Rates of RNA synthesis during early embryogenesis in Drosophila melanogaster

We determined the absolute rates of RNA synthesis during embryogenesis in Drosophila melanogaster by measuring the incorporation of ³H-5-orotic acid into RNA, and the specific activity of the UTP pool. Initially (preblastoderm) the rate of RNA synthesis is relatively high, but declines to a lower level by gastrulation. The data suggest that RNA synthesis is initiated during very early embryogenesis.     

mRNA sequence diversity during early embryogenesis in Drosophila melanogaster.

The structure and metabolism of membrane glycoprotein carbohydrate units were studied in three clonal strains of contact-deficient mouse L cells as compared with several contact-competent rodent fibroblasts. The L cells appeared deficient in the formation of stable adhesive and communicating contacts. Formation of these contacts could not be induced by artificial adhesion induced by Sendai virus. The absence of functional contacts in L cells was associated with synthesis of incomplete, dialysable fucosyl glycopeptides exposed at the cell surface. Somatic cell hybrids between communication incompetent L cells and mouse leukemia cells synthesized membrane carbohydrates of near-normal size distribution, but these cells remained deficient in functional contacts. However, in hybrids of L cells and normal human fibroblasts or lymphocytes, glycoprotein synthesis and formation of functional contacts were concomitantly restored.     

The mitotic arrest in response to hypoxia and of polar bodies during early embryogenesis requires Drosophila Mps1

Mps1 kinase plays an evolutionary conserved role in the mitotic spindle checkpoint. This system precludes anaphase onset until all chromosomes have successfully attached to spindle microtubules via their kinetochores. Mps1 overexpression in budding yeast is sufficient to trigger a mitotic arrest, which is dependent on the other mitotic checkpoint components, Bub1, Bub3, Mad1, Mad2, and Mad3. Therefore, Mps1 might act at the top of the mitotic checkpoint cascade. Moreover, in contrast to the other mitotic checkpoint components, Mps1 is essential for spindle pole body duplication in budding yeast. Centrosome duplication in mammalian cells might also be controlled by Mps1 , but the fission yeast homolog is not required for spindle pole body duplication. Our phenotypic characterizations of Mps1 mutant embryos in Drosophila do not reveal an involvement in centrosome duplication, while the mitotic spindle checkpoint is defective in these mutants. In addition, our analyses reveal novel functions. We demonstrate that Mps1 is also required for the arrest of cell cycle progression in response to hypoxia. Finally, we show that Mps1 and the mitotic spindle checkpoint are responsible for the developmental cell cycle arrest of the three haploid products of female meiosis that are not used as the female pronucleus.     

Abnormal behavior of the yolk centrosomes during early embryogenesis of Drosophila melanogaster

After the 10th nuclear cycle the yolk centrosomes follow an irregular pathway. Unlike the somatic centrosomes, which move to the opposite poles of the nuclei to form the bipolar spindles, the yolk centrosomes remain as pairs at one pole of the yolk nuclei or shift feebly and nucleate irregular spindles, most of which have only one main pole. The yolk centrosomes are no longer observed near the yolk nuclei, but progressively move away into the surrounding cytoplasm. Despite the irregular behavior of the centrosomes and although the yolk nuclei cease to divide, the yolk centrosome duplication cycle continues. The early development of Drosophila thus provides an excellent natural system for the study of the uncoupling of the nuclear and centrosomal cycles.     

Analysis of twin of eyeless regulation during early embryogenesis in Drosophila melanogaster

The Drosophila Pax6 homolog twin of eyeless (toy) is so far the first zygotically expressed gene involved in eye morphogenesis in Drosophila. The study of its expression during embryogenesis is therefore informative of the initial events of eye development in Drosophila. We have analyzed how the initial expression domain of toy at cellular blastoderm is regulated. We show that the three maternal patterning systems active in the cephalic region (the anterior, terminal and dorsal-ventral systems) cooperate with zygotically activated gap genes to shape the initial expression domain of toy. Whereas Bicoid, Dorsal and Torso signaling synergistically act as activators, Hunchback, Knirps and Decapentaplegic act as repressors.     

Rates of ribosomal protein and total protein synthesis during Drosophila early embryogenesis

Embryos at various stages of early development from 1.5 to 5 hr after oviposition were made permeable with octane and labeled for 1 hr with [³H]phenylalanine. Measurements of the rate of incorporation of [³H]phenylalanine into ribosomal proteins and total protein were made using these synchronized Drosophila embryos. The rate of synthesis of those ribosomal proteins incorporated into ribosomes increases until 3 to 4 hr after fertilization (550 pg/embryo-hr) then declines later in embryonic development. The rate of total protein synthesis is maximal as early during embryonic development as could be measured. During the period between 1.5 and 2.5 hr after fertilization this rate is 9.4 ng/embryo-hr and then also declines. The synthesis of ribosomal proteins accounts for a substantial portion (4.5%–8.9%) of total protein synthesis in early embryos. These results indicate that ribosome formation is a significant activity during the earliest stages of Drosophila development.     

The function of PS integrins during Drosophila embryogenesis

The Drosophila position-specific (PS) antigens are homologous to the vertebrate fibronectin receptor family, or integrins. A Drosophila gene required for embryonic morphogenesis, l(1)myospheroid, codes for a product homologous to the beta subunit of the vertebrate integrins. l(1)myospheroid mutants die during embryogenesis. We show here that they lack the beta subunit of the PS antigens. In the absence of the beta subunit in mutant embryos, the PS alpha subunits are not expressed on the cell surface. We conclude that the l(1)myospheroid phenotype represents the lack-of-function phenotype for these Drosophila integrins. In wild-type embryos, PS antigens are found at the interface between mesoderm and ectoderm, and later mainly at the attachment sites of muscles to the epidermis and gut. Together these results indicate that during embryogenesis, Drosophila integrins are used to attach mesoderm to ectoderm, and are required for the proper assembly of the extracellular matrix and for muscle attachment.     

The study of proteins during early stages of embryogenesis of sea urchins

Summary The distribution of the preexisting and of thede novo synthesized proteins among soluble, and insoluble fractions, as well as between immunoprecipitable and non-immunoprecipitable soluble proteins has been studied in sea urchin embryos at different stages of development.In the S-100 fraction, which represented about 20% of total proteins, only a minor part of radioactivity was found. The majority of newly synthesized proteins was insoluble at neutral pH. Such distribution was practically invariant for all investigated stages of development, and was not markedly affected by Dactinomycin nor by 5-azacytidine.Only a small percentage of the total radioactivity of the S-100 fraction was found in the antigen-antibody complexes of soluble proteins. No shift of newly synthesized proteins towards the type of old, preexisting antigenic proteins was detected, and the majority of soluble newly synthesized proteins was found to be related to the non-immunoprecipitable soluble proteins.The authors gratefully acknowledge a generous gift of 5-azacytidine from Drs. Doskoil and ponar. We are also indebted to Dr. Miroslav Simi for kind interest and discussion.This work was supported in part by grants No 3111/1 from Federal Research Fund of Yugoslavia and No 02-020-1 from the National Institute of Health, U. S. Department of Health, Education and Welfare (PL-480 programme).     

The use of counterflow centrifugation to enrich gonadotropes and somatotropes.

Counterflow centrifugation produces populations of gonadotropes or growth hormone (GH) cells enriched to 90% in a Beckman elutriator. The pituitary populations are first separated by size into three fractions applying different flow rates, stimulated with either gonadotropin-releasing hormone (GnRH) to enlarge the gonadotropes or growth hormone-releasing hormone (GHRH) to enlarge the somatotropes for 3 hr. The fractions are re-eluted, first at the original flow rates and then at higher flow rates to separate enlarged gonadotropes or somatotropes. Most other cell types are reduced to less than 5%. However, co-storage of GH and gonadotropin antigens is seen in either population. Enriched gonadotropes or somatotropes can be used in studies of proliferation, autocrine or paracrine regulation, or ion channel functions.(J Histochem Cytochem 49:663-664, 2001)     

Analysis of cell movements and fate mapping during early embryogenesis in Drosophila melanogaster

Four spatially differentiated surface regions, called aeropyle crown, flat, stripe, and micropyle, are found on the mature eggshell (chorion). Specializations of the apical surfaces of the secretory follicular epithelial cells are implicated in the formation of regional patterns on the chorion. Some of these specializations are restricted to cells overlying certain regions; others are shared by more than one region. Differences between regions are more apparent on the surface than within the bulk of the chorion. Evidence is presented that distinct cell populations, corresponding to the regions, are present long before the start of choriogenesis. One hundred eighty-six chorion-specific polypeptides have been resolved by two-dimensional gel electrophoresis. Fifteen of these are found entirely or predominantly in the aeropyle crown and stripe regions, while eight others are restricted to the aeropyle crown region. Certain of the spatially restricted components are quite unusual in their amino acid compositions when compared with previously analyzed chorion components. Others are closely related, although clearly distinct.     

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.     

A stage-specific factor confers Fab-7 boundary activity during early embryogenesis in Drosophila

The Fab-7 boundary is required to ensure that the iab-6 and iab-7 cis-regulatory domains in the Drosophila Bithorax complex can function autonomously. Though Fab-7 functions as a boundary from early embryogenesis through to the adult stage, this constitutive boundary activity depends on subelements whose activity is developmentally restricted. In the studies reported here, we have identified a factor, called early boundary activity (Elba), that confers Fab-7 boundary activity during early embryogenesis. The Elba factor binds to a recognition sequence within a Fab-7 subelement that has enhancer-blocking activity during early embryogenesis, but not during mid-embryogenesis or in the adult. We found that the Elba factor is present in early embryos but largely disappears during mid-embryogenesis. We show that mutations in the Elba recognition sequence that eliminate Elba binding in nuclear extracts disrupt the early boundary activity of the Fab-7 subelement. Conversely, we find that early boundary activity can be reconstituted by multimerizing the Elba recognition site.     

The function of type IV collagen during Drosophila embryogenesis

A collagen gene (Dcg1) was characterized in Drosophila melanogaster and shown to encode a peptide related to vertebrate basement membrane type IV collagen chains. To study the function of type IV collagen during Drosophila development, we transformed flies with a partially truncated Dcg1 gene under the control of a heat-shock promotor. This construct induced synthesis of shortened pro- chains which associated with normal ones and thereby caused degradation of the shortened and normal pro- chains through a process called pro-collagen suicide. A large proportion of embryos expressing the transgene developed a phenotype exhibiting absence or partial retraction of the germ band with defects in nerve cord condensation and dorsal closure. Together these results indicated that, during embryogenesis, type IV collagen was an essential guiding factor for cell-matrix interactions in morphogenetic events.     

A study of Drosophila spinster expression and its functions during embryogenesis

During the development of the Drosophila nervous system, the programmed cell death (PCD) regulates the cell number. The spinster (spin) encodes a multiple transmembrane protein and females showed a strong rejection response against the courting males. Mutation in spin interferes with the PCD of neurons, which subsequently induce the degeneration of adult neural cells. However, this spin functions has not been investigated yet during embryogenesis. In this study we first examined spin expression in detail and its function during embryonic development. Spin was primarily expressed in surface glial cells, including subperineurial glial cells and exit glia, but not in neural cells. In spin loss-of-function mutant embryos, Glial cells increased in number, and neural overgrowth occurred. In spin gain-of-function mutant embryos, PNS was predominantly degenerated at late embryonic stages. These results indicate that spin is involved in neurogenesis via cell death during embryogenesis.