Axis formation during Drosophila oogenesis.

Recent advances shed light on the cellular processes that cooperate during oogenesis to produce a fully patterned egg, containing all the maternal information required for embryonic development. Progress has been made in defining the early steps in oocyte specification and it has been shown that progression of oogenesis is controlled by a meiotic checkpoint and requires active maintenance of the oocyte cell fate. The function of Gurken signalling in patterning the dorsal-ventral axis later in oogenesis is better understood. Anterior-posterior patterning of the embryo requires activities of bicoid and oskar mRNAs, localised within the oocyte. A microtubule motor, Kinesin, is directly implicated in localisation of oskar mRNA to the posterior pole of the oocyte.     

The salvador-warts-hippo pathway is required for epithelial proliferation and axis specification in Drosophila

In Drosophila, the body axes are specified during oogenesis through interactions between the germline and the overlying somatic follicle cells [1-5]. A Gurken/TGF-alpha signal from the oocyte to the adjacent follicle cells assigns them a posterior identity [6, 7]. These posterior cells then signal back to the oocyte, thereby inducing the repolarization of the microtubule cytoskeleton, the migration of the oocyte nucleus, and the localization of the axis specifying mRNAs [8-10]. However, little is known about the signaling pathways within or from the follicle cells responsible for these patterning events. We show that the Salvador Warts Hippo (SWH) tumor-suppressor pathway is required in the follicle cells in order to induce their Gurken- and Notch-dependent differentiation and to limit their proliferation. The SWH pathway is also required in the follicle cells to induce axis specification in the oocyte, by inducing the migration of the oocyte nucleus, the reorganization of the cytoskeleton, and the localization of the mRNAs that specify the anterior-posterior and dorsal-ventral axes of the embryo. This work highlights a novel connection between cell proliferation, cell growth, and axis specification in egg chambers.     

Opposing effects of Notch-signaling in maintaining the proliferative state of follicle cells in the telotrophic ovary of the beetle Tribolium

ABSTRACT: INTRODUCTION: Establishment of distinct follicle cell fates at the early stages of Drosophila oogenesis is crucial for achieving proper morphology of individual egg chambers. In Drosophila oogenesis, Notch-signaling controls proliferation and differentiation of follicular cells, which eventually results in the polarization of the anterior-posterior axis of the oocyte. Here we analyzed the functions of Tribolium Notch-signaling factors during telotrophic oogenesis, which differs fundamentally from the polytrophic ovary of Drosophila. RESULTS: We found Notch-signaling to be required for maintaining the mitotic cycle of somatic follicle cells. Upon Delta RNAi, follicle cells enter endocycle prematurely, which affects egg-chamber formation and patterning. Interestingly, our results indicate that Delta RNAi phenotypes are not solely due to the premature termination of cell proliferation. Therefore, we monitored the terminal /stalk cell precursor lineage by molecular markers. We observed that upon Delta RNAi terminal and stalk cell populations were absent, suggesting that Notch-signaling is also required for the specification of follicle cell populations, including terminal and stalk precursor cells. CONCLUSIONS: We demonstrate that with respect to mitotic cycle/endocycle switch Notch-signaling in Tribolium and Drosophila has opposing effects. While in Drosophila a Delta-signal brings about the follicle cells to leave mitosis, Notch-signaling in Tribolium is necessary to retain telotrophic egg-chambers in an "immature" state. In most instances, Notch-signaling is involved in maintaining undifferentiated (or preventing specialized) cell fates. Hence, the role of Notch in Tribolium may reflect the ancestral function of Notch-signaling in insect oogenesis. The functions of Notch-signaling in patterning the follicle cell epithelium suggest that Tribolium oogenesis may - analogous to Drosophila - involve the stepwise determination of different follicle cell populations. Moreover, our results imply that Notch-signaling may contribute at least to some aspects of oocyte polarization and AP axis also in telotrophic oogenesis.     

Drosophila par-1 is required for oocyte differentiation and microtubule organization

BACKGROUND: Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. RESULTS: In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which we used a protein null mutation, reveal that Drosophila par-1 (par-1) is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The PAR-1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, PAR-1 colocalizes with microtubules along the basolateral membrane. However, in either case PAR-1 localization is independent of microtubules. CONCLUSIONS: The Drosophila par-1 gene plays at least two essential roles during oogenesis; it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian PAR-1 homologs.     

鲻鱼早期卵子发生的超微结构研究

用电子显微镜技术观察鲻鱼早期卵子发生进入第一次成熟分裂前期联会丝复合体期、粗线期、双线期和网状期卵原细胞和初级卵母细胞生发泡和胸质的超微结构特点。在联会丝复合体期,生发泡内同源染色体配对,联会丝复合体中央出现重组节,胞质中不同发育类型的核仁样及其相关线粒体的分布及其数量可作为划分鲻鱼早期卵子发生各个时期的依据。另外,首次观察到靠近膜细胞有一种不规则形细胞,推测是分泌成熟抑制肽细胞。     

Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence

Homologous gap junctions are generally recognized as a means of coordinating cellular behavior under developmental and homeostatic conditions. In the mammalian ovary, heterologous gap junctions between the oocyte and the granulosa cells have been widely implicated in the regulation of meiotic maturation late in oogenesis. However, the role of oocyte-granulosa cell gap junctions at earlier stages of oogenesis is poorly understood. Stage-specific defects in both oocyte and follicle development have been identified in juvenile mice deficient in heterologous oocyte-granulosa cell gap junctions due to targeted deletion of Gja4, the gene encoding connexin-37. Follicle development arrests at the type 4 preantral stage and although oocytes commence growth, oocyte growth ceases at a diameter of 52 microm (74.3% of control size). Analysis of cell cycle and cytoskeletal markers indicates that oocytes arrest in a G(2) state based on uniform decondensed GV chromatin, interphase microtubule arrays, and nonphosphorylated cytoplasmic centrosomes. Functional assays of meiotic competence confirm that oocytes from connexin-37-deficient mice are unable to enter M phase (initiate meiotic maturation) unless treated with the phosphatase inhibitor okadaic acid (OA). Unlike growing oocytes from heterozygous control animals, OA-treated oocytes from connexin-37-deficient mice respond acutely and progress rapidly to the circular bivalent stage of meiosis I and upon removal from OA rapidly revert to an interphase state. In contrast, OA-treated control incompetent oocytes are slow to respond, exhibit a lower proportion of chromosomal bivalent stage oocytes, but remain in and progress into meiotic M phase upon removal from OA. This study demonstrates that heterologous gap-junctional communication is required for the completion of oocyte growth and the acquisition of cytoplasmic meiotic competence.     

Par-1 and Tau regulate the anterior-posterior gradient of microtubules in Drosophila oocytes

The formation of an anterior-posterior (AP) gradient of microtubules in Drosophila oocytes is essential for specification of the AP axis. Proper microtubule organization in the oocyte requires the function of serine/threonine kinase Par-1. The N1S isoform of Par-1 is enriched at the posterior cortex of the oocyte from stage 7 of oogenesis. Here we report that posterior restriction of Par-1 (N1S) kinase activity is critical for microtubule AP gradient formation. Egg chambers with excessive and ectopic Par-1 (N1S) kinase activity in the germline cells display phenotypes similar to those of egg chambers treated with the microtubule-depolymerizing drug colcemid: depolymerization of microtubules in the oocyte and disruption of oocyte nucleus localization. A phosphorylation target of Par-1, the microtubule-associated protein Tau, is also involved in oocyte polarity formation, and overexpression of Tau alleviates the phenotypes caused by ectopic Par-1 (N1S) kinase activity, suggesting that Par-1 regulates oocyte polarity at least partly through Tau. Our findings reveal that maintaining proper levels of Par-1 at correct position in the oocyte is key to oocyte polarity formation and that the conserved role of Par-1 and Tau is crucial for the establishment of an AP gradient of microtubules and for AP axis specification.     

Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila

Oogenesis in Drosophila involves specification of both germ cells and the surrounding somatic follicle cells, as well as the determination of oocyte polarity. We found that two neurogenic genes, Notch and Delta, are required in oogenesis. These genes encode membrane proteins with epidermal growth factor repeats and are essential in the decision of an embryonic ectodermal cell to take on the fate of neuroblast or epidermoblast. In oogenesis, mutation in either gene leads to an excess of posterior follicle cells, a cell fate change reminiscent of the hyperplasia of neuroblasts seen in neurogenic mutant embryos. Furthermore, the Notch mutation in somatic cells causes mislocalization of bicoid in the oocyte. These results suggest that the neurogenic genes Notch and Delta are involved in both follicle cell development and the establishment of anterior-posterior polarity in the oocyte.     

Organizer activity of the polar cells during Drosophila oogenesis

Patterning of the Drosophila egg requires the establishment of several distinct types of somatic follicle cells, as well as interactions between these follicle cells and the oocyte. The polar cells occupy the termini of the follicle and are specified by the activation of Notch. We have investigated their role in follicle patterning by creating clones of cells mutant for the Notch modulator fringe. This genetic ablation of polar cells results in cell fate defects within surrounding follicle cells. At the anterior, the border cells, the immediately adjacent follicle cell fate, are absent, as are the more distant stretched and centripetal follicle cells. Conversely, increasing the number of polar cells by expressing an activated form of the Notch receptor increases the number of border cells. At the posterior, elimination of polar cells results in abnormal oocyte localization. Moreover, when polar cells are mislocalized laterally, the surrounding follicle cells adopt a posterior fate, the oocyte is located adjacent to them, and the anteroposterior axis of the oocyte is re-oriented with respect to the ectopic polar cells. Our observations demonstrate that the polar cells act as an organizer that patterns surrounding follicle cells and establishes the anteroposterior axis of the oocyte. The origin of asymmetry during Drosophila development can thus be traced back to the specification of the polar cells during early oogenesis.     

哺乳动物卵泡卵母细胞发生的分子调控

哺乳动物卵泡卵母细胞发生的研究一直是发育生物学研究的重点之一。简要叙述了哺乳动物卵泡卵母细胞发生的一般过程,重点分析了原始生殖细胞向卵母细胞分化过程中gdf9、c-kti、BMP4及TGF家族关键基因的表达调控对卵母细胞发生的影响,以及卵母细胞与颗粒细胞间的相互调节作用,介绍了卵母细胞体外发生的最新研究进展及面临的难题等,为进一步研究原始生殖细胞向卵母细胞分化以及卵泡生长发育的机制提供了理论基础。     

Premeiotic aster as a device to anchor the germinal vesicle to the cell surface of the presumptive animal pole in starfish oocytes

The germinal vesicle (GV) of starfish oocytes stays just beneath the oocyte cortex at the presumptive animal pole during the long period of oogenesis. We subjected oocytes to a centrifugal force field to detach the GV from the cortex. The association between the cortex and the GV persisted and withstood a small amount of centrifugal acceleration at 200 g. The GV was eventually separated from the cortex at 700 g. The amount of acceleration sufficient for the GV separation was lowered when the oocytes were pretreated with Nocodazole and was increased by Taxol pretreatment. Observation of microtubular structures with an anti-alpha-tubulin antibody revealed the presence of a complex of spots and radiating arrays as was described by J. J. Otto and T. E. Schroeder (1984, Dev. Biol. 101, 274-281) and called the premeiotic aster. Nocodazole shortened the astral arrays, and Taxol enhanced them. These observations indicate that the premeiotic aster works as a device to hold the GV in an eccentric position just beneath the oocyte cortex.     

Structure of the germinal vesicle during oogenesis in leech Glossiphonia heteroclita (Annelida, Hirudinea, Rhynchobdellida)

Oogenesis in the glossiphoniid leech Glossiphonia heteroclita (Hirudinea, Rhynchobdellida) is nutrimental, i.e., the growing oocyte is supported by specialized germline cells, the nurse cells. The main function of the nurse cells is to provide oocytes with cell organelles and RNAs (mainly rRNA). However, in studied leech species, irrespective of the nutrimental mode of oogenesis, the germinal vesicle (GV = oocyte nucleus) seems to be very active in rRNA production. As shown in the present study, during early previtellogenesis in the GV the meiotic chromosomes and prominent primary nucleoli occur. In late previtellogenesis the chromosomes condense and occupy a limited space of nucleoplasm in close vicinity to primary nucleolus, forming a karyosome. At the onset of vitellogenesis several prominent extrachromosomal DNA bodies appear in close association with the karyosome. At the same time, the primary nucleolus is no longer visible in the GV. As vitellogenesis proceeds the extrachromosomal DNA bodies undergo fragmentation and numerous spherical, RNA- and AgNOR-positive inclusions occur in the nucleoplasm. They are regarded as multiple nucleoli. Finally, in late oogenesis numerous accessory nuclei are formed in close proximity to the nuclear envelope. They usually contain one dense body, morphologically similar to multiple nucleoli. The amplification of rDNA genes, the occurrence of extrachromosomal DNA bodies, as well as the presence of multiple nucleoli and accessory nuclei are described for the first time in the phylum Annelida.     

哺乳动物卵母细胞生发泡移植

生发泡(GV)移植是指将GV期卵母细胞的GV移入到去核的受体细胞(GV期卵母细胞、MII期卵母细胞或受精卵)透明带下,经融合形成一个重组卵的过程。GV移植对研究卵母细胞的细胞周期调控、成熟及受精时细胞核与细胞质之间的相互作用非常重要,可用于研究卵母细胞减数分裂异常和与年龄相关变化之间的关系及细胞质衰老与卵母细胞非整倍性之间的关系。现简要介绍了GV移植的基本程序,GV核体与胞质体的融合,重组卵的培养条件,重组卵成熟后的受精、人工激活和胚胎发育能力以及GV移植的意义。     

Cell polarity and oocyte determination in Drosophila melanogaster

During early oogenesis, one cell from a cyst of 16 germ cells is selected to become the oocyte. Recent data suggest that the choice of this cell within the cyst is strongly biased as early as the cyst itself forms. However, it was further shown that, although selected, the oocyte fate needs to be maintained. The maintenance of the oocyte identity requires the activity of the Drosophila homologues of the Caenorhabditis elegans par genes. It was shown that the par genes are required for the first polarisation of the oocyte as early as in region 3 of the germarium. This reveals a striking conservation between the polarisation along the antero-posterior axis of the Caenorhabditis elegans one-cell embryo and the Drosophila oocyte.     

Fer tyrosine kinase is required for germinal vesicle breakdown and meiosis-I in mouse oocytes

The control of microtubule and actin-mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Our previous studies demonstrated a role for FYN tyrosine kinase in chromosome and spindle organization and in cortical polarity of the mature mammalian oocyte. In addition to Fyn, mammalian oocytes express the protein tyrosine kinase Fer at high levels relative to other tissues. The objective of the present study was to determine the function of this kinase in the oocyte. Feline encephalitis virus (FES)-related kinase (FER) protein was uniformly distributed in the ooplasm of small oocytes, but became concentrated in the germinal vesicle (GV) during oocyte growth. After germinal vesicle breakdown (GVBD), FER associated with the metaphase-I (MI) and metaphase-II (MII) spindles. Suppression of Fer expression by siRNA knockdown in GV stage oocytes did not prevent activation of cyclin dependent kinase 1 activity or chromosome condensation during in vitro maturation, but did arrest oocytes prior to GVBD or during MI. The resultant phenotype displayed condensed chromosomes trapped in the GV, or condensed chromosomes poorly arranged in a metaphase plate but with an underdeveloped spindle microtubule structure or chromosomes compacted into a tight sphere. The results demonstrate that FER kinase plays a critical role in oocyte meiotic spindle microtubule dynamics and may have an additional function in GVBD.