Mutant Egg Research Articles

Cup regulates oskar mRNA stability during oogenesis

The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form.

Drosophila Cyclin G Is a Regulator of the Notch Signalling Pathway during Wing Development.

Notch signalling regulates a multitude of differentiation processes during Drosophila development. For example, Notch activity is required for proper wing vein differentiation which is hampered in mutants of either the receptor Notch, the ligand Delta or the antagonist Hairless. Moreover, the Notch pathway is involved in several aspects of Drosophila oogenesis as well. We have identified Drosophila Cyclin G (CycG) as a molecular interaction partner of Hairless, the major antagonist in the Notch signalling pathway, in vitro and in vivo. Loss of CycG was shown before to cause female sterility and to disturb the architecture of the egg shell. Nevertheless, Notch dependent processes during oogenesis appeared largely unaffected in cycG mutant egg chambers. Loss of CycG modified the dominant wing phenotypes of Notch, Delta and Hairless mutants. Whereas the Notch loss of function phenotype was ameliorated by a loss of CycG, the phenotypes of either Notch gain of function or of Delta or Hairless loss of function were enhanced. In contrast, loss of CycG had only a minor effect on the wing vein phenotype of mutants affecting the EGFR signalling pathway emphasizing the specificity of the interaction of CycG and Notch pathway members.

A Mutation in fat2 Uncouples Tissue Elongation from Global Tissue Rotation.

Global tissue rotation was proposed as a morphogenetic mechanism controlling tissue elongation. In Drosophila ovaries, global tissue rotation of egg chambers coincides with egg chamber elongation. Egg chamber rotation was put forward to result in circumferential alignment of extracellular fibers. These fibers serve as molecular corsets to restrain growth of egg chambers perpendicular to the anteroposterior axis, thereby leading to the preferential egg chamber elongation along this axis. The atypical cadherin Fat2 is required for egg chamber elongation, rotation, and the circumferential alignment of extracellular fibers. Here, we have generated a truncated form of Fat2 that lacks the entire intracellular region. fat2 mutant egg chambers expressing this truncated protein fail to rotate yet display normal extracellular fiber alignment and properly elongate. Our data suggest that global tissue rotation, even though coinciding with tissue elongation, is not a necessary prerequisite for elongation.

The Structural Variation Is Associated with the Embryonic Lethality of a Novel Red Egg Mutant Fuyin-lre of Silkworm, Bombyx mori.

Bombyx mori presents several types of egg color mutations, all of which have been extensively discussed in sericulture. While the red egg mutation has been previously observed, lethal red-egg mutants have not been reported. In the present work, the red egg mutant Fuyin-lre (Fuyin-lethal red egg) was discovered from the Fuyin germplasm resource of B. mori. This mutant features red-colored eggs and embryonic lethality. Genetic analysis showed that Fuyin-lre follows recessive inheritance, with the red egg gene re governing the egg color, and the embryonic lethality of Fuyin-lre may be caused by mutations of other genes closely linked to re. Digital gene expression (DGE) was employed to compare the transcription profiles of Fuyin and Fuyin-lre eggs after 24 and 48 h of incubation. A total of 48 differentially expressed genes followed the same expression patterns in both groups at both time points (FDR < 0.01 and log 2 Ratio ≥ 1). Further analyses indicated that 8 out of the 48 genes (including re) were closely linked to re. These 8 genes were highly expressed in wild-type Fuyin and the red egg mutant re but showed nearly absent expression in Fuyin-lre. Sequencing of the re gene confirmed that the re gene itself does not induce embryonic lethality, and structure analysis showed that the structural variation of the region where the 8 genes were located may be associated with the embryonic lethality of Fuyin-lre. The present work provides a good foundation for future studies on the mechanism of embryonic lethality and embryonic development in Fuyin-lre.

Riboflavin Binding Protein and Riboflavin Transport to Hen Eggs as an Educational Tool

Riboflavin Binding Protein (RBP) is a stable glycophosphoprotein abundant in the eggs of many oviparous species, including chickens. The function of the protein is to transfer riboflavin (Rf) to the developing embryo, and recent evidence suggests it also binds copper. Because of its bright yellow color and fluorescence, Rf transport serves as an excellent model system. A natural mutant strain of white leghorn chickens, containing a site mutation, is unable to synthesize RBP and thus the eggs are devoid of Riboflavin. We are currently investigating the protein and mutant egg use for both an undergraduate biochemistry lab (1) as well as an outreach project for K-12 students (2). The undergraduate research-style experiment for advanced biochemistry lab students is a useful exercise in learning protein purification techniques, including dialysis, salt precipitation and chromatography. Because of the holoRBP yellow color, it is easy to follow, and its stability renders it possible to carry out steps over time at room temperature. We have modified a standard procedure and students have successfully purified protein. Using the mutant and regular chicken eggs we have modified an education series as an informative and didactic approach towards problem-based learning. Our main motive was to perform outreach to K-12 with an interactive experiment featuring the importance of vitamins and the mechanisms of molecular transport.

Border Cell Migration: A Model System for Live Imaging and Genetic Analysis of Collective Cell Movement.

Border cell migration in the Drosophila ovary has emerged as a genetically tractable model for studying collective cell movement. Over many years border cell migration was exclusively studied in fixed samples due to the inability to culture stage 9 egg chambers in vitro. Although culturing late-stage egg chambers was long feasible, stage 9 egg chambers survived only briefly outside the female body. We identified culture conditions that support stage 9 egg chamber development and sustain complete migration of border cells ex vivo. This protocol enables one to compare the dynamics of egg chamber development in wild-type and mutant egg chambers using time-lapse microscopy and taking advantage of a multiposition microscope with a motorized imaging stage. In addition, this protocol has been successfully used in combination with fluorescence resonance energy transfer biosensors, photo-activatable proteins, and pharmacological agents and can be used with wide-field or confocal microscopes in either an upright or an inverted configuration.

Endophilin B is required for the Drosophila oocyte to endocytose yolk downstream of Oskar

The nutritional environment is crucial for Drosophila oogenesis in terms of controlling hormonal conditions that regulate yolk production and the progress of vitellogenesis. Here, we discovered that Drosophila Endophilin B (D-EndoB), a member of the endophilin family, is required for yolk endocytosis as it regulates membrane dynamics in developing egg chambers. Loss of D-EndoB leads to yolk content reduction, similar to that seen in yolkless mutants, and also causes poor fecundity. In addition, mutant egg chambers exhibit an arrest at the previtellogenic stage. D-EndoB displayed a crescent localization at the oocyte posterior pole in an Oskar-dependent manner; however, it did not contribute to pole plasm assembly. D-EndoB was found to partially colocalize with Long Oskar and Yolkless at the endocytic membranes in ultrastructure analysis. Using an FM4-64 dye incorporation assay, D-EndoB was also found to promote endocytosis in the oocyte. When expressing the full-length D-endoB(FL) or D-endoB(ΔSH3) mutant transgenes in oocytes, the blockage of vitellogenesis and the defect in fecundity in D-endoB mutants was restored. By contrast, a truncated N-BAR domain of the D-EndoB only partially rescued these defects. Taken together, these results allow us to conclude that D-EndoB contributes to the endocytic activity downstream of Oskar by facilitating membrane dynamics through its N-BAR domain in the yolk uptake process, thereby leading to normal progression of vitellogenesis.

A Meiosis-Specific Form of the APC/C Promotes the Oocyte-to-Embryo Transition by Decreasing Levels of the Polo Kinase Inhibitor Matrimony

Author SummaryDespite their many differences, the meiotic and mitotic divisions of the early embryo take place within the same cytoplasmic space. The oocyte-to-embryo transition is the process by which an oocyte, which initially undergoes meiosis, becomes “adapted” to support the rapid mitotic divisions of embryogenesis. This involves fertilization as well as the stockpiling of proteins and mRNA for the transcriptionally silent early embryo. The Anaphase Promoting Complex/Cyclosome (APC/C) is a large protein complex that is active during both mitosis and meiosis and is responsible for targeting certain proteins for degradation. The discovery of the existence of APC/C activators that are present only during meiosis hinted at the possibility that this complex also functions to regulate protein degradation during the oocyte-to-embryo transition. Here we study Cortex, a female- and meiosis-specific activator of the APC/C in the fruit fly Drosophila melanogaster. We find that Cortex activity is necessary for the degradation of Matrimony, a key regulator of female meiosis in Drosophila. Matrimony itself inhibits Polo kinase, another important regulator of both mitosis and meiosis that also functions in chromosome segregation, centrosome dynamics, and cytokinesis. When excess Matrimony protein is not removed from the early embryo, developmental defects arise. Together our findings demonstrate that the precise regulation of Matrimony levels in the egg is necessary for the switch from meiosis to mitosis.

MiR-989 Is Required for Border Cell Migration in the Drosophila Ovary

microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by destabilizing target transcripts and/or inhibiting their translation. miRNAs are thought to have roles in buffering gene expression to confer robustness. miRNAs have been shown to play important roles during tissue development to control cell proliferation, differentiation and morphogenesis. Many miRNAs are expressed in the germ line of Drosophila, and functions have been reported for a few miRNAs in maintenance of stem cell proliferation during oogenesis. Here, we analyse the function of Drosophila miR-989 in oogenesis. miR-989 is abundant in ovaries. Mutants lacking miR-989 did not display gross abnormalities affecting egg chamber formation or maturation. However, the migration of the border cell cluster was severely delayed in miR-989 mutant egg chambers. We demonstrate that miR-989 function is required in the somatic cells in the egg chamber, not in germ line cells for border cell migration. Loss of miR-989 from a fraction of the border cell cluster was sufficient to impair cluster migration as a whole, suggesting a role in border cells. Gene ontology analysis reveals that many predicted miR-989 target mRNAs are implicated in regulating cell migration, cell projection morphogenesis, cell adhesion as well as receptor tyrosine kinase and ecdysone signalling, consistent with an important regulatory role for miR-989 in border cell migration.

Drosophila CIAPIN1 homologue is required for follicle cell proliferation and survival

The conserved cytokine-induced apoptosis inhibitor-1 (CIAPIN1) gene has been implicated in several processes, such as apoptosis, cell division, angiogenesis and Fe/S protein biogenesis. In this study, we identified the Drosophila CIAPIN1 homologue (D-CIAPIN1) and studied its role in ovarian development. We found that D-CIAPIN1 is conserved as it can complement the nonviability of the yeast CIAPIN1-deletion strain. Several D-CIAPIN1 alleles were identified, including one allele in which that codon encoding the highly conserved twin cysteine CX2 C motif is mutated, demonstrating for the first time the importance of this motif to protein function. We demonstrated D-CIAPIN1 is an essential gene required for ovarian development. We found that D-CIAPIN1 female mutants are sterile, containing rudimentary ovaries. We noted a decrease in follicle cell numbers in D-CIAPIN1 mutant egg chambers. We further demonstrated that the decrease in follicle cell numbers in D-CIAPIN1 mutants is due to a reduced mitotic index and enhanced cell death. Our study reveals that D-CIAPIN1 is essential for egg chamber development and is required for follicle cell proliferation and survival.

Mitochondrial GCD1 Dysfunction Reveals Reciprocal Cell-to-Cell Signaling during the Maturation of Arabidopsis Female Gametes

Cell-to-cell communication in embryo sacs is thought to regulate the development of female gametes in flowering plants, but the details remain poorly understood. Here, we report a mitochondrial protein, GAMETE CELL DEFECTIVE 1 (GCD1), enriched in gametophytes that is essential for final maturation of female gametes. Using Arabidopsis gcd1 mutants, we found that final maturation of the egg and central cells is not required for double fertilization but is necessary for embryogenesis initiation and endosperm development. Furthermore, nonautonomous effects, observed when GCD1 or AAC2 function is disrupted, suggest that mitochondrial function influences reciprocal signaling between central and egg cells to regulate maturation of the partner (egg or central) cell. Our findings confirm that cell-to-cell communication is important in functional maturation of female gametic cells and suggest that both egg and central cells sense and transmit their mitochondrial metabolic status as animportant cue that regulates the coordination of gamete maturation.

Drosophila Argonaute 1 and its miRNA biogenesis partners are required for oocyte formation and germline cell division

Argonaute 1 (Ago1) is a member of the Argonaute/PIWI protein family involved in small RNA-mediated gene regulation. In Drosophila, Ago1 plays a specific role in microRNA (miRNA) biogenesis and function. Previous studies have demonstrated that Ago1 regulates the fate of germline stem cells. However, the function of Ago1 in other aspects of oogenesis is still elusive. Here we report the function of Ago1 in developing egg chambers. We find that Ago1 protein is enriched in the oocytes and is also highly expressed in the cytoplasm of follicle cells. Clonal analysis of multiple ago1 mutant alleles shows that many mutant egg chambers contain only 8 nurse cells without an oocyte which is phenocopied in dicer-1, pasha and drosha mutants. Our results suggest that Ago1 and its miRNA biogenesis partners play a role in oocyte determination and germline cell division in Drosophila.

Conserved requirement for DEAD-box RNA helicase Gemin3 in Drosophila oogenesis

BackgroundDEAD-box RNA helicase Gemin3 is an essential protein since its deficiency is lethal in both vertebrates and invertebrates. In addition to playing a role in transcriptional regulation and RNA silencing, as a core member of the SMN (survival of motor neurons) complex, Gemin3 is required for the biogenesis of spliceosomal snRNPs (small nuclear ribonucleoproteins), and axonal mRNA metabolism. Studies in the mouse and C. elegans revealed that loss of Gemin3 function has a negative impact on ovarian physiology and development.FindingsThis work reports on the generation and characterisation of gemin3 mutant germline clones in Drosophila adult females. Gemin3 was found to be required for the completion of oogenesis and its loss led to egg polarity defects, oocyte mislocalisation, and abnormal chromosome morphology. Canonical Cajal bodies were absent in the majority of gemin3 mutant egg chambers and histone locus bodies displayed an atypical morphology. snRNP distribution was perturbed so that on gemin3 loss, snRNP cytoplasmic aggregates (U bodies) were only visible in wild type.ConclusionsThese findings establish a conserved requirement for Gemin3 in Drosophila oogenesis. Furthermore, in view of the similarity to the phenotypes described previously in smn mutant germ cells, the present results confirm the close functional relationship between SMN and Gemin3 on a cellular level.

Histone H3K9 Trimethylase Eggless Controls Germline Stem Cell Maintenance and Differentiation

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms.

Differentially expressed genes in white egg 2 mutant of silkworm, Bombyx mori, at early embryo development stages

White egg 2 is one of white egg mutants in silkworm, whose molecular mechanism remains unknown so far. In order to obtain an overall view on gene expression profiles at early embryo development stages, the white egg 2 near-isogenic line was constructed and the whole-genome of silkworm microarray system containing 21375 predicted genes from the silkworm whole genome sequence was employed to investigate gene expression profiles at 0, 24 and 48 h post oviposition between white egg 2 mutant and normal black egg strain. At 24 h post oviposition, 49 genes exhibited at least 2.0 fold differences at expression level, including 24 up-regulated genes and 25 down-regulated genes while at 48 h post oviposition, 52 genes, including 23 up-regulated genes and 29 down-regulated genes were expressed differentially over 2.0 change fold. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that nine differentially expression genes were involved in nine significant (p<0.05) pathways at 24 h post oviposition and 24 significant pathways at 48 h post oviposition, respectively. These pathways were related to amino acid metabolism, sugar metabolism, and series of major physiological metabolism. Our results hopefully shed light on the further study of molecular mechanism of white egg 2 mutant. Key words : Bombyx mori , white egg 2 mutant, microarray, embryo, differentially expressed gene.

Maelstrom coordinates microtubule organization during Drosophila oogenesis through interaction with components of the MTOC

The establishment of body axes in multicellular organisms requires accurate control of microtubule polarization. Mutations in Drosophila PIWI-interacting RNA (piRNA) pathway genes often disrupt the axes of the oocyte. This results from the activation of the DNA damage checkpoint factor Checkpoint kinase 2 (Chk2) due to transposon derepression. A piRNA pathway gene, maelstrom (mael), is critical for the establishment of oocyte polarity in the developing egg chamber during Drosophila oogenesis. We show that Mael forms complexes with microtubule-organizing center (MTOC) components, including Centrosomin, Mini spindles, and γTubulin. We also show that Mael colocalizes with αTubulin and γTubulin to centrosomes in dividing cyst cells and follicle cells. MTOC components mislocalize in mael mutant germarium and egg chambers, leading to centrosome migration defects. During oogenesis, the loss of mael affects oocyte determination and induces egg chamber fusion. Finally, we show that the axis specification defects in mael mutants are not suppressed by a mutation in mnk, which encodes a Chk2 homolog. These findings suggest a model in which Mael serves as a platform that nucleates other MTOC components to form a functional MTOC in early oocyte development, which is independent of Chk2 activation and DNA damage signaling.

A systematic analysis ofDrosophilaTUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors

PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) form the core of a gonad-specific small RNA silencing pathway that protects the animal genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology as TUDOR domains of various proteins bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analysed the Drosophila TUDOR protein family and identified four previously not characterized TUDOR domain-containing proteins (CG4771, CG14303, CG11133 and CG31755) as essential piRNA pathway factors. We characterized CG4771 (Vreteno) in detail and demonstrate a critical role for this protein in primary piRNA biogenesis. Vreteno physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Vreteno also interacts with the Tdrd12 orthologues CG11133 (Brother of Yb) and CG31755 (Sister of Yb), which are essential for the primary piRNA pathway in the germline and probably replace the function of the related but soma-specific factor Yb.

Genetic evidence for antagonism between Pak protein kinase and Rho1 small GTPase signaling in regulation of the actin cytoskeleton during Drosophila oogenesis.

During Drosophila oogenesis, basally localized F-actin bundles in the follicle cells covering the egg chamber drive its elongation along the anterior-posterior axis. The basal F-actin of the follicle cell is an attractive system for the genetic analysis of the regulation of the actin cytoskeleton, and results obtained in this system are likely to be broadly applicable in understanding tissue remodeling. Mutations in a number of genes, including that encoding the p21-activated kinase Pak, have been shown to disrupt organization of the basal F-actin and in turn affect egg chamber elongation. pak mutant egg chambers have disorganized F-actin distribution and remain spherical due to a failure to elongate. In a genetic screen to identify modifiers of the pak rounded egg chamber phenotype several second chromosome deficiencies were identified as suppressors. One suppressing deficiency removes the rho1 locus, and we determined using several rho1 alleles that removal of a single copy of rho1 can suppress the pak phenotype. Reduction of any component of the Rho1-activated actomyosin contractility pathway suppresses pak oogenesis defects, suggesting that Pak counteracts Rho1 signaling. There is ectopic myosin light chain phosphorylation in pak mutant follicle cell clones in elongating egg chambers, probably due at least in part to mislocalization of RhoGEF2, an activator of the Rho1 pathway. In early egg chambers, pak mutant follicle cells have reduced levels of myosin phosphorylation and we conclude that Pak both promotes and restricts myosin light chain phosphorylation in a temporally distinct manner during oogenesis.

MIRO1 influences the morphology and intracellular distribution of mitochondria during embryonic cell division in Arabidopsis

Regulating the morphology and intracellular distribution of mitochondria is essential for embryo development in animals. However, the importance of such regulation is not clearly defined in plants. The evolutionarily conserved Miro proteins are known to be involved in the regulation of mitochondrial morphology and motility. We previously demonstrated that MIRO1, an Arabidopsis thaliana orthologue of the Miro protein, is required for embryogenesis. An insertional mutation in the MIRO1 gene causes arrest of embryonic cell division, leading to abortion of the embryo at an early stage. Here we investigated the role of MIRO1 in the regulation of mitochondrial behaviour in egg cells and early-stage embryos using GFP-labeled mitochondria. Two-photon laser scanning microscopy revealed that, in miro1 mutant egg cells, mitochondria are abnormally enlarged, although egg cell formation is nearly unaffected. After fertilization and subsequent zygotic cell division, the homozygous miro1 mutant two-celled embryo contained a significantly reduced number of mitochondria in its apical cell compared with the wild type, suggesting that the miro1 mutation inhibits proper intracellular distribution of mitochondria, leading to an arrest of embryonic cell division. Our findings suggest that proper mitochondrial morphology and intracellular distribution are maintained by MIRO1 and are vital for embryonic cell division.

Characteristics of the vit mutant egg in Bombyx mori

Characteristics of the vit mutant egg in Bombyx mori