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PubMed2000

Region-specific activation of the Xenopus brachyury promoter involves active repression in ectoderm and endoderm: a study using transgenic frog embryos.

Tissue specification in the early embryo requires the integration of spatial information at the promoters of developmentally important genes. Although several response elements for signalling pathways have been identified in Xenopus promoters, it is not yet understood what defines the sharp borders that restrict expression to a specific tissue. Here we use transgenic frog embryos to study the spatial and temporal regulation of the Xbra promoter. Deletion analysis and point mutations in putative transcription factor-binding sites identified two repressor modules, which exert their main effects at different stages during gastrulation. One module is defined by a bipartite binding site for a Smad-interacting protein (SIP1) of the deltaEF1 repressor family and acts to confine expression to the marginal zone early in gastrulation. The other module is defined by two homeodomain-binding sites and is responsible for repression in dorsal mesoderm and ectoderm at mid-gastrula stages. In addition, an upstream region of the promoter is necessary to repress expression in neural tissues later in development. Together, our results show that repression plays an important role in the restriction of Xbra expression to the mesoderm, and we suggest that similar mechanisms may be involved in the spatial regulation of other genes in early embryonic development.

Animals↗
PubMed2000

Characterization of blue-light and developmental regulation of the photolyase gene phr1 in Trichoderma harzianum.

Blue light and development regulate the expression of the phr1 gene of the filamentous fungus Trichoderma harzianum. The predicted product of phr1, the DNA repair enzyme photolyase, is likely to help protect Trichoderma, which grows in the soil as a mycoparasite or saprophyte, from damage upon emergence and exposure to ultraviolet-c. phr1 is transiently expressed in mycelium and conidiophores after illumination. phr1 mRNA also accumulates in conidiophores during development and spore maturation. As no other genes displaying rapid, direct light regulation have been described previously in this organism, we have characterized the fluence and time dependence of phr1 induction, and its relation to sporulation and photoreactivation. Induction is transient following a pulse, and, with slower decay, in continuous light. This implies that the photoreceptor, transducers or response are capable of adaptation. About two-fold more light is required to induce phr1 than conidiation, but this difference is modest, so both responses could use the same or similar chromophore. Adenosine 3':5'-cyclic monophosphate bypasses the requirement for light for sporulation, while atropine prevents sporulation even after photoinduction. Light regulation of phr1, however, is indifferent to both these effectors. Induction of photolyase expression behaves as a direct, rapid response to light, independent of the induction of sporulation. Indeed, illumination of mature spores increases their capacity for photoreactivation. Blue light seems to warn the organism against the harmful effects of short wave-lengths, inducing phr1 expression and sporulation by pathways that are, at least in part, distinct.

Deoxyribodipyrimidine Photo-Lyase↗
PubMed2002

Developmental expression of pIgR gene in sheep mammary gland and hormonal regulation.

Secretory IgA found in external secretions are constituted by polymeric IgA (pIgA) bound to the extra-cellular part of the polymeric immunoglobulin receptor (pIgR). The receptor mediates transcytosis of pIgA across epithelial cells. The aim of the present study was to analyse the evolution of pIgR expression in the sheep mammary gland during the development of the mammary gland and to analyse its hormonal regulation. Gene expression of the pIgR was analysed in sheep mammary gland during pregnancy and lactation. By Northern Blot analysis, we observed that low levels of pIgR mRNA are expressed until day 70 of pregnancy. Accumulation of pIgR mRNA started during the third part of pregnancy and intensified 3 d after parturition to reach highest levels during established lactation (day 70). In situ hybridization analysis was used to confirm the increase in pIgR gene expression per mammary epithelial cell. In order to examine the hormonal regulation of the pIgR expression, virgin ewes were hormonally treated. Treatment with oestradiol and progesterone increased pIgR mRNA levels slightly. Subsequent addition of gluocotricoids induced a significant accumulation of pIgR mRNA in the mammary gland of the treated animals. Immunohistochemical analysis was performed to verify that the increase of pIgR mRNA level was associated with enhancement of the pIgR protein in mammary cells. No increase of pIgR mRNA levels were observed if PRL secretion was blocked by bromocryptine injections throughout the hormonal procedure. In conclusion, the present experiments suggest that the enhancement of pIgR levels during lactation result from combined effects of both prolactin and glucocorticoids.

Animals↗
PubMed1996

Developmental regulation of myeloid gene expression and demethylation during ex vivo culture of peripheral blood progenitor cells.

Expression of tissue- and development-specific genes is coordinately regulated during maturation of hematopoietic precursor cells toward functional, end-stage peripheral blood (PB) cells. To study the expression and methylation of several myeloid-specific genes during in vitro differentiation of normal hematopoietic progenitor cells, we used a model of CD34+ selected PB progenitor cells (PBPCs). PBPCs from six patients with solid tumors were recruited by standard-dose chemotherapy and subsequent administration of recombinant granulocyte colony-stimulating factor (G-CSF). PBPCs were collected and CD34+ cells selected by immunoadsorption columns using a biotinylated anti-CD34 monoclonal antibody. Enriched cells contained between 78% and 90% (median, 84%) CD34+ cells as determined by fluorescence-activated cell sorting analysis. Cell preparations were cultured in the presence of interleukin-1 beta (IL-1 beta), IL-3, IL-6 and stem cell factor and with or without G-CSF for various time intervals up to 20 days. Genes for CD34 surface antigen, lysozyme (LZM) and myeloperoxidase (MPO) were examined by RNA and DNA analyses. A rapid and early downregulation of CD34 transcripts was observed, with concomitant, time-dependent upregulation of expression of both the LZM and MPO genes. These effects were enhanced in the presence of G-CSF. Analysis of the DNA methylation status at key sites within these genes showed a pattern of differentiation- and expression-associated demethylation of the LZM gene, which was also enhanced by G-CSF, and constitutive and unaltered demethylation at key regions of the CD34 and MPO genes. In conclusion, the genes for CD34, LZM, and MPO are regulated during in vitro culture of very immature PBPCs in the presence of stem cell factor, IL-1, IL-3, IL-6; their effects are enhanced by G-CSF.

Antigens, CD34↗
PubMed1998

Identification and cloning of a developmentally regulated Cryptosporidium parvum gene by differential mRNA display PCR.

To identify Cryptosporidium parvum genes expressed during intracellular development, differential mRNA display was used to detect differences in gene expression between mock-infected and C. parvum-infected human epithelial cells. A reproducible band present only in C. parvum-infected cells, ddHC-23, was isolated and cloned. Southern blot analysis demonstrated that ddHC-23 represented a C. parvum gene. RT-PCR revealed that HC-23 mRNA levels decreased from 6 to 12h post-infection (pi), were maximally expressed at 24h pi, and returned to low levels at 48 and 72h pi. Northern blot analysis determined that the approx. 3.6kb transcript is expressed by sporozoites prior to invasion of epithelial cells. Screening of a C. parvum genomic library with ddHC-23 isolated a genomic subclone which contained a 2790bp ORF, uninterrupted by introns. Sequence analysis indicated that the encoded protein, which displayed no similarity to any sequences in the public databases, contained a high proportion of polar amino acids, with the most abundant being Asp (17.3%), Ser (15.8%) and Gly (8.1%). Numerous potential sites for posttranslational modification were present including: casein kinase II and protein kinase C phosphorylation sites, N-myristolation sites and N-glycosylation sites. These findings demonstrate the usefulness of differential mRNA display for identifying developmentally regulated C. parvum genes within the background of genes expressed by the host cell. 1998 Elsevier Science B.V.

Amino Acid Sequence↗
PubMed1993

Conservation and change of the developmentally crucial fushi tarazu gene in Drosophila.

We have studied the evolutionary changes occurring in the noncoding regions around the developmentally important fushi tarazu (ftz) gene in a total of 11 species in the genus Drosophila. Previous molecular developmental studies have identified DNA elements both 3' and 5' to the coding region which are important in proper regulation of expression of the Drosophila melanogaster ftz gene. We show here that these same elements are the most evolutionarily conserved regions in the vicinity of the gene homologs. Parts of some control elements are more conserved than exonic sequences. Not only is there sequence conservation, but the relative position, orientation, and distances among the control elements remain conserved. One quite significant difference does exist between the two major subgenera studied, Sophophora and Drosophila: namely, an inversion of the ftz unit with respect to other genes in the Antennapedia complex, ANT-C. As a comparison, we applied similar analysis to a "housekeeping" gene-rosy (ry), or Xdh. In contrast, DNA sequences 5' to the ry coding region revealed little evolutionary conservation. These studies bear out the proposition that functionally important DNA sequences remain more conserved through evolutionary time than do less functionally important sequences. This proposition could be tested in the present case because we could predict a priori from the developmental studies which DNA regions should be most conserved.

Animals↗
PubMed1986

Developmental regulation of two genes encoding ribulose-bisphosphate carboxylase small subunit in pea and transgenic petunia plants: Phytochrome response and blue-light induction.

We describe the effects of light quality on the light-induced expression of two genes (rbcS-3A and rbcS-3C) encoding the ribulose-1,5-bisphosphate carboxylase small subunit of pea plants. These two genes exhibit distinctive photoresponses depending on the developmental stages of the leaves. In etiolated primary leaves, changes in the rbcS-3A and rbcS-3C transcript levels are regulated by phytochrome. In mature green leaves, on the other hand, the transcript levels are modulated by a putative blue-light photoreceptor working in concert with phytochrome. We transferred the two pea rbcS genes into the petunia genome and found that their phytochrome (rbcS-3A and -3C) and blue-light responses (rbcS-3C) are recapitulated in the appropriate developmental stages of the transgenic plants. Moreover, the transgenic plants express the pea rbcS genes in leaves at levels close to those in pea leaves. Our results show that 0.4 kilobases of the rbcS-3A and 2 kilobases of the rbcS-3C upstream regions are sufficient for these photoresponses.

Journal Article↗
PubMed2000

A novel gene family with a developmentally regulated expression in Xenopus laevis.

We have isolated a new maternal gene called 4G2. 4G2 cDNA encodes a predicted protein of 501 amino acids, and its apparent molecular mass of 61 kDa was determined by SDS-PAGE of 4G2 recombinant protein expressed in E. coli or in vitro translated in rabbit reticulocyte lysate. Amino acid analysis of 4G2 revealed the RGD and LDV motif with a potential cell attachment activity. The open reading frames (ORF) also contained a consensus bipartite nuclear localization signal (NLS). There were number of expressed tag sequences (ESTs) from Drosophila, zebrafish, chicken, mouse, and human origin that encode a high degree of identity to the predicted 4G2 protein, thereby suggesting that 4G2 may constitute a novel gene family whose function has not been elucidated. We also present evidence that 4G2 transcript is maternally synthesized in stage IV oocyte, localized to animal hemisphere of egg, and zygotically reactivated in mid-neurula stage.

Amino Acid Motifs↗
PubMed1993

Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.

We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth. The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide.

Amino Acid Sequence↗
PubMed1995

Repression of Pax-2 by WT1 during normal kidney development.

The developmental, regulatory gene Pax-2 is activated during early kidney morphogenesis and repressed in mature renal epithelium. Persistent Pax-2 expression is also observed in a variety of kidney tumors. Yet, little is known about the signals regulating this transient expression pattern in the developing kidney. We have examined the spatial and temporal expression patterns of Pax-2 and the Wilm's tumor suppresser protein WT1 with specific antibodies in developing mouse kidneys. A marked increase in WT1 protein levels coincided precisely with down-regulation of the Pax-2 gene in the individual precursor cells of the visceral glomerular epithelium, suggesting a direct effect of the WT1 repressor protein on Pax-2 regulatory elements. To examine whether WT1 could directly repress Pax-2 transcription, binding of WT1 to three high affinity sites in the 5' untranslated Pax-2 leader sequence was demonstrated by DNAseI footprinting analysis. Furthermore, co-transfection assays using CAT reporter constructs under the control of Pax-2 regulatory sequences demonstrated WT1-dependent transcriptional repression. These three WT1 binding sites were also able to repress transcription, in a WT1-dependent manner, when inserted between a heterologous promoter and the reporter gene. The data indicate that Pax-2 is a likely target gene for WT1 and suggest a direct link, at the level of transcriptional regulation, between a developmental control gene, active in undifferentiated and proliferating cells, and a known tumor suppressor gene.

Animals↗
PubMed2006

High glucose alters the expression of genes involved in proliferation and cell-fate specification of embryonic neural stem cells.

AIMS/HYPOTHESIS: Maternal diabetes induces neural tube defects during embryogenesis. Since the neural tube is derived from neural stem cells (NSCs), it is hypothesised that in diabetic pregnancy neural tube defects result from altered expression of developmental control genes, leading to abnormal proliferation and cell-fate choice of NSCs. MATERIALS AND METHODS: Cell viability, proliferation index and apoptosis of NSCs and differentiated cells from mice exposed to physiological or high glucose concentration medium were examined by a tetrazolium salt assay, 5-bromo-2'-deoxyuridine incorporation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and immunocytochemistry. Expression of developmental genes, including sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4), neurogenin 1/2 (Neurog1/2), achaete-scute complex-like 1 (Ascl1), oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte lineage transcription factor 2 (Olig2), hairy and enhancer of split 1/5 (Hes1/5) and delta-like 1 (Dll1), was analysed by real-time RT-PCR. Proliferation index and neuronal specification in the forebrain of embryos at embryonic day 11.5 were examined histologically. RESULTS: High glucose decreased the proliferation of NSCs and differentiated cells. The incidence of apoptosis was increased in NSCs treated with high glucose, but not in the differentiated cells. High glucose also accelerated neuronal and glial differentiation from NSCs. The decreased proliferation index and early differentiation of neurons were evident in the telencephalon of embryos derived from diabetic mice. Exposure to high glucose altered the mRNA expression levels of Shh, Bmp4, Neurog1/2, Ascl1, Hes1, Dll1 and Olig1 in NSCs and Shh, Dll1, Neurog1/2 and Hes5 in differentiated cells. CONCLUSIONS/INTERPRETATION: The changes in proliferation and differentiation of NSCs exposed to high glucose are associated with altered expression of genes that are involved in cell-cycle progression and cell-fate specification during neurulation. These changes may form the basis for the defective neural tube patterning observed in embryos of diabetic pregnancies.

Animals↗
PubMed2001

Expression of the developmental and oncogenic PAX2 gene in human prostate cancer.

PURPOSE: In the human prostate cancer cell lines LNCaP, DU145 and PC3, 27 primary prostate cancers, 10 benign prostatic hyperplasia specimens and 5 normal prostates we investigated the expression pattern of PAX2, a member of the PAX family of developmental control genes. PAX2 is expressed at high levels in developing undifferentiated cells of the urogenital system and is repressed upon terminal differentiation with no expression in normal adult cells. It is also been shown to be a proto-oncogene in mice and is expressed in human renal cell carcinoma. MATERIALS AND METHODS: PAX2 expression was assessed at the RNA level by reverse transcriptase-polymerase chain reaction and Southern blot analysis using specific sets of nucleotides. The expression pattern of PAX2 was reconfirmed at the protein level by immunofluorescence in the cell lines, and by Western blot analysis in primary human prostate cancers and benign prostatic tissue. RESULTS: Using reverse transcription-polymerase chain reaction combined with Southern hybridization PAX2 expression was detected in 52% of primary cancers and all 3 cell lines. PAX2 expression in these samples was confirmed at a protein level using immunoblotting and immunofluorescence. PAX2 messenger RNA was not detected in any benign or normal prostatic samples. Immunoblotting of protein from benign prostatic hyperplasia samples confirmed the lack of expression of PAX2 protein. CONCLUSIONS: The expression of PAX2 in prostate cancer compared to nonmalignant prostates is statistically significant (Fisher's exact test p = 0.0004). These results suggest a possible role for PAX2 in prostate cancer. Although previous studies have suggested a role for PAX2 for supporting proliferation in undifferentiated cells, no correlation of PAX2 expression with Gleason score was found in prostate cancer.

Blotting, Western↗
PubMed1994

Developmental expression of the genes encoding transforming growth factor alpha and its receptor in the hypothalamus of female rhesus macaques.

Studies in female rats have shown that transforming growth factor alpha (TGF alpha) stimulates release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual maturation, and that expression of the TGF alpha gene in the hypothalamus increases during both the initiation of normal puberty and after hypothalamic lesions that induce sexual precocity. Since blockade of epidermal growth factor receptors (EGFR), which mediate TGF alpha actions, delayed the normal timing of puberty, it was postulated that TGF alpha/EGFR contributes to the neuroendocrine process that underlies the initiation of normal female puberty. The present study was undertaken to examine the hypothesis that hypothalamic expression of the TGF alpha gene and its receptor changes in relation to the stage of sexual development in nonhuman primates, and to determine whether these changes are accompanied by corresponding alterations in LHRH gene expression. DNA fragments complementary to the coding regions of the rhesus monkey TGF alpha, EGFR and LHRH genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR), sequenced and used to prepare monkey-specific antisense RNA probes. A quantitative RT-PCR was developed in which the cloned sequences were utilized to prepare RNA standards for the quantitation of tissue mRNA levels. Both TGF alpha and EGFR mRNA levels in the medial basal hypothalamus and preoptic area of female monkeys were elevated during neonatal life (1 week to 6 months of age), when FSH secretion is also high, decreased during juvenile development (8-18 months of age), when secretion of both FSH and LH is low, and markedly increased during the expected time of puberty (30-36 months of age). No such changes were observed in either the cerebellum or the cerebral cortex, two brain regions irrelevant to neuroendocrine reproductive control. In contrast to the pronounced alterations in hypothalamic TGF alpha/EGFR gene expression observed during sexual development, LHRH mRNA levels did not vary significantly during this time. Hybridization histochemistry revealed the presence of both TGF alpha and EGFR mRNAs in cells scattered throughout the hypothalamus, but more predominantly in the median eminence, suprachiasmatic nuclei, optic chiasm and cells along the wall of the third ventricle. These results demonstrate that increases in TGF alpha and EGFR gene expression, specific to the neuroendocrine brain, occur during developmental phases in which gonadotropin output is also elevated--most noticeably at the time of puberty.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2002

Developmental events and shoot apical meristem gene expression patterns during shoot development in Arabidopsis thaliana.

Critical developmental and gene expression profiles were charted during the formation of shoots from root explants in Arabidopsis tissue culture. Shoot organogenesis is a two-step process involving pre-incubation on an auxin-rich callus induction medium (CIM) during which time root explants acquire competence to form shoots during subsequent incubation on a cytokinin-rich shoot induction medium (SIM). At a histological level, the organization of shoot apical meristems (SAMs) appears to occur during incubation on SIM about the time of shoot commitment, i.e. the transition from hormone-dependent to hormone-independent shoot development. Genes involved in SAM formation, such as SHOOTMERISTEMLESS (STM) and CLAVATA1 (CLV1), were upregulated at about the time of shoot commitment, while WUSCHEL (WUS) was upregulated somewhat earlier. Genes required for STM expression, such as CUP-SHAPED COTYLEDON 1 and 2 (CUC1 and 2) were upregulated prior to shoot commitment. Gene expression patterns were determined for two GFP enhancer trap lines with tissue-specific expression in the SAM, including one line reporting on CUC1 expression. CUC1 was generally expressed in callus tissue during early incubation on SIM, but later CUC1 was expressed more locally in presumptive sites of shoot formation. In contrast, the expression pattern of the enhancer trap lines during zygotic embryogenesis was more localized to the presumptive SAM even in early stages of embryogenesis.

Arabidopsis↗
PubMed1995

Exposure to lead-acetate modulates the developmental expression of myelin genes in the rat frontal lobe.

Postnatal exposure to high levels (4%) of lead (Pb) have been shown to disrupt myelin formation and result in abnormal conduction of nerve impulses, components necessary for information processing in the CNS. To investigate whether the pathological changes in myelin, due to Pb exposure, might be partially mediated by modulations of the expression of genes involved in CNS myelin, we have examined the developmental profiles of the proteolipid protein (PLP) and myelin basic protein (MBP), two major structural constituents of CNS myelin and 2',3'-cyclic nucleotide 3' phosphodiesterase (CNP), a non-structural enzyme associated with myelin formation. Rat pups were postnatally exposed, from birth to weaning, to moderate amounts of Pb (0.2%), in the drinking water of the dam, and their frontal cortices were assayed for changes in the expression profile of the above genes by Northern Analysis. On PND 20, Pb resulted in a dramatic stimulation of the mRNA levels of PLP and a small increase in MBP mRNA levels, but had no effect on the CNP message. These data suggest that moderate levels of Pb selectively interfere with the gene expression of structural proteins of CNS myelin and may thus influence the composition of myelin in this way.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed2000

Developmental regulation of eukaryotic gene loci: which cis-regulatory information is required?

It is becoming increasingly accepted that gene loci comprise an extensive cis-regulatory system that encodes different layers of regulatory information, all of which are necessary to achieve and maintain tissue-specific gene expression in ontogeny. To gain a detailed understanding of developmental processes, it is clearly necessary to unravel the molecular basis behind the different regulatory processes that control gene expression. This information is also of utmost importance for any practical application that uses gene transfer technology.

Animals↗
PubMed1993

The tyrosine phosphatase cdc25 selectively inhibits transcription of the Xenopus oocyte-type tRNAtyrC gene.

The Xenopus tyrosine tRNAtyrC (TyrC) genes are developmentally regulated. These multicopy genes are expressed in early oocytes and inactivated as oocytes reach maturity. As shown here, this developmental regulation can be reproduced in vitro in extracts of early and late stage oocytes: the TyrC gene is transcribed in early oocyte extracts but is virtually inactive in mature oocyte extracts. The inability to transcribe the TyrC gene is not due to the lack of functional pol III transcriptional components, since the somatic-type TyrD gene is fully active in mature oocyte extracts. Instead, the loss of TyrC transcription appears to be due to a change in the template specificity of transcription factor TFIIIC: addition of TFIIIC from immature extracts restores TyrC transcription in mature extracts. In mixtures of immature and mature extracts, the transcriptional activity of the TyrC gene is reduced. The presence of sodium vanadate, an inhibitor of tyrosine phosphatases, increases the level of TyrC transcription in the extract mixtures. Also, cdc25 phosphatase treatment of immature extracts causes a decrease in TyrC transcription which is reversed by addition of exogenous TFIIIC. These findings indicate that changes in phosphorylation state alters the template specificity of TFIIIC leading to the selective inactivation of oocyte type TyrC genes.

Animals↗