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PubMed1988

Expression of lipid body protein gene during maize seed development. Spatial, temporal, and hormonal regulation.

The storage lipids of seeds are packaged into lipid bodies, simple organelles containing a triacylglycerol core surrounded by a layer of phospholipids and proteins. A cDNA encoding the major protein from lipid bodies of maize (L3) was used as a hybridization probe to analyze the regulation of lipid body biosynthesis during seed development and germination. Immunofluorescent microscopy demonstrates that L3 protein accumulation is tissue-specific, confined to the embryo (scutellum and embryonic axis) and the aleurone layer of developing seed. Northern analyses show that L3 mRNA also accumulates to high levels in the embryo and is not found in any nonseed tissue. The steady-state level of L3 mRNA is developmentally regulated, increasing during early seed development and peaking at about the midpoint of seed development. It then decreases slowly, to 20% of the peak level, in the embryo of the mature seed and declines rapidly to undetectable levels as the seed germinates. The high level of L3 gene expression during seed development is not due to amplification of the L3 gene, since Southern analyses of maize genomic DNA indicate that the L3 gene has the same relatively low copy number throughout development (1-4 copies/haploid) genome). Rather, the increase in the steady-state level of L3 mRNA during seed development is transcriptionally regulated. The developmental changes in L3 mRNA levels are paralleled by changes in the transcription of the L3 gene, as measured by run-off transcription in isolated nuclei. The role of the plant growth regulator abscisic acid in regulating L3 gene expression was investigated. When the mature seed is allowed to imbibe in the presence of exogenous abscisic acid, germination is retarded, and the period of L3 gene expression is extended. The extended period of L3 gene expression in the presence of exogenous abscisic acid is due, at least in part, to a dramatic and specific increase in L3 gene transcription.

Abscisic Acid↗
PubMed1988

In situ hybridization patterns of zebrafish homeobox genes homologous to Hox-2.1 and En-2 of mouse.

Vertebrate homeobox genes have been identified on the basis of sequence homology to Drosophila segmentation and homeotic genes. We have analyzed the spatial distribution patterns in hatching larvae of transcripts from two different types of zebrafish (Brachydanio rerio) homeobox genes. Tritiated DNA fragments from one zebrafish gene (ZF-21) of the Antennapedia-class and one (ZF-EN) of the engrailed-type were used as probes to detect transcripts in larval tissue sections. Both zebrafish genes are expressed mainly in spatially restricted regions of the central nervous system. The transcripts from ZF-21 was detected in the posterior part of the hindbrain, and ZF-EN was found to be most intensively expressed in a restricted domain located in the region of the junction between the mid- and hindbrain. These neural expression patterns are very similar to those of the homologous murine genes Hox-2.1 and En-2 and clearly suggest a strong functional conservation.

Animals↗
PubMed2005

Identification of genes differentially expressed in extraradical mycelium and ectomycorrhizal roots during Paxillus involutus-Betula pendula ectomycorrhizal symbiosis.

The development of ectomycorrhizal symbiosis leads to drastic changes in gene expression in both partners. However, little is known about the spatial regulation of symbiosis-regulated genes. Using cDNA array profiling, we compared the levels of expression of fungal genes corresponding to approximately 1,200 expressed sequenced tags in the ectomycorrhizal root tips (ECM) and the connected extraradical mycelium (EM) for the Paxillus involutus-Betula pendula ectomycorrhizal association grown on peat in a microcosm system. Sixty-five unique genes were found to be differentially expressed in these two fungal compartments. In ECM, a gene coding for a putative phosphatidylserine decarboxylase (Psd) was up-regulated by 24-fold, while genes coding for urea (Dur3) and spermine (Tpo3) transporters were up-regulated 4.1- and 6.2-fold in EM. Moreover, urea was the major nitrogen compound found in EM by gas chromatography-mass spectrometry analysis. These results suggest that (i) there is a spatial difference in the patterns of fungal gene expression between ECM and EM, (ii) urea and polyamine transporters could facilitate the translocation of nitrogen compounds within the EM network, and (iii) fungal Psd may contribute to membrane remodeling during ectomycorrhiza formation.

Basidiomycota↗
PubMed2005

Somatic transgenesis in the avian model system.

The chick embryo is a versatile model system, in which classical embryology can be combined with modern molecular approaches. In the last two decades, several efficient methods have been developed to introduce exogenous genes into the chick embryo. These techniques allow alteration of gene expression levels in a spatially and temporally restricted manner, thereby circumventing embryonic lethality and/or eliminating secondary effects in other tissues. Here, we present the current status of avian somatic transgenic techniques, focusing on electroporation and retrovirus-mediated gene transfer. Electroporation allows quick and efficient gain-of-function studies based on transient misexpression of genes. Retroviral vectors, which are capable of integrating exogenous genes into the host chromosome, permit analysis of long-term effects of gene misexpression. The variety of methods available for somatic transgenesis, along with the recent completion of the chicken genome, are transforming the chick embryo into one of the most attractive model systems to examine function of genes that are important for embryonic development.

Animals↗
PubMed2002

Subchromosomal positioning of the epidermal differentiation complex (EDC) in keratinocyte and lymphoblast interphase nuclei.

The epidermal differentiation complex (EDC) at 1q21 is host to many structurally and functionally related genes coding for proteins involved in the differentiation process of keratinocytes. The grouping together of these genes which share spatial and temporal expression and interrelated functions is a remarkable genomic feature which has led to suggestions that the region may have a coordinated transcription control mechanism. With the growing awareness that the organization of the genome within the interphase nucleus is relevant to transcriptional activity, we have investigated the spatial organization of the EDC in the nuclei of keratinocytes, where the EDC genes are highly expressed, and lymphoblasts, where they are silent. Using 2D and 3D FISH we find that in keratinocyte nuclei the EDC is frequently positioned external to the chromosome 1 territory compared to lymphoblasts where the EDC more often adopts a peripheral or internal location. It has been previously shown that the MHC region can extend from the chromosome 6 territory in relation to transcriptional activity. This study of the EDC thus provides a further example of a gene-dense complex capable of assuming extraterritorial positioning in relation to cell type/transcription status.

B-Lymphocytes↗
PubMed1996

Enterocytic gene expression in intestinal adaptation: evidence for a specific cellular response.

After massive small bowel resection, the remnant gut epithelium undergoes an adaptive response marked by an increase in villus height, crypt depth, and crypt cell production rate. Although morphological features of gut adaptation have been well characterized, the differentiation status and response of epithelial cells populating the adaptive villus is unclear. To address these issues, cell-specific and spatial patterns of expression of a set of enterocytic genes were characterized in rats after 70% small bowel resection. The liver and intestinal (I) fatty acid binding protein (FABP) and apolipoprotein A-I (apo A-I) and apo A-IV genes were studied because they exhibit unique regional and cell-specific patterns of expression in the developing and adult gut. At 48 h after surgery, apo A-IV and I-FABP mRNA levels were increased up to 3.5-fold in adaptive remnant ileum compared with sham-operated or sham-resected control ileum. In situ hybridization and immunohistochemical analyses revealed a marked increase in enterocytic apo A-IV mRNA and protein expression in the adaptive ileum, from villus base to tip but not in crypts. By 1 wk after resection, apo A-IV, but not I-FABP, mRNA levels remained elevated in remnant ileum, although duodenal I-FABP mRNA levels were still increased. In contrast, apo A-I mRNA levels were not significantly induced. These results indicate that the enterocyte can respond acutely to loss of small bowel surface area by increasing expression of several genes. This compensatory enterocytic response is spatially (from duodenum to ileum) and temporally regulated. These results suggest initiation of the adaptive response occurs by way of a complex set of molecular pathways involving villus and crypt cells.

Adaptation, Physiological↗
PubMed2004

Unique contribution of heat shock transcription factor 4 in ocular lens development and fiber cell differentiation.

Mammalian ocular lens development results via a differentiation program that is highly regulated by tissue-specific transcription factors. Central to this is the terminal differentiation of fiber cells, which develop from epithelial cells on the anterior surface of the lens, accompanied by a change in cell shape and expression of structural proteins (such as membrane proteins MP19, MIP26, connexin 43, 46, and 50, cytoskeletal proteins CP49, CP115, and alpha, beta, and gamma crystallins), creating a transparent, refractive index gradient in the lens. Mutations in genes controlling eye development and in lens structural protein genes are associated with multiple ocular developmental disorders, including cataracts and other opacities of the lens. Here we show that heat shock transcription factor 4 (HSF4) expression in the developing lens is required for correct lens development and that inactivation of hsf4 leads to early postnatal cataract formation with primary effects specific to terminal fiber cell differentiation. These data suggest that HSF4 acts as a critical transcription factor for lens-specific target gene expression, in particular regulating the small 25 kDa heat shock protein that acts as a modifier for lens opacity and cataract development. Thus, HSF4 fulfills a central role in controlling spatial and temporal expression of genes critical for correct development and function of the lens.

Animals↗
PubMed2003

Estrogen receptors in Xenopus: duplicate genes, splice variants, and tissue-specific expression.

The estrogenic steroid hormones, acting primarily through the nuclear estrogen receptors ERalpha and ERbeta, regulate sexual differentiation in a wide variety of vertebrates. In the frog Xenopus laevis, estrogen regulates the strength of vocal neuromuscular synapses and contributes to the physiological basis of sexually differentiated songs. To understand the mechanisms by which estrogen produces these effects, we have characterized the ERs of X. laevis and their expression in laryngeal muscle and other tissues. We found a remarkable molecular diversity in the estrogen receptor population within individuals. First, we have identified two distinct ERalpha genes, xlERalpha1 and xlERalpha2, which represent, to our knowledge, the first discovery of retained duplicates of the ERalpha gene in any species. These two genes are highly conserved at the amino acid level but have distinct nucleotide sequences; moreover, ERalpha2 has no N-terminal domain. Cloning of ERalpha and ERbeta in the related species Xenopus tropicalis and phylogenetic analysis indicate that the two xlERalpha loci were generated by a duplication specific to the X. laevis lineage-most likely the genome duplication that led to a doubling of the X. laevis chromosome number about 30 million years ago. The primary ER expressed in X. laevis laryngeal muscle is the novel gene xlERalpha2; ERalpha1 is primarily expressed in liver, forebrain, and oviduct. Alternatively spliced transcripts of both xlERalpha1 and xlERalpha2 are also expressed in a tissue-specific manner. We propose that complementary spatial expression of these two genes and their alternatively spliced transcripts contributes to their conservation over such a long period of time, consistent with the subfunctionalization model for evolution after gene duplication.

Amino Acid Sequence↗
PubMed1998

Cloning and expression pattern of Xenopus prx-1 (Xprx-1) during embryonic development.

Homeobox genes are expressed both temporally and spatially during vertebrate development, and regulate the tissue-specific expression of other genes. A Xenopus paired-related homeobox- 1 (Xprx-1) cDNA was cloned. Xprx-1 had a paired-related homeodomain, but did not contain a paired-box. The sequence of Xprx-1 had a high level of homology with K-2(mouse) and Prx-1 (chicken), thus Xprx-1 is assumed to be the Xenopus homolog of these genes. Xprx-1 transcripts were maternally restricted, in Xenopus embryos, and a decrease in the late blastula stage was followed by an increase in zygotic transcripts after gastrulation. The transcripts were localized to the animal hemisphere of the late blastula and were concentrated in the branchial arches of the tail-bud stage embryo. In animal cap experiments, Activin A dose-dependently induced Xprx-1 gene expression. These results suggest that Xprx-1 plays a role in early Xenopus development similar to other species.

Activins↗
PubMed1999

Intron-independent association of splicing factors with active genes.

The cell nucleus is organized as discrete domains, often associated with specific events involved in chromosome organization, replication, and gene expression. We have examined the spatial and functional relationship between the sites of heat shock gene transcription and the speckles enriched in splicing factors in primary human fibroblasts by combining immunofluorescence and fluorescence in situ hybridization (FISH). The hsp90alpha and hsp70 genes are inducibly regulated by exposure to stress from a low basal level to a high rate of transcription; additionally the hsp90alpha gene contains 10 introns whereas the hsp70 gene is intronless. At 37 degrees C, only 30% of hsp90alpha transcription sites are associated with speckles whereas little association is detected with the hsp70 gene, whose constitutive expression is undetectable relative to the hsp90alpha gene. Upon exposure of cells to heat shock, the heavy metal cadmium, or the amino acid analogue azetidine, transcription at the hsp90alpha and hsp70 gene loci is strongly induced, and both hsp transcription sites become associated with speckles in >90% of the cells. These results reveal a clear disconnection between the presence of intervening sequences at specific gene loci and the association with splicing factor-rich regions and suggest that subnuclear structures containing splicing factors are associated with sites of transcription.

Azetidines↗
PubMed2000

Functional characterization of a cis-acting DNA antisilencer region that modulates myelin proteolipid protein gene expression.

Regulation of myelin proteolipid protein (PLP:) gene expression is tightly controlled, both spatially and temporally. Previously, we have shown with transgenic mice that a PLP:-lacZ fusion gene (which includes the entire sequence for PLP: intron 1 DNA) is regulated in a similar manner to endogenous PLP: gene expression. Furthermore, by deletion-transfection analyses using assorted PLP:-lacZ constructs with partial deletion of PLP: intron 1 sequences, we have shown that the first intron possesses an antisilencer region that is capable of over-coming repression mediated by two distinct regions located elsewhere within intron 1 DNA. Here, we report the ability of various fragments encompassing the antisilencer region to restore beta-galactosidase activity when inserted into PLP:-lacZ constructs, which originally exhibited low levels of beta-galactosidase activity. Additional constructs were generated to test the effects of these antisilencer-containing fragments in constructs that are missing either one or both of the negative regulatory regions that are overridden during antisilencing. Transfection analyses, in conjunction with protein-DNA binding assays, suggest that several nuclear factors are necessary for derepression of PLP: gene activity in an oligodendroglial cell line. Moreover, either the "core" or complete antisilencing region can act in an additive or synergistic fashion when multiple copies are inserted into the Plp-lacZ constructs.

Animals↗
PubMed2007

alpha7 Nicotinic receptor gene delivery into mouse hippocampal neurons leads to functional receptor expression, improved spatial memory-related performance, and tau hyperphosphorylation.

Brain alpha7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal alpha7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)-derived vectors. In order to determine whether alpha7 receptor-mediated cytotoxicity was dependent on receptor density, rat alpha7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [3H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an alpha7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat alpha7 nicotinic receptors increased [3H]MLA binding in this region in wild type and alpha7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no alpha7 receptor response did so following alpha7 receptor gene delivery in wild type mice. Recovery of alpha7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat alpha7 receptor gene compared with green fluorescent protein-transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether alpha7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of alpha7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.

Aconitine↗
PubMed2001

Tet-system for the regulation of gene expression during embryonic development.

The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTACMV(M1) and rTACMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and beta-galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTACMV(M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTACMV-3/NZL-2 embryos at E13.5. Doxycycline abolished beta-gal expression in tTACMV(M1)/NZL-2 but induced it in rTACMV-3/NZL-2 embryos including late stages of embryo-genesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that 'double reporter' animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.

Aging↗
PubMed1994

Developmental cell-specific regulation of Na(+)-K(+)-ATPase alpha 1-, alpha 2-, and alpha 3-isoform gene expression.

Na(+)-K(+)-activated adenosine triphosphatase (Na(+)-K(+)-ATPase) is the integral membrane protein that maintains the Na(+)-K(+) electrochemical gradient across the plasma membrane. Because of the importance of the Na(+)-K(+) electrochemical gradient to fundamental and specialized cell functions, we investigated the cell-specific modulation of Na(+)-K(+)-ATPase alpha-subunit isoform (alpha 1, alpha 2, and alpha 3) gene expression in different stages of postimplantation mouse embryos and neonatal rat tissues by in situ hybridization with use of isoform-specific rat-derived antisense RNA probes. At early organogenesis (9.5-10.5 days postcoitus), we demonstrated generalized coexpression of alpha 1- and alpha 2-isoforms throughout the mouse embryo with greater levels in the developing but already functional heart, in contrast to the distinct spatially restricted alpha 3-isoform gene expression in the early developing neural tube. At midorganogenesis (15.5-16.5 days postcoitus), differential spatial variation in alpha 1-, alpha 2-, and alpha 3-isoform gene expression was already evident in all organs. Interestingly, region-specific expression patterns within single cell types were noted throughout development and were exemplified by 1) alpha 3-isoform gene expression in marginal cells of the 10.5-day-postcoitus developing neural tube; 2) alpha 1-, alpha 2-, and alpha 3-isoform gene expression in cerebellar granular cells of the 4-day-old rat brain; and 3) alpha 1- and alpha 3-isoform gene expression in 4-day-old rat ventricular cardiomyocytes. These isoform-specific changes in cellular and regional Na(+)-K(+)-ATPase alpha-isoform gene expression may play an active role in development and specialized cell functions.

Animals↗
PubMed2006

angaGEDUCI: Anopheles gambiae gene expression database with integrated comparative algorithms for identifying conserved DNA motifs in promoter sequences.

BACKGROUND: The completed sequence of the Anopheles gambiae genome has enabled genome-wide analyses of gene expression and regulation in this principal vector of human malaria. These investigations have created a demand for efficient methods of cataloguing and analyzing the large quantities of data that have been produced. The organization of genome-wide data into one unified database makes possible the efficient identification of spatial and temporal patterns of gene expression, and by pairing these findings with comparative algorithms, may offer a tool to gain insight into the molecular mechanisms that regulate these expression patterns. DESCRIPTION: We provide a publicly-accessible database and integrated data-mining tool, angaGEDUCI, that unifies 1) stage- and tissue-specific microarray analyses of gene expression in An. gambiae at different developmental stages and temporal separations following a bloodmeal, 2) functional gene annotation, 3) genomic sequence data, and 4) promoter sequence comparison algorithms. The database can be used to study genes expressed in particular stages, tissues, and patterns of interest, and to identify conserved promoter sequence motifs that may play a role in the regulation of such expression. The database is accessible from the address http://www.angaged.bio.uci.edu. CONCLUSION: By combining gene expression, function, and sequence data with integrated sequence comparison algorithms, angaGEDUCI streamlines spatial and temporal pattern-finding and produces a straightforward means of developing predictions and designing experiments to assess how gene expression may be controlled at the molecular level.

Algorithms↗
PubMedDate not supplied

Control of transgene expression using local hyperthermia in combination with a heat-sensitive promoter.

BACKGROUND: Local production of therapeutic proteins, e.g. for cancer treatments, is based on gene therapy approaches and requires tight spatial and temporal control of gene expression. Here we demonstrate the use of local hyperthermia of varying intensity and duration to control the expression of a transgene under control of the thermoinducible hsp70 (heat shock protein) promoter. METHODS: Heat-induced expression of the EGFP (green fluorescent protein) reporter gene was characterized using a stably transfected glioma C6 cell line expressing the EGFP gene under control of the heat inducible minimal hsp70 promoter both in vitro and in vivo for subcutaneous tumors in immunodeficient mice. RESULTS: A heat shock of 20-30 min at 43 degrees C in cell culture led to a maximum EGFP concentration at about 24 h. Heat treatments at higher temperature (up to 48 degrees C) but with shorter durations (down to 30 s) also induced strong EGFP expression. Local heating in situ led to gradients in EGFP expression which decreased with increasing distance from the heat source. CONCLUSION: Local hyperthermia, in combination with a heat sensitive promoter, represents a method for the spatial and temporal control of transgene expression.

Animals↗
PubMed1995

Adenovirus-mediated expression of a reporter gene in thalamocortical cocultures.

Organotypic cocultures of thalamic and cortical explants have recently been used to study the development of the thalamocortical axonal network in the mammalian neocortex. To explore the possibility of genetically manipulating organotypic explants, rat thalamocortical (TC) cocultures were infected with the recombinant adenovirus, Adv/RSV beta gal. Infection of the cortical explants resulted in long-term expression (2 weeks) of the reporter gene (beta-galactosidase) with no significant alterations to the structural integrity of the explants. By micro-injecting the adenoviruses into cortical explants a significant degree of spatial control over reporter gene expression was obtained. DiI-labeled axonal projections from thalamic explants into infected (n = 116) and control cortical (n = 120) explants were also analyzed. There was no significant difference in the extent or degree of TC ingrowth into infected or control cortical explants. Thalamic explants were also efficiently infected with the Adv/RSV beta gal virus. While the pattern and extent of TC ingrowth from infected thalamic explants was similar to controls, the percentage of viable, infected thalamic explants was decreased. These experiments were necessary precursors for future studies using recombinant adenoviruses and organotypic cocultures. Genetic manipulation of these cocultures should enable the dissection of proteins involved in the development of axonal networks in the mammalian neocortex, using a system amenable to direct manipulation and observation.

Adenoviridae↗
PubMed2026

DREAMS illuminates spatial DNA and RNA modification landscapes.

DNA and RNA modifications regulate gene expression and RNA processing, but their spatial organization in complex tissues remains elusive. Here we developed DNA RNA Elements Areal Mass Spectrometry (DREAMS), a mass spectrometry imaging platform that spatially maps diverse nucleic acid modifications simultaneously. Applying DREAMS to TET-deficient mouse brains (Tet1Δ/Δ and triple Tet1/2/3Δ/Δ), we uncover TET1's unexpected role in modulating N1-methyladenosine (m1A), a pivotal RNA modification. While DREAMS reveals broad modification landscapes altered across TET knockouts, we identify TET1-mediated changes in m1A that correlate with transcriptome alterations. Our work establishes DREAMS as a transformative tool for spatial epigenomics/epitranscriptomics and suggests that TET enzymes could influence multiple DNA and RNA modifications with potential gatekeeping roles in nucleic acid regulation.

Animals↗