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PubMed2000

Photoactivated gene expression for cell fate mapping and cell manipulation.

A long-standing goal of developmental biologists is to create developmental fate maps by tracking individual cells through development. Another objective is to perturb the behavior of selected cells and follow the ensuing effects. To this end, we have developed a technique that allows for spatial and temporal control of gene expression in single cells or patches of cells using light to induce gene expression. This technique relies on "caging" the activity of the potent transcriptional activator GAL4VP16 with a photolabile compound, which can be removed with a brief exposure to long-wavelength ultraviolet (UV) light. The caged GAL4VP16 is injected into early-stage embryos, which are aged to the desired point in development, and the cell(s) of interest are irradiated with a brief pulse of long-wavelength UV light. This method has been used extensively in Drosophila, Xenopus, and Zebrafish embryos. The methods for purifying, caging, injection, and photoactivation of the GAL4VP16 protein, and methods for the visualization of marked cells are described in detail.

Animals↗
PubMed1999

Gene regulation by patterned electrical activity during neural and skeletal muscle development.

Patterned neural activity modifies central synapses during development and the physiological properties of skeletal muscle by selectively repressing or stimulating transcription of distinct genes. The effects of neural activity are mostly mediated by calcium. Of particular interest are the cellular mechanisms that may be used to sense and convert changes in calcium into specific alterations in gene expression. Recent studies have addressed the importance of spatial heterogeneity or of temporal changes in calcium levels for the regulation of gene expression.

Action Potentials↗
PubMed2001

The Isolation of differentially expressed mRNA sequences by selective amplification via biotin and restriction-mediated enrichment.

Molecular analysis of development frequently implies the isolation and characterization of genes with specific spatial and temporal expression patterns. Several methods have been developed to identify such DNA sequences. The most comprehensive technique involves the genomewide probing of DNA sequence microarrays with mRNA sequences. However, at present this technology is limited to the few organisms for which the entire genome has been sequenced. Here, we describe a subtractive hybridization technique, called selective amplification via biotin and restriction-mediated enrichment (SABRE), which allows the selective amplification of cDNA fragments representing differentially expressed mRNA species. The method involves the competitive hybridization of an excess of driver cDNA fragments (D) to a trace of tester cDNA fragments (T), and the subsequent purification of tester homohybrids (in which both strands are contributed by the tester cDNA). After competitive hybridization, cDNA fragments that are more abundant in the tester than in the driver are enriched in the tester homohybrids. However, as the fraction of tester homohybrids is very small [T(2)/(D + T)(2)], their purification requires highly efficient procedures. In SABRE, the isolation of tester homohybrids is afforded by a combination of three successive steps: removal of biotinylated terminal sequences from most of the heterohybrids by S1 nuclease digestion, capture of biotinylated hybrids with streptavidin-coated paramagnetic beads, and specific release of homohybrids from the beads by restriction nuclease digestion. If several rounds of SABRE selection are performed in series, even relatively rare differentially expressed mRNA sequences may result in the production of predominant cDNA fragments in the final tester homohybrid population.

Animals↗
PubMed2003

Selection of normalizer genes in conducting relative gene expression analysis of embryos.

BACKGROUND: In relative gene expression analysis, a reference gene for sample normalization is required for determining target expression changes among experimental treatment groups. Since some developmental toxicants secondarily cause general growth retardation and/or other general biological changes, commonly used housekeeping genes may not serve as accurate normalizers. METHODS: We conducted real-time polymerase chain reaction (PCR) with normalization to calculate relative target transcriptional change, using housekeeping and structure-specific expression genes as normalizers. Relative levels of Hoxb1 expression were measured in cultured rodent embryos at 24 hr post retinoic acid (RA) administration. Transcriptional response was also evaluated using two novel compounds that produced posterior axial and growth defects in rat whole-embryo culture. Embryos treated with these compounds were evaluated for general biological processes, and their respective biological states were considered in the context of the relative gene expression change calculated with the housekeeping normalizers. RESULTS: Normalized RA-induced Hoxb1 expression demonstrated that only some reference genes accurately quantitated the expected 1.5- to 2-fold increase in Hoxb1 expression. Evaluation of the test compounds demonstrated that only normalization with the spatially-restricted hindbrain gene, Krox-20, calculated significant expression decreases of T-gene, a gene known to be functionally relevant in posterior axial development. Reduction in T-gene expression was confirmed qualitatively by whole-mount in situ hybridization. CONCLUSIONS: Prudent reference gene selection is important in evaluating relative gene expression in embryos. An experimental control design is proposed to facilitate the identification of normalizing genes that will accurately calculate relative gene expression change in treated embryos.

Animals↗
PubMed1998

Control of anteroposterior and dorsoventral domains of Nkx-6.1 gene expression relative to other Nkx genes during vertebrate CNS development.

Here we report the isolation, sequence and developmental expression in the central nervous system of several members of the chicken and mouse Nkx gene family. These are among the earliest genes to be regionally expressed in the neural plate; they are expressed just above the axial mesendoderm (prechordal mesendoderm and notochord). Each Nkx gene has a distinct spatial pattern of expression along the anterior-posterior axis of the ventral central nervous system: Nkx-2. 2 is expressed along the entire axis, whereas Nkx-2.1 is restricted to the forebrain, and Nkx-6.1 and Nkx-6.2 are largely excluded from the forebrain. They are also expressed in distinct patterns along the dorsal-ventral axis. These genes are expressed in both the ventricular and mantle zones; in the mantle zone Nkx-6.1 is co-expressed with Islet-1 in a subset of motor neurons. Like other Nkx genes, expression of Nkx-6.1 is induced by the axial mesendoderm and by sonic hedgehog protein. BMP-7 represses Nkx-6.1 expression. While the notochord can induce Nkx-6.1 expression in the anterior neural plate, sonic hedgehog protein does not, suggesting that the notochord produces additional molecules that can regulate ventral patterning.

Amino Acid Sequence↗
PubMed1992

Analysis of the murine Hox-2.7 gene: conserved alternative transcripts with differential distributions in the nervous system and the potential for shared regulatory regions.

In this study we have investigated the organization and regulation of the mouse Hox-2.7 gene. There are several alternative transcripts some of which are conserved between mouse and humans. By Northern and in situ analysis we are able to identify at least three types of transcripts which are different in size and splicing pattern and have distinctly different boundaries of expression in the nervous system. One subset of the endogenous transcripts has a boundary of expression that corresponds to the adjacent Hox-2.8 gene instead of Hox-2.7. In another type of transcript there is an alternative reading frame which predicts a protein that has homology to an enzyme ATPase and suggests that a non-homeobox containing gene may be located in the Hox-2 cluster. A Hox-2.7-lacZ transgene is expressed in a similar pattern to the endogenous gene in that spatially-restricted domains of expression are seen in the branchial arches, neural tube, paraxial mesoderm (somites), cranial ganglia, neural crest and gut. However, the anterior boundaries of transgene expression only correspond to the subset of Hox-2.7 transcripts which map to the Hox-2.8 boundary. The proximity of a Hox-2.7 promoter to regions which regulate the adjacent Hox-2.6 gene and the expression of transgenic and endogenous transcripts in a Hox-2.8 pattern, suggest that regulatory elements may be shared by neighbouring genes to establish the complete expression pattern.

Amino Acid Sequence↗
PubMed2004

Multiple regulatory elements with spatially and temporally distinct activities control the expression of the epithelial differentiation gene lin-26 in C. elegans.

Epithelial differentiation is a very early event during development of most species. The nematode Caenorhabditis elegans, with its well-defined and invariant lineage, offers the possibility to link cell lineage, cell fate specification and gene regulation during epithelial differentiation. Here, we focus on the regulation of the gene lin-26, which is required for proper differentiation of epithelial cells in the ectoderm and mesoderm (somatic gonad). lin-26 expression starts in early embryos and remains on throughout development, in many cell types originating from different sublineages. Using GFP reporters and mutant rescue assays, we performed a molecular dissection of the lin-26 promoter and could identify almost all elements required to establish its complex spatial and temporal expression. Most of these elements act redundantly, or synergistically once combined, to drive expression in cells related by function. We also show that lin-26 promoter elements mediate activation in the epidermis (hypodermis) by the GATA factor ELT-1, or repression in the foregut (pharynx) by the FoxA protein PHA-4. Taken together, our data indicate that lin-26 regulation is achieved to a large extent through tissue-specific cis-regulatory elements.

Animals↗
PubMed1996

Developmental expression of synthetic cis-regulatory systems composed of spatial control elements from two different genes.

Synthetic cis-regulatory systems consisting of positively and negatively acting cis-regulatory modules of the Endo16 gene were combined with the lineage-specific regulatory element of the SM50 gene associated with a reporter and injected into eggs of sea urchins. We show here that synthetic cis-regulatory systems consisting of the positive Endo16 regulatory elements linked with the SM50 regulatory element are expressed spatially exactly as the sum of the individual endodermal and skeletogenic expression patterns. In combination, both lineage-specific positive regulatory elements function autonomously. However, addition of the Endo16 regulatory module that represses ectopic skeletogenic expression of Endo16 receptor constructs does not affect expression driven by the SM50 regulatory elements in the same skeletogenic cells. The repression function of this element is thus dedicated to control of the positive spatial output of the Endo16 regulatory system.

Animals↗
PubMed1990

Interactions of Polycomb and trithorax with cis regulatory regions of Ultrabithorax during the development of Drosophila melanogaster.

The activity of the Ultrabithorax gene is continuously required during imaginal development to maintain the morphogenetic identity of the third thoracic segment of Drosophila. The spatial pattern of Ultrabithorax gene expression depends on certain cis regulatory regions and several trans regulatory genes. Amongst the latter the Polycomb gene is necessary to maintain Ultrabithorax repressed in cells where it was not initially activated and the trithorax gene is required for maintaining the expression of the gene where initially active. We have studied genetic interactions between several Ultrabithorax mutations in coding and cis regulatory regions in combination with Polycomb and trithorax mutations. Our results suggest that Polycomb and trithorax gene products do not interact with Ultrabithorax protein products but interact (directly or indirectly) with specific and discrete cis regulatory regions such as those where anterobithorax and postbithorax but not bithorax mutations map. We discuss possible mechanisms of these interactions.

Alleles↗
PubMed2000

Chamber formation and morphogenesis in the developing mammalian heart.

In this study we challenge the generally accepted view that cardiac chambers form from an array of segmental primordia arranged along the anteroposterior axis of the linear and looping heart tube. We traced the spatial pattern of expression of genes encoding atrial natriuretic factor, sarcoplasmic reticulum calcium ATPase, Chisel, Irx5, Irx4, myosin light chain 2v, and beta-myosin heavy chain and related these to morphogenesis. Based on the patterns we propose a two-step model for chamber formation in the embryonic heart. First, a linear heart forms, which is composed of "primary" myocardium that nonetheless shows polarity in phenotype and gene expression along its anteroposterior and dorsoventral axes. Second, specialized ventricular chamber myocardium is specified at the ventral surface of the linear heart tube, while distinct left and right atrial myocardium forms more caudally on laterodorsal surfaces. The process of looping aligns these primordial chambers such that they face the outer curvature. Myocardium of the inner curvature, as well as that of inflow tract, atrioventricular canal, and outflow tract, retains the molecular signature originally found in linear heart tube myocardium. Evidence for distinct transcriptional programs which govern compartmentalization in the forming heart is seen in the patterns of expression of Hand1 for the dorsoventral axis, Irx4 and Tbx5 for the anteroposterior axis, and Irx5 for the distinction between primary and chamber myocardium.

Animals↗
PubMed1993

Border disputes: do boundaries play a role in growth-cone guidance?

One of the earliest indications of regional patterning in the CNS is the spatially restricted expression of regulatory genes within the neuroepithelium. Many of these genes encode transcription factors and, although little is known of their downstream targets, it seems likely that they control the identity of cells in different regions of the CNS. This review discusses how the expression of these patterning genes might influence the location at which the first axon pathways in the CNS are pioneered. Evidence is described that suggests that the boundary regions between adjacent domains of regulatory gene expression influence where the first axons will extend.

Animals↗
PubMed2004

An artificial promoter construct for heat-inducible misexpression during fish embryogenesis.

Beside spatial distribution, timing of gene expression is a key parameter controlling gene function during embryonic development. Gain-of-function experiments can therefore have quite opposing results, depending on the time of gene activation. Induction techniques are necessary to control timing in these experiments from outside of the organism. Natural heat shock promoters constitute a simple inducible misexpression system, the main disadvantage is a high background level of expression. We present here a new heat stress-inducible bidirectional promoter consisting of multimerized heat shock elements (HSE). The simplified architecture of this promoter largely improves the properties needed for an efficient induction system: dramatically reduced background activity, improved inducibility, and loss of all tissue specific components. Based on this new artificial promoter, we present a transient induction system for fish embryos. Application of this new induction system for Fgf8 misexpression during embryonic development reveals different windows of competence during eye development. A dramatic early phenotype resulting in loss of the eyes is observed for conventional mRNA injection. Later activation, by using our inducible promoter, uncovers different eye phenotypes like cyclopic eyes. Even after 14 days, an efficient heat stress response could be evoked in the injected embryos. The HSE promoter therefore represents a new artificial heat shock promoter with superior properties, making possible transient experiments with inducible misexpression at various stages of development.

Animals↗
PubMed2006

Embryonic expression profiles and conserved localization mechanisms of pem/postplasmic mRNAs of two species of ascidian, Ciona intestinalis and Ciona savignyi.

In many animals, the first cue for development is transcripts and/or proteins that are provided maternally and are localized at specific regions of fertilized eggs and early embryos. The ascidian is known to exhibit a mosaic mode of development, which is largely dependent on localized maternal factors. In early Ciona intestinalis embryos, the posterior-most localization appears to be the major specialized pattern of maternal transcripts. The present study examined the temporal and spatial expression pattern of 40 genes known as pem/postplasmic genes, for which maternal mRNAs are localized at the posterior-most region during early Ciona embryogenesis. Ten of these genes showed redistribution to B8.12-line cells, which are known to give rise to germ cells in ascidians. In addition 23 orthologues were newly identified in a related ascidian species, Ciona savignyi, and 16 of them showed the mRNA localization pattern at the posterior-most region. Furthermore, the localized pattern of exogenous mRNA, which comprised the 3' UTR of C. intestinalis pem/postplasmic genes conjugated with the LacZ ORF, showed the localization at the posterior-most region in C. savignyi embryos. Likewise, the 3' UTR of C. savignyi pem/postplasmic genes conjugated with the LacZ ORF showed localization at the posterior most region in C. intestinalis embryos, suggesting that localization mechanisms are conserved between the two species. The present study therefore provides basic information for future functional analyses of these pem/postplasmic genes and for exploring the mechanisms of localization of mRNAs.

Animals↗
PubMed2006

In vivo enhancer analysis of human conserved non-coding sequences.

Identifying the sequences that direct the spatial and temporal expression of genes and defining their function in vivo remains a significant challenge in the annotation of vertebrate genomes. One major obstacle is the lack of experimentally validated training sets. In this study, we made use of extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements, and characterized the in vivo enhancer activity of a large group of non-coding elements in the human genome that are conserved in human-pufferfish, Takifugu (Fugu) rubripes, or ultraconserved in human-mouse-rat. We tested 167 of these extremely conserved sequences in a transgenic mouse enhancer assay. Here we report that 45% of these sequences functioned reproducibly as tissue-specific enhancers of gene expression at embryonic day 11.5. While directing expression in a broad range of anatomical structures in the embryo, the majority of the 75 enhancers directed expression to various regions of the developing nervous system. We identified sequence signatures enriched in a subset of these elements that targeted forebrain expression, and used these features to rank all approximately 3,100 non-coding elements in the human genome that are conserved between human and Fugu. The testing of the top predictions in transgenic mice resulted in a threefold enrichment for sequences with forebrain enhancer activity. These data dramatically expand the catalogue of human gene enhancers that have been characterized in vivo, and illustrate the utility of such training sets for a variety of biological applications, including decoding the regulatory vocabulary of the human genome.

Animals↗
PubMed2005

The human VE-cadherin promoter is subjected to organ-specific regulation and is activated in tumour angiogenesis.

Vascular endothelial (VE)-cadherin is exclusively expressed at interendothelial junctions of normal and tumour vessels. In this report, we characterized the transcriptional activity of the human VE-cadherin promoter. Transient transfection assays revealed that sequences at positions --1135/-744 and -166/-5 base pairs are critical for promoter activity in endothelial cells. We show that specific sequences in the proximal region interact with Ets and Sp1 family members. Transgenic mice were created and the human VE-cadherin promoter was able to confer correct temporal and spatial expression on the LacZ gene in embryos. In adults, the transgene was specifically and strongly expressed in the lung, heart, ovary, spleen and kidney glomeruli, whereas expression was weak or absent in the vasculature of other organs, including the brain, thymus, liver and skeletal muscle. Neovessels in tumour grafts and Matrigel implants harboured strong stainings, indicating that promoter activity is enhanced in angiogenic situations. Furthermore, Matrigel and transfection assays showed that VE-cadherin promoter is subjected to bFGF induction. Transgene expression was also noticed in extravascular sites of the central nervous system, suggesting that silencer elements may be located elsewhere in the gene. These results are a first step towards addressing the organ- and tumour-specific regulation of the VE-cadherin gene.

Animals↗
PubMed1992

Molecular characterization and nephron distribution of a family of transcripts encoding the pore-forming subunit of Ca2+ channels in the kidney.

Active, transepithelial, Ca2+ reabsorption in kidney occurs primarily in the distal convoluted tubule. Recent evidence suggests that entry of Ca2+ at the apical membrane through channels bearing resemblance to those of the voltage-dependent L type may be the rate-determining step in Ca2+ reabsorption. To determine the molecular identity of the pore-forming subunit of voltage-dependent Ca2+ channel(s) in the kidney, a homology-based PCR cloning strategy was employed. Nondegenerate primers, based on conserved regions of the published cDNA sequences of voltage-dependent Ca2+ channel alpha 1 subunits, were used to amplify cDNA from rat kidney, and the products were subcloned and sequenced. A family of molecular species was identified, representing alternatively spliced transcripts of four known genes encoding these channel subunits. Northern blot analysis indicated that the expression of each of the genes exhibits a distinct spatial distribution within the kidney. One gene, CaCh4, is expressed primarily in the cortex, and by microdissected-tubule PCR was found predominantly in the distal convoluted tubule, consistent with a role in transepithelial Ca2+ reabsorption at this site.

Amino Acid Sequence↗
PubMed1998

The cell and molecular biology of fracture healing.

Fracture healing is a complex physiologic process that involves the coordinated participation of several cell types. By using a reproducible model of experimental fracture healing in the rat, it is possible to elucidate the integrated cellular responses that signal the pathways and the role of the extracellular matrix components in orchestrating the events of fracture healing. Histologic characterization of fracture healing shows that intramembranous ossification occurs under the periosteum within a few days after an injury. Events of endochondral ossification occur adjacent to the fracture site and span a period of up to 28 days. Remodeling of the woven bone formed by intramembranous and endochondral ossification proceeds for several weeks. Spatial and temporal expression of genes for major collagens (Types I and II), minor fibrillar collagens (Types IV and XI), and several extracellular matrix components (osteocalcin, osteonectin, osteopontin, fibronectin and CD44) are detected by in situ hybridization. Immunohistochemical studies show that expression of proliferating cell nuclear antigen is both time and space dependent and differentially expressed in the callus tissues formed by the intramembranous and endochondral processes. Chondrocytes involved in endochondral ossification undergo apoptosis (programmed cell death), and early events in fracture healing may be initiated by the expression of early response genes such as c-fos. Additional characterization and elucidation of fracture healing will lay the foundation for subsequent studies aimed at identifying mechanisms for enhancing skeletal repair.

Animals↗