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At least 289 records · Page 16Linked to original sources
PubMed2004

Rat tyrosine hydroxylase promoter directs tetracycline-inducible foreign gene expression in dopaminergic cell types.

A prerequisite for creating animal models in which gene expression is spatially and temporally controlled is the development of promoters to target genetic switches to specific populations of cells. Here we used the dopaminergic biosynthetic enzyme, tyrosine hydroxylase (TH) to test various combinations of tetracycline (Tet) system elements to determine the optimal configuration for inducible, tissue-specific expression. The present study shows that the degree of expression and level of leakiness associated with the Tet transactivators rtTA, rtTA2S-M2, tTS/rtTA or tTS/rtTA2S-M2 was dependent upon both the promoter and cell type utilized. Specifically, CMV-driven tTS/rtTA2S-M2 exhibited the highest level of inducibility in HEK cells (approximately 1000-fold) versus the dopaminergic cell line, MN9D (approximately 70-fold). In contrast, TH-driven rtTA2S-M2 yielded the highest level of expression with the least background in dopaminergic cell types versus HEK cells. Moreover, the TH promoter could be combined with the bi-directional Tet response system, BiTetO, allowing for the co-expression and regulation of two genes in the same cell. To further test the feasibility of this system we replaced the reporter gene with human Bcl-2. Consistent with previous studies, induction of Bcl-2 expression in dopaminergic cell types attenuated cell death due to the neurotoxin, MPP+. Taken together, these data suggest that targeted, inducible gene expression can be achieved in dopaminergic cell types.

Animals↗
PubMed1986

Spatial regulation of Antennapedia and bithorax gene expression by the Polycomb locus in Drosophila.

Transcripts encoded by six of the homeotic lethal complementation groups within the Antennapedia and bithorax gene complexes (ANT-C and BX-C) accumulate primarily in nonoverlapping regions of the embryonic central nervous system (CNS). It has been previously proposed that the maintenance of these selective patterns of expression involve hierarchical cross-regulatory interactions among ANT-C and BX-C loci. Selective expression of the ANT-C and BX-C also involves regulation by a number of unlinked genetic loci, including the Polycomb (Pc) locus. Here we show that the ANT-C and BX-C transcript distribution patterns are dramatically altered in the CNS of Pc- embryos. We discuss these altered patterns of expression in the context of different models for the spatial regulation of ANT-C and BX-C gene expression.

Animals↗
PubMed1990

A retinoic acid responsive gene MK found in the teratocarcinoma system is expressed in spatially and temporally controlled manner during mouse embryogenesis.

A newly identified gene MK is transiently expressed in early stages of retinoic acid-induced differentiation of embryonal carcinoma cells (Kadomatsu, K., M. Tomomura, and T. Muramatsu, 1988. Biochem. Biophys. Res. Commun. 151:1312-1318). MK gene has been predicted to code a polypeptide that is rich in basic amino acids and cysteine and is not related to any other peptides so far reported. In the present study, we investigated MK expression during mouse embryogenesis by in situ hybridization. The MK transcript was detected all over the embryo proper of the 7-d embryo, while it was not detectable in the 5-d embryo. The ubiquitous expression continued in the 9-d embryo proper. On the 11th-13th d of gestation, the sites where MK gene was intensely expressed became progressively restricted; these sites were the brain ectoderm around the lens and brain ventricles, the anterior lobe of the pituitary gland, the upper and lower jaw, the caudal sclerotomic half of vertebral column, the limbs, the stomach, and the epithelial tissues of the lung, the pancreas, the small intestine, and the metanephros. These areas include the region where secondary embryonic induction is prominent. In the 15-d embryo, only the kidney expressed MK significantly. These data suggest that MK gene plays a fundamental role in the differentiation of a wide variety of cells; MK gene may also play some specific roles in generation of epithelial tissues, and remodeling of mesoderm.

Animals↗
PubMed1997

Promoter trap markers differentiate structural and positional components of polar development in Arabidopsis.

To investigate mechanisms involved in establishing polar organization in Arabidopsis embryos and seedlings, we used promoter trapping to identify molecular markers (beta-glucuronidase fusion genes) expressed in spatially restricted patterns along the apical-basal axis. Three markers were identified that are expressed, respectively, in the embryonic and seedling root tip (POLARIS), cotyledons and shoot and root apices (EXORDIUM), and root cap (COLUMELLA). Each marker was crossed into the mutants hydra and emb30, which are defective in embryonic and seedling morphogenesis. All three markers were expressed in hydra mutants in patterns similar to those observed in phenotypically wild-type embryos and seedlings. In emb30 mutants, the EXORDIUM marker was expressed in cotyledons but not in the expected position of shoot and root meristems, and the marker COLUMELLA was not expressed at all, which is consistent with the view that the emb30 mutant, but not hydra, lacks shoot and root meristems. However, POLARIS was expressed in the basal part of hydra embryos lacking an embryonic root and in the basal parts of both hydra and emb30 seedlings. Expression of POLARIS is inducible by exogenous auxin and suppressed by cytokinin but is unaffected by inhibitors of polar auxin transport or cell division. We conclude that POLARIS differentiates positional aspects of polar development from structural aspects.

Arabidopsis↗
PubMed2001

SPACRCAN in the developing retina and pineal gland of the rat: spatial and temporal pattern of gene expression and protein synthesis.

SPACRCAN is a hyaluronan-binding proteoglycan that is present in the pineal gland and interphotoreceptor matrix of the retina. Here, we evaluate the pattern of SPACRCAN gene expression and protein appearance during retinal and pineal gland development in the rat. In situ hybridization histochemistry with SPACRCAN riboprobes indicates that hybridization signals are first evident in the retina over developing photoreceptor cells at embryonic day 16 (E16) and in the pineal gland at E21. Immunocytochemistry using a SPACRCAN antibody shows localization of SPACRCAN protein in the developing interphotoreceptor matrix by Postnatal day 5 (P5) and in the pineal gland by P6. These studies suggest that SPACRCAN mRNA expression may occur substantially earlier than the time when SPACRCAN protein is detectable in both the retina and the pineal gland. The period of retinal histogenesis when SPACRCAN is detected first is coincident with the time photoreceptors begin to extend from the outer retinal surface, suggesting that SPACRCAN may participate in the maturation and maintenance of the light-sensitive photoreceptor outer segment.

Age Factors↗
PubMed2007

Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous system.

Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell-cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement.

Animals↗
PubMed2006

How do genes make teeth to order through development?

This introduction to new patterning theories for the vertebrate dentition outlines the historical concepts to explain graded sequences in tooth shape in mammals (incisors, canines, premolars, molars) which change in evolution in a linked manner, constant for each region. The classic developmental models for shape regulation, known as the 'regional field' and 'dental clone' models, were inspired by the human dentition, where it is known that the last tooth in each series is the one commonly absent. The mouse, as a valuable experimental model, has provided data to test these models and more recently, based on spatial-temporal gene expression data, the 'dental homeobox code' was proposed to specify regions and regulate tooth shape. We have attempted to combine these hypotheses in a new model of the combinatorial homeobox gene expression pattern with the clone and field theories in one of 'co-operative genetic interaction'. This also explains the genetic absence of teeth in humans ascribed to point mutations in mesenchymally expressed genes, which affect tooth number in each series.

Animals↗
PubMed2004

Novel NMR approach to assessing gene transfection: 4-fluoro-2-nitrophenyl-beta-D-galactopyranoside as a prototype reporter molecule for beta-galactosidase.

Gene therapy holds great promise for the treatment of diverse diseases. However, widespread implementation is hindered by difficulties in assessing the success of transfection in terms of spatial extent, gene expression, and longevity of expression. The development of noninvasive reporter techniques based on appropriate molecules and imaging modalities may help to assay gene expression. 4-Fluoro-2-nitrophenyl-beta-D-galactopyranoside (PFONPG) is a novel prototype NMR-sensitive molecule, which is highly responsive to the action of beta-galactosidase (beta-gal), the product of the lacZ gene. The molecule is stable in solution and with respect to wild-type cells, but the enzyme causes very rapid liberation of the aglycone, accompanied by color formation and a 19F NMR chemical shift of 5-10 ppm, depending on pH. Since the product is pH-sensitive, this opens the possibility for direct pH determinations at the site of enzyme activity. Molecular and 19F NMR characteristics of PFONPG in solution, blood, and prostate tumor cells are presented. This prototype molecule facilitates a novel approach for assaying gene activity in vivo.

Adenoviridae↗
PubMed1999

Lack of regulation in the heart forming region of avian embryos.

The ability to regenerate a heart after ablation of cardiogenic mesoderm has been demonstrated in early stage fish and amphibian embryos but this type of regulation of the heart field has not been seen in avians or mammals. The regulative potential of the cardiogenic mesoderm was examined in avian embryos and related to the spatial expression of genes implicated in early cardiogenesis. With the identification of early cardiac regulators such as bmp-2 and nkx-2.5, it is now possible to reconcile classical embryological studies with molecular mechanisms of cardiac lineage determination in vivo. The most anterior lateral embryonic cells were identified as the region that becomes the heart and removal of all or any subset of these cells resulted in the loss of corresponding cardiac structures. In addition, removal of the lateral heart forming mesoderm while leaving the lateral endoderm intact also results in loss of cardiac structures. Thus the medial anterior mesoderm cannot be recruited into the heart lineage in vivo even in the presence of potentially cardiac inducing endoderm. In situ analysis demonstrated that genes involved in early events of cardiogenesis such as bone morphogenetic protein 2 (bmp-2) and nkx-2.5 are expressed coincidentally with the mapped far lateral heart forming region. The activin type IIa receptor (actR-IIa) is a potential mediator of BMP signaling since it is expressed throughout the anterior mesoderm with the highest level of expression occurring in the lateral prospective heart cells. The posterior boundary of actR-IIa is consistent with the posterior boundary of nkx-2.5 expression, supporting a model whereby ActR-IIa is involved in restricting the heart forming region to an anterior subset of lateral cells exposed to BMP-2. Analysis of the cardiogenic potential of the lateral plate mesoderm posterior to nkx-2.5 and actR-IIa expression demonstrated that these cells are not cardiogenic in vitro and that removal of these cells from the embryo does not result in loss of heart tissue in vivo. Thus, the region of the avian embryo that will become the heart is defined medially, laterally, and posteriorly by nkx-2.5 gene expression. Removal of all or part of the nkx-2.5 expressing region results in the loss of corresponding heart structures, demonstrating the inability of the chick embryo to regenerate cardiac tissue in vivo at stages after nkx-2.5 expression is initiated.

Activin Receptors, Type II↗
PubMed1996

Centromeres reposition to the nuclear periphery during L6E9 myogenesis in vitro.

To test the hypothesis that genome architecture in interphase is related to nuclear function, we have compared the disposition of centromeres in nuclei of undifferentiated rat L6E9 myoblasts with that in nuclei of L6E9 myotubes differentiated in vitro. Immunofluorescence labeling showed that centromeres repositioned to the nuclear periphery during differentiation, and condensed chromatin was more prominent at the myotube nuclear envelope by electron microscopy. These data indicate that, in parallel with considerable changes in gene expression, the spatial order of the genome undergoes substantial rearrangement during myogenesis.

Animals↗
PubMed2005

WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development.

Histone H3 lysine 4 (K4) methylation has been linked to the transcriptional activation in a variety of eukaryotic species. Here we show that a common component of MLL1, MLL2, and hSet1 H3 K4 methyltransferase complexes, the WD40-repeat protein WDR5, directly associates with histone H3 di- and trimethylated at K4 and with H3-K4-dimethylated nucleosomes. WDR5 is required for binding of the methyltransferase complex to the K4-dimethylated H3 tail as well as for global H3 K4 trimethylation and HOX gene activation in human cells. WDR5 is essential for vertebrate development, in that WDR5-depleted X. laevis tadpoles exhibit a variety of developmental defects and abnormal spatial Hox gene expression. Our results are the first demonstration that a WD40-repeat protein acts as a module for recognition of a specific histone modification and suggest a mechanism for reading and writing an epigenetic mark for gene activation.

Animals↗
PubMed2004

Local protein synthesis during axon guidance and synaptic plasticity.

mRNA localization and regulated translation take central roles in axon guidance and synaptic plasticity. By spatially restricting gene expression within neurons, local protein synthesis provides growth cones and synapses with the capacity to autonomously regulate their structure and function. Studies in a variety of systems have provided insight into the specific roles of local protein synthesis during axonal navigation and during synaptic plasticity, and have begun to delineate the mechanisms underlying mRNA localization and regulated translation. Several powerful new tools have recently been developed to visualize each of these processes.

Animals↗
PubMed2006

Synapse formation and mRNA localization in cultured Aplysia neurons.

mRNA localization and regulated translation provide a means of spatially restricting gene expression within neurons during axon guidance and long-term synaptic plasticity. Here we show that synapse formation specifically alters the localization of the mRNA encoding sensorin, a peptide neurotransmitter with neurotrophin-like properties. In isolated Aplysia sensory neurons, which do not form chemical synapses, sensorin mRNA is diffusely distributed throughout distal neurites. Upon contact with a target motor neuron, sensorin mRNA rapidly concentrates at synapses. This redistribution only occurs in the presence of a target motor neuron and parallels the distribution of sensorin protein. Reduction of sensorin mRNA, but not protein, with dsRNA inhibits synapse formation. Our results indicate that synapse formation can alter mRNA localization within individual neurons. They further suggest that translation of a specific localized mRNA, encoding the neuropeptide sensorin, is required for synapse formation between sensory and motor neurons.

Analysis of Variance↗
PubMed2004

Spatial expression of a sunflower SERK gene during induction of somatic embryogenesis and shoot organogenesis.

Organogenesis or somatic embryogenesis can be induced on immature zygotic embryos (IZE) of sunflower depending on the culture conditions. Both morphogenic processes originate from the same group of cells and show identical kinetics. Using real-time PCR and in situ hybridisation, we showed that somatic embryogenesis receptor-like kinase (SERK) transcripts accumulate early after the beginning of the culture in the morphogenic zone of IZE explants whatever the induction conditions used, i.e. organogenic, embryogenic or highly embryogenic conditions. Quantitative analyses failed to show any correlation between the SERK expression level during the period decisive for the orientation of the morphogenic pathway, i.e. the first 2 days of culture, and the type of morphogenesis induced. However, after 2 days of culture on the organogenic medium, the SERK gene expression level was severely down-regulated in the IZE explants. At 4 days of culture, SERK transcripts were no longer detectable by in situ hybridisation in the developing shoot structures whereas they still continued to accumulate in the embryonic structures induced on both embryogenic and highly embryogenic culture media. The significance of these expression analyses was addressed by transfer medium experiments. Results revealed that IZE cultured on the organogenic medium were able to form somatic embryos when transferred on the highly embryogenic medium as long as the SERK transcripts accumulated at a high level in their morphogenic zone, i.e. first 2 days of culture. Passt this delay, explants rapidly lost their embryogenic competence. Indeed, after 4 days of culture on the organogenic medium, IZE were definitely oriented towards shoot organogenesis. Taken together, these data suggest that reactive cells of IZE develop the competence to somatic embryogenesis during the first day of culture whatever the morphogenic induction conditions used.

Culture Media↗
PubMed2006

Wnt/PCP signaling: a veritable polar star in establishing patterns of polarity in embryonic tissues.

Embryonic patterning has traditionally been viewed as the establishment of spatially significant gene expression in response to secreted signals. Recent work has highlighted the role of the Wnt/planar cell polarity (PCP) pathway in patterning tissues. Rather than establishing characteristic arrays of gene expression, however, this pathway functions to institute uniform polarity of cells within a tissue. Cells thus polarized can undergo directed migrations, cell divisions, etc., which are essential for normal morphogenesis. In this review, I will highlight the similarities between mechanisms that establish patterns of polarity between Drosophila and vertebrates. Further, I will discuss recent advances with regard to Wnt/PCP signaling in vertebrates.

Animals↗
PubMed2002

Insulin-like growth factor-I improves cellular and molecular aspects of healing in a collagenase-induced model of flexor tendinitis.

Flexor tendinitis is a common and debilitating injury of elite and recreational athletes. Healing may be improved through intratendinous injection of insulin-like growth factor-I (IGF-I), which has been shown in vitro to stimulate mitogenesis and enhance tendon matrix production. This study investigated the effects of intratendinous injection of IGF-I on tendon healing in an equine model of flexor tendinitis. Collagenase-induced lesions were created in the tensile region of theflexor digitorum superficialis tendon of both forelimbs of eight horses. Treated tendons were injected with 2 microg rhlGF-I intralesionally every other day for 10 injections, while controls received 0.9% NaCl. Tendon fiber deposition and organization were evaluated serially using ultrasonography throughout the 8 week trial period. Following euthanasia, the tendons were harvested and DNA, hydroxyproline, and glycosaminoglycan content determined, mechanical strength and stiffness evaluated, gene expression and spatial arrangement of collagen types I and III assessed by northern blot and in situ hybridization, and tendon fiber architecture assessed by polarized light microscopy. Local soft tissue swelling was reduced in the IGF-I treated limbs. Similarly, lesion size in IGF-I treated tendons was smaller 3 and 4 weeks after initiation of treatment. Cell proliferation and collagen content of the IGF-I treated tendons were increased compared to controls. Mechanically, IGF-I treated tendons showed a trend toward increased stiffness compared to saline treated controls. Considered together with the decreased soft tissue swelling and improved sonographic healing, these data support the potential use of intralesional IGF-I for treatment of debilitating tendon injuries.

Animals↗
PubMed1994

Complex spatial and temporal expression of lipoxygenase genes during Phaseolus vulgaris (L.) development.

A new member (Lox1) of the lipoxygenase (LOX) gene family in French bean was isolated and its specific expression compared to the general expression pattern of related LOX genes. LOX transcripts detected by a French bean cDNA LOX probe were generally abundant in young, developing tissues, and LOX protein, detected on immunoblots showed a similar distribution. Tissue prints showed that cell-specific, locally high amounts of LOX protein were observed in some tissues, for example in parts of the starch sheath in the hypocotyl and a cell layer in the pericarp. The Lox1 gene from Phaseolus vulgaris was shown in genomic Southerns to be present as a single copy per haploid genome. RNase protection studies showed that the gene is active and the transcription start site was mapped by cDNA primer extension. RNase protection studies showed that Lox1 mRNA was present only in flowers and embryonic primary leaves, in stems and most strongly, in young secondary leaves but not in any of the other organs tested (i.e. not in roots, nodules, hypocotyls, cotyledons, older secondary leaves, pericarp or seeds). Lox1 gene transcripts did not accumulate in leaves in response to methyl jasmonate or pathogen inoculation, even though these treatments caused the accumulation of other LOX transcripts. Thus, the various lipoxygenase genes are regulated differentially during French bean development and in response to different stress stimuli, and possible functions are discussed in view of the specific expression patterns observed.

Amino Acid Sequence↗
PubMed2001

P[Switch], a system for spatial and temporal control of gene expression in Drosophila melanogaster.

We have developed a method for turning on and off the expression of transgenes within Drosophila in both time and space. Two different enhancer detector elements carrying an RU486-inducible form of the yeast transcription factor GAL4 were constructed and used to generate enhancer detector lines. These lines were screened for RU486-inducible reporter gene expression in the adult head. We identified lines that exhibit inducible expression in many cell and tissue types, verifying that the elements respond to nearby enhancers. No expression was detected in the absence of the ligand. The P[Switch1] element responded to genomic enhancers less efficiently than P[Switch2] but produced more specific patterns of expression. Two P[Switch] lines were used to ablate fat body tissue in adult females through the induced expression of diphtheria toxin. These females were sterile, which correlates with fat body loss, and they died prematurely.

Animals↗