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Mutations in centrosomin reveal requirements for centrosomal function during early Drosophila embryogenesis.

BACKGROUND: Although centrosomes serve as the primary organizing centers for the microtubule-based cytoskeleton in animal cells, various studies question the requirements for these organelles during the formation of microtubule arrays and execution of microtubule-dependent processes. Using a genetic approach to interfere with centrosomal function, we present an assessment of this issue, in the context of early embryogenesis of the fruit fly Drosophila melanogaster. RESULTS: We identified mutant alleles of the centrosomin (cnn) locus, which encodes a core component of centrosomes in Drosophila. The cnn mutant flies were viable but sterile. The normal course of early embryonic development was arrested in all progeny of cnn mutant females. Our analysis identified a failure to form functional centrosomes and spindle poles as the primary mutant phenotype of cnn embryos. Various aspects of early development that are dependent on cytoskeletal control were disrupted in cnn mutant embryos. In particular, structural rearrangements of cortical microfilaments were strongly dependent on proper centrosomal function. CONCLUSIONS: Centrosomin is an essential core component of early embryonic centrosomes in Drosophila. Microtubule-dependent events of early embryogenesis display differential requirements for centrosomal function.

Alleles↗

Protein phosphorylation during Rhodnius prolixus embryogenesis: protein kinase casein kinase II activity.

Protein kinase casein kinase II (CK II) activity was assayed during Rhodnius prolixus embryogenesis. Vitellin (VT) is the main endogenous substrate during the whole development. It is maximally phosphorylated at the third day of embryogenesis by CK II and then its phosphorylation decreases to a basal level by the time of first instar eclosion. When dephosphorylated casein was used as an exogenous substrate a different profile of enzyme activity was obtained. CK II activity increases on day 1 after fertilization and reaches a plateau on day 7 and its activity remains elevated until eclosion. Extracts obtained from oocytes or from 3-day old eggs were fractionate through gel filtration chromatography. CK II activity was assayed in each fraction and the enzyme obtained from the 3-day old eggs was shown to be three times more active than that obtained from oocytes, although the amount of enzyme present in the fractions was the same. These enriched CK II fractions were assayed against different effectors, such as: cAMP, H-8, H-89, calphostin C, sphingosine, polylysine and heparin. Heparin was the most effective one. When CK II activity was assayed in non-fertilized eggs, no activation of the enzyme was observed when compared to fertilized eggs. These data indicate that CK II is activated in a fertilization dependent process. The decrease in CK II activity against VT coincides with the beginning of VT proteolysis processing suggesting a possible relationship between protein phosphorylation and yolk degradation.

Animals↗

Rhythmic contractions in chick amnio-yolk sac and snake amnion during embryogenesis.

The rhythmic movements of fetal membranes in chick and reptile embryos were studied to explore the developmental role of the extra-embryonic motor activity. In the snakes Lamprophis fuliginosus and Elaphe radiata, rhythmic contractions of amnion inside the developing egg were recorded from the 11th incubation day until pre-hatching stages (ca. day 60-72). The duration of these contractions averaged 2.02+/-0.27 min. The frequency ranged from 2 to 6 per 10 min and averaged 4.61+/-0.57 per 10 min. A tendency of frequency to increase toward the end of embryogenesis was observed. Lowering the temperature from 28 to 20 degrees C significantly decreased the frequency of amnion contractions to 2.85+/-0.91 per 10 min. The isolated snake amnion retained its capacity for spontaneous contraction. Noradrenaline inhibited, acetylcholine stimulated and serotonin did not affect the rhythmic activity of the isolated snake amnion. Similar effects were found when these agents were applied into the snake amniotic cavity. In the chick, yolk sac rhythmic contractions were recorded from the fifth until the 12th incubation days. The duration of these contractions ranged from 15 to 60 s, their frequency averaged 11.8+/-3.18 per 10 min and depended on temperature. The low temperature threshold was approximately 30 degrees C. After surgical removal of the amnion and embryo, the yolk sac continued contracting inside the egg. The yolk sac rhythmic contractions likely participate in the space movement of the embryo inside the egg during embryogenesis.

Amnion↗

Up, down and up again is a signature global gene expression pattern at the beginning of gymnosperm embryogenesis.

Somatic embryogenesis of a gymnosperm, Picea abies, represents a sequence of specifically regulated developmental stages including proembryogenic mass (PEM), PEM-to-embryo transition, and early and late embryogeny. Here, we report cDNA array analysis of expression patterns of 373 genes in the beginning of P. abies embryo development. The analysis revealed a group of 107 genes (29% of arrayed cDNAs) which were upregulated upon PEM-to-embryo transition, then downregulated during early embryogeny and finally upregulated again at the beginning of late embryogeny. This major gene expression pattern was abrogated in a developmentally arrested cell line that is unable to pass through the PEM-to-embryo transition. Thirty-five genes (9.4% of arrayed cDNAs) were found to be differentially expressed during normal embryonic pattern formation. Among them, 22 genes (5.9% of arrayed cDNAs) were directly associated with embryo pattern formation and can be considered as marker genes for early stages of P. abies embryogenesis. The majority of the marker genes encode for proteins involved in translation and posttranslational modification. Among them, 18 genes displayed the major expression pattern.

Gene Expression↗

PCP4 is highly expressed in ectoderm and particularly in neuroectoderm derivatives during mouse embryogenesis.

PCP4 (PEP-19) belongs to a family of proteins involved in calcium transduction signals and binds calmodulin via an IQ motif, in a calcium independent manner. PCP4 gene maps to murine chromosome 16 and in human to chromosome 21. Murine PCP4 expression in the brain has been detected by Northern blot analysis to be mainly post-natal and in the adult to have a neuronal pattern. To investigate if it might have a role earlier in development, we analyzed its expression during mouse embryogenesis by in situ hybridization from E7.5 post-coitum (p.c.) to E17.5 p.c., and in P0 brain. Early, at E7.5, a high expression is restricted to the extra embryonic ectoderm. Embryonic expression starts at E9.5. At E10.5, PCP4 shows a strong signal in the post-mitotic cells of the diencephalon, the metencephalon and the myelencephalon and in the dorsal and cranial ganglia. The floor plate is also densely labelled. At E17.5, PCP4 is expressed in the central nervous system, in the myenteric plexus, and in other ectoderm derivatives, for instance the lens, the hairy cells of the cochlea, the enamel organ and the hair follicles. Thus, during embryogenesis PCP4 is mainly expressed in ectoderm and neuroectoderm comprising neural crest derived cells.

Animals↗

Lessons from the space experiment SL-J/FMPT/L7: the effect of microgravity on chicken embryogenesis and bone formation.

Thirty fertilized chicken eggs preincubated for 0, 7, and 10 days on earth (10 eggs each) were flown in the space shuttle "Endeavour" and further incubated for 7 days under microgravity. Twenty out of 30 eggs (nine out of ten 10-day-old eggs; 10 out of ten 7-day-old eggs; 1 out of ten 0-day-old eggs) were recovered alive after landing. The only living embryo of the 0-day-old egg died 24 days after launch, and was comparable to a 16-day-old embryo when it died. The high mortality of the 0-day-old eggs appeared to be related to the specific inner structure of the egg. The yolk (specific gravity, 1.029) would not have separated from the albumen (1.040) during space flight. The subtle difference in specific gravity between the yolk and the albumen appeared to play a critical role in early chick embryogenesis. All the tissues, including cartilage and bone, were formed normally in 7- and 10-day-old chicken embryos during space flight. When the 7- and 10-day-old embryos recovered at Kennedy Space Center (KSC) were further incubated on earth, they hatched normally. No appreciable changes were recognized in these chicks. The reason for these unexpected results is not known. Further space experiments are needed to explain the failure of the effects of microgravity on chicken embryogenesis.

Alkaline Phosphatase↗

Spatially regulated expression of retrovirus-like transposons during Drosophila melanogaster embryogenesis.

Over twenty distinct families of long terminal direct repeat (LTR)-containing retrotransposons have been identified in Drosophila melanogaster. While there have been extensive analyses of retrotransposon transcription in cultured cells, there have been few studies of the spatial expression of retrotransposons during normal development. Here we report a detailed analysis of the spatial expression patterns of fifteen families of retrotransposons during Drosophila melanogaster embryogenesis (17.6, 297, 412, 1731, 3S18, blood, copia, gypsy, HMS Beagle, Kermit/flea, mdg1, mdg3, opus, roo/B104 and springer). In each case, analyses were carried out in from two to four wild-type strains. Since the chromosomal insertion sites of any particular family of retrotransposons vary widely among wild-type strains, a spatial expression pattern that is conserved among strains is likely to have been generated through interaction of host transcription factors with cis-regulatory elements resident in the retrotransposons themselves. All fifteen families of retrotransposons showed conserved patterns of spatially and temporally regulated expression during embryogenesis. These results suggest that all families of retrotransposons carry cis-acting elements that control their spatial and temporal expression patterns. Thus, transposition of a retrotransposon into or near a particular host gene-possibly followed by an excision event leaving behind the retrotransposon's cis-regulatory sequences-might impose novel developmental control on such a host gene. Such a mechanism would serve to confer evolutionarily significant alterations in the spatio-temporal control of gene expression.

Animals↗

Embryogenesis in Litomosoides carinii from pyridoxine deficient cotton rats.

Pyridoxine (vitamin B6) deficiency was induced in cotton rats which were then infected with the filarial parasite Litomosoides carinii. Embryogenesis was assessed microscopically in worms taken from pyridoxine deficient cotton rats and from various categories of control animals. Embryogenesis was retarded in worms from pyridoxine deficient hosts and more abnormal embryos were present in such worms than in those from control animals.

Animals↗

Immunocytochemical localisation of the nucleolar protein fibrillarin and RNA polymerase I during mouse early embryogenesis.

We have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA polymerase I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar precursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.

Animals↗

Characterization of somatic embryogenesis-related cDNAs from alfalfa (Medicago sativa L.).

Messenger RNAs from cultures of embryogenic and non-embryogenic alfalfa (Medicago sativa L.) genotypes were used to differentially screen a cDNA library prepared from embryogenic cell masses of somatic embryo cultures to identify early-stage embryo transcripts. The three alfalfa somatic embryogenesis-specific transcripts cDNAs (ASET1, ASET2 and ASET3) identified by this screen were enriched in RNA samples from embryogenic tissues of the embryogenic genotype but were absent from petioles or mature embryos of an embryogenic genotype as well from tissue cultures of a nonembryogenic genotype. The ASET clones did not cross-hybridize and showed different patterns of expression in northerns of RNA from various fractions of alfalfa somatic embryo cultures. The ASET clones did not hybridize with the soybean embryogenesis-specific clone (Sbh1) which was shown to be expressed in embryogenic and non-embryogenic alfalfa tissue cultures. Sequencing showed ASET1 to be a partial transcript 595 nucleotides long. ASET2 was a complete transcript of 1193 nucleotides. From a comparison of the predicted open reading frame with the GenBank protein database it was concluded that ASET2 was a novel transcript. The protein predicted by the ASET2 sequence has several potential membrane-spanning domains and a potential phosphorylation site. In addition, the ASET2 cDNA had a long 5' region that contained two upstream reading frames (URFs) which could potentially code for 30 and 6 amino acid polypeptides.

Amino Acid Sequence↗

Traffic of the tick embryo basic protein during embryogenesis of the camel tick Hyalomma dromedarii (Acari: Ixodidae).

The tick embryo basic protein (TEBP) is present in the nucleus as a counterpart of histones at early embryonic stages of the tick Hyalomma dromedarii. The sharp drop in the TEBP nuclear level and elimination of the N-terminal dipeptide (leucine-serine) between days 12 and 15 after oviposition suggested the transport of TEBP to the cytoplasm for protein turnover. The traffic of TEBP during tick embryogenesis was examined. The level of TEBP was detected in the cytoplasm from the different embryonic stages by the established enzyme-linked immunosorbent assay (ELISA) and confirmed by immunoblotting. At day 12, a 2-fold increase in the cytoplasmic TEBP level coincided with its decrease in the nucleus. This result indicates that TEBP starts to leave the nucleus for the cytoplasm at day 12. The changes in the cytoplasmic leucine aminopeptidase (LAP)-specific activity were followed during tick embryogenesis. The LAP activity started to increase at day 12 and reached its maximum level at day 21. The enzyme displayed an optimum pH between 7.5 and 8.8 and a K(m) value of 0.5 microM for leucine-p-nitroanilide. The involvement of the exopeptidase activity in the TEBP turnover after its translocation to the cytoplasm is discussed.

Amino Acid Sequence↗

Two novel MYB homologues with changed expression in late embryogenesis-defective Arabidopsis mutants.

Two novel MYB genes (ATMYBR1 and ATMYBR2) were isolated from Arabidopsis thaliana. Binding to a conserved MYB recognition sequence is demonstrated for the ATMYBR1 protein. The expression of both genes is affected by the fus3, lec1 and abi3 mutations causing pleiotropic defects during late embryogenesis and seed maturation including the loss of dormancy and desiccation tolerance. The strong increase of the transcript levels of both MYB genes during very late stages of embryogenesis typically found in wild type is missing in the mutants. Furthermore, the expression of both MYB genes is developmentally regulated in vegetative tissues. The highly conserved repeats (R2 and R3) of the DNA binding MYB domain of both proteins represent chimeric structures combining features typical of plant and animal derived proteins. This demonstrates the existence of a distinct subfamily of animal-like MYB factors in plant genomes.

Amino Acid Sequence↗

Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis.

For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severely affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action.

Amino Acid Sequence↗

Changes in cytokinin distribution in the ovule and ovary of Taraxacum officinale Web. in early stages of embryogenesis.

Quantitative changes in the hormonal status of the ovules and ovaries were first studied in Taraxacum officinale Web. at the early stages of embryogenesis. The plant material was analyzed by ELISA using labeled anti-rabbit antibodies. A new procedure for differential and quantitative determination of the main endogenous cytokinins based on the estimation of the effective zeatin and zeatin riboside concentrations from calibration curves constructed using zeatin and zeatin riboside as standard antigens was developed. It was shown that, at the three initial stages of embryogenesis examined, the concentration of zeatin uniformly increased in T. officinale ovules. The concentration of zeatin riboside, conversely, uniformly decreased. However, their total concentration changed insignificantly. A gradual increase in the concentration of the active and storage hormone forms from the ovary to the ovule was shown.

Asteraceae↗

Induction of direct somatic embryogenesis in garlic (Allium sativum).

An efficient novel method of direct somatic embryogenesis from basal tissue of garlic clove was developed. The influence of plant growth regulators, basal medium and explant type on somatic embryo induction was examined. The best plant growth regulator combination was, 2,4-D and kinetin at 1.0 mg/L and 0.5 mg/L respectively, inducing direct somatic embryogenesis in 60% of explants. White's medium was used as basal medium and somatic embryos developed on explants after six weeks. The technique has potential applicability for problems associated with plant regeneration and virus elimination in garlic.

2,4-Dichlorophenoxyacetic Acid↗

Mechanical modulation of cartilage structure and function during embryogenesis in the chick.

The mechanical behavior of cartilage is intimately related to its biochemical composition, and tissue composition is known to be influenced by its local mechanical loading environment. Although this phenomenon has been well-studied in adult cartilage, few investigations have examined such structure-function relationships in embryonic cartilage. The goal of this work was to elucidate the role of mechanical loading on the development of cartilage composition during embryogenesis. Using an embryonic chick model, cartilage from the tibiofemoral joints of immobilized embryos was compared to that of controls. The normal time course of changes in glycosaminoglycan/DNA and hydroxyproline/DNA were significantly influenced by loading history, with the most pronounced effects observed between days 9 and 14 during the period of most rapid increase in motility in control embryos. Stress-relaxation tests conducted on samples from day 14 indicate that the effects of embryonic immobilization on cartilage matrix composition have direct consequences for the mechanical behavior of the tissue, resulting in compromised material properties (e.g. 50% reduction in E(inst)). Because embryogenesis provides a unique model for identifying key factors which influence the establishment of functional biomechanical tissues in the skeleton, these data suggest that treating mechanical loading as an in vitro culture variable for tissue engineering approaches to cartilage repair is likely to be a sound approach.

Adaptation, Physiological↗

Nuclear localization of beta-catenin in vegetal pole cells during early embryogenesis of the starfish Asterina pectinifera.

Recently, beta-catenin has been reported to control the expression of morphogenetic genes through the Wnt signaling pathway in invertebrate embryogenesis. In this study, the distribution pattern of beta-catenin during starfish embryogenesis was investigated using immunohistochemistry. In 16-cell stage embryos, beta-catenin began to accumulate in some nuclei at the vegetal pole. During the early cleavage stage, the cells expressing nuclear beta-catenin increased in number in the vegetal pole region of the embryos, and the beta-catenin signal increased in intensity in each nucleus. At the blastula stage, signal for beta-catenin was also found in the cytoplasm of the cells with nuclear beta-catenin. At the vegetal plate stage, almost all vegetal plate cells expressed beta-catenin in both the nucleus and cytoplasm. When the embryos developed to early gastrulae, cells with nuclear beta-catenin were restricted to the archenteron tip, and the signal gradually faded in later stages. The localization and temporal change of beta-catenin expression suggests that beta-catenin has a pivotal role in archenteron formation in starfish embryos.

Animals↗

Identification of tightly regulated groups of genes during Drosophila melanogaster embryogenesis.

Time-series analysis of whole-genome expression data during Drosophila melanogaster development indicates that up to 86% of its genes change their relative transcript level during embryogenesis. By applying conservative filtering criteria and requiring 'sharp' transcript changes, we identified 1534 maternal genes, 792 transient zygotic genes, and 1053 genes whose transcript levels increase during embryogenesis. Each of these three categories is dominated by groups of genes where all transcript levels increase and/or decrease at similar times, suggesting a common mode of regulation. For example, 34% of the transiently expressed genes fall into three groups, with increased transcript levels between 2.5-12, 11-20, and 15-20 h of development, respectively. We highlight common and distinctive functional features of these expression groups and identify a coupling between downregulation of transcript levels and targeted protein degradation. By mapping the groups to the protein network, we also predict and experimentally confirm new functional associations.

Analysis of Variance↗