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PubMed1977

Embryogenesis of Xyleborus ferrugineus (Fabr.) (Coleoptera, Scolytidae). I. External morphogenesis of male and female embryos.

A description of the external morphogenesis of male and female embryos of X. ferrugineus, derived from in vivo observation, is presented here. The embryogenesis of this beetle is typical of the Coleoptera, and is also similar in most respects to the generalized insect plan. Observed unusual aspects of X. ferrugineus embryogenesis include the precocious formation of primordial g/rm cells and their temporary exclusion from somatic blastoderm; the precocious in situ delineation of gnathal metameres, the closing of the gastral groove beginning at both ends and proceeding towards its middle, the formation of several paired lateral amnio-serosal folds, the formation of cellular processes between the amnion and serosa, and the absorption of the thoracic limbs to produce an apodous larva. The embryonic developmental picture provided here, and the previously established means of rearing X. ferrugineus in controlled laboratory culture jointly provide a promising basis for the further use of this insect in developmental studies.

Age Factors↗

PubMed1977

Embryogenesis of Xyleborus ferrugineus (Fabr.). (Coleoptera, Scolytidae) II. Developmental rates of male and female embryos.

The developmental rates of male and female embryos of Xyleborus ferrugineus were compared by charting for each sex the mean age for each of ten discrete morphological stages of embryogenesis from pole cell exclusion to eclosion male and female embryos developed synchronously from stage 1 (which begins with pole cell exclusion) through stage 4 (which ends with the completion of germ band extension and metamerization). After stage 4 and throughout the remainder of embryogenesis, the mean ages per morphological stage of male embryos were significantly greater than those for female embryos. The expected physiological consequences of a haploid genome in the male embryo as compared to a diploid condition in the female embryo are discussed as the possible basis for the persistent lag observed in male developmental rate after stage 4.

Animals↗

PubMed2001

Germ cell specific promoter drives ectopic transgene expression during embryogenesis.

In this study, we used the male germ cell-specific phosphoglycerate kinase 2 (Pgk2) promoter to generate Pgk2Cre transgenic mice to allow investigation of genes critically involved in meiosis. The Pgk2 promoter had been used previously to target transgene expression to spermatocytes and spermatids in several laboratories including ours. In several Cre targeting experiments using other promoters, ectopic Cre expression had been observed, but the timing and extent of this expression was not analyzed. We demonstrate that in adult mice the Pgk2Cre transgene is expressed specifically in spermatocytes and spermatids, as expected. However, in offspring from matings of Pgk2Cre mice and an H19loxP indicator strain, we discovered that recombination events had occurred in several, but not all, tissues to varying extents. The lacZ-loxP transgenic indicator strain was next used to uncover ectopic Cre expression even in single cells, which indicated that the Pgk2Cre transgene is expressed between days 11 and 15 during embryogenesis in several tissues and organs. Using an RT PCR assay we were unable to detect endogenous Pgk2 mRNA during embryogenesis or in adult tissues other than testis. In conclusion, the Pgk2 promoter is a valid choice for targeting gene expression to meiotic male germ cells, since transient ectopic expression is unlikely to have a discernable effect in most studies, but it may be inappropriate for utilization with Cre recombinase.

Animals↗

PubMed1993

The expression of the H-19 and IGF-2 genes during human embryogenesis and placental development.

The H-19 gene in mice is maternally imprinted and its ectopic expression causes prenatal lethality. We have recently identified H-19 transcript in differentiating human placental cells and showed that its expression increases concomitantly with differentiation of cytotrophoblasts in vitro. Placental and embryonal specimens were collected from conception products derived from normal first and second trimester pregnancy terminations. We investigated the abundance of H-19 mRNA throughout placental development in vivo and compared it to the expression of other genes linked to placental differentiation. Furthermore, the expression of H-19 transcript in different organs of human fetuses, aborted during the second trimester, was examined by RNA isolation from separated fetal organs. Since IGF-2 is known to play an important role in embryogenesis, identical blots were hybridized with IGF-2 probe. H-19 expression in human placenta from the different trimesters of pregnancy remains practically constant. A high amount of H-19 gene product was found in the fetoplacental unit with the highest level measured in the adrenal gland. These findings argue that H-19 gene may play a role in human embryogenesis.

Adrenal Glands↗

PubMed2005

Proteome analysis of early somatic embryogenesis in Picea glauca.

Forestry is a valuable natural resource for many countries. Rapid production of large quantities of genetically improved and uniform seedlings for restocking harvested lands is a key component of sustainable forest management programs. Clonal propagation through somatic embryogenesis has the potential to meet this need in conifers and can offer the added benefit of ensuring consistent seedling quality. Although in commercial use, mass production of conifers through somatic embryogenesis is relatively new and there are numerous biological unknowns regarding this complex developmental pathway. To aid in unravelling the embryo developmental process, two-dimensional electrophoresis was employed to quantitatively assess the expression levels of proteins across four stages of somatic embryo maturation in white spruce (0, 7, 21 and 35 days post abscisic acid treatment). Forty-eight differentially expressed proteins have been identified, which display a significant change in abundance as early as day 7 of embryo development. These proteins are involved in a variety of cellular processes, many of which have not previously been associated with embryo development. The identification of these proteins was greatly assisted by the availability of a substantial expressed sequence tag (EST) resource developed for white, sitka and interior spruce. The combined use of these spruce ESTs in conjunction with GenBank accessions for other plants improved the rate of protein identification from 38% to 62% when compared with GenBank alone using automated, high-throughput techniques. This underscores the utility of EST resources in a proteomic study of any species for which a genome sequence is unavailable.

Cell Line↗

PubMed1993

Stage-specific effects of teratogens on sea urchin embryogenesis.

The effect of direct (chlorambucil and allopurinol) and indirect (cyclophosphamide) teratogens on the fertilization and early development of sea urchin embryos has been investigated. Fertilization was affected by none of the drugs tested. Continuous exposure of embryos to chlorambucil (10(-6) to 3 x 10(-4) M) starting after fertilization delayed the first cleavage and hatching. Developmental defects in chlorambucil-treated embryos consisted mainly of blastula and gastrula-arrested embryos and in a limited number (25%) of plutei with malformed gut or skeleton. Post-hatching exposure to chlorambucil led to malformed plutei only. Early (pre-hatching) exposure to allopurinol (10(-6) to 10(-3) M) did not affect cleavage but induced developmental defects in a ratio comparable to chlorambucil. Post-hatching exposure to allopurinol failed to affect the embryogenesis. The indirect teratogen cyclophosphamide (10(-6) to 3 x 10(-5) M) had no effect on the early embryogenesis. Results were discussed in view of using sea urchin embryos to detect and analyze the early mechanisms of teratogenic action.

Allopurinol↗

PubMed1996

Molecular cloning of Xenopus activin type I receptor and the analysis of its expression during embryogenesis.

We cloned cDNAs coding for the Xenopus counterparts of a type I activin receptor. The cDNA clones were predicted to encode 56kd proteins, namely XAR3 and XAR4 proteins. The two proteins are highly homologous to each other, showing 96% identity in the amino acid sequences. The GST-fused kinase domain of XAR3 autophosphorylated itself in vitro on threonine residues. The expression of XAR4 was found throughout embryogenesis, from oocytes to tailbud embryos, and also in adult tissues. By whole mount in situ hybridization, the XAR4 transcripts were detected in the animal half of blastulae and dorsally in gastrulae and neurulae. In tadpoles, the transcript was seen in the brain and around the otic vesicles. These results show that the activin type I receptor is expressed during Xenopus embryogenesis and suggest that the type I and II receptors are expressed together both temporally and spatially, supporting the idea that activin induces mesoderm in the embryo through activation of the two types of receptors.

Activin Receptors↗

PubMed1999

Electromagnetic fields in combination with elevated temperatures affect embryogenesis of Drosophila.

The effect of electromagnetic fields (50 Hz, 100 microT magnetic flux density) on Drosophila embryogenesis was tested under conditions of mild thermal stress (temperatures between 34 and 37 degrees C). When exposed to these stressor(s) for 30 min during early embryogenesis those embryos which were subjected to both electromagnetic fields and elevated temperature (costress) showed pattern anomalies more frequently than embryos subjected to thermal stress alone. Furthermore, under costress conditions development was considerably delayed in three different strains tested. The use of transgenic strains with a lacZ reporter being expressed in segmental patterns facilitated the identification and quantification of the pattern anomalies.

Animals↗

PubMed2002

Physical interactions of Dmnk with Orb: implications in the regulated localization of Orb by Dmnk during oogenesis and embryogenesis.

The Dmnk (Drosophila maternal nuclear kinase) gene, encoding a nuclear protein serine/threonine kinase, is expressed predominantly in the germline cells during embryogenesis, suggesting its possible role in the establishment of germ cells. We report here that Dmnk interacts physically with Drosophila RNA binding protein Orb, which plays crucial roles in the establishment of Drosophila oocyte by regulating the distribution and translation of several maternal mRNAs. Considering similar spatiotemporal expression pattern of Dmnk and orb during oogenesis and early embryogenesis, it is suggested that Dmnk plays a role in establishment of germ cells by interacting with Orb. Although there are two forms of Dmnk proteins, Dmnk-L (long) and Dmnk-S (short) via the developmentally regulated alternative splicing, Orb can associate with both forms of Dmnk proteins when expressed in culture cells. However, immunohistochemical analysis revealed that Dmnk-S, but not Dmnk-L, can affect the subcellular localization of Orb in a kinase activity-dependent manner, suggesting differential functions of Dmnk-S and Dmnk-L in the regulation of Orb.

Alternative Splicing↗

PubMed1996

The Xenopus GATA-4/5/6 genes are associated with cardiac specification and can regulate cardiac-specific transcription during embryogenesis.

The GATA family of nuclear factors has been implicated in the regulation of cell type-specific transcription. We report the isolation of the Xenopus GATA-4 and GATA-6 cDNA clones and characterize the expression patterns of the xGATA-4/5/6 genes. By comparing the sequence of the cDNAs with those previously reported from chick and mammalian sources, we conclude that each is conserved across vertebrate evolution as a distinct gene product. Each gene is expressed in differentiated adult heart and gut, but maintains distinct transcript patterns in various other adult tissues. During embryogenesis, each gene displays a similar overlapping distribution of transcripts localized throughout the developing cardiogenic region. The xGATA-4 gene can be detected in dorsal cardiac progenitor rudiments prior to migration. Axis disruption experiments were used to demonstrate that transcription of these genes is intimately associated with the specification of cardiac progenitors. Ectopic expression of each gene is specifically capable of activating during embryogenesis the transcription of the cardiac genes encoding actin and myosin heavy chain alpha. The data are consistent with a primary role for the GATA-4/5/6 genes in regulating heart development.

Amino Acid Sequence↗

PubMed1997

Twist protein in mouse embryogenesis.

The genes in the twist family of bHLH transcription factors are essential for embryogenesis of both vertebrates and invertebrates, as demonstrated by the embryonic lethal phenotypes of the Drosophila and mouse twist-null mutants. Presented here is the embryonic distribution of murine Twist protein (MTwist). Its appearance and presence in the course of early embryogenesis was followed with a monoclonal antibody, alphaTwiMab-1, the first antibody generated against any of the vertebrate Twist proteins. The specificity for MTwist was demonstrated in comparative Western blot experiments, reticulocyte lysate assays, and immunohistochemistry of embryonic mouse samples. Consistent with its probable role as a transcription factor, MTwist localized to the nuclei. MTwist signal first appeared in 8- to 10-somite embryos at 8.25 dpc in the cranial neural crest cells and branchial arches, in the limb-bud mesenchyme and the somatic lateral plate, and in the sclerotome and the dermatome of the somites. The presence of MTwist protein in these tissues corroborates the reported MTwist RNA distribution and the phenotype of the MTwist-null mutants. There also emerged, however, an unexpected difference between MTwist RNA and protein expression. No protein could be detected prior to 8.25 dpc, despite the reported high levels of transcripts as early as 7.0 dpc. Also, the presomitic mesoderm, epithelial somites, and anterior mesoderm expressed abundant MTwist RNA, but no protein. The results suggest posttranscriptional downregulation of MTwist in these regions. A proposed role of MTwist in somite formation and maturation is inhibition of myogenic bHLH and MEF2 genes and thus prevention of premature and/or ectopic differentiation in the presomitic mesoderm and epithelial somites. The absence of MTwist protein from these areas indicates that its role in somitogenesis must be reevaluated.

Animals↗

PubMed1998

The paternal effect gene ms(3)sneaky is required for sperm activation and the initiation of embryogenesis in Drosophila melanogaster.

Although a large number of maternal factors are known to be essential for fertilization or the earliest stages of embryogenesis in Drosophila melanogaster, the role of paternally supplied products is not clearly understood. Paternal effect mutations provide a means to identify factors specifically required by the sperm after its entry into the egg. Here we describe the third strict paternal effect gene to be identified in Drosophila ms(3)sneaky(snky), which defines the earliest developmental arrest phenotype so far described. Characterization of two independently isolated snky mutations showed that they affected male fertility, but not viability or female fertility. Cytological analyses showed that spermatogenesis proceeded normally in snky males. However, the snky defect was evident after sperm entry into the egg; snky sperm did not undergo nuclear decondensation, form a functional male pronucleus, or initiate mitotic divisions in the egg. Immunolocalization of tubulin and Drosophila Centrosomin, a known centrosomal component, showed that snky-inseminated eggs failed to reconstitute a microtubule-organizing center. In addition, snky sperm chromatin retained the histochemical properties of mature sperm chromatin for several hours after sperm entry, showed reduced staining with membrane-impermeant nuclear dyes, and failed to replicate. We conclude that the snky+ product is required for the initial response of the sperm to cytoplasmic cues in the egg and for the subsequent initiation of embryogenesis in Drosophila. We suggest that all of the snky defects can be explained by the failure of the sperm plasma membrane to break down after entry into the egg.

Animals↗

PubMed2000

A role for cyclin J in the rapid nuclear division cycles of early Drosophila embryogenesis.

The nuclear division cycles of early Drosophila embryogenesis have a number of unique features that distinguish them from later cell cycles. These features include the lack of some checkpoints that operate in later cell cycles, the absence of gap phases, and very rapid DNA synthesis phases. The molecular mechanisms that control these rapid nuclear division cycles are poorly understood. Here we describe analysis of cyclin J, a previously uncharacterized cyclin which has an RNA expression pattern that suggests a possible role in early embryogenesis. We show that the cyclin J protein is present in early embryos where it forms active kinase complexes with cyclin-dependent kinase (Cdk) 2. To determine whether cyclin J plays a role in controlling the early nuclear cycles we isolated peptide aptamers that specifically bind to cyclin J and inhibit its ability to activate Cdks. We injected the inhibitory aptamers into syncytial Drosophila embryos and demonstrated that they caused defects in chromosome segregation and progression through mitosis. We obtained similar results by injecting cyclin J antibodies into embryos. Our results suggest that a cyclin J-associated kinase activity is required for the early embryonic division cycles.

Amino Acid Sequence↗

PubMed2002

Differential expression of insulin-like growth factor I and II mRNAs during embryogenesis and early larval development in rabbitfish, Siganus guttatus.

In rodents, the expression of insulin-like growth factor II (IGF-II) is higher than that of insulin-like growth factor I (IGF-I) during fetal life while the reverse is true after birth. We wanted to examine whether this is also true in fish and whether IGF-I and IGF-II are differentially regulated during different stages of embryogenesis and early larval development in rabbitfish. We first cloned the cDNAs of rabbitfish IGF-I and IGF-II from the liver. Rabbitfish IGF-I has an open reading frame of 558 bp that codes for a signal peptide of 44 amino acids (aa), a mature protein of 68 aa, and a single form of E domain of 74 aa. Rabbitfish IGF-II, on the other hand, has an open reading frame of 645 bp that codes for a signal peptide of 47 aa, a mature protein of 70 aa, and an E domain of 98 aa. On the amino acid level, rabbitfish IGF-I shares 68% similarity with IGF-II. We then examined the relative expression of the two IGFs in unfertilized eggs, during different stages of embryogenesis, and in early larval stages of rabbitfish by a semiquantitative reverse transcription-polymerase chain reaction. Primers that amplify the mature peptide region of both IGFs were used and PCR for both peptides was done simultaneously, with identical PCR conditions for both. The identity of the PCR products was confirmed by direct sequencing. Contrary to published reports for seabream and rainbow trout, IGF-I mRNA was not detected in rabbitfish unfertilized eggs; it was first expressed in larvae soon after hatching. IGF-II mRNA, however, was expressed in unfertilized eggs, albeit weakly, and was already strongly expressed during the cleavage stage. mRNAs for both peptides were strongly expressed in the larvae, although IGF-II mRNA expression was higher than IGF-I expression.

Amino Acid Sequence↗

PubMed2001

Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein.

The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene is expressed in developing ovules and early embryos. AtSERK1 is also transiently expressed during somatic embryogenesis. The predicted AtSERK1 protein contains an extracellular domain with a leucine zipper motif followed by five leucine-rich repeats, a proline-rich region, a single transmembrane region and an intracellular kinase domain. The AtSERK1 cDNA was fused to two different variants of green fluorescent protein (GFP), a yellow-emitting GFP (YFP) and a cyan-emitting GFP (CFP), and transiently expressed in both plant protoplasts and insect cells. Using confocal laser scanning microscopy it was determined that the AtSERK1-YFP fusion protein is targeted to plasma membranes in both plant and animal cells. The extracellular leucine-rich repeats, and in particular the N-linked oligosaccharides that are present on them appear to be essential for correct localization of the AtSERK1-YFP protein. The potential for dimerization of the AtSERK1 protein was investigated by measuring the YFP/CFP fluorescence emission ratio using fluorescence spectral imaging microscopy. This ratio will increase due to fluorescence resonance energy transfer if the AtSERK1-CFP and AtSERK1-YFP fusion proteins interact. In 15 % of the cells the YFP/CFP emission ratio for plasma membrane localized AtSERK1 proteins was enhanced. Yeast-protein interaction experiments confirmed the possibility for AtSERK1 homodimerization. Elimination of the extracellular leucine zipper domain reduced the YFP/CFP emission ratio to control levels indicating that without the leucine zipper domain AtSERK1 is monomeric.

Amino Acid Motifs↗

PubMed1994

Signal molecules involved in plant embryogenesis.

In plant embryogenesis, inductive interactions mediated by diffusable signal molecules are most likely of great importance. Evidence has been presented that at late globular stages in plant embryogenesis, perturbation of the polar auxin transport results in abberrant embryo morphology. Rhizobium lipooligosaccharides or Nod factors are a newly discovered class of bacterial molecules that are able to trigger initial steps in root nodule development in legumes. Part of the activity of Nod factors may be directed towards alteration of endogenous plant growth regulator balance. The same bacterial Nod factors promoted the formation of globular embryos in the carrot cell line ts11. Whether there exist plant analogues of the Nod factors and whether these molecules are active as a more universal control system perhaps designed to initiate and or mediate gradients in auxin and cytokinin remains to be determined.

Cell Communication↗

PubMed1992

Spatial and temporal expression patterns directed by the Agrobacterium tumefaciens T-DNA gene 5 promoter during somatic embryogenesis in carrot.

We have analysed the patterns of expression of a gene encoding beta-glucuronidase (GUS) fused to the promoter of the Agrobacterium tumefaciens T-DNA gene 5 during embryogenesis in carrot, Daucus carota L. Gene expression was monitored by a histochemical assay of beta-glucuronidase activity. The gene 5 promoter, although of bacterial origin, conferred expression upon the marker gene in all stages of embryo development. The patterns of expression however, differed between embryos in different stages of development. In the globular stage expression was confined to the basal part of the embryo, suggesting that the promoter is sensitive to regulatory functions active in the primary establishment of polarity in the radially symmetric globular embryo. In the heart and torpedo stages of development GUS expression was high in the entire embryonic axis, but not in the cotyledons. During germination expression was reduced in the elongating hypocotyl and radicle, and high levels of expression were detected only in the shoot and root apices. Among the transformed cell lines analysed, one was found that showed an aberrant pattern of GUS expression during embryogenesis, in that expression in the upper part of the embryo was undetectable, and expression was restricted to the root apex in later stages of development. This difference in organ specificity of expression is likely due to a large deletion of the promoter.

Agrobacterium tumefaciens↗

PubMed1994

Influence of low temperature preincubation on somatic embryogenesis and ethylene emanation from orchardgrass leaves.

The objectives of this study were to determine the effects of low temperature (4 degrees C) preincubation on somatic embryogenesis from orchardgrass (Dactylis glomerata L.) leaf cultures and to relate these effects to ethylene emanation during the preincubation and incubation periods. Experiments were also conducted with an ethylene biosynthesis inhibitor aminooxyacetic acid (AOA). Segments from the innermost two leaves were cultured on SH medium with 30 micromoles dicamba at 4 degrees C for 1 to 7 d before transfer to 21 degrees C. Results from a paired design showed that the embryogenic response of leaf segments preincubated at 4 degrees C was equal or superior to nonpreincubated leaves at all time periods. Ethylene emanation was decreased during the low temperature incubation. Transfer of leaf segments from 4 degrees C to 21 degrees C was accompanied by a burst of ethylene which rose to control levels within 30 min. AOA at 20 and 40 micromoles decreased ethylene emanation but did not stimulate the embryogenic response. We conclude that the stimulation of somatic embryogenesis by low temperature is probably due to factors other than suppression of ethylene biosynthesis.

Aminooxyacetic Acid↗