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PubMed1996

Comparison of the expression patterns of five developmentally regulated genes in Manduca sexta and their regulation by 20-hydroxyecdysone in vitro.

A previous study, using subtractive hybridization, identified five genes (esr16, esr20, Mng10, Mng14 and tps9) whose transcripts were up-regulated prior to metamorphosis in Manduca sexta nervous tissue. The developmental time points chosen for subtraction suggested that expression could be coordinately regulated and should be negatively regulated by the steroid hormones, the ecdysteroids. In the present paper, we present an analysis of the expression patterns of these five genes, using reverse transcription-polymerase chain reaction (RT-PCR), at various times during development and assess the effects of 20-hydroxyecdysone and cycloheximide on their expression in vitro. This analysis revealed that with the exception of esr20 all transcripts were detectable at all times and that all but one of the genes were up-regulated in vivo and in vitro in the absence or in the presence of low levels of the ecdysteroids. In the absence of ecdysteroids, cycloheximide blocked the accumulation of only two transcripts, esr16 and esr20. These results reveal a much more complex pattern of gene expression in the central nervous system prior to metamorphosis than previously imagined.

Animals↗
PubMed1989

Evidence that the Myxococcus xanthus frz genes are developmentally regulated.

The frizzy (frz) mutants of Myxococcus xanthus are unable to form fruiting bodies. Instead of forming discrete mounds, these strains aggregate as filaments which have a circular and tangled appearance. Mutations leading to this phenotype have been mapped to five complementation groups, frzA, frzB, frzCD, frzE, and frzF. All have been found to be involved in the control of directional movement of the bacteria and, except for frzB, to be homologous to the chemotaxis genes of enteric bacteria. In this report we present a study of the regulation of expression of the first four genes of the frz gene cluster (frzA, frzB, frzCD, and frzE) by using Tn5-lac transcriptional fusions as reporters of gene expression. We found that these frz genes are developmentally regulated, with their transcription peaking at about the time of early mound formation (12 to 18 h). Analysis of FrzCD expression by enzyme-linked immunosorbent assay showed a 10-fold greater induction at 15 h of development over the level of vegetative cell expression. Northern blot hybridization analysis suggested that the frz genes were arranged as an operon. To test this hypothesis, double mutants were constructed which contained Tn5-132 either upstream or downstream of the reporter Tn5-lac. The expression of the frz genes in the double mutants was consistent with the hypothesis that the first four genes (frzA, frzB, frzCD, and frzE) are organized as an operon with an internal promoter. Insertion mutations in frzCD lowered gene expression whether they were upstream or downstream of the reporter Tn5-lac, suggesting that the FrzCD protein regulates transcription of the entire operon from a promoter upstream of frzA. Evidence is presented suggesting that FrzE is required for induction of transcription as well. When frz mutations were placed in strains that were unable to aggregate (tag), the frz genes were expressed at an elevated level on fruiting agar; this high level of expression was maintained for several days. These results suggest that the tag gene products interact with the frz functions.

DNA Transposable Elements↗
PubMed1996

repE--the Dictyostelium homolog of the human xeroderma pigmentosum group E gene is developmentally regulated and contains a leucine zipper motif.

We have cloned and characterized the Dictyostelium discoideum repE gene, a homolog of the human xeroderma pigmentosum (XP) group E gene which encodes a UV-damaged DNA binding protein. The repE gene maps to chromosome 4 and it is the first gene identified in Dictyostelium that is homologous to those involved in nucleotide excision repair and their related XP diseases in humans. The predicted protein encodes a leucine zipper motif. The repE gene is not expressed by mitotically dividing cells, and repE mRNA is first detected during the aggregation phase of development when the cells have ceased dividing and replicating genomic DNA. The mRNA level plateaus by the time the developing cells have entered multicellular aggregates and remains at the same steady-state level for the remainder of development. In addition, we have demonstrated that the level of mRNA is very low in developing cells. These observations suggest that repE may play a regulatory role in development. The data indicate that potential developmental roles for XP-related genes can be profitably studied in this system.

Amino Acid Sequence↗
PubMed1998

Distinct molecular phenotypes in murine cardiac muscle development, growth, and hypertrophy.

The onset of cardiac hypertrophy is associated with characteristic changes in myocardial gene expression that are thought to recapitulate a developmental gene program. We report here the first gene expression profile of the murine myocardium, using a rapid method of quantitative expression analysis based on real-time analytical RT-PCR. This assay was used to measure expression levels of 29 genes in (1) late stage development as represented by day 1 neonatal ventricles, (2) normal cardiac growth in 3 and 18 month old mice, and (3) cardiac hypertrophy following pressure overload by aortic constriction. For males and females normal growth is not associated with differential expression although there is elevated expression of skeletal and smooth muscle actin mRNA's in males compared to females. Using normal adult ventricles as a reference, there are many qualitative and quantitative differences between the day 1 neonatal myocardium and experimental cardiac hypertrophy. These data suggest that the response to POL involves a subset of re-expressed developmental genes together with altered expression of genes not necessarily associated with cardiac development.

Animals↗
PubMed1991

A developmentally regulated gene encodes the dictyostelium homolog of yeast ribosomal protein S4 and mammalian LLRep3 proteins.

We report the sequence and expression of a single-copy gene from Dictyostelium discoideum which encodes the homolog of yeast ribosomal protein S4, a protein located on the small ribosomal subunit and known to play an important role in maintaining translational fidelity. Over a highly conserved central region, the Dictyostelium protein has 78% sequence similarity to the yeast protein and 83% sequence similarity to mammalian S4 protein homologs, the LLRep3 proteins. The Dictyostelium gene encodes a polypeptide 28,717 Da in size and hence this ribosomal protein has been named rp29. The N-terminal sequence of the Dictyostelium rp29 protein is extended by 61 amino acids and 14 amino acids compared to the mammalian and yeast proteins, respectively, and the C-terminus is correspondingly 15 amino acids or 2 amino acids shorter. Although the coding region of the rp29 gene is present on a single exon, a 157bp intron interrupts the 5' untranslated region and unusually contains four direct repeats of the sequence TCAATCT. The gene is expressed maximally during vegetative growth but a second peak of expression also occurs late in development which is restricted to prestalk cells; rp29 is the first Dictyostelium ribosomal protein gene reported which shows prestalk-specific developmental expression. During each round of expression, only a single 0.9kb transcript is produced which is similar in size to the yeast S4 ribosomal protein transcript (0.8kb) but markedly smaller than the mammalian LLRep3 mRNA (1.7kb) due to a much shorter 5' untranslated region.

Amino Acid Sequence↗
PubMed2005

The evolutionary origins of vertebrate midbrain and MHB: insights from mouse, amphioxus and ascidian Dmbx homeobox genes.

Comparative studies on developmental gene expression suggest that the ancestral chordate central nervous system comprised anterior, midbrain-hindbrain boundary (MHB) and posterior regions. The most anterior region consists of both forebrain and midbrain in vertebrates. It remains, however, unresolved when or how the vertebrate midbrain was established from this anterior zone. I previously reported a mouse PRD-class homeobox gene, Dmbx1, expressed in the presumptive midbrain at early developmental stages, and in the hindbrain at later stages, with exclusion from the MHB. To investigate the evolution of midbrain development, I have cloned Dmbx genes from amphioxus and from Ciona, representing the two most closely related lineages to the vertebrates, and examined embryonic Dmbx expression in these species. In amphioxus, no Dmbx expression is observed in the neural tube, supporting previous arguments that the MHB equivalent region has been secondarily lost in evolution. In Ciona, the CiDmbx gene is detected in neural cells posterior to Pax-2/5/8-positive cells (MHB homologue), but not in any cells anterior to them. These results support the lack of a midbrain homologue in Ciona, and suggest that midbrain development is a vertebrate innovation. Here, I report the full sequences of these genes and discuss the evolution of midbrain development in relation to the tripartite neural ground plan and the origin of the MHB organizer.

Amphibians↗
PubMed1997

Treatment of Grape Berries, a Nonclimacteric Fruit with a Synthetic Auxin, Retards Ripening and Alters the Expression of Developmentally Regulated Genes.

Treatment of grape (Vitis vinifera L.) berries with the synthetic auxin-like compound benzothiazole-2-oxyacetic acid (BTOA) caused a delay in the onset of ripening of approximately 2 weeks. This was manifested as a retardation of the increases in berry weight, color, deformability, and hexose concentration. BTOA treatment also delayed by 2 weeks the increase in abscisic acid level that normally accompanies ripening and altered the expression of a number of developmentally regulated genes. A putative vacuolar invertase, which is normally expressed from berry set until ripening and turned off after ripening commences, remained expressed throughout development in BTOA-treated grape berries. This elevated expression resulted in increased levels of invertase activity. In contrast, the up-regulation of four other genes normally switched on at the time of ripening was delayed in BTOA-treated fruit. These included chalcone synthase and UDP-glucose-flavonoid 3-O-glucosyl transferase, both of which are involved in anthocyanin synthesis, a chitinase, and a ripening-related gene of an unknown function. These observations support the view that auxins (perhaps in conjunction with abscisic acid) may have a role in the control of grape berry ripening by affecting the expression of genes involved in the ripening process.

Journal Article↗
PubMed1998

Targeted gene deletion of Leishmania major genes encoding developmental stage-specific leishmanolysin (GP63).

The major surface glycoprotein of Leishmania major is a zinc metalloproteinase of 63 kDa referred to as leishmanolysin or GP63, which is encoded by a family of seven genes. Targeted gene replacement was used to delete gp63 genes 1-6 encoding the highly expressed promastigote and constitutively expressed GP63. In the L. major homozygous mutants deficient in gp63 genes 1-6, there was no expression of GP63 as detected by reverse transcription-polymerase chain reaction (RT-PCR) or fluorescent staining in promastigotes from the procyclic stage (logarithmic growth phase). The remaining L. major gP63 gene 7 was shown to be developmentally regulated, as it was expressed exclusively in infectious metacyclic stage (late stationary growth phase) promastigotes and in lesion amastigotes. The gp63 genes 1-6-deficient mutants showed increased sensitivity to complement-mediated lysis. The sensitivity to lysis was greater in procyclics than in metacyclics when compared with the equivalent wild-type stages. Increased resistance of the mutant metacyclic promastigotes correlated with the expression of gp63 gene 7 and was restored to the same levels as wild-type promastigotes by transfection with gp63 gene 1. Thus, expression of GP63 is clearly involved in conferring resistance to complement-mediated lysis. The L. major GP63 1-6 mutants were capable of infecting mouse macrophages and differentiating into amastigotes. Similar levels of infection and subsequent intracellular survival were observed when mouse macrophages were infected in vitro with wild type, GP63 1-6 mutants and mutants transfected with gp63 gene 1. The GP63 1-6 mutants were capable of lesion formation in BALB/c mice and, thus, gp63 genes 1-6 do not play a role in the survival of the parasite within mouse macrophages. The role of gp63 genes 1-6 in parasite development within the sandfly vector was studied. GP63 1-6 mutants grew normally in the blood-engorged midgut of both Phlebotomus argentipes and P. papatasi However, both wild-type and mutant promastigotes were lost after 2 days' growth in P. papatasi. The complete developmental pathway in P. argentipes was observed for wild-type promastigotes, GP63 1-6 mutants and mutants transfected with gp63 gene 1. Normal stage differentiation from amastigotes to procyclics, to nectomonads, to haptomonads and to infectious metacyclics was observed. Thus, the highly expressed promastigote forms of GP63, encoded by gp63 genes 1-6, do not appear to be required for nutrient utilization in the bloodmeal during the early stages of development in the sandfly or for midgut attachment and further development. gp63 1-6 genes do, however, play a major protective role against complement-mediated lysis when promastigotes are introduced into the mammalian host.

Animals↗
PubMed1996

Aggression from a developmental perspective: genes, environments and interactions.

Genetic influences on the social behaviours of non-human mammals are ubiquitous, powerful and readily detected. But demonstrations that social behaviours are influenced by genes constitute only part of the story. Developmental findings have helped complete the picture. Specifically, these studies show that: (1) genetic effects for aggressive behaviours are highly malleable over the course of development; (2) genetic influences on aggressive behaviour are more dynamic, easily achieved and open to rapid manipulation than has been recognized in current models of social evolution and behavioural genetics; (3) developmental timing has a significant impact upon the nature of the genetic effects observed in aggressive behaviours. These empirical results are consistent with the view that social behaviours are among the first features to be influenced by genetic selection and by environmental experience. Social actions have distinctive properties in adaptation because they organize the space between the organism and the environment and promote rapid, selective and novel accommodations. The modern integrative view of the development of individual-in-context brings attention to the correlation between constraints within and external to individuals. This paper reviews findings on how these processes become integrated over time in individuals and species, and their implications for the nurture of nature.

Aggression↗
PubMed1986

The Drosophila virilis dopa decarboxylase gene is developmentally regulated when integrated into Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) has been isolated from Drosophila virilis and introduced into the germ-line of Drosophila melanogaster by P-element mediated transformation. The integrated gene is induced at the correct stages during development with apparently normal tissue specificity, indicating that cis-acting elements required for regulation are functionally conserved between the two species. A comparison of the DNA sequences from the 5' flanking regions reveals a cluster of small (8-16 bp) conserved sequence elements within 150 bp upstream of the RNA startpoint, a region required for normal expression of the D. melanogaster Ddc gene.

Animals↗
PubMed2001

Drosophila Med6 is required for elevated expression of a large but distinct set of developmentally regulated genes.

Mediator is the evolutionarily conserved coactivator required for the integration and recruitment of diverse regulatory signals to basal transcription machinery. To elucidate the functions of metazoan Mediator, we isolated Drosophila melanogaster Med6 mutants. dMed6 is essential for viability and/or proliferation of most cells. dMed6 mutants failed to pupate and died in the third larval instar with severe proliferation defects in imaginal discs and other larval mitotic cells. cDNA microarray, quantitative reverse transcription-PCR, and in situ expression analyses of developmentally regulated genes in dMed6 mutants showed that transcriptional activation of many, but not all, genes was affected. Among the genes found to be affected were some that play a role in cell proliferation and metabolism. Therefore, dMed6 is required in most cells for transcriptional regulation of many genes important for diverse aspects of Drosophila development.

Alleles↗
PubMed1998

Krüppel acts as a developmental switch gene that mediates Notch signalling-dependent tip cell differentiation in the excretory organs of Drosophila.

Cell proliferation in the excretory organs of Drosophila, the Malpighian tubules (MT), is under the control of a neural tip cell. This unique cell is singled out from equivalent MT primordial cells in response to Notch signalling. We show that the gene Krüppel (Kr), best known for its segmentation function in the early embryo, is under the control of the Notch-dependent signalling process. Lack-of-function and gain-of-function experiments demonstrate that Kr activity determines the neural fate of tip cells by acting as a direct downstream target of proneural basic helix-loop-helix (bHLH) proteins that are restricted in response to Notch signalling. We have identified a unique cis-acting element that mediates all spatial and temporal aspects of Kr gene expression during MT development. This element contains functional binding sites for the restricted proneural bHLH factors and Fork head protein which is expressed in all MT cells. Our results suggest a mechanism in which these transcription factors cooperate to set up a unique cell fate within an equivalence group of cells by restricting the activity of the developmental switch gene Kr in response to Notch signalling.

Animals↗
PubMed2003

Homotypic regulatory clusters in Drosophila.

Cis-regulatory modules (CRMs) are transcription regulatory DNA segments (approximately 1 Kb range) that control the expression of developmental genes in higher eukaryotes. We analyzed clustering of known binding motifs for transcription factors (TFs) in over 60 known CRMs from 20 Drosophila developmental genes, and we present evidence that each type of recognition motif forms significant clusters within the regulatory regions regulated by the corresponding TF. We demonstrate how a search with a single binding motif can be applied to explore gene regulatory networks and to discover coregulated genes in the genome. We also discuss the potential of the clustering method in interpreting the differential response of genes to various levels of transcriptional regulators.

Animals↗
PubMed2004

LIM-homeodomain genes as developmental and adult genetic markers of Xenopus forebrain functional subdivisions.

We have investigated the expression patterns of five LIM-homeodomain (LIM-hd) genes, x-Lhx1, x-Lhx2, x-Lhx5, x-Lhx7, and x-Lhx9 in the forebrain of the frog Xenopus laevis during larval development and in the adult. The results were analyzed in terms of neuromeric organization of the amphibian brain and of combinatorial LIM-hd code and showed that LIM-hd developmental transcription factors are particularly powerful to highlight the coherence of several groups or nuclei, to delineate subdivisions, and/or to clarify structures that are still a matter of debate. Among other findings, we bring substantial evidence for the following: (1) a dual origin of olfactory bulb neurons, based on x-Lhx5 expression; (2) the existence of a ventral pallium in frog, based on x-Lhx9 expression; (3) a multiple (pallial and subpallial) origin for the nuclei of the amygdaloid complex, based on distinct combinations of the five studied genes; (4) a clear homology between the Xenopus medial pallium and the mammalian hippocampus, based on x-Lhx2 pattern; and (5) a confirmed prosomeric organization of the diencephalon, based on alternating x-Lhx1/5 and x-Lhx2/9 expressions. In addition, the important expression levels for LIM-hd factors found throughout development and in the adult brain suggest a role for these genes in development and maintenance of neuronal specification and phenotype, as for example in the case of x-Lhx7 and cholinergic neurons. Moreover, following LIM-hd patterns throughout development points out to some of the migrations and morphogenetic movements, which give rise to the adult structures. Finally, the detailed description of the LIM-hd code in the developing and adult Xenopus forebrain provides interesting cues for the possible mechanisms of evolution of the vertebrate forebrain.

Animals↗
PubMed1997

A mutation that affects fibril protein, development, cohesion and gene expression in Myxococcus xanthus.

Extracellular matrix fibrils are involved in the cell-cell interactions of the social prokaryote, Myxococcus xanthus. The fibrils are composed of a carbohydrate backbone and a set of five integral fibrillar proteins (IFPs) ranging from 14 to 66 kDa. As part of an attempt to understand the function(s) of the IFPs, a mutant (ifp-1:20) was generated that lacks IFP-1:20, one of the fibril proteins, as shown by Western blot analysis of both whole cells and isolated fibrils. Unlike those of the parent strain, the fibrils of the mutant were removed easily from the cells by shear forces. Development in ifp-1:20 was aberrant--aggregation and early mound formation were delayed by 6-10 h and mature fruiting bodies never formed. Myxospore production was also greatly reduced. Additionally, fibril-mediated cohesion in ifp-1:20 was changed. Cohesion resulted in chains of cells rather than the characteristic clumps of cells seen for the parent strain. Isolated ifp-1:20 fibrils, unlike wild-type fibrils, could not rescue cohesion of non-cohesive, fibril-negative dsp cells, supporting the notion that the fibrils were functionally altered. The mutation also reduced developmental gene expression by three- to fourfold in omega 4521, a transposon insertion mutant expressed early in development. Expression of a later developmental gene fusion was not affected, suggesting that the fibrils may not be required for later developmental gene expression. These data suggest that intact fibrils may function early in development to facilitate close cell proximity for signal exchange.

Bacterial Adhesion↗
PubMed1984

Effects of the muscular dysgenesis gene on developmental stability in the mouse mandible.

Muscular dysgenesis (mdg) is an autosomal recessive gene in mice affecting primarily the skeletal musculature. mdg/mdg mice exhibit developmental arrest of myogenesis and degenerative changes in all skeletal muscles. In addition, there are pronounced abnormalities in skeletal traits, including the shape of the skull and mandible. Herein, we examine the phenotypic consequences of a single mdg allele in the heterozygous condition (+/mdg) on the size, shape, and developmental stability in 14 osteometric traits from the mouse mandible. Developmental stability in the mandible is measured by fluctuating asymmetry in bilateral traits. There are no statistically significant differences in the size or shape of the mandible between +/+ and +/mdg mice. However, compared to +/+ mice, +/mdg individuals exhibit less developmental stability for several mandible traits. The more unstable traits include height at the mandibular notch, height at the incisive process, condyloid width, height and area of the coronoid process, and size of the tooth-bearing region. All of these latter traits are closely associated with areas of muscle attachment and/or the muscular dysgenesis phenotype, suggesting that the presence of a single mdg allele is sufficient to alter developmental pathways. Traits not showing significantly increased instability in +/mdg mice bear no clear relationship to either muscle attachment areas or to the mdg/mdg phenotype.

Abnormalities, Multiple↗
PubMed1990

P-element-mediated enhancer detection allows rapid identification of developmentally regulated genes and cell specific markers in Drosophila.

We have employed a new technique in Drosophila that allows in vivo detection of genomic regulatory elements using a beta-galactosidase reporter gene. A translational fusion of the reporter gene to the P-transposase gene, which is encoded by the P-transposon of Drosophila, places the expression of beta-galactosidase under the control of the weak P-transposase promoter. Flies carrying single insertions of this P-element construct at different locations in the Drosophila genome frequently stain for beta-galactosidase activity in a temporally and spatially restricted fashion in embryos, larvae and adult ovaries, reflecting the influence of nearby genomic regulatory elements on the P-transposase promoter. This technique is a powerful tool as it can be used to produce very many different cell markers and to isolate developmentally regulated genes in Drosophila. We discuss the implications of our results and the applications of the technique to further the study of Drosophila development.

Animals↗
PubMed2000

The development and application of molecular markers for abiotic stress tolerance in barley.

This article represents some current thinking and objectives in the use of molecular markers to abiotic stress tolerance. Barley has been chosen for study as it is an important crop species, as well as a model for genetic and physiological studies. It is an important crop and, because of its well-studied genetics and physiology, is an excellent candidate in which to devise more efficient breeding methods. Abiotic stress work on cultivated gene pools of small grain cereals frequently shows that adaptive and developmental genes are strongly associated with responses. Developmental genes have strong pleiotropic effects on a number of performance traits, not just abiotic stresses. One concern is that much of the genetic variation for improving abiotic stress tolerance has been lost during domestication, selection and modern breeding, leaving pleiotropic effects of the selected genes for development and adaptation. Such genes are critical in matching cultivars to their target agronomic environment, and since there is little leverage in changing these, other sources of variation may be required. In barley, and many other crops, greater variation to abiotic stresses exists in primitive landraces and related wild species gene pools. Wild barley, Hordeum spontaneum C. Koch is the progenitor of cultivated barley, Hordeum vulgare L. and is easily hybridized to H. vulgare. Genetic fingerprinting of H. spontaneum has revealed genetic marker associations with site-of-origin ecogeographic factors and also experimentally imposed stresses. Genotypes and collection sites have been identified which show the desired variation for particular stresses. Doubled haploid and other segregating populations, including landrace derivatives have been used to map genetically the loci involved. These data can be used in molecular breeding approaches to improve the drought tolerance of barley. One strategy involves screening for genetic markers and physiological traits for drought tolerance, and the associated problem of drought relief-induced mildew susceptibility in naturally droughted fields of North Africa.

Adaptation, Physiological↗