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Analysis of carbohydrate metabolism enzymes and cellular contents of sugars and proteins during spruce somatic embryogenesis suggests a regulatory role of exogenous sucrose in embryo development.

Carbohydrate metabolism was investigated during spruce somatic embryogenesis. During the period of maintenance corresponding to the active phase of embryogenic tissue growth, activities of soluble acid invertase and alkaline invertase increased together with cellular glucose and fructose levels. During the same time, sucrose phosphate synthase (SPS) activity increased while sucrose synthase (SuSy) activity stayed constant together with the cellular sucrose level. Therefore, during maintenance, invertases were thought to generate the hexoses necessary for embryogenic tissue growth while SuSy and SPS would allow cellular sucrose to be kept at a constant level. During maturation on sucrose-containing medium, SuSy and SPS activities stayed constant whereas invertase activities were high during the early stage of maturation before declining markedly from the second to the fifth week. This decrease of invertase activities resulted in a decreased hexose:sucrose ratio accompanied by starch and protein deposition. Additionally, carbohydrate metabolism was strongly modified when sucrose in the maturation medium was replaced by equimolar concentrations of glucose and fructose. Essentially, during the first 2 weeks, invertase activities were low in tissues growing on hexose-containing medium while cellular glucose and fructose levels increased. During the same period, SuSy activity increased while the SPS activity stayed constant together with the cellular sucrose level. This metabolism reorganization on hexose-containing medium affected cellular protein and starch levels resulting in a decrease of embryo number and quality. These results provide new knowledge on carbohydrate metabolism during spruce somatic embryogenesis and suggest a regulatory role of exogenous sucrose in embryo development.

Carbohydrate Metabolism↗

Origin of alimentary tract in embryogenesis of Ixodes ricinus (Acari: Ixodidae).

We studied the embryos of Ixodes ricinus (L.) in the second and third trimester of embryonic development, by using light and transmission electron microscopy. At the beginning of the second trimester, the formation of the foregut and rectal sac, by a process of invagination, was observed. The invagination, which develops into the primordium of the hindgut, forms only in the third trimester. The rectum forms in the last phase of embryogenesis. The development of the midgut is incomplete during embryogenesis. The yolk is surrounded by a wall, formed of an amorphous basal lamina and flattened cells, that gradually accumulate deutoplasmic material. These cells do not acquire the typical features of the gut epithelium until after larval hatching. These features are, however, found in the cells forming the rectal sac.

Animals↗

Effect of low carbohydrate diets during pregnancy on embryogenesis and fetal growth and development in rats.

Effects in pregnant rats of feeding diets specifically deficient in carbohydrate were studied. The dietary nonprotein energy source was lipid, provided as intact fat (soybean oil) or a fatty acid mixture (edible oleic acid) or a combination of these. These diets provided 9.5% casein protein, which was shown to be minimally adequate in both the lipid-based experimental diets and the high carbohydrate control diet. The diets were fed from mating through d 21 of pregnancy, and pups were delivered by cesarean section. The soybean oil-based zero-carbohydrate diet supported embryogenesis and produced at term normal numbers of normal appearing pups of body weight lower than that of pups from the high carbohydrate control diet. In contrast, the oleic acid-based zero-carbohydrate diet failed to maintain pregnancy, indicating a requirement for carbohydrate or intact fat or both. To maintain pregnancy to term required both 5-10% intact fat and 4% carbohydrate as glucose or its equivalent amount of glycerol from lipid. From feeding graded levels of glucose in fatty acid based diets containing 5-10% intact fat as soybean oil, the carbohydrate requirement was found to be 6-8% glucose to sustain maternal food intake and weight gain and to produce normal fetal weight at term, and 12% glucose to provide approximately half the fetal liver glycogen levels in controls fed a high carbohydrate diet. These experiments have produced the first evidence of the quantitative requirement for carbohydrate for embryogenesis and fetal growth and development in the pregnant rat dam.

Animals↗

Expression of the viviparous 1 (Pavp1) and p34cdc2 protein kinase (cdc2Pa) genes during somatic embryogenesis in Norway spruce (Picea abies [L.] Karst).

Detailed expression analysis of the Norway spruce (Picea abies [L.] Karst) Viviparous 1 (Pavp1) and p34cdc2 (cdc2Pa) genes was carried out during somatic embryogenesis. Pavp1, a gene associated with embryo development, was expressed in proliferating embryogenic suspension cultures in the absence of exogenous ABA. When somatic embryo formation was promoting by blocking proliferation, Pavp1 expression was reduced. During maturation, exogenous ABA induced increased Pavp1 expression, which peaked at the early cotyledonary stage of somatic embryogenesis. Following partial desiccation of mature somatic embryos at high relative humidity, Pavp1 expression persisted under germination conditions. Pavp1 expression was also detected in non-dormant immature male strobili and dormant terminal buds. These data confirm the functional conservation of Pavp1 during the evolution of seed plants and extend its function beyond the embryo. Cdc2Pa, a gene associated with the cell cycle, was up-regulated when the proliferation of embryogenic cells was blocked. Expression was again up-regulated in early embryogeny and again during germination. The implications of this up-regulation of cdc2Pa are discussed.

Abscisic Acid↗

Analysis of mRNAs under translational control during Xenopus embryogenesis: isolation of new ribosomal protein clones.

We have analyzed several randomly selected mRNAs, of the relatively abundant category, on the basis of maternal or zygotic origin and translational efficiency at different developmental stages. For this purpose, clones from a Xenopus embryo cDNA library were hybridized with cDNA probes prepared with poly(A)+RNA from polysomes and from mRNPs of embryos at different stages. The results obtained indicate that the majority of the relatively abundant mRNAs (38 out of 61) is subject to some kind of translational regulation during embryogenesis. Moreover, 30 clones have been selected as corresponding to mRNAs that behave, from the point of view of transcriptional and translational regulation, similarly to previously studied ribosomal protein (r-protein) mRNAs. Sequence analysis of 20 of these selected cDNAs has shown that half of them are in fact homologous to already sequenced r-protein mRNAs. Unexpectedly we have found that also the mRNA for alpha-cardiac actin and another mRNA homologous to creatine kinase M mRNA have a similar translational regulation during embryogenesis.

Actins↗

Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus.

The orthodenticle-related protein (HpOtx) gene derived from the sea urchin Hemicentrotus pulcherrimus encodes two distinct isoforms, HpOtxE and HpOtxL, which are differentially expressed during early embryogenesis and are driven by TATA-less and TATA-containing promoters, respectively. In order to determine if the TATA element is involved in the establishment of the temporally specific expression profile of the HpOtx gene, reporter genes under the control of modified or wild-type HpOtxE/L promoters were introduced into fertilized eggs. When the activities of the different promoter constructs were examined, we found that deletion of the TATA element from the HpOtxL promoter causes early expression, whereas addition of the TATA element to the HpOtxE promoter causes delayed expression. This suppressive action of the TATA element on transcription from the HpOtxE/L promoters requires the presence of upstream CACGTG elements. These results indicate that the presence or absence of the TATA element determines, at least in part, the expression profile of the HpOtxE/L promoters, in concert with the transcription factor(s) that binds to the upstream CACGTG element. Immunoblot and gel retardation analyses suggest that functional interaction between CACGTG binding factor(s) and TATA factor(s) may be regulated by an unidentified third factor(s) during early embryogenesis in the sea urchin.

Amino Acid Sequence↗

Stage-specific inhibition of Xenopus embryogenesis by aprotinin, a serine protease inhibitor.

We examined the effects of various protease inhibitors on Xenopus laevis embryogenesis. Aprotinin, a serine protease inhibitor, was found to inhibit embryogenesis markedly, but other protease inhibitors had virtually no effect. The inhibitory effect of aprotinin was specific for embryos at the blastula or gastrula stage. These results suggest that an aprotinin-sensitive protease involved in embryonic development is secreted from the embryos or appears on the surface of embryonic cells at these stages. We found that various serine proteases are in fact secreted from the embryos with their development and that some of them are sensitive to aprotinin.

Amino Acid Sequence↗

Development of myokinase mRNA during embryogenesis of the chick.

During chick embryogenesis, the mRNA for myokinase, as determined by in vitro translation, appears on the 16th day. The mRNA levels do not change drastically in succeeding stages and increase immediately after hatching. In contrast, myokinase activity and its protein gradually increase from the 16th day. Thus, myokinase mRNA that is translatable in vitro seems to accumulate transiently in the late stage of embryogenesis. In sucrose density gradient centrifugation, the mRNA sedimented as one peak with a coefficient of 10S.

Adenylate Kinase↗

One type of chalcone synthase gene expressed during embryogenesis regulates the flavonoid accumulation in citrus cell cultures.

To elucidate the relationship between the expression of chalcone synthase (CHS) genes and the production of flavonoid in citrus cell cultures, two cDNA clones encoding CHS were isolated (CitCHS1 and CitCHS2) from the citrus. The accumulation of CitCHS2 mRNA was notably induced by embryogenesis but CitCHS1 mRNA was not. There was no detectable accumulation of flavonoid in the undifferentiated calli, but flavonoid accumulated after the morphological changes to embryoids. These results indicate that two CHS genes differentially expressed during citrus somatic embryogenesis and CitCHS2 may regulate the accumulation of flavonoid in citrus cell cultures.

Acyltransferases↗

Purification from conditioned medium and chemical identification of a factor that inhibits somatic embryogenesis in carrot.

Somatic embryogenesis is strongly inhibited in cultures of carrot (Daucus carota L.) cells when the cell density is high. The inhibition is caused by factors that are released by cells into the medium of such cultures. In this study, we purified and identified one of the inhibitory factors found in the medium of high-cell-density cultures of carrot cells. The inhibitory factor with the strongest apparent activity was purified by fractionation with ethylacetate, chromatography on an octadecylsilyl (ODS) silica gel-column and HPLC. The inhibitory factor had a single peak of absorbance at 280 nm and was identified as 4-hydroxybenzyl alcohol by mass spectrometry and 1H- and 13C-NMR spectroscopy. Authentic 4-hydroxybenzyl alcohol strongly inhibited the formation of somatic embryos at a concentration equal to that in high-cell-density cultures. These results suggest that 4-hydroxybenzyl alcohol is a major factor that accumulates in high-cell-density cultures of carrot cells and inhibits somatic embryogenesis.

Benzyl Alcohols↗

Rice globular embryo 4 (gle4) mutant is defective in radial pattern formation during embryogenesis.

In higher plants, the main elements of the fundamental body plan, the apical-basal and radial patterns, are established during embryogenesis. We have isolated several globular embryo (gle) mutants of rice that fail to develop any embryonic organs. We expected that these gle mutants might include mutants defective in their radial pattern formation ability. We developed two markers specifically staining the L2 and L3 layers (OsSCR and OsPNH1, respectively) and characterized the gle mutants by using these markers in addition to the already developed markers Roc1 (marker for the L1 layer), Ramy1A (marker of the L1 layer of the epithelium), and OSH1 (marker of the apical region). One of the gle mutants, gle4, expressed Roc1 and Ramy1A at the normal positions, but other markers exhibited an abnormal expression pattern; that is, both OsPNH1 and OsSCR were expressed in the central region of the embryo and OSH1 expression was not observed. Calli from the gle4 epithelium regenerated plants with abnormal morphologies. These results indicate that the GLE4 gene is involved in radial pattern formation during rice embryogenesis to differentiate the L2 and L3 layers, but is not involved in the establishment of the L1 layer or in the formation of embryonic organs.

Genetic Markers↗

Sequential exposure to cytokines reflecting embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells.

Differentiation of adult bone marrow stem cells (BMSC) into hepatocyte-like cells is commonly performed by continuous exposure to a cytokines-cocktail. Here, it is shown that the differentiation efficacy in vitro can be considerably enhanced by sequential addition of liver-specific factors (fibroblast growth factor-4, hepatocyte growth factor, insulin-transferrin-sodium selenite, and dexamethasone) in a time-dependent order that closely resembles the secretion pattern during in vivo liver embryogenesis. Quantitative RT-PCR analysis and immunocytochemistry showed that, upon sequential exposure to liver-specific factors, different stages of hepatocyte differentiation, as seen during liver embryogenesis, can be mimicked. Indeed, expression of the early hepatocyte markers alpha-fetoprotein and hepatocyte nuclear factor (HNF)3beta decreased as differentiation progressed, whereas levels of the late liver-specific markers albumin (ALB), cytokeratin (CK)18, and HNF1alpha were gradually upregulated. In contrast, cocktail treatment did not significantly alter the expression pattern of the hepatic markers. Moreover, sequentially exposed cells featured highly differentiated hepatic functions, including ALB secretion, glycogen storage, urea production, and inducible cytochrome P450-dependent activity, far more efficiently compared to the cocktail condition. In conclusion, sequential induction of the differentiation process, analogous to in vivo liver development, is crucial for in vitro differentiation of adult rat BMSC into functional hepatocyte-like cells. This model may not only be applicable for in vitro studies of endoderm differentiation but it also provides a "virtually unlimited" source of functional hepatocytes, suitable for preclinical pharmacological research and testing, and cell and organ development.

Animals↗

Early selection improves clonal performance and reduces intraclonal variation of Norway spruce plants propagated by somatic embryogenesis.

Height growth during the first and second growth periods (i.e., the June-September period in consecutive years) and intraclonal variation were assessed in 13 Norway spruce (Picea abies (L.) Karst.) clones propagated by somatic embryogenesis. The plants were acclimatized and grown in a greenhouse until mid-July and then transferred outdoors. The clonal mean heights after the first and second growth periods were lower for somatic embryo plants than for seedlings from corresponding families sown at the time of somatic embryo plant ex vitro transfer, because a large proportion of somatic embryo plants were small. We determined whether certain selection criteria at ex vitro transfer can be used to identify somatic embryo plants with height growth characteristics comparable with those of seedlings. Epicotyl length and presence of lateral roots proved to be important parameters for selection, whereas main root length was less useful. A combined selection for somatic embryo plants with lateral roots and with an epicotyl length exceeding 8 mm resulted in taller plants and reduced intraclonal variation after the first and second growth periods. The growth of somatic embryo plants selected in this way was similar to that of seedlings from the corresponding families. We conclude that selection according to these criteria at ex vitro transfer can result in improved performance of clonal stock propagated by somatic embryogenesis.

Cloning, Organism↗

High stability of nuclear microsatellite loci during the early stages of somatic embryogenesis in Norway spruce.

Somatic embryos of Norway spruce (Picea abies (L.) Karst.) differentiate from proembryogenic masses (PEMs), which are subject to autodestruction through programmed cell death. In PEMs, somatic embryo formation and activation of programmed cell death are interrelated processes. We sought to determine if activation of programmed cell death in PEMs is caused by genetic aberrations during somatic embryogenesis. Based on the finding that withdrawal of auxin and cytokinin induces programmed cell death in PEMs, 1-week-old cell suspensions were cultured in medium either with or without auxin and cytokinin and then transferred to maturation medium containing abscisic acid. We analyzed the stability of three nuclear simple sequence repeat (SSR) microsatellite markers at successive stages of somatic embryogenesis in two cell lines. There were no mutations at the SSR loci at any of the successive developmental stages from PEMs to cotyledonary embryos, irrespective of whether or not the proliferation medium in which cell suspensions had been cultured contained auxin or cytokinin. The morphologies of plants regenerated from the cultures were similar, although withdrawal of auxin and cytokinin significantly stimulated the yield of both embryos and plants. We conclude, therefore, that the high genetic stability of somatic embryos in Norway spruce is unaffected by the induction of programmed cell death caused by withdrawal of auxin and cytokinin.

Cell Line↗

Developmental expression of 2489 gene clusters during pig embryogenesis: an expressed sequence tag project.

Identification of mRNAs that are present at early stages of embryogenesis is critical for a better understanding of development. To this end, cDNA libraries were constructed from germinal vesicle-stage oocytes, in vivo-produced four-cell- and blastocyst-stage embryos, and from in vitro-produced four-cell- and blastocyst-stage embryos. Randomly picked clones (10 848) were sequenced from the 3' end and those of sufficient quality (8066, 74%) were clustered into groups of sequence similarity (>95% identity), resulting in 2489 clusters. The sequence of the longest representative expressed sequence tag (EST) of each cluster was compared with GenBank and TIGR. Scores below 200 were considered unique, and 1114 (44.8%) did not have a match in either database. Sequencing from the 5' end yielded 12 of 37 useful annotations, suggesting that one third of the 1114 might be identifiable, still leaving over 700 unique ESTs. Virtual Northerns compared between the stages identified numerous genes where expression appears to change from the germinal vesicle oocyte to the four-cell stage, from the four-cell to blastocyst stage, and between in vitro- and in vivo-derived four-cell- and blastocyst-stage embryos. This is the first large-scale sequencing project on early pig embryogenesis and has resulted in the discovery of a large number of genes as well as possible stage-specific expression. Because many of these ESTs appear to not be in the public databases, their addition will be useful for transcriptional profiling experiments conducted on early pig embryos.

Animals↗

Effects of a 1.5-Tesla static magnetic field on spermatogenesis and embryogenesis in mice.

RATIONALE AND OBJECTIVES: There is a trend toward the use of higher magnetic field strengths in magnetic resonance imaging procedures. Considering this trend and the lack of consensus on the biologic effects of static magnetic fields, it is of considerable interest to examine the biologic effects of a 1.5-tesla (T) static magnetic field on spermatogenesis and embryogenesis in mice. METHODS: Male and pregnant female Swiss Webster mice were exposed to a 1.5-T static magnetic field for 30 minutes. Effects on spermatogenesis in male mice were investigated by counting testicular spermheads and epididymal spermhead shape-abnormalities as a function of time after exposure. Pregnant female mice were exposed to the field at the two-cell embryo stage, sacrificed immediately, and the ability of these preimplantation embryos to mature into blastocysts was examined in vitro. RESULTS: Exposure to the static 1.5-T magnetic field caused a statistically significant reduction (15%) in testicular sperm on the 16th and 29th days after exposure. However, the increase in spermhead shape abnormalities above normal control values was minimal. A substantial effect was noted on the development of preimplantation embryos with a survival fraction of 0.56 compared with controls. CONCLUSIONS: A 30-minute exposure to a 1.5-T static magnetic field appears to cause some deleterious effects on spermatogenesis and embryogenesis in mice.

Animals↗

Extradural endodermal cyst of posterior fossa: case report, review of the literature, and embryogenesis.

OBJECTIVE AND IMPORTANCE: Posterior fossa endodermal cysts are rare. They are located in the midline, in ventral or ventrolateral locations, or intrinsic to the neural axis. Accordingly, various theories of embryogenesis have been proposed. We report the first case of an extradural, dorsolaterally situated endodermal cyst. CLINICAL PRESENTATION: An adult male patient presented with a short history of headache and cerebellar ataxia. Neuroimaging revealed an extra-axial cystic posterior fossa mass. INTERVENTION: An entirely extradural cyst was found and was totally excised. Immunohistochemistry confirmed the diagnosis of endodermal cyst. CONCLUSION: The extradural, dorsal location of the endodermal cyst suggests gaps at the cranial end of the notochord causing ectodermal-endodermal adhesions during early gastrulation and the persistence of endodermal remnants in the dorsal mesenchyme of the blastemal cranium. The literature is reviewed, and proposed theories of embryogenesis are discussed.

Adult↗

Differences in DNA methylation during oogenesis and spermatogenesis and their persistence during early embryogenesis in the mouse.

We have examined the relative methylation levels of several dispersed repeated and low-copy-number gene sequences during gametogenesis and early embryogenesis. Southern blot analyses revealed that L1, intercisternal A particle (IAP), and major urinary protein (MUP) sequences were undermethylated extensively at MspI sites in DNA from diplotene oocytes. In contrast, the same sequences were highly methylated in DNA from pachytene spermatocytes, round spermatids, and epididymal sperm. These results indicate that there are genome-wide DNA methylation differences between oogenesis and spermatogenesis. Repeated sequences in DNA from cleavage-stage embryos and inner cell masses (ICM) were methylated at intermediate levels, consistent with transient maintenance of gametic methylation levels during early embryogenesis. Gametic differences in DNA methylation observed here indicate that methylation could provide a mechanism for imprinting maternal and paternal genomes resulting in differential regulation of parental genomes during early development.

Animals↗