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PubMed1993

Identification of genomic regions required for DNA replication during Drosophila embryogenesis.

A collection of Drosophila deficiency stocks was examined by bromodeoxyuridine (BrdU) labeling of embryos to analyze the DNA replication patterns in late embryogenesis. This permitted us to screen 34% of the genome for genes that when absent in homozygous deficiencies affect the cell cycle or DNA replication. We found three genomic intervals that when deleted result in cessation of DNA replication in the embryo, 39D2-3;E2-F1, 51E and 75C5-7;F1. Embryos deleted for the 75C5-7;F1 region stop DNA replication at the time in embryogenesis when a G1 phase is added to the mitotic cell cycle and the larval tissues begin to become polytene. Thus, this interval may contain a gene controlling these cell cycle transitions. DNA replication arrests earlier in embryos homozygous for deletions for the other two regions. Analysis of the effects of deletions in the 39D2-3;E2-F1 region on DNA replication showed that the block to DNA replication correlates with deletion of the histone genes. We were able to identify a single, lethal complementation group in 51E, l(2)51Ec, that is responsible for the cessation of replication observed in this interval. Deficiencies that removed one of the Drosophila cdc2 genes and the cyclin A gene had no effect on replication during embryogenesis. Additionally, our analysis identified a gene, pimples, that is required for the proper completion of mitosis in the post-blastoderm divisions of the embryo.

Animals↗

PubMed1996

Expression of the von Hippel-Lindau disease tumour suppressor gene during human embryogenesis.

The von Hippel-Lindau (VHL) disease product is thought to down-regulate transcription by antagonizing elongin-enhanced transcriptional elongation. Germline VHL gene mutations predispose to the development of retinal, cerebellar and spinal haemangioblastomas, renal cell carcinoma and phaeochromocytoma. In addition, somatic Inactivation of the VHL gene is frequent in sporadic renal cell carcinoma and haemangioblastoma. Regulation of transcript elongation is an important control mechanism for gene expression and the VHL gene might modify the expression of proto-oncogenes and growth suppressor genes during embryogenesis. We therefore investigated the expression of VHL mRNA during human embryogenesis by in situ hybridization studies at 4, 6 and 10 weeks post conception. Although VHL mRNA was expressed in all three germ layers, strong expression was noted in the central nervous system, kidneys, testis and lung. Within the kidney, VHL mRNA was differentially expressed within renal tubules suggesting that the VHL gene product may have a specific role in kidney development. Two alternatively spliced VHL mRNAs characterized by inclusion (isoform I) or exclusion (isoform II) of exon 2 are transcribed in adult tissues. To investigate if the two isoforms are differentially expressed during embryogenesis, VHL mRNA was reverse transcribed from 13 fetal tissues (8-10 weeks gestation). The quantitative distribution of VHL mRNA within fetal tissues reflected that seen by in situ hybridization and the ratio of the two VHL isoforms was similar between tissues. Although the genes regulated by the VHL gene product have not yet been identified, our findings are compatible with the hypothesis that VHL-mediated control of transcriptional elongation may have a role in normal human development.

Adult↗

PubMed1992

Dormancy, embryogenesis, and hatching of eggs of Prosimulium kiotoense (Diptera: Simuliidae) under natural and experimental conditions.

The egg dormancy and embryogenesis of the univoltine black fly, Prosimulium kiotoense Shiraki, in southwestern Japan were examined under natural and laboratory conditions. Eggs laid in April remained dormant until August (approximately 14 wk after oviposition). Hatching commenced in early October (after 23 wk), when air and water temperatures decreased to 20 and 15 degrees C, respectively, and continued until mid-December (32 wk) in the field. Laboratory experiments showed that lower temperatures (10 and 14 degrees C) supported embryogenesis similar to that observed in the field. In contrast, higher temperatures (18 and 22 degrees C) retarded or completely suppressed embryogenesis after the termination of dormancy. However, completion of dormancy was accelerated when eggs were exposed experimentally to these higher temperatures for 3-12 wk, then returned to a constant low temperature (14 degrees C). Daylength did not affect the maintenance and termination of dormancy. Therefore, temperature played an important role differentially affecting completion of dormancy and postdormancy development. Variable lengths of developmental periods among egg batches under the same conditions indicated possible genetic control of dormancy intensity.

Animals↗

PubMed2005

Mature embryo axis-based high frequency somatic embryogenesis and plant regeneration from multiple cultivars of barley (Hordeum vulgare L.).

A highly reproducible regeneration system through somatic embryogenesis from the excised mature embryos (MEs) of dry seeds of a range of European barley cultivars was developed. By minimizing the germination of plated MEs, primary callus could be obtained with high frequency which permitted efficient embryogenesis and regeneration of a large number of green plants. Different approaches were tested to reduce or prevent normal germination: (i) the use of a well defined balance of maltose and 2,4-D in the induction medium, (ii) soaking of seeds in water containing 2,4-D solution, (iii) direct culture of excised embryonic axes, (iv) longitudinally bisected MEs giving two halves, and (v) complete removal of the elongated main shoot including any roots within a week of culture initiation. Culturing of bisected MEs and whole embryonic axes gave the best responses with respect to large amounts of callus combined with minimal germination. The incorporation of BAP at low levels in the medium was found to be most effective for embryogenesis and the maintenance of long-term morphogenic capacity (more than 11 months up to now). This procedure allows the complete regeneration of plants in 16-20 weeks, from the initial isolation of MEs through all the steps to the development of plants ready to be transferred to the soil. The protocol was first developed for cv. Golden Promise and successfully applied to commercial cultivars. All cultivars tested formed embryogenic callus, with overall rates ranging from 22-55% and an average number of green plants per embryogenic callus from 1.5 to 7.5 across the genotypes.

Germination↗

PubMed2005

Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective.

Embryogenesis in plants is a unique process in the sense that it can be initiated from a wide range of cells other than the zygote. Upon stress, microspores or young pollen grains can be switched from their normal pollen development towards an embryogenic pathway, a process called androgenesis. Androgenesis represents an important tool for research in plant genetics and breeding, since androgenic embryos can germinate into completely homozygous, double haploid plants. From a developmental point of view, androgenesis is a rewarding system for understanding the process of embryo formation from single, haploid microspores. Androgenic development can be divided into three main characteristic phases: acquisition of embryogenic potential, initiation of cell divisions, and pattern formation. The aim of this review is to provide an overview of the main cellular and molecular events that characterize these three commitment phases. Molecular approaches such as differential screening and cDNA array have been successfully employed in the characterization of the spatiotemporal changes in gene expression during androgenesis. These results suggest that the activation of key regulators of embryogenesis, such as the BABY BOOM transcription factor, is preceded by the stress-induced reprogramming of cellular metabolism. Reprogramming of cellular metabolism includes the repression of gene expression related to starch biosynthesis and the induction of proteolytic genes (e.g. components of the 26S proteasome, metalloprotease, cysteine, and aspartic proteases) and stress-related proteins (e.g. GST, HSP, BI-1, ADH). The combination of cell tracking systems with biochemical markers has allowed the key switches in the developmental pathway of microspores to be determined, as well as programmed cell death to be identified as a feature of successful androgenic embryo development. The mechanisms of androgenesis induction and embryo formation are discussed, in relation to other biological systems, in special zygotic and somatic embryogenesis.

Apoptosis↗

PubMed2003

Heat-shock proteins 70 kDa and 19 kDa are not required for induction of embryogenesis of Brassica napus L. cv. topas microspores.

It is currently accepted that 'stress' triggers induction of microspore embryogenesis, and for Brassica napus L. cv. Topas it is heat-shock. It has been postulated that the heat-shock proteins (HSPs) generated during heat stress have a central role in the induction mechanism. To test this hypothesis we developed a microspore induction procedure, using colchicine instead of heat treatment. The level of HSP70 increased significantly during and following the microspore heat treatment while sHSP19 expression was induced at the onset of heat-shock and declined after 8 h. In contrast, induction of embryogenesis with colchicine was not accompanied by elevation of HSP70 nor by induction of sHSP19, indicating that these HSPs are not required for induction of microspore embryogensis in this model system. These data refute the current hypothesis that HSPs have an essential role in triggering microspore embryogenesis.

Adaptation, Physiological↗

PubMed2005

Identification of a novel factor, vanillyl benzyl ether, which inhibits somatic embryogenesis of Japanese larch (Larix leptolepis Gordon).

In contrast to angiosperms, some gymnosperms form well-developed suspensors in somatic embryogenesis. This characteristic makes it easy to study suspensor biology. In cultures with high cell densities, somatic embryogenesis of Japanese larch, especially the suspensor development, is strongly inhibited due to factor(s) that are released by the cells into the culture medium. In this study, we purified and identified one of the inhibitory factors present in high-cell-density conditioned medium (HCM) of larch cells. The factor with the strongest inhibitory activity was purified by dialysis, extraction by ethyl acetate, octadecylsilyl (ODS) column chromatography and high-performance liquid chromatography (HPLC). The inhibitory factor was identified as vanillyl benzyl ether (VBE) by physicochemical analysis. This compound was first isolated from natural resources. Authentic VBE inhibited somatic embryo formation in Japanese larch, and the inhibitory effect in the suspensor was stronger than in the embryo proper. Furthermore, quantification of VBE by HPLC demonstrated that VBE accumulates at high concentrations in HCM. These results suggest that VBE is a novel negative regulator of somatic embryogenesis.

Benzaldehydes↗

PubMed2003

Tissue distribution of prolactin receptor mRNA during late stage embryogenesis of the chick.

Serum prolactin increases during late embryogenesis. In order to elucidate the function of prolactin at this period, tissue distribution of prolactin receptor mRNA was examined by RNase protection assay. The mRNA was detected strongly in the kidney, intestine, and allantoic membrane; weakly detected in the brain; but not detected in the liver. The expression levels of the prolactin receptor mRNA in the kidney, intestine, and allantoic membrane were retained at constant levels during later stages of embryogenesis (Days 17 and 19) and posthatch periods (2 and 28 d after hatching). These results suggest that prolactin is mainly involved in the osmoregulation during the later stage of embryogenesis and that the expression of prolactin receptor mRNA in the kidney, intestine, and allantoic membrane is regulated by a serum prolactin-independent manner.

Allantois↗

PubMed2004

Thermal manipulations during broiler chick embryogenesis: effects of timing and temperature.

This study aimed at elucidating the effects of thermal manipulation (TM) during different periods of embryogenesis on hatchability, body weight, and thermoregulation of Ross chicks at hatch, and on the chick's ability to cope with thermal challenge (TC) at 3 d of age. Control embryos were incubated at 37.8 degrees C and 56% RH, whereas the TM embryos were treated for 3 h at 39.5 (1) or 41.0 degrees C (2) and 65% RH, during early embryogenesis (EE), E8 to E10, and late embryogenesis (LE) at E16 to E18. At hatch, after feather drying, BW and body temperature (Tb) were measured, blood was drawn from the jugular vein to measure plasma concentrations of thyroxine (T4), triiodothyronine (T3), and corticosterone. These parameters were also measured in 3-d-old chicks during exposure to TC at 41 degrees C for 6 h. Hatchability was significantly higher in the LE1-treated group than in the other treatments. EE1- and LE1-treated embryos exhibited significantly lower Tb at hatch than the LE2 chicks. Significant hyperthermia was noted in the control chicks during 6 h of TC; it coincided with a higher plasma T3 concentration (P = 0.074). During TC, the LE1-treated chicks exhibited the lowest T3 to T4 ratio and a significantly reduced plasma corticosterone concentration. It can be concluded that TM at 39.5 degrees C for 3 h during E16 to E18 of incubation improved chick's thermotolerance acquisition and reduced the corticosterone level of chicks exposed to TC at the age of 3 d.

Adaptation, Physiological↗

PubMed2004

Variation in transcript abundance during somatic embryogenesis in gymnosperms.

Somatic embryogenesis of Norway spruce (Picea abies L.) is a versatile model system to study molecular mechanisms regulating embryo development because it proceeds through defined developmental stages corresponding to specific culture treatments. Normal embryonic development involves early differentiation of proembryogenic masses (PEMs) into somatic embryos, followed by early and late embryogeny leading to the formation of mature cotyledonary embryos. In some cell lines there is a developmental arrest at the PEM-somatic embryo transition. To learn more about the molecular mechanisms regulating embryogenesis, we compared the transcript profiles of two normal lines and one developmentally arrested line. Ribonucleic acid, extracted from these cell lines at successive developmental stages, was analyzed on DNA microarrays containing 2178 expressed sequence tags (ESTs) (corresponding to 2110 unique cDNAs) from loblolly pine (Pinus taeda L.). Hybridization between spruce and pine species on microarrays has been shown to be effective (van Zyl et al. 2002, Stasolla et al. 2003). In contrast to the developmentally arrested line, the early phases of normal embryo development are characterized by a precise pattern of gene expression, i.e., repression followed by induction. Comparison of transcript levels between successive stages of embryogenesis allowed us to identify several genes that showed unique expression responses during normal development. Several of these genes encode proteins involved in detoxification processes, methionine synthesis and utilization, and carbohydrate metabolism. The potential role of these genes in embryo development is discussed.

Cycadopsida↗

PubMed2000

Differential regulation of three thyroid hormone-responsive matrix metalloproteinase genes implicates distinct functions during frog embryogenesis.

Matrix metalloproteinases (MMPs) are a family of Zn(2+)-dependent extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix (ECM). They are expressed in developmental and pathological processes such as postlactation mammary gland involution and tumor metastasis. Relatively few studies have been carried out to investigate the function of MMPs during embryogenesis and postembryonic organ development. Using Xenopus development as a model system, we and others have previously isolated three MMP genes as thyroid hormone response genes. They have distinct temporal and organ-specific regulations during thyroid hormone-dependent metamorphosis. We demonstrate here that three MMPs-stromelysin-3 (ST3), collagenases-3 (Col3), and collagenases-4 (Col4)-also have distinct spatial and temporal expression profiles during embryogenesis. Consistent with earlier suggestions that ST3 is a direct thyroid hormone response gene whereas Col3 and Col4 are not, we show that precocious overexpression of thyroid hormone receptors in the presence of thyroid hormone lead to increased expression of ST3, but not Col3. Furthermore, our whole-mount in situ hybridizations reveal a tight but distinct association of individual MMPs with tissue remodeling in different regions of the animal during embryogenesis. These results suggest that ST3 is likely to play a role in ECM remodeling that facilitate apoptotic tissue remodeling or resorption, whereas Col3 and Col4 appear to participate in connective tissue degradation during development.

Animals↗

PubMed2006

Three-dimensional computed tomography pancreatography of an annular pancreas with special reference to embryogenesis.

OBJECTIVES: Several hypotheses have been proposed to explain the embryogenesis of an annular pancreas. Three-dimensional (3D) imaging of the annular pancreas may be useful to elucidate the embryogenesis. The aim of this case report is to clarify the pancreatic ducts in the annular pancreas by 3D computed tomography pancreatography (3D-CTP), stereographically. METHODS: Three-dimensional CTP under endoscopic retrograde pancreatography using a balloon catheter was performed with a helical CT scanner. Three-dimensional images of the pancreatic duct were obtained with simple threshold, shaped surface display. RESULTS: Wirsung duct that surrounds the ventral side of the descending portion of the duodenum and Santorini duct that surrounds the dorsal side of the descending portion of the duodenum were reconstructed by 3D-CTP, stereographically, and the presence of an annular pancreas was confirmed. These findings suggest that the formation of the annular pancreas in the current case may be attributable not only to abnormal development of the right lobe of the ventral pancreatic bud, but also to overgrowth of the dorsal pancreatic bud toward the ventral aspect of the duodenum or excessive rotation of the duodenum following fusion of the ventral bud to the dorsal bud. CONCLUSION: Three-dimensional CTP is expected to contribute usefully toward clarifying the embryogenesis of an annular pancreas.

Aged↗

PubMed1987

Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis.

Expression of the gene encoding atrial natriuretic factor (ANF) during mouse cardiac embryogenesis was studied using in situ hybridization to histologic sections. ANF mRNA was first detected in a subpopulation of myocardial cells at day 8 of embryogenesis. During day 9, abundant hybridization of the ANF probe to the atrium and the primitive ventricle was found. At day 14, strong labeling of cells in both atria and the trabeculated regions of the left ventricle was seen, whereas fewer cells in the right ventricle expressed the ANF gene. The developmental regulation of ANF gene expression in atrial and ventricular cells suggests an important role for this peptide hormone in the embryo. Localized expression of the ANF gene provides a marker for discerning subpopulations of cardiac cells during embryogenesis. Identification of embryonic ventricular ANF mRNA transcripts may have implications with respect to pathologic reexpression of the ANF gene in adult ventricles.

Animals↗

PubMed1994

Development of Desiccation Tolerance during Embryogenesis in Rice (Oryza sativa) and Wild Rice (Zizania palustris) (Dehydrin Expression, Abscisic Acid Content, and Sucrose Accumulation).

The ability of seeds to withstand desiccation develops during embryogenesis and differs considerably among species. Paddy rice (Oryza sativa L.) grains readily survive dehydration to as low as 2% water content, whereas North American wild rice (Zizania palustris var interior [Fasset] Dore) grains are not tolerant of water contents below 6% and are sensitive to drying and imbibition conditions. During embryogenesis, dehydrin proteins, abscisic acid (ABA), and saccharides are synthesized, and all have been implicated in the development of desiccation tolerance. We examined the accumulation patterns of dehydrin protein, ABA, and soluble saccharides (sucrose and oligosaccharides) of rice embryos and wild rice axes in relation to the development of desiccation tolerance during embryogenesis. Dehydrin protein was detected immunologically with an antibody raised against a conserved dehydrin amino acid sequence. Both rice and wild rice embryos accumulated a 21-kD dehydrin protein during development, and an immunologically related 38-kD protein accumulated similarly in rice. Dehydrin protein synthesis was detected before desiccation tolerance had developed in both rice embryos and wild rice axes. However, the major accumulation of dehydrin occurred after most seeds of both species had become desiccation tolerant. ABA accumulated in wild rice axes to about twice the amount present in rice embryos. There were no obvious relationships between ABA and the temporal expression patterns of dehydrin protein in either rice or wild rice. Wild rice axes accumulated about twice as much sucrose as rice embryos. Oligosaccharides were present at only about one-tenth of the maximum sucrose concentrations in both rice and wild rice. We conclude that the desiccation sensitivity displayed by wild rice grains is not due to an inability to synthesize dehydrin proteins, ABA, or soluble carbohydrates.

Journal Article↗

PubMed2006

Consistency of polyamine profiles and expression of arginine decarboxylase in mitosis during zygotic embryogenesis of Scots pine.

In this study, we show that both arginine decarboxylase (ADC) protein and mRNA transcript are present at different phases of mitosis in Scots pine (Pinus sylvestris) zygotic embryogenesis. We also examined the consistency of polyamine (PA) profiles with the effective temperature sum, the latter indicating the developmental stage of the embryos. PA metabolism was analyzed by fitting statistical regression models to the data of free and soluble conjugated PAs, to the enzyme activities of ADC and ornithine decarboxylase (ODC), as well as to the gene expression of ADC. According to the fitted models, PAs typically had the tendency to increase at the early stages but decrease at the late stages of embryogenesis. Only the free putrescine fraction remained stable during embryo development. The PA biosynthesis strongly preferred the ADC pathway. Both ADC gene expression and ADC enzyme activity were substantially higher than putative ODC gene expression or ODC enzyme activity, respectively. ADC gene expression and enzyme activity increased during embryogenesis, which suggests the involvement of transcriptional regulation in the expression of ADC. Both ADC mRNA and ADC protein localized in dividing cells of embryo meristems and more specifically within the mitotic spindle apparatus and close to the chromosomes, respectively. The results suggest the essential role of ADC in the mitosis of plant cells.

Carboxy-Lyases↗

PubMed1995

Differential Accumulation of Sunflower Tetraubiquitin mRNAs during Zygotic Embryogenesis and Developmental Regulation of Their Heat-Shock Response.

We have isolated and sequenced Ha UbiS, a cDNA for a dry-seed-stored mRNA that encodes tetraubiquitin. We have observed differential accumulation of tetraubiquitin mRNAs during sunflower (Helianthus annuus L.) zygotic embryogenesis. These mRNAs were up-regulated during late embryogenesis and reached higher prevalence in the dry seed, where they were found to be associated mainly with provascular tissue. UbiS mRNA, as confirmed by Rnase A protection experiments, accumulated also in response to heat shock, but only in leaves and later during postgerminative development. These novel observations demonstrate expression during seed maturation of specific plant polyubiquitin transcripts and developmental regulation of their heat-shock response. Using ubiquitin antibodies we also detected discrete, seed-specific proteins with distinct temporal expression patterns during zygotic embryogenesis. Some of these patterns were concurrent with UbiS mRNA accumulation in seeds. The most abundant ubiquitin-reacting proteins found in mature seeds were small (16-22 kD) and acidic (isoelectric points of 6.1-7.4). Possible functional implications for UbiS expression elicited from these observations are discussed.

Journal Article↗

PubMed2000

Calcium-mediated signaling during sandalwood somatic embryogenesis. Role for exogenous calcium as second messenger.

The possible involvement of Ca(2+)-mediated signaling in the induction/regulation of somatic embryogenesis from pro-embryogenic cells of sandalwood (Santalum album) has been investigated. (45)Ca(2+)-uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca(2+)](cyt) of pro-embryogenic cells in response to culture conditions conducive for embryo development. Sandalwood pro-embryogenic cell masses (PEMs) are obtained in the callus proliferation medium that contains the auxin 2,4-dichlorophenoxyacetic acid. Subculture of PEMs into the embryo differentiation medium, which lacks 2,4-dichlorophenoxyacetic acid and has higher osmoticum, results in a 4-fold higher (45)Ca(2+) incorporation into the symplast. Fura-2 ratiometric analysis corroboratively shows a 10- to 16-fold increase in the [Ca(2+)](cyt) of PEMs, increasing from a resting concentration of 30 to 50 nM to 650 to 800 nM. Chelation of exogenous Ca(2+) with ethyleneglycol-bis(aminoethyl ether)-N,N'-tetraacetic acid arrests such an elevation in [Ca(2+)](cyt). Exogenous Ca(2+) when chelated or deprived also arrests embryo development and inhibits the accumulation of a sandalwood Ca(2+)-dependent protein kinase. However, such culture conditions do not cause cell death as the PEMs continue to proliferate to form larger cell clumps. Culture treatment with N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide reduced embryogenic frequency by 85%, indicating that blockage of Ca(2+)-mediated signaling pathway(s) involving sandalwood Ca(2+)-dependent protein kinase and/or calmodulin causes the inhibition of embryogenesis. The observations presented are evidence to suggest a second messenger role for exogenous Ca(2+) during sandalwood somatic embryogenesis.

Calcimycin↗

PubMed1977

Effects of ethephon, ethylene, and 2,4-dichlorophenoxyacetic Acid on asexual embryogenesis in vitro.

Asexual embryogenesis in Daucus carota L. ;Queen Anne's Lace' callus was suppressed by Ethephon, ethylene, and 2,4-dichlorophenoxyacetic acid (2,4-D). The Ethephon effect could be attributed to volatile and nonvolatile substances. The volatile component was probably entirely ethylene. Ethylene was liberated in the cultures in direct proportion to Ethephon added to the medium. Autoclaving of Ethephon caused a substantial decrease of measurable ethylene. Continuous exposure of callus to 5 mul/l ethylene depressed somatic cell embryogenesis, but not markedly. Depression of embryogenesis by 2,4-D was unrelated to ethylene evolution.

Journal Article↗