Akiyo Takahashi

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.

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.

Visualization of RP58 promoter activity in neural progenitor cells using luminescence live cell imaging system

Although del Río Hortega originally reported that leptomeningeal cells are the direct source of ramified microglia in the developing brain, recent views seem not to pay much attention to this notion. In this study, in vitro experiments were conducted to determine whether leptomeninges generate ramified microglia. The leptomeninges of neonatal rats containing Iba1+ macrophages were peeled off the brain surface. Leptomeningeal macrophages expressed CD68 and CD163 strongly, but not microglia in the brain parenchyma. Leptomeningeal macrophages expressed epidermal growth factor receptor (EGFR) as revealed by RT-PCR and immunohistochemical staining. Cells obtained from the peeled-off leptomeninges were cultured in a serum-free medium containing EGF for 4 or 6 d resulting in the formation of large cell aggregates, in which many proliferating macrophages were present. In contrast to EGF, macrophage-colony stimulating factor (M-CSF) did not enhance the generation of Iba1+ cells from the leptomeningeal culture. The cell aggregates generated ramified Iba1+ cells in the presence of fetal calf serum, which express CD68 and CD163 at lower levels than primary microglia isolated from a mixed glial culture. Therefore, the leptomeningederived cells resembled parenchymal microglia better than the primary microglia. Furthermore, it was found that cultured leptomeninges gave rise to not only microglia but also other types of cells including NG2+ ramified cells resembling NG2 cells (or oligodendrocytes progenitor cells) in vivo. To see if similar events to these in vitro observations occur in vivo, introduction of lentiviral vector carrying GFP-gene into subdural space is now conducting. This study suggests that microglial progenitors expressing EGFR reside in the leptomeninges and that there is a population of microglia that grow independently of M-CSF, and also suggests the possibility that the leptomeninges function as a source of multiple types of cells in the brain parenchyma.

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.