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PubMed2000

Coordinate expression of novel genes during osteoblast differentiation.

To achieve new insights into the coordinate regulation of gene expression during osteoblast differentiation we utilized an approach involving global analysis of gene expression to obtain the identities of messenger RNAs (mRNAs) expressed using an established in vitro model of bone development. MC3T3-E1 osteoblast-like cells were induced to differentiate by the addition of beta-glycerophosphate (beta-GP) and ascorbic acid. RNA samples derived from induced and uninduced control MC3T3-E1 cells were used to prepare complementary DNA (cDNA) for serial analysis of gene expression (SAGE). A preliminary SAGE database was produced and used to prepare a hybridization array to further facilitate the characterization of changes in the expression levels of 92 of the SAGE-mRNA assignments after induction of osteoblast differentiation, specifically after 6 days and 14 days of ascorbate treatment. SAGE-array hybridization analysis revealed coordinate induction of a number of mRNAs including Rab24, calponin, and calcyclin. Levels of MSY-1, SH3P2, fibronectin, alpha-collagen, procollagen, and LAMPI mRNAs, present at day 6 postinduction, were markedly reduced by day 14 postinduction. A number of unanticipated and potentially important developmental genes were identified including the transforming growth factor beta (TGF-beta) superfamily member Lefty-1. Lefty-1 transcript and translation product were found to be induced during the course of MC3T3-E1 cell differentiation. We present evidence, using transient transfection and antibody neutralization approaches, that Lefty-1 modulates the induction of alkaline phosphatase (ALP) after treatment of MC3T3-E1 cells with ascorbate and beta-GP. These data should provide useful new information for future analysis of transcriptional events in osteoblast differentiation and mineralization.

Alkaline Phosphatase↗
PubMed1997

HSP gene expression and HSF2 in mouse development.

During the pre-implantation phase of development, the mouse embryo synthesizes HSC70, and HSP90 alpha and beta at a very high rate. After implantation, the expression of HSPs appears non-coordinated and is not uniform in the different tissues. The expression of inducible HSPs appears later in development than that of constitutive members of the family. HSP25 is highly expressed early in heart and muscle development, but also in some structure of the central nervous system. HSC70 and HSP90 beta are expressed ubiquitously, but their expression reaches very high levels in the nervous system (neural tracks) and during bone morphogenesis (in the hypertrophic chondrocytes). The mechanisms involved in HSP expression during mouse embryogenesis are probably diverse, involving tissue-specific sequences. Although the DNA-binding activity and expression of the second heat shock transcription factor, HSF2, seems to be developmentally regulated, becoming detectable at the blastocyst stage and reaching a peak at day 10 of development, there is no obvious correlation between the level of this factor and the expression of HSPs. HSF2 might be involved in the onset of expression of HSPs, regulate (inhibit) their expression, or control the expression of other developmental genes yet to be discovered.

Animals↗
PubMedDate not supplied

Abd-B expression in Porcellio scaber Latreille, 1804 (Isopoda: Crustacea): conserved pattern versus novel roles in development and evolution.

The Hox genes are intimately involved in patterning the animal body during development and are considered to have had a pivotal role in the evolution of different body plans among the metazoans. From this perspective, crustaceans, a group that has evolved an extreme diversity of body structures, represent a choice group in which to study the evolution of these genes and their expression. The expression of one of these genes, Abdominal-B (Abd-B), has only been studied in two distantly related crustaceans, Artemia and Sacculina, where it shows dissimilar patterns, highly differentiated from the one described in other arthropods. Moreover, we have no information for the Malacostraca. Thus, we cloned the gene Abd-B and followed its expression through development by in situ hybridization in the isopod Porcellio scaber. We found a highly dynamic expression pattern of PsAbd-B during embryonic development. In early stages, it is expressed in the posterior-most part of the germ band, in a domain common to several arthropods studied to date, and later it is expressed in the developing limb buds of the pleon and still later in the endopodites of the third to fifth pleopodites. This raises the interesting possibility of the involvement of this gene in the later respiratory specialization of these appendages. In association with the above expression domain, Abd-B appears to be expressed in later stages also in the ventral ectoderm, raising the further suggestion of its possible involvement in patterning the developing nervous system. Moreover, we show that the first pleopod and the endopodite of the second pleopod, whereas present as limb buds in early embryonic stages, are later reduced and actually absent in the first postembryonic stage, although they reappear again in adults. These appendages thus represent an example of Lazarus appendages. Our data show strong plasticity in the use of a key developmental gene and point out the necessity of further research that may end with a revision of the current understanding of its role in animal evolution.

Animals↗
PubMed2006

A distal enhancer and an ultraconserved exon are derived from a novel retroposon.

Hundreds of highly conserved distal cis-regulatory elements have been characterized so far in vertebrate genomes. Many thousands more are predicted on the basis of comparative genomics. However, in stark contrast to the genes that they regulate, in invertebrates virtually none of these regions can be traced by using sequence similarity, leaving their evolutionary origins obscure. Here we show that a class of conserved, primarily non-coding regions in tetrapods originated from a previously unknown short interspersed repetitive element (SINE) retroposon family that was active in the Sarcopterygii (lobe-finned fishes and terrestrial vertebrates) in the Silurian period at least 410 million years ago (ref. 4), and seems to be recently active in the 'living fossil' Indonesian coelacanth, Latimeria menadoensis. Using a mouse enhancer assay we show that one copy, 0.5 million bases from the neuro-developmental gene ISL1, is an enhancer that recapitulates multiple aspects of Isl1 expression patterns. Several other copies represent new, possibly regulatory, alternatively spliced exons in the middle of pre-existing Sarcopterygian genes. One of these, a more than 200-base-pair ultraconserved region, 100% identical in mammals, and 80% identical to the coelacanth SINE, contains a 31-amino-acid-residue alternatively spliced exon of the messenger RNA processing gene PCBP2 (ref. 6). These add to a growing list of examples in which relics of transposable elements have acquired a function that serves their host, a process termed 'exaptation', and provide an origin for at least some of the many highly conserved vertebrate-specific genomic sequences.

Animals↗
PubMed2005

CisMols Analyzer: identification of compositionally similar cis-element clusters in ortholog conserved regions of coordinately expressed genes.

Combinatorial interactions of sequence-specific trans-acting factors with localized genomic cis-element clusters are the principal mechanism for regulating tissue-specific and developmental gene expression. With the emergence of expanding numbers of genome-wide expression analyses, the identification of the cis-elements responsible for specific patterns of transcriptional regulation represents a critical area of investigation. Computational methods for the identification of functional cis-regulatory modules are difficult to devise, principally because of the short length and degenerate nature of individual cis-element binding sites and the inherent complexity that is generated by combinatorial interactions within cis-clusters. Filtering candidate cis-element clusters based on phylogenetic conservation is helpful for an individual ortholog gene pair, but combining data from cis-conservation and coordinate expression across multiple genes is a more difficult problem. To approach this, we have extended an ortholog gene-pair database with additional analytical architecture to allow for the analysis and identification of maximal numbers of compositionally similar and phylogenetically conserved cis-regulatory element clusters from a list of user-selected genes. The system has been successfully tested with a series of functionally related and microarray profile-based co-expressed ortholog pairs of promoters and genes using known regulatory regions as training sets and co-expressed genes in the olfactory and immunohematologic systems as test sets. CisMols Analyzer is accessible via a Web interface at http://cismols.cchmc.org/.

Base Sequence↗
PubMed2005

Mutations affecting predation ability of the soil bacterium Myxococcus xanthus.

Myxococcus xanthus genetic mutants with characterized phenotypes were analysed for the ability to prey on susceptible bacteria. Quantification of predatory ability was scored by a newly developed method under conditions in which prey bacteria provided the only source of nutrients. These results were corroborated by data derived using a previously published protocol that measures predation in the presence of limited external nutrients. First, early developmental regulatory mutants were examined, because their likely functions in assessing the local nutrient status were predicted to be also important for predation. The results showed that predation efficiency is reduced by 64-80 % for mutants of three A-signalling components, AsgA, AsgC and AsgE, but not for AsgB. This suggests that an Asg regulon function that is separate from A-signal production is needed for predation. Besides the Asg components, mutations in the early developmental genes sdeK and csgA were also consistently observed to reduce predatory efficacy by 36 and 33 %, respectively. In contrast, later developmental components, such as DevRS, 4406 and PhoP4, did not appear to play significant roles in predation. The predatory abilities of mutants defective for motility were also tested. The data showed that adventurous, but not social, motility is required for predation in the assay. Also, mutants for components in the chemotaxis-like Frz system were found to be reduced in predation efficiency by between 62 and 85 %. In sum, it was demonstrated here that defects in development and development-related processes affect the ability of M. xanthus to prey on other bacteria.

Bacterial Proteins↗
PubMed1997

Mutants of Neurospora crassa defective in regulation of blue light perception.

A new selection system was used to isolate mutants deficient in regulation of blue light perception in Neurospora crassa. This selection system has two possible applications and was used for the isolation of either blind or constitutive mutants. We isolated 17 UV-induced mutants that showed the pleiotropic white collar (wc) phenotype and were completely blocked in transduction of the light signal. From the segregation pattern in sexual crosses, we tentatively assigned the 17 mutants to either the wc-1 or wc-2 gene. Furthermore, two Neurospora mutants, ccb-1 and ccb-2, were isolated that showed constitutive carotenoid biosynthesis in the dark. Analysis of these mutants for transcripts of the carotenoid biosynthesis genes al-3 and al-1 revealed no higher steady-state levels in the dark than in the control strain. The mutant cch-2 showed major differences in mRNA levels only for conidiation and developmental genes. The lack of a specific change in mRNA levels in response to light, and the mutant phenotype, which seems to reflect a step in conidiation, indicate a role for the cch-2 product in a developmental process, such as conidiation. In contrast, the mutant cch-1 showed a threefold overinduction of carotenoid biosynthesis genes in response to light. This effect was not observed for the conidiation genes examined. The recessive nature of the ccb-1 mutation, together with its specific effect on light induction of the carotenoid biosynthesis genes, indicate that the gene product of ccb-1 acts as a repressor of transcription in some light-regulated processes, but not others.

Carotenoids↗
PubMed2002

Diabetic nephropathy: renal development gone awry?

Nephrogenesis is controlled by a sequence of inductive signals between different areas of the developing kidney. As these signals are being elucidated, it has become clear that many important developmental genes are re-expressed in the mature organ following injury, possibly as part of repair and regeneration. While this reuse of developmental pathways may contribute to healing and repair, it may alternatively result in scar formation if specific components of the pathways are missing, if the temporal correlation of various elements is faulty, or if an injurious stimulus persists. In the review we will use diabetic nephropathy as an example to illustrate this paradigm in renal disease. The pathogenesis of diabetic nephropathy is complex and characterized by altered expression of many genes, including growth factors, apoptotic regulators, cellular matrix components, and cytoskeletal proteins. Many of these factors also function during kidney development. The elucidation of the roles these genes play in nephrogenesis and of their array of molecular partners and modulators may ultimately shed light on the pathogenesis of disease (and indeed vice versa), and may even suggest new therapeutic strategies.

Animals↗
PubMed1997

Junk DNA and sectorial gene repression.

Transcriptional repression in eukaryotes often involves tens or hundreds of kilobase pairs, two to three orders of magnitude more than the bacterial operator/repressor model does. Classical repression, represented by this model, was maintained over the whole span of evolution under different guises, and consists of repressor factors interacting primarily with promoters and, in later evolution, also with enhancers. The use of much larger amounts of DNA in the other mode of repression, here called the sectorial mode ('superrepression'), results in the conceptual transfer of so-called junk DNA to the domain of functional DNA. This contribution to the solution of the c-value paradox involves perhaps 15% of genomic 'junk,' and encompasses the bulk of the introns, thought to fill a stabilizing role in sectorially repressed chromatin structures. In the case of developmental genes, such structures appear to be heterochromatoid in character. However, solid clues regarding general structural features of superrepressed terminal differentiation genes remain elusive. The competition among superrepressible DNA sectors for sectorially binding factors offers, in principle, a molecular mechanism for developmental switches. Position effect variegation may be considered an abnormal manifestation of normal processes that underly development and involve heterochromatoid sectorial repression, which is apparently required for local elimination or modulation of morphological features (morpholysis). Sectorial repression of genes participating either in development or in terminal differentiation is considered instrumental in establishing stable cell types, and provides a basis for the distinction between determination and cell type specification. The gamut of possible stable cell types may have been broadened by the appearance in evolution of heavy isochores. Additional types of relatively frequent GC-rich cis-acting DNA motifs may offer reiterated binding sites to factors endowed with a selective (though not individually strong) affinity for these motifs. The majority of sequence motifs thought to be used in superrepression need not be individually maintained by natural selection. It is re-emphasized that the dispensability of sequences is not an indicator of their nonfunctionality and that in many cases, along noncoding sequences, nucleotides tend to fill functions collectively, rather than individually.

Animals↗
PubMed2025

Genome-Wide Mining of lncRNAs Reveals Their Potential Regulatory Role in the Evolution of Viviparity.

Reproduction in vertebrates usually involves egg-laying (oviparity) or live-bearing (viviparity). Oviparity is the ancestral trait from which viviparity has independently evolved more than 100 times in squamate reptiles. This transition involves a series of physiological and structural changes, including the degeneration of eggshell and the evolution of a placenta and differences in the temporal and spatial expression patterns of some functional genes that drive the structural transformation. Long non-coding RNAs (lncRNAs) play important roles in the regulation of gene expression, yet it remains unclear whether they participate in gene expression shifts during the transition from oviparity to viviparity, and if so how. Therefore, we employ deep mining to identify novel lncRNAs of a closely related oviparous-viviparous pair of lizards (Phrynocephalus przewalskii and P. vlangalii). We construct cis- and trans-regulatory networks between lncRNAs and target genes using the transcriptomic data of oviduct or uteri tissues across reproductive periods. Results show that lncRNAs that regulate eggshell gland developmental genes in the oviparous lizard are lost or less expressed in the viviparous lizard. A number of lncRNAs involved in the regulation of placental development and embryo attachment in viviparous species have no orthologs in oviparous species, and others show little or no expression. Accordingly, lncRNAs may play important regulatory roles in the physiological and structural changes in the transition from oviparity to viviparity. These results open doors to the further elucidation of genetic regulatory networks.

Animals↗
PubMed2005

The transcriptomes of Trypanosoma brucei Lister 427 and TREU927 bloodstream and procyclic trypomastigotes.

We describe developmentally regulated genes in two strains of Trypanosoma brucei: the monomorphic strain Lister 427 and the pleomorphic strain TREU927. Expression patterns were obtained using an array of 24,567 genomic fragments. Probes were prepared from bloodstream-form or procyclic-form trypanosomes. Fourteen procyclic-specific and 77 bloodstream-specific signals were obtained from sequences matching variant surface glycoprotein or associated genes, and a further 17 regulated sequences were repetitive or transposable-element-related. Two hundred and eighty-six regulated spots corresponded to mRNAs from other protein-coding genes; these spots represent 191 different proteins. Regulation of 113 different genes (79 from procyclic forms, 34 from bloodstream-forms) was supported by at least two independent experiments or criteria; of these, about 60 were novel. Only two genes -- encoding HSP83 and an importin-related protein -- appeared to be regulated in the TREU927 strain only. Our results confirmed previous estimates that 2% of trypanosome genes show developmental regulation at the mRNA level.

Animals↗
PubMed2004

Species-related differences in blastocyst quality are associated with differences in relative mRNA transcription.

The objective of this study was to compare the relative transcript abundance of several important candidate genes between ovine and bovine blastocysts. Blastocysts were produced by in vitro maturation, fertilization, and subsequent culture in one of two formulations of synthetic oviduct fluid medium (SOF1 and SOF2). From each IVF replicate groups of 10 bovine and 10 ovine blastocysts from each of the two media were used for analysis of mRNA relative abundance. Transcript levels for mitochondrial Mn-superoxide dismutase (MnSOD), survivin, and glucose transport 5 (Glut-5) were significantly higher in ovine blastocysts than bovine (P < 0.05), while transcripts for Connexin 31 (Cx31), interferon tau (IFN-tau), and sarcosine oxidase (SOX) were significantly more abundant in bovine blastocysts (P < 0.01). For the two remaining transcripts, E-cadherin (E-cad) and Na/K ATPase (Na/K), there was no difference. Culture of bovine embryos in SOF2 resulted in a significant increase in the level of expression of MnSOD and Glut-5 (P < 0.05) compared to those bovine embryos cultured in SOF1. For all the other transcripts, except survivin, there was a significant decrease in the relative abundance. Culture of sheep embryos in either SOF1 or SOF2 did not have a major influence on transcript abundance; of the eight transcripts examined, the relative abundance of only one, SOX, was significantly altered. Bovine blastocysts produced in SOF2 had significantly higher survival rates at 24, 48, and 72 hr and significantly higher hatching rates following vitrification and warming than those cultured in SOF1 (P < 0.001). In conclusion, we have quantified for the first time the mRNA expression of a set of important developmental genes in sheep blastocysts and we have demonstrated that these differences between species in their adaptability to culture conditions, manifested in differences in embryo morphology and cryotolerance, are related to differences in mRNA relative abundance. The results also highlight the usefulness of transcript analysis as a marker of embryo quality.

Animals↗
PubMed1991

Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice.

Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region.

Adult↗
PubMed2005

Nervous and muscle system development in Phascolion strombus (Sipuncula).

Recent interpretations of developmental gene expression patterns propose that the last common metazoan ancestor was segmented, although most animal phyla show no obvious signs of segmentation. Developmental studies of non-model system trochozoan taxa may shed light on this hypothesis by assessing possible cryptic segmentation patterns. In this paper, we present the first immunocytochemical data on the ontogeny of the nervous system and the musculature in the sipunculan Phascolion strombus. Myogenesis of the first anlagen of the body wall ring muscles occurs synchronously and not subsequently from anterior to posterior as in segmented spiralian taxa (i.e. annelids). The number of ring muscles remains constant during the initial stages of body axis elongation. In the anterior-posteriorly elongated larva, newly formed ring muscles originate along the entire body axis between existing myocytes, indicating that repeated muscle bands do not form from a posterior growth zone. During neurogenesis, the Phascolion larva expresses a non-metameric, paired, ventral nerve cord that fuses in the mid-body region in the late-stage elongated larva. Contrary to other trochozoans, Phascolion lacks any larval serotonergic structures. However, two to three FMRFamide-positive cells are found in the apical organ. In addition, late larvae show commissure-like neurones interconnecting the two ventral nerve cords, while early juveniles exhibit a third, medially placed FMRFamidergic ventral nerve. Although we did not find any indications for cryptic segmentation, certain neuro-developmental traits in Phascolion resemble the conditions found in polychaetes (including echiurans) and myzostomids and support a close relationship of Sipuncula and Annelida.

Animals↗
PubMed2004

Use of beta-glucuronidase reporter gene for gene expression analysis in turfgrasses.

The beta-glucuronidase (GUS) gene has been successfully used as a reporter gene in innumerable number of plant species. The functional GUS gene produces blue coloration in plants upon integration into the plant genome. Because of the ease it provides to analyze the gene expression (as no expensive equipment is needed), GUS gene is surely plant biotechnologist's first choice as a reporter gene. The turfgrass family contains the world's most economically important horticultural crops. There is a world-wide drive for genetic modification of grasses due to its huge economic importance. GUS gene can be transiently or stably expressed in grasses for the purpose of promoter analysis and to study tissue-specific and developmental gene expression. This paper summarizes the use of GUS gene for transient and stable expression studies in various turfgrass species.

Gene Expression Profiling↗
PubMed1986

Spatial pattern of catalase (Cat2) gene activation in scutella during postgerminative development in maize.

The scutellum of maize is a fully differentiated, nondividing, diploid embryonic tissue. Two distinct structural genes (Cat1 and Cat2) encoding the enzyme catalase (CAT) are differentially expressed in this tissue during postgerminative development. As development proceeds, the expression of Cat1 diminishes, while that of Cat2 is enhanced, leading to the disappearance of the CAT-1 protein and the gradual accumulation of the CAT-2 protein. The present investigation was undertaken to determine whether all scutellar cells may be genetically programmed to activate expression of Cat2 synchronously or whether there is an asynchronous spatial gradient of Cat2 activation. By using immunofluorescence microscopy and anti-CAT-2 IgG, we have found that a gradient of Cat2 activation occurs within the scutellar cell mass during postgerminative development. The gradient of Cat2 activation occurs from the outer perimeter of the tissue inward toward the embryonic axis. To determine a potential site of origin for any putative "triggering signal" for Cat2 activation, we demonstrated that Cat2 is expressed in the single layer of aleurone cells prior to its expression in any other tissue during kernel development. To our knowledge, this is the first observation of a gradient-type spatial pattern of a eukaryote gene activation occurring in a stable, virtually nondividing tissue such as the maize scutellum. The significance of these results with respect to developmental gene regulation is discussed.

Catalase↗
PubMed1998

Cardiac and extracardiac expression of Csx/Nkx2.5 homeodomain protein.

Csx/Nkx2.5 is an evolutionary conserved homeobox gene related to the Drosophila tinman gene, which is essential for the dorsal mesoderm formation. Expression of Csx/Nkx2.5 mRNA is the earliest marker for heart precursor cells in all vertebrates so far examined. Previous studies have demonstrated that Csx/Nkx2.5 mRNA is highly expressed in the heart and at lower levels in the spleen, tongue, stomach, and thyroid in the murine embryo. Since some developmental genes are regulated by posttranscriptional mechanisms, we analyzed the developmental pattern of Csx protein expression at the single-cell level using Csx-specific antibodies. Immunohistochemical analysis of murine embryos at 7.8 days post coitum revealed that Csx protein is strongly expressed in the nucleus of endodermal and mesodermal cells in the cardiogenic plate. Subsequently, in the heart, Csx protein was detected only in the nucleus of myocytes of the atrium and the ventricle through the adult stage. During the fetal period, Csx protein expression in the nucleus was also noted in the spleen, stomach, liver, tongue, and anterior larynx. Unexpectedly, confocal microscopy revealed that Csx immunoreactivity was detected only in the cytoplasm of a subset of cranial skeletal muscles. Csx protein was not detected in the thyroid glands. The expression of Csx protein in all organs was markedly downregulated after birth except in the heart. These results raise the possibility that Csx/Nkx2.5 may play a role in the early developmental process of multiple tissues in addition to its role in early heart development.

Animals↗
PubMed1989

Search for Drosophila genes encoding a conserved domain present in the achaete-scute complex and myc proteins.

Several genes of the achaete-scute complex (AS-C) of Drosophila melanogaster encode a 60 amino acids long conserved domain which shares a significant homology with a region of the vertebrate myc proteins. Based on these results, the existence of a family of Drosophila genes that would share both this conserved domain and the neurogenic function of the AS-C has been postulated. To test this proposal, we have searched a D. melanogaster genomic library with a probe that encodes the conserved domain. Only under very low stringency hybridization conditions, clones not belonging to the AS-C cross-hybridized with the probe. Those that gave the strongest signals were characterized. Sequencing of the cross-hybridizing regions showed that they had no significant homology with the conserved domain, the sequence similarity extending at the most for 37 nucleotides. Although our results do not conclusively disprove the existence of a family of AS-C-like genes, they indicate that the conservation of the domain would be lower than that found for shared motifs in other families of Drosophila developmental genes.

Amino Acid Sequence↗