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PubMed2006

Fabrication methods of an engineered microenvironment for analysis of cell-biomaterial interactions.

Success in tissue engineering requires an understanding of how cells integrate the signals presented from the microenvironment created by biomaterial scaffolds to alter their responses. Besides the presence of chemical stimuli, there is growing evidence that the spatial organization of cells and tissue within a 3-dimensional (3-D) extracellular matrix (ECM) context is a critical element in controlling cellular function. Therefore, in order to direct cells toward a desirable tissue structure, it is necessary to engineer biomaterials to have spatiotemporal control of the presentation of regulatory signals. Given that, micro-patterning techniques have profited by combining micro-fabrication technology with the chemical conjugation of biologically active molecules to provide new culture systems where cells can be cultured within a specific geometry. The micro-engineered environments have been developed as 2- and 3-D structures, which have proven greatly useful as versatile platforms to study cell, biomaterial, and ECM interactions on both macroscopic and microscopic levels. The main focus of this review is a brief summary of the use of micro-engineered substrates in the analysis of cell-biomaterial interactions with the aim to provide an introductory overview of practical applications available in the literature. In particular, topics regarding (1) the soft-lithography technique to prepare micro-patterned substrates for the spatial control of cell adhesion, (2) biomaterials stiffness-dependent cellular responses, and (3) the microarray techniques for analysis of cell/biomaterials interactions are discussed.

Animals↗
PubMed2004

The fan effect in fMRI: left hemisphere specialization in verbal working memory.

We studied the fan effect of verbal working memory using functional magnetic resonance imaging (fMRI). Participants were presented with a sentence-pair matching task that described semantic relationships (e.g. classroom-school). Working memory load and semantic processing were manipulated by increasing the number of sentences to be remembered and varying whether they matched expectation. Increased working memory load elicited activation in left dorsal frontal and left inferior parietal regions, and also delayed the hemodynamic responses. Convergent results from semantic matches occurred in the left parietal lobe, whereas, left ventral frontal activation from mismatches diverged from working memory results. The findings were consistent with behavioural and electrophysiological evidence, with the fan effect in fMRI providing novel insight into the spatiotemporal nature of verbal working memory in the left hemisphere.

Adult↗
PubMed2003

Left inferior parietal cortex integrates time and space during collision judgments.

Left inferior parietal lobe lesions can cause perturbation of the space-time plans underlying skilled actions. But does the perceptual integration of spatiotemporal information use the same neural substrate or is the role of the left inferior parietal cortex restricted to visuomotor transformations? We use fMRI and a collision judgment paradigm to examine whether the left inferior parietal cortex integrates temporal and spatial variables in situations in which no complex action and no visuomotor transformation is required. We used a perceptual task in which healthy subjects indicated by simple button presses whether two moving objects (of the same or different size) would or would not collide with each other. This task of interest was contrasted with a control task that employed the same stimuli and identical motor responses but in which the size of the two moving objects had to be compared. To assess putative differential eye-movement effects both tasks were performed with and without central fixation. Analysis of the fMRI data (employing a random-effects model and SPM99) showed that collision judgments (relative to size judgments) provoked a significant increase in neural activity in the left inferior parietal cortex (supramarginal gyrus) only. These results show that left inferior parietal cortex is involved in the integration of perceptual spatiotemporal information and thus provide a neural correlate for the use of space-time plans (whose perturbation can lead to apraxia as originally hypothesized by Liepmann). Furthermore, the data suggest that the left supramarginal gyrus combines temporal and spatial variables more widely than previously supposed.

Adolescent↗
PubMed1984

Torpedo electromotor system development: a quantitative analysis of synaptogenesis.

Synaptogenesis in the electric organ of Torpedo marmorato has been studied quantitatively at the ultrastructural level of observation. In addition to establishing the normal developmental time course for this event we were interested in determining whether a gradient of synaptogenesis might be present because the electric organ produces several morphologically recognizable spatiotemporal gradients during its early ontogeny. These gradients genesis of electrocyte columns, both gradients of which are operative for periods of weeks. No gradient of synaptogenesis was found, indicating this to be a synchronous process. The idea is advanced that synaptogenesis in the electric organ is modulated by extrinsic influences, many of which may originate from the target electrocytes which, by this time, have become synchronized in their development.

Acetylcholinesterase↗
PubMed1999

Cloning of rat fibrillin-2 cDNA and its role in branching morphogenesis of embryonic lung.

Fibrillin-2 is an extracellular matrix protein. It is associated with elastic fibers in several tissues and is believed to serve as a ligand for alphavbeta3 integrin, the latter being a known morphogen. In this study, the role of fibrillin-2 in lung development was investigated. Also, rat fibrillin-2 cDNA was isolated and sequenced and its spatiotemporal expression determined. It had approximately 88% homology with human fibrillin-2 and had Ca(2+) binding epidermal growth factor-like domains, transforming growth factor-beta binding protein motifs, and two RGD binding sites. Northern blot analysis revealed an approximately 10-kb transcript, and fibrillin-2 expression was developmentally regulated, and it paralleled that of tropoelastin. At day 13 of gestation, fibrillin-2 was expressed in the mesenchyme and at the epithelial:mesenchymal interface. From day 13 to 19 of gestation, its expression intensified and was confined around the tracheobronchial airways, while it lessened during the postnatal period. Immunoprecipitation revealed an approximately 350-kDa band by SDS-PAGE. Treatment with fibrillin-2 antisense oligodeoxynucleotide induced dysmorphogenesis of the lung explants. They were smaller and had rudimentary lung bud branches, collapsed conducting airways, and loose expanded mesenchyme. Concomitantly, fibrillin-2 mRNA, antibody reactivity in the explants, and fibrillin-2-specific radioincorporation were reduced. Anti-alphav and -laminin antibody reactivity and their respective incorporated specific radioactivities were unaltered. These data indicate that fibrillin-2 modulates organogenesis of the lung in the context of epithelial:mesenchymal interactions. Conceivably, the collapse of the conducting airways may also be related to the perturbed biology of the fibrillin-2 interacting protein, i.e., elastin, the latter being critical for the normal biophysiology of the lungs.

Actins↗
PubMed2001

Type 1 and type 3 ryanodine receptors generate different Ca(2+) release event activity in both intact and permeabilized myotubes.

In this investigation we use a "dyspedic" myogenic cell line, which does not express any ryanodine receptor (RyR) isoform, to examine the local Ca(2+) release behavior of RyR3 and RyR1 in a homologous cellular system. Expression of RyR3 restored caffeine-sensitive, global Ca(2+) release and causes the appearance of relatively frequent, spontaneous, spatially localized elevations of [Ca(2+)], as well as occasional spontaneous, propagating Ca(2+) release, in both intact and saponin-permeabilized myotubes. Intact myotubes expressing RyR3 did not, however, respond to K(+) depolarization. Expression of RyR1 restored depolarization-induced global Ca(2+) release in intact myotubes and caffeine-induced global release in both intact and permeabilized myotubes. Both intact and permeabilized RyR1-expressing myotubes exhibited relatively infrequent spontaneous Ca(2+) release events. In intact myotubes, the frequency of occurrence and properties of these RyR1-induced events were not altered by partial K(+) depolarization or by application of nifedipine, suggesting that these RyR1 events are independent of the voltage sensor. The events seen in RyR1-expressing myotubes were spatially more extensive than those seen in RyR3-expressing myotubes; however, when analysis was limited to spatially restricted "Ca(2+) spark"-like events, events in RyR3-expressing myotubes were larger in amplitude and duration compared with those in RyR1. Thus, in this skeletal muscle context, differences exist in the spatiotemporal properties and frequency of occurrence of spontaneous release events generated by RyR1 and RyR3. These differences underscore functional differences between the Ca(2+) release behavior of RyR1 and RyR3 in this homologous expression system.

Animals↗
PubMed1998

Isolation of rat fibrillin-1 cDNA and its relevance in metanephric development.

The role of fibrillin-1 in metanephrogenesis was investigated. Fibrillin-1 cDNA was isolated from the rat kidney cDNA library and sequenced, and its spatiotemporal expression was studied. It had approximately 88% homology with human fibrillin-1 and had Ca2+ binding epidermal growth factor-like domains, transforming growth factor-beta binding protein motifs, and an RGD binding site. Northern blot analysis revealed an approximately 10-kb transcript, and fibrillin-1 expression was developmentally regulated. In situ hybridization and immunofluorescence studies indicated that at day 15 of gestation, fibrillin-1 is expressed in the metanephric mesenchyme. At day 18, its expression was confined to nascent blood vessels and glomeruli, and it increased in the newborn and neonatal kidneys. Immunoprecipitation revealed an approximately 300-kDa band by SDS-PAGE. Treatment with fibrillin-1 antisense oligodeoxynucleotide induced marked dysmorphogenesis of the embryonic metanephroi. Concomitantly, the fibrillin-1 mRNA, antibody reactivity in the metanephroi, and fibrillin-1-specific radioincorporation were reduced. These data indicate that, like alphavbeta3 integrin, a known morphogen and a putative receptor of fibrillin-1, the fibrillin-1 modulates events related to early organogenesis and possibly also the vascularization of the rat kidney.

Amino Acid Sequence↗
PubMed2005

Different neural correlates of reward expectation and reward expectation error in the putamen and caudate nucleus during stimulus-action-reward association learning.

To select appropriate behaviors leading to rewards, the brain needs to learn associations among sensory stimuli, selected behaviors, and rewards. Recent imaging and neural-recording studies have revealed that the dorsal striatum plays an important role in learning such stimulus-action-reward associations. However, the putamen and caudate nucleus are embedded in distinct cortico-striatal loop circuits, predominantly connected to motor-related cerebral cortical areas and frontal association areas, respectively. This difference in their cortical connections suggests that the putamen and caudate nucleus are engaged in different functional aspects of stimulus-action-reward association learning. To determine whether this is the case, we conducted an event-related and computational model-based functional MRI (fMRI) study with a stochastic decision-making task in which a stimulus-action-reward association must be learned. A simple reinforcement learning model not only reproduced the subject's action selections reasonably well but also allowed us to quantitatively estimate each subject's temporal profiles of stimulus-action-reward association and reward-prediction error during learning trials. These two internal representations were used in the fMRI correlation analysis. The results revealed that neural correlates of the stimulus-action-reward association reside in the putamen, whereas a correlation with reward-prediction error was found largely in the caudate nucleus and ventral striatum. These nonuniform spatiotemporal distributions of neural correlates within the dorsal striatum were maintained consistently at various levels of task difficulty, suggesting a functional difference in the dorsal striatum between the putamen and caudate nucleus during stimulus-action-reward association learning.

Adult↗
PubMed1990

Expression of the neuronal surface glycoprotein Thy-1 is under post-transcriptional control, and is spatially regulated, in the developing olfactory system.

Expression of the neuronal cell surface glycoprotein Thy-1 has been studied during the development of the olfactory bulb in mice and rats, using in situ hybridisation and immunohistochemistry to follow the appearance of Thy-1 mRNA and protein, respectively. The mRNA was first detected 4 days before birth on all mitral cells, the main projection neuron of the bulb, as they formed a distinct layer and grew dendrites. At no stage was any spatial gradient of expression of Thy-1 mRNA evident around the mitral cell layer. Thy-1 protein, on the other hand, was first detectable 2 days later on a group of mitral cells immediately adjacent the point of entry of the olfactory nerve. The numbers of immunoreactive cells spread, over the next 7 days, to include all mitral cells, those located rostrally and laterally in the bulb being slowest to express Thy-1 protein. Thus there was a spatiotemporal gradient of expression of Thy-1 protein, which was not apparent in the earlier general expression of its mRNA, suggesting that some further inductive signal was required after transcription in order to get effective production of protein. Analysis of the growth of the mitral cell axons in the lateral olfactory tract suggested this signal was related to the cessation of axonogenesis, as Thy-1 immunoreactivity became detectable on these axons only when their expression of the transient epitope detected by the G10 antibody, present on microtubule-associated protein (MAP)1x only during axonal growth, declined. For the first week after Thy-1 protein appeared on mitral cells, it was not distributed uniformally on their surface. Immunoreaction was relatively weak on the somatic surface, and the molecule appeared to be entirely excluded from the distal regions of its main dendrite, above the outer plexiform layer. Here the dendrite reaches up to the synaptic glomeruli formed with the incoming olfactory nerve axons. These distal regions of the dendritic shaft became immunoreactive only after the periglomerular cells had first begun to express Thy-1 protein in the glomeruli. Immunolabelling of the somatic membrane then increased, to give the adult pattern of uniform Thy-1 labelling of the neuronal membrane by the end of the second postnatal week. It is suggested that some of the molecular features of Thy-1, and anatomical features of the main bulb, could interact to produce this initial restriction of Thy-1 to particular parts of the mitral cell surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Neurothelin: molecular characteristics and developmental regulation in the chick CNS.

Neurothelin has recently been identified as a cell surface protein specific for chick endothelial cells forming the blood-brain barrier. Neurons of the adult brain are essentially devoid of neurothelin. In contrast, neurons of the chick retina, which lack blood vessels and accessory astrocytes, express neurothelin. Here we demonstrate that during chick brain development initially neurothelin is expressed probably in all neuroblasts. With proceeding cytodifferentiation, such as vascularization and gliogenesis, brain neurons become neurothelin negative. Coincidentally the endothelial cells forming the blood-brain barrier start to synthesize neurothelin. In contrast to brain neurons, in retina neurons, neurothelin expression increases by one order of magnitude during the course of histogenesis. Coculturing of chick retinal cells with purified rat astrocytes in vitro results in reduction of neural neurothelin expression as quantified by ELISA. Conversely, disruption of the glia-neuron interactions by culturing brain neurons as individualized cells in vitro leads to a reexpression of neurothelin. This is consistent with the hypothesis that astrocytes inhibit neurothelin expression in neurons. Biochemical characterization classifies neurothelin as an integral membrane protein. Temperature-induced-detergent phase separation, phospholipase C digestion and sodium carbonate treatment were employed to distinguish between integral membrane proteins, lipid-anchored proteins and peripheral membrane proteins. Two-dimensional gel electrophoresis reveals an isoelectric point of about 6.4 for neurothelin. Polysaccharide analysis by glycosidase digestion and lectin binding indicates that neurothelin is highly glycosylated. The relative molecular mass of glycosylated neurothelin is 41 x 10(3), whereas the peptide backbone is only 25 x 10(3). The very strict spatiotemporal regulation of neurothelin expression in the central nervous system suggests that neurothelin fulfils possibly a crucial function such as transport of low relative molecular mass components that are essential for neuronal metabolism. The proposed biological activity of neurothelin might be specifically affected by some of its distinct biochemical features.

Animals↗
PubMed2004

Adaptive temporal integration of motion in direction-selective neurons in macaque visual cortex.

Direction-selective neurons in the primary visual cortex (V1) and the extrastriate motion area MT/V5 constitute a critical channel that links early cortical mechanisms of spatiotemporal integration to downstream signals that underlie motion perception. We studied how temporal integration in direction-selective cells depends on speed, spatial frequency (SF), and contrast using randomly moving sinusoidal gratings and spike-triggered average (STA) analysis. The window of temporal integration revealed by the STAs varied substantially with stimulus parameters, extending farther back in time for slow motion, high SF, and low contrast. At low speeds and high SF, STA peaks were larger, indicating that a single spike often conveyed more information about the stimulus under conditions in which the mean firing rate was very low. The observed trends were similar in V1 and MT and offer a physiological correlate for a large body of psychophysical data on temporal integration. We applied the same visual stimuli to a model of motion detection based on oriented linear filters (a motion energy model) that incorporated an integrate-and-fire mechanism and found that it did not account for the neuronal data. Our results show that cortical motion processing in V1 and in MT is highly nonlinear and stimulus dependent. They cast considerable doubt on the ability of simple oriented filter models to account for the output of direction-selective neurons in a general manner. Finally, they suggest that spike rate tuning functions may miss important aspects of the neural coding of motion for stimulus conditions that evoke low firing rates.

Action Potentials↗
PubMed1981

Neurogenetic gradients in the superior and inferior colliculi of the rhesus monkey.

The spatiotemporal patterns of neurogenesis in the superior colliculus (SC) and inferior colliculus (IC) were analyzed by plotting the positions of heavily labeled neurons in autoradiograms from a series of rhesus monkeys that had been exposed to 3H-thymidine at various embryonic (E) days and killed either shortly thereafter or at 2-3 months postnatally. Tectal neurons in this species are generated in the ventricular and subventricular zones surrounding the mesocoele (future Sylvian aqueduct) within the first third of the 165-day gestation period. Cells destined for the SC undergo final mitosis from E30 to E56, with peak proliferation from E38 to E43. These cells exhibit only a weak ventrodorsal gradient of neurogenesis and virtually no mediolateral or rostrocaudal gradients. Genesis of the neurons of the central nucleus (CN) of the IC also occurs between E30 and E56 and peaks around E43; but in contrast to cells of the SC, CN cells exhibit distinct rostrocaudal, lateromedial, and ventrodorsal gradients of genesis. Quantitative analysis revealed that whereas neurogenesis of the SC proceeds with about equal strength along the three cardinal axes, in the CN the lateral-medial gradient is significantly stronger than the anterior-posterior gradient. In addition, both the lateral-medial and ventral-dorsal IC gradients are significantly stronger than any SC gradients. Analysis of embryos sacrificed at short intervals following 3H-thymidine injection shows that the gradients are not caused by secondary lateral cell movement or by selective cell death. Thus, these findings suggest that within the generative zone of the midbrain tectum significant regional differences exist which result in distinctive spatiotemporal patterns of proliferation for the neurons of the primate superior and inferior colliculi.

Animals↗
PubMed2006

Kinematic analysis of unimanual reaching and grasping movements in children with hemiplegic cerebral palsy.

BACKGROUND: Kinematic investigations of prehension movements made by children with cerebral palsy are few and mainly focus on the reaching phase. To increase understandings of how both reaching and grasping movements are organized in these children, the aim was to investigate their spatiotemporal characteristics and additionally, how different kinematic parameters are related to the level of hand functions. METHODS: Eleven children with hemiplegic cerebral palsy; categorized as "mild" and "moderate", in their age of 5.8-12 years and eleven typically developing, "control" children participated. Reaching and grasping made with both the affected/non-preferred and unaffected/preferred sides were recorded and several kinematic parameters investigated. FINDINGS: In comparison to the control and the mild hemiplegic children, the moderate children exhibited more segmented reaches, longer reach- and grasp durations, and with no anticipatory shaping of the fingers when grasping with the affected hand. Their reaching path with the "unaffected" hand was also more segmented. The mild hemiplegic children showed ability for anticipatory hand closure and performed reaches with similar duration and trajectory as controls. The velocity at hand-object-contact and the quality of their grasping was however affected in comparison to the controls. INTERPRETATION: Findings from the investigated kinematics provide evidence of how spatiotemporal organization of reaching and grasping are affected in children with hemiplegia and related to the level of impaired hand functions. The outcomes clearly show that if these children are treated as one homogeneous group much information about their prehension capacity described from the kinematics will be concealed. Thus, observations of importance for developing effective, individual interventions adjusted to different level of impairments.

Biomechanical Phenomena↗
PubMed2003

Time-series analysis of MRI intensity patterns in multiple sclerosis.

In progressive neurological disorders, such as multiple sclerosis (MS), magnetic resonance imaging (MRI) follow-up is used to monitor disease activity and progression and to understand the underlying pathogenic mechanisms. This article presents image postprocessing methods and validation for integrating multiple serial MRI scans into a spatiotemporal volume for direct quantitative evaluation of the temporal intensity profiles. This temporal intensity signal and its dynamics have thus far not been exploited in the study of MS pathogenesis and the search for MRI surrogates of disease activity and progression. The integration into a four-dimensional data set comprises stages of tissue classification, followed by spatial and intensity normalization and partial volume filtering. Spatial normalization corrects for variations in head positioning and distortion artifacts via fully automated intensity-based registration algorithms, both rigid and nonrigid. Intensity normalization includes separate stages of correcting intra- and interscan variations based on the prior tissue class segmentation. Different approaches to image registration, partial volume correction, and intensity normalization were validated and compared. Validation included a scan-rescan experiment as well as a natural-history study on MS patients, imaged in weekly to monthly intervals over a 1-year follow-up. Significant error reduction was observed by applying tissue-specific intensity normalization and partial volume filtering. Example temporal profiles within evolving multiple sclerosis lesions are presented. An overall residual signal variance of 1.4% +/- 0.5% was observed across multiple subjects and time points, indicating an overall sensitivity of 3% (for axial dual echo images with 3-mm slice thickness) for longitudinal study of signal dynamics from serial brain MRI.

Algorithms↗
PubMedDate not supplied

Spatiotemporal structure of olfactory inputs to the mushroom bodies.

A requirement to understand mushroom body (MB) function is to characterize the operations (or transformations) that they impose on incoming signals. Understanding the nature of these integrative operations requires an understanding of the inputs from other brain areas. By inputs we mean not only the anatomical pathways leading to the MBs, but also the dynamic structure of the inflow of sensory (and other) signals. Neurons are complex, capacitative, and generally nonlinear devices that transform barrages of neurochemical packets into electrical waveforms. Their modes of operation are intrinsically time dependent and therefore, their functions or roles in a circuit cannot be inferred only from structural data. Thanks to elegant anatomical, behavioral, genetic, and molecular (for review, see Crittenden et al. 1998; Hammer and Menzel 1998; Heisenberg 1998; Wolf et al. 1998) studies, there is convincing evidence that MB circuits are involved, at least in fruit flies and honeybees, in some forms of odor integration and learning. In vivo electrophysiological studies of MB neurons, however, are rare and mainly restricted to individual (or small populations of) so-called extrinsic neurons, that is, those whose processes link MBs with other brain areas (Schildberger 1983, 1984; Homberg 1984; Hammer 1993; Mauelshagen 1993; Li and Strausfeld 1997). Kaulen et al. (1984) examined extracellular potentials in the MBs of bees, using current source density analysis, and more recently, Laurent and Naraghi (1994) provided a description of stimulus-evoked activity in Kenyon cells (KCs), the intrinsic neurons of the MBs, using intracellular recordings. In this short review we will summarize the recent results from our laboratory in an attempt to provide a description of the spatiotemporal structure of olfactory inputs to the MBs and their intrinsic neurons. We will focus only on the encoding of odor quality. We will then speculate on the possible role of MB circuits for olfactory processing.

Animals↗
PubMed1995

Expression of receptor protein tyrosine phosphatase-sigma (RPTP-sigma) in the nervous system of the developing and adult rat.

The expression of receptor protein tyrosine phosphatase-sigma (RPTP-sigma) mRNA during rat development was examined by Northern blot and in situ hybridization analyses. Northern blot analysis revealed that the two transcripts (5.7 kb and 6.9 kb) had different spatial and temporal patterns of expression. The 6.9-kb transcript was more abundant during embryonic development, whereas the 5.7-kb transcript was more abundant during postnatal development and in the adult. In situ hybridization revealed that RPTP-sigma mRNA was widely expressed throughout the central and peripheral nervous system during embryonic development. Very high levels were seen in the ventricular zone, subventricular zone, cortex, dorsal root ganglia, cranial nerve ganglia, olfactory epithelium, and retina. During postnatal development the level of expression decreased in most brain regions. However, high levels continued to be seen in the hippocampus. Emulsion autoradiography revealed that the majority of RPTP-sigma mRNA is expressed in neurons. Northern analysis showed that cultured glial cells expressed the 6.9-kb transcript, but not the 5.7-kb. RPTP-sigma mRNA expression profiles were clearly distinct from those of leukocyte antigen-related protein (LAR), a closely related RPTP. The spatiotemporal pattern of RPTP-sigma mRNA expression indicates that RPTP-sigma may play a role in the development of the nervous system.

Age Factors↗
PubMed2025

Identifying genomic surveillance gaps in Africa for the global public health response to West Nile virus: a systematic review.

West Nile virus (WNV) is a priority pathogen that poses a high risk for public health emergencies of global concern. Although WNV is endemic to Africa, only few (n=63) whole genomic sequences are available from the continent. In this Review, we examined the status of the molecular testing and genomic sequencing of WNV across Africa and mapped its global spatiotemporal spread. WNV has been detected in 39 African countries, the Canary Islands, and Réunion Island. Although publications, including those with molecular data, originated from 24 of these countries, genomic sequences were available from only 16 countries. Our analysis identified regions with detected viral circulation but without molecular surveillance. The current literature has substantial knowledge gaps in terms of the disease burden, molecular epidemiology, and distribution of WNV in Africa. Addressing these gaps requires an integrated One Health surveillance approach, which is challenging to establish. We propose three key surveillance needs that could improve the current understanding of the WNV disease burden in Africa, to strengthen the global public health response to this vector-borne disease.

West Nile Fever↗
PubMed2006

Nuclear and chromatin reorganization in the MHC-Oct3/4 locus at developmental phases of embryonic stem cell differentiation.

Epigenetic gene control is involved in mechanisms of development. Little is known about the cooperation of nuclear and chromatin events in programmed differentiation from mouse embryonic stem cells (ESC). To address this, Oct3/4-positive ESC and differentiated progenies, Sox1-positive neural precursor cells (NPC) and post-mitotic neurons (PMN), were isolated using a stage-selected culture system. We first investigated global nuclear organization at the each stage. Chromocenter preexists in ESC, disperses in NPC and becomes integrated into large heterochromatic foci in PMN, while the formation of PML bodies markedly decreases in neural differentiation. We next focused on the gene-dense MHC-Oct3/4 region. Oct3/4 gene is expressed preferentially adjacent to PML bodies in ESC and are repressed in the absence of chromocenter association in NPC and PMN. Histone deacetylation in NPC, demethylation of lysine 4 of histone H3 (H3K4), tri-methylation of H3K27, and CpG methylation in PMN are targeted for the Oct3/4 promoter within the region. Interestingly, di-methyl H3K4 mark is present in Oct3/4 promoter in NPC as well as ESC. These findings provide insights into the molecular basis of global nuclear reorganization and euchromatic gene silencing in differentiation through the spatiotemporal order of epigenetic controls.

Animals↗