Orbit/Mast,the CLASP orthologue of Drosophila,is required for asymmetric stem cell and cystocyte divisions and development of the polarised microtubule network that interconnects oocyte and nurse cells during oogenesis

Drosophila oocyte differentiation is preceded by the formation of a polarised 16-cell cyst from a single progenitor stem cell as a result of four rounds of asymmetric mitosis followed by incomplete cytokinesis. We show that the Orbit/Mast microtubule-associated protein is required at several stages in the formation of such polarised 16-cell cysts. In wild-type cysts, the Orbit/Mast protein not only associates with the mitotic spindle and its poles, but also with the central spindle (spindle remnant), ring canal and fusome, suggesting it participates in interactions between these structures. In orbit mutants, the stem cells and their associated fusomes are eventually lost as Orbit/Mast protein is depleted. The mitotic spindles of those cystocytes that do divide are either diminutive or monopolar, and do not make contact with the fusome. Moreover, the spindle remnants and ring canals fail to differentiate correctly in such cells and the structure of fusome is compromised. The Orbit/Mast protein thus appears to facilitate multiple interactions of the fusome with mitotic spindles and ring canals. This ensures correct growth of the fusome into a branched asymmetrically distributed organelle that is pre-determinative of 16-cell cyst formation and oocyte fate specification. Finally the Orbit/Mast protein is required during mid-oogenesis for the organisation of the polarised microtubule network inside the 16-cell cyst that ensures oocyte differentiation. The localisation of CLIP-190 to such microtubules and to the fusome is dependent upon Orbit/Mast to which it is complexed.     

The fusome organizes the microtubule network during oocyte differentiation in Drosophila

Differentiation of the Drosophila oocyte takes place in a cyst of 16 interconnected germ cells and is dependent on a network of microtubules that becomes polarized as differentiation progresses (polarization). We have investigated how the microtubule network polarizes using a GFP-tubulin construct that allows germ-cell microtubules to be visualized with greater sensitivity than in previous studies. Unexpectedly, microtubules are seen to associate with the fusome, an asymmetric germline-specific organelle, which elaborates as cysts form and undergoes complex changes during cyst polarization. This fusome-microtubule association occurs periodically during late interphases of cyst divisions and then continuously in 16-cell cysts that have entered meiotic prophase. As meiotic cysts move through the germarium, microtubule minus ends progressively focus towards the center of the fusome, as visualized using a NOD-lacZ marker. During this same period, discrete foci rich in gamma tubulin that very probably correspond to migrating cystocyte centrosomes also associate with the fusome, first on the fusome arms and then in its center, subsequently moving into the differentiating oocyte. The fusome is required for this complex process, because microtubule network organization and polarization are disrupted in hts(1) mutant cysts, which lack fusomes. Our results suggest that the fusome, a specialized membrane-skeletal structure, which arises in early germ cells, plays a crucial role in polarizing 16-cell cysts, at least in part by interacting with microtubules and centrosomes.     

Orbit/CLASP Is Required for Myosin Accumulation at the Cleavage Furrow in Drosophila Male Meiosis

Peripheral microtubules (MTs) near the cell cortex are essential for the positioning and continuous constriction of the contractile ring (CR) in cytokinesis. Time-lapse observations of Drosophila male meiosis showed that myosin II was first recruited along the cell cortex independent of MTs. Then, shortly after peripheral MTs made contact with the equatorial cortex, myosin II was concentrated there in a narrow band. After MT contact, anillin and F-actin abruptly appeared on the equatorial cortex, simultaneously with myosin accumulation. We found that the accumulation of myosin did not require centralspindlin, but was instead dependent on Orbit, a Drosophila ortholog of the MT plus-end tracking protein CLASP. This protein is required for stabilization of central spindle MTs, which are essential for cytokinesis. Orbit was also localized in a mid-zone of peripheral MTs, and was concentrated in a ring at the equatorial cortex during late anaphase. Fluorescence resonance energy transfer experiments indicated that Orbit is closely associated with F-actin in the CR. We also showed that the myosin heavy chain was in close proximity with Orbit in the cleavage furrow region. Centralspindlin was dispensable in Orbit ring formation. Instead, the Polo-KLP3A/Feo complex was required for the Orbit accumulation independently of the Orbit MT-binding domain. However, orbit mutations of consensus sites for the phosphorylation of Cdk1 or Polo did not influence the Orbit accumulation, suggesting an indirect regulatory role of these protein kinases in Orbit localization. Orbit was also necessary for the maintenance of the CR. Our data suggest that Orbit plays an essential role as a connector between MTs and the CR in Drosophila male meiosis.     

Rab11 maintains connections between germline stem cells and niche cells in the Drosophila ovary

All stem cells have the ability to balance their production of self-renewing and differentiating daughter cells. The germline stem cells (GSCs) of the Drosophila ovary maintain such balance through physical attachment to anterior niche cap cells and stereotypic cell division, whereby only one daughter remains attached to the niche. GSCs are attached to cap cells via adherens junctions, which also appear to orient GSC division through capture of the fusome, a germline-specific organizer of mitotic spindles. Here we show that the Rab11 GTPase is required in the ovary to maintain GSC-cap cell junctions and to anchor the fusome to the anterior cortex of the GSC. Thus, rab11-null GSCs detach from niche cap cells, contain displaced fusomes and undergo abnormal cell division, leading to an early arrest of GSC differentiation. Such defects are likely to reflect a role for Rab11 in E-cadherin trafficking as E-cadherin accumulates in Rab11-positive recycling endosomes (REs) and E-cadherin and Armadillo (beta-catenin) are both found in reduced amounts on the surface of rab11-null GSCs. The Rab11-positive REs through which E-cadherin transits are tightly associated with the fusome. We propose that this association polarizes the trafficking by Rab11 of E-cadherin and other cargoes toward the anterior cortex of the GSC, thus simultaneously fortifying GSC-niche junctions, fusome localization and asymmetric cell division. These studies bring into focus the important role of membrane trafficking in stem cell biology.     

New components of the Drosophila fusome suggest it plays novel roles in signaling and transport

The fusome plays an essential role in prefollicular germ cell development within insects such as Drosophila melanogaster. Alpha-spectrin and the adducin-like protein Hu-li tai shao (Hts) are required to maintain fusome integrity, synchronize asymmetric cystocyte mitoses, form interconnected 16-cell germline cysts, and specify the initial cell as the oocyte. By screening a library of protein trap lines, we identified 14 new fusome-enriched proteins, including many associated with its characteristic vesicles. Our studies reveal that fusomes change during development and contain recycling endosomal and lysosomal compartments in females but not males. A significant number of fusome components are dispensable, because genetic disruption of tropomodulin, ferritin-1 heavy chain, or scribble, does not alter fusome structure or female fertility. In contrast, rab11 is required to maintain the germline stem cells, and to maintain the vesicle content of the spectrosome, suggesting that the fusome mediates intercellular signals that depend on the recycling endosome.     

Intercellular bridges and factors determining their patterns in the grasshopper testis

Intercellular bridges joining cells contained in cysts of Chortophaga viridifasciata testes were studied with light and electron microscopy. Preparations consisted of expressed whole cells (living, or fixed and stained) as well as sections. The secondary spermatogonia of each cyst are joined centrally by persisting fused interzonal bodies (fusomes) of incompletely cleaved cells. Shifts in cell orientation during anaphase are apparently responsible for central as opposed to chain linkage of cells. In the primary spermatocytes, the central fusome is replaced by a chain linkage, apparently resulting from the breakdown of the fusome into its original interzonal body components. Intercellular bridges are also present in spermatids, but there is no evidence to indicate the time of their formation (in the immediately preceding meiotic divisions or in the secondary spermatogonial divisions). The function of the compact centrally situated fusome in the secondary spermatogonial cyst is discussed as it relates to synchrony, number of cell divisions, spermatodesm formation, and fertility.     

Centrosome inheritance in the male germ line of Drosophila requires hu-li tai-shao function

Cytokinesis partitions a centrosome to each daughter cell at cell division that will duplicate and assemble a bipolar spindle in the subsequent M phase. Cytokinesis is incomplete in proliferating germ cells in Drosophila and cytoplasmic channels connect sibling germ cells. Although centrosomes are essential to male fertility, the molecular mechanism that retains centrosomes in parental germ cells is not known. Cortical cytoplasmic structures known as fusomes extend through ring canals and connect cells within the cyst. Fusome assembly in males requires function of hu-li tai-shao (hts), an adducin like protein found in fusomes and in the cortical membrane cytoskeleton of somatic cells. This work used immunological and cytological methods to place hts mutants in an allelic series. Male fertile hts mutants express hts protein and generate apparently normal or fragmented fusomes. A male sterile allele does not express hts protein or show fusome structures. Gonial cells in all hts mutants showed 2 centrosomes and mitotic spindles were bipolar. Yet, primary spermatocytes, with and without fusome structures, frequently contained too many or too few centrosomes. Although spindle structures were not found in spermatocytes without centrosomes, meiotic spermatocytes with centrosomes generated bipolar, monopolar, and multipolar spindles. Collectively, these results indicate that hts function is necessary for centrosome inheritance in spermatocytes as well as for male fertility.     

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.     

A spectraplakin is enriched on the fusome and organizes microtubules during oocyte specification in Drosophila

BACKGROUND: During Drosophila oogenesis a membranous organelle called the fusome has a key function in the establishment of oocyte fate and polarity, ultimately leading to the establishment of the major body axes of the animal. The fusome is necessary for the microtubule-driven restriction of markers of oocyte fate to the oocyte, but the mechanism by which the fusome organizes the microtubules is not known.RESULTS: We have identified the spectraplakin Short stop (Shot) as a new component of the fusome. Spectraplakins are giant cytoskeletal linker proteins, with multiple isoforms produced from each gene. Shot is the sole spectraplakin in Drosophila. The phenotype caused by the absence of Shot is not similar to that of other components of the fusome but instead is similar to the absence of the downstream components that interact with microtubules: the dynein/dynactin-complex-associated proteins Egalitarian and BicaudalD. Shot is required for the association of microtubules with the fusome and the subsequent specification of the oocyte in 16-cell cysts. Shot is also required for the concentration of centrosomes into the oocyte, a process thought to be independent of microtubules because it still occurs in the presence of microtubule depolymerizing drugs. This suggests that Shot may protect some microtubules from depolymerization and that these microtubules are sufficient for this process.CONCLUSIONS: Shot provides the missing link between the fusome and microtubules within meiotic cysts, which is essential for the establishment of the oocyte. Shot associates with the fusome and is required for microtubule organization. We suggest that it does this directly, via its microtubule binding GAS2 domain.     

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.     

Microtubule dynamics differentially regulates Rho and Rac activity and triggers Rho-independent stress fiber formation in macrophage polykaryons

Multinucleated giant cells (MNGC) derived from avian peripheral blood monocytes present a dense microtubular network emanating from peripherally located centrosomes. We were interested to study how microtubule and F-actin cytoskeletons cooperate in MNGC to maintain cell shape. Microtubule depolymerization by nocodazole triggered the reorganization of the F-actin cytoskeleton in MNGC that is normally organized into podosomes, cortical actin filaments and membrane ruffles. After nocodazole treatment, F-actin was redistributed into unusual transverse fibers associated with focal adhesion plaques. When microtubules were allowed to repolymerize after nocodazole removal, F-actin appeared transiently, together with the small GTPase Rac, in large membrane ruffles. Using affinity precipitation assays, we show that microtubule depolymerization leads to activation of Rho and inhibition of Rac, whereas microtubule repolymerization induces Rac activation and Rho inhibition. Thus, the level of microtubule polymerization inversely regulates Rho and Rac activity in MNGC. Moreover, using C3 exoenzyme, a known inhibitor of Rho, we demonstrate that both the F-actin fiber formation in response to microtubule depolymerization and the formation of membrane ruffles after microtubule repolymerization occur in C3-treated MNGC, indicating that Rho is not required for these events.     

A ZYG-12–dynein interaction at the nuclear envelope defines cytoskeletal architecture in the C. elegans gonad

Changes in cellular microtubule organization often accompany developmental progression. In the Caenorhabditis elegans embryo, the centrosome, which is attached to the nucleus via ZYG-12, organizes the microtubule network. In this study, we investigate ZYG-12 function and microtubule organization before embryo formation in the gonad. Surprisingly, ZYG-12 is dispensable for centrosome attachment in the germline. However, ZYG-12–mediated recruitment of dynein to the nuclear envelope is required to maintain microtubule organization, membrane architecture, and nuclear positioning within the syncytial gonad. We examined γ-tubulin localization and microtubule regrowth after depolymerization to identify sites of nucleation in germ cells. γ-Tubulin localizes to the plasma membrane in addition to the centrosome, and regrowth initiates at both sites. Because we do not observe organized microtubules around zyg-12(ct350) mutant nuclei with attached centrosomes, we propose that gonad architecture, including membrane and nuclear positioning, is determined by microtubule nucleation at the plasma membrane combined with tension on the microtubules by dynein anchored at the nucleus by ZYG-12.     

Microtubules remodel actomyosin networks in Xenopus egg extracts via two mechanisms of F-actin transport

Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid ( approximately 250-300 nm/s) jerking and slow ( approximately 50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings.     

Study of the potential spermatogonial stem cell compartment in dogfish testis, <Emphasis Type="Italic">Scyliorhinus canicula</Emphasis> L.

In the lesser-spotted dogfish (Scyliorhinus canicula), spermatogenesis takes place within spermatocysts made up of Sertoli cells associated with stage-synchronized germ cells. As shown in testicular cross sections, cysts radiate in maturational order from the germinative area, where they are formed, to the opposite margin of the testis, where spermiation occurs. In the germinative zone, which is located in a specific area between the tunica albuginea of the testis and the dorsal testicular vessel, individual large spermatogonia are surrounded by elongated somatic cells. The aim of this study has been to define whether these spermatogonia share characteristics with spermatogonial stem cells described in vertebrate and non-vertebrate species. We have studied their ultrastructure and their mitotic activity by 5′-bromo-2′-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) immunodetection. Additionally, immunodetection of c-Kit receptor, a marker of differentiating spermatogonia in rodents, and of α- and β-spectrins, as constituents of the spectrosome and the fusome, has been performed. Ultrastructurally, nuclei of stage I spermatogonia present the same mottled aspect in dogfish as undifferentiated spermatogonia nuclei in rodents. Moreover, intercellular bridges are not observed in dogfish spermatogonia, although they are present in stage II spermatogonia. BrdU and PCNA immunodetection underlines their low mitotic activity. The presence of a spectrosome-like structure, a cytological marker of the germline stem cells in Drosophila, has been observed. Our results constitute the first step in the study of spermatogonial stem cells and their niche in the dogfish. G.L. is supported by a CIFRE grant (ANRT and C.RIS Pharma).     

Dorsomorphin Promotes Survival and Germline Competence of Zebrafish Spermatogonial Stem Cells in Culture

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs) in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species.     

Sterile Testis Complementation with Spermatogonial Lines Restores Fertility to DAZL-Deficient Rats and Maximizes Donor Germline Transmission

Despite remarkable advances in assisted reproductive capabilities ∼4% of all couples remain involuntarily infertile. In almost half of these cases, a lack of conception can in some measure be attributed to the male partner, wherein de novo Y-chromosomal deletions of sperm-specific Deleted-in-Azoospermia (DAZ) genes are particularly prevalent. In the current study, long-term cultures of rat spermatogonial stem cells were evaluated after cryo-storage for their potential to restore fertility to rats deficient in the DAZ-like (DAZL) gene. Detailed histological analysis of DAZL-deficient rat testes revealed an apparently intact spermatogonial stem cell compartment, but clear failure to produce mature haploid gametes resulting in infertility. After proliferating >1 million-fold in cell number during culture post-thaw, as few as 50,000 donor spermatogonia transplanted into only a single testis/recipient effectively restored fecundity to DAZL-deficient rats, yielding 100% germline transmission to progeny by natural mating. Based on these results, the potency and efficacy of this donor stem cell line for restoring fertility to azoospermic rodents is currently unprecedented. Prospectively, similar successes in humans could be directly linked to the feasibility of obtaining enough fully functional spermatogonial stem cells from minimal testis biopsies to be therapeutically effective. Thus, regeneration of sperm production in this sterile recipient provides an advanced pre-clinical model for optimizing the efficacy of stem cell therapies to cure a paradoxically increasing number of azoospermic men. This includes males that are rendered infertile by cancer therapies, specific types of endocrine or developmental defects, and germline-specific de novo mutations; all of whom may harbor healthy sources of their own spermatogonial stem cells for treatment.     

Cyclin A associates with the fusome during germline cyst formation in the Drosophila ovary

Regulated changes in the cell cycle underlie many aspects of growth and differentiation. Prior to meiosis, germ cell cycles in many organisms become accelerated, synchronized, and modified to lack cytokinesis. These changes cause cysts of interconnected germ cells to form that typically contain 2(n) cells. In Drosophila, developing germ cells during this period contain a distinctive organelle, the fusome, that is required for normal cyst formation. We find that the cell cycle regulator Cyclin A transiently associates with the fusome during the cystocyte cell cycles, suggesting that fusome-associated Cyclin A drives the interconnected cells within each cyst synchronously into mitosis. In the presence of a normal fusome, overexpression of Cyclin A forces cysts through an extra round of cell division to produce cysts with 32 germline cells. Female sterile mutations in UbcD1, encoding an E2 ubiquitin-conjugating enzyme, have a similar effect. Our observations suggest that programmed changes in the expression and cytoplasmic localization of key cell cycle regulatory proteins control germline cyst production.     

Centrosome migration into the Drosophila oocyte is independent of BicD and egl, and of the organisation of the microtubule cytoskeleton

During early Drosophila oogenesis, one cell from a cyst of 16 germ cells is selected to become the oocyte, and accumulates oocyte-specific proteins and the centrosomes from the other 15 cells. Here we show that the microtubule cytoskeleton and the centrosomes follow the same stepwise restriction to one cell as other oocyte markers. Surprisingly, the centrosomes still localise to one cell after colcemid treatment, and in BicD and egl mutants, which abolish the localisation of all other oocyte markers and the polarisation of the microtubule cytoskeleton. In contrast, the centrosomes fail to migrate in cysts mutant for Dynein heavy chain 64C, which disrupts the fusome. Thus, centrosome migration is independent of the organisation of the microtubule cytoskeleton, and seems to depend instead on the polarity of the fusome.     

BimC motor protein KLP61F cycles between mitotic spindles and fusomes in Drosophila germ cells.

KLP61F in Drosophila is a member of the BimC family of kinesins and, as for other family members [1], is required for spindle assembly [2] [3]. KLP61F is a bipolar homotetramer that cross-links spindle microtubules [4]. It is not known, however, whether the function of KLP61F is dedicated to mitosis or whether KLP61F interacts exclusively with microtubules. Previous work suggested that KLP61F functions during interphase in proliferating germ cells [3]. Cytokinesis is incomplete in germ cells and a branched cortical structure known as a fusome extrudes through intercellular bridges called ring canals. Here I show that, in germ cells, KLP61F cycles between spindles during mitosis and fusomes during interphase. Inspection of fusome-deficient hu-li tai shao (hts) mutants indicated that KLP61F gains fusome-dependent interactions near telophase that mediate its incorporation into these structures. KLP61F proved to be maintained in fusomes by microtubule-independent, detergent-resistant interactions. Inspection of KLP61F mutants indicated that KLP61F is required to recruit fusome material to spindle midbodies near telophase and for normal fusome organization. These observations suggest that KLP61F is bifunctional in germ cells, with microtubule-dependent functions in spindle assembly and microtubule-independent functions in fusome organization. Cytological analyses with antibodies against phosphorylated Eg5 peptide [4] suggest that cycling of KLP61F might reflect phosphorylation.     

Protein Phosphatase 1? Limits Ring Canal Constriction during Drosophila Germline Cyst Formation

Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well.