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PubMed2005

[Changes in polyamine levels in Citrus sinensis Osb. cv. Valencia callus during somatic embryogenesis].

Somatic embryogenetic capability and changes in polyamine level and their relationship were analyzed using the long-term (8 years) subcultured calli of Citrus sinensis Osb. cv. Valencia as materials. The results showed that endogenous polyamine contents in embryogenic calli were higher than those in non-embryogenic calli, and the embryogenetic capability was positively correlated to the levels of endogenous polyamines. When the calli were transferred to a differentiation medium, the putrescine content rapidly increased and reached a peak, then fell gradually. Applying exogenous putrescine raised the embryogenesis frequency and endogenous putrescine level. It indicated that increase in putrescine content at early stage of differentiation promoted embryogenesis. With the development of somatic embryo, spermidine content reached its the highest level at globular embryo stage, spermine content rose and reached a peak at a later stage of globular embryo development. Furthermore, changes of the putrescine, spermidine and spermine contents during somatic embryogenesis were similar in Valencia calli which had different ploidy levels, but their contents decreased following the increasing of ploidy level. Changes in arginine decarboxylase activity were positively correlated to the polyamine levels, which suggest that the later is a key factor in regulating the polyamine levels during somatic embryogenesis in citrus plants.

Citrus sinensis↗

PubMed1991

Heat shock and thermotolerance in plant and animal embryogenesis.

Although the strategies of early embryogenesis differ greatly among multicellular eukaryotes, there are certain parallels in structure, form, and function that cross even kingdom lines: the extreme heat sensitivity of zygotes and very early embryos, followed by the acquisition of thermotolerance during subsequent development, is one such parallel. The heat sensitivity may be so extreme that even moderate increases in temperature result in lethality (generally associated with the earliest phases of embryogenesis), or the effects may be less severe, resulting in defects in development but not in lethality. Mechanistically, and molecularly, these two forms of thermosensitivity appear to have different origins. On the one hand, outright lethality appears to result from an inability to induce heat shock genes and proteins; on the other hand, heat-induced developmental defects appear to result from an alteration in expression of non-heat shock genes and from a delay in the overall developmental program that generally accompanies the cell's response to heat shock. This review is focused on the developmental regulation of the heat shock response during early embryogenesis and on the impact of this regulation on the development of both animal and plant embryos. The two basic issues that we address here are (i) the expression of heat shock genes in the absence of heat shock during embryogenesis and (ii) the expression (or lack of expression) of heat shock genes after deliberate exposure of the embryos to heat shock and the consequences of this expression on its subsequent survival and development.

Animals↗

PubMed1988

Changes in nucleus, nucleolus and cell size accompanying somatic embryogenesis of Theobroma cacao L. II. Relation between basic protein content and size of nucleus, nucleolus and cell.

Embryo formation from callus of Theobroma cacao L. was associated with the changes in relationship between nuclear, nucleolar and cell sizes and the content of basic proteins (FG-FCF-stained). Together with the increase in nuclear size of callus and proembryo cells the increase in the amount of nuclear basic proteins was found. In the callus cells the increase in nucleolar protein content exceeded that in nucleolus size, which led to the rise in basic protein concentration in the nucleolus. However, in the early stage of embryogenesis the increase in protein content was not so marked as that in callus, which indicated that embryogenesis involved a decrease in concentration of nucleolar basic proteins. Differences between callus and proembryo cells were also observed in the concentration of cytoplasmic proteins. The increase in size of callus cells was the same as the increasing amount of cytoplasmic proteins. In proembryos a significant increase in cell size was accompanied by only slight changes in cytoplasmic proteins. The stimulation of embryogenesis by 2,4-D resulted in an increase of nuclear concentration of basic proteins in proembryos. The intensification of embryogenesis involved the decrease of the concentration of nucleolar proteins together with the increase in concentration of basic cytoplasmic proteins.

Cacao↗

PubMed1989

The contents of proteins, carbohydrates, lipids and DNA during the embryogenesis of Drosophila.

The concentration of proteins, sugars, lipids and DNA has been determined during the embryogenesis of Drosophila. The protein content decreases after fertilization, being in the late embryo only 60% the value of the oocyte. The total sugar increases about 2.5-fold, from 3.5 h on until the end of embryogenesis. The lipids increase with a sharp peak at about 4 h and decrease during the rest of embryogenesis. DNA increases exponentially from the beginning of embryogenesis.

Animals↗

PubMed1987

Cardiac embryogenesis: a three-dimensional approach.

In order to aid students in the understanding of cardiac embryogenesis, seven three-dimensional fiberglass models depicting stages eleven to eighteen of cardiac embryogenesis, and one model of a mature heart were constructed. The details of these models, and a review of cardiac embryogenesis is presented. The results we have had with these models in the teaching of cardiac embryogenesis to medical students, cardiac technologists, and nurses have been encouraging, and this approach to teaching is highly recommended.

Heart↗

PubMed1985

Acquisition of insulin-dependent protein tyrosine kinase activity during Drosophila embryogenesis.

We have previously reported (Petruzzelli, L., Herrera, R., Garcia, R., and Rosen, O. M. (1985) Cancer Cells 3, 115-121) that adult Drosophila melanogaster contain a specific, high-affinity insulin-binding protein. Insulin-dependent protein tyrosine kinase activity has now been identified in Drosophila. Activity first appears at 6-12 h of embryogenesis, increases during the 12-18-h period and falls to low levels in the adult. 125I-insulin was cross-linked specifically and with high affinity to a protein (Mr = 135,000) throughout embryogenesis and in the adult. However, during the 6-12- and 12-18-h periods of embryogenesis when insulin-dependent protein tyrosine kinase activity is expressed, another protein (Mr = 100,000) becomes cross-linked to 125I-insulin. Crosslinking to both proteins was competitively inhibited by the addition of 100 nM insulin. We conclude that the insulin-binding and insulin-dependent protein tyrosine kinase activities of the mammalian insulin receptor are conserved in Drosophila. However, the insulin-dependent protein tyrosine kinase activity of the receptor is detected only during specific times in embryogenesis.

Amino Acids↗

PubMed1993

Developmental analysis of primary and secondary somatic embryogenesis in soybean tissue culture.

Somatic embryogenesis from immature soybean cotyledons was induced on Murashige & Skoog medium, supplemented with 2,4-dichlorophenoxyacetic acid. The full developmental analyses of primary and secondary somatic embryogenesis was monitored by scanning electron microscopy. The developmental stages of embryogenesis from the first unequal cell division through the three- and four-cell embryos, and multi-cell globular and heart shape embryos, to the fully developed plants were determined. When the globular stage embryos were incubated on the embryo maturation medium for 4 to 6 weeks a secondary embryogenesis was observed and scanned. Chimeric embryos with epidermis zones similar to mature leaf were characterized.

Culture Media↗

PubMed2026

CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and zygotic genome activation during goat embryogenesis.

Timely and efficient degradation of maternal mRNA is essential for early embryonic development, which occurs from fertilization through the initiation of zygotic genome activation (ZGA). Yet, the regulatory mechanisms governing this process remain poorly characterized. In the present study, we investigated the function of CCR4-NOT transcription complex subunit 1 (CNOT1) during goat embryogenesis. We found that CNOT1 was upregulated during mammalian ZGA, and that its knockdown led to developmental arrest and a marked reduction in blastocyst formation. Moreover, CNOT1 knockdown impaired nascent RNA activity, resulting in 814 upregulated and 1014 downregulated genes, which were enriched for RNA splicing, regulation of chromosome organization, and RNA localization. RNA splicing analysis revealed differential splicing events in 2959 genes, of which 259 were downregulated following CNOT1 knockdown. Notably, CNOT1 was predicted to crosstalk with the m6A reader YTHDF2. Knockdown of YTHDF2 resulted in CNOT1 downregulation at the 8-cell stage in goats and increased transcription levels around polyadenylation sites during ZGA in mice. Together, these findings indicate that CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and ZGA during goat embryogenesis. Our work provides new insight into the complex regulatory landscape underlying ZGA and may inform strategies to improve the efficiency of goat embryogenesis.

Animals↗

PubMed1997

Differential expression of galectin-1 and galectin-3 during first trimester human embryogenesis.

Development of complex organisms requires specific temporospatial differentiation and expression of the correct phenotype through activation of a variety of genes. Galectins are mammalian lectins able to interact with various extracellular matrix glycoconjugates and have been implicated in several biological events including cell attachment, differentiation, apoptosis, embryogenesis, and cancer invasion and metastasis. In this study, we have examined the expression of galectin-1 and galectin-3 during human first trimester embryogenesis using immunohistochemistry and Western blotting. Variable amounts of galectin-1 and galectin-3 were detected in all tissue protein extracts. Galectin-1 expression was demonstrated in the connective tissue and derived tissues such as smooth and striated muscle cells, and in some epithelia, such as in the basal layers of the skin after 14 weeks and in the epithelial cells of the gonads. Galectin-3 was detected mainly in epithelia, such as the skin, epithelial lining of the digestive and respiratory tract, and urothelium and excretory tubes of the kidney, but also in the myocardial cells, in the peripheral and preossifying hypertrophic chondrocytes, and in the notochord and in the liver. Our study constitutes the first demonstration of galectin-1 and galectin-3 during human embryogenesis. The differential expression of these two lectins suggests that they could participate in the complex processes of tissue differentiation.

Antigens, Differentiation↗

PubMed1998

Inhibition of hsp70-1 and hsp70-3 expression disrupts preimplantation embryogenesis and heightens embryo sensitivity to arsenic.

Mouse 70-kDa heat shock proteins Hsp70-1 and Hsp70-3 (Hsp70-1/3) are stress-inducible protein chaperones thought to protect embryonic cells and tissues from the effects of a wide range of environmental exposures. Hsp70-1/3 are expressed constitutively, and at times are stress-inducible during various stages of preimplantation embryogenesis. In order to elucidate the functions of constitutive and stress-inducible Hsp70 expression in mouse preimplantation embryos, the consequences of inhibiting expression with antisense oligonucleotides complementary to the mRNAs of hsp70-1 and hsp70-3 (A070-1/3) were evaluated. Transfection of preimplantation embryos (four-cell stage) with 2.5 microM A070-1/3 had no effect on in vitro blastocoel formation. However, transfection with 5 or 10 microM A070-1/3 reduced in vitro blastocyst development to 30% and 0%, respectively (approximately 90% control embryos developed to blastocyst). Thus constitutive expression of Hsp70-1/3 appears significant to preimplantation embryogenesis. Limiting expression of Hsp70-1/3 with 5 microM A070-1/3 also heightened embryo sensitivity to arsenic, resulting in less than 5% in vitro development to blastocyst in the presence of the subtoxic dose of 0.4 microM sodium arsenite. Whether the combined effect of A070-1/3 and arsenic is due to blocking inducible expression of the Hsp70s, or due to further reducing the amount of constitutively expressed Hsp70s available to the embryo is not known at this time. However, these results clearly indicate that some minimal amount of Hsp70-1 and/or Hsp70-3 is required for preimplantation embryogenesis, and that increasing the demand for Hsp70s by arsenic exposure heightens this requirement.

Animals↗

PubMed2002

A key developmental switch during Norway spruce somatic embryogenesis is induced by withdrawal of growth regulators and is associated with cell death and extracellular acidification.

The biotechnology of somatic embryogenesis holds considerable promise for clonal propagation and breeding programs in forestry. To efficiently regulate the whole process of plant regeneration through somatic embryogenesis, it is of outmost importance to understand early developmental events when somatic embryos are just formed. In Norway spruce, somatic embryos transdifferentiate from proembryogenic masses (PEMs). This work describes the developmental dynamics (frequency distribution of PEMs and early somatic embryos) of the whole embryogenic suspension culture growing in the presence and absence of plant growth regulators (PGRs), auxin and cytokinin. The experiments have shown that PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos and ultimately plant production. This switch was induced by the withdrawal of PGRs in cell suspension leading to a rapid accumulation of early somatic embryos (to a maximum of 75% of the entire population of suspension culture) and concomitant degradation of PEMs. The latter was evident from increased level of cell death measured through spectrophotometric Evans blue staining assay. Proembryogenic mass-to-embryo transition and concomitant activation of cell death were mediated by strong extracellular acidification. Therefore, buffering PGR-free culture medium at high (pH 5.8) or low (pH 4.5) levels of pH inhibited both PEM-to-embryo transition and cell death. The yield of mature somatic embryos on abscisic acid (ABA)-containing medium was increased up to 10-fold if the suspension culture had been pretreated for 1 to 9 days in unbuffered PGR-free medium. In this case a large proportion (75%) of the total number of mature embryos was formed within a short, 5-week, contact with ABA. The latter is practically important because prolonged contact with ABA suppresses the growth of somatic embryo plants. Based on these results, an improved method for regulating somatic embryogenesis was set up and tested for nine genotypes of Norway spruce. Over 800 plants regenerated from all tested genotypes demonstrated a good performance in the greenhouse and they were transferred to the field.

Apoptosis↗

PubMed1995

Morphogenetic reorganization of the brain during embryogenesis in the grasshopper.

We have studied the morphogenetic reorganization that occurs in the grasshopper brain during embryogenesis. We find that morphogenetic movements occur at three organizational levels during brain development. First, the entire developing brain changes its orientation with respect to the segmental chain of ventral ganglia. A 90 degrees shift in the attitude of the brain neuraxis occurs during embryogenesis due to a gradual upward movement of the cerebral structures in the head. Second, the clusters of proliferating neuroblasts and progeny that generate the neuroarchitecture of the mature brain move relative to one another and to nonneural structures such as the stomodeum. This is especially pronounced for the pars intercerebralis and for the tritocerebrum, as shown by annulin and engrailed immunoreactivity. Third, individual neuroblasts within a given proliferative cluster undergo positional reorganization during embryogenesis. Identified neuroblasts of the tritocerebrum and the pars intercerebralis are displaced within the brain. We conclude that the transformation of the simple sheet-like structure of the early embryonic brain into the highly differentiated structure of the mature brain involves a series of morphogenetic movements that occur in virtually all parts of the brain.

Animals↗

PubMed2002

Zebrafish yolk-specific not really started (nrs) gene is a vertebrate homolog of the Drosophila spinster gene and is essential for embryogenesis.

By using retroviral insertional mutagenesis in zebrafish, we have identified a recessive lethal mutation in the not really started (nrs) gene. The nrs mutation disrupts a gene located in linkage group 3 that is highly homologous to the spinster gene identified in Drosophila and to spinster orthologs identified in mammals. In flies, spinster encodes a membrane protein involved in lysosomal metabolism and programmed cell death in the central nervous system and in the ovary. In nrs mutant fish embryos, we detect an opaque substance in the posterior yolk cell extension at approximately 1 day after fertilization. This material progressively accumulates and by 48 hr after fertilization fills the entire yolk. By day 3 of embryogenesis, mutant embryos are severely reduced in size compared with their wild-type siblings and they die a few hours later. By in situ hybridization, we show that the nrs mRNA is expressed in the yolk cell at the time the mutant phenotype becomes apparent. In wild-type embryos, nrs message is present maternally and zygotically throughout embryogenesis and is also detected in adult animals. In nrs homozygous mutant embryos, nrs transcripts are undetectable at the time the phenotype becomes apparent, indicating that the retroviral insertion has most likely abolished expression of the nrs gene. Finally, the nrs phenotype can be partially rescued by microinjection of nrs encoding DNA. These results suggest that the nrs mutation affects an essential gene encoding a putative membrane-bound protein expressed specifically in the yolk cell during zebrafish embryogenesis.

Amino Acid Sequence↗

PubMed2002

Expression of the boc gene during murine embryogenesis.

BOC is a receptor-like protein that, with the related factor CDO, belongs to a newly recognized subfamily within the immunoglobulin superfamily of cell-surface molecules. BOC and CDO form complexes that positively regulate myogenesis in vitro. To gain a better understanding of the role of boc during vertebrate embryogenesis and whether it could cooperate with cdo in vivo, we carried out an extensive in situ hybridization analysis of boc expression in mouse embryos from E7.0 to E17.5. Our results show that boc is widely expressed during murine embryogenesis, in a spatially and temporally restricted pattern. Overall, the highest levels of boc expression are detected between E10.5 and E15.5, with the strongest signals observed in the developing musculoskeletal and central nervous systems. At late stages of development, boc expression becomes more restricted and is limited primarily to regions harboring proliferating cells, undifferentiated cells, or both. This expression pattern is strikingly similar to that described for cdo (Mulieri et al., 2000). The overlapping expression patterns of cdo and boc in vivo, combined with the promotion of myogenesis by CDO/BOC complexes in cultured cells, strongly suggests that these proteins play a role together in the determination, differentiation, or both, of numerous cell types during vertebrate embryogenesis.

Animals↗

PubMed2005

Expression of rck/p54, a DEAD-box RNA helicase, in gametogenesis and early embryogenesis of mice.

rck/p54 is a DEAD-box RNA helicase protein with ATP-dependent RNA-unwinding activity. Its ortholog is required for sexual reproduction in yeast and for oocyte survival and sperm fertility in Caenorhabditis elegans. In the current study, we investigated the expression of rck/p54 in mouse gametogenesis and early embryogenesis. Western blot analysis revealed that rck/p54 was highly expressed in both the ovary and testis. In the ovary, maturing oocytes strongly expressed rck/p54 in their cytoplasm. In contrast, in the testis, spermatogonia and primary spermatocytes highly expressed rck/p54 in their cytoplasm, but its expression decreased in the spermatids. Interestingly, rck/p54 was concentrated in the heads of spermatozoa; and then its expression gradually decreased as these cells matured along the epididymal duct. After fertilization, rck/p54 protein and its mRNA remained present in the pronucleus phase; and then their expression levels slightly but definitely decreased in morulae and blastocytes. The injection of a CMV-based rck/p54 expression vector into the pronuclei of fertilized eggs caused a delay in early embryogenesis. In generating RCK transgenic mice, the birth rate of the mice was significantly lower than those of other gene transgenic mice. These findings indicate that rck/p54 may play an important role in gametogenesis and early embryogenesis in mammals.

Animals↗

PubMed1990

Cellular and developmental biological aspects of bovine meiotic maturation, fertilization, and preimplantation embryogenesis in vitro.

Cellular aspects of reinitiated meiosis, fertilization, and early preimplantation embryogenesis in the bovine species were examined under in vitro conditions. An analysis of the cytoplasmic distribution of mitochondria, lipid droplets and vesicles in over 5,000 living GV-stage oocytes, with subsequent examination by electron microscopy, indicated that the organization of cytoplasm was pleomorphic and that five distinct cellular phenotypes could be identified. Inspection of oocytes during the resumption of arrested meiosis in vivo and in vitro demonstrated that the subcellular organization of the bovine oocyte cytoplasm remains unchanged during maturation to metaphase II. The influence of culture conditions and media on the frequency of maturation, cytoplasmic organization, fertilizability, and early preimplantation embryogenesis was also determined. The findings indicate that meiotic maturation and fertilization in the bovine species can occur at high frequency under comparatively simple and defined conditions. However, the acquisition of developmental competence for fertilization, and the ability of the egg to develop progressively after fertilization appears to be related to the organization of the cytoplasm at the GV stage. The relationship between cytoplasmic organization and conditions of maturation, fertilization, presence or absence of cumulus cells, and the acquisition of developmental competence is discussed with respect to 1) cell biological aspects of mammalian oocyte maturation, 2) the potential influence of extrinsic factors (e.g., differential intrafollicular biochemistry and morphophysiology) on subcellular organization of the GV-stage oocyte, and 3) the finding that morphologically equivalent bovine embryos derived from the in vitro fertilization of in vitro-matured oocytes may be developmentally heterogeneous. The studies also revealed that nuclear and cytoplasmic aberrations which could preclude normal embryogenesis can develop shortly after fertilization. The significance of this finding with respect to cytoplasmic phenotype of the oocyte and conditions of maturation, fertilization, and early embryo culture is discussed.

Animals↗

PubMed1988

Microtubule formation from maternal tubulins during sea urchin embryogenesis: measurement of soluble and insoluble tubulin pools.

The mass of tubulin protein in developing embryos of the sea urchin Lytechinus pictus was measured using a radiodilution immunoassay based on densitometric analysis of immunoprecipitated tubulins resolved electrophoretically. The tubulins constitute an average of 360 +/- 35 pg per egg, or 0.66% of the total protein, and there is no significant change in their concentration during embryogenesis. The masses of soluble and polymerized tubulin were measured for extracts prepared under conditions that stabilize microtubules. In eggs, a maximum of 14% of the tubulin is insoluble, and this increases throughout embryogenesis to 67% at pluteus stage (72 hr). The concentration of tubulin in eggs is at least 500 micrograms/ml, well above the critical concentration for tubulin assembly in vitro, yet microtubules have not been observed in eggs. The mass of newly synthesized tubulin, estimated from the mass of tubulin mRNA per embryo, accounts for a small fraction of the total tubulin by the end of gastrulation but for over half of the tubulin by the 72-hr pluteus stage. These observations are consistent with a model in which the declining level of unpolymerized tubulin controls the stability of tubulin mRNa, providing an autogenous regulation of the ontogenetic pattern of tubulin synthesis during sea urchin embryogenesis (Gong and Brandhorst, Development 102: 31-43).

Animals↗

PubMed1995

Alternative mRNAs encode multiple isoforms of transcription factor AP-2 during murine embryogenesis.

Transcription factor AP-2 has been implicated as an important regulator of gene expression during vertebrate embryogenesis. We report here the cDNA cloning and analysis of mouse embryonic mRNA splice variants encoding four AP-2 isoforms. Isoform 1 is the homolog of the previously known human (HeLa) AP-2. The three new AP-2 isoforms all share the same DNA binding/dimerization domain as isoform 1 but either lack the proline-rich transcriptional activation domain encoded by exon 2 (isoform 2) or have different amino-termini encoded by two previously unknown alternative first coding exons for AP-2 (isoforms 3 and 4). All four AP-2 mRNA variants are present at significant levels between Days 11.5 and 17.5 of mouse embryogenesis. Variants 1, 3, and 4 show qualitatively but not quantitatively similar restricted expression patterns in 8.5-12.5 dpc embryos examined by in situ hybridization. At mid-embryogenesis, variant 3 is the major AP-2 mRNA species in the nervous system and in total embryo RNA but is less prevalent than variants 1 and 4 in the epidermis. The four mRNAs are all induced, although unequally, during differentiation of P19 cells into neural cell types and by cAMP stimulation of primary astrocytes. Variants 1-3 are coexpressed in different ratios in HeLa cells and in three human glioblastoma cell lines. These findings reveal that transcriptional regulation by AP-2 is likely to be more complex than previously assumed given the potential for multiple AP-2 homo- and heterodimeric DNA binding forms.

Alternative Splicing↗