Expression and cellular localization of the Toucan protein during Drosophila oogenesis

The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.     

Somatic signaling mediated by fs(1)Yb is essential for germline stem cell maintenance during Drosophila oogenesis

Drosophila oogenesis starts when a germline stem cell divides asymmetrically to generate a daughter germline stem cell and a cystoblast that will develop into a mature egg. We show that the fs(1)Yb gene is essential for the maintenance of germline stem cells during oogenesis. We delineate fs(1)Yb within a 6.4 kb genomic region by transgenic rescue experiments. fs(1)Yb encodes a 4.1 kb RNA that is present in the third instar larval, pupal and adult stages, consistent with its role in regulating germline stem cells during oogenesis. Germline clonal analysis shows that all fs(1)Yb mutations are soma-dependent. In the adult ovary, fs(1)Yb is specifically expressed in the terminal filament cells, suggesting that fs(1)Yb acts in these signaling cells to maintain germline stem cells. fs(1)Yb encodes a novel hydrophilic protein with no potential signal peptide or transmembrane domains, suggesting that this protein is not itself a signal but a key component of the signaling machinery for germline stem cell maintenance.     

Femcoat, a novel eggshell protein in Drosophila: functional analysis by double stranded RNA interference.

In Drosophila oogenesis, follicle cells derived from somatic tissue surround the oocyte and play key roles in generating properly polarized oocytes. During the later steps of oogenesis, follicle cells are involved in secretion of proteins that make the eggshell, an essential protective layer for the oocyte. Although studies on the signaling processes to make polarized oocytes have been progressed very far, studies on the mechanisms for eggshell formation is not clear yet. To elucidate the underlying mechanism in eggshell formation, we used a differential display screen to isolate genes that are specifically expressed during the later stages of oogenesis, and isolated a novel gene, Femcoat. Femcoat encodes a putative chorion membrane protein that contains many highly charged residues and has a putative signal peptide. Femcoat is expressed specifically in the follicle cells with a punctate staining pattern typical of secreted proteins, and becomes cross-linked heavily at the final steps of oogenesis. To identify the developmental role of Femcoat in eggshell formation, we performed an inducible double stranded RNA mediated interference (dsRNAi) method to specifically reduce Femcoat expression during oogenesis in adult flies. Electron microscopy analysis of egg chambers from these flies showed defects in chorion formation. These pieces of evidence demonstrated that Femcoat is necessary for eggshell formation, especially during chorion synthesis. Our results demonstrate that inducible dsRNAi analysis can be effective in determining the developmental function of novel genes.     

Involvement of EDTP, an egg-derived tyrosine phosphatase, in the early development of Drosophila melanogaster

Previously, we purified a novel protein tyrosine phosphatase from eggs of the flesh fly, Sarcophaga peregrina. This protein tyrosine phosphatase, named egg-derived tyrosine phosphatase (EDTP), is expressed during oogenesis and early embryogenesis but is rapidly degraded in middle embryogenesis by lysosomal cathepsin L. Here, we demonstrate the requirement of EDTP in the development of the fruit fly, Drosophila melanogaster. Deletion of the Drosophila EDTP gene using transposase-catalyzed imprecise excision resulted in homozygous lethals during embryogenesis. Additionally, germline clones generated using the FLP-FRT-ovo(D) system showed severe defects in ovarian development during oogenesis. These results indicate that the Drosophila EDTP gene is crucial in oogenesis and embryogenesis.     

SH3 domain-mediated binding of the Drk protein to Dos is an important step in signaling of Drosophila receptor tyrosine kinases

The vasa gene (vas) is essential for germline development in Drosophila melanogaster. Zygotic vas is expressed in pole cells earlier than any other pole cell-expressing genes thus far identified, and VAS protein is continuously present in germline cells throughout development. Here, we report the identification of a regulatory region that directs germline-specific vas expression. A genomic fragment containing the vas locus was linked to enhanced green fluorescent protein (egfp)-vas fusion gene, and the resulting gene was introduced into fly genome. Developmental vas expression was assessed by monitoring the expression of EGFP-VAS in these transformants. The spatio-temporal expression pattern of EGFP-VAS is essentially identical to that of endogenous VAS throughout germline development. By dissecting the vas promoter, we identified a 40-bp regulatory element, which is necessary and sufficient for germline-specific expression during oogenesis. This region interacts specifically with ovarian protein(s). Furthermore, this region is also required for vas expression in pole cells during embryogenesis. These results suggest that a similar mechanism regulates vas expression both in oogenesis and embryogenesis.     

Germline cell death is inhibited by P-element insertions disrupting the dcp-1/pita nested gene pair in Drosophila

Germline cell death in Drosophila oogenesis is controlled by distinct signals. The death of nurse cells in late oogenesis is developmentally regulated, whereas the death of egg chambers during mid-oogenesis is induced by environmental stress or developmental abnormalities. P-element insertions in the caspase gene dcp-1 disrupt both dcp-1 and the outlying gene, pita, leading to lethality and defective nurse cell death in late oogenesis. By isolating single mutations in the two genes, we have found that the loss of both genes contributes to this ovary phenotype. Mutants of pita, which encodes a C2H2 zinc-finger protein, are homozygous lethal and show dumpless egg chambers and premature nurse cell death in germline clones. Early nurse cell death is not observed in the dcp-1/pita double mutants, suggesting that dcp-1+ activity is required for the mid-oogenesis cell death seen in pita mutants. dcp-1 mutants are viable and nurse cell death in late oogenesis occurs normally. However, starvation-induced germline cell death during mid-oogenesis is blocked, leading to a reduction and inappropriate nuclear localization of the active caspase Drice. These findings suggest that the combinatorial loss of pita and dcp-1 leads to the increased survival of abnormal egg chambers in mutants bearing the P-element alleles and that dcp-1 is essential for cell death during mid-oogenesis.     

PAP- and GLD-2-type poly(A) polymerases are required sequentially in cytoplasmic polyadenylation and oogenesis in Drosophila

Cytoplasmic polyadenylation has an essential role in activating maternal mRNA translation during early development. In vertebrates, the reaction requires CPEB, an RNA-binding protein and the poly(A) polymerase GLD-2. GLD-2-type poly(A) polymerases form a family clearly distinguishable from canonical poly(A) polymerases (PAPs). In Drosophila, canonical PAP is involved in cytoplasmic polyadenylation with Orb, the Drosophila CPEB, during mid-oogenesis. We show that the female germline GLD-2 is encoded by wispy. Wispy acts as a poly(A) polymerase in a tethering assay and in vivo for cytoplasmic polyadenylation of specific mRNA targets during late oogenesis and early embryogenesis. wispy function is required at the final stage of oogenesis for metaphase of meiosis I arrest and for progression beyond this stage. By contrast, canonical PAP acts with Orb for the earliest steps of oogenesis. Both Wispy and PAP interact with Orb genetically and physically in an ovarian complex. We conclude that two distinct poly(A) polymerases have a role in cytoplasmic polyadenylation in the female germline, each of them being specifically required for different steps of oogenesis.     

Cytotype control of Drosophila P element transposition: the 66 kd protein is a repressor of transposase activity

Drosophila P transposable elements encode two proteins, an 87 kd transposase protein and a 66 kd protein that has been hypothesized to repress transposition. We have made germline transformants carrying modified P element derivatives that encode only the 66 kd protein and shown that these elements repress transposase activity in both the germline and the soma. The position of these elements in the genome quantitatively affected their ability to negatively regulate transposase and to express the 66 kd protein. Single 66 kd element-containing strains did not exhibit the maternal inheritance of P cytotype characteristic of P strains. However, we demonstrated that a true P strain produced higher levels of the 66 kd protein during oogenesis than single 66 kd P elements. Thus, the expression of the 66 kd repressor during oogenesis may be a major determinant of the maternal effect of P cytotype.     

Soma-to-germline interactions during Drosophila oogenesis are influenced by dose-sensitive interactions between cut and the genes cappuccino, ovarian tumor and agnostic.

The cut gene of Drosophila melanogaster encodes a homeodomain protein that regulates a soma-to-germline signaling pathway required for proper morphology of germline cells during oogenesis. cut is required solely in somatic follicle cells, and when cut function is disrupted, membranes separating adjacent nurse cells break down and the structural integrity of the actin cytoskeleton is compromised. To understand the mechanism by which cut expression influences germline cell morphology, we determined whether binucleate cells form by defective cytokinesis or by fusion of adjacent cells. Egg chambers produced by cut, cappuccino, and chickadee mutants contained binucleate cells in which ring canal remnants stained with antibodies against Hu-li tai shao and Kelch, two proteins that are added to ring canals after cytokinesis is complete. In addition, defects in egg chamber morphology were observed only in middle to late stages of oogenesis, suggesting that germline cell cytokineses were normal in these mutants. cut exhibited dose-sensitive genetic interactions with cappuccino but not with chickadee or other genes that regulate cytoskeletal function, including armadillo, spaghetti squash, quail, spire, Src64B, and Tec29A. Genomic regions containing genes that cooperate with cut were identified by performing a second-site noncomplementing screen using a collection of chromosomal deficiencies. Sixteen regions that interact with cut during oogenesis and eight regions that interact during the development of other tissues were identified. Genetic interactions between cut and the ovarian tumor gene were identified as a result of the screen. In addition, the gene agnostic was found to be required during oogenesis, and genetic interactions between cut and agnostic were revealed. These results demonstrate that a signaling pathway regulating the morphology of germline cells is sensitive to genetic doses of cut and the genes cappuccino, ovarian tumor, and agnostic. Since these genes regulate cytoskeletal function and cAMP metabolism, the cut-mediated pathway functionally links these elements to preserve the cytoarchitecture of the germline cells.     

Translational complexity of the fragile x mental retardation protein: insights from the fly

Through the awesome power of Drosophila genetics, two recent studies reveal novel mechanisms by which the Fragile X Mental Retardation protein regulates selective mRNA translation, controlling key steps of germline development during oogenesis and neuronal development during synaptogenesis.     

Stage-specific localization of the small heat shock protein Hsp27 during oogenesis inDrosophila melanogaster

The developmental and heat shock-induced expression of the small heat shock protein Hsp27 was investigated by confocal microscopy of whole-mount immunostained preparations of ovarioles during oogenesis inDrosophila melanogaster. In unstressed flies, Hsp27 was mainly associated with germline nurse cells throughout egg development. A small group of somatic follicle cells also expressed Hsp27 specifically at stages 8 to 10 of oogenesis. Interestingly, this Hsp showed a different intracellular localization depending on the stages of egg chamber development. Thus Hsp27 was localized in the nucleus of nurse cells during the first stages of oogenesis (from germarium to stage 6) whereas it showed a perinuclear and cytoplasmic localization from stage 8. After a heat shock, Hsp27 accumulated in somatic follicle cells surrounding the egg chamber whereas the expression of this small Hsp did not seem to be enhanced in nurse cells. The stage-dependent pattern of intracellular localization of Hsp27 observed in nurse cells of unstressed flies was also observed following heat shock. At late stages of oogenesis, Hsp27 also showed a perinuclear distribution in follicle and nurse cells after heat stress. These observations suggest that different factors may modulate the expression and intracellular distribution of Hsp27. This modulation may be associated with the specific activities occurring in each particular cell type throughout oogenesis during both normal development and under heat shock conditions. Edited by: E.R. Schmidt     

Mago Nashi and Tsunagi/Y14, respectively, regulate Drosophila germline stem cell differentiation and oocyte specification

A protein complex consisting of Mago Nashi and Tsunagi/Y14 is required to establish the major body axes and for the localization of primordial germ cell determinants during Drosophila melanogaster oogenesis. The Mago Nashi:Tsunagi/Y14 heterodimer also serves as the core of the exon junction complex (EJC), a multiprotein complex assembled on spliced mRNAs. In previous studies, reduced function alleles of mago nashi and tsunagi/Y14 were used to characterize the roles of the genes in oogenesis. Here, we investigated mago nashi and tsunagi/Y14 using null alleles and clonal analysis. Germline clones lacking mago nashi function divide but fail to differentiate. The mago nashi null germline stem cells produce clones over a period of at least 11 days, suggesting that mago nashi is not necessary for stem cell self-renewal. However, germline stem cells lacking tsunagi/Y14 function are indistinguishable from wild type. Additionally, in tsunagi/Y14 null germline cysts, centrosomes and oocyte-specific components fail to concentrate within a single cell and oocyte fate is not restricted to a single cell. Together, our results suggest not only that mago nashi is required for germline stem cell differentiation but that surprisingly mago nashi functions independently of tsunagi/Y14 in this process. On the other hand, Tsunagi/Y14 is essential for restricting oocyte fate to a single cell and may function with mago nashi in this process.     

Stage-specific regulation of caspase activity in drosophila oogenesis

In Drosophila oogenesis, the programmed cell death of germline cells occurs predominantly at three distinct stages. These cell deaths are subject to distinct regulatory controls, as cell death during early and midoogenesis is stress-induced, whereas the cell death of nurse cells in late oogenesis is developmentally regulated. In this report, we show that the effector caspase Drice is activated during cell death in both mid- and late oogenesis, but that the level and localization of activity differ depending on the stage. Active Drice formed localized aggregates during nurse cell death in late oogenesis; however, active Drice was found more ubiquitously and at a higher level during germline cell death in midoogenesis. Because Drice activity was limited in late oogenesis, we examined whether another effector caspase, Dcp-1, could drive the unique morphological events that occur normally in late oogenesis. We found that premature activation of the effector caspase, Dcp-1, resulted in a disappearance of filamentous actin, rather than the formation of actin bundles, suggesting that Dcp-1 activity must also be restrained in late oogenesis. Overexpression of the caspase inhibitor DIAP1 suppressed cell death induced by Dcp-1 but had no effect on cell death during late oogenesis. This limited caspase activation in dying nurse cells may prevent destruction of the nurse cell cytoskeleton and the connected oocyte.     

普通针毛蕨颈卵器和卵的发育

蕨类植物卵发生是有性生殖研究的重要内容。真水龙骨类II的卵发生尚无人研究, 本文对该类群中的普通针毛蕨(Macrothelypteris torresiana)卵发生过程进行了光镜和透射电镜研究。结果显示: 卵细胞刚形成时, 与腹沟细胞紧密相连, 随着卵进一步发育, 卵细胞和腹沟细胞之间逐渐形成分离腔, 但孔区处卵细胞和腹沟细胞始终相连。随后, 卵细胞上表面有不定型物质堆积在质膜外, 形成一层加厚的卵膜, 孔区处没有卵膜覆盖的位置最后形成受精孔。在卵发育后期, 卵细胞核变得不规则, 近成熟时卵核产生大量核外突。卵发育过程中卵膜的出现、受精孔的产生以及核外突等特征, 与进化的真水龙骨类中其他蕨类植物的卵发生研究结果相似, 与薄囊蕨类中原始基部类群的卵发生现象有显著差别。由此可见, 普通针毛蕨属于进化类型。依据卵发生中卵膜和受精孔等特征推测原始薄囊蕨类经过里白类、桫椤类, 最终演化为水龙骨类。     

Deadlock, a novel protein of Drosophila, is required for germline maintenance, fusome morphogenesis and axial patterning in oogenesis and associates with centrosomes in the early embryo

The deadlock gene is required for a number of key developmental events in Drosophila oogenesis. Females homozygous for mutations in the deadlock gene lay few eggs and those exhibit severe patterning defects along both the anterior-posterior and dorsal-ventral axis. In this study, we analyzed eggs and ovaries from deadlock mutants and determined that deadlock is required for germline maintenance, stability of mitotic spindles, localization of patterning determinants, oocyte growth and fusome biogenesis in males and females. Deadlock encodes a novel protein which colocalizes with the oocyte nucleus at midstages of oogenesis and with the centrosomes of early embryos. Our genetic and immunohistological experiments point to a role for Deadlock in microtubule function during oogenesis.     

The shut-down gene of Drosophila melanogaster encodes a novel FK506-binding protein essential for the formation of germline cysts during oogenesis

In Drosophila melanogaster, the process of oogenesis is initiated with the asymmetric division of a germline stem cell. This division results in the self-renewal of the stem cell and the generation of a daughter cell that undergoes four successive mitotic divisions to produce a germline cyst of 16 cells. Here, we show that shut-down is essential for the normal function of the germline stem cells. Analysis of weak loss-of-function alleles confirms that shut-down is also required at later stages of oogenesis. Clonal analysis indicates that shut-down functions autonomously in the germline. Using a positional cloning approach, we have isolated the shut-down gene. Consistent with its function, the RNA and protein are strongly expressed in the germline stem cells and in 16-cell cysts. The RNA is also present in the germ cells throughout embryogenesis. shut-down encodes a novel Drosophila protein similar to the heat-shock protein-binding immunophilins. Like immunophilins, Shut-down contains an FK506-binding protein domain and a tetratricopeptide repeat. In plants, high-molecular-weight immunophilins have been shown to regulate cell divisions in the root meristem in response to extracellular signals. Our results suggest that shut-down may regulate germ cell divisions in the germarium.     

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.     

Clonal analysis of cmp44E,which encodes a conserved putative transmembrane protein,indicates a requirement for cell viability in Drosophila

We have identified the cmp44E gene which encodes a putative multi-pass transmembrane protein that is conserved from yeast to humans. The expression of cmp44E during embryogenesis is ubiquitous with notably higher levels in the CNS and brain. It is also expressed in the germline during the germarial stages as well as several later stages of oogenesis. Utilizing a P-element insertion at the 5′ end of cmp44E we have isolated several deletions, created by imprecise excision events, which eliminate most or all of its coding region. Analysis of these deficiencies has revealed that cmp44E is an essential gene required for embryogenesis. Results obtained from germline clone analysis indicate that cmp44E is not only required in the germline stem cells early in oogenesis, but is also required in other tissues probably due to it being required for cell viability. Finally, using germline transformation, we have identified a minimal genomic fragment capable of fully rescuing a null allele of cmp44E. Dev. Genet. 23:264–274, 1998. © 1998 Wiley-Liss, Inc.