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PubMed2005

Temporal and spatial changes in the expression pattern of multiple red and green subtype opsin genes during zebrafish development.

Zebrafish have two red, LWS-1 and LWS-2, and four green, RH2-1, RH2-2, RH2-3 and RH2-4, opsin genes encoding photopigments with distinct absorption spectra. Occurrence of opsin subtypes by gene duplication is characteristic of fish but little is known whether the subtypes are expressed differently in the retina, either spatially or temporally. Here we show by in situ hybridization the dynamic expression patterns of the opsin subtypes in the zebrafish retina. Expression of red type opsins is initiated with the shorter-wavelength subtype LWS-2, followed by the longer-wavelength subtype LWS-1. In the adult retina, LWS-2 was expressed in the central to dorsal area and LWS-1 in the ventral and peripheral areas. Expression patterns of green type opsins were similar to those of the red type opsins. The expression started with the shortest wavelength subtype RH2-1 followed by the longer wavelength ones, and in the adult retina, the shorter wavelength subtypes (RH2-1 and RH2-2) were expressed in the central to dorsal area and longer wavelength subtypes (RH2-3 and RH2-4) in the ventral and peripheral areas. These results provide the framework for subsequent studies of opsin gene regulation and for probing functional rationale of the developmental changes by using the power of zebrafish genetics.

Animals↗
PubMed2003

Practical procedures for ectopic induction of gene expression in zebrafish embryos using Bhc-diazo-caged mRNA.

We previously reported mRNA caging technology as a novel and simple technique for photo-mediated temporal and spatial control of gene activation in zebrafish embryos and as an alternative to the 'gene knockdown' approach using antisense morpholino oligonucleotides. The caging reagent used is 6-bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo), which forms a covalent bond with the phosphate moiety of the sugar-phosphate backbone of RNA. Mainly because of the reduced solubility of caged mRNA in aqueous solutions, special care in handling is needed. The Bhc-diazo group binds to the phosphate moieties of RNA and abolishes the translational activity of the latter. The translational activity of Bhc-caged mRNA is restored by photolysis/uncaging when exposed to long-wave UV light (350 approximately 365 nm). In this paper we describe the technique and detailed procedures for spatially and temporally controlled induction of gene expression in zebrafish embryos.

Animals↗
PubMed1993

Replication-competent retroviral vectors encoding alkaline phosphatase reveal spatial restriction of viral gene expression/transduction in the chick embryo.

Replication-competent avian retroviruses, capable of transducing and expressing up to 2 kb of nonviral sequences, are now available to effect widespread gene transfer in chicken (chick) embryos (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We have constructed novel avian retroviral vectors that encode human placental alkaline phosphatase as a marker whose expression can be histochemically monitored. These vectors have been tested for expression by introducing them into the embryonic chick nervous system. They have revealed that the expression of retrovirally transduced genes can be spatially and temporally limited without the need for tissue-specific promoters. By varying the site and time of infection, targeted gene transfer can be confined to selected populations of neural cells over the course of several days, a time window that is sufficient for many key developmental processes. The capability of differentially infecting specific target populations may avoid confounding variables such as detrimental effects of a transduced gene on processes unrelated to the cells or tissue of interest. These vectors and methods thus should be useful in studies of the effect of transduced genes on the development of various organs and tissues during avian embryogenesis. In addition, the vectors will facilitate studies aimed at an understanding of viral infection and expression patterns.

Age Factors↗
PubMed1997

Light modulates the spatial patterns of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in Arabidopsis thaliana.

Although the coordinated biosynthesis of isoprenoid compounds is thought to be essential to the normal processes of plant growth and development, the mechanisms that regulate the mevalonate pathway in plants are not well understood. As the first committed step in the pathway, the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonic acid by HMG CoA reductase and the regulation of the genes encoding this enzyme have been implicated in the network that controls isoprenoid biosynthesis in higher plants. Using histochemical staining for beta-glucuronidase, as well as conventional RNA hybridization analysis, the temporal and spatial regulation of HMG1, one of the genes encoding HMG CoA reductase in the crucifer Arabidopsis thaliana, has been characterized. Furthermore, the HMG1 promoter is shown to be differentially responsive to illumination in different organs, and promoter activation by light deprivation is confined primarily to immature leaves. In contrast, expression of the HMG1 gene in roots is confined to the elongation zone and is not responsive to illumination. Light-mediated regulation of HMG1 expression is shown to be an organ-autonomous response that depends on direct illumination, and environmental cues regarding light do not appear to be exchanged between different organs in Arabidopsis. These studies reveal several new features of HMG1 regulation, and indicate that the high levels of HMG CoA reductase expression detected in immature leaves may be primarily attributed to the dark-induced expression of HMG1, and that HMG1 is expressed at low levels throughout the plant in response to light. Thus, environmental cues interact with the developmental program to define the pattern of HMG1 gene expression in Arabidopsis.

Arabidopsis↗
PubMed2006

Three-dimensional morphology and gene expression in the Drosophila blastoderm at cellular resolution I: data acquisition pipeline.

BACKGROUND: To model and thoroughly understand animal transcription networks, it is essential to derive accurate spatial and temporal descriptions of developing gene expression patterns with cellular resolution. RESULTS: Here we describe a suite of methods that provide the first quantitative three-dimensional description of gene expression and morphology at cellular resolution in whole embryos. A database containing information derived from 1,282 embryos is released that describes the mRNA expression of 22 genes at multiple time points in the Drosophila blastoderm. We demonstrate that our methods are sufficiently accurate to detect previously undescribed features of morphology and gene expression. The cellular blastoderm is shown to have an intricate morphology of nuclear density patterns and apical/basal displacements that correlate with later well-known morphological features. Pair rule gene expression stripes, generally considered to specify patterning only along the anterior/posterior body axis, are shown to have complex changes in stripe location, stripe curvature, and expression level along the dorsal/ventral axis. Pair rule genes are also found to not always maintain the same register to each other. CONCLUSION: The application of these quantitative methods to other developmental systems will likely reveal many other previously unknown features and provide a more rigorous understanding of developmental regulatory networks.

Animals↗
PubMed1990

The use of lacZ gene fusions in the studies of mammalian development: developmental regulation of mammalian homeobox genes in the CNS.

1. Mammalian cells can produce bacterial beta-galactosidase when they carry the lacZ gene under the control of mammalian regulatory elements. The single cell resolution of the beta-galactosidase histochemical detection method makes this molecule an excellent marker in studies of development at the cellular and molecular level. Different lacZ fusion genes can be engineered to study the histological diversification of cell lineages, the developmental regulation of isolated genes, or to recognize and clone genes with new expression profile. 2. Transgenic mice carrying lacZ gene fusions provide information on the cell type, developmental stage and spatial specificity of cis-acting regulatory regions linked to a mammalian homeobox gene. We describe our strategy for designing the gene fusions. 3. The scord region of the Hox 1.3 gene is sufficient to determine spatially restricted expression of a heterologous protein in the midgestational spinal cord. We propose to use this region to alter the expression pattern of other homeobox gene products. Developmental alterations due to a variant expression pattern would point to the function of the misexpressed gene.

Animals↗
PubMed1998

Opioid gene expression in the developing and adult rat heart.

Opioid peptides are known to play a role in the function and growth of the mammalian heart. Although some information about gene expression of opioids in the heart is available, there is no data on the cellular location of opioid gene expression during development or in the adult. Using in situ hybridization and rat heart ranging from embryonic day 14 (E14) to adulthood, we have evaluated the distribution of gene expression for proenkephalin, proopiomelanocortin, and prodynorphin. With respect to preproenkephalin mRNA (PPE mRNA), message in the ventricle was abundant from E14 (the first time point examined) until shortly after birth, with a marked reduction noted on postnatal days 5, 10, and 21. Adults displayed considerable message, though less than in preparations of embryonic and neonatal heart. PPE mRNA was detected in epicardial, myocardial, and endocardial cells, as well as the walls of blood vessels, capillaries, and fibroblasts. Preproopiomelanocortin (POMC) mRNA was only found in adults, and was localized to the myocardium. Message for preprodynorphin could not be observed in the ventricles of developing or adult rats. These results are the first to define the temporal and spatial ontogeny of opioid gene expression with regard to the emergence of cardiac architecture. The data suggest that gene expression for proenkephalin is especially prevalent in embryonic and neonatal rats and may be related to the modulatory activity of the opioid growth factor, [Met5]-enkephalin, on cell proliferation and differentiation. The role of PPE and POMC mRNA in adult rat heart requires elucidation.

Animals↗
PubMed1994

The mouse ileal lipid-binding protein gene: a model for studying axial patterning during gut morphogenesis.

Normal, chimeric-transgenic, and transgenic mice have been used to study the axial patterns of ileal lipid-binding protein gene (Ilbp) expression during and after completion of gut morphogenesis. Ilbp is initially activated in enterocytes in bidirectional wave that expands proximally in the ileum and distally to the colon during late gestation and the first postnatal week. This activation occurs at the same time that a wave of cytodifferentiation of the gut endoderm is completing its unidirectional journey from duodenum to colon. The subsequent contraction of Ilbp's expression domain, followed by its reexpansion from the distal to proximal ileum, coincides with a critical period in gut morphogenesis (postnatal days 7-28) when its proliferative units (crypts) form, establish their final stem cell hierarchy, and then multiply through fission. The wave of reactivation is characterized by changing patterns of Ilbp expression: (a) at the proximal most boundary of the wave, villi contain a mixed population of scattered ileal lipid-binding protein (ILBP)-positive and ILBP-negative enterocytes derived from the same monoclonal crypt; (b) somewhat more distally, villi contain vertical coherent stripes of wholly ILBP-positive enterocytes derived from monoclonal crypts and adjacent, wholly ILBP-negative stripes of enterocytes emanating from other monoclonal crypts; and (c) more distally, all the enterocytes on a villus support Ilbp expression. Functional mapping studies of Ilbp's promoter in transgenic mice indicate that nucleotides -145 to +48 contain cis-acting elements sufficient to produce an appropriately directed distal-to-proximal wave of Ilbp activation in the ileum, to maintain an appropriate axial distribution of monophenotypic wholly reporter-positive villi in the distal portion of the ileum, as well as striped and speckled villi in the proximal portion of its expression domain, and to correctly support reporter production in villus-associated ileal enterocytes. Nucleotides -417 to -146 of Ilbp contain a "temporal" suppressor that delays initial ileal activation of the gene until the second postnatal week. Nucleotides -913 to -418 contain a temporal suppressor that further delays initial activation of the gene until the third to fourth postnatal week, a spatial suppressor that prohibits gene expression in the proximal quarter of the ileum and in the proximal colon, and a cell lineage suppressor that prohibits expression in goblet cells during the first two postnatal weeks.

Amino Acid Sequence↗
PubMed2003

A Hes1-based oscillator in cultured cells and its potential implications for the segmentation clock.

During somitogenesis an oscillatory mechanism termed the "segmentation" clock generates periodic waves of gene expression, which translate into the periodic spatial pattern manifest as somites. The dynamic expression of the clock genes shares the same periodicity as somitogenesis. Notch signaling is believed to play a role in the segmentation clock mechanism. The paper by Hirata et al.(1) identifies a biological clock in cultured cells that is dependent upon the Notch target gene Hes1, and which shows a periodicity similar to that of the segmentation clock. This finding opens the possibility that the same oscillator mechanism might also operate in other tissues or cell types.

Animals↗
PubMed1996

Disperse versus compact elements for the regulation of runt stripes in Drosophila.

The segmented body pattern of the Drosophila embryo is established through a hierarchical network of interacting genes. At each successive step in this pathway, transcriptional regulation is used to convert coarse positional information into finer patterns of gene expression. Central to this process are the cis-regulatory regions that drive the dynamic spatial expression of the different segmentation genes. Here we describe the cis-regulatory region of the runt gene. As found for both other primary pair-rule genes, hairy and even-skipped, there are stripe-specific elements which mediate the initial regulation of runt stripes by gap genes. We did not find autoregulatory elements as described for even-skipped and fushi tarazu. The regulation of runt by other pair-rule genes is mediated by a large region, extending over 5 kb upstream and downstream of the transcription start site. This "disperse" element cannot be subdivided into functionally independent subelements or minimal elements. Such disperse elements mediating pair-rule gene interactions may have escaped detection in other segmentation genes and may involve molecular mechanisms different from those mediating regulation by gap genes.

Animals↗
PubMed2002

Plzf mediates transcriptional repression of HoxD gene expression through chromatin remodeling.

The molecular mechanisms that regulate coordinated and colinear activation of Hox gene expression in space and time remain poorly understood. Here we demonstrate that Plzf regulates the spatial expression of the AbdB HoxD gene complex by binding to regulatory elements required for restricted Hox gene expression and can recruit histone deacetylases to these sites. We show by scanning forced microscopy that Plzf, via homodimerization, can form DNA loops and bridge distant Plzf binding sites located within HoxD gene regulatory elements. Furthermore, we demonstrate that Plzf physically interacts with Polycomb proteins on DNA. We propose a model by which the balance between activating morphogenic signals and transcriptional repressors such as Plzf establishes proper Hox gene expression boundaries in the limb bud.

Animals↗
PubMed1990

Developmental regulation of myosin gene expression in mouse cardiac muscle.

Expression of the two isoforms of cardiac myosin heavy chain (MHC), MHC alpha and MHC beta, in mammals is regulated postnatally by a variety of stimuli, including serum hormone levels. Less is known about the factors that regulate myosin gene expression in rapidly growing cardiac muscle in embryos. Using isoform-specific 35S-labeled cRNA probes corresponding to the two MHC genes and the two myosin alkali light chain (MLC) genes expressed in cardiac muscle, we have investigated the temporal and spatial pattern of expression of these different genes in the developing mouse heart by in situ hybridization. Between 7.5 and 8 d post coitum (p.c.), the newly formed cardiac tube begins to express MHC alpha, MHC beta, MLC1 atrial (MLC1A), and MLC1 ventricular (MLC1V) gene transcripts at high levels throughout the myocardium. As a distinct ventricular chamber forms between 8 and 9 d p.c., MHC beta mRNAs begin to be restricted to ventricular myocytes. This process is complete by 10.5 d p.c. During this time, MHC alpha mRNA levels decrease in ventricular muscle cells but continue to be expressed at high levels in atrial muscle cells. MHC alpha transcripts continue to decrease in ventricular myocytes until 16 d p.c., when they are detectable at low levels, but then increase, and finally replace MHC beta mRNAs in ventricular muscle by 7 d after birth. Like MHC beta, MLC1V transcripts become restricted to ventricular myocytes, but at a slower rate. MLC1V mRNAs continue to be detected at low levels in atrial cells until 15.5 d p.c. MLC1A mRNA levels gradually decrease but are still detectable in ventricular cells until a few days after birth. This dynamic pattern of changes in the myosin phenotype in the prenatal mouse heart suggests that there are different regulatory mechanisms for cell-specific expression of myosin isoforms during cardiac development.

Aging↗
PubMed2002

Kindling induces the mRNA expression of methyl DNA-binding factors in the adult rat hippocampus.

We have investigated the gene expression responses of a family of methyl CpG-binding domain-containing factors (MeCP2, MBD1, MBD2, and MBD3) in the hippocampus of electrically kindled rats. Expression was examined in both amygdala- and partial perforant-pathway-kindled subjects, 24 h and 28 days following the final stimulation. In general, the responses of MBDs 2 and 3 paralleled each another, both temporally and spatially. The expression of both genes was significantly elevated in all hippocampal subfields at 24 h following either the fifth stage 5 seizure (amygdala kindling) or the 15th stimulation of the perforant pathway. This induced expression was transient, however, as the expression of both genes returned to control levels by 28 days. This pattern of response contrasted to that observed for MeCP2 and MBD1. MeCP2 displayed no change in expression either 24 h or 28 days after amygdala kindling, but did display a late-developing, significant increase in expression in the dentate gyrus at 28 days following perforant-pathway kindling. The expression of MBD1 was unchanged by partial perforant-pathway kindling, but was induced in the dentate gyrus 28 days after amygdala kindling. These results demonstrate that electrical kindling alters the hippocampal expression of methyl DNA-binding factors, but does not affect each factor equivalently. The responsive patterns observed suggest that this family of transcriptional regulators can be differentially altered in the hippocampus by seizure activity.

Amygdala↗
PubMed2002

Overlapping of MINK and CHRNE gene loci in the course of mammalian evolution.

Overlapping of genes, especially in an anti-parallel fashion, is quite rare in eukaryotic genomes. We have found a rare instance of exon overlapping involving CHRNE and MINK gene loci on chromosome 17 in humans. CHRNE codes for the epsilon subunit of the nicotinic acetylcholine receptor (AChRepsilon) whereas MINK encodes a serine/threonine kinase belonging to the GCK family. To elucidate the evolutionary trail of this gene overlapping event, we examined the genomes of a number of primates and found that mutations in the polyadenylation signal of the CHRNE gene in early hominoids led to the overlap. Upon extending this analysis to genomes of other orders of placental mammals, we observed that the overlapping occurred at least three times independently during the course of mammalian evolution. Because CHRNE and MINK are differentially expressed, the potentially hazardous mutations responsible for the exon overlap seem to have escaped evolutionary pressures by differential temporo-spatial expression of the two genes.

Animals↗
PubMed1998

Cooperating morphogens control hoxd gene expression in the developing vertebrate limb.

A mechanism was recently proposed which controls the expression of Hoxa genes in the developing vertebrate limb. This is a three-dimensional diffusion model of a morphogen Ma secreted at the apical ectodermal ridge. The morphogen Ma is at the same time degraded by first order chemical kinetics. The emerging diffusion gradient, supplemented with a natural sequence of thresholds, can explain the spatial and temporal collinearities of the Hoxa gene expression domains. In the present model it is assumed that a second diffusing morphogen Mz is produced at the zone of polarizing activity. A concentration superposition of morphogens Ma and Mz can then explain the observed pattern of expression of Hoxd-10, 11, 12, and 13 during normal limb development (spatial and temporal collinearity). The associated thresholds for gene expression increase sequentially from Hoxd-10 to Hoxd-13 (threshold collinearity). The observed deformations or deletions of these expression domains in manipulated limbs are also explained by the model. Additional model predictions are proposed to test the validity of the model. In patterning two mechanisms are mainly considered as responsible for signalling: (i) a gradient of a long-range morphogen created by passive diffusion; and (ii) a short-range inducer initiating a cascade of consecutive signals. A time-test is proposed which distinguishes the modes of specific gene expressions for these distinct signalling mechanisms.

Animals↗
PubMed1994

Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and HNF-1 beta regulate transcription via two elements in an intestine-specific promoter.

Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and HNF-1 beta are transcription factors that contain a divergent homeodomain, bind to DNA as homo- or heterodimers, and act to regulate transcription of many genes that are expressed primarily in liver. We show that HNF-1 alpha and HNF-1 beta act as regulatory factors for transcription of sucrase-isomaltase (SI), the gene for which is expressed exclusively in absorptive enterocytes of the small intestine. HNF-1 alpha and HNF-1 beta bind to two evolutionarily conserved sites in the SI promoter, previously named SIF2 and SIF3, with different relative affinities. HNF-1 alpha binds to the SIF3 element with greater affinity than to the SIF2 element, whereas HNF-1 beta binds to both elements equally. Co-transfection experiments demonstrated that HNF-1 alpha is able to increase transcriptional initiation from a minimal SI intestine-specific promoter. In contrast, HNF-1 beta has no functional effect on transcription of the SI promoter. The functional and DNA binding differences of HNF-1 alpha and HNF-1 beta on elements in the SI promoter suggest that these transcription factors may play a role in the complex spatial patterns of SI gene expression in the intestine.

Base Sequence↗
PubMed1998

Developmental expression and biochemical analysis of the Arabidopsis atao1 gene encoding an H2O2-generating diamine oxidase.

A copper amine oxidase encoding gene, atao1, has been isolated and characterized from Arabidopsis thaliana. Sequence analysis reveals that atao1 encodes a 668 amino acid polypeptide (ATAO1) with 48% identity to copper amine oxidases from pea and lentil. The promoter region of atao1 was transcriptionally fused with the reporter genes encoding beta-glucuronidase and modified green fluorescent protein. Analysis of transgenic Arabidopsis together with in situ hybridization of wild-type plants reveals temporally and spatially discrete patterns of gene expression in lateral root cap cells, vascular tissue of roots, developing leaves, the hypocotyl, and in the style/stigmatal tissue. Enzyme activity assays show that ATAO1 preferentially oxidizes the aliphatic diamine putrescine with production of the corresponding aldehyde, ammonia and hydrogen peroxide, a recognized plant signal molecule and substrate for peroxidases. Histochemical analysis reveals that atao1 expression in developing tracheary elements precedes and overlaps with lignification and therefore is a good marker for vascular development. In both vascular tissue and the root cap, atao1 expression occurs in cells destined to undergo programmed cell death.

Amine Oxidase (Copper-Containing)↗
PubMed1995

Spatial and temporal patterns of lin-12 expression during C. elegans hermaphrodite development.

The lin-12 gene encodes a receptor that mediates certain cell-cell interactions during Caenorhabditis elegans development. We have examined the expression of a lin-12::lacZ reporter gene in individual cells during the development of C. elegans hermaphrodites. lin-12::lacZ is expressed in a discrete spatial and temporal pattern during development and teh lin-12::lacZ reporter gene will provide a useful marker for other studies, particularly of somatic gonadal and vulval development. In general, the cells that express lin-12:: lacZ correspond to cells whose fates are known to be altered in lin-12 mutants implying that restriction of lin-12 expression may be an important regulatory mechanism; the exceptions to this statement may reveal the cellular defects that underlie aspects of the lin-12 phenotype that have not been previously explained. For decisions that are not naturally variable, lin-12::lacZ expression does not appear to change before or upon commitment to a cell fate implying that in these cases postranscriptional regulation of lin-12 activity may control cell fate specification.

Animals↗