas et al., 2008). However, little is known about the cues coming from the CNS that are responsible for securing the transition zone.

Here we propose a novel role for the radial glia as a CNS-derived population that confines the motor neurons. At the MEPs, the spinal radial glia develop an extensive web of glial endfeet which only allows motor axons to pass but not cell bodies (Barry et al., 2012). We show that in reeler mice, the organization of the radial glial endfeet and their attachment to the basement membrane are disrupted at the MEPs, coincident with the presence of extraspinal motor neurons. The identities of the ectopic motor neurons suggest that the Reelin signal affects most motor neurons, especially the lateral motor column (LMC) neurons at the brachial level, and the hypaxial motor column (HMC) neurons at the thoracic level. Furthermore, when we enhanced Notch activity by misexpressing the notch intracellular domain (NICD) in reeler mice the radial glia phenotype was rescued so that the glia limitans, the endfeet structure of the radial glia, became regular and cohesive as in the wild type. We observed that the amount of NICD protein was reduced in reeler spinal cords, suggesting a link between Reelin and Notch signaling. Our observations suggest that Reelin signaling activates the Notch pathway and promotes radial glia formation, which plays a critical role in constraining motor neurons within the neural tube.