Cep120 and TACCs control interkinetic nuclear migration and the neural progenitor pool

Centrosome- and microtubule-associated proteins have been shown to be important for maintaining the neural progenitor pool during neocortical development by regulating the mitotic spindle. It remains unclear whether these proteins may control neurogenesis by regulating other microtubule-dependent processes such as nuclear migration. Here, we identify Cep120, a centrosomal protein preferentially expressed in neural progenitors during neocortical development. We demonstrate that silencing Cep120 in the developing neocortex impairs both interkinetic nuclear migration (INM), a characteristic pattern of nuclear movement in neural progenitors, and neural progenitor self-renewal. Furthermore, we show that Cep120 interacts with transforming acidic coiled-coil proteins (TACCs) and that silencing TACCs also causes defects in INM and neural progenitor self-renewal. Our data suggest a critical role for Cep120 and TACCs in both INM and neurogenesis. We propose that sustaining INM may be a mechanism by which microtubule-regulating proteins maintain the neural progenitor pool during neocortical development.     

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

A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. During S to G2 progression, the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2-phase by altering microtubule organization. Thus, Tpx2 links cell-cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.     

Hex homeobox gene controls the transition of the endoderm to a pseudostratified, cell emergent epithelium for liver bud development

Little is known about the mechanism by which embryonic liver, lung, and pancreas progenitor cells emerge from the endodermal epithelium to initiate organogenesis. Understanding this process and its genetic control provides insight into ontogeny, developmental abnormalities, and tissue regeneration. We find that shortly after hepatic endoderm cells are specified, they undergo a transition from a columnar, gut morphology to a pseudostratified morphology, with concomitant "interkinetic nuclear migration" (INM) during cell division. INM is a hallmark of pseudostratified epithelia and the process used by neural progenitors to emerge from the neural epithelium. We find that the transition of the hepatic endoderm, but not the neural epithelium, to a pseudostratified epithelium is dependent upon the cell-autonomous activity of the homeobox gene Hex. In the absence of Hex, hepatic endoderm cells survive but maintain a columnar, simple epithelial phenotype and ectopically express Shh and other genes characteristic of the midgut epithelium. Thus, Hex promotes endoderm organogenesis by promoting the transition to a pseudostratified epithelium, which in turn allows hepatoblasts to emerge into the stromal environment and continue differentiating.     

Maternal sevoflurane exposure induces temporary defects in interkinetic nuclear migration of radial glial progenitors in the fetal cerebral cortex through the Notch signalling pathway

ObjectivesThe effects of general anaesthetics on fetal brain development remain elusive. Radial glial progenitors (RGPs) generate the majority of neurons in developing brains. Here, we evaluated the acute alterations in RGPs after maternal sevoflurane exposure.MethodsPregnant mice were exposed to 2.5% sevoflurane for 6 hours on gestational day 14.5. Interkinetic nuclear migration (INM) of RGPs in the ventricular zone (VZ) of the fetal brain was evaluated by thymidine analogues labelling. Cell fate of RGP progeny was determined by immunostaining using various neural markers. The Morris water maze (MWM) was used to assess the neurocognitive behaviours of the offspring. RNA sequencing (RNA‐Seq) was performed for the potential mechanism, and the potential mechanism validated by quantitative real‐time PCR (qPCR), Western blot and rescue experiments. Furthermore, INM was examined in human embryonic stem cell (hESC)‐derived 3D cerebral organoids.ResultsMaternal sevoflurane exposure induced temporary abnormities in INM, and disturbed the cell cycle progression of RGPs in both rodents and cerebral organoids without cell fate alternation. RNA‐Seq analysis, qPCR and Western blot showed that the Notch signalling pathway was a potential downstream target. Reactivation of Notch by Jag1 and NICD overexpression rescued the defects in INM. Young adult offspring showed no obvious cognitive impairments in MWM.ConclusionsMaternal sevoflurane exposure during neurogenic period temporarily induced abnormal INM of RGPs by targeting the Notch signalling pathway without inducing long‐term effects on RGP progeny cell fate or offspring cognitive behaviours. More importantly, the defects of INM in hESC‐derived cerebral organoids provide a novel insight into the effects of general anaesthesia on human brain development.     

A role for CK2 upon interkinetic nuclear migration in the cell cycle of retinal progenitor cells

In developing retina, the nucleus of the elongated neuroepithelial cells undergoes interkinetic nuclear migration (INM), that is it migrates back and forth across the proliferative layer during the cell cycle. S-phase occurs at the basal side, while M-phase occurs at the apical margin of the retinal progenitors. G1 and G2-phases occur along the nuclear migration pathway. We tested whether this feature of the retinal cell cycle is controlled by CK2, which, among its many substrates, phosphorylates both molecular motors and cytoskeletal components. Double immunolabeling showed that CK2 is contained in BrdU-labeled retinal progenitors. INM was examined after pulse labeling the retina of newborn rats with BrdU, by plotting nuclear movement from basal to apical sides of the retinal progenitors during G2. The CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole inhibited the activity of rat retinal CK2, and blocked nuclear movement proper in a dose-dependent way. No apoptosis was detected, and total numbers of BrdU-labeled nuclei remained constant following treatment. Immunohistochemistry showed that, following inhibition of CK2, the tubulin cytoskeleton is disorganized, with reduced acetylated and increased tyrosinated tubulin. This indicates a reduction in stable microtubules, with accumulation of free tubulin dimers. The results show that CK2 activity is required for INM in retinal progenitor cells.     

Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient

The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.     

Endothelin/endothelin-B receptor signals regulate ventricle-directed interkinetic nuclear migration of cerebral cortical neural progenitors

We determined the expression profile of ～300 G protein-coupled receptors (GPCRs) in embryonic cortical neural progenitor cells (NPCs) and identified a number of highly expressed GPCRs, among which endothelin-B receptor (ET(B)-R) was expressed at the highest level. We also revealed that endothelins (ETs) were predominantly expressed in CD31-positive endothelial cells of the embryonic cerebral cortex. Activation of ET(B)-R induced NPC assembly in vitro by promoting fibronectin-dependent-motility and N-cadherin-associated cell contact. NPC assembly also required a Rho-family GTPase(s) and phosphatidylinositol-3-kinase. In the embryonic cerebral cortex, a specific ET(B)-R agonist, IRL-1620, accelerated interkinetic nuclear migration (INM) of NPCs toward the ventricular wall (VW) ex vivo. Conversely, a specific ET(B)-R antagonist, BQ788, slowed INM, thereby inducing mislocalization of phospho-histone H3-positive M-phase nuclei in the ventricular zone (VZ) and decreasing the number of Tuj1-positive newborn neurons. Our results suggest that ET(B)-R-mediated assembly signals drive INM that precedes neurogenesis.     

Development of three-dimensional architecture of the neuroepithelium: Role of pseudostratification and cellular 'community'

This review discusses the development of the neuroepithelium (NE) and its derivative ventricular zone (VZ), from which the central nervous system (CNS) is formed. First, the histological features of the NE and VZ are summarized, highlighting the phenomenon of pseudostratification, which is achieved by polarization and interkinetic nuclear migration (INM) of neural progenitor cells. Next, our current understanding of the cellular and molecular mechanisms and biological significance of INM and pseudostratification are outlined. The recent three-dimensional time-lapse observations revealing heterogeneity in cell lineages within the NE and VZ are also described, focusing on the neuronal lineage. Finally, the necessity of comprehensive studies on cell-cell interactions in the NE/VZ is discussed, as well as the importance of electrophysiological and biomechanical approaches. In particular, we suggest that a systems biology approach to the NE/VZ as a cellular 'community' may be fruitful.     

Neural progenitors isolated from newborn rat spinal cords differentiate into neurons and astroglia

Permanent functional deficit in patients with spinal cord injury (SCI) is in part due to severe neural cell death. Therefore, cell replacement using stem cells and neural progenitors that give rise to neurons and glia is thought to be a potent strategy to promote tissue repair after SCI. Many studies have shown that stem cells and neural progenitors can be isolated from embryonic, postnatal and adult spinal cords. Recently, we isolated neural progenitors from newborn rat spinal cords. In general, the neural progenitors grew as spheres in culture, and showed immunoreactivity to a neural progenitor cellular marker, nestin. They were found to proliferate and differentiate into glial fibrillary acidic protein-positive astroglia and multiple neuronal populations, including GABAergic and cholinergic neurons. Neurotrophin 3 and neurotrophin 4 enhanced the differentiation of neural progenitors into neurons. Furthermore, the neural progenitors that were transplanted into contusive spinal cords were found to survive and have migrated in the spinal cord rostrally and caudally over 8 mm to the lesion center 7 days after injury. Thus, the neural progenitors isolated from newborn rat spinal cords in combination with neurotrophic factors may provide a tool for cell therapy in SCI patients.     

Expression of yeast INM1 encoding inositol monophosphatase is regulated by inositol, carbon source and growth stage and is decreased by lithium and valproate

Inositol monophosphatase plays a vital role in the de novo biosynthesis of inositol and in the phosphoinositide second messenger signalling pathway. We cloned the Saccharomyces cerevisiae open reading frame (ORF) YHR046c (termed INM1), which encodes inositol monophosphatase, characterized the protein Inm1p and analysed expression of the INM1 gene. INM1 was expressed in bacteria under the control of the lacZ promoter. The purified protein has inositol monophosphatase activity that is inhibited by the antibipolar drug lithium, but not valproate. In the inm1Delta:URA3 null mutant, inositol monophosphatase activity was reduced but not eliminated. The disruption had little effect on growth in the presence of lithium or valproate and no effect on growth in the absence of inositol. To characterize the regulation of INM1, we examined the effects of inositol, carbon source, growth phase, and the antibipolar drugs lithium and valproate on INM1 expression using an INM1-lacZ reporter gene. Unlike all other phospholipid biosynthetic enzyme-encoding genes studied, which contain the UASINO regulatory element, INM1 expression is increased in the presence of inositol. In addition, INM1 expression was repressed during growth in glycerol and derepressed as glucose-grown cells entered stationary. Both lithium and valproate, which cause a decrease in intracellular inositol, effect a decrease in INM1 expression. A model is presented to account for regulation of INM1 expression.     

Identification of neural progenitors in the adult mammalian eye

We have shown that the embryonic mammalian retina contains neural progenitors which display stem cell properties in vitro. Here we report the characterization of neural progenitors isolated from the adult mammalian eye. These quiescent cells, located in the pigmented ciliary bodies, proliferate in the presence of FGF2 and express the neuroectodermal marker nestin. The proliferating cells give rise to neural spheres and are multipotential; they express cell type-specific markers corresponding to neurons and glia. In addition, neural progenitors can generate secondary neural spheres, thus displaying potential to self-renew. The ciliary body-derived neural progenitors display retina-specific properties; the undifferentiated cells express Chx10, a retinal progenitor marker, and upon differentiation express markers corresponding to specific retinal cell types. Therefore, the pigmented ciliary body in the adult mammalian eye harbors neural progenitors that display stem cell properties and have the capacity to give rise to retinal neurons in vitro.     

Mechanism and a Peptide Motif for Targeting Peripheral Proteins to the Yeast Inner Nuclear Membrane

Trm1 is a tRNA specific m₂²G methyltransferase shared by nuclei and mitochondria in Saccharomyces cerevisiae . In nuclei, Trm1 is peripherally associated with the inner nuclear membrane (INM). We investigated the mechanism delivering/tethering Trm1 to the INM. Analyses of mutations of the Ran pathway and nuclear pore components showed that Trm1 accesses the nucleoplasm via the classical nuclear import pathway. We identified a Trm1 cis-acting sequence sufficient to target passenger proteins to the INM. Detailed mutagenesis of this region uncovered specific amino acids necessary for authentic Trm1 to locate at the INM. The INM information is contained within a sequence of less than 20 amino acids, defining the first motif for addressing a peripheral protein to this important subnuclear location. The combined studies provide a multi-step process to direct Trm1 to the INM: (i) translation in the cytoplasm; (ii) Ran-dependent import into the nucleoplasm; and (iii) redistribution from the nucleoplasm to the INM via the INM motif. Furthermore, we demonstrate that the Trm1 mitochondrial targeting and nuclear localization signals are in competition with each other, as Trm1 becomes mitochondrial if prevented from entering the nucleus.     

Trafficking to uncharted territory of the nuclear envelope

The nuclear envelope (NE) in eukaryotic cells serves as the physical barrier between the nucleus and cytoplasm. Until recently, mechanisms for establishing the composition of the inner nuclear membrane (INM) remained uncharted. Current findings uncover multiple pathways for trafficking of integral and peripheral INM proteins. A major route for INM protein transport occurs through the nuclear pore complexes (NPCs) with additional requirements for nuclear localization sequences, transport receptors, and Ran-GTP. Studies also reveal a putative NPC-independent vesicular pathway for NE trafficking. INM perturbations lead to changes in nuclear physiology highlighting the potential human disease impacts of continued NE discoveries.     

Evaluation of respiratory model employing conventional NIH mice to access the immunity induced by cellular and acellular pertussis vaccines

The increasing number of pertussis cases reported on the last twenty years and the existence of new acellular vaccines reinforce the need of research for experimental models to assure the quality of available pertussis vaccines. In this study, allotments of whole-cell and acellular pertussis vaccines were tested through the Intranasal Challenge Model (INM) using conventional NIH mice. The results have been compared to those achieved by the "Gold standard" Intracerebral Challenge Model (ICM). In contrast to ICM, INM results did not show intralaboratorial variations. Statistical analysis by Anova and Ancova tests revealed that the INM presented reproducibility and allowed identification and separation of different products, including three-component and four-component accellular pertussis vaccines. INM revealed differences between pertussis vaccines. INM provides lower distress to the mice allowing the reduction of mice number including the possibility of using conventional mice (less expensive) under non-aseptic environment. Thus, INM may be used as an alternative method of verifying the consistence of allotment production, including acellular pertussis vaccines.     

Genome-wide screen for inner nuclear membrane protein targeting in Saccharomyces cerevisiae: roles for N-acetylation and an integral membrane protein

Appropriate nuclear membrane structure is important for all eukaryotic organisms as evidenced by the numerous human diseases and alterations in gene expression caused by inappropriate targeting of proteins to the inner nuclear membrane (INM). We report here the first genome-wide screen to identify proteins functioning in INM targeting. We transformed to near completion the 4850 members of the Saccharomyces cerevisiae deletion collection of unessential genes in the 96-well format with a plasmid encoding a reporter protein, Trm1-II-GFP, which normally resides at the INM. We found that deletion of genes encoding subunits of the N-terminal acetyltransferase, NatC, cause mislocation of Trm1-II-GFP from the INM to the nucleoplasm. Mass spectroscopic analysis indicates that Trm1-II-GFP is N-acetylated. N-terminal mutations of Trm1-II-GFP predicted to ablate N-acetylation cause nucleoplasmic location, whereas a variant with an N-terminal alteration predicted to allow N-acetylation by NatC is located at the INM, providing genetic support that Trm1p-II N-acetylation is necessary for its subnuclear INM location. However, because N-acetylation appears not to be sufficient for INM targeting, it may provide a necessary role for INM targeting by affecting Trm1-II-GFP structure and exposure of cis-acting INM targeting motifs. We also discovered that YIL090W/Ice2p, an integral membrane protein located in the endoplasmic reticulum, is necessary for efficient targeting of Trm1-II-GFP to the INM. YIL090W/Ice2p may serve as a tether for INM proteins or as a regulator of INM tethers. Our methodology can be extrapolated to obtain genome-wide perspectives of mechanisms necessary to achieve appropriate subcellular and/or suborganellar location for any resident protein.     

人胚胎干细胞定向分化为神经前体细胞

采用单层贴壁分化的方法在无血清条件下诱导同源饲养层培养的人胚胎干细胞定向分化,得到了高比例的神经前体细胞(97.5±0.83)%(P<0.05)。这些神经前体细胞具有分化为神经元、星形胶质细胞和少突胶质细胞的能力。在长期的传代培养中发现,随着培养时间的延长,nestin阳性的神经前体细胞比例下降,同时发育能力也发生了变化。在传代培养的早期,神经前体细胞发育为神经元的比例很高,几乎没有胶质细胞分化出来。随着培养时间的延长,胶质细胞的比例逐渐上升。这与体内神经系统的发育过程非常相似。进一步研究发现具有bHLH(basic helix-loop-helix)结构域的转录因子neurogenein2(Ngn2)和Olig2可能在这一变化中起重要作用。因此,人胚胎干细胞来源的神经前体细胞能够模拟体内神经发育的模式,为在体外研究人的神经发育和再生医学奠定了基础。     

Making and breaking the inner nuclear membrane proteome

The nuclear envelope (NE) is the defining feature of eukaryotic cells, separating the nucleus from the cytoplasm. It has a complex architecture consisting of two lipid bilayers that, despite being continuous between them and with the endoplasmic reticulum, have different protein compositions consistent with their distinct functions. In particular, the unique composition of the inner nuclear membrane (INM), facing the nucleoplasm and its underlying nuclear lamina, is critical for the organisation and function of nuclear processes, from cell fate to gene regulation and DNA repair. Mutations in INM proteins affecting this organisation are associated with muscular dystrophies and premature ageing syndromes highlighting the role of INM architecture in cell homeostasis. Here, we discuss recent progress in understanding how specific proteins concentrate at the INM, as well as the quality control mechanisms involved in remodelling and maintaining INM protein homeostasis.     

Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore

Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.     

Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces

Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860). In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN), the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the cell cytosol (CC). In contrast to Endoplasmic Reticulum (ER) which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM) of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA). The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of the lipids in the ER, in the region adjacent to nucleus, is defining nuclear outer and inner biomembrane composition, is responsible for transport of the cytosolic protein into the nucleus and, replenishment of ER membrane used for vesicular transport.