scholarly journals Effect of heritable symbionts on maternally-derived embryo transcripts

2019 ◽  
Author(s):  
Mariana Mateos ◽  
Nadisha O. Silva ◽  
Paulino Ramirez ◽  
Victor M. Higareda-Alvear ◽  
Rodolfo Aramayo ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Cytoplasmic Incompatibility ◽  
Developmental Stages ◽  
Early Embryo ◽  
Male Embryo ◽  
Ribosome Inactivating Proteins ◽  
Male Killing ◽  
Early Embryos ◽  
Early Developmental ◽  
Mrna Transcriptome

AbstractMaternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. In the present study, we investigated whether heritable endosymbionts that cause different reproductive phenotypes in Drosophila melanogaster influence the mRNA transcriptome of early embryos. We used mRNA-seq to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. We found no consistent evidence of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, we capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. We found small but statistically significant signals of depurination of Drosophila rRNA in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.

Morphological and molecular modifications induced by heat shock in Drosophila melanogaster embryos

Development ◽  
1983 ◽  
Vol 77 (1) ◽  
pp. 167-182
Author(s):  
Giorgio Graziosi ◽  
Franco de Cristini ◽  
Angelo di Marcotullio ◽  
Roberto Marzari ◽  
Fulvio Micali ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Dna Synthesis ◽  
Histological Analysis ◽  
Developmental Stages ◽  
Early Embryo ◽  
Direct Relation ◽  
Hsp 70 ◽  
Survival Pattern ◽  
Shock Proteins

The early embryo of Drosophila melanogaster did not survive treatment at 37 °C (heat shock) for 25 min. The histological analysis of eggs treated in this way showed that the heat shock caused disintegration of nuclei and of cytoplasmic islands, displacement and swelling of nuclei and blocked mitoses. These effects were not observed in embryos treatedafter blastoderm formation. After this stage, we noticed that development was slowed down. The heat shock proteins (hsp 83,70 and 68) were, under shock, synthesized at all developmental stages. There was little or no synthesis of hsp 70 and 68 in unfertilized eggs, but synthesis increased in proportion to the number of nuclei present. Most probably, hsp 70 synthesis was directed by zygotic mRNA. DNA synthesis was not blocked by the heat shock though the overall incorporation of [3H]thymidine was substantially reduced, presumably because of the block of mitoses. We did not find a direct relation between survival pattern and hsp synthesis. We concluded that some, at least, of the heat shock genes can be activated at all developmental stages and that heat shock could be used for synchronizing mitoses.

scholarly journals Transgenic Testing Does Not Support a Role for Additional Candidate Genes in Wolbachia Male Killing or Cytoplasmic Incompatibility

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jessamyn I. Perlmutter ◽  
Jane E. Meyers ◽  
Seth R. Bordenstein
Keyword(s):  
Drosophila Melanogaster ◽  
Copy Number ◽  
Cytoplasmic Incompatibility ◽  
Functional Study ◽  
Content Type ◽  
Transgenic Expression ◽  
Additional Gene ◽  
Reproductive Parasitism ◽  
Transcript Length ◽  
Male Killing

ABSTRACT Endosymbiotic bacteria in the genus Wolbachia remarkably infect nearly half of all arthropod species. They spread in part because of manipulations of host sexual reproduction that enhance the maternal transmission of the bacteria, including male killing (death of infected males) and unidirectional cytoplasmic incompatibility (CI; death of offspring from infected fathers and uninfected mothers). Recent discoveries identified several genes in prophage WO of Wolbachia (wmk, cifA, and cifB) that fully or partially recapitulate male killing or CI when transgenically expressed in Drosophila melanogaster. However, it is not yet fully resolved if other gene candidates contribute to these phenotypes. Here, we transgenically tested 10 additional gene candidates for their involvement in male killing and/or CI. The results show that despite sequence and protein architecture similarities or comparative associations with reproductive parasitism, transgenic expression of the candidates does not recapitulate male killing or CI. Sequence analysis across Wmk and its closest relatives reveals amino acids that may be important to its function. In addition, evidence is presented to propose new hypotheses regarding the relationship between wmk transcript length and its ability to kill a given host, as well as copy number of wmk homologs within a bacterial strain, which may be predictive of host resistance. Together, these analyses continue to build the evidence for identification of wmk, cifA, and cifB as the major genes that have thus far been shown to cause reproductive parasitism in Wolbachia, and the transgenic resources provide a basis for further functional study of phage WO genes. IMPORTANCE Wolbachia are widespread bacterial endosymbionts that manipulate the reproduction of diverse arthropods to spread through a population and can substantially shape host evolution. Recently, reports identified three prophage WO genes (wmk, cifA, and cifB) that transgenically recapitulate many aspects of reproductive manipulation in Drosophila melanogaster. Here, we transgenically tested 10 additional gene candidates for CI and/or male killing in flies. The results yield no evidence for the involvement of these gene candidates in reproductive parasitism, bolstering the evidence for identification of the cif and wmk genes as the major factors involved in their phenotypes. In addition, evidence supports new hypotheses for prediction of male-killing phenotypes or lack thereof based on wmk transcript length and copy number. These experiments inform efforts to understand the full basis of reproductive parasitism for basic and applied purposes and lay the foundation for future work on the function of an interesting group of Wolbachia and phage WO genes.

scholarly journals Microarray analysis of gene expression during early development: a cautionary overview

Reproduction ◽  
2010 ◽  
Vol 140 (6) ◽  
pp. 787-801 ◽  
Author(s):  
Claude Robert
Keyword(s):  
Gene Expression ◽  
Early Development ◽  
Microarray Analysis ◽  
Developmental Stages ◽  
Sample Handling ◽  
Early Embryos ◽  
High Throughput Method ◽  
Full Benefit ◽  
Key Steps ◽  
Early Developmental

The rise of the ‘omics’ technologies started nearly a decade ago and, among them, transcriptomics has been used successfully to contrast gene expression in mammalian oocytes and early embryos. The scarcity of biological material that early developmental stages provide is the prime reason why the field of transcriptomics is becoming more and more popular with reproductive biologists. The potential to amplify scarce mRNA samples and generate the necessary amounts of starting material enables the relative measurement of RNA abundance of thousands of candidates simultaneously. So far, microarrays have been the most commonly used high-throughput method in this field. Microarray platforms can be found in a wide variety of formats, from cDNA collections to long or short oligo probe sets. These platforms generate large amounts of data that require the integration of comparative RNA abundance values in the physiological context of early development for their full benefit to be appreciated. Unfortunately, significant discrepancies between datasets suggest that direct comparison between studies is difficult and often not possible. We have investigated the sample-handling steps leading to the generation of microarray data produced from prehatching embryo samples and have identified key steps that significantly impact the downstream results. This review provides a discussion on the best methods for the preparation of samples from early embryos for microarray analysis and focuses on the challenges that impede dataset comparisons from different platforms and the reasons why methodological benchmarking performed using somatic cells may not apply to the atypical nature of prehatching development.

scholarly journals Physical constraints on early blastomere packings

2021 ◽  
Vol 17 (1) ◽  
pp. e1007994
Author(s):  
James Giammona ◽  
Otger Campàs
Keyword(s):  
Cell Division ◽  
Developmental Stages ◽  
Physical Parameters ◽  
Cell Stage ◽  
Physical Constraints ◽  
Equilibrium Dynamics ◽  
Deformable Particles ◽  
Early Embryos ◽  
Early Developmental Stages ◽  
Early Developmental

At very early embryonic stages, when embryos are composed of just a few cells, establishing the correct packing arrangements (contacts) between cells is essential for the proper development of the organism. As early as the 4-cell stage, the observed cellular packings in different species are distinct and, in many cases, differ from the equilibrium packings expected for simple adherent and deformable particles. It is unclear what are the specific roles that different physical parameters, such as the forces between blastomeres, their division times, orientation of cell division and embryonic confinement, play in the control of these packing configurations. Here we simulate the non-equilibrium dynamics of cells in early embryos and systematically study how these different parameters affect embryonic packings at the 4-cell stage. In the absence of embryo confinement, we find that cellular packings are not robust, with multiple packing configurations simultaneously possible and very sensitive to parameter changes. Our results indicate that the geometry of the embryo confinement determines the packing configurations at the 4-cell stage, removing degeneracy in the possible packing configurations and overriding division rules in most cases. Overall, these results indicate that physical confinement of the embryo is essential to robustly specify proper cellular arrangements at very early developmental stages.

The casein kinase 1 alpha gene of Drosophila melanogaster is developmentally regulated and the kinase activity of the protein induced by DNA damage

1996 ◽  
Vol 109 (7) ◽  
pp. 1847-1856 ◽  
Author(s):  
J.A. Santos ◽  
E. Logarinho ◽  
C. Tapia ◽  
C.C. Allende ◽  
J.E. Allende ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Kinase Activity ◽  
Early Embryo ◽  
Casein Kinase ◽  
Early Embryogenesis ◽  
Kinase Gene ◽  
Casein Kinase 1 ◽  
Adult Females ◽  
Early Embryos

We report the molecular cloning and characterisation of the first CK1(casein kinase) gene of Drosophila melanogaster (dmCK1). The protein sequence (DMCK1) shares significant homology with other mammalian CK1 protein kinases of the alpha sub-class. The dmCK1 gene is expressed only in adult females and during early embryonic development as a single transcript. Western blot analysis of total protein extracts of different stages of development show that the gene product is likewise present during early embryogenesis and in adult females. Kinase activity studies show that DMCK1 is active when in vitro translated but inactive when immunoprecipitated from total early embryo extracts. However, after dephosphorylation treatment the immunoprecipitates show high kinase activity. More significantly, DMCK1 kinase activity present in the immunoprecipitates can be specifically activated by gamma-irradiation of early embryos. Also, when DMCK1 is immunoprecipitated after irradiation it appears to undergo phosphorylation. Immunolocalization of DMCK1 in early embryos shows that the protein is predominantly cytoplasmic but after irradiation there is a significant relocalization to the interphase nucleus. The results suggest a possible requirement of the Drosophila CK1 alpha for mechanisms associated with DNA repair during early embryogenesis.

scholarly journals Chromosome damage and early developmental arrest caused by the Rex element of Drosophila melanogaster.

Genetics ◽  
1994 ◽  
Vol 138 (2) ◽  
pp. 401-411 ◽  
Author(s):  
L G Robbins ◽  
S Pimpinelli
Keyword(s):  
Drosophila Melanogaster ◽  
Ribosomal Dna ◽  
Maternal Effect ◽  
Cell Biology ◽  
Chromosome Breakage ◽  
Chromosome Damage ◽  
Specific Chromosome ◽  
Developmental Arrest ◽  
Early Embryos ◽  
Early Developmental

Abstract Rex (Ribosomal exchange) is a genetically identified repeated element within the ribosomal DNA (rDNA) of Drosophila melanogaster. Rex has a semidominant maternal effect that promotes exchange between and within rDNA arrays in the first few embryonic mitoses. Several of Rex's genetic properties suggest that its primary effect is rDNA-specific chromosome breakage that is resolved by recombination. We report here that rDNA crossovers are only a small, surviving minority of Rex-induced events. Cytology of embryos produced by Rex-homozygous females reveals obvious chromosome damage in at least a quarter of the embryos within the first three mitotic divisions. More than half of the embryos produced by Rex females die, and the developmental arrest is among the earliest reported for any maternal-effect lethal. The striking lethal phenotype suggests that embryos with early chromosome damage could be particularly fruitful subjects for analysis of the cell biology of early embryos.

scholarly journals Cytoplasmic incompatability in Drosophila melanogaster due to different Wolbachia genotypes

2009 ◽  
Vol 7 (2) ◽  
pp. 11-18
Author(s):  
Yury Y Ilinsky ◽  
Ilya K Zakharov
Keyword(s):  
Drosophila Melanogaster ◽  
Host Species ◽  
Cytoplasmic Incompatibility ◽  
Male Killing ◽  
Do So

Wolbachia is an endosymbiont prevalent among different arthropoda and in some Nematoda species. The bacteria ads across the populations of host species via induction of reproductive abnormalities: cytoplasmic incompatability, parthenogenesis, feminization, and male killing. we estimated the level of cytoplasmic incompatability in Drosophila melanogaster caused by three most prevalent Wolbachia genotypes, namely wmel, wmelCS и wmelCS2. wmel and wmelCS genotypes were shown to cause mild cytoplasmic incompatibility (< 10 %), while wmelCS Wolbachia were unable to do so. Possible mechanisms of sustainability of Wolbachia in the populations of Drosophila melanogaster are discussed.<table border="0" cellpadding="3" cellspacing="0"> <tbody> </tbody></table>

scholarly journals Transcription and splicing dynamics during early Drosophila development

RNA ◽  
2021 ◽  
pp. rna.078933.121
Author(s):  
Pedro Prudencio ◽  
Rosina Savisaar ◽  
Kenny Rebelo ◽  
Rui Goncalo Martinho ◽  
Maria Carmo-Fonseca
Keyword(s):  
Rna Polymerase Ii ◽  
Developmental Stages ◽  
Early Embryo ◽  
Drosophila Embryo ◽  
Dynamic Features ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Cleavage And Polyadenylation ◽  
Dna Template ◽  
Early Developmental

Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nucleotides of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

scholarly journals Programmable RNA Targeting using CasRx in Flies

2020 ◽  
Author(s):  
Anna Buchman ◽  
Dan J. Brogan ◽  
Ruichen Sun ◽  
Ting Yang ◽  
Patrick Hsu ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Genome Engineering ◽  
Developmental Stages ◽  
Comprehensive Evaluation ◽  
Model Organism ◽  
Endogenous Expression ◽  
Current State ◽  
Rna Targeting ◽  
Transcript Cleavage ◽  
Early Developmental

AbstractCRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof-of-principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on Cas ribonuclease function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact in the field. Here we report on our efforts to develop a programmable platform for RNA-targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster. By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster, paving the way for future optimization of the system.

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