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[Determination of the dorso-ventral polarity of the Drosophila embryo].

Embryonic pattern formation has been studied extensively in many organisms. In Drosophila, the powerful combination of genetics cytoplasm transplantation experiments, as well as recent molecular data, have helped to elucidate the mechanisms responsible for the establishment of embryonic polarity. A small number of genes, most of them maternally expressed, are involved in this process and participate in four independent systems--three for the antero-posterior axis (A/P) and one for dorsoventral axis (D/V)--which define various embryonic territories by specifically localized cues. This review concerns the definition of the dorsoventral polarity responsible for the establishment of the germ layers of the embryo. Dorsoventral development is regulated by a single group of maternally expressed genes: the "dorsal group" of genes. It includes 11 genes, the loss of function of any of which results in a dorsalized development, whereas mutation of the 12th gene, cactus, results in a ventralized development. These genes are arranged according to a functional hierarchy, and have been shown to cooperate in the formation of a graded nuclear concentration of the dorsal gene product. The dorsal product corresponds to the dorsoventral morphogen and is homologous to the transcription factor NF-kappa B. Among the 11 genes of the dorsal group, 3 are required in the somatic line. This suggests the existence of inductive signals originating during oogenesis from the follicle cells that surround the developing oocyte. This somatically expressed spatial information probably controls dorsoventral development by defining the polarity of a signal transducing pathway that specifically activates the nuclear uptake of the dorsal product. This model, highlights the importance of the polarity of the egg chamber, and suggests that it is the oocyte nucleus due to its asymmetrical localization, that determines the dorsoventral pattern formation of the embryo.

Animals↗

Identifying the molecular phenotype of renal progenitor cells.

Although many of the molecular interactions in kidney development are now well understood, the molecules involved in the specification of the metanephric mesenchyme from surrounding intermediate mesoderm and, hence, the formation of the renal progenitor population are poorly characterized. In this study, cDNA microarrays were used to identify genes enriched in the murine embryonic day 10.5 (E10.5) uninduced metanephric mesenchyme, the renal progenitor population, in comparison with more rostral derivatives of the intermediate mesoderm. Microarray data were analyzed using R statistical software to determine accurately genes differentially expressed between these populations. Microarray outliers were biologically verified, and the spatial expression pattern of these genes at E10.5 and subsequent stages of early kidney development was determined by RNA in situ hybridization. This approach identified 21 genes preferentially expressed by the E10.5 metanephric mesenchyme, including Ewing sarcoma homolog, 14-3-3 theta, retinoic acid receptor-alpha, stearoyl-CoA desaturase 2, CD24, and cadherin-11, that may be important in formation of renal progenitor cells. Cell surface proteins such as CD24 and cadherin-11 that were strongly and specifically expressed in the uninduced metanephric mesenchyme and mark the renal progenitor population may prove useful in the purification of renal progenitor cells by FACS. These findings may assist in the isolation and characterization of potential renal stem cells for use in cellular therapies for kidney disease.

Animals↗

Experimental manipulation of transforming growth factor-beta isoforms significantly affects adhesion formation in a murine surgical model.

Transforming growth factor-beta (TGF-beta), a multifunctional growth factor, represents three mammalian isoforms, TGF-beta1, TGF-beta2, and TGF-beta3. In cutaneous wound healing, combined neutralization of TGF-beta1 and -beta2 or addition of TGF-beta3 reduces scar formation. Here, we investigated whether experimental manipulation of TGF-beta isoforms reduced adhesion formation after injury to the peritoneum. Adhesions were produced in mice by surgical abrasion of adjacent serosa followed by close apposition. In the first part of this study, a detailed analysis of TGF-beta isoform distribution was performed through immunolocalization. TGF-beta isoforms clearly showed a unique temporal and spatial pattern of expression after peritoneal wounding. Based on this pharmacokinetic data, we next administered neutralizing antibodies to TGF-beta1 and -beta2 or exogenous TGF-beta3 peptide by local application and intraperitoneal injection at various times before and after surgery. At day 7 after surgery, addition of neutralizing antibodies to both TGF-beta1 and -beta2 significantly reduced the number and size of adhesions (P < 0.05) compared with the vehicle control. By contrast, exogenous addition of TGF-beta3 either had no effect or increased adhesion formation compared to the vehicle control. In conclusion, these results show that by blocking both TGF-beta1 and TGF-beta2 using neutralizing antibodies, it is possible to prevent abdominal adhesion formation.

Animals↗

Hierarchical structure formation and pattern replication induced by an electric field.

Several techniques based on soft lithography have emerged to replicate micrometre-sized patterns. Similar to most other lithographic methods, these techniques structure a single layer of photo resist. For many applications, however, it is desirable to control the spatial arrangement of more than one component. With traditional methods, this requires an iterative, multistep procedure, making the replication process more complex and less reliable. Here, a replication process is described where multiple materials are processed simultaneously. Using a bilayer formed by two different polymers, electrohydrodynamic instabilities at both polymer surfaces produce a hierarchic lateral structure that exhibits two independent characteristic dimensions. A lateral modulation of the electric field enables replication with a resolution down to 100 nanometres. This approach might provide a simple strategy for large-area, sub-100-nanometre lithography.

Air↗

Subitizing, magnitude representation, and magnitude retrieval in deaf and hearing adults.

This study examines basic number processing (subitizing, automaticity, and magnitude representation) as the possible underpinning of mathematical difficulties often evidenced in deaf adults. Hearing and deaf participants completed tasks to assess the automaticity with which magnitude information was activated and retrieved from long-term memory (using a Stroop-like paradigm to assess congruity effects), the representational format of magnitude information (by analysis of distance and Spatial Numerical Association of Response Codes effects), and the ability to rapidly enumerate small sets (subitizing). Both groups showed distance effects taken to indicate the use of a visual-spatial analog number line representing approximate quantity. Furthermore, both groups showed similar patterns of performance on the subitizing tasks and showed similar amounts of interference in an analysis of congruity effects. This is taken as evidence against the notion that idiosyncratic differences in basic number processing account for mathematical difficulties experienced by deaf individuals.

Adolescent↗

Synapse formation molecules in muscle and autonomic ganglia: the dual constraint hypothesis.

In 1970 it was thought that if the motor-nerve supply to a muscle was interrupted and then allowed to regenerate into the muscle, motor-synaptic terminals most often formed presynaptic specializations at random positions over the surface of the constituent muscle fibres, so that the original spatial pattern of synapses was not restored. However, in the early 1970s a systematic series of experiments were carried out showing that if injury to muscles was avoided then either reinnervation or cross-reinnervation reconstituted the pattern of synapses on the muscle fibres according to an analysis using the combined techniques of electrophysiology, electronmicroscopy and histology on the muscles. It was thus shown that motor-synaptic terminals are uniquely restored to their original synaptic positions. This led to the concept of the synaptic site, defined as that region on a muscle fibre that contains molecules for triggering synaptic terminal formation. However, nerves in developing muscles were found to form connections at random positions on the surface of the very short muscle cells, indicating that these molecules are not generated by the muscle but imprinted by the nerves themselves; growth in length of the cells on either side of the imprint creates the mature synaptic site in the approximate middle of the muscle fibres. This process is accompanied at first by the differentiation of an excess number of terminals at the synaptic site, and then the elimination of all but one of the terminals. In the succeeding 25 years, identification of the synaptic site molecules has been a major task of molecular neurobiology. This review presents an historical account of the developments this century of the idea that synaptic-site formation molecules exist in muscle. The properties that these molecules must possess if they are to guide the differentiation and elimination of synaptic terminals is considered in the context of a quantitative model of this process termed the dual-constraint hypothesis. It is suggested that the molecules agrin, ARIA, MuSK and S-laminin have suitable properties according to the dual-constraint hypothesis to subserve this purpose. The extent to which there is evidence for similar molecules at neuronal synapses such as those in autonomic ganglia is also considered.

Animals↗

Restoration of cholinergic circuitry in the hippocampus by foetal grafts.

The pathway from medial septum to hippocampus is one of the major and most well-documented cholinergic connections in the rodent brain. Interruption of this pathway by either direct destruction of the cells of origin in the medial septum or by transection of the fimbria-fornix, the fibre tract along which the septohippocampal axons traverse, results in a virtually complete depletion of cholinergic markers within the hippocampal formation. Previous experiments have shown that grafts of foetal rat septal-diagonal band region placed into the denervated hippocampus can restore acetylcholinesterase (AChE) fibre density to 85-90% of control values (Björklund et al., Acta physiol. scand. Suppl. 522 (1983) 49-58). More recently, it has been demonstrated using the more specific technique of choline acetyltransferase (ChAT) immunocytochemistry in combination with electron microscopy that septal grafts are also able to restore the cholinergic connectivity at the synaptic level in the dorsal hippocampal formation. However, we have demonstrated that this restoration of both AChE and ChAT fibre density represents a specific mechanism and that the source of the foetal cholinergic neurons is crucial to the extent of reinnervation and pattern of connectivity achieved. In aged rats, judged as being behaviourally impaired with respect to their spatial memory, there appears to be an intrinsic denervation of the septohippocampal pathway such that the hippocampus is depleted of cholinergic markers. In these cases, transplantation can again restore cholinergic innervation but without the requirement of prior denervation by a fimbria-fornix transection--grafts are placed into the intact hippocampus. Results show that the grafts survive well in the aged, intact hippocampus and are able to ameliorate the behavioural impairments, perhaps by the formation of substantial numbers of cholinergic synapses between the graft and host brain. In conclusion, therefore, neural grafting of cholinergic neurons of appropriate type and origin is able to reinnervate the hippocampal formation previously denervated either by mechanical transection of the fimbria-fornix or as a result of an age-dependent deterioration.

Acetylcholine↗

Heteroplasmy and organelle gene dynamics.

This study assesses factors that influence the rates of change of organelle gene diversity and the maintenance of heteroplasmy. Losses of organelle gene diversity within individuals via vegetative segregation during ontogeny are paramount to resultant spatial and temporal patterns. Steady-state losses of organelle variation from the zygote to the gametes are determined by the effective number of organelles, which will be approximately equal to the number of intracellular organelles if random segregation prevails. Both rapid increases in organelle number after zygote formation and reductions at germ lines will reduce variation within individuals. Terminal reductions in organelles must be to very low copy numbers (<5) for substantial losses in variation to occur rapidly. Nonrandom clonal expansion and vegetative segregation during gametogenesis may be effective in reducing genetic variation in gametes. If organelles are uniparentally inherited, the asymptotic expectations for effective numbers of gametes and spatial differentiation will be identical for homoplasmic and heteroplasmic conditions. The rate of attainment of asymptote for heteroplasmic organelles, however, is governed by the rate of loss of variation during ontogeny. With sex-biased dispersal, the effective number of gametes is maximized when the proportional contributions of the sex having the higher dispersal rate are low.

Animals↗

Parallel somatic and synaptic processing in the induction of intermediate-term and long-term synaptic facilitation in Aplysia.

The induction of different phases of memory depends on the amount and patterning of training, raising the question of whether specific training patterns engage different cellular mechanisms and whether these mechanisms operate in series or in parallel. We examined these questions by using a cellular model of memory formation: facilitation of the tail sensory neuron-motor neuron synapses by serotonin (5-hydroxytryptamine, 5-HT) in the CNS of Aplysia. We studied facilitation in two temporal domains: intermediate-term facilitation (1.5-3 h) and long-term facilitation (LTF, >24 h). Both forms can be induced by using several different temporal and spatial patterns of 5-HT, including (i) repeated, temporally spaced pulses of 5-HT to both the sensory neuron soma and the sensory neuron-motor neuron synapse, and (ii) temporally asymmetric exposure of 5-HT to the soma and synapse under conditions in which neither exposure alone induces LTF. We first examined the protein and RNA synthesis requirements for LTF induced by these two patterns and found that asymmetric (but not repeated) 5-HT application induced LTF that required postsynaptic protein and RNA synthesis. We next focused on the patterning and protein synthesis requirements for intermediate-term facilitation. We found that intermediate-term facilitation (i) is induced locally at the synapse, (ii) requires multiple pulses of 5-HT, and (iii) requires synaptic protein synthesis. Our findings show that different temporal and spatial patterns of 5-HT induce specific temporal phases of long-lasting facilitation in parallel by engaging different cellular and molecular mechanisms.

Animals↗

The spatial and temporal immunolocalization of TGF-beta 1 and bone morphogenetic protein-2/-4 in phallic bone formation in inbred Sprague Dawley male rats.

In male rats and mice, the membranous bone is rapidly formed in the proximal segment (p-segment) of the os penis, and then endochondral ossification proceeds in the proximal half. The spatial and temporal patterns of expression of transforming growth factor-beta 1 (TGF-beta 1) and bone morphogentic protein-2/-4 (BMP-2/-4) were examined immunohistochemically until 14 days after birth and compared. BMP-2/-4 expression was weak in the mesenchymal condensation just at birth, and up-regulated in the membranous bone and cartilage 3 days after birth. BMP-2/-4 was strongly expressed in proliferating and mature chondrocytes 7 days after birth. At birth, TGF-beta 1 expression was diffuse and very strong in mesenchymal condensation. Thereafter, TGF-beta 1 expression was continuously strong in membranous bone and cartilage. Seven days after birth, TGF-beta 1 was actively expressed in proliferating chondrocytes. TGF-beta 1 and BMP-2/-4 seemed to act co-operatively. In particular, TGF-beta 1 appeared to strongly regulate immature stage and BMP-2/-4 may regulate the mature stage.

Animals↗

Functional thalamocortical synapse reorganization from subplate to layer IV during postnatal development in the reeler-like mutant rat (shaking rat Kawasaki).

Transient synapse formation between thalamic axons and subplate neurons is thought to be important in thalamocortical targeting. Shaking rat Kawasaki (SRK), having reversed cortical layering similarly observed in reeler mouse, provides an interesting model system to test this idea. The spatial and temporal pattern of excitation was investigated using optical recording with voltage-sensitive dyes in thalamocortical slice preparations from SRK. At postnatal day 0 (P0), a strong optical response was elicited within the superplate of the SRK in the cell layer corresponding to subplate in wild-type (WT) rats. By P3, this response rapidly descended into deep cortical layers comprised of layer IV cells, as identified with 5-bromo-2'-deoxyuridine birthdating at embryonic day 17. During the first 3 postnatal days, both the subplate and cortical plate responses were present, but by P7, the subplate response was abolished. Tracing individual axons in SRK revealed that at P0-P3, a large number of thalamocortical axons reach the superplate, and by P7-P10, the ascending axons develop side branches into the lower or middle cortical layers. Synaptic currents were also demonstrated in WT subplate cells and in SRK superficial cortical cells using whole-cell recording. These currents were elicited monosynaptically, because partial AMPA current blockade did not modify the latencies. These results suggest that the general developmental pattern of synapse formation between thalamic axons and subplate (superplate) neurons in WT and SRK is very similar, and individual thalamic arbors in cortex are considerably remodeled during early postnatal development to find layer IV equivalent neurons.

Action Potentials↗

Pattern formation and cell differentiation: trichomes in Arabidopsis as a genetic model system.

Arabidopsis trichomes are single-celled hairs that originate from epidermal cells and are distributed regularly on most aerial body parts. During the last decade, trichome formation in Arabidopsis has been established as a genetic and molecular model system to study various general developmental and cellular mechanisms. This review summarizes progress in the investigation of several aspects of trichome development: the spatial regulation of cell fate determination, the regulation of cell differentiation in response to exogenous signals and plant hormones, and the regulation of endoreplication, cell growth, and cell morphogenesis.

Arabidopsis↗

Spatial characteristics of neurons in the central mesencephalic reticular formation (cMRF) of head-unrestrained monkeys.

Prior studies of the central portion of the mesencephalic reticular formation (cMRF) have shown that in head-restrained monkeys, neurons discharge prior to saccades. Here, we provide a systematic analysis of the patterns of activity in cMRF neurons during head unrestrained gaze shifts. Two types of cMRF neurons were found: presaccadic neurons began to discharge before the onset of gaze movements, while postsaccadic neurons began to discharge after gaze shift onset and typically after the end of the gaze shift. Presaccadic neuronal responses were well correlated with gaze movements, while the discharge of postsaccadic neurons was more closely associated with head movements. The activity of presaccadic neurons was organized into gaze movement fields, while the activity of postsaccadic neurons was better organized into movement fields associated with head displacement. We found that cMRF neurons displayed both open and closed movement field responses. Neurons with closed movement fields discharged before a specific set of gaze (presaccadic) or head (postsaccadic) movement amplitudes and directions and had a clear distal boundary. Neurons with open movement fields discharged for gaze or head movements of a specific direction and also for movement amplitudes up to the limit of measurement (70 degrees). A subset of open movement field neurons displayed an increased discharge with increased gaze shift amplitudes, similar to pontine burst neurons, and were called monotonically increasing open movement field neurons. In contrast, neurons with non-monotonically open movement fields demonstrated activity for all gaze shift amplitudes, but their activity reached a plateau or declined gradually for gaze shifts beyond specific amplitudes. We suggest that presaccadic neurons with open movement fields participate in a descending pathway providing gaze signals to medium-lead burst neurons in the paramedian pontine reticular formation, while presaccadic closed movement field neurons may participate in feedback to the superior colliculus. The previously unrecognized group of postsaccadic cMRF neurons may provide signals of head position or velocity to the thalamus, cerebellum, or spinal cord.

Action Potentials↗

DNA delivery from hyaluronic acid-collagen hydrogels via a substrate-mediated approach.

Efficient and controlled gene delivery from biodegradable materials can be employed to stimulate cellular processes that lead to tissue regeneration. In this report, a substrate-mediated approach was developed to deliver DNA from hyaluronic acid-collagen hydrogels. The hydrogels were formed by crosslinking HA with poly(ethylene glycol) diglycidyl ether. Poly(ethylene imine)(PEI)/DNA complexes were immobilized to the substrate using either biotin/neutravidin or non-specific adsorption. Complexes were formed in the presence or absence of salt to regulate complex size, and resulted in complexes with z-average diameters of 1221.7 +/- 152.3 and 139.4 +/- 1.3 nm, respectively. During 48-h incubation in PBS or hyaluronidase, DNA was released slowly from the hydrogel substrate (<30% of immobilized DNA), which was enhanced by incubation with conditioned media (approximately 50% of immobilized DNA). Transgene expression mediated by immobilized, large diameter complexes was 3 to 7-fold greater than for small diameter complexes. However, the percentage of cells expressing the transgene was greater for small diameter complexes (48.7%) than for large diameter complexes (22.3%). Spatially controlled gene transfer was achieved by topographically patterning the hydrogel to pattern cell adhesion. Biomaterial-based gene delivery can be applicable to numerous tissue engineering applications, or as a tool to examine tissue formation.

Absorbable Implants↗

Mechanical environment alters tissue formation patterns during fracture repair.

Fracture repair has previously been shown to be sensitive to mechanical environment, yet the specific relationship between strain character, magnitude and frequency, as well as other mechanical parameters, and tissue formation is not well understood. This study aimed to correlate strain distribution within the healing fracture gap with patterns of tissue formation using a rat model of a healing osteotomy subject to mechanical stimulation in bending. Finite element models based on realistic tissue distributions were used to estimate both the magnitude and spatial distribution of strains within the fracture gap. The spatial distribution of regenerating tissue was determined by microcomputed tomography and histology, and was confirmed using reverse transcription-polymerase chain reaction (RT-PCR). Results suggest that tensile strains suppress chondrogenesis during the mechanical stimulation period. After stimulation ends, however, tensile strains increased chondrogenesis followed by rapid bone formation. In contrast, in compressive environments, bone is formed primarily via intramembranous ossification. Taken together, these results suggest that intermittent tensile strains during fracture repair stimulate endochondral ossification and promote eventual bone healing compared to intermittent compressive strains and unstimulated fractures. Further understanding of these relationships may allow proposal of optimal therapeutic strategies for improvement of the fracture repair process.

Animals↗

New experimental data and mechanistic studies on the bromate-dual substrate-dual catalyst batch oscillator.

The bromate-hypophosphite-acetone-Mn(II)-Ru(bpy)(3)(2+) batch oscillator was recently suggested for studying two-dimensional pattern formation. The system meets all major requirements that are needed for generation of good quality traveling waves in a thin solution layer. The serious drawback of using the system for studying temporal and spatial dynamical phenomena is its unknown chemical mechanism. In order to develop a mechanism that explains the observed long-lasting batch oscillations the bromate-hypophosphite-acetone-Mn(II)-Ru(bpy)(3)(2+) oscillator was revisited. We studied the dynamics both in the total system and in some composite reactions, and kinetic measurements were carried out in three subsystems. From the new experimental results we concluded that the two oscillatory sequences observed in the full system are originated from two oscillatory subsystems, the Mn(II)-catalyzed bromate-hypophosphite-acetone and the Ru(bpy)(3)(2+)-catalyzed bromate-bromoacetone reactions. Here we propose a mechanism which is capable of simulating the dynamical features that appeared in the complex system.

Journal Article↗

Precursor tissue analogs as a tissue-engineering strategy.

Natural tissues are composed of functionally diverse cell types that are organized in spatially complex arrangements. Organogenesis of complex tissues requires a coordinated sequential transformation process, with individual stages involving time-dependent expression of cell-cell, cell-matrix, and cell-signal interactions in three dimensions. The common theme of temporal-spatial patterning of these cellular interactions is also observed in other physiological processes, such as growth and development, wound healing, and tumor migration. The "precursor tissue analog" (PTA) applies the temporal-spatial patterning theme to tissue engineering. The goal of PTA in tissue engineering is not to fabricate the final transplantable tissue but rather to guide the dynamic organization, maturation, and remodeling leading to the formation of normal and functional tissues. We describe the critical design principles of PTA. First, structural, mechanical, and physiological requirements of the PTA as a temporary scaffold must be met by a fabrication method with flexibility. The fabrication potential incorporating biological materials such as living cells and plasmid DNA has been addressed. Second, the PTA concept is considered suitable for future tissue engineering in light of the use of undifferentiated stem cells, and may possess a capability to guide stem cells toward diverse differentiation characteristics in situ. To this end, the behavior of the engineered cell and tissue must be monitored in detail. The development of a practical phenotype monitoring system such as a DNA microarray may be integral to the fabrication strategies of PTA. Third, the microtopographical and microenvironmental control on the liquid-solid interaction may lead to a critical design for PTA to provide soluble factors, nutrients, and gases to the cells embedded within the scaffold. We suggest that the level set numerical simulation method may be utilized to engineer the consistent circulation of bioactive liquid throughout the PTA microenvironment.

Animals↗

Coordinated expression of noggin and bone morphogenetic proteins (BMPs) during early skeletogenesis and induction of noggin expression by BMP-7.

Coordinated regulation of the activities of bone morphogenetic protein (BMP) and its inhibitors is essential for skeletal development since loss-of-function experiments show that both BMPs and BMP inhibitory signals, such as noggin, are required to establish proper formation of skeletal tissues. In this paper, we asked how and when noggin would be functional to interact with BMPs during skeletogenesis in mammals. For this purpose, we first analyzed the spatial and temporal patterns of noggin, BMP-2, BMP-4, and BMP-7 expression during early skeletogenesis in mouse embryos. In situ hybridization study revealed that noggin expression was detected at a low level in limb mesenchyme, whereas BMP-7 was expressed at a high level throughout limb mesenchyme 10.5 days postcoitum (dpc) in mouse embryos. One day later, noggin mRNA was expressed at a high level in the prechondrogenic condensations in appendicular and axial skeletal primordia, where sox9 transcripts were also expressed. At this stage, noggin-expressing cells were surrounded by those expressing BMP-7. The chondrogenic cell condensation continued to express noggin transcripts in 12.5 dpc and 13.5 dpc embryos, and again the noggin-expressing cells within the cartilaginous tissue were surrounded by those expressing BMP-7. We further examined interaction of noggin and BMPs by using organ cultures of 11.5 dpc mouse forelimbs and found that implantation of carriers containing BMP-7 protein into the forelimb explants induced noggin expression in the limb mesenchyme. BMP-7 also induced type II collagen and sox9 mRNAs in the same cell population, indicating that noggin induction occurred in the chondrogenic precursor cells. BMP-7 effects on noggin expression were observed in a dose-dependent manner within a dose range of 10-100 ng/microliter. These results suggest that BMP-7 induced expression of noggin transcripts within skeletal cell condensation and that this noggin expression in turn could act antagonistically to attenuate BMP action in the early skeletogenesis.

Animals↗