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At least 667 records · Page 37Linked to original sources
PubMed2003

Temporal and spatial control of gene silencing in transgenic plants by inducible expression of double-stranded RNA.

Downregulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. The recent discovery that double-stranded RNA (dsRNA) is an extremely effective trigger of gene silencing greatly enhanced the predictability of this approach. However, strong constitutive silencing often leads to pleiotropic effects, which make it difficult to directly relate phenotype to gene function, or even interferes with the recovery of viable transgenic plants. Here, we show that strong genetic interference can be achieved in a chemically inducible fashion, allowing for temporal and spatial control of gene silencing in transgenic plants. To this end, transgenic tobacco plants were established expressing dsRNA in the form of intron-spliced hairpin structures under the control of the ethanol-inducible alc gene expression system. Targeting magnesium (Mg)-chelatase subunit I (Chl I) and glutamate 1-semialdehyde aminotransferase (GSA), both involved in chlorophyll (chl) biosynthesis, resulted in rapid and specific mRNA degradation upon induction with ethanol. Ethanol-inducible silencing of the target genes caused strong but transient phenotypical alterations featured by a progressive loss of chl in young leaves, which persisted for about 7-9 days before newly growing leaves completely recovered. About 10-30% of the primary transformants showed phenotype development upon induction. Local silencing of Chl I could be achieved by confined ethanol treatment of a single leaf without affecting any other part of the plant. Inducible gene silencing using the alc system promises to obviate the problems associated with constitutive RNA silencing and enables to dissect primary and secondary effects of PTGS at temporal and spatial resolution.

Enzymes↗
PubMed2006

Dpp signaling down-regulates the expression of VM32E eggshell gene during Drosophila oogenesis.

Among the members of the Drosophila melanogaster vitelline membrane protein gene family, VM32E has the unique feature of being a component of both the vitelline and the endochorion layers. The VM32E gene is expressed at stage 10 of egg chamber development in the main body follicle cells, and it is repressed in the anterior and posterior follicle cells. Here, we show that this spatial restriction of VM32E gene expression is conserved in the D. pseudoobscura orthologous gene, suggestive of a conserved function of VM32E protein. The VM32E gene is not expressed in the centripetal migrating follicle cells, where the Decapentaplegic (Dpp) pathway is active in patterning the anterior eggshell structures. By analyzing the native VM32E gene and the activity of specific VM32E regulatory regions, in genetic backgrounds altering the Dpp pathway, we show that VM32E gene is negatively regulated by the Dpp signaling. Therefore, it appears that the Dpp signaling pathway executes its control on eggshell morphogenesis also by controlling the expression of eggshell structural genes.

Animals↗
PubMed2005

Temporal and spatial expression profile of the novel armadillo-related gene, Alex2, during testicular differentiation in the mouse embryo.

In a screen for transcripts differentially expressed during gonadal development in mouse embryos, we identified the novel armadillo-related gene, Alex2. The armadillo (arm) family of proteins share a 42 amino acid tandem repeat motif called the arm domain, through which they interact with different binding partners. These intracellular proteins are implicated in a variety of developmental processes, including cell proliferation, migration, maintenance of tissue integrity, and tumorigenesis. Alex2 is a member of a novel subgroup within the arm family, encoding a protein with a single arm domain and a putative transmembrane or signal sequence. Alex2 has a developmentally regulated expression profile during embryogenesis in the mouse. In the urogenital system, it is strongly expressed in the developing testis but is down-regulated during ovarian development. Alex2 expression is localized within the interstitial cell lineage of the developing testis, which gives rise to peritubular myoid, endothelial, and fetal Leydig cells. Alex2 is also expressed in the developing forebrain and somites and in dorsal root ganglia. In testicular cell lines, Alex2 fusion proteins localize to membrane structures within the cell. The expression profile of Alex2 suggests that it plays a role in the development of several tissues during embryogenesis, notably testicular differentiation. In the developing testis, its expression profile suggests that Alex2 has a role in specification or development of the interstitial cell lineage.

Amino Acid Sequence↗
PubMed1996

Progressive spatial restriction of Sek-1 and Krox-20 gene expression during hindbrain segmentation.

After segmentation of the vertebrate hindbrain, expression of the zinc-finger gene Krox-20 and the receptor tyrosine kinase gene Sek-1 is precisely restricted to rhombomeres (r) 3 and 5. This precise segmental expression is likely to reflect a critical requirement for these rhombomeres to acquire a distinct and homogeneous identity and raises the question as to how this relates to the intermingling and restriction of cell movement during segmentation. We have analysed Krox-20 and Sek-1 expression in the mouse and chick hindbrain at single-cell resolution using whole-mount in situ hybridisation and immunocytochemistry. We find that, in the mouse, the presumptive r3 and r5 expression domains each arise as narrow stripes that then broaden, suggestive of a recruitment of cells to an r3/r5 identity and/or a segmental regulation of cell proliferation. In addition, we find that expression of these genes initially occurs in fuzzy domains, and that these are progressively restricted to segmental domains, although occasional "violating" cells are observed even after segmentation. We propose that the establishment and maintenance of these segmental domains may involve both a dynamic regulation of r3/r5 identity and the restriction of cell movement across rhombomere boundaries.

Animals↗
PubMed1995

Efficient isolation of novel mouse genes differentially expressed in early postimplantation embryos.

Most genes with regulatory functions in embryogenesis are expressed in highly specific patterns, suggesting that expression patterns can serve as criteria to define potential candidates for developmentally relevant genes. To isolate such genes, we selected and partially sequenced 80 cDNA clones from a 10.5-day mouse embryo library. Forty-one clones that represented novel mouse genes were analyzed for expression in embryos of the same stage by whole-mount in situ hybridization. A high proportion (24%) of these genes, including a homologue of the Drosophila Delta gene, were expressed in specific spatially restricted patterns, suggesting that selection based on expression patterns is a useful strategy to isolate novel genes that may play pivotal roles in mammalian development.

Animals↗
PubMed2004

Spatial and temporal expression patterns of Xenopus Nkx-2.3 gene in skin epidermis during metamorphosis.

Using PCR cloning, the mRNA of XNkx-2.3 gene, a Xenopus tinman homologue, was identified in a cDNA library prepared from thyroid hormone (T(3))-treated tadpole skin. Quantitative RT-PCR and RNase Protection Assay confirmed the expression of XNkx-2.3 in adult frog skin and its amount was similar to the amount found in heart. In situ hybridization indicated that XNkx-2.3 was expressed in the frog epidermis. Further analysis of XNkx-2.3 expression patterns demonstrates that it shares great similarities with a 63 kDa keratin, a well-characterized marker for skin maturation, in the following aspects. First, XNkx-2.3 was expressed in tadpole skin during metamorphosis (stages 55-59), but not in pre-metamorphic (stage 54) skin. Secondly, XNkx-2.3 expression in skin responded to T(3) stimulation because it could be precociously induced by T(3) at pre-metamorphic stage, both in tadpoles and in cultures of skin explants. Finally, the T(3)-induced appearance of XNkx-2.3 in head skin occurred earlier and at higher level than that in tail skin. These data suggest that XNkx-2.3 may be an important factor for skin maturation and may also serve as a good marker to indicate the maturation of Xenopus epidermis.

Animals↗
PubMed1999

Temporal and spatial expression of EmH-3, a homeobox-containing gene isolated from the freshwater sponge Ephydatia muelleri.

Homeoboxes have been particularly valuable in identifying genes involved in development. This prompted us to look for homeobox-containing genes in sponges, the most primitive metazoans, and to explore the potential role of these genes in their development. Using the reverse transcription polymerase reaction (RT-PCR), we analyzed the expression of EmH-3 homeobox-containing gene at different stages of development, and in different cell-type populations. The patterns of EmH-3 expression show that this gene is expressed differentially in the course of development and in a cell-type specific manner. The level of transcripts increases from undetectable levels in resting gemmules to higher levels at the moment of hatching and throughout the sponge's life. EmH-3 is strongly expressed in the pluripotent archaeocytes, whether isolated from fully differentiated sponges (adult archaeocytes) or from HU-treated sponges (embryonic archaeocytes). Conversely, in differentiated cells such as pinacocytes and choanocytes, EmH-3 expression is very weak and similar to that found in the resting gemmules. On the other hand, another freshwater sponge homeobox-containing gene, prox1 from Ephydatia fluviatilis is expressed almost at the same level at all stages of development and in all the investigated cell populations. Together, these results suggest that EmH-3 plays a role in cell determination and/or differentiation. In particular EmH-3 would determine which archaeocytes will multiply and undergo differentiation and which ones will remain undifferentiated.

Amino Acid Sequence↗
PubMedDate not supplied

Two structurally distinct and spatially compartmentalized adenylate kinases are expressed from the AK1 gene in mouse brain.

Adenylate kinases (AK, EC 2.7.4.3) have been considered important enzymes for energy homeostasis and metabolic signaling. To gain a better understanding of their cell-specific significance we studied the structural and functional aspects of products of one adenylate kinase gene, AK1, in mouse tissues. By combined computer database comparison and Northern analysis of mRNAs, we identified transcripts of 0.7 and 2.0 kilobases with different 5' and 3' non-coding regions which result from alternative use of promoters and polyadenylation sites. These mRNAs specify two distinct proteins, AK1 and a membrane-bound AK1 isoform (AK1beta), which differ in their N-terminal end and are co-expressed in several tissues with high-energy demand, including the brain. Immunohistochemical analysis of brain tissue and primary neurons and astrocytes in culture demonstrated that AK1 isoforms are expressed predominantly in neurons. AK1beta, when tested in transfected COS-1 and N2a neuroblastoma cells, located at the cellular membrane and was able to catalyze phosphorylation of ADP in vitro. In addition, AK1beta mediated AMP-induced activation of recombinant ATP-sensitive potassium channels in the presence of ATP. Thus, two structurally distinct AK1 isoforms co-exist in the mouse brain within distinct cellular locations. These enzymes may function in promoting energy homeostasis in the compartmentalized cytosol and in translating cellular energetic signals to membrane metabolic sensors.

Adenylate Kinase↗
PubMed2001

Spatial and temporal expression of two cytosolic glutamine synthetase genes in Scots pine: functional implications on nitrogen metabolism during early stages of conifer development.

Ammonium assimilation during the initial stages of Scots pine growth involves two cytosolic glutamine synthetase (GS, EC: 6.3.1.2) isoenzymes encoded by separate genes, GS1a and GS1b. GS1a was most exclusively expressed in photosynthetic tissues of the seedling whereas GS1b was expressed ubiquitously showing higher levels in non-photosynthetic tissues such as root and hypocotyl. Temporal expression analysis has shown that when germination starts GS1b is the predominant form in the embryo, however, its relative abundance in the tissue decreased in the postgerminative stages when green cotyledons are developed. In contrast GS1a was present at a low level in the embryo but its abundance increased markedly during germination and seedling growth. These data suggest that GS1a and GS1b genes display different and non-redundant roles in the nitrogen metabolism of conifers. The precise localization of individual transcripts by in situ hybridization strongly supports this possibility. GS1 gene products are mainly expressed in different cellular types: GS1a in chlorophylic parenchyma and GS1b in the vascular bundles of all tissues examined in the seedling. Our data support that glutamine biosynthesis in pine seedlings follows a different pattern related to angiosperms involving two cytosolic GS proteins: one of them a typical cytosolic GS which may be involved in the generation of glutamine for N transport and a second cytosolic GS generating amino donors for the biosynthesis of major N compounds in photosynthetic tissues, a closer role to angiosperm chloroplastic GS. The results are discussed with regard to recent studies on N mobilization and metabolism during the initial stages of conifer development.

Cycadopsida↗
PubMed1997

Divergent and conserved features in the spatial expression of the Drosophila pseudoobscura esterase-5B gene and the esterase-6 gene of Drosophila melanogaster.

The regulatory regions of homologous genes encoding esterase 6 (Est-6) of Drosophila melanogaster and esterase 5B (Est-5B) of Drosophila pseudoobscura show very little similarity. We have undertaken a comparative study of the pattern of expression directed by the Est-5B and Est-6 5'-flanking DNA to attempt to reveal conserved elements regulating tissue-specific expression in adults. Esterase regulatory sequences were linked to a lacZ reporter gene and transformed into D. melanogaster embryos. Est-5B, 5' upstream elements, give rise to a beta-galactosidase expression pattern that coincides with the wild-type expression of Est-5B in D. pseudoobscura. The expression patterns of the Est-5B/lacZ construct are different from those of a fusion gene containing the upstream region of Est-6. Common sites of expression for both kinds of constructs are the third segment of antenna, the maxillary palps, and salivary glands. In vitro deletion mutagenesis has shown that the two genes have a different organization of regulatory elements controlling expression in both the third segment of antenna and maxillary palps. The results suggest that the conservation of the expression pattern in genes that evolved from a common ancestor may not be accompanied by preservation of the corresponding cis-regulatory elements.

Animals↗
PubMed2000

Spatially and temporally regulated expression of rice MADS box genes with similarity to Arabidopsis class A, B and C genes.

The floral morphology of grass species is distinct from that of typical dicot plants. In order to achieve a better understanding of the molecular basis for this diversion, we isolated RAP1A, RAP1B and RAG, putative rice orthologs for the Arabidopsis class A gene APETALA1 (AP1) and class C gene AGAMOUS (AG). The expression patterns of RAP1A, RAG and OsMADS2, a rice ortholog of the class B gene, were analyzed by in situ hybridization. RAP1A mRNA was expressed in the apical region of the floral meristem at an early stage of spikelet development, and then its expression was localized in developing lemma, palea and lodicules. The OsMADS2 transcript was first observed in the region where stamen primordia are formed. Soon after, OsMADS2 mRNA appeared in the lodicule primordia as well as the stamen primordia, and this RNA accumulation pattern persisted until late stages of floral development. The expression of RAG was observed in stamens and pistils of wild-type young spikelets. These RNA accumulation patterns are mostly similar to those of Arabidopsis class A, B, C genes, supporting the notion that the ABC model may be extended to rice.

Amino Acid Sequence↗
PubMed2004

Temporal and spatial patterns of Kv1.1 and Kv1.2 protein and gene expression in spinal cord white matter after acute and chronic spinal cord injury in rats: implications for axonal pathophysiology after neurotrauma.

After spinal cord injury (SCI), surviving white matter axons display axonal dysfunction associated with demyelination and altered K+ channel activity. To clarify the molecular basis of posttraumatic axonal pathophysiology after SCI, we investigated the changes in expression and distribution of the axonal K+ channel subunits Kv1.1 and Kv1.2 in spinal cord white matter after in vivo SCI in the rat. Using Western blot analysis, we found an increased expression of Kv1.1 and Kv1.2 at 2 and 6 weeks after SCI. By real-time PCR we observed an increase in Kv1.1 and Kv1.2 mRNA levels 1 day after SCI, which persisted until 6 weeks. Confocal immunohistochemistry showed a markedly dispersed labelling of Kv1.1 and Kv1.2 along the injured axons, in contrast to the tight localization of these channels to the juxtaparanodes of noninjured axons. This redistribution of Kv1.1 and Kv1.2 occurred as early as 1 h postinjury along some injured axons, and persisted at 6 weeks postinjury. In parallel with the redistribution of Kv1.1 and 1.2, contactin-associated protein (Caspr), which is normally confined to a paranodal location, also displayed a more diffuse distribution along the injured spinal cord axons. Our results suggest that the increased expression of Kv1.1 and Kv1.2 proteins is transcriptionally regulated. In contrast, the redistribution of the axonal K+ channel subunits occurs very early postinjury and probably reflects a disruption of the juxtaparanodal axonal region due to physical trauma, as shown by altered localization of Caspr.

Actins↗
PubMed1990

Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues.

The messenger RNAs and core proteins of the two small chondroitin/dermatan sulfate proteoglycans, biglycan and decorin, were localized in developing human bone and other tissues by both 35S-labeled RNA probes and antibodies directed against synthetic peptides corresponding to nonhomologous regions of the two core proteins. Biglycan and decorin expression and localization were substantially divergent and sometimes mutually exclusive. In developing bones, spatially restricted patterns of gene expression and/or matrix localization of the two proteoglycans were identified in articular regions, epiphyseal cartilage, vascular canals, subperichondral regions, and periosteum, and indicated the association of each molecule with specific developmental events at specific sites. Study of non-skeletal tissues revealed that decorin was associated with all major type I (and type II) collagen-rich connective tissues. Conversely, biglycan was expressed and localized in a range of specialized cell types, including connective tissue (skeletal myofibers, endothelial cells) and epithelial cells (differentiating keratinocytes, renal tubular epithelia). Biglycan core protein was localized at the cell surface of certain cell types (e.g., keratinocytes). Whereas the distribution of decorin was consistent with matrix-centered functions, possibly related to regulation of growth of collagen fibers, the distribution of biglycan pointed to other function(s), perhaps related to cell regulation.

Adrenal Glands↗
PubMed1994

The spatial and temporal dynamics of Sax1 (CHox3) homeobox gene expression in the chick's spinal cord.

Sax1 (previously CHox3) is a chicken homeobox gene belonging to the same homeobox gene family as the Drosophila NK1 and the honeybee HHO genes. Sax1 transcripts are present from stage 2 H&H until at least 5 days of embryonic development. However, specific localization of Sax1 transcripts could not be detected by in situ hybridization prior to stage 8-, when Sax1 transcripts are specifically localized in the neural plate, posterior to the hindbrain. From stages 8- to 15 H&H, Sax1 continues to be expressed only in the spinal part of the neural plate. The anterior border of Sax1 expression was found to be always in the transverse plane separating the youngest somite from the yet unsegmented mesodermal plate and to regress with similar dynamics to that of the segregation of the somites from the mesodermal plate. The posterior border of Sax1 expression coincides with the posterior end of the neural plate. In order to study a possible regulation of Sax1 expression by its neighboring tissues, several embryonic manipulation experiments were performed. These manipulations included: removal of somites, mesodermal plate or notochord and transplantation of a young ectopic notochord in the vicinity of the neural plate or transplantation of neural plate sections into the extraembryonic area. The results of these experiments revealed that the induction of the neural plate by the mesoderm has already occurred in full primitive streak embryos, after which Sax1 is autonomously regulated within the spinal part of the neural plate.

Animals↗
PubMed1992

Regulation of TIMP gene expression in cell culture and during mouse embryogenesis.

We have identified elements in the 5' region of the murine tissue inhibitor of metalloproteinase (TIMP) gene which control the response to serum, phorbol esters and transforming growth factor beta (TGF-beta) in cell culture. These elements lie between -858 and -601 relative to the translation initiation start site in the gene and are distinct from those that control expression in response to virus induction. The temporal and spatial expression of the TIMP gene was also analysed during mouse embryogenesis using in situ hybridization. A transgenic mouse line was constructed which expressed a TIMP/lac Z fusion gene. Using in situ beta-galactosidase assays we were able to compare the expression of the transgene with the endogenous gene. Thus we concluded that most of the sequences controlling in vivo expression of TIMP were present in the transgene and could be localised to the 5' region of the gene.

3T3 Cells↗
PubMed1994

Spatial and temporal expression patterns of two sodium channel genes in Drosophila.

Genetic and molecular studies have identified two different sodium channel genes in Drosophila, para and DSC1. The functional contributions of the para-encoded channel have been inferred from analysis of mutant phenotypes. However, no mutations of DSC1 have been identified, so the in vivo functions of the channel it encodes are not yet known. To learn more about the possible functions of DSC1 in the Drosophila nervous system compared with those of para, we have characterized the expression patterns of these two sodium channel genes at embryonic, larval, pupal, and adult stages by tissue in situ hybridization, para encodes the predominant type of sodium channel and is ubiquitously transcribed throughout the CNS and PNS at all developmental stages. The expression pattern of DSC1 is very different from para during embryonic and larval stages during which there are very few DSC1-expressing cells in either the CNS or PNS. Double-labeling studies suggest that some of these cells are non-neuronal. However, in pupal and adult stages, para and DSC1 have completely overlapping patterns of expression in the CNS and retina. In the pupal and adult, PNS expression of these genes is still distinct because only para transcripts are detected in wing sensory neurons. The strong and widespread expression of DSC1 in the CNS of pupae and adults suggests that the DSC1 channels are likely to provide an important function in neurons during these stages. Since most, if not all, neurons in the pupal and adult CNS express both para and DSC1, these two sodium channel genes probably subserve distinct functions within these cells. Our results provide the background for elucidating the respective in vivo contributions of para and DSC1 to neuronal excitability and for dissecting the regulatory mechanisms that underlie their different patterns of expression.

Animals↗
PubMed1997

Spatial and temporal expression of the period and timeless genes in the developing nervous system of Drosophila: newly identified pacemaker candidates and novel features of clock gene product cycling.

The circadian timekeeping system of Drosophila functions from the first larval instar (L1) onward but is not known to require the expression of clock genes in larvae. We show that period (per) and timeless (tim) are rhythmically expressed in several groups of neurons in the larval CNS both in light/dark cycles and in constant dark conditions. Among the clock gene-expressing cells there is a subset of the putative pacemaker neurons, the "lateral neurons" (LNs), that have been analyzed mainly in adult flies. Like the adult LNs, the larval ones are also immunoreactive to a peptide called pigment-dispersing hormone. Their putative dendritic trees were found to be in close proximity to the terminals of the larval optic nerve Bolwig's nerve, possibly receiving photic input from the larval eyes. The LNs are the only larval cells that maintain a strong cycling in PER from L1 onward, throughout metamorphosis and into adulthood. Therefore, they are the best candidates for being pacemaker neurons responsible for the larval "time memory" (inferred from previous experiments). In addition to the LNs, a subset of the larval dorsal neurons (DNLs) expresses per and tim. Intriguingly, two neurons of this DNL group cycle in PER and TIM immunoreactivity almost in antiphase to the other DNLs and to the LNs. Thus, the temporal expression of per and tim are regulated differentially in different cells. Furthermore, the light sensitivity associated with levels of the TIM protein is different from that in the heads of adult Drosophila.

Aging↗
PubMed1988

Expression of the gene for main intrinsic polypeptide (MIP): separate spatial distributions of MIP and beta-crystallin gene transcripts in rat lens development.

The main intrinsic polypeptide (MIP) is the major protein present in the lens fiber cell membrane and is the product of a gene which, as far as is known, is expressed only in the lens. We have used in situ hybridization and immunofluorescence microscopy to characterize the expression of this gene during the course of development in the rat. At progressive stages of lens morphogenesis, we find that synthesis of the protein is closely tied to the accumulation of MIP mRNA in cells that are committed to terminal differentiation, first in the elongating presumptive primary lens fibers and later in the secondary fibers as they differentiate from the anterior epithelial cells. The transcripts accumulate in the basal cytoplasm of the primary fibers and in the cytoplasm which surrounds the cell nucleus in the secondary fibers. We have compared this pattern of expression with that of a gene for a cytoplasmic protein, beta-crystallin beta-A1/A3. In sharp contrast to the localized concentrations seen for the MIP mRNA, beta-A1/A3 transcripts are relatively uniformly distributed throughout the cytoplasm. Neither MIP nor crystallin gene appears to be transcriptionally active in the undifferentiated epithelial cell, but transcripts from the beta-A1/A3 gene appear earlier in fiber cell differentiation than do those from the gene for MIP.

Animals↗