Ring Canals Research Articles

Lineage-based scaling of germline intercellular bridges during oogenesis.

Using the fruit fly egg chamber as a model, this study demonstrates that the size and scaling of germline intercellular bridges vary based on lineage.

Cell cycle variants during Drosophila male accessory gland development.

The Drosophila melanogaster male accessory gland (AG) is a functional analog of the mammalian prostate and seminal vesicles containing two secretory epithelial cell types, termed main and secondary cells. This tissue is responsible for making and secreting seminal fluid proteins and other molecules that contribute to successful reproduction. The cells of this tissue are binucleate and polyploid, due to variant cell cycles that include endomitosis and endocycling during metamorphosis. Here, we provide evidence of additional cell cycle variants in this tissue. We show that main cells of the gland are connected by ring canals that form after the penultimate mitosis, and we describe an additional post-eclosion endocycle required for gland maturation that is dependent on juvenile hormone signaling. We present evidence that the main cells of the D. melanogaster AG undergo a unique cell cycle reprogramming throughout organ development that results in step-wise cell cycle truncations culminating in cells containing two octoploid nuclei with under-replicated heterochromatin in the mature gland. We propose this tissue as a model to study developmental and hormonal temporal control of cell cycle variants in terminally differentiating tissues.

Coenocystic oogenesis - modification of or deviation from the germ cell cyst paradigm?

Invertebrate and vertebrate species have many unusual cellular structures, such as long- or short-lived cell-in-cell structures and coenocytes. Coenocytes (often incorrectly described as syncytia) are multinuclear cells derived, unlike syncytia, not from the fusion of multiple cells but from multiple nuclear divisions without cytokinesis. An example of a somatic coenocyte is the coenocytic blastoderm in Drosophila. An astonishing property of coenocytes is the ability to differentiate the nuclei sharing a common cytoplasm into different subpopulations with different fate trajectories. An example of a germline coenocyte is the oogenic precursor of appendicularian tunicates, which shares many features with the somatic coenocyte of Drosophila. The germline coenocyte (coenocyst) is quite an unexpected structure because in most animals, including Drosophila, Xenopus, and mice, oogenesis proceeds within a group (cyst, nest) of sibling cells (cystocytes) connected by the intercellular bridges (ring canals, RCs) derived from multiple divisions with incomplete cytokinesis of a progenitor cell called the cystoblast. Here, I discuss the differences and similarities between cystocyte-based and coenocyst-based oogenesis, and the resemblance of coenocystic oogenesis to coenocytic somatic blastoderm in Drosophila. I also describe cell-in-cell structures that although not mechanistically, cytologically, or molecularly connected to somatic or germline coenocytes, are both unorthodox and intriguing cytological phenomena rarely covered by scientific literature.

The dynamic duo of microtubule polymerase Mini spindles/XMAP215 and cytoplasmic dynein is essential for maintaining Drosophila oocyte fate

In many species, only one oocyte is specified among a group of interconnected germline sister cells. In Drosophila melanogaster, 16 interconnected cells form a germline cyst, where one cell differentiates into an oocyte, while the rest become nurse cells that supply the oocyte with mRNAs, proteins, and organelles through intercellular cytoplasmic bridges named ring canals via microtubule-based transport. In this study, we find that a microtubule polymerase Mini spindles (Msps), the Drosophila homolog of XMAP215, is essential for maintenance of the oocyte specification. mRNA encoding Msps is transported and concentrated in the oocyte by dynein-dependent transport along microtubules. Translated Msps stimulates microtubule polymerization in the oocyte, causing more microtubule plus ends to grow from the oocyte through the ring canals into nurse cells, further enhancing nurse cell-to-oocyte transport by dynein. Knockdown of msps blocks the oocyte growth and causes gradual loss of oocyte determinants. Thus, the Msps-dynein duo creates a positive feedback loop, ensuring oocyte fate maintenance by promoting high microtubule polymerization activity in the oocyte, and enhancing dynein-dependent nurse cell-to-oocyte transport.

Evolutionarily conserved midbody remodeling precedes ring canal formation during gametogenesis.

How canonical cytokinesis is altered during germ cell division to produce stable intercellular bridges, called "ring canals," is poorly understood. Here, using time-lapse imaging in Drosophila, we observe that ring canal formation occurs through extensive remodeling of the germ cell midbody, a structure classically associated with its function in recruiting abscission-regulating proteins in complete cytokinesis. Germ cell midbody cores reorganize and join the midbody ring rather than being discarded, and this transition is accompanied by changes in centralspindlin dynamics. The midbody-to-ring canal transformation is conserved in the Drosophila male and female germlines and during mouse and Hydra spermatogenesis. In Drosophila, ring canal formation depends on Citron kinase function to stabilize the midbody, similar to its role during somatic cell cytokinesis. Our results provide important insights into the broader functions of incomplete cytokinesis events across biological systems, such as those observed during development and disease states.

Ovary micromorphology in hormogastrid earthworms with a particular emphasis on the organization of the germline cysts

There is a gap in our knowledge of microorganization and the functioning of ovaries in earthworms (Crassiclitellata) and allied taxa. Recent analyses of ovaries in microdriles and leech-like taxa revealed that they are composed of syncytial germline cysts accompanied by somatic cells. Although the pattern of cyst organization is conserved across Clitellata – each cell is connected via one intercellular bridge (ring canal) to the central and anuclear cytoplasmic mass termed the cytophore – this system shows high evolutionary plasticity. In Crassiclitellata, only the gross morphology of ovaries and their segmental localization is well known, whereas ultrastructural data are limited to lumbricids like Dendrobaena veneta. Here we present the first report about ovarian histology and ultrastructure in Hormogastridae, a small family of earthworms inhabiting the western parts of the Mediterranean sea basin. We analyzed three species from three different genera and showed that the pattern of ovary organization is the same within this taxon. Ovaries are cone-like, with a broad part connected to the septum and a narrow distal end forming an egg string. Ovaries are composed of numerous cysts uniting a small number of cells, eight in Carpetania matritensis. There is a gradient of cysts development along the long ovary axis, and three zones can be distinguished. In zone I, cysts develop in complete synchrony and unite oogonia and early meiotic cells (till diplotene). Then (zone II), the synchrony is lost, and one cell (prospective oocyte) grows faster than the rest (prospective nurse cells). In zone III, oocytes pass the growth phase and gather nutrients; at this time, their contact with the cytophore is lost. Nurse cells grow slightly, eventually die via apoptosis, and are removed by coelomocytes. The most characteristic feature of hormogastrid germ cysts is the inconspicuous cytophore in the form of thread-like thin cytoplasmic strands (reticular cytophore). We found that the ovary organization in studied hormogastrids is very similar to that described for D. veneta and propose the term "Dendrobaena" type of ovaries. We expect the same microorganization of ovaries will be found in other hormogastrids and lumbricids.

Integrative Systematics and Biogeography of the Hydrozoans (Leptothecata: Eirenidae) Eirene menoni Kramp, 1953 and Eirene lacteoides Kubota and Horita, 1992 from Japan and China with Comments on Pacific Ocean Distributions.

The hydrozoan family Eirenidae is known scientifically for its morphological plasticity and challenges in species identification. We used an integrative taxonomic approach based on morphological, molecular and life history evidence to systematically assess field-collected medusae of Eirene menoni Kramp 1953 and captive raised polyps of both E. menoni and E. lacteoides Kubota and Horita 1992. Following morphological review, we updated the genus description to include the presence of rudimentary bulbs (warts) on the ring canal in at least eight of the 24 valid Eirene species. We propose the potential for the mature E. menoni hydrotheca to develop into a gonotheca. However, this proposal will require additional study for verification. We provide validated distribution records from the Indo-Pacific Ocean for E. menoni,and updated collection records for E. lacteoides from the Yellow and East China Seas, and public aquaria-cultured specimens from Japan and Hawaii, using cytochrome c oxidase I (COI) sequences that we generated and compared with those from GenBank. The COI gene reliably separated four species, each forming a monophyletic clade with strong bootstrap support and low mean intraspecific molecular divergences (≤ 1%) within clades. However, some of the deeper nodes of the tree remained poorly resolved, and our analysis failed to demonstrate monophyly among eirenid genera Eirene and Tima. Our integrative taxonomic approach is essential in confirming species identity within the family Eirenidae and genus Eirene,and we have also identified a likely range expansion of E. lacteoides to Hawaii.

Formation of Coelomic Cavities during Abbreviated Development of the Brittle Star Ophioplocus esmarki.

AbstractIn brittle stars, the coelomic cavities that form during embryogenesis contribute to most of the internal organ systems of the juvenile. In the ancestral mode of development, the coelomic cavities begin with bilateral symmetry and play a minor role in the function of the ophiopluteus larva. However, the coelomic cavities undergo extensive changes during metamorphosis to set up the body systems of the juvenile brittle star. Many lineages of brittle stars have evolved life histories without the ophiopluteus larva. The non-feeding vitellaria larva has rapid development of juvenile structures. This work demonstrates the modifications to the origin and early development of the coelomic cavities in a vitellaria larva. Much of the archenteron forms an unpaired axocoel, hydrocoel, and somatocoel. The posterior-most portion of the archenteron forms the rudiment of the juvenile stomach. The right somatocoel and a portion of the left somatocoel form as invaginations of the lateral ectoderm. Later morphogenesis of the axocoel, the hydrocoel, and the two somatocoels is similar to what has been shown for brittle stars with an ophiopluteus larva. Confocal microscopy and three-dimensional modeling were used to show new details for the later morphogenesis of the coelomic cavities. The stone canal originates as an outgrowth of the hydrocoel between lobes 4 and 5. The hydrocoel lobes have minimal migration after they meet to complete the ring canal. The right somatocoel contributes to a component of the axial complex and perihemal system. A detailed description is given for how the left somatocoel contributes to multiple organ systems.

A Diaphanous and Enabled-dependent asymmetric actin cable array repositions nuclei during Drosophila oogenesis.

Cells reposition their nuclei for diverse specialized functions through a wide variety of cytoskeletal mechanisms. During Drosophila oogenesis, 15 nurse cells connected by ring canals to each other and the oocyte contract, 'dumping' their cytoplasm into the oocyte. Prior to dumping, actin cables initiate from the nurse cell cortex and elongate toward their nuclei, pushing them away from ring canals to prevent obstruction. How the cable arrays reposition nuclei is unknown. We found that these arrays are asymmetric, with regional differences in actin cable growth rate dependent on the differential localization of the actin assembly factors Enabled and Diaphanous. Enabled mislocalization produces a uniform growth rate. In oocyte-contacting nurse cells with asymmetric cable arrays, nuclei move away from ring canals. With uniform arrays, these nuclei move toward the adjacent ring canal instead. This correlated with ring canal nuclear blockage and incomplete dumping. Our data suggest that nuclear repositioning relies on the regulated cortical localization of Diaphanous and Enabled to produce actin cable arrays with asymmetric growth that push nuclei away from ring canals, enabling successful oogenesis.

Bidirectional communication in oogenesis: a dynamic conversation in mice and Drosophila.

In most animals, the oocyte is the largest cell by volume. The oocyte undergoes a period of large-scale growth during its development, prior to fertilization. At first glance, tissues that support the development of the oocyte in different organisms have diverse cellular characteristics that would seem to prohibit functional comparisons. However, these tissues often act with a common goal of establishing dynamic forms of two-way communication with the oocyte. We propose that this bidirectional communication between oocytes and support cells is a universal phenomenon that can be directly compared across species. Specifically, we highlight fruit fly and mouse oogenesis to demonstrate that similarities and differences in these systems should be used to inform and design future experiments in both models.

Aged mice ovaries harbor stem cells and germ cell nests but fail to form follicles

BackgroundWe recently published evidence to suggest that two populations of stem cells including very small embryonic-like stem cells (VSELs) and ovarian stem cells (OSCs) in ovary surface epithelium (OSE) undergo proliferation/differentiation, germ cell nests (GCN) formation, meiosis and eventually differentiate into oocytes that assemble as primordial follicles on regular basis during estrus cycle. Despite presence of stem cells, follicles get exhausted with advancing age in mice and result in senescence equivalent to menopause in women. Stem cells in aged ovaries can differentiate into oocytes upon transplantation into young ovaries, however, it is still not well understood why follicles get depleted with advancing age despite the presence of stem cells. The aim of the present study was to study stem cells and GCN in aged ovaries.MethodsOSE cells from aged mice (> 18 months equivalent to > 55 years old women) were enzymatically separated and used to study stem cells. Viable (7-AAD negative) VSELs in the size range of 2–6 µm with a surface phenotype of Lin−CD45−Sca-1+ were enumerated by flow cytometry. Immuno-fluorescence and RT-PCR analysis were done to study stem/progenitor cells (OCT-4, MVH, SCP3) and transcripts specific for VSELs (Oct-4A, Sox-2, Nanog), primordial germ cells (Stella), germ cells (Oct-4, Mvh), early meiosis (Mlh1, Scp1) and ring canals (Tex14).ResultsPutative VSELs and OSCs were detected as darkly stained, spherical cells with high nucleo-cytoplasmic ratio along with germ cells nests (GCN) in Hematoxylin & Eosin stained OSE cells smears. Germ cells in GCN with distinct cytoplasmic continuity expressed OCT-4, MVH and SCP3. Transcripts specific for stem cells, early meiosis and ring canals were detected by RT-PCR studies.ConclusionRather than resulting as a consequence of accelerated loss of primordial follicle and their subsequent depletion, ovarian senescence/menopause occurs as a result of stem cells dysfunction. VSELs and OSCs exist along with increased numbers of GCNs arrested in pre-meiotic or early meiotic stage in aged ovaries and primordial follicle assembly is blocked possibly due to age-related changes in their microenvironment.

A novel mechanism of bulk cytoplasmic transport by cortical dynein in Drosophila ovary.

Cytoplasmic dynein, a major minus-end directed microtubule motor, plays essential roles in eukaryotic cells. Drosophila oocyte growth is mainly dependent on the contribution of cytoplasmic contents from the interconnected sister cells, nurse cells. We have previously shown that cytoplasmic dynein is required for Drosophila oocyte growth and assumed that it simply transports cargoes along microtubule tracks from nurse cells to the oocyte. Here, we report that instead of transporting individual cargoes along stationary microtubules into the oocyte, cortical dynein actively moves microtubules within nurse cells and from nurse cells to the oocyte via the cytoplasmic bridges, the ring canals. This robust microtubule movement is sufficient to drag even inert cytoplasmic particles through the ring canals to the oocyte. Furthermore, replacing dynein with a minus-end directed plant kinesin linked to the actin cortex is sufficient for transporting organelles and cytoplasm to the oocyte and driving its growth. These experiments show that cortical dynein performs bulk cytoplasmic transport by gliding microtubules along the cell cortex and through the ring canals to the oocyte. We propose that the dynein-driven microtubule flow could serve as a novel mode of fast cytoplasmic transport.

Microtubule and Actin Cytoskeletal Dynamics in Male Meiotic Cells of Drosophila melanogaster.

Drosophila dividing spermatocytes offer a highly suitable cell system in which to investigate the coordinated reorganization of microtubule and actin cytoskeleton systems during cell division of animal cells. Like male germ cells of mammals, Drosophila spermatogonia and spermatocytes undergo cleavage furrow ingression during cytokinesis, but abscission does not take place. Thus, clusters of primary and secondary spermatocytes undergo meiotic divisions in synchrony, resulting in cysts of 32 secondary spermatocytes and then 64 spermatids connected by specialized structures called ring canals. The meiotic spindles in Drosophila males are substantially larger than the spindles of mammalian somatic cells and exhibit prominent central spindles and contractile rings during cytokinesis. These characteristics make male meiotic cells particularly amenable to immunofluorescence and live imaging analysis of the spindle microtubules and the actomyosin apparatus during meiotic divisions. Moreover, because the spindle assembly checkpoint is not robust in spermatocytes, Drosophila male meiosis allows investigating of whether gene products required for chromosome segregation play additional roles during cytokinesis. Here, we will review how the research studies on Drosophila male meiotic cells have contributed to our knowledge of the conserved molecular pathways that regulate spindle microtubules and cytokinesis with important implications for the comprehension of cancer and other diseases.

Prognosis Analysis and Clinical Features of Orbital Cavernous Venous Malformations With Refractory Insidious Onset.

ObjectiveThe present study aims to analyse the clinical presentation, treatment and prognosis of a group of patients with orbital cavernous venous malformation (OCVM) with an insidious onset.MethodThe clinical data of 35 patients with OCVM treated at our centre between 2003 and 2020 were retrospectively analysed. The OCVMs were classified as one of six types (I–VI) according to the orbital position of the tumour. The clinical characteristics, treatment methods and follow-up results were recorded.ResultsA total of 35 patients with OCVM under the optic nerve sheath in the orbital apex area or the common tendon ring (Types I and II) were included in the present study. In 20 cases (57.1%), patients were misdiagnosed with optic neuritis, and in 20 cases (57.1%), the tumour was not identified based on imaging. The presentation was acute or subacute in 23 cases (65.7%). All patients underwent surgery: transnasal surgery in 22 cases (62.9%) and craniotomy in 13 cases (37.1%). A total of 9 patients (25.7%) experienced postoperative complications, and 17 patients (48.6%) experienced vision improvement. The average patient age at first diagnosis was 43.3 ± 10.3 years, and the median follow-up period was 64.5 months. Overall, 14 patients (40%) experienced postoperative complications: postoperative blindness in 6 cases, postoperative vision loss in 8 cases and orbital apex syndrome in 7 cases.ConclusionPatients with Type I and Type II OCVMs are the most complex cases. They have an insidious onset and are associated with a high rate of misdiagnosis and missed diagnosis. Acute and subacute decreases in visual acuity are mainly caused by OCVM haemorrhage. The difficulty of surgical treatment and the poor prognosis of postoperative vision are characteristics of this tumour. Transnasal surgery and craniotomy can be used to remove OCVMs located in the common tendon ring or optic canal as well as those involving the intracranial area through the supraorbital fissure. Meanwhile, the orbital approach (orbitotomy) has proven to be an effective method of treating OCVMs not involving the deep orbital apex and intracranial area.

Stem Cells in Adult Mice Ovaries Form Germ Cell Nests, Undergo Meiosis, Neo-oogenesis and Follicle Assembly on Regular Basis During Estrus Cycle.

Very small embryonic-like (VSELs) and ovarian (OSCs) stem cellsare located in adult mammalian ovary surface epithelium (OSE). OSCs can expand long-term and differentiate into oocyte-like structures in vitro and have resulted in birth of fertile pups. Lineage tracing studies have provided evidence to suggest OSCs differentiation into oocytes in vivo. But how these stem cells function under normal physiological conditions has not yet been well worked out. Besides studying STRA-8 and SCP-3 expression in enzymatically isolated OSE cells smears, mice were injected BrdU to track mitosis, meiosis and follicle assembly. H&E stained OSE cells during late diestrus and proestrus showed VSELs undergoing asymmetrical cell divisions to give rise to slightly bigger OSCs which in turn underwent symmetrical cell divisions followed by clonal expansion (rapid expansion with incomplete cytokinesis) during early estrus to form germ cell nests (GCN). OCT-4, SSEA-1, MVH and DAZL positive cells in GCN expressed Erα, Erβ and FSHR, were interconnected by ring canals (TEX-14), showed mitochondrial aggregation (Cytochrome C) and Balbiani Body (TRAL). Apoptosis in 'nurse' cells was marked by PARP and putative oocytes were clearly visualized. BrdU was detected in cells undergoing mitosis/meiosis and also in an oocyte of secondary follicle. FACS sorted, green fluorescent protein (GFP) positive VSELs upon transplantation resulted in GFP positive GCN suggesting crucial role for VSELs in adult ovaries. Results suggest that various events described during oogenesis and follicle assembly in fetal ovaries are recapitulated on regular basis in adult ovary and result in the formation of follicles.

Gatekeeper function for Short stop at the ring canals of the Drosophila ovary

Growth of the Drosophila oocyte requires transport of cytoplasmic materials from the interconnected sister cells (nurse cells) through ring canals, the cytoplasmic bridges that remained open after incomplete germ cell division. Given the open nature of the ring canals, it is unclear how the direction of transport through the ring canal is controlled. In this work, we show that a single Drosophila spectraplakin Short stop (Shot) controls the direction of flow from nurse cells to the oocyte. Knockdown of shot changes the direction of transport through the ring canals from unidirectional (toward the oocyte) to bidirectional. After shot knockdown, the oocyte stops growing, resulting in a characteristic small oocyte phenotype. In agreement with this transport-directing function of Shot, we find that it is localized at the asymmetric actin baskets on the nurse cell side of the ring canals. In wild-type egg chambers, microtubules localized in the ring canals have uniform polarity (minus ends toward the oocyte), while in the absence of Shot, these microtubules have mixed polarity. Together, we propose that Shot functions as a gatekeeper directing transport from nurse cells to the oocyte via the organization of microtubule tracks to facilitate the transport driven by the minus-end-directed microtubule motor cytoplasmic dynein. VIDEO ABSTRACT.

Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution.

Understanding cellular organization demands the best possible spatial resolution in all three dimensions (3D). In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved isoSTED nanoscope, which utilizes an advanced adaptive optics strategy to achieve sub-50 nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

Precise levels of the Drosophila adaptor protein Dreadlocks maintain the size and stability of germline ring canals.

ABSTRACTIntercellular bridges are essential for fertility in many organisms. The developing fruit fly egg has become the premier model system to study intercellular bridges. During oogenesis, the oocyte is connected to supporting nurse cells by relatively large intercellular bridges, or ring canals. Once formed, the ring canals undergo a 20-fold increase in diameter to support the movement of materials from the nurse cells to the oocyte. Here, we demonstrate a novel role for the conserved SH2/SH3 adaptor protein Dreadlocks (Dock) in regulating ring canal size and structural stability in the germline. Dock localizes at germline ring canals throughout oogenesis. Loss of Dock leads to a significant reduction in ring canal diameter, and overexpression of Dock causes dramatic defects in ring canal structure and nurse cell multinucleation. The SH2 domain of Dock is required for ring canal localization downstream of Src64 (also known as Src64B), and the function of one or more of the SH3 domains is necessary for the strong overexpression phenotype. Genetic interaction and localization studies suggest that Dock promotes WASp-mediated Arp2/3 activation in order to determine ring canal size and regulate growth.This article has an associated First Person interview with the first author of the paper.

First person – Kara Stark

First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Kara Stark is first author on 'Precise levels of the Drosophila adaptor protein Dreadlocks maintain the size and stability of germline ring canals', published in JCS. Kara conducted the research described in this article while an undergraduate research assistant in Dr Lindsay Lewellyn's lab at Butler University, Indianapolis, IN. She is now a PhD student in the lab of Dr Zhao Zhang at Duke University, Durham, NC, investigating how transposon mobilization interacts with the immune system during development.

Zebrafish dazl regulates cystogenesis and germline stem cell specification during the primordial germ cell to germline stem cell transition.

Fertility and gamete reserves are maintained by asymmetric divisions of the germline stem cells to produce new stem cells or daughters that differentiate as gametes. Before entering meiosis, differentiating germ cells (GCs) of sexual animals typically undergo cystogenesis. This evolutionarily conserved process involves synchronous and incomplete mitotic divisions of a GC daughter (cystoblast) to generate sister cells connected by intercellular bridges that facilitate the exchange of materials to support rapid expansion of the gamete progenitor population. Here, we investigated cystogenesis in zebrafish and found that early GCs are connected by ring canals, and show that Deleted in azoospermia-like (Dazl), a conserved vertebrate RNA-binding protein (Rbp), is a regulator of this process. Analysis of dazl mutants revealed the essential role of Dazl in regulating incomplete cytokinesis, germline cyst formation and germline stem cell specification before the meiotic transition. Accordingly, dazl mutant GCs form defective ring canals, and ultimately remain as individual cells that fail to differentiate as meiocytes. In addition to promoting cystoblast divisions and meiotic entry, dazl is required for germline stem cell establishment and fertility.