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PubMed2002

Spatial repression of PCNA by p53 during kidney development.

Transcriptional repression is a key mechanism for the spatial specification of gene expression and cell fate determination. During kidney development, proliferating cell nuclear antigen (PCNA) is expressed in the nephrogenic zone and is downregulated rapidly as renal epithelial cells enter terminal differentiation and acquire functional characteristics. Our laboratory reported that the transcription factor p53 stimulates the terminal differentiation of renal epithelial cells by means of transcriptional activation of renal function genes (Saifudeen Z, Dipp S, and El-Dahr SS. J Clin Invest 109: 1021-1030, 2002). Because p53-induced growth arrest correlates with downregulation of PCNA gene expression, we examined the impact of p53 inactivation on PCNA expression in mice and evaluated the effect of p53 on PCNA transcription. Immunohistochemistry revealed that the transition from nephrogenesis to terminal epithelial cell differentiation correlates with accumulation of the transcription factor p53. Importantly, the spatially restricted pattern of PCNA expression is disrupted in kidneys of p53-deficient pups, in which there was a redistribution of PCNA expression into the differentiation zone (without a change in total kidney PCNA content) and distortion of the tubular architecture. Electrophoretic mobility shift assays revealed that the binding of kidney nuclear extracts to the p53 response elements in human and rat PCNA promoters is developmentally regulated. Transient transfection assays performed in p53-deficient HeLa cells revealed that exogenous p53 strongly represses transcription from human PCNA promoter-reporter constructs. Interestingly, deletion of the p53-binding site confers enhanced responsiveness to p53-mediated repression, suggesting that transcriptional repression of PCNA by p53 is achieved by a mechanism other than direct DNA binding. On the basis of these results, we propose the hypothesis that p53-mediated transcriptional repression plays a role in the spatial restriction of PCNA gene expression during normal renal development.

Amino Acid Sequence↗
PubMed1999

Spatial and temporal changes in myosin heavy chain gene expression in skeletal muscle development.

Seven myosin heavy chains (MyHC) are expressed in mammalian skeletal muscle in spatially and temporally regulated patterns. The timing, distribution, and quantitation of MyHC expression during development and early postnatal life of the mouse are reported here. The three adult fast MyHC RNAs (IIa, IIb, and IId/x) are expressed in the mouse embryo and each mRNA has a distinct temporal and spatial distribution. In situ hybridization analysis demonstrates expression of IIb mRNA by 14.5 dpc, which proceeds developmentally in a rostral to caudal pattern. IId/x and IIa mRNAs are detectable 2 days later. Ribonuclease protection assays demonstrate that the three adult fast genes are expressed at approximately equal levels relative to each other in the embryo but at quite low levels relative to the two developmental isoforms, embryonic and perinatal. Just after birth major changes in the relative proportions of different MyHC RNAs and protein occur. In all cases, RNA expression and protein expression appear coincident. The changes in MyHC RNA and protein expression are distinct in different muscles and are restricted in some cases to particular regions of the muscle and do not always reflect their distribution in the adult.

Animals↗
PubMed1997

Zonal distribution of transcripts of four hepatic transcription factors in the mature rat liver.

Within the liver acinus the majority of genes are expressed in an ascending or descending gradient from the portal to the central vein. The regulatory factors involved in this spatial pattern of gene expression are still poorly understood. Many liver genes are regulated by liver-enriched transcription factors. Here we report on mRNA distribution of four transcription factors in cell lysates obtained from either the periportal or the perivenous region after zone-specific digitonin infusion and by in-situ hybridization. Northern blot analysis revealed that there were slightly more transcripts of C/EBP, HNF1 beta (n.s.) and HNF4 (p < 0.05), but fewer of HNF3 gamma (n.s.), in perivenous than in periportal lysates. A somewhat stronger staining in the perivenous region of HNF4 was also seen by in-situ hybridization. The moderate acinar zonation of the mRNAs of these transcription factors suggests that at best they could modulate but not govern the zonated expression of liver genes in the mature liver.

Animals↗
PubMed2004

A cluster translocation model may explain the collinearity of Hox gene expressions.

A model is proposed that deals with the observed collinearities (spatial, temporal and quantitative) of Hox gene expression during pattern formation along the primary and secondary axes of vertebrates. In particular, in the proximodistal axis of the developing limb, it is assumed that a morphogen gradient is laid down with its source at the distal tip of the bud. The extracellular signals in every cell of the morphogenetic field are transduced and uniformly amplified so that molecules are produced in the nucleus with appropriate physicochemical properties. These molecules can exert a concentration-dependent force on the Hox cluster. It is assumed that, before activation, the Hox cluster is packaged as an elongated rigid body inside the chromatin and is covered by a coat that prevents the transcription factors reaching the genes of the cluster. The transcription factors are confined to the interchromatin domain and their density decreases with their distance from the chromatin surface. A gradual increase in the extracellular morphogen concentration causes a corresponding increase in the number of the nuclear molecules and the resulting bigger force pushes the Hox cluster toward the interchromatin domain. The step-by-step translocations of the Hox cluster initiate the consecutive exposure of genes to their transcription factors. The model explains how gene activation is triggered and it describes spatial, temporal and quantitative collinearities at the initial stages of gene expression. Some recent experiments of Hox deletions and duplications are accounted for by the model.

Animals↗
PubMed1987

Establishment and refinement of segmental pattern in the Drosophila embryo: spatial control of engrailed expression by pair-rule genes.

We are examining the development of the segmental body pattern of Drosophila by immunolocalization of engrailed, a developmental regulatory protein that maintains segmental subdivisions in the embryo, and is expressed in a spatially restricted (striped) manner that persists while the body pattern is being established and refined. A regulatory network among pair-rule segmentation genes establishes the striped pattern of engrailed expression. In general, mutations in particular pair-rule genes affect either even- or odd-numbered engrailed stripes. For example, fushi tarazu or odd-paired mutations delete even-numbered stripes, whereas paired mutations delete odd-numbered stripes. An analysis of engrailed expression in other mutants, including even-skipped odd-skipped double mutants, indicates that some pair-rule genes play a rule in establishing the correct width and position of engrailed stripes. Overall, the changes in engrailed pattern have consequences for final embryonic body pattern. Thus, the pair-rule loci, acting through engrailed, establish an early, general outline of body pattern. However, in several pair-rule mutants, engrailed patterns are dynamic, suggesting that as later events build upon this general rule to form the final body pattern, adjustments are made in response to the earlier pair-rule defect--that is, the pattern regulates.

Animals↗
PubMed1994

Spatially regulated expression of three receptor tyrosine kinase genes during gastrulation in the zebrafish.

We describe the isolation and early developmental expression of three novel zebrafish genes (rtk1-3) that encode members of the eph family of receptor tyrosine kinases. At the onset of gastrulation, rtk1 is expressed in the shield region corresponding to the future dorsal side of the embryo. As gastrulation proceeds, both rtk1 and rtk2 are expressed within the axial hypoblast along the entire axis of the embryo. After the gastrula stage is complete, expression of both genes is maintained in precursor cells of the notochord in the tail bud but is downregulated in other regions of the axial hypoblast, rtk3 is expressed in anterior axial hypoblast including the 'pillow' at the anterior tip of the hypoblast and in paraxial tissue in posterior regions of the embryo. We show that the precise spatial regulation of expression of rtk genes, ntl and goosecoid along the anteroposterior axis is maintained in embryos that have no dorsoventral axis. This indicates that the mechanisms that regulate gene expression along the anteroposterior and dorsoventral axes of the hypoblast may be independent.

Amino Acid Sequence↗
PubMed1998

Spatial specificity of H2O2-generating oxalate oxidase gene expression during wheat embryo germination.

Germin, a molecular marker of wheat embryo germination, is a protease-resistant, apoplastic, homopentameric glycoprotein with peroxide-generating oxalate oxidase activity. The spatial specificity of germin-like oxalate oxidase (gl-OXO) gene expression has been determined in tissues of germinating wheat embryos by a combination of histochemical, immunocytochemical and in situ hybridization techniques. The synthesis and accumulation of gl-OXO mRNA and protein is localised within the enveloping tissues of the embryonic axis (particularly the coleorhiza) during the first 24 h of imbibition. By 48 h germination, gl-OXO accumulation is detected throughout the root, with the exception of the postmitotic zone of cell elongation, where accumulation of its transcript is restricted to outer cell layers. At this time in the elongating shoot, gl-OXO is restricted to the coleoptile where it is detected only in the epidermal cell layer, the vascular bundles and bundle sheath cells. In older seedlings (approximately 9 days post-imbibition) gl-OXO activity is detected in leaves, but only within the vascular bundles. These patterns of expression are consistent with the hypothesis that the biological function of gl-OXO is to restrict cell growth by participating in cell-wall restructuring through the local provision of hydrogen peroxide for cross-linking of wall components.

Gene Expression Regulation, Developmental↗
PubMed1989

A chromosome rearrangement suggests that donor and recipient sites are associated during Tam3 transposition in Antirrhinum majus.

We describe the structure of a chromosome rearrangement which changes the spatial pattern of expression of the pallida gene of Antirrhinum majus. The rearrangement involves a chromosome inversion of ~6 map units with one breakpoint at the end of a copy of the transposable element Tam3 located in the promoter region of the pallida locus. The sequence at the breakpoints shows that 5-7 bp, present once in the progenitor, has been duplicated and flanks both ends of the inversion. We propose that this structure arose from an aberrant Tam3 transposition, suggesting a model for normal transposition which involves physical association between donor and recipient sites. This may explain why transposition of plant transposable elements such as Ac in maize occurs preferentially to recipient sites closely linked to the donor site. Excision of the Tam3 copy located at the end of the chromosome inversion, results in a unique spatial pattern of pallida gene expression as a consequence of replacing all sequence 70 bp upstream of transcription by a new sequence. This pattern may be the result of deleting specific upstream components which regulate pallida expression and/or of changing the relative chromosome position of the pallida gene.

Journal Article↗
PubMed2026

Genome-wide identification of CHY zinc finger and RING finger (CHYR) genes in pepper and functional characterization of CaCHYR5 in response to Phytophthora capsici infection.

CHY zinc finger and RING finger (CHYR) proteins play crucial roles in the growth and development, as well as stress response. To date, no systematic or comprehensive analysis of the CHYR gene family has been performed in pepper (Capsicum annuum L.). In this study, we identified 8 CaCHYR genes (CaCHYR1-CaCHYR8), which were classified into 3 groups based on phylogenetic relationships. CaCHYR members within the same group exhibited similar distributions of conserved motifs and exon-intron structures. Chromosomal localization analysis showed that 8 CaCHYR genes were unevenly distributed on 6 chromosomes. Segmental duplication, rather than tandem duplication, was found to be the major contributor to the expansion of this gene family. CaCHYR genes feature a variety of cis-elements involved in developmental processes, phytohormone responses, and stress adaptation. Expression analysis based on RNA-seq data revealed that CaCHYR genes exhibited distinct spatial expression patterns across different tissues and in response to Phytophthora capsici infection (PCI), and quantitative real-time PCR (qRT-PCR) further confirmed that three of them (CaCHYR2, CaCHYR3, and CaCHYR5) exhibited altered expression under PCI. Furthermore, transient overexpression of CaCHYR5 in pepper leaves increased susceptibility to PCI, suggesting its potential negative regulatory role in pepper defense against P. capsici. Collectively, these findings reveal the expression patterns and regulatory functions of pepper CHYR genes in growth and development, laying a groundwork for breeding pepper cultivars tolerant to PCI.

Phytophthora capsici infection (PCI)↗
PubMed2002

Transcriptional integration of competence modulated by mutual repression generates cell-type specificity within the cardiogenic mesoderm.

The way in which spatially patterned cellular identities are generated is a central question of organogenesis. In the case of Drosophila heart formation, the cardiac progenitors are specified in precise mesodermal positions, giving rise to multiple cell types in a highly ordered arrangement. Here, we study the mechanisms by which positional information conveyed by signaling pathways and a combinatorial code of activating and repressing transcription factors work together to confine the expression of the homeobox gene even-skipped (eve) to a small region of the dorsal mesoderm. By manipulating both expression patterns and binding sites for transcription factors, we show that a complex combination of regulatory activities converge on a single enhancer of eve to generate precisely targeted gene expression within the cardiac mesoderm. In particular, ladybird early (lbe), a homeobox gene expressed adjacent to eve, restricts the positive actions of factors downstream of wingless, decapentaplegic, and ras to generate the eve pattern. Mutation of a Lbe binding site causes dramatic expansion of expression and abolishes the responsiveness to repression by lbe. Conversely, eliminating eve in the mesoderm expands lbe expression into the normal eve-expressing territory, suggesting that mutual repression between eve and lbe is essential for delineating the spatial patterns of gene expression that specify cell types within the cardiac mesoderm.

Animals↗
PubMed2006

The Dictyostelium bZIP transcription factor DimB regulates prestalk-specific gene expression.

The ecmA gene is specifically expressed in prestalk cells and its transcription is induced by the chlorinated hexaphenone DIF-1. We have purified a novel bZIP transcription factor, DimB, by affinity chromatography on two spatially separated ecmA promoter fragments. Mutagenesis of the cap-site proximal DimB-binding site (the -510 site) greatly decreases ecmA expression in the pstO cells, which comprise the rear half of the prestalk zone, and also in the Anterior-Like Cells, which lie scattered throughout the prespore region. However, DimB is not essential for normal expression of the ecmA gene, instead it spatially limits its expression; ecmA is relatively highly expressed in the subset of prestalk cells that coats the prestalk zone, but in slugs of a DimB-null strain, ecmA is highly expressed throughout the prestalk zone. Because the -510 site is required for correct ecmA expression, we posit a separate activator protein that competes with DimB for binding to the -510 site. DimB rapidly accumulates in the nucleus when cells are exposed to DIF-1, and ChIP analysis shows that, in the presence of extracellular cAMP, DIF-1 causes DimB to associate with the ecmA promoter in vivo. Thus, DIF-1 regulates DimB activity to generate a gradient of ecmA expression in the prestalk zone of the slug.

Amino Acid Sequence↗
PubMed2004

Gene expression systems in Drosophila: a synthesis of time and space.

Until recently, ectopic gene expression in Drosophila was largely accomplished through the use of the heat-shock promoter, which provides the experimenter with temporal control over transgene induction, or the GAL4-UAS system, which provides the experimenter with spatial control over transgene expression. But significant advances have now been made in combining the attributes of temporal and spatial control over gene expression into a single system. In this article, we review the progress on the development and implementation of several gene expression systems that offer control in time and space. These include systems employing the yeast FLP recombinase gene and FRT sites (FLP and/or FRT), tetracycline-responsive transcription factors (Tet-On and Tet-Off), steroid hormone responsive transcription factors (GeneSwitch and ER-GAL4) and temperature-sensitive repressors of the classical GAL4-UAS system (TARGET).

Animals↗
PubMed1993

Patterns of gene expression in Bacillus subtilis colonies.

Bacillus subtilis 5:7, a derivative of macrofiber-producing strain FJ7, carries the lacZ reporter gene within Tn917 at an unknown location in the host genome. Expression of the host gene carrying lacZ within colonies of 5:7 was observed by examining growth under different conditions in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). At a high plating density small colonies arose that expressed the host gene early and throughout the colony, whereas at a low density large colonies were produced that expressed the host gene late in development and only in cells forming a ring pattern close to the colony periphery. A highly regulated spatial and temporal gene expression pattern was observed in growth from cross-streaks, suggesting that gene expression is responsive to concentration gradient fields established by neighboring growth. Colonies cultured on agar blocks revealed that expression was governed by depletion of a medium component and also by the geometry of the substrate upon which the colonies grew. At least three factors influenced the control of expression: (i) the concentration of a diffusible component of the medium exhausted by cell growth, (ii) a spatial-temporal factor related to growth within the colony, and (iii) the geometry of the growth substrate.

Bacillus subtilis↗
PubMed2002

Understanding inner ear development with gene expression profiling.

Understanding the development of the inner ear requires knowing the spatial and temporal pattern of gene expression, and the functions of those gene products. In the last decade, hearing research has benefited tremendously from the progress of the human and mouse genome projects, as amply illustrated by the identification of many deafness genes in both human and mouse. However, the sheer amount of information generated from the genome project has far outpaced the rate at which it is utilized. Microarray technology offers a means to quantify the expression level of transcripts at a whole-genome scale. Cross-tissue comparisons will identify genes unique to the inner ear, which will expedite the identification of new deafness genes. Microdissection and subtraction after ablation of cell types can reveal genes expressed in certain cells, such as hair cells. Expression profiling of both inner ear and other tissues, under a variety of conditions (such as during development, with drug treatment or in knock-out animals), can be used for cluster analysis to group genes of similar expression. Coexpression can suggest functional pathways and interactions between known genes, and can identify new genes in a structure or pathway. In this review we give examples for both transcription factors and cochlear structures.

Animals↗
PubMed1995

A nuclear GFP that marks nuclei in living Drosophila embryos; maternal supply overcomes a delay in the appearance of zygotic fluorescence.

The central role of gene expression in regulating development has largely been studied by in situ hybridization and antibody staining techniques in fixed material. However, rapid temporal and spatial changes in gene expression are often difficult to correlate with complex morphogenetic movements. A green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, can be used as a real-time reporter for gene expression and could aid analysis of dynamic events during embryogenesis. Here, we describe a transgenic Drosophila line ubiquitously expressing a nuclear GFP fusion protein that highlights morphogenesis, cell movement, and mitosis in living embryos. The fusion protein is highly fluorescent when maternally supplied, but there is a long delay between its zygotic expression and the appearance of fluorescence. GFP is thus an excellent marker for the expression of stable gene products, but a poor reporter for dynamic zygotic gene expression in early Drosophila embryos.

Animals↗
PubMed1990

Developmental regulation and spatial pattern of expression of the structural genes for nitrogenase in the cyanobacterium Anabaena.

Depriving the cyanobacterium Anabaena of fixed nitrogen induces the differentiation of heterocysts at intervals along its filaments. To test whether the oxygen-deficient conditions believed to prevail within mature heterocysts are sufficient, in the absence of fixed nitrogen, to elicit the expression of nitrogenase, PnifHDK was fused transcriptionally to luxAB (encoding luciferase). Expression, monitored from individual cells as light emission, was localized (with a resolution of approximately 1 micron) to differentiated cells, whether or not oxygen was present. Anabaena PCC 7118 is a heterocystless mutant strain that is known to fix nitrogen when deprived of combined nitrogen under anaerobic conditions. Three lines of evidence indicate that the mutant has retained the ability to develop a pattern despite its inability to make heterocysts. First, morphologically distinct cells appear at nonrandom intervals when filaments are starved of nitrogen. Second, these cells, like heterocysts, have little or no phycocyanin-dependent fluorescence. Third, nitrogen-starved filaments fragment, with fragment lengths similar to the spacing normally seen between heterocysts. Expression of PnifHDK-luxAB was largely confined to differentiated cells in the mutant as in the wild-type strain. These results provide evidence for a causal relationship between development and transcriptional events in Anabaena.

Aerobiosis↗
PubMed2001

A physical force may expose Hox genes to express in a morphogenetic density gradient.

In both invertebrates and vertebrates, a set of homeobox genes is involved in the primary pattern formation along the anterior-posterior axis of the developing organism. In particular, the genes of the Hox/HOM complex are located in a physical order in the 3' to 5' direction of the gene clusters. Furthermore, the vertebrate genes of the Hoxa and Hoxd clusters are expressed following the empirical rules of temporal and spatial collinearities: the genes are expressed one after the other according to their positional order and their domains of expression start anteriorly and move gradually towards more posterior locations along the developmental axis. The mechanism that controls this remarkable expression behaviour remains elusive. A proposed morphogen gradient model could justify the serial gene expression in space and time during vertebrate limb development. It is therefore likely that a morphogen concentration ordering might cause the sequential gene expression. I put forward this hypothesis and explore some possibilities that concentration-dependent physical forces might push the Hoxa,d clusters to an environment where the transcriptional activity of the genes is possible. The suggested mechanisms offer satisfactory concentration resolution for differential gene expression. Some experiments are proposed to test the presence of such forces. The verification of this hypothesis would provide a solution to the interpretation problem of the positional information theory in development. Furthermore, it would broaden our knowledge of how gene transcription can be triggered.

Animals↗
PubMed2006

A comprehensive study of the spatial and temporal expression of the col5a1 gene in mouse embryos: a clue for understanding collagen V function in developing connective tissues.

Collagen V is a quantitatively minor component of collagen I fibrils and the defective product of classic Ehlers-Danlos syndrome (EDS). To provide new insights into its embryonic function, a continuous evaluation of the expression pattern of proalpha1(V), a chain common to all collagen V molecular forms, was performed by in situ hybridization of developing mouse from 7.5 days after conception (dpc) to birth. Proalpha1(V) transcripts were first detected at 8.5 dpc, signals being considerably augmented at 16.5 dpc and declining at birth. Hybridization signals were, at first, exclusively detected in the dorsal aorta wall, heart, and adnexa. At 10.5 dpc, col5a1 expression was found in the heart, dorsal aorta wall, branchial arches, mesonephrotic tubules, and intestinal mesenchyme and coincided with proalpha1(I) developmental expression. Later stages exhibited an intense signal in more restricted regions, notably the skin, the bones and vertebral column, the cornea, the tendons and ligaments, the peritoneal membranes, the umbilical cord, and the salivary gland. The data revealed the important contribution of collagen V to the development of functional connective tissues. Proalpha1(V) signals were exclusively detected in the flattened cells of the surface ectoderm at 10.5 dpc. By 12.5 dpc, when cells had become cuboidal, the signal switched to the dermal fibroblasts. Thus, type V collagen appears to contribute to epidermis differentiation. Our data also suggest that collagen V participates in bone formation and/or mineralization and in the renewal of stromal cells in the cornea. The results underscore the role of collagen V in developing embryos and provide important clues for analyzing the phenotype of mouse models for EDS.

Animals↗