Yukako Yokota

Essential Roles for GSK-3s and GSK-3-Primed Substrates in Neurotrophin-Induced and Hippocampal Axon Growth

Glycogen synthase kinase-3beta (GSK-3beta) is thought to mediate morphological responses to a variety of extracellular signals. Surprisingly, we found no gross morphological deficits in nervous system development in GSK-3beta null mice. We therefore designed an shRNA that targeted both GSK-3 isoforms. Strong knockdown of both GSK-3alpha and beta markedly reduced axon growth in dissociated cultures and slice preparations. We then assessed the role of different GSK-3 substrates in regulating axon morphology. Elimination of activity toward primed substrates only using the GSK-3 R96A mutant was associated with a defect in axon polarity (axon branching) compared to an overall reduction in axon growth induced by a kinase-dead mutant. Consistent with this finding, moderate reduction of GSK-3 activity by pharmacological inhibitors induced axon branching and was associated primarily with effects on primed substrates. Our results suggest that GSK-3 is a downstream convergent point for many axon growth regulatory pathways and that differential regulation of primed versus all GSK-3 substrates is associated with a specific morphological outcome.

α3β1 integrin modulates neuronal migration and placement during early stages of cerebral cortical development

We show that α3 integrin mutation disrupts distinct aspects of neuronal migration and placement in the cerebral cortex. The preplate develops normally in α3 integrin mutant mice. However, time lapse imaging of migrating neurons in embryonic cortical slices indicates retarded radial and tangential migration of neurons, but not ventricular zone-directed migration. Examination of the actin cytoskeleton of α3 integrin mutant cortical cells reveals aberrant actin cytoskeletal dynamics at the leading edges. Deficits are also evident in the ability of developing neurons to probe their cellular environment with filopodial and lamellipodial activity. Calbindin or calretinin positive upper layer neurons as well as the deep layer neurons ofα 3 integrin mutant mice expressing EGFP were misplaced. These results suggest that α3β1 integrin deficiency impairs distinct patterns of neuronal migration and placement through dysregulated actin dynamics and defective ability to search and respond to migration modulating cues in the developing cortex.

Disrupted-in-Schizophrenia-1 expression is regulated by β-site amyloid precursor protein cleaving enzyme-1-neuregulin cascade

Neuregulin-1 (NRG1) and Disrupted-in-Schizophrenia-1 (DISC1) are promising susceptibility factors for schizophrenia. Both are multifunctional proteins with roles in a variety of neurodevelopmental processes, including progenitor cell proliferation, migration, and differentiation. Here, we provide evidence linking these factors together in a single pathway, which is mediated by ErbB receptors and PI3K/Akt. We show that signaling by NRG1 and NRG2, but not NRG3, increase expression of an isoform of DISC1 in vitro. Receptors ErbB2 and ErbB3, but not ErbB4, are responsible for transducing this effect, and PI3K/Akt signaling is also required. In NRG1 knockout mice, this DISC1 isoform is selectively reduced during neurodevelopment. Furthermore, a similar decrease in DISC1 expression is seen in β-site amyloid precursor protein cleaving enzyme–1 (BACE1) knockout mice, in which NRG1/Akt signaling is reportedly impaired. In contrast to neuronal DISC1 that was reported and characterized, expression of DISC1 in other types of cells in the brain has not been addressed. Here we demonstrate that DISC1, like NRG and ErbB proteins, is expressed in neurons, astrocytes, oligodendrocytes, microglia, and radial progenitors. These findings may connect NRG1, ErbBs, Akt, and DISC1 in a common pathway, which may regulate neurodevelopment and contribute to susceptibility to schizophrenia.

The Adenomatous Polyposis Coli Protein Is an Essential Regulator of Radial Glial Polarity and Construction of the Cerebral Cortex

Radial glia are highly polarized cells that serve as neuronal progenitors and as scaffolds for neuronal migration during construction of the cerebral cortex. How radial glial cells establish and maintain their morphological polarity is unknown. Using conditional gene targeting in mice, we demonstrate that adenomatous polyposis coli (APC) serves an essential function in the maintenance of polarized radial glial scaffold during brain development. In the absence of APC, radial glial cells lose their polarity and responsiveness to the extracellular polarity maintenance cues, such as neuregulin-1. Elimination of APC further leads to marked instability of the radial glial microtubule cytoskeleton. The resultant changes in radial glial function and loss of APC in radial glial progeny lead to defective generation and migration of cortical neurons, severely disrupted cortical layer formation, and aberrant axonal tract development. Thus, APC is an essential regulator of radial glial polarity and is critical for the construction of cerebral cortex in mammals.

Radial Glial Dependent and Independent Dynamics of Interneuronal Migration in the Developing Cerebral Cortex

Interneurons originating from the ganglionic eminence migrate tangentially into the developing cerebral wall as they navigate to their distinct positions in the cerebral cortex. Compromised connectivity and differentiation of interneurons are thought to be an underlying cause in the emergence of neurodevelopmental disorders such as schizophrenia. Previously, it was suggested that tangential migration of interneurons occurs in a radial glia independent manner. Here, using simultaneous imaging of genetically defined populations of interneurons and radial glia, we demonstrate that dynamic interactions with radial glia can potentially influence the trajectory of interneuronal migration and thus the positioning of interneurons in cerebral cortex. Furthermore, there is extensive local interneuronal migration in tangential direction opposite to that of pallial orientation (i.e., in a medial to lateral direction from cortex to ganglionic eminence) all across the cerebral wall. This counter migration of interneurons may be essential to locally position interneurons once they invade the developing cerebral wall from the ganglionic eminence. Together, these observations suggest that interactions with radial glial scaffold and localized migration within the expanding cerebral wall may play essential roles in the guidance and placement of interneurons in the developing cerebral cortex.

Arl13b-regulated cilia activities are essential for polarized radial glial scaffold formation

The construction of cerebral cortex begins with the formation of radial glia. Once formed, polarized radial glial cells divide either symmetrically or asymmetrically to balance appropriate production of progenitor cells and neurons. Following birth, neurons use the processes of radial glia as scaffolding for oriented migration. Radial glia therefore provide an instructive structural matrix to coordinate the generation and placement of distinct groups of cortical neurons in the developing cerebral cortex. We found that Arl13b, a cilia-enriched small GTPase that is mutated in Joubert syndrome, was critical for the initial formation of the polarized radial progenitor scaffold. Using developmental stage–specific deletion of Arl13b in mouse cortical progenitors, we found that early neuroepithelial deletion of ciliary Arl13b led to a reversal of the apical–basal polarity of radial progenitors and aberrant neuronal placement. Arl13b modulated ciliary signaling necessary for radial glial polarity. Our findings indicate that Arl13b signaling in primary cilia is crucial for the initial formation of a polarized radial glial scaffold and suggest that disruption of this process may contribute to aberrant neurodevelopment and brain abnormalities in Joubert syndrome–related ciliopathies.

Nap1-regulated neuronal cytoskeletal dynamics is essential for the final differentiation of neurons in cerebral cortex.

The cytoskeletal regulators that mediate the change in the neuronal cytoskeletal machinery from one that promotes oriented motility to one that facilitates differentiation at the appropriate locations in the developing neocortex remain unknown. We found that Nck-associated protein 1 (Nap1), an adaptor protein thought to modulate actin nucleation, is selectively expressed in the developing cortical plate, where neurons terminate their migration and initiate laminar-specific differentiation. Loss of Nap1 function disrupts neuronal differentiation. Premature expression of Nap1 in migrating neurons retards migration and promotes postmigratory differentiation. Nap1 gene mutation in mice leads to neural tube and neuronal differentiation defects. Disruption of Nap1 retards the ability to localize key actin cytoskeletal regulators such as WAVE1 to the protrusive edges where they are needed to elaborate process outgrowth. Thus, Nap1 plays an essential role in facilitating neuronal cytoskeletal changes underlying the postmigratory differentiation of cortical neurons, a critical step in functional wiring of the cortex.

MARCKS modulates radial progenitor placement, proliferation and organization in the developing cerebral cortex

The radial glial cells serve as neural progenitors and as a migratory guide for newborn neurons in the developing cerebral cortex. These functions require appropriate organization and proliferation of the polarized radial glial scaffold. Here, we demonstrate in mice that the myristoylated alanine-rich C-kinase substrate protein (MARCKS), a prominent cellular substrate for PKC, modulates radial glial placement and expansion. Loss of MARCKS results in ectopic collection of mitotically active radial progenitors away from the ventricular zone (VZ) in the upper cerebral wall. Apical restriction of key polarity complexes [CDC42, β-catenin (CTNNB1), N-cadherin (CDH2), myosin IIB (MYOIIB), aPKCζ, LGL, PAR3, pericentrin, PROM1] is lost. Furthermore, the radial glial scaffold in Marcks null cortex is compromised, with discontinuous, non-radial processes apparent throughout the cerebral wall and deformed, bulbous, unbranched end-feet at the basal ends. Further, the density of radial processes within the cerebral cortex is reduced. These deficits in radial glial development culminate in aberrant positioning of neurons and disrupted cortical lamination. Genetic rescue experiments demonstrate, surprisingly, that phosphorylation of MARCKS by PKC is not essential for the role of MARCKS in radial glial cell development. By contrast, the myristoylation domain of MARCKS needed for membrane association is essential for MARCKS function in radial glia. The membrane-associated targeting of MARCKS and the resultant polarized distribution of signaling complexes essential for apicobasal polarity may constitute a critical event in the appropriate placement, proliferation and organization of polarized radial glial scaffold in the developing cerebral cortex.

Cdc42 and Gsk3 modulate the dynamics of radial glial growth, inter-radial glial interactions and polarity in the developing cerebral cortex

Polarized radial glia are crucial to the formation of the cerebral cortex. They serve as neural progenitors and as guides for neuronal placement in the developing cerebral cortex. The maintenance of polarized morphology is essential for radial glial functions, but the extent to which the polarized radial glial scaffold is static or dynamic during corticogenesis remains an open question. The developmental dynamics of radial glial morphology, inter-radial glial interactions during corticogenesis, and the role of the cell polarity complexes in these activities remain undefined. Here, using real-time imaging of cohorts of mouse radial glia cells, we show that the radial glial scaffold, upon which the cortex is constructed, is highly dynamic. Radial glial cells within the scaffold constantly interact with one another. These interactions are mediated by growth cone-like endfeet and filopodia-like protrusions. Polarized expression of the cell polarity regulator Cdc42 in radial glia regulates glial endfeet activities and inter-radial glial interactions. Furthermore, appropriate regulation of Gsk3 activity is required to maintain the overall polarity of the radial glia scaffold. These findings reveal dynamism and interactions among radial glia that appear to be crucial contributors to the formation of the cerebral cortex. Related cell polarity determinants (Cdc42, Gsk3) differentially influence radial glial activities within the evolving radial glia scaffold to coordinate the formation of cerebral cortex.

Generation and characterization of brain lipid-binding protein promoter-based transgenic mouse models for the study of radial glia.

Radial glia play an essential role in the generation of the cerebral cortex through their function as neuronal precursors and as neuronal migration guides. A molecular marker for radial glia in the developing central nervous system is the brain lipid‐binding protein (BLBP). To generate mouse models for the visualization and study of radial glia, we expressed EGFP, EYFP, or dsRed2 in transgenic mice under the control of the BLBP promoter. In these transgenic lines, fluorescent protein expression is restricted to radial glia in the embryonic cortex and to astrocytes in the adult brain. Electroporation of the transgenes into embryonic cortex also resulted in radial glia‐specific transgene expression. These BLBP promoter driven transgenic mice and organotypic brain slices expressing different fluorescent markers in a radial glia‐specific manner will be useful tools to further study the differentiation and function of radial glia in distinct regions of the developing CNS.