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A 20-nucleotide element in the intestinal fatty acid binding protein gene modulates its cell lineage-specific, differentiation-dependent, and cephalocaudal patterns of expression in transgenic mice.

A sequence of epithelial cell proliferation, allocation to four principal lineages, migration-associated differentiation, and cell loss occurs along the crypt-villus axis of the mouse intestine. The sequence is completed in a few days and is recapitulated throughout the life-span of the animal. We have used an intestine-specific fatty acid binding protein gene, Fabpi, as a model for studying regulation of gene expression in this unique developmental system. Promoter mapping studies in transgenic mice identified a 20-bp cis-acting element (5'-AGGTGGAAGCCATCACACTT-3') that binds small intestinal nuclear proteins and participates in the control of Fabpi's cephalocaudal, differentiation-dependent, and cell lineage-specific patterns of expression. Immunocytochemical studies using confocal and electron microscopy indicate that it does so by acting as a suppressor of gene expression in the distal small intestine/colon, as a suppressor of gene activation in proliferating and nonproliferating cells located in the crypts of Lieberkühn, and as a suppressor of expression in the growth factor and defensin-producing Paneth cell lineage. The 20-bp domain has no obvious sequence similarities to known transcription factor binding sites. The three functions modulated by this compact element represent the types of functions required to establish and maintain the intestine's remarkably complex spatial patterns of gene expression. The transgenes described in this report also appear to be useful in characterizing the crypt's stem cell hierarchy.

Animals↗

A leader intron and 115-bp promoter region necessary for expression of the carnation S-adenosylmethionine decarboxylase gene in the pollen of transgenic tobacco.

The expression of CSDC9 encoding S-adenosylmethionine decarboxylase (SAMDC) is developmentally and spatially regulated in carnation. To examine the regulation of the SAMDC gene, we analyzed the spatial expression of CSDC9 with a 5'-flanking beta-glucuronidase fusion in transgenic tobacco plants. GUS was strongly expressed in flower, pollen, stem and vein of cotyledons. Expression in both anther and stigma was under developmental control; analysis of a series of mutants with deletions of the 5'-flanking region demonstrated differential activation in petal, anther, stigma and pollen grains. All the major cis-regulatory elements required for pollen-specific transcription were located in the upstream region between -273 and -158. This region contains four putative elements related to gibberellin induction (pyrimidine boxes, TTTTTTCC and CCTTTT) and pollen-specific expression (GTGA and AGAAA). In addition, the first 5'-leader intron was necessary for tissue-specific expression.

5' Flanking Region↗

Spatial and temporal expression pattern during sea urchin embryogenesis of a gene coding for a protease homologous to the human protein BMP-1 and to the product of the Drosophila dorsal-ventral patterning gene tolloid.

A cDNA clone coding for a sea urchin embryonic protein was isolated from a prehatching blastula lambda gt11 library. The predicted translation product is a secreted 64 x 10(3) Mr enzyme designated as BP10. The protein contains several domains: a signal peptide, a putative propeptide, a catalytic domain with an active center typical of a Zn(2+)-metalloprotease, an EGF-like domain and two internal repeats similar to repeated domains found in the C1s and C1r serine proteases of the complement cascade. The BP10 protease is constructed with the same domains as the human bone morphogenetic protein BMP-1, a protease described as a factor involved in bone formation, and as the recently characterized product of the tolloid gene which is required for correct dorsal-ventral patterning of the Drosophila embryo. The transcription of the BP10 gene is transiently activated around the 16- to 32-cell stage and the accumulation of BP10 transcripts is limited to a short period at the blastula stage. By in situ hybridization with digoxygenin-labelled RNA probes, the BP10 transcripts were only detected in a limited area of the blastula, showing that the transcription of the BP10 gene is also spatially controlled. Antibodies directed against a fusion protein were used to detect the BP10 protein in embryonic extracts. The protein is first detected in early blastula stages, its level peaks in late cleavage, declines abruptly before ingression of primary mesenchyme cells and remains constant in late development. The distribution of the BP10 protein during its synthesis and secretion was analysed by immunostaining blastula-stage embryos. The intracellular localization of the BP10 staining varies with time. The protein is first detected in a perinuclear region, then in an apical and submembranous position just before its secretion into the perivitelline space. The protein is synthesized in a sharply delimited continuous territory spanning about 70% of the blastula. Comparison of the size and orientation of the labelled territory in the late blastula with the fate map of the blastula stage embryo shows that the domain in which the BP10 gene is expressed corresponds to the presumptive ectoderm. Developing embryos treated with purified antibodies against the BP10 protein and with synthetic peptides derived from the EGF-like domain displayed perturbations in morphogenesis and were radialized to various degrees. These results are consistent with a role for BP10 in the differentiation of ectodermal lineages and subsequent patterning of the embryo. On the basis of these results, we speculate that the role of BP10 in the sea urchin embryo might be similar to that of tolloid in Drosophila. We discuss the idea that the processes of spatial regulation of gene expression along the animal-vegetal in sea urchin and dorsal-ventral axes in Drosophila might have some similarities and might use common elements.

Amino Acid Sequence↗

A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana.

The S-locus glycoprotein gene of Brassica is derived from the genetic locus that controls the self-incompatibility response and the specific recognition between pollen and stigma. The promoter of this gene was used to direct expression of the diphtheria toxin A chain gene and the Escherichia coli beta-glucuronidase gene in transgenic Nicotiana tabacum. Expression of the promoter in cells of the pistil and in pollen suggests that a single gene may direct the self-incompatibility response in the two interacting cell types. Additionally, the fusion genes were expressed gametophytically in the heterologous host species, Nicotiana, rather than sporophytically as expected for Brassica. Thus, although the genes involved in self-incompatibility in Brassica and Nicotiana are not homologous in their coding regions, signals for expression of these genes are apparently conserved between the two genera. Our analysis of toxic gene fusion transformants shows that genetic ablation is useful for probing developmental processes and for studying temporal and spatial patterns of gene expression in plants.

Brassica↗

Novel transcriptional mechanisms are involved in regulating preproenkephalin gene expression in vivo.

For the dissection of the temporal and spatial patterns of cell- and tissue-specific gene expression an understanding of the contributing regulating mechanisms is required. We now confirm that there are novel mechanisms regulating preproenkephalin gene expression in basal as well as cholinergic agonist treated rats. Moreover, we demonstrate that these novel transcriptional mechanisms are consistent with RNA intragenic elongation pausing, alternate promoter usage, and small sense and antisense RNA transcription from the preproenkephalin gene locus. We report that while basal striatal and olfactory bulb proenkephalin RNA transcripts are initiated from the "normal" proximal promoter, in cerebellum de novo RNA transcription appears to be initiated from the distal so-called "germ-cell" promoter. Furthermore, "normally" initiated olfactory bulb proenkephalin RNA transcripts appear to be down-regulated by the time the RNA polymerase II complex reaches the first preproenkephalin intron, in a way that is consistent with RNA elongation pausing. As the pattern of small sense and antisense transcripts found associated with this gene's expression is tissue-specific, we suggest that they may also play a role in regulating gene expression. The understanding of this gene's regulation should have widespread importance, not only to those interested in opioid gene expression, but also to those interested in gene regulation, in general.

Adrenal Medulla↗

Temperature and the expression of seven muscle-specific protein genes during embryogenesis in the Atlantic cod Gadus morhua L.

Seven cDNA clones coding for different muscle-specific proteins (MSPs) were isolated from the fast muscle tissue of Atlantic cod Gadus morhua L. In situ hybridization using cRNA probes was used to characterize the temporal and spatial patterns of gene expression with respect to somite stage in embryos incubated at 4 degrees C, 7 degrees C and 10 degrees C. MyoD transcripts were first observed in the presomitic mesoderm prior to somite formation, and in the lateral compartment of the forming somites. MyoD expression was not observed in the adaxial cells that give rise to the slow muscle layer, and expression was undetectable by in situ hybridization in the lateral somitic mesoderm after the 35-somite stage, during development of the final approximately 15 somites. RT-PCR analysis, however, confirmed the presence of low levels of the transcript during these later stages. A phylogenetic comparison of the deduced aminoacid sequences of the full-length MyoD cDNA clone and those from other teleosts, and inference from the in situ expression pattern suggested homology with a second paralogue (MyoD2) recently isolated from the gilthead seabream Sparus aurata. Following MyoD expression, alpha-actin was the first structural gene to be switched on at the 16-somite stage, followed by myosin heavy chain, troponin T, troponin I and muscle creatine kinase. The final mRNA in the series to be expressed was troponin C. All genes were switched on prior to myofibril assembly. The troponin C sequence was unusual in that it showed the greatest sequence identity with the rainbow trout Oncorhynchus mykiss cardiac/slow form, but was expressed in the fast myotomal muscle and not in the heart. In addition, the third TnC calcium binding site showed a lower level of sequence conservation than the rest of the sequence. No differences were seen in the timing of appearance or rate of posterior progression (relative to somite stage) of any MSP transcripts between embryos raised at the different temperatures. It was concluded that myofibrillar genes are activated asynchronously in a distinct temporal order prior to myofibrillar assembly and that this process was highly canalized over the temperature range studied.

Actins↗

Strategies of conditional gene expression in myocardium: an overview.

The use of specialized reporter genes to monitor real-time, tissue-specific transgene expression in animal models offers an opportunity to circumvent current limitations associated with the establishment of transgenic mouse models. The Cre-loxP and the tetracycline (Tet)-inducible systems are useful methods of conditional gene expression that allow spatial (cell-type-specific) and temporal (inducer-dependent) control. Most often, the alpha-myosin heavy chain (alpha-MHC) promoter is used in these inducible systems to restrict expression of reporter genes and transgenes to the myocardium. An overview of each inducible system is described, along with suggested reporter genes for real-time, noninvasive imaging in the myocardium. Effective gene delivery of the inducible gene expression system is carried out by lentiviral vectors, which offer high transduction efficiency, long-term transgene expression, and low immunogenicity. This chapter outlines the packaging of myocardium-specific inducible expression systems into lentiviral vectors, in which a transgene and a reporter gene are transduced into cardiomyocytes. In doing so, transgene and reporter expression can be monitored/tracked with bioluminescence imaging (BLI) and positron emission tomography (PET).

Animals↗

Developmental expression of the four plasma membrane calcium ATPase (Pmca) genes in the mouse.

The plasma membrane calcium ATPases are critical components in the regulation of cellular calcium homeostasis and signaling. In mammals, there are 4 Pmca genes, and information on the cellular and tissue distribution of their expression during development will provide insight into the regulation and possible function of each Pmca isoform. Using specific probes and in situ hybridization, we found that the four Pmca genes are expressed in spatially overlapping but distinct patterns in the mouse embryo. The dynamic temporal patterns of expression indicate that the individual isoforms are subject to both positive and negative regulation. The differential and restricted expression of Pmca genes supports the notion that they play unique functional roles in mammalian development.

Animals↗

Use of suppression subtractive hybridization for differential gene expression in stroke: discovery of CD44 gene expression and localization in permanent focal stroke in rats.

BACKGROUND AND PURPOSE: CD44 is a transmembrane glycoprotein involved in endothelial cell recognition, lymphocyte trafficking, and regulation of cytokine gene expression in inflammatory diseases. The present report describes the discovery of upregulated CD44 gene expression and its spatial and temporal distribution in the brain after focal stroke. METHODS: Rats were subjected to permanent occlusion of the middle cerebral artery (MCAO). Suppression subtractive hybridization (SSH) strategy was used to identify differentially expressed genes. Northern blotting and real-time polymerase chain reaction were used to evaluate the expression of CD44 and hyaluronan synthase 2 (HAS-2) mRNA. Western blotting and immunohistochemistry were used to examine CD44 expression and cellular distribution. RESULTS: CD44 upregulation after focal stroke was discovered by the SSH approach and confirmed by DNA sequencing. Northern blot using a pooled poly(A)+ RNA revealed 3 splice variants of CD44 mRNA, and their inducible expression started at 6 hours (5.3-fold increase over sham operation), peaked at 24 hours (28.6-fold increase), and persisted up to 72 hours (17.8-fold increase) after MCAO. A parallel induction profile of HAS-2 mRNA was observed in the ischemic brain tissue. The levels of CD44 were markedly elevated at 6 hours (1.8-fold increase over sham; n=3), 24 hours (2.9-fold, peak induction; P<0.01), and 72 hours (2.4-fold increase; P<0.05) after MCAO by means of Western analysis. Immunohistochemical and confocal microscopy confirmed that constitutive expression of CD44 is limited to microvessels in normal brain but is strongly induced after ischemia, where the immunoreactive signal mainly resided in endothelial cells and monocytes. Double-labeling immunohistochemistry demonstrated that a marked induction of CD44 in the ischemic lesion is dominantly located in microglia and a subset of macrophages. CONCLUSIONS: The discovery of concomitant induction of CD44 and HAS-2 mRNA expression and the localization of CD44 in the microglia, macrophages, and microvessels of the ischemic brain tissue suggest that an active interaction between CD44 and hyaluronan may occur and play a role in the known inflammatory response and tissue remodeling after stroke.

Alternative Splicing↗

Control of segmental asymmetry in Drosophila embryos.

During Drosophila development, an important aspect of body patterning is the division of the embryo into repeating morphological units referred to as parasegments. The parasegmental domains are first defined at the blastoderm stage by alternating stripes of transcripts encoded by the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) and later by stripes encoded by the segment polarity genes engrailed (en) and wingless. Here, we show that the runt gene (run) is required to generate asymmetries within these parasegmental domains. Using a heat-shock-inducible run transgene, we found that ectopic run expression leads to rapid repression of eve stripes and a somewhat delayed expansion of ftz stripes. Unexpectedly, we also found that ectopic run was a rapid and potent repressor of odd-numbered en stripes. Two remarkably different segmental phenotypes were generated as a consequence of these effects. In solving the mechanisms underlying these phenotypes, we discovered that the positioning of en stripes is largely determined by the actions of negative regulators. Our data indicate that run is required to limit the domains of en expression in the odd-numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of run and odd by the product of the eve gene. The spatial restriction of gene expression via negative and double negative pathways such as these is likely to be a common theme during development.

Animals↗

Identification of genes induced in regenerating Xenopus tadpole tails by using the differential display method.

To identify candidate gene(s) involved in the tail regeneration of Xenopus laevis tadpoles, we used the differential display method to isolate four genes (clones 1, 2, 13a, and 13b) whose expression is induced in regenerating tadpole tails. Among them, clones 13a and 13b were found to encode the Xenopus homologues of the alpha1 chain of type XVIII collagen and neuronal pentraxin I, respectively. Expression of clone 2 and neuronal pentraxin I genes increased dramatically in the blastema 3 days after amputation, whereas that for the clone 1 and type XVIII collagen genes was induced gradually after amputation. In situ hybridization revealed that the neuronal pentraxin I gene is expressed specifically in the regenerating tail epidermis but not in the normal tail epidermis or the most distal margin of the tail blastema, suggesting that it has a tissue-inductive role in tail regeneration. Expression of the four genes was induced in the limb and in the tail blastema, suggesting that they are involved in the regeneration of both organs. Finally, expression of clone 2 and neuronal pentraxin I genes was scarce during embryonic stages in comparison to the tail blastema, suggesting that their main functions are in organ regeneration. Our results demonstrate unique features of spatial and temporal gene expression patterns during Xenopus tadpole tail regeneration.

Aging↗

Upregulation of cathepsin D expression in the dedifferentiating salamander limb regenerates and enhancement of its expression by retinoic acid.

In the regenerating urodele limb, catheptic activity has been known to be present in the stump tissue undergoing histolysis, and it has been implicated in the modification of some intracellular and extracellular matrix molecules. In this study, we compared spatial and temporal gene expression profiles of cathepsin D in normal, retinoic acid-treated, or denervated larval limb regenerates of Hynobius leechii and compared cathepsin D activities between normal and retinoic acid-treated limb regenerates. The results showed that the expression of cathepsin D increased markedly in the vicinity of an amputation site under the wound epidermis at dedifferentiation stage, whereas the expression level of cathepsin D was low in the denervated limb. With retinoic acid treatment, the expression of cathepsin D was elevated in terms of both level and duration. In addition, the profile of cathepsin D activity coincided well with the expression profile of cathepsin D in normal and retinoic acid-treated limb regenerates. These results suggest that the increase of cathepsin D activity during the dedifferentiation period is due to the upregulation of cathepsin D transcription, and nerve factors are involved in this process. Furthermore, retinoic acid appears to upregulate cathepsin D expression, which might be linked to the enhanced dedifferentiation in the retinoic acid-treated limb regenerates.

Amputation, Surgical↗

Spatial- and temporal-restricted pattern for amelogenin gene expression during mouse molar tooth organogenesis.

Position- and time-restricted amelogenin gene transcription was analysed in developing tooth organs using in situ hybridization with asymmetric complementary RNA probes produced from a cDNA specific to the mouse 26 x 10(3) Mr amelogenin. In situ analysis was performed on developmentally staged fetal and neonatal mouse mandibular first (M1) and maxillary first (M1) molar tooth organs using serial sections and three-dimensional reconstruction. Amelogenin mRNA was first detected in a cluster of ameloblasts along one cusp of the M1 molar at the newborn stage of development. In subsequent developmental stages, amelogenin transcripts were detected within foci of ameloblasts lining each of the five cusps comprising the molar crown form. The number of amelogenin transcripts appeared to be position-dependent, being more abundant on one cusp surface while reduced along the opposite surface. Amelogenin gene transcription was found to be bilaterally symmetric between the developing right and left M1 molars, and complementary between the M1 and M1 developing molars; indicating position-restricted gene expression resulting in organ stereoisomerism. The application of in situ hybridization to forming tooth organ geometry provides a novel strategy to define epithelial-mesenchymal signal(s) which are believed to be responsible for organ morphogenesis, as well as for temporal- and spatial-restricted tissue-specific expression of enamel extracellular matrix.

Amelogenin↗

Spatially deranged though temporally correct expression of Strongylocentrotus purpuratus actin gene fusion in transgenic embryos of a different sea urchin family.

We report the unexpected observation that cis-regulatory sequences of a Strongylocentrotus purpuratus actin gene, which direct a particular, lineage-specific pattern of embryonic expression, confer a completely different spatial pattern of expression when introduced into embryos of another sea urchin species. We utilized a fusion gene construct in which the bacterial chloramphenicol acetyl transferase (CAT) reporter gene is driven by CyIIIa actin regulatory sequences. We previously showed that the regulatory region that is included suffices to promote the accumulation of CAT mRNA in transgenic S. purpuratus embryos, on the same developmental schedule and in the same embryonic region, the aboral ectoderm, in which the CyIIIa actin gene is normally expressed (Flytzanis et al. 1987; Hough-Evans et al. 1987). When injected into zygotes of Lytechinus variegatus, which belongs to a different echinoid family, the expected temporal pattern of expression of CAT enzyme was observed. Thus, in both S. purpuratus and L. variegatus embryos, expression is activated at the early blastula stage, although this stage is attained several hours sooner in L. variegatus embryo cultures. Similar kinetics of CAT enzyme accumulation were obtained whether the gene was introduced directly into the L. variegatus zygote nucleus or into the cytoplasm. However, when examined by in situ hybridization, the transgenic L. variegatus embryos were found to display a totally new pattern of CAT mRNA accumulation. Copious CAT transcripts were detected not only in aboral ectoderm cells, but also in skeletogenic mesenchyme cells, gut cells, and oral ectoderm, all cell types that in the transgenic S. purpuratus controls are invariably devoid of detectable CAT transcripts.

Acetyltransferases↗

Chromatin domains and nuclear compartments: establishing sites of gene expression in eukaryotic nuclei.

Establishing sites of transcription in the nuclei of higher eukaryotic cells is a very complex process. Before transcription can begin, a series of transcription factors must associate with their recognition motifs, within promoters and more remote activating sequences. Once bound, these factors and associated proteins are believed to form a complex that positions the RNA polymerase holoenzyme so that transcription can commence. As a consequence, active genes assume a specialized chromatin state across regions that define functional domains. Global nuclear architecture appears to stabilize these active domains by providing local environments dedicated to gene expression. As the spatial organization of these sites is unaffected by the removal of most chromatin they must be associated with a structural network. This nucleoskeleton, the associated transcription 'factories' and chromatin loops that arise as DNA binds proteins within factories now appear to be fundamental features of nuclear structure in higher eukaryotes. I argue that concentrating proteins needed to perform different steps of RNA synthesis within specialized nuclear compartments will be important in orchestrating events required for efficient gene expression.

Cell Nucleus↗

A streamlined method for systematic, high resolution in situ analysis of mRNA distribution in plants.

BACKGROUND: In situ hybridisation can provide cellular, and in some cases sub-cellular, resolution of mRNA levels within multicellular organisms and is widely used to provide spatial and temporal information on gene expression. However, standard protocols are complex and laborious to implement, restricting analysis to one or a few genes at any one time. Whole-mount and reverse transcriptase-PCR (RT-PCR) based protocols increase throughput, but can compromise both specificity and resolution. With the advent of genome-wide analysis of gene expression, there is an urgent need to develop high-throughput in situ methods that also provide high resolution. RESULTS: Here we describe the development of a method for performing high-throughput in situ hybridisations that retains both the high resolution and the specificity of the best manual versions. This refined semi-automated protocol has the potential for determining the spatial and temporal expression patterns of hundreds of genes in parallel on a variety of tissues. We show how tissue sections can be organized on microscope slides in a manner that allows the screening of multiple probes on each slide. Slide handling, hybridisation and processing steps have been streamlined providing a capacity of at least 200 probes per week (depending on the tissue type). The technique can be applied easily to different species and tissue types, and we illustrate this with wheat seed and Arabidopsis floral meristems, siliques and seedlings. CONCLUSION: The approach has the high specificity and high resolution of previous in situ methods while allowing for the analysis of several genes expression patterns in parallel. This method has the potential to provide an analysis of gene expression patterns at the genome level.

Journal Article↗

Modified intranuclear organization of regulatory factors in human acute leukemias: reversal after treatment.

Acute leukemias arise secondary to chromosomal aberrations that cause dysfunctions in gene regulation and regulatory factors. Significant differences in morphology between acute leukemic and nonleukemic hematopoietic cells are readily observed. How morphologic changes of the nuclei of acute leukemic cells relate to the underlying functional alterations of gene expression is minimally understood. Spatial modifications in the representation and/or organization of regulatory factors may be functionally linked to perturbations of gene expression in acute leukemic cells. Using in situ immunofluorescence microscopy, we addressed the interrelationships of modifications in nuclear morphology with the intranuclear distribution of leukemia-related regulatory factors (including ALL-1, PML, and AF-9) in cells from patients with acute leukemia. We compared the localization of leukemia-associated proteins with various factors involved in gene transcription and RNA processing (e.g., RNA polymerase II and SC-35). Our findings suggest that there are leukemia-associated aberrations in mechanisms that direct regulatory factors to sites within the nucleus. This misplacement of key cognate factors may contribute to perturbations in gene expression characteristic of leukemias.

Cell Nucleus↗

Identification of novel elements which regulate the cell-type specificity of Dictyostelium 7E gene expression.

Previously, we have identified the Dictyostelium 7E gene promoter and shown that it is capable of driving expression in the same temporal and cAMP responsive manner as the endogenous gene during development. Furthermore, we have mapped the corresponding transcriptional regulatory sequences within the promoter. In the present study we used the lacZ reporter gene system to examine the role of 7E promoter elements in regulating cell-type specific expression during Dictyostelium morphogenesis. In situ detection of beta-galactosidase activity revealed that expression was induced within anterior prestalk cells at approximately 18 h of development. Subsequently, we found that promoter activity was independently regulated in subpopulations of prestalk cells. Element(s) upstream of position - 532 were necessary for expression in pstA cells while more proximal elements (located downstream of position - 426) were capable of directing expression in pstO cells. Deletion of a G-rich element ('GGT' box; 5'-GGT GAT GA-3') located between positions - 159 and - 152 resulted both in a loss of expression in pstA cells and aberrant expression in the prespore zone. Furthermore, the spatial organisation of reporter gene expression directed by this construct during culmination delineated a population of cells that have not been previously defined. These data suggest that the 7E gene is independently regulated in subpopulations of prestalk cells during development.

ATP-Binding Cassette Transporters↗