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Results for “spatial gene expression”

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PubMed2005

Protein synthesis-dependent LTP in isolated dendrites of CA1 pyramidal cells.

Local dendritic protein synthesis provides a mechanism by which the effects of gene expression can be spatially restricted within synaptodendritic compartments of neurons. In the present study, we show that long-term potentiation (LTP), induced by two strong tetanic trains, can be produced in isolated CA1 apical dendrites and that new protein synthesis is critical for this dendritic LTP. LTP in isolated dendrites is also blocked by rapamycin, an inhibitor of growth-related protein synthesis, similar to that observed for LTP in the intact hippocampal slice. These results show that increased dendritic translation of proteins is a critical molecular mechanism for hippocampal LTP.

Animals↗
PubMed2006

Gene expression in cortex and hippocampus during acute pneumococcal meningitis.

BACKGROUND: Pneumococcal meningitis is associated with high mortality (approximately 30%) and morbidity. Up to 50% of survivors are affected by neurological sequelae due to a wide spectrum of brain injury mainly affecting the cortex and hippocampus. Despite this significant disease burden, the genetic program that regulates the host response leading to brain damage as a consequence of bacterial meningitis is largely unknown. We used an infant rat model of pneumococcal meningitis to assess gene expression profiles in cortex and hippocampus at 22 and 44 hours after infection and in controls at 22 h after mock-infection with saline. To analyze the biological significance of the data generated by Affymetrix DNA microarrays, a bioinformatics pipeline was used combining (i) a literature-profiling algorithm to cluster genes based on the vocabulary of abstracts indexed in MEDLINE (NCBI) and (ii) the self-organizing map (SOM), a clustering technique based on covariance in gene expression kinetics. RESULTS: Among 598 genes differentially regulated (change factor > or = 1.5; p < or = 0.05), 77% were automatically assigned to one of 11 functional groups with 94% accuracy. SOM disclosed six patterns of expression kinetics. Genes associated with growth control/neuroplasticity, signal transduction, cell death/survival, cytoskeleton, and immunity were generally upregulated. In contrast, genes related to neurotransmission and lipid metabolism were transiently downregulated on the whole. The majority of the genes associated with ionic homeostasis, neurotransmission, signal transduction and lipid metabolism were differentially regulated specifically in the hippocampus. Of the cell death/survival genes found to be continuously upregulated only in hippocampus, the majority are pro-apoptotic, while those continuously upregulated only in cortex are anti-apoptotic. CONCLUSION: Temporal and spatial analysis of gene expression in experimental pneumococcal meningitis identified potential targets for therapy.

Acute Disease↗
PubMed1994

DET1, a negative regulator of light-mediated development and gene expression in arabidopsis, encodes a novel nuclear-localized protein.

The mechanisms by which plants integrate light signals to modify endogenous developmental programs are largely unknown. One candidate for a signal transduction component that may integrate light with developmental pathways is the Arabidopsis DET1 gene product. Here we report the positional cloning of the DET1 locus and show that DET1 is a unique nuclear-localized protein. An analysis of a number of det1 mutants indicates that mutants with partial DET1 activity develop as light-grown plants in the dark. det1 null mutants share this phenotype, but also display severe defects in temporal and spatial regulation of gene expression. These results suggest that DET1 acts in the nucleus to control the cell type-specific expression of light-regulated promoters.

Amino Acid Sequence↗
PubMed1994

Overlapping patterns of IGF2 and H19 expression during human development: biallelic IGF2 expression correlates with a lack of H19 expression.

The spatial patterns of IGF2 and H19 gene expression are strikingly similar during parts of human embryonic/fetal and early postnatal development. Notable exceptions were found with the ciliary anlage of the embryonic retina and the choroid plexus/leptomeninges, where transcripts from the IGF2 but not the H19 locus could be detected. Moreover, in contrast to the other tissue samples examined, the choroid plexus/leptomeninges expressed both parental IGF2 alleles. Whilst RNase protection analysis revealed a weak activity of the P1 promoter in the choroid plexus/leptomeninges, the P2, P3 and P4 promoters were all active wherever IGF2 was expressed. We discuss these observations with respect to a hypothesized coordinated control of the reciprocally imprinted and closely linked IGF2 and H19 loci.

Alleles↗
PubMed2026

Genetic Deletion of Cis-Regulatory Elements to Dissect the Function of the Non-coding Genome in human Preimplantation Models.

Cis-regulatory elements coordinate gene expression in a spatially and temporally controlled manner and contribute to the establishment of distinct cellular states during development. A substantial proportion of transcriptionally active cis-regulatory elements in primate embryos originated from ancient retroviral integrations into the germline. These endogenous retroviruses, also known as long terminal repeat retrotransposons, retain intrinsic regulatory activity and are often species-specific, making them strong candidates for regulating species-divergent aspects of embryonic development. Ethical and legal restrictions on human embryo research have historically limited direct investigation of gene regulation during human embryogenesis. Human naive pluripotent stem cells and three-dimensional stem cell-based blastocyst models provide alternative systems for studying early developmental processes. This protocol describes the CRISPR-Cas9-mediated deletion of endogenous retrovirus-derived cis-regulatory elements in human naive pluripotent stem cells. Preassembled Cas9 and single-guide RNA ribonucleoprotein complexes are delivered by nucleofection, followed by single-cell cloning, PCR-based genotyping, Sanger sequencing, expansion, cryopreservation, and genomic stability assessment of the edited lines. The resulting wild-type, heterozygous, and homozygous or hemizygous deletion clones provide a platform for investigating the contribution of individual endogenous retrovirus-derived elements to gene regulation in human preimplantation models. This method enables direct functional interrogation of species-specific non-coding regulatory sequences and supports the study of transcriptional mechanisms involved in early human development.

Humans↗
PubMed1999

Developmental bioinformatics: linking genetic data to virtual embryos.

This paper discusses current efforts to produce databases of gene expression for the major model embryos used in developmental biology. The efforts to build these resources were motivated by the need for immediate internet access to all types of research data, and the production of these databases is a major and new challenge for bioinformatics. Thus far bioinformatics has mainly been concerned with textually oriented resources and data, much of it concerned with gene and protein sequences. Because the genetic basis of developmental biology is integrated with developmental anatomy, these databases require the use of images to link molecular data with spatial information. In order to standardise database formats, digital atlases of some model systems are being produced that include integrated anatomical descriptions and these are being linked to appropriate genetic data. Integrating such image-based, searchable data into databases makes new demands on the field of bioinformatics and we consider here the imaging modalities that are used to obtain information and we discuss in particular the production of 3D images from serial sections. Next, we consider how to integrate textual and spatial descriptions of gene expression and the key tool needed to make this possible, i.e. anatomical nomenclature. A short review of internet resources on developmental biology is also given and future prospects for the development of these databases are discussed.

Animals↗
PubMed2000

Nonreceptor tyrosine protein kinase pp60c-src in spatial learning: synapse-specific changes in its gene expression, tyrosine phosphorylation, and protein-protein interactions.

c-src is a nonreceptor tyrosine protein kinase that is highly concentrated in synaptic regions, including synaptic vesicles and growth cones. Here, we report that the mRNA signal of pp60c-src is widely distributed in the rat brain with particularly high concentrations in the hippocampus. After spatial maze learning, up-regulation of c-src mRNA was observed in the CA3 region of the hippocampus, which was accompanied by increases in pp60c-src protein in hippocampal synaptosomal preparations. Training also triggered an increase in c-src protein tyrosine kinase activity that was correlated with its tyrosine dephosphorylation in the synaptic membrane fraction. After training, pp60c-src from hippocampus showed enhanced interactions with synaptic proteins such as synapsin I, synaptophysin, and the type 2 N-methyl-d-aspartate receptor, as well as the cytoskeletal protein actin. The association of pp60c-src with insulin receptor in the synaptic membrane fraction, however, was temporally decreased after training. Furthermore, in vitro results showed that Ca(2+) and protein kinase C might be involved in the regulation of protein-protein interactions of pp60c-src. These results suggest, therefore, that pp60c-src participates in the regulation of hippocampal synaptic activity during learning and memory.

Animals↗
PubMed2006

Identification of a novel pituitary-specific chicken gonadotropin-releasing hormone receptor and its splice variants.

In all vertebrates, GnRH regulates gonadotropin secretion through binding to a specific receptor on the surface of pituitary gonadotropes. At least two forms of GnRH exist within a single species, and several corresponding GnRH receptors (GNRHRs) have been isolated with one form being pituitary specific. In chickens, only one type of widely expressed GNRHR has previously been identified. The objectives of this study were to isolate a chicken pituitary-specific GNRHR and to determine its expression pattern during a reproductive cycle. Using a combined strategy of PCR and rapid amplification of cDNA ends (RACE), a new GNRHR (chicken GNRHR2) and two splice variants were isolated in domestic fowl (Gallus gallus domesticus). Full-length GNRHR2 and one of its splice variant mRNAs were expressed exclusively in the pituitary, whereas mRNA of the other splice variant was expressed in most brain tissues examined. The deduced amino acid sequence of full-length chicken GNRHR2 reveals a seven transmembrane domain protein with 57%-65% homology to nonmammalian GNRHRs. Semiquantitative real-time PCR revealed that mRNA levels of full-length chicken GNRHR2 in the pituitary correlate with the reproductive status of birds, with maximum levels observed during the peak of lay and 4 wk postphotostimulation in females and males, respectively. Furthermore, GnRH stimulation of GH3 cells that were transiently transfected with cDNA that encodes chicken GNRHR2 resulted in a significant increase in inositol phosphate accumulation. In conclusion, we isolated a novel GNRHR and its splice variants in chickens, and spatial and temporal gene expression patterns suggest that this receptor plays an important role in the regulation of reproduction.

Amino Acid Sequence↗
PubMed1998

Maternal and zygotic expression of the endoderm-specific alkaline phosphatase gene in embryos of the ascidian, Halocynthia roretzi.

Alkaline phosphatase (AP) activity is expressed by endodermal cells of ascidian larvae. It was reported previously that the expression of AP activity is resistant to treatment with actinomycin D, a transcription inhibitor that inhibits the appearance of several other tissue-specific molecules and morphological markers of tissue formation in developing ascidians. The resistance of AP expression to actinomycin D treatment suggests that endodermal AP activity does not depend on zygotic transcription and that its appearance is mediated by the translational activation of maternal AP mRNA present in ascidian eggs. However, it was also shown that anucleate merogons do not develop AP activity. To directly examine whether maternal AP transcripts are present in the cytoplasm of eggs, we isolated a cDNA of an endoderm-specific AP in Halocynthia roretzi and examined the temporal and spatial expressions of this gene during embryogenesis using Northern blots and in situ hybridization. Maternal AP transcripts were detected in oocytes, cleaving-stage embryos, and in gastrulae, and endoderm-specific AP transcripts dramatically increased about 14 times from the neurula stage to the larval stage in endoderm precursor cells. These results suggest that the differentiation of endoderm is primarily correlated with the activation of zygotic transcription of the AP gene, presumably by egg cytoplasmic factors, similar to how muscle and epidermis are believed to develop.

Alkaline Phosphatase↗
PubMed1986

Hybrid dysgenesis-induced revertants of insertions at the 5' end of the rudimentary gene in Drosophila melanogaster: transposon-induced control mutations.

Mutations in the 5' control region of the rudimentary (r) gene of Drosophila melanogaster were generated by using hybrid dysgenesis to mobilize P elements that were already inserted within this region. Eighteen new mutations out of 7793 chromosomes were isolated. Among the mutations were small insertions, deletions and inversions. All five of the deletions deleted into the 5' coding region of the r gene resulting in a severe mutant phenotype. The inversions and small insertions left the coding region intact, but altered the 5' control region. Northern analyses of the message levels in adult females indicated that these mutations alter the amount of the wild-type rudimentary message. These data also indicated that there is a region about 800 bp upstream of the start of transcription which is necessary for normal r expression. This region is not involved with the temporal and spatial regulation of r gene expression, but with the quantitative expression. An analysis of the DNA changes associated with each of the mutations demonstrates that P elements usually mobilize in an imprecise manner. Of the 22 mutations that have been isolated to date, only three are precise excisions. This means that P elements are potent mutators of the genome.

Animals↗
PubMed2000

Spatial and temporal expression of a Polysphondylium spore-specific gene.

In the cellular slime mold Polysphondylium spherical masses of cells are periodically released from the base of the culminating sorogen. These whorls undergo a morphogenetic transformation from spherical to radial symmetry, marked by the early emergence of a radially symmetric prepattern on the whorl surface. In previous experiments, morphogenesis was followed by observing prestalk cell markers. Here we describe the isolation and characterization of a spore coat gene whose expression pattern is the negative image of the prestalk pattern. To study the molecular mechanism of sp-45 gene regulation, we have cloned and analyzed the sp-45 promoter. Deletion analysis localized a single positive regulatory element (PRE) to a 106-bp fragment between positions -246 and -352 of the upstream coding sequence. This fragment can be further divided into a promoter-proximal and promoter-distal PRE and a 29-bp sequence between them. The distal PRE can regulate prespore expression when fused to a nonfunctioning basal promoter. The distal PRE contains two adjacent essential elements, a Gr box (GTGATATAGTGG) and a TA box (TAATATATT). Each element can drive prespore cell-specific reporter gene expression independently when incorporated into a nonfunctional promoter. Our results also show that prespore cell-specific gene expression is solely under positive regulation, with no evidence for spore-specific enhancers or cis-acting negative regulatory elements. By fusing GFP to the C-terminus of sp-45, we have demonstrated that the graded gene expression of SP45 in the sorogen is regulated by a sequence lying within the sp-45 coding sequence. The temporal and spatial expression pattern of this protein, taken together with the prestalk expression pattern, demonstrates unambiguously that the radial symmetries that emerge in the whorl are established by a system of positional coordinates and that cell sorting plays little if any role in this process.

Amino Acid Sequence↗
PubMed2005

Expression of mRNAs encoding insulin-like growth factor (IGF) ligands, IGF receptors and IGF binding proteins during follicular growth and atresia in the ovine ovary throughout the oestrous cycle.

The components of the insulin-like growth factor (IGF) system appear to be involved in the regulation of ovarian follicular growth and atresia in sheep. However, previous studies have only investigated a select few components of the system. The aim of the present study was to investigate the expression of mRNA encoding all of the components of the sheep IGF system among follicles of varying size and health status throughout the oestrous cycle using sheep-specific ribonucleotide probes and in situ hybridisation. For all IGF components, gene expression was unaffected by stage of oestrous cycle. IGF-I mRNA expression in all classes of follicle was generally low throughout the oestrous cycle, while IGFBP-1 mRNA expression could not be demonstrated at all. In contrast, there was relatively intense follicular expression of mRNAs encoding all remaining IGF system components. For IGF-II, both IGF receptors and IGFBP-2, -3, -4, -5, and -6, gene expression decreased as follicles increased in diameter (P < 0.01). IGF-II, type I IGF-R and IGFBP-2, -3, -4, and -6 mRNA expression significantly decreased as follicles progressed from healthy to atretic status (P < 0.01), whereas gene expression for type II IGF-R and IGFBP-5 was greater in atretic follicles (P < 0.01). This study demonstrates the spatial patterns of follicular gene expression for all of the IGF system components in cycling sheep for the first time. These results further highlight the potential functional role of IGF-II, in contrast to IGF-I, in the autocrine and/or paracrine regulation of follicle growth in sheep.

Animals↗
PubMed1999

Towards a molecular anatomy of the Xenopus pronephric kidney.

Kidney development is distinguished by the sequential formation of three structures of putatively equivalent function from the intermediate mesoderm, the pronephros, mesonephros, and metanephros. While these organs differ morphologically, their basic structural organization exhibits important similarities. The earliest form of the kidney, the pronephros, is the primary blood filtration and osmoregulatory organ of fish and amphibian larvae. Simple organization and rapid formation render the Xenopus pronephric kidney an ideal model for research on the molecular and cellular mechanisms dictating early kidney organogenesis. A prerequisite for this is the identification of genes critical for pronephric kidney development. This review describes the emerging framework of genes that act to establish the basic components of the pronephric kidney: the corpuscle, tubules, and the duct. Systematic analysis of marker gene expression, in temporal and spatial resolution, has begun to reveal the molecular anatomy underlying pronephric kidney development. Furthermore, the emerging evidence indicates extensive conservation of gene expression between pronephric and metanephric kidneys, underscoring the importance of the Xenopus pronephric kidney as a simple model for nephrogenesis. Given that Xenopus embryos allow for easy testing of gene function, the pathways that direct cell fate decisions in the intermediate mesoderm to make the diverse spectrum of cell types of the pronephric kidney may become unraveled in the future.

Animals↗
PubMed1998

Expression of the tobacco anionic peroxidase gene is tissue-specific and developmentally regulated.

Transcriptionally regulated expression of tobacco anionic peroxidase was investigated with regard to tissue specificity and developmental regulation. Two tobacco species, Nicotiana sylvestris and Nicotiana tabacum cv. Xanthi, were stably transformed with a gene chimera composed of 3 kb of the tobacco anionic peroxidase promoter, the Escherichia coli beta-glucuronidase (GUS) coding region and the nopaline synthase terminator. Gene expression was regulated spatially and developmentally in all organs, and generally increased with age and maturity of the plant, tissue or organ. In the aerial portions of the plant, GUS activity was strongly expressed in trichomes and epidermis at nearly all developmental stages. In later stages of development, activity was also detected in ground tissue and parenchyma cells associated with vascular tissues. Activity in roots was limited to cortical cells and vascular-associated parenchyma cells. In reproductive tissue, expression was observed in sepals and petals before anthesis, and in all floral organs after anthesis. Expression was never detected in vascular tissue and was poorly correlated with lignification except in the cells surrounding primary xylem and pericyclic fibers in N. sylvestris. These studies suggest that this peroxidase isoenzyme is only limitedly involved in lignification but may be important in plant defense, growth and development.

Cell Wall↗
PubMed1994

The expression of the receptor-protein tyrosine kinase gene, eck, is highly restricted during early mouse development.

Cell-cell interactions during embryogenesis have been shown to be important for establishing developmental fates in a number of organisms such as Drosophila and Caenorhabditis. One class of genes shown to mediate this process are receptor-protein tyrosine kinases (R-PTKs). To examine whether R-PTKs might participate in similar mechanisms operating in the mammalian embryo, an important prerequisite is to show that these genes are expressed in spatial and temporal patterns consistent with such a role. Here, we analyze the expression of eck, a member of the eph family of R-PTKs, during gastrulation and early organogenesis by in situ hybridization. eck transcripts are first detected in gastrulation stage embryos (6.5-7.5 days post coitum (dpc)) in ectodermal cells adjacent to the distal region of the primitive streak. By the neural plate stage (approximately 7.5 dpc), eck expression becomes restricted to the extreme distal end or node of the primitive streak. After the beginning of somitogenesis (approximately 8.0 dpc), eck expression persists in the node as this structure regresses toward the caudal end of the embryo. In addition, beginning at the mid head fold stage (approximately 7.75 dpc), we observe that eck exhibits a dynamic and spatially restricted expression pattern in the prospective hindbrain region. eck transcripts are initially detected in a 5-cell wide strip of mesodermal cells underlying prospective rhombomere 4 (r4). Subsequently at the beginning of somitogenesis, eck mRNA expression is observed in prospective r4. At the 4--8-somite stage, eck transcripts are observed in r4, mesenchymal cells underlying r4, and surface ectoderm in the vicinity of the developing second branchial arch. By the 10-somite stage, eck mRNA expression in these cells is downregulated. Additionally, at the 5--8-somite stage, eck transcripts are detected initially in the lateral mesenchyme immediately underlying the surface ectoderm adjacent to r5 and r6, and subsequently in surface ectoderm overlying the developing third branchial arch. These data suggest that eck may be involved in cell-cell interactions guiding early hindbrain development.

Amino Acid Sequence↗
PubMed1995

A rapid method for mRNA detection in single-cell biopsies from preimplantation-stage bovine embryos.

Major questions concerning the control of development and gene expression at the cellular level are still unanswered. Nowhere is this more evident than during the earliest stages of development and embryogenesis. This study describes the detection of specific gene transcripts in single cells derived from bovine embryos. Following in vitro fertilization (IVF) and in vitro culture (IVC) of bovine embryos, small groups of cells and even single blastomeres from 32 to 64-cell embryos were micromanipulated into individual tubes for analysis of cytoplasmic RNAs. Reverse transcriptase-PCR was applied to cell lysates for the amplification of beta-actin mRNA transcripts. Primers were designed to flank an intron expected to be present within genomic DNA sequences, thus allowing for simple differentiation between DNA- and RNA-derived amplification products. Using a 50-cycle amplification profile, a 260 bp band could be seen as a PCR product derived from a single blastomere following electrophoresis in an ethidium bromide-stained agarose gel. The identity of the band was verified by DNA sequence determination and diagnostic restriction digestion. Lysates derived from single blastomeres in this way have been used for simultaneously phenotyping multiple RNA products. This capability allows the spatial analysis of gene expression and development within embryos from the earliest stages of cellular differentiation.

Journal Article↗
PubMed2005

Linking structural assembly to gene expression: a novel mechanism for regulating the activity of a sigma54 transcription factor.

In Caulobacter crescentus, the temporal and spatial expression of late flagellar genes is regulated by the sigma54 transcriptional activator, FlbD. Genetic experiments have indicated that the trans-acting factor FliX regulates FlbD in response to the progression of flagellar assembly, repressing FlbD activity until an early flagellar basal body structure is assembled. Following assembly of this structure, FliX is thought to function as an activator of FlbD. Here we have investigated the mechanism of FliX-mediated regulation of FlbD activity. In vitro transcription experiments showed that purified FliX could function as a repressor of FlbD-activated transcription. Transcription activated by a gain-of-function mutant of FlbD (FlbD-1204) that is active in vivo in the absence of an early flagellar structure, was resistant to the repressive effects of FliX. DNA binding studies showed that FliX inhibited the interaction of wild-type FlbD with enhancer DNA but did not effect FlbD-catalysed ATPase activity. DNA binding activity of FlbD-1204 was relatively unaffected by FliX indicating that this mutant protein bypasses the transcriptional requirement for early flagellar assembly by escaping FliX-mediated negative regulation. Gel filtration and co-immunoprecipitation experiments indicated that FliX formed a stable complex with FlbD. These experiments demonstrate that regulation of FlbD activity is unusual among the well-studied sigma54 transcriptional activators, apparently combining a two-component receiver domain with additional control imposed via interaction with a partner protein, FliX.

Adenosine Triphosphatases↗
PubMed1999

Clustered cadherin genes: a sequence-ready contig for the desmosomal cadherin locus on human chromosome 18.

We describe the assembly of a cosmid and PAC contig of approximately 700 kb on human chromosome 18q12 spanning the DSC and DSG genes coding for the desmocollins and desmogleins. These are members of the cadherin superfamily of calcium-dependent cell adhesion proteins present in the desmosome type of cell junction found especially in epithelial cells. They provide the strong cell-cell adhesion generated by this type of cell junction for which expression of both a desmocollin and a desmoglein is required. In the autoimmune skin diseases pemphigus foliaceous and pemphigus vulgaris (PV), where the autoantigens are, respectively, encoded by the DSG1 and DSG3 genes, severe areas of acantholysis (cell separation), potentially life-threatening in the case of PV, are evident. Dominant mutations in the DSG1 gene causing striate palmoplantar keratoderma result in hyperkeratosis of the skin on the parts of the body where pressure and abrasion are greatest, viz., on the palms and soles. These genes are also candidate tumor suppressor genes in squamous cell carcinomas and other epithelial cancers. We have screened two chromosome 18-specific cosmid libraries by hybridization with previously isolated YAC clones and DSC and DSG cDNAs, and a whole genome PAC library, both by hybridization with the YACs and by screening by PCR using cDNA sequences and YAC end sequence. The contigs were extended by further PCR screens using STSs generated by vectorette walking from the ends of the cosmids and PACs, together with sequence from PAC ends. Despite screening of two libraries, the cosmid contig still had four gaps. The PAC contig filled these gaps and in fact covered the whole locus. The positions of 45 STSs covering the whole of this region are presented. The desmocollin and desmoglein genes, which are about 30-35 kb in size, are quite well separated at approximately 20-30 kb apart and are arranged in two clusters, one DSC cluster and one DSG cluster, which are transcribed outward from the interlocus region. The order of the genes is correlated with the spatial order of gene expression in the developing mouse embryo, and this, and previous transgenic experiments, suggests that long-range genetic elements that coordinate expression of these genes may be present. The complete bacterial clone contig described in this paper is thus a resource not only for future sequencing but also for investigations into the control of expression of these clustered genes.

Bacteriophage P1↗