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At least 973 records · Page 54Linked to original sources

PubMed2003

Comparing protein stabilities during zebrafish embryogenesis.

The stabilities of many key proteins are regulated, e.g. via ubiquitination and proteasomal degradation, with important biological consequences. We present a convenient method that allows the analysis and comparison of protein stabilities during embryogenesis using early zebrafish development as a model system. Basically, this method involves ectopic overexpression of epitope-tagged proteins via mRNA injections in one-to-four-cell stage embryos and subsequent protein detection after various time points. Indeed, the protein stability of the ubiquitin ligase RLIM, which is able to autoubiquitinate and target itself for proteasomal degradation, was much shorter when compared to a protein consisting of a Myc epitope-tag and a nuclear localization domain. Thus, this method may be used more widely for the study of developmental protein stability.

Animals↗

PubMed2001

Effects of light quality on somatic embryogenesis in Araujia sericifera.

The effects of photoperiod, light quality and end-of-day (EOD) phytochrome photoconversion on somatic embryogenesis (SE) of Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 8- and 16-h photoperiods using Gro-lux fluorescent lamps. The photon fluence rate was 90-100 µmol m-2 s-1 and the red (R):far-red (FR) ratio was 98. R, FR, R followed by FR (R-FR) and FR followed by R (FR-R) light treatments were applied for 3 weeks at the end of the photoperiods. In a set of experiments, DL-alpha-difluoromethylarginine (DFMA) or methylglyoxal bis(guanylhydrazone) (MGBG), both inhibitors of polyamine biosynthesis, were added to the culture medium in order to study the involvement of polyamine metabolism. The level of SE was the same in long (LD) and short (SD) days. Thus, the light effect was accomplished after 8 h. All EOD treatments that decreased the Pfr level inhibited SE when applied after SD, but not after LD. The FR-R treatment after LD caused an additional stimulatory effect on SE, even in the presence of polyamine inhibitors. DFMA inhibited SE in both SD and LD, but MGBG did not modify SE in either SD or LD. The R, FR and R-FR treatments did not alter the level of SE when applied after LD in the presence of DFMA or MGBG. However, these treatments decreased SE after SD when the medium contained polyamine inhibitors. Our results suggest that Gro-lux lamps, which produce an extremely high R:FR ratio, promote SE in A. sericifera and a timing response to phytochrome photoconversion during photoperiodic induction. Thus, our data corroborate the involvement of phytochromes and polyamines in SE in A. sericifera, which responded as a light-dominant long-day plant.

Journal Article↗

PubMed2001

Effect of the tetrapeptide RGDS on somatic embryogenesis in Daucus carota.

The present work examines the requirement for RGD-binding sites, such as those in the plasma membrane protein integrin during somatic embryogenesis in carrot (Daucus carota L. cv. Scarlet nantes). It is possible to assay for this requirement by competitively inhibiting binding of the site to the natural RGD-containing ligand by adding an excess of synthetic RGDS. We found that treatment of carrot callus cultures with RGDS (2.7 mM) inhibited the development of a normal shoot pole in carrot somatic embryos. The structures that formed contained separate zones of highly cytoplasmic and vacuolate cells and no evidence of embryonic organization, although occasionally a root-like structure was observed. If the aspartic acid residue in the peptide was replaced by a chemically similar amino acid (glutamic acid), the resultant somatic embryos were indistinguishable from those developing in untreated cultures. These effects are similar to those reported in animal systems where the protein receptor involved has been identified as integrin. Our results are thus consistent with the conclusion that a binding site for a motif similar to the integrin-binding site in a variety of animal extracellular matrix proteins exists in plants and appears to be important in somatic embryo development in carrot.

Journal Article↗

PubMed1988

Two-tiered regulation of spatially patterned engrailed gene expression during Drosophila embryogenesis.

A regulatory cascade, initiated during the syncytial stage of embryogenesis, culminates in the striped pattern of engrailed gene expression at the cellular blastoderm stage. The early regulatory genes, for example the pair-rule genes, are expressed transiently and as their products decay a distinct regulatory programme involving segment polarity genes takes over. This late programme maintains and perhaps modifies the striped pattern of engrailed expression through interactions that may involve cell communication.

Animals↗

PubMed2001

macho-1 encodes a localized mRNA in ascidian eggs that specifies muscle fate during embryogenesis.

Maternal information stored in particular regions of the egg cytoplasm has an important function in the determination of developmental fate during early animal development. Ascidians show mosaic development; such autonomous development has been taken as evidence that prelocalized ooplasmic factors specify tissue precursor cells during embryogenesis. Interest has been concentrated on the mechanisms underlying the formation of muscle cells in the tail, as yellow-coloured myoplasm in eggs is preferentially segregated into muscle-lineage blastomeres. Here we show that maternal messenger RNA of the macho-1 gene is a determinant of muscle fate in the ascidian Halocynthia roretzi. The macho-1 mRNA encodes a zinc-finger protein, and the mRNA is localized to the myoplasm of eggs. Depletion of the mRNA specifically resulted in the loss of primary muscle cells in the tail, as shown by the expression of muscle-specific molecular markers. The myoplasm of macho-1-deficient eggs lost its ability to promote muscle formation. Injection of synthesized macho-1 mRNA caused ectopic muscle formation in non-muscle-lineage cells. Our results indicate that macho-1 maybe both required and sufficient for specification of muscle fate, and that the mRNA is a genuine, localized muscle determinant.

Amino Acid Sequence↗

PubMed1994

Ventralizing signal determined by protease activation in Drosophila embryogenesis.

Specification of dorsal-ventral cell fate during Drosophila embryogenesis is mediated by a signal transduction pathway. Asymmetry of cell fates arises through the spatially restricted production of a ligand in an extracellular compartment called the perivitelline space. The snake and easter genes are required for the production of the ligand and they encode the proenzyme form of secreted extracellular serine proteases. We have examined the effect of producing a preactivated form of the snake protease on the generation of dorsal-ventral polarity. SP6 RNA microinjection experiments reveal that different cell fates acquired at cellular blastoderm can be specified by the amount and spatial distribution of activated snake protein. Our results support a protease cascade model in which localized activation of uniformly distributed protease proenzymes leads to the spatially restricted production of ligand in the perivitelline space on the ventral side of the embryo.

Animals↗

PubMed1997

Functional expression of opioid receptor-like receptor and its endogenous specific agonist nociceptin/orphanin FQ during mouse embryogenesis.

Expression of opioid receptor-like receptor (ORL1) and its endogenous peptide agonist nociceptin/orphanin FQ (N/OFQ) during mouse embryogenesis have been investigated. Transcripts of ORL1 and N/OFQ were detected by RT-PCR in mouse brain of day 8 embryo (E8) and the expression continued afterwards. Northern blot analysis revealed abundant expression of ORL1 at postnatal day 1 (P1) and N/OFQ at E17 and P1 in the brain but none was detected in other embryonic tissues. The presence of functional ORL1 in mouse embryonic brain was also confirmed by specific binding of [3H] N/OFQ (kd = 1.3 +/- 0.5 nM and Bmax = 72 +/- 9 fmol/mg protein) as well as by N/OFQ-stimulated G protein activation.

Animals↗

PubMed1996

Expression of the recombinase-activating gene (RAG-1) in murine early embryogenesis.

The recombinase activation genes, RAG-1 and RAG-2, are expressed together in immature T or B lymphocytes and possess activity to induce V(D)J rearrangement in T cell receptor (TCR) and Ig genes. In vertebrates, only Ig and TCR molecules are reported to have recombination in their development using multiple V, D, J component gene segments. Thus, expression of RAG genes are localized only in lymphoid organs and sites of extrathymic T cell differentiation. In this study, we have used RAG-1 and RAG-2 genes as markers of possible genetic recombination in developing murine preimplantation embryos, using the highly sensitive reverse transcriptase polymerase chain reaction (RT-PCR) technique and in situ hybridization. From 40 preimplantation embryos of various developmental stages we extracted RNA, reverse-transcribed it into cDNA and used it in RT-PCR studies. A PCR of 35 cycles disclosed expression of RAG-1 but not RAG-2 in morulae and blastocysts. Southern blot hybridization using a specific synthetic oligonucleotide probe for RAG-1 and RT-PCR with another primer pair identified RAG-1 expression in developing embryos. In situ hybridization using a cooled CCD camera also revealed localization of RAG-1 mRNA in blastocysts. We propose possible genetic recombination during late preimplantation murine embryogenesis which may contribute to the loss of totipotency and differentiation of inner cell mass and trophoectoderm.

Animals↗

PubMed1997

Platelet-derived growth factor: a key regulator of connective tissue cells in embryogenesis and pathogenesis.

Studies of PDGF-A, PDGF-B and PDGF receptor-beta knock-out mice have revealed critical functions for the PDGF-PDGF receptor signaling systems in the ontogeny of connective tissue cells: the mesangial cells of kidney glomeruli and the alveolar smooth muscle cells (SMC) of the lung. The phenotypes of the PDGF mutant mice have also shed light on the identity and functions of these cell types, as well as revealed analogies suggesting that common morphogenetic principles have evolved for use in different organs, involving related growth factors and cell types. Although the lethality of PDGF knock-out mice has not allowed an investigation of the role of PDGF in SMC of the vessel wall, regulation of PDGF and its receptors in adult vessels following injury is consistent with a role for PDGF in the fibroproliferative response in the intima that occurs as part of the pathogenesis of atherosclerosis. PDGF modulation of connective tissue synthesis may thus be critical to connective tissue phenotype and proliferation in both embryogenesis and pathogenesis.

Animals↗

PubMed2006

Vascular defects and liver damage by the acute inactivation of the VHL gene during mouse embryogenesis.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene leads to the development of central nervous system hemangioblastomas, pheochromocytomas and renal cell carcinomas. The biological role of the VHL gene during development is poorly understood because of early lethality of VHL-null embryos. To overcome early embryo lethality observed in the conventional knockout mouse, we introduced a tamoxifen-inducible Cre (CreER(TM)) transgene for the stage specific inactivation of the VHL gene. Acute tamoxifen-induced inactivation of the VHL gene at E10.5 resulted in embryonic lethality between E14.5 and E15.0 with extensive hemorrhage and necrosis, while littermate controls showed normal development. Examination of the VHL-inactivated embryos between E10.5 and E14.5 revealed dilated blood vessels, hemorrhage and necrotizing liver damage. Concomitant with severe hemorrhage and abnormal vasculature at E15.0, blood circulation in the yolk sac was impaired in the VHL-inactivated embryos, which may be the cause of embryo death. Placental development looked normal before embryo death (E14.5); however, at E16.5 following embryo death, we observed reduced growth of the placental labyrinthine layer. Inactivation of the VHL gene resulted in hypoxia-inducible factor (HIF)-1alpha stabilization and induction of its target genes, VEGF and CAIX, in mouse embryonic fibroblasts (MEFs). In addition, we observed lactate overproduction and acidification of culture media by the inactivation of the VHL gene. Thus, by using a novel conditional VHL knockout mouse model, we could show that the VHL gene plays an important role in the developing vasculature and liver during embryogenesis through regulation of HIF-1alpha and its target genes. This mouse model will be useful for the screening of anti-HIF or anti-VEGF drugs in vivo. Additionally, this acute VHL inactivation system may provide a useful tool for the in vivo study of genes that cause early embryonic lethality.

Animals↗

PubMed2000

E-selectin expression and stimulation by inflammatory mediators are developmentally regulated during embryogenesis.

Leukocyte recruitment during inflammation is specified, in part, by the spatial distribution and temporal regulation of endothelial adhesion molecules. In this study we investigated the developmental onset of E-selectin and intercellular adhesion molecule-1 (ICAM-1) basal expression and inducibility by inflammatory mediators as indices of lineage-restricted endothelial adhesion molecule expression. We studied both murine embryos and embryoid bodies (EB), derived from differentiated embryonic stem cells, to examine a broad range of endothelial ontogeny. Our results reveal that E-selectin and ICAM-1 are differentially regulated during development and that three stages define the ontogeny of the E-selectin-inducible response. The earliest endothelial lineage cells in Day 4 and Day 5 EB did not express E-selectin in the basal state or after stimulation. A second stage, observed between embryonic Day 9.5 (E9.5) and E11.5 to E12.5 in cultured embryo cells and transiently at Day 6 of EB differentiation, was characterized by basal expression that was not stimulated by inflammatory mediators. A third stage was characterized by both basal and inducible expression of E-selectin and was observed beginning at E12.5 to E13.5 in cultured embryo cells and at Day 7 in EB. In contrast ICAM-1 was stimulated at all of the embryonic stages examined and before the onset of E-selectin inducibility in both embryos and EB. E-selectin expression in embryos was also stimulated by introducing endotoxin into the embryonic, but not the maternal, peritoneum. This suggests that embryos are protected from inflammatory insults present in the maternal circulation. The developmentally regulated acquisition of E-selectin inducibility during embryogenesis likely involves changes in signal transduction cascades, transcription factors, and/or chromatin accessibility that specify inducible expression within the endothelial lineage and further restrict inducibility to particular endothelial subpopulations.

Animals↗

PubMed2003

Caenorhabditis elegans early embryogenesis and vulval morphogenesis require chondroitin biosynthesis.

Defects in glycosaminoglycan biosynthesis disrupt animal development and can cause human disease. So far much of the focus on glycosaminoglycans has been on heparan sulphate. Mutations in eight squashed vulva (sqv) genes in Caenorhabditis elegans cause defects in cytokinesis during embryogenesis and in vulval morphogenesis during postembryonic development. Seven of the eight sqv genes have been shown to control the biosynthesis of the glycosaminoglycans chondroitin and heparan sulphate. Here we present the molecular identification and characterization of the eighth gene, sqv-5. This gene encodes a bifunctional glycosyltransferase that is probably localized to the Golgi apparatus and is responsible for the biosynthesis of chondroitin but not heparan sulphate. Our findings show that chondroitin is crucial for both cytokinesis and morphogenesis during C. elegans development.

Alleles↗

PubMed2006

PGC7/Stella protects against DNA demethylation in early embryogenesis.

DNA methylation is an important means of epigenetic gene regulation and must be carefully controlled as a prerequisite for normal early embryogenesis. Although global demethylation occurs soon after fertilization, it is not evenly distributed throughout the genome. Genomic imprinting and epigenetic asymmetry between parental genomes, that is, delayed demethylation of the maternal genome after fertilization, are clear examples of the functional importance of DNA methylation. Here, we show that PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. After determining that PGC7/Stella binds to Ran binding protein 5 (RanBP5; a nuclear transport shuttle protein), mutant versions of the two proteins were used to examine exactly when and where PGC7/Stella functions within the cell. It is likely that PGC7/Stella protects the maternal genome from demethylation only after localizing to the nucleus, where it maintains the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogramming after fertilization.

Animals↗

PubMed2005

Genetic interaction between DNA polymerase beta and DNA-PKcs in embryogenesis and neurogenesis.

DNA polymerase beta (Polbeta) has been implicated in base excision repair. Polbeta knockout mice exhibit apoptosis in postmitotic neuronal cells and die at birth. Also, mice deficient in nonhomologous end-joining (NHEJ), a major pathway for DNA double-strand break repair, cause massive neuronal apoptosis. Severe combined immunodeficiency (SCID) mice have a mutation in the gene encoding DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the component of NHEJ, and exhibit defective lymphogenesis. To study the interaction between Polbeta and DNA-PKcs, we generated mice doubly deficient in Polbeta and DNA-PKcs. Polbeta(-/-)DNA-PKcs(scid/scid) embryos displayed greater developmental delay, more extensive neuronal apoptosis, and earlier lethality than Polbeta(-/-) and DNA-PKcs(scid/scid) embryos. Furthermore, to study the involvement of p53 in the phenotype, we generated Polbeta(-/-)DNA-PKcs(scid/scid)p53(-/-) triple-mutant mice. The mutants did not exhibit apoptosis but were lethal with defective neurulation at midgestation. These results suggest a genetic interaction between Polbeta and DNA-PKcs in embryogenesis and neurogenesis.

Animals↗

PubMed2005

EndoG is dispensable in embryogenesis and apoptosis.

The mitochondrial protein, endonuclease G (EndoG), is one of the endonucleases implicated in DNA fragmentation during apoptosis. It has been shown to translocate from the mitochondria to the nucleus upon cell death stimuli. These observations suggest that EndoG is a mitochondrial cell death effector, and that it possibly acts as a cell death nuclease, similar to DNA fragmentation factor. To better understand the role of EndoG in development and apoptosis, we generated EndoG null mice by homologous gene targeting without disruption of D2Wsu81e. EndoG null mice are viable and develop to adulthood with no obvious abnormalities. Fibroblasts generated from the EndoG null mice show no difference in susceptibility when induced to die by a variety of intrinsic and extrinsic apoptotic stimuli. Additionally, EndoG null mice are equally sensitive to excitotoxic stress. These data suggest that EndoG is not essential for early embryogenesis and apoptosis.

Animals↗

PubMed2001

Transgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system.

After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.

Anti-Bacterial Agents↗

PubMed2001

Auxin distribution and transport during embryogenesis and seed germination of Arabidopsis.

Auxin distribution during embryogenesis and seed germination were studied with transgenic Arabidopsis plants expressing GUS gene driven by a synthetic DR5 promoter, an auxin responsive promoter. The results showed that GUS activity is higher in ends of hypophysis and cotyledon primordia of heart-, torpedo- and cotyledon-stage embryos, leaf tip area, lateral root primordia, root apex and cotyledon of young seedlings. And GUS accumulated in root apex of the seedlings grown on auxin transport inhibitor containing media. All these suggested that above-mentioned part of the organs and tissues have a higher level of auxin, and auxin polar transport inhibitor could cause the accumulation of auxin in root apex. And auxin transport inhibitor also resulted in aberration of Arabidopsis leaf pattern formation, root gravitropism and elongation.

Arabidopsis↗

PubMed2005

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation.

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.

Animals↗