Chiaki Ohtaka-Maruyama

RP58 Regulates the Multipolar-Bipolar Transition of Newborn Neurons in the Developing Cerebral Cortex

Accumulating evidence suggests that many brain diseases are associated with defects in neuronal migration, suggesting that this step of neurogenesis is critical for brain organization. However, the molecular mechanisms underlying neuronal migration remain largely unknown. Here, we identified the zinc-finger transcriptional repressor RP58 as a key regulator of neuronal migration via multipolar-to-bipolar transition. RP58(-/-) neurons exhibited severe defects in the formation of leading processes and never shifted to the locomotion mode. Cre-mediated deletion of RP58 using in utero electroporation in RP58(flox/flox) mice revealed that RP58 functions in cell-autonomous multipolar-to-bipolar transition, independent of cell-cycle exit. Finally, we found that RP58 represses Ngn2 transcription to regulate the Ngn2-Rnd2 pathway; Ngn2 knockdown rescued migration defects of the RP58(-/-) neurons. Our findings highlight the critical role of RP58 in multipolar-to-bipolar transition via suppression of the Ngn2-Rnd2 pathway in the developing cerebral cortex.

The transcriptional repressor RP58 is crucial for cell-division patterning and neuronal survival in the developing cortex.

The neocortex and the hippocampus comprise several specific layers containing distinct neurons that originate from progenitors at specific development times, under the control of an adequate cell-division patterning mechanism. Although many molecules are known to regulate this cell-division patterning process, its details are not well understood. Here, we show that, in the developing cerebral cortex, the RP58 transcription repressor protein was expressed both in postmitotic glutamatergic projection neurons and in their progenitor cells, but not in GABAergic interneurons. Targeted deletion of the RP58 gene led to dysplasia of the neocortex and of the hippocampus, reduction of the number of mature cortical neurons, and defects of laminar organization, which reflect abnormal neuronal migration within the cortical plate. We demonstrate an impairment of the cell-division patterning during the late embryonic stage and an enhancement of apoptosis of the postmitotic neurons in the RP58-deficient cortex. These results suggest that RP58 controls cell division of progenitor cells and regulates the survival of postmitotic cortical neurons.

Spatial and temporal expression of RP58, a novel zinc finger transcriptional repressor, in mouse brain.

RP58, a novel zinc finger protein containing a POZ domain, is a sequence‐specific transcriptional repressor. To understand the role of this protein, we examined RP58 gene expression in the developing mouse brain by quantitative polymerase chain reaction (PCR) and in situ hybridization. RP58 mRNA expression was detected at embryonic day (E) 10 in the neuroepithelium, and subsequently in the ventricular zones of the cerebral cortex in the E12 embryo. Strong expression was observed in the preplate in the cerebral cortex from this stage onward. High levels of expression continued to be detected in the cortical plate and subventricular zone of the neocortex, hippocampus, and parts of the amygdala, but not in the thalamus or striatum. These results suggest that RP58 plays a crucial role in neuronal proliferation, migration, and differentiation in the developing cerebral cortex. RP58 is also expressed in the adult mouse neocortex, hippocampus, parts of the amygdala, and granule cells in the cerebellum. Double in situ hybridization using GAD67 or VGLUT1 probes revealed that RP58 is expressed in glutamatergic excitatory neurons. J. Comp. Neurol. 502:1098–1108, 2007.

Corrigendum: Molecular Pathways Underlying Projection Neuron Production and Migration during Cerebral Cortical Development

Glutamatergic neurons of the mammalian cerebral cortex originate from radial glia (RG) progenitors in the ventricular zone (VZ). During corticogenesis, neuroblasts migrate toward the pial surface using two different migration modes. One is multipolar (MP) migration with random directional movement, and the other is locomotion, which is a unidirectional movement guided by the RG fiber. After reaching their final destination, the neurons finalize their migration by terminal translocation, which is followed by maturation via dendrite extension to initiate synaptogenesis and thereby complete neural circuit formation. This switching of migration modes during cortical development is unique in mammals, which suggests that the RG-guided locomotion mode may contribute to the evolution of the mammalian neocortical 6-layer structure. Many factors have been reported to be involved in the regulation of this radial neuronal migration process. In general, the radial migration can be largely divided into four steps; (1) maintenance and departure from the VZ of neural progenitor cells, (2) MP migration and transition to bipolar cells, (3) RG-guided locomotion, and (4) terminal translocation and dendrite maturation. Among these, many different gene mutations or knockdown effects have resulted in failure of the MP to bipolar transition (step 2), suggesting that it is a critical step, particularly in radial migration. Moreover, this transition occurs at the subplate layer. In this review, we summarize recent advances in our understanding of the molecular mechanisms underlying each of these steps. Finally, we discuss the evolutionary aspects of neuronal migration in corticogenesis.

RP58 controls neuron and astrocyte differentiation by downregulating the expression of Id1–4 genes in the developing cortex

Appropriate number of neurons and glial cells is generated from neural stem cells (NSCs) by the regulation of cell cycle exit and subsequent differentiation. Although the regulatory mechanism remains obscure, Id (inhibitor of differentiation) proteins are known to contribute critically to NSC proliferation by controlling cell cycle. Here, we report that a transcriptional factor, RP58, negatively regulates all four Id genes (Id1–Id4) in developing cerebral cortex. Consistently, Rp58 knockout (KO) mice demonstrated enhanced astrogenesis accompanied with an excess of NSCs. These phenotypes were mimicked by the overexpression of all Id genes in wild‐type cortical progenitors. Furthermore, Rp58 KO phenotypes were rescued by the knockdown of all Id genes in mutant cortical progenitors but not by the knockdown of each single Id gene. Finally, we determined p57 as an effector gene of RP58‐Id‐mediated cell fate control. These findings establish RP58 as a novel key regulator that controls the self‐renewal and differentiation of NSCs and restriction of astrogenesis by repressing all Id genes during corticogenesis.

Co-localization of a novel transcriptional repressor simiRP58 with RP58

We have cloned a novel transcriptional repressor protein, termed simiRP58, which has high homology to RP58. Both simiRP58 and RP58 belong to the POZ domain and Kruppel Zn finger (POK) family of proteins. Using the luciferase assay system, we found that simiRP58 also has transcriptional repressor activity like RP58. Northern blotting and quantitative RT-PCR showed that simiRP58 was expressed in testes at the highest level. In situ hybridization of testes showed that simiRP58 is expressed by spermatocytes in only a portion of the seminiferous tubules. In contrast, expression of RP58 by spermatocytes was ubiquitous in all seminiferous tubules. Using COS-7 cells, we observed that simiRP58 was localized in the cytoplasm, which is in contrast to RP58 that was localized in the nucleus. Interestingly, co-transfection with simiRP58 and RP58 induced changes in the localization patterns of both proteins.

Synaptic transmission from subplate neurons controls radial migration of neocortical neurons

Transient instruction changes migration The brain neocortex is built by waves of neurons migrating from deep within the brain to the surface layers. Ohtaka-Maruyama et al. found that a layer of neurons that multipolar neurons encounter on their travels instructs the migrating neurons to change phenotype and direction (see the Perspective by Schinder and Lanuza). These subplate neurons form transient glutamatergic synapses with the immature migrants. This results in the migrating multipolar neurons becoming bipolar, more directed, and faster in their final migrations. Science, this issue p. 313; see also p. 265 In the developing mouse neocortex, subplate neurons form transient synapses on immature migrating multipolar neurons. The neocortex exhibits a six-layered structure that is formed by radial migration of excitatory neurons, for which the multipolar-to-bipolar transition of immature migrating multipolar neurons is required. Here, we report that subplate neurons, one of the first neuron types born in the neocortex, manage the multipolar-to-bipolar transition of migrating neurons. By histochemical, imaging, and microarray analyses on the mouse embryonic cortex, we found that subplate neurons extend neurites toward the ventricular side of the subplate and form transient glutamatergic synapses on the multipolar neurons just below the subplate. NMDAR (N-methyl-d-aspartate receptor)–mediated synaptic transmission from subplate neurons to multipolar neurons induces the multipolar-to-bipolar transition, leading to a change in migration mode from slow multipolar migration to faster radial glial-guided locomotion. Our data suggested that transient synapses formed on early immature neurons regulate radial migration.

Editorial: Mechanisms of Neuronal Migration during Corticogenesis

The mammalian neocortex shows an extremely well-organized structure that underlies higher brain functions such as cognition, language, and memory. The neocortex consists of a six-layered structure, in which excitatory and inhibitory neurons form complex neural circuits in concert with glial cells. As a result of recent technological innovations in live imaging and in utero electroporation, the processes involved in neocortical development, especially the mechanism of neuronal migration, have been successively revealed. Furthermore, it has been recognized recently that defects in neuronal migration lead to brain malformations and diverse psychiatric and neurological disorders including schizophrenia, epilepsy, and autism. Accordingly, it is important to elucidate the molecular mechanism of neuronal migration in the neocortex, in order to understand not only the basic principles of brain development but also the pathological processes of these disorders. In this special issue, we attempt to cover topics ranging from the basic mechanisms of neocortical development to the malformation and evolution of the neocortex, with a special focus on neuronal migration. Radial glial cells (RGCs) are primary progenitors capable of generating various types of neurons and glial cells, which include Cajal-Retzius cells, subplate neurons, pyramidal neurons, interneurons, oligodendrocytes, and astrocytes. Thus, it is important to know how these diverse types of cells are generated from RGCs and integrated into complex neocortical circuits. Toma and Hanashima reviewed the mechanisms that regulate the changes in RGC competency and neuronal subtype transitions, focusing on the regulatory networks of various transcription factors including Foxg1. At the earlier stage of neocortical development, RGCs predominantly produce a large number of neurons, but later they change into glia-restricted progenitors. After the discovery of the importance of astrocytes in synaptic plasticity and blood flow, the mechanisms of glial development have attracted increasing interest for many neuroscientists. Tabata reviewed the mechanism controlling the production of diverse types of astrocytes and their migration behavior, demonstrating the multiple origins of glial cells in the neocortex. Neocortical circuits consist of highly interconnected excitatory glutamatergic and inhibitory GABAergic neurons, which are generated from distinct pools of RGCs. The excitatory neurons are generated from RGCs localized in the ventricular zone of the dorsal telencephalon and migrate radially toward the pial surface in an inside-out manner (radial migration). On the other hand, inhibitory neurons mainly originate from the ventral telencephalon and migrate tangentially into the neocortex (tangential migration). In spite of such different developmental origins, both excitatory and inhibitory neurons go through the multipolar stage with several minor processes in the neocortex before axon extension. Then, they undergo dramatic morphological changes to initiate axon formation, namely, neuronal polarization. Sakakibara and Hatanaka reviewed the sequential events in polarization processes of both excitatory and inhibitory neurons, and they discussed the underlying molecular mechanisms. At the multipolar stage, the excitatory neurons transiently use a multipolar migration mode, namely migration with no fixed direction, in the subventricular and intermediate zones. Then, they adopt a bipolar shape during neuronal polarization and migrate quickly toward the pial surface along RGC processes, which is called locomotion mode. Many kinds of molecules are involved in these dynamic changes in the morphology and behavior of neurons. Small GTP binding proteins belonging to the Rho family play critical roles in cytoskeletal regulation during such dynamic processes. Azzarelli et al. reviewed the roles of Rnd proteins, “atypical” Rho family members, in neuronal migration and discussed its upstream and downstream pathways. The functions of many cytoplasmic proteins including cytoskeletal components are regulated by phosphorylation and dephosphorylation processes. Ohshima focused on protein kinases, including CDK5 and JNKs, and reviewed their regulatory roles in cytoskeletal organization during multipolar-bipolar transition and radial migration. Ohtaka-Maruyama and Okado comprehensively summarized the molecular pathways involved in these developmental processes, emphasizing the importance of subplate neurons in the development and evolution of the six-layered neocortical structure. It is apparent that neuronal migration and wiring are regulated by various secreted factors such as growth factors, chemokines, and extracellular matrix molecules, although their mechanisms are poorly understood. Kondo et al. demonstrated that subplate neurons transiently express high levels of secretary proteins such as connective tissue growth factor, neuroserpin, and insulin-like growth factor binding protein 5, which may be involved in cortical circuit formation. Greenman et al. reported a novel finding that autotaxin (ENPP2), a secretary enzyme bearing lysophospholipase D activity, regulates the localization and adhesion of neural progenitor cells independent of its catalytic activity. Maeda reviewed the roles of proteoglycans in neuronal polarization and migration and discussed the possibility that extracellular matrix regulates the distribution and activity of multiple secreted factors in the developing neocortex. In addition to the long-range gradient of secreted factors, axon pathfinding is also regulated by short-range guidance cues and direct cell-cell contacts mediated by guidepost cells. Squarzoni et al. reviewed the roles of already known guideposts such as Cajal-Retzius cells for entorhinal-hippocampal axons and corridor cells for thalamocortical axons, and further proposed a new class of guidepost cells, microglia, in the cortex. Hippocampal formation has a close relationship with the neocortex both functionally and structurally, but it shows a distinct arrangement of pyramidal neurons from that of the neocortex. Hayashi et al. reviewed the differences in the migratory behaviors of neocortical and hippocampal neurons, which lead to the formation of distinct layered structures in these two cortical regions. Defects in the migration of excitatory and inhibitory neurons can lead to the various neurological and psychiatric disorders. Kato reviewed recent development in the understanding of the genetic bases of neuronal migration disorders in terms of genotype-phenotype correlations, focusing mainly on lissencepahaly. Muraki and Tanigaki discussed the possible relationship between neuronal migration defects and behavioral abnormalities relevant to schizophrenia based on studies using genetically defined animal models. The evolutionary approaches should greatly deepen our understanding of the mechanisms underlying neocortical development. Nomura et al. established the method of in ovo electroporation and ex ovo culture of reptilian embryos. Comparative studies using this method will provide significant insights into the origin of the mammalian neocortex. It is hoped that the special issue entitled “Mechanisms of Neuronal Migration during Corticogenesis” will serve as a valuable resource for many neuroscientists to promote their research perspectives. Finally, as topic editors, we would like to express our sincere appreciation to all the authors for their outstanding contributions and to all the reviewers for their insightful comments on the papers. We also thank the editorial office and the production staff for their unceasing efforts and dedication.

RP58 represses Id3 gene promoter activity

Anatomical segregation of the functionally distinct central and peripheral nervous system (CNS and PNS) and the establishment of precise connectivity between them are fundamental in the development of the nervous system. Yet, the mechanisms underlying these processes are poorly understood. We provide evidence here that the signalling between the chemokine SDF-1 and its receptor CXCR4 contributes to the segregation and connection between the CNS and PNS in mice. SDF-1 or CXCR4 knockout mice show mis-positioned PNS boundary cap cells inside the spinal cord, disrupted glial limiting membrane and misrouted peripheral axons. Analysis of knockout mice of the second SDF-1 receptor CXCR7 showed phenotypes that are suggestive of CXCR7 being an integral component of SDF-1 signalling that controls the normal development of the boundary cap cells and the radial glial fibres.