Cyclin dependent kinase inhibitorによる神経前駆細胞の分化誘導と脳形成

大脳皮質は知覚,認知,思考等の高次機能を担う中枢である.哺乳類では大脳皮質は6層構造をなし,それぞれの層では機能的,形態的に類似した細胞集団が異なる領域からの投射をやり取りしている.神経細胞およびグリア細胞は,胎生期から生後にかけて神経前駆細胞(Neural progenitor cell,NPC)から分裂し産生される.NPCは時期に応じて神経細胞,続いてグリア細胞へ分化し,最終的な脳の構造が完成する.NPCから分化する細胞運命の決定には,細胞分裂を中止して分化を始める機構が必要であり,その中でもCyclin dependent kinase inhibitors(CKIs)の働きが注目されている.今回我々はこのCKIsのひとつであるp18遺伝子を用いて胎児マウス脳の発生に与える影響を解析した.方法としては胎生13～17日目(E13-17)のマウス胎児脳にp18遺伝子を子宮内エレクトロポレーション法によって強制発現させ,その後に脳組織内での分布を観察した.またCre-LoxP を用いた蛍光蛋白発現システムで個々の細胞形態の立体再構築を行った.その結果,p18遺伝子発現群はコントロール群に比べて多く脳室面側に留まり,特にE14, 15において顕著に認められた.更に遺伝子導入した細胞において分化後の形態を観察したところ,生後脳においてp18遺伝子発現群ではアストロサイトの形態をした細胞が増えており,免疫組織学的染色からもこれらの細胞がアストロサイトであることが示唆された.この細胞はE13～E17いずれの時期においても増加していたが,特にE14,15で顕著に認めたことから,この時期にニューロンの分化からアストロサイトの分化に切り替わる何らかのメカニズムが存在していると考えられた.今回の研究によってNPCがニューロンへの分化からアストロサイトへの分化に切り替わるメカニズムにCKIsが関与している可能性が示唆された.アストロサイトはグルタミントランスポータの発現により,二次的脳損傷から神経細胞を保護しており,その発生が解明されることで低酸素性虚血性脳症の治療への応用も期待される.The cerebral cortex plays a central role in high-level functions such as thinking, perception, and cognition. In mammals, the cerebral cortex has a six-layered structure, and each layer contains cells with similar functions and shapes that exchange signals from different regions of the brain. Neurons and glial cells are produced by differentiation from neural progenitor cells(NPC)from the embryonic stage up to the postnatal stage. NPCs are differentiated into neurons at the embryonic stage, and switch into glial cells at birth, before the structure of the brain is mature. Some molecules have been proposed to determine the cell fate of NPCs by stopping cell division and initiating differentiation. Cyclindependent kinase inhibitors(CKIs)are considered to be important molecular factors in this process. Here we used a CKI, the p18 gene, and analyzed its effect on the formation of the fetal mouse brain. For this purpose, we expressed the p18 gene in the mouse fetal brain at embryonic day 13 to day 17(E13-17), and observed the distribution of the electroporated cells in the cortical tissue. Furthermore, we performed three-dimensional reconstruction of the individual cellular morphology by using the Cre-LoxP fluorescent protein expression system. As a result, the p18-gene expressing cells remained in the ventricular zone more than the control cells. The difference was particularly remarkable at E14 and E15. Importantly, we observed a differentiated cell shape in electroporated cells, and found that the proportion of astrocyte-like cells was greatly increased in the p18 gene-expressing group compared to the control group. We confirmed that these cells were indeed astrocytes with immunohistochemical staining. The number of such astrocyte-like cells increased at any time of electroporation from E13 to E17, especially at E14 and E15. Therefore, we propose that there might be some mechanisms that control the switch from neurons to astrocytes at this stage of development. In conclusion, our study suggests that CKIs are involved in the differentiation program of NPCs from neurons to astrocytes. Astrocytes are known to protect neurons from secondary brain damage by the expression of glutamine transporter. Therefore, elucidating the mechanism of their generation is expected to have applications for the treatment of hypoxic-ischemic encephalopathy in the future

Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain

In proliferating neural epithelia, cells undergo interkinetic nuclear migration: stereotyped cell cycle-dependent movements in the apico-basal plane. The microtubule-binding protein Tpx2 is here shown to regulate the G2-phase basal-to-apical migration, while passive displacement effects are responsible for basally directed movements

Deubiquitinating enzymes regulate Hes1 stability and neuronal differentiation.

Hairy and enhancer of split 1 (Hes1), a basic helix-loop-helix transcriptional repressor protein, regulates the maintenance of neural stem/progenitor cells by repressing proneural gene expression via Notch signaling. Previous studies showed that Hes1 expression oscillates in both mouse embryonic stem cells and neural stem cells, and that the oscillation contributes to their potency and differentiation fates. This oscillatory expression depends on the stability of Hes1, which is rapidly degraded by the ubiquitin/proteasome pathway. However, the detailed molecular mechanisms governing Hes1 stability remain unknown. We analyzed Hes1-interacting deubiquitinases purified from mouse embryonic stem cells using an Hes1-specific antibody, and identified the ubiquitin-specific protease 27x (Usp27x) as a new regulator of Hes1. We found that Hes1 was deubiquitinated and stabilized by Usp27x and its homologs ubiquitin-specific protease 22 (Usp22) and ubiquitin-specific protease 51 (Usp51). Knockdown of Usp22 shortened the half-life of Hes1, delayed its oscillation, and enhanced neuronal differentiation in mouse developing brain, whereas mis-expression of Usp27x reduced neuronal differentiation. These results suggest that these deubiquitinases modulate Hes1 protein dynamics by removing ubiquitin molecules, and thereby regulate neuronal differentiation of stem cells

Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells

At the onset of neurogenesis in the mammalian central nervous system, neuroepithelial cells switch from symmetric, proliferative to asymmetric, neurogenic divisions. In analogy to the asymmetric division of Drosophila neuroblasts, this switch of mammalian neuroepithelial cells is thought to involve a change in cleavage plane orientation from perpendicular (vertical cleavage) to parallel (horizontal cleavage) relative to the apical surface of the neuroepithelium. Here, we report, using TIS21-GFP knock-in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes. Remarkably, however, neurogenic divisions of neuroepithelial cells, but not proliferative ones, involve an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1–2%) of the entire neuroepithelial cell plasma membrane. Our results support a novel concept for the cell biological basis of asymmetric, neurogenic divisions of neuroepithelial cells in the mammalian central nervous system

Novel and robust transplantation reveals the acquisition of polarized processes by cortical cells derived from mouse and human pluripotent stem cells

Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apico-basal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Novel and robust transplantation reveals the acquisition of polarized processes by cortical cells derived from mouse and human pluripotent stem cells

Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apico-basal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.status: publishe

Lamin B1 as a key modulator of the developing and aging brain

Lamin B1 is an essential protein of the nuclear lamina that plays a crucial role in nuclear function and organization. It has been demonstrated that lamin B1 is essential for organogenesis and particularly brain development. The important role of lamin B1 in physiological brain development and aging has only recently been at the epicenter of attention and is yet to be fully elucidated. Regarding the development of brain, glial cells that have long been considered as supporting cells to neurons have overturned this representation and current findings have displayed their active roles in neurogenesis and cerebral development. Although lamin B1 has increased levels during the differentiation of the brain cells, during aging these levels drop leading to senescent phenotypes and inciting neurodegenerative disorders such as Alzheimer's and Parkinson's disease. On the other hand, overexpression of lamin B1 leads to the adult-onset neurodegenerative disease known as Autosomal Dominant Leukodystrophy. This review aims at highlighting the importance of balancing lamin B1 levels in glial cells and neurons from brain development to aging.Summary of the involvement of lamin B1 in the different processes of brain development and aging. Created with BioRender.com