The Interactive Fly

Maternally transcribed genes

Cellularization and maternal-to-zygotic transition

  • Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo
  • The BAR domain of amphiphysin is required for cleavage furrow tip-tubule formation during cellularization in Drosophila embryos
  • Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles
  • An Arf-GEF regulates antagonism between endocytosis and the cytoskeleton for Drosophila blastoderm development
  • The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport
  • Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster
  • Membrane supply and demand regulates F-Actin in a cell surface reservoir
  • Pre-assembled nuclear pores insert into the nuclear envelope during early development
  • The Arf GAP Asap promotes Arf1 function at the Golgi for cleavage furrow biosynthesis in Drosophila
  • Geometric constraints alter cell arrangements within curved epithelial tissues
  • Syndapin promotes pseudocleavage furrow formation by actin organization in the syncytial Drosophila embryo

  • Number of nuclear divisions in the Drosophila blastoderm controlled by onset of zygotic transcription
  • Calpain A controls mitotic synchrony in the Drosophila blastoderm embryo
  • Co-activation of microRNAs by Zelda is essential for early Drosophila development
  • Drak is required for actomyosin organization during Drosophila cellularization
  • Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila
  • The Smaug RNA-binding protein is essential for microRNA synthesis during the Drosophila maternal-to-zygotic transition
  • Differentially-dimensioned furrow formation by zygotic gene expression and the MBT
  • Essential function of the serine hydroxymethyl transferase (SHMT) gene during rapid syncytial cell cycles in Drosophila
    Cytoskeletal components

    Others

    Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo

    Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). This study used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. It is proposed that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo (Mavrakis, 2008).

    To study the organization of the PM and the spatiotemporal dynamics of membrane components in living Drosophila embryos, transgenic animals were generated expressing different PM proteins tagged with Cerulean or Venus fluorescent proteins. The proteins were selected because they have different modes of membrane attachment and potentially different PM distributions. They included: (1) Venus fused to the first 20 amino acids of growth-associated protein 43 (GAP43), which contain a dual palmitoylation signal that tightly anchors the protein to the inner leaflet of the PM, (2) Cerulean fused to the pleckstrin-homology domain of phospholipase C delta 1, PH(PLCδ1), which binds specifically to the phosphoinositide PI(4,5)P2, and (3) Venus fused to full-length Toll receptor, a type I transmembrane protein that is required for dorsal-ventral embryonic polarity (Mavrakis, 2008).

    This study provides evidence that the plasma membrane of the fly syncytial blastoderm exhibits a polarized, epithelial-like organization prior to cellularization. Previously, it was thought that the PM of the blastoderm had no specialized organization prior to the formation of cell boundaries at cellularization. The results show that despite the absence of cell boundaries, the PM of the syncytial blastoderm has apical- and basolateral-like domains surrounding individual cortical nuclei and that PM proteins do not exchange between PM regions surrounding adjacent nuclei. This organization is maintained throughout syncytial mitotic division cycles and is dependent on an intact F-actin network (Mavrakis, 2008).

    Support for these conclusions came from live imaging and fluorescent highlighting experiments in living embryos. Using a variety of membrane markers, two distinct PM regions were distinguished. One region was above individual nuclei and had apical-like characteristics, including the presence of microvilli and an enrichment in PI(4,5)P2, a key determinant of apical PM biogenesis, as well as in GAP43, a protein that localizes to raft-like membranes, which typically compose apical PM surfaces in epithelial cells. The second PM region was lateral to nuclei, and was enriched in markers typically associated with basolateral membranes and junctions, including the cell-cell adhesion molecule E-cadherin, the multi-PDZ domain scaffolding protein DPatj. FRAP experiments showed that the molecules could freely diffuse in the PM domains surrounding individual nuclei but did not diffuse outside them, suggesting the presence of a diffusion barrier between the domains during interphase. Moreover, optical pulse-chase experiments showed that these components did not diffuse outside PM domains surrounding mitotic units throughout the time period of syncytial divisions. Thus, during mitosis, the polarized organization and restricted diffusion pattern of proteins in the PM did not change. Finally, the requirement of an intact F-actin network was supported by drug-induced actin depolymerization, which disrupted PM association of DPatj and Peanut and abolished the restricted diffusion pattern in the PM (Mavrakis, 2008).

    The finding that the PM of the syncytial blastoderm is organized as a pseudoepithelium prior to cellularization has several important implications for understanding many aspects of embryo development. First, it directly impacts on how dorsal-ventral and terminal patterning are set up prior to cellularization. These are dependent on Toll and Torso membrane receptors. Toll is distributed uniformly along the syncytial PM, but is activated only ventrally. Similarly, Torso is uniformly expressed along the surface membrane of early embryos, but its activation occurs only at the anterior and posterior poles. Given that membrane receptors have the capacity to diffuse across the PM, it has been unclear why the activation zones of these receptors do not spread widely across the PM. The results revealing the compartmentalized character of the PM during interphase and syncytial nuclear divisions now provide a potential answer. Receptors diffuse locally within the PM surrounding a particular nucleus, but they do not diffuse to PM regions associated with other nuclei. Consequently, activation zones of receptors (set up by the localized spatial signal of ligands) do not spread, allowing robust downstream signaling events in particular regions of the embryo. This possibility is supported by the spreading of the Dorsal gradient to more anterior and posterior regions in embryos treated with latA. LatA-induced actin depolymerization abolished the confined diffusion pattern in the PM suggesting that an intact actin network is likely to be important for containing activated Toll diffusion and thus maintaining a robust downstream Dorsal gradient (Mavrakis, 2008).

    The molecular basis for the compartmentalized diffusion in the PM of the syncytial embryo appears to be due to the presence of bona fide diffusion barriers in the PM regions directly between adjacent nuclei. The finding that septins and components of junctions are specifically enriched in this PM region raises the possibility that these molecules together with other cytoskeletal components organize a barrier to diffusion in the plane of the PM in a way similar either to the organization of septin rings at the yeast bud neck or of adherens junctions in epithelial cells. Moreover, the loss of PM association of DPatj and Peanut, as well as the abolishment of the restricted diffusion pattern in latA-treated embryos, suggest that an intact F-actin network is required both to localize and/or maintain septins and junctional components to specialized PM regions and to contain diffusion of proteins in PM units around individual syncytial nuclei. An intact F-actin network was recently shown to be required for compartmentalizing furrow canals during cellularization further supporting that F-actin organizes lateral diffusion of proteins in the PM. Future studies will need to genetically dissect the molecular machineries involved in organizing such diffusion barriers (Mavrakis, 2008).

    A second implication of the observed PM dynamics during syncytial mitoses relates to the machinery driving PM invagination. It was found that the PM was organized into highly convoluted microvillous membrane buds over interphase nuclei and these flattened out as soon as nuclei entered mitosis before reorganizing again into microvillous buds upon re-entry into the next interphase. Furthermore, the rate at which PM invaginated (~1.5-2 μm/min) was twice as fast as during the fast phase of cellularization, which involves de novo membrane delivery. Although endocytosis was recently shown to accompany metaphase furrow ingression, the current observations support a mechanism for PM invagination in mitosis that involves contractile machinery which transiently redistributes PM from microvilli caps into transient furrows surrounding mitotic units rather than an internal membrane source (Mavrakis, 2008).

    A final implication of these findings relates to cellularization, which produces the primary epithelial cells of the embryo. Polarization of the invaginating PM during cellularization has been reported, and it is during cellularization that PM polarity is first thought to be achieved in early fly embryogenesis. Because the data demonstrate that the PM is already polarized prior to cellularization, it is likely that the embryo uses this organization to initiate and organize the cellularization process. Consistent with this, it was found that the junctional proteins E-cadherin and DPatj, the septin protein Peanut, and Toll are all highly enriched in the PM at sites between adjacent nuclei during syncytial interphases, which reflects the PM organization between nuclei right at the onset of cellularization (first few minutes of interphase 14). Indeed, these are precisely the PM sites that become further differentiated within the first 5 min into cellularization, with the formation of an invaginating membrane front that contains Peanut and DPatj, basal adherens junctions directly adjacent to the invaginating front that contain E-cadherin, and the extension of the lateral membranes that are positive for Toll. The epithelial polarization occurring during cellularization is thus already reflected in the organization of the syncytial blastoderm PM (Mavrakis, 2008).

    In summary, these findings that the syncytial blastoderm PM exhibits an epithelial-like polarization prior to cellularization, and that distinct PM domains do not significantly exchange membrane components, point to an as yet unexplored mechanism for how the embryo maintains and generates morphogen gradients at this stage. By preventing activation zones of membrane receptors on the PM from spreading, robust downstream signaling events within the cytoplasm and nuclei of the embryo can be established. This mechanism would work in conjunction with nuclear-cytoplasmic shuttling of transcription factors, and a compartmentalized secretory pathway, to generate the dorsal-ventral and terminal patterning systems of the blastoderm fly embryo (Mavrakis, 2008).

    The BAR domain of amphiphysin is required for cleavage furrow tip-tubule formation during cellularization in Drosophila embryos

    De novo formation of cells in the Drosophila embryo is achieved when each nucleus is surrounded by a furrow of plasma membrane. Remodeling of the plasma membrane during cleavage furrow ingression involves the exocytic and endocytic pathways, including endocytic tubules that form at cleavage furrow tips (CFT-tubules). The tubules are marked by amphiphysin but are otherwise poorly understood. This study identified the septin family of GTPases as new tubule markers. Septins do not decorate CFT-tubules homogeneously: instead, novel septin complexes decorate different CFT-tubules or different domains of the same CFT-tubule. Using these new tubule markers, it was determined that all CFT-tubule formation requires the BAR domain of amphiphysin. In contrast, dynamin activity is preferentially required for the formation of the subset of CFT-tubules containing the septin Peanut. The absence of tubules in amphiphysin-null embryos correlates with faster cleavage furrow ingression rates. In contrast, upon inhibition of dynamin, longer tubules formed, which correlated with slower cleavage furrow ingression rates. These data suggest that regulating the recycling of membrane within the embryo is important in supporting timely furrow ingression (Su, 2013).

    Cellularization in the Drosophila embryo involves de novo generation of 6000 columnar epithelial cells, which are generated by the ingression of plasma membrane furrows (cleavage furrows) that enclose each nucleus. At the tip of ingressing cleavage furrows, CFT-tubules form. This study demonstrated the existence of three populations of CFT-tubules, which can de defined by different septin family members. The different populations of CFT-tubules are differentially regulated, and their presence or absence correlates with changes in cleavage furrow ingression kinetics (Su, 2013).

    Septins were identified as additional factors localizing to the CFT-tubules. Of interest, not all septins localize to the same CFT-tubules or the same domain within a single CFT-tubule. This suggests that although the CFT-tubules are formed by an endocytic pathway (Sokac, 2008), the tubules are not homogeneous. Instead, tubules can contain different domains that may have different functions. Three distinct types of tubules were identified: those that contain only amphiphysin and the septins Sep1 and Sep2, those that contain only the septins Peanut, Sep4, and Sep5, and those that possess heterogeneous subdomains each defined by a distinct composition of these various components. Of importance, localization studies suggest that distinct septin complexes localize to different structures. Because Peanut, Sep4, and Sep5 do not colocalize with Sep1 and Sep2 on CFT-tubules, it is predicted that Peanut, Sep4, and Sep5 form a novel septin complex. This new septin complex may resemble the previously isolated complex of Peanut, Sep1, and Sep2, as Sep2 is most closely related to Sep5 (72% identity) and Sep1 is most closely related to Sep4 (47% identity). It was not possible to isolate individual septin complexes by immunoprecipitation, as all septins coimmunoprecipitated. This finding is consistent with studies in mammalian cells and reflects either the heterogeneous nature of septin complexes within the entire embryo or that, in part, partial septin filaments were being immunoprecipitated. Unexpectedly, Peanut did not colocalize with Sep1 and Sep2 on CFT-tubules. This observation raises the possibility that Sep1 and Sep2 alone form a complex. Septin filaments in yeast and mammalian systems are generated from octamers containing two copies of four different septins arranged in an inverted repeat; however, this may not be true for all systems. In the case of Drosophila a hexamer of Peanut, Sep1, and Sep2 has been isolated, and in Caenorhabditis elegans there are only two septin genes (Su, 2013).

    Septins have predominantly been implicated in modulating events at the plasma membrane in conjunction with the actin cytoskeleton. In mammalian cells, septins have also been linked to potential roles in membrane trafficking, especially in the exocytic pathway, possibly by regulating vesicle fusion. It seems unlikely that the septins on CFT-tubules are regulating exocytosis, as all evidence suggests that exocytosis occurs at distinct apical sites in the syncytial embryo). In contrast, one study suggests a role for septins in the endocytic pathway by regulating recruitment of the coat protein complex AP-3 to lysosomal membranes (Baust, 2008). The precise roles for septins in this process are unclear. In CFT-tubules, it is possible that septins exert an effect directly on the membrane. Septins can tubulate membranes containing phosphatidylinositol (4,5)-bisphosphate, a lipid that has a key role in cytokinesis. However, the current data demonstrate that CFT-tubule formation is dependent on amphiphysin. Septins have been proposed to stabilize membranes. Therefore septins could stabilize the CFT-tubules once formed. Indeed, reduced recruitment of septins to cleavage furrows destabilizes the entire cleavage cleavage furrow. Furthermore, embryos depleted of Peanut form unstable yolk channels at the end of cellularization, further supporting the model that septins can stabilize membrane structures to which they localize. These findings also suggest that mutations that deplete septins will not allow examination of the role of septins in CFT-tubule organization and function (Su, 2013).

    This study found that CFT-tubule formation requires the BAR domain of amphiphysin. The N-BAR subfamily, to which amphiphysin belongs, can bind to membranes and promote their curvature. Amphiphysin is also involved in t-tubule formation in Drosophila indirect flight muscles and mouse heart muscle. These findings suggest a conserved role for amphiphysin in promoting tubule formation and organization (Su, 2013).

    Loss of amphiphysin and the prevention of CFT-tubule formation did not inhibit furrow ingression, suggesting that amphiphysin is not required for remodeling of the membrane to drive furrow ingression. Instead, loss of amphiphysin increased the rate of furrow ingression. Because amphiphysin localizes to the tip of the furrow, it is possible that amphiphysin acts as a negative regulator of furrow ingression. Alternatively, by preventing CFT-tubule formation, amphiphysin may render more plasma membrane accessible for furrow ingression, and therefore the rate of furrow ingression increases. Consistent with this model, when CFT-tubules become longer upon disruption of dynamin, the rate of cleavage furrow ingression is reduced. One potential consequence of inhibiting endocytosis at the furrow tip would be to reduce the amount of membrane available for the expansion of the plasma membrane and the ingression of the furrow. In such a scenario membrane derived from endocytosis at the tip of the furrow would be recycled back to the plasma membrane through the exocytic pathway, thereby providing sufficient membrane for the expansion and ingression of the furrow. This reduced availability of membrane could manifest itself as a reduced rate of furrow ingression seen in shibirets embryos at the nonpermissive temperature, where CFT-tubules elongate due to a failure to pinch off. The additional membrane may be especially important for the rapid increase in furrow ingression that is seen once the furrow has ingressed ∼10 μm, a depth of ingression where CFT-tubules normally become shorter and disappear (Su, 2013).

    Changes in tubule parameters correlate with changes in cleavage furrow ingression kinetics, especially in the fast phase of ingression; longer, more persistent tubules correlate with slower ingression kinetics, and the absence of tubules correlates with faster ingression kinetics. If the fast phase of cleavage furrow ingression were dependent upon new membrane being inserted into the plasma membrane, then restricting membrane insertion would suppress the fast phase. If membrane was recycled by endocytosis at the cleavage furrow tips through an endocytic compartment back to the plasma membrane, then changes in CFT-tubule parameters might be expected to affect cleavage furrow ingression kinetics (Su, 2013).

    In the models outlined in this study CFT-tubules would function to buffer the amount of available membrane that is accessible for efficient cleavage furrow ingression. However, no comparable measurements have been made with respect to t-tubules in muscles. Therefore it remains unclear whether the tubules in these different systems have a common function, whether they are examples of specialized endocytosis, or whether the creation of extra membrane surface area facilitates specialized functions in these different systems (Su, 2013).

    Mavrakis, M., Azou-Gros, Y., Tsai, F. C., Alvarado, J., Bertin, A., Iv, F., Kress, A., Brasselet, S., Koenderink, G. H. and Lecuit, T. (2014). Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles. Nat Cell Biol 16(4): 322-34. PubMed ID: 24633326

    Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles

    Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. This problem was addressed by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. The cleavage of the Drosophila melanogaster embryo was used as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings (Mavrakis, 2014).

    These findings demonstrate that septins are required during cellularization for generating curved and tightly packed actin filament networks at the tips of cellularization furrows termed furrow canals (FCs). Given that the F-actin bundling and bending activity is conserved for fly and human septins, it is predicted that septins contribute to the assembly, stabilization and contractility of cytokinetic rings. Septin depletion in dividing cultured cells, where anillin is localized to the cleavage furrow, leads to furrow instability, suggesting that the septin F-actin bundling activity could be required for proper cortical tension at the equator or/and the poles, which is in turn critical for the positioning of the cytokinetic ring46. Defective formation or stabilization of curved F-actin could further contribute to furrow shape instability. Decreased cytokinetic ring contractility was also recently confirmed in dividing Drosophila septin mutant epithelia, where anillin localizes to the cleavage furrow (Mavrakis, 2014).

    Septins could also potentially contribute to the shape of FCs in actin-independent ways. Recent studies provide compelling evidence that septins regulate the mechanical properties of the cortex in non-dividing mammalian cells. Septins were also proposed to provide the cortical rigidity and membrane curvature necessary for rice blast fungal infection. It will be important to investigate whether septins bind membranes at the FC, and how this might synergize with actin crosslinking for cortex organization (Mavrakis, 2014).

    The current findings indicate that the F-actin bending activity of septins requires septin hexamers and not septin filaments, although it cannot be excluded that they both function together in vivo. Septin post-translational modifications might also promote septin filament ring formation, which might in turn organize F-actin in curved bundles. Actin filaments could also potentially act in septin filament nucleation, even under conditions where septins alone do not form filaments. It will be important to compare the actin remodelling activity of septin hexamers and octamers, given that human septins (unlike Drosophila septins form octamers with hSep9 at the ends (Mavrakis, 2014).

    A striking feature of septins is that they bend actin into rings and other highly curved geometries. Actin circularization is energetically unfavourable owing to the large bending rigidity of actin filaments. Thus far, stable actin circles have been reported only under strong adhesion mediated by divalent cations or positively charged lipid monolayers. The adhesion energy mediated by septins seems sufficiently high to overcome the large bending energy associated with such a highly curved geometry. In cells, septin-mediated actin curving may act in synergy with myosin-induced actin filament buckling (Mavrakis, 2014).

    Septins could conceivably crosslink F-actin into loose contractile networks in processes that do not involve contractile F-actin rings, depending on the local septin and actin filament concentration and turnover, and septin post-translational modifications. Although no direct actin-septin interaction is known in interphase cells, their interplay has been reported in non-dividing cells, despite anillin's nuclear confinement. This study suggests that septins, alone or together with myosin-II, could contribute to these biological processes through their F-actin bundling activity (Mavrakis, 2014).

    Mammalian septins are known to interact with exocyst components and SNAREs and to regulate membrane fusion. Membrane growth during cellularization relies on vesicular trafficking, and the exocyst is necessary for this process. As membrane growth is delayed in septin mutants, it will be important to investigate whether septins mediate trafficking events that act together with actin remodelling at the FC to drive membrane growth (Mavrakis, 2014).

    An Arf-GEF regulates antagonism between endocytosis and the cytoskeleton for Drosophila blastoderm development

    Actin cytoskeletal networks push and pull the plasma membrane (PM) to control cell structure and behavior. Endocytosis also regulates the PM and can be promoted or inhibited by cytoskeletal networks. However, endocytic regulation of the general membrane cytoskeleton is undocumented. This study provides evidence for endocytic inhibition of actomyosin networks. Specifically, it was found that Steppke, a cytohesin Arf-guanine nucleotide exchange factor (GEF), controls initial PM furrow ingression during the syncytial nuclear divisions and cellularization of the Drosophila embryo. Acting at the tips of ingressing furrows, Steppke promotes local endocytic events through its Arf-GEF activity and in cooperation with the AP-2 clathrin adaptor complex. These Steppke activities appear to reduce local Rho1 protein levels and ultimately restrain actomyosin networks. Without Steppke, Rho1 pathways linked to actin polymerization and myosin activation abnormally expand the membrane cytoskeleton into taut sheets emanating perpendicularly from the furrow tips. These expansions lead to premature cellularization and abnormal expulsions of nuclei from the forming blastoderm. Finally, consistent with earlier reports, it was also found that actomyosin activity can act reciprocally to inhibit the endocytosis at furrow tips. It is proposed that Steppke-dependent endocytosis keeps the cytoskeleton in check as early PM furrows form. Specifically, a cytohesin Arf-GEF-Arf G protein-AP-2 endocytic axis appears to antagonize Rho1 cytoskeletal pathways to restrain the membrane cytoskeleton. However, as furrows lengthen during cellularization, the cytoskeleton gains strength, blocks the endocytic inhibition, and finally closes off the base of each cell to form the blastoderm (Lee, 2013).

    Coupling actomyosin networks to the plasma membrane (PM) is essential for cells to migrate, interact, change shape, and divide. As examples, actin networks form and function at the leading edge of migratory cells, at cell-substrate adhesion complexes, and at cell-cell adhesion complexes in multicellular tissues. To assemble these complexes, receptors can physically engage the actin cytoskeleton and also induce cytoskeletal assembly via Rho- family guanosine triphosphate (GTP)ases and phosphoinositide signaling. Inversely, endocytosis can remove receptors from the PM promoting the turnover of adhesion and signaling complexes. More generally, the close links of both actin networks and endocytic machinery with the PM suggest possible crosstalk between these subsystems. Indeed, endocytic signaling nucleates local actin networks to help drive membrane invagination and scission (Mooren, 2012; Anitei, 2012). In contrast, more widespread membrane cytoskeleton activity can create tension that inhibits membrane invagination. Conceivably, endocytosis could also inhibit the membrane cytoskeleton, but such activity is undocumented (Lee, 2013).

    The syncytial Drosophila embryo is a well-established model for studying actomyosin networks and membrane trafficking during PM furrow ingression. In the early syncytial embryo, nuclei divide synchronously just beneath the PM. At each division cycle, the activities of Rho-family GTPases, the Arp2/3 complex, and the formin Diaphanous (Dia) organize actomyosin-based PM ingressions (pseudocleavage furrows) that surround each nucleus to prevent nuclear collision and loss. Once ~6,000 nuclei form, similar mechanisms induce a final round of PM ingressions. These furrows persist and elongate through membrane trafficking to apical and lateral sites, and with support of actomyosin networks at their basal tips (the furrow canals). This massive PM growth cellularizes the first embryonic epithelium, a process completed with constriction of actomyosin rings formed at the base of each cell. Recently, endocytic events were detected at the tips of pseudocleavage furrows and early cellularization furrow canals by the presence of Amphiphysin (Amph)-positive tubules and the internalization of labeled PM (Sokac, 2008). These events have provided a model for studying how the actin cytoskeleton can both promote and inhibit endocytosis (Sokac, 2008; Yan, 2013). However, the role of this endocytosis is unclear, and paradoxically, it would appear to counteract membrane growth. This study examined how Arf G protein (Arf) activation might be involved. In other contexts, Arfs promote endocytosis by recruiting coat proteins, activating lipid signaling, and triggering actin polymerization. Like other G proteins, Arfs are activated by guanine nucleotide exchange factors (GEFs). Cytohesins are a major class of PM Arf-GEFs (Donaldson, 2011), and roles for cytohesin Arf-GEFs have been documented at migratory leading edges, focal adhesions, and adherens junctions in mammalian cell culture (Santy, 2005; Torii, 2010; Ikenouchi, 2010). Drosophila contains one cytohesin, called Steppke (Step). Step is known to function in postembryonic insulin and EGF signaling, which mammalian cytohesins do as well, but its contributions to the Drosophila embryo and to other cellular processes are unknown. This study shows that Step promotes endocytosis at pseudocleavage furrows and furrow canals to restrain actomyosin networks at these sites (Lee, 2013).

    These data provide the first description of cytohesin function in a developing embryo. Drosophila Step promotes a subset of endocytic events at the tips of ingressing PM furrows during embryo cellularization. Endocytosis has been documented previously at these sites (Sokac, 2008), but its role has been unclear. By manipulating a conserved upstream activator of endocytosis, this study has identified an important role of endocytosis in controlling the membrane cytoskeleton. The data argue that Step acts at furrow tips to induce local Arf-dependent endocytosis, which in turn antagonizes Rho1-dependent actomyosin network assembly at these sites. It was also found that the cytoskeleton can inhibit endocytosis at the furrow tips, as has been previously shown in this system (Sokac, 2008; Yan, 2013) and in other contexts. An overall model is proposed in which this reciprocal relationship is one-sided at specific developmental stages. At newly forming PM furrows, Step dominates, promoting endocytosis that keeps cytoskeleton activity in check for proper pseudocleavage and cellularization furrow architecture and growth. During later cellularization, the cytoskeleton dominates. Zygotic expression of actin regulators such as Nullo normally increases actomyosin activity as cellularization proceeds and appears to work in conjunction with Dia to block endocytic events at the furrow tips (Sokac, 2008; Yan, 2013)]. By counteracting the inhibitory endocytosis, cytoskeletal activity would elevate further but at these later stages is locally restrained by a distinct mechanism requiring Bottleneck (Schejter, 1993). To form the blastoderm, this second restraint mechanism is removed, and contractile rings close off the base of each cell. In the absence of the initial step-mediated restraint mechanism, it is proposed that the cytoskeleton abnormally dominates the relationship at all early PM furrows. Without Step-based endocytic inhibition, it is speculated that actomyosin networks abnormally expand and inhibit other endocytic events leading to coexpansion of cytoskeletal polymers and PM from the furrow tips (Lee, 2013).

    An important element of the model is the local induction of endocytic events. The data localize Step to the tips of ingressing PM furrows, and both the loss and overexpression of Step alter membrane organization specifically at these sites. This localized Step-regulated activity occurs in a dynamic global membrane trafficking system within each forming cell. During the peripheral nuclear divisions, each nucleus acquires its own endoplasmic reticulum and Golgi apparatus that function with recycling endosomes to direct exocytosis to growing PM furrows at cellularization. Simultaneously, endocytic events occur over the apical PM and at the furrow tips, with endocytosed material recycled to the growing furrows. Thus, the overall membrane system is in continual flux, and coordination by local regulation would be expected. The data identify a polarized endocytic activator required for the process. Step Arf-GEF activity is critical for restraining the membrane cytoskeleton at furrow tips, and a subset of AP-2 activities is involved as well (Lee, 2013).

    How could endocytosis and actomyosin networks impact each other at the tips of PM furrows or elsewhere? This question can be considered from several levels of organization. First, a simple and direct connection could be endocytic removal of one or more PM actomyosin regulators. This work identifies Rho1 or an upstream regulator as a candidate. Intriguingly, membrane trafficking has been previously linked to the Rho1 pathway in this context. Specifically, recycling endosomes have been implicated in the trafficking of RhoGEF2 to the PM (Cao, 2008). It was hypothesized that RhoGEF2 might also be a target of Step for its removal from the PM but no difference was found in RhoGEF2 levels at furrow canals in step loss-of-function embryos. Thus, Rho1 may be a more specific target of Step, although a direct connection to the Rho1 pathway remains to be determined. Of note, a number of septins have also recently been observed on the Amph-positive tubules (Lee, 2013).

    Second, interplay between different pools of actin is possible. For example, actin contributes to the invagination and scission of endocytic vesicles, and thus, endocytic actin and other PM actin networks could compete for regulators or components. Additionally, there could be signaling crosstalk between regulators of the different networks. For example, Arf signaling often elicits local Rac or Cdc42 activity, and this might trigger crosstalk affecting Rho activity. Interestingly, overexpression of Cdc42-interacting protein 4 (Cip4) appears to antagonize Dia at furrow canals, although Cip4 mutants have no cellularization phenotype on their own. Significantly, however, this study found that Step acts with AP-2 to control the membrane cytoskeleton. This Step-AP-2 cooperation suggests that clathrin-coated pits are involved in the antagonism, although it does not exclude the possibility of separate cytoskeletal crosstalk (Lee, 2013).

    Third, larger scale interactions should be considered. Endocytosis could remove membrane in bulk that would otherwise support the membrane cytoskeleton, although observations of residual furrow canal endocytic activity with step loss of function suggest a more specific mechanism. Inversely, the membrane cytoskeleton could block endocytosis by elevating PM tensio or possibly by sterically blocking endocytic machinery from accessing the PM (Lee, 2013).

    Endocytic-cytoskeletal crosstalk is relevant to many cellular processes. For example, receptor endocytosis occurs in proximity to actomyosin networks in various contexts, including migratory leading edges, focal adhesions, and adherens junctions. However, these endocytic events and actomyosin networks have mainly been studied independently, and thus their functional integration is not understood. This study highlights the possibility that endocytic activity at such assemblies could simultaneously remove receptors and antagonize local cytoskeletal networks, with both effects promoting complex turnover and cellular dynamics (Lee, 2013).

    The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport

    Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. This study shows that mutations in drop out (dop; CG6498) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. This study shows that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein (Hain, 2014).

    This study is the first mutational analysis of a MAST kinase in any organism and demonstrates that the MAST kinase Dop plays an important role in plasma membrane cortex compartmentalisation during the generation of epithelial polarity in the fly. The results reported in this study demonstrate a requirement of Dop in the establishment of the furrow canal and the bAJ at the cycle 14 transition. The defect in bAJ formation is likely to be a consequence of a failure in the initial specification of the incipient furrows. It is proposed that Dop acts upstream in furrow canal formation by controlling the formation of F-actin-rich foci, which initiate the assembly of a specific furrow membrane cortex (Hain, 2014).

    In mid-cellularisation stages, dop mutant phenotypes are reminiscent of embryos lacking the early zygotic gene bottleneck (bnk). In bnk mutants the initial formation of the cleavage furrows is normal, but then furrows close prematurely. Although it cannot be excluded that bnk might play a role in later defects associated with dop mutations, the primary defect in dop mutants concerned the lack of regular F-actin-rich furrows during the onset of cellularisation. Another early zygotic gene, nullo, is required for the proper recruitment of F-actin during furrow canal formation. Nullo and the actin regulator RhoGEF2 have been proposed to act in parallel pathways controlling processes that are distinct but both essential for F-actin network formation during the establishment of the furrow canal. Since early F-actin rearrangements are largely normal in nullo and RhoGEF2 single mutants, it is proposed that Dop is essential for the initial early focussing of F-actin, whereas Nullo and RhoGEF2 are required to elaborate and maintain F-actin levels to stabilise the furrows. The actin regulator enabled (ena) has been shown to act downstream of Abelson tyrosine kinase (Abl) in the redistribution of F-actin from the plasma membrane cortex into the furrows in both syncytial stages and cellularisation. Although ena would provide a good candidate for acting downstream of dop in the redistribution of F-actin, ena is already required for syncytial cleavages and the F-actin phenotypes in Abl mutants are much more severe than those that were found for dop mutants (Hain, 2014).

    The similarity of syncytial cleavage furrows and the cleavage furrows at cellularisation raises the question of how they differ from each other. The molecular basis of the hexagonal pattern of the F-actin-rich cell cortex at the cleavage furrow relies upon the recycling endosome components Rab11 and Nuclear fallout (Nuf) and the actin polymerisation factors Dia and Scar/Arp2/3. In contrast to dop mutants, nuf, dia or Scar mutants indicate that these genes are required also for the dynamic redistribution of F-actin during syncytial development. Since Dop is a maternally supplied protein, its activity might be regulated by events triggered during the cycle 13-14 transition. The major difference between the furrows in syncytial stages and cellularisation is that metaphase furrows are formed during M phase, whereas cellularisation furrows are formed during G2 phase. Since Dop is a maternally supplied gene product, one would have to implicate regulation of Dop by zygotic factors to explain its phenotype at the cycle 13-14 transition. An alternative possibility is that Dop is regulated by phosphorylation or other post-translational modification through the cell cycle machinery and that, in the absence of Cdk1-dependent phosphorylation, its phosphorylation state is changed. This study provided evidence that Dop is indeed differentially post-translationally modified during syncytial versus cellular blastoderm stages. It is proposed that such cell cycle-dependent regulation of Dop may be crucial in transforming syncytial cleavages into persistent cellularisation furrows. Furthermore, the data suggest that this transition could require Dop-dependent regulation of dynein-associated microtubule transport (Hain, 2014).

    The mechanisms for the initial localisation of Baz and E-cadherin are still unclear but, interestingly, dop is required for the localisation of both proteins. At the cycle 14 transition, E-cadherin and Arm puncta are associated with apical membrane projections and the homophilic association of these cadherin puncta is strengthened by membrane flow and is dependent on actin. Baz function allows these puncta to become tightened into sAJs. Thus, Dop might affect the stabilisation of the weakly interacting puncta either through cortical actin organisation or membrane flow. In addition to this early requirement for Baz localisation, Dop is also involved in clearing Baz from the basal cytoplasm during late cellularisation. The mechanism that eventually clears Baz from the basal cytoplasm depends on dynein-based transport. Therefore, Dop is required for dynein-based transport of different cargoes during cellularisation: lipid droplets, mRNA particles, Golgi and Baz. It is proposed that the main function of Dop in cellularisation is in regulating dynein-mediated transport of important cargos along microtubules (Hain, 2014).

    This study presents the first evidence for regulation of dynein-mediated transport by a MAST family kinase. Dop is shown to controls phospho-Dic levels in a direct or indirect manner. The data are consistent with a model in which the initiation of furrow formation involves dynein-dependent transport that is controlled by Dop. In support of a role in membrane formation, this study found defects in the distribution of the recycling endosome and Golgi compartments in dop mutants. Interference with Rab11 function causes similar defects in Slam distribution as those shown by dop mutants. Therefore, Dop might control the transport of endomembrane compartments, which drive membrane growth. In addition, F-actin redistribution plays a major role in membrane cortical compartmentalisation in the initial stages of cellularisation. The focussing of F-actin to incipient furrows might involve a dynein-dependent shift of actin regulators or existing actin filaments to the furrow. An attractive hypothesis is that the translocation of F-actin and/or its regulators is coupled to an endomembrane compartment that is transported via microtubules towards the incipient furrow canals. Future studies should aim to determine which dynein cargos contribute to furrow formation and how Dop regulates Dic phosphorylation at the molecular level (Hain, 2014).

    Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster

    One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminates in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events have remained unclear. This study shows that Rab8 has striking dynamic behaviors in vivo. As furrows ingress, cytoplasmic Rab8 puncta are depleted and Rab8 accumulates at the plasma membrane in a location that coincides with known regions of directed membrane addition. CRISPR/Cas9 technology was used to N-terminally tag Rab8, which is then used to address both endogenous localization and function. Endogenous Rab8 displays partial coincidence with Rab11 and the Golgi, and this colocalization is enriched during the fast phase of cellularization. When Rab8 function is disrupted, furrow formation in the early embryo is completely abolished. Rab8 behaviors require the function of the exocyst complex subunit Sec5 as well as the recycling endosome Rab11. Active, GTP-locked Rab8 is primarily associated with dynamic membrane compartments and the plasma membrane, while GDP-locked Rab8 forms large cytoplasmic aggregates. These studies suggest a model in which active Rab8 populations direct furrow ingression by guiding the targeted delivery of cytoplasmic membrane stores to the cell surface through exocyst tethering complex interactions (Mavor, 2016).

    Membrane supply and demand regulates F-Actin in a cell surface reservoir

    Cells store membrane in surface reservoirs of pits and protrusions. These membrane reservoirs facilitate cell shape change and buffer mechanical stress, but how reservoir dynamics are regulated is not known. During cellularization, the first cytokinesis in Drosophila embryos, a reservoir of microvilli unfolds to fuel cleavage furrow ingression. This study found that regulated exocytosis adds membrane to the reservoir before and during unfolding. Dynamic F-actin deforms exocytosed membrane into microvilli. Single microvilli extend and retract in ~20 s, while the overall reservoir is depleted in sync with furrow ingression over 60-70 min. Using pharmacological and genetic perturbations, this study shows that exocytosis promotes microvillar F-actin assembly, while furrow ingression controls microvillar F-actin disassembly. Thus, reservoir F-actin and, consequently, reservoir dynamics are regulated by membrane supply from exocytosis and membrane demand from furrow ingression (Figard, 2016).

    Pre-assembled nuclear pores insert into the nuclear envelope during early development

    Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. This study shows that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. This paper delineates a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. This unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation (Hampoelz, 2016).

    The Arf GAP Asap promotes Arf1 function at the Golgi for cleavage furrow biosynthesis in Drosophila

    Biosynthetic traffic from the Golgi drives plasma membrane growth. For Drosophila embryo cleavage, this growth is rapid, but regulated, for cycles of furrow ingression and regression. The highly conserved small G protein Arf1 organizes Golgi trafficking. Arf1 is activated by guanine nucleotide exchange factors, but essential roles for Arf1 GTPase activating proteins (GAPs) are less clear. This study reports that the conserved Arf GAP Asap is required for cleavage furrow ingression in the early embryo. Since Asap can affect multiple sub-cellular processes, genetic approaches were used to dissect the primary effect of Asap. The data argue against cytoskeletal or endocytic involvement, and reveal a common role for Asap and Arf1 in Golgi organization. Although Asap lacked Golgi enrichment, it was necessary and sufficient for Arf1 accumulation at the Golgi, and a conserved Arf1-Asap binding site was required for Golgi organization and output. Notably, Asap re-localized to the nuclear region at metaphase, a shift that coincided with subtle Golgi re-organization preceding cleavage furrow regression. It is concluded that Asap is essential for Arf1 to function at the Golgi for cleavage furrow biosynthesis. Asap may recycle Arf1 to the Golgi from post-Golgi membranes, providing optimal Golgi output for specific stages of the cell cycle (Rodrigues, 2016).

    Geometric constraints alter cell arrangements within curved epithelial tissues

    Organ and tissue formation are complex three-dimensional processes involving cell division, growth, migration, and rearrangement, all of which occur within physically constrained regions. However, analyzing such processes in three dimensions in vivo is challenging. This study focused on the process of cellularization in the anterior pole of the early Drosophila embryo to explore how cells compete for space under geometric constraints. Using microfluidics combined with fluorescence microscopy, quantitative information was extracted on the three-dimensional epithelial cell morphology. A cellular membrane rearrangement was observed in which cells exchange neighbors along the apical-basal axis. Such apical-to-basal neighbor exchanges were observed more frequently in the anterior pole than in the embryo trunk. Furthermore, cells within the anterior pole skewed toward the trunk along their long axis relative to the embryo surface, with maximum skew on the ventral side. A vertex model was constructed for cells in a curved environment. The observed cellular skew was reproduced in both wild-type embryos and embryos with distorted morphology. Further, such modeling showed that cell rearrangements were more likely in ellipsoidal, compared with cylindrical, geometry. Overall, it was demonstrated that geometric constraints can influence three-dimensional cell morphology and packing within epithelial tissues (Rupprecht, 2017).

    Syndapin promotes pseudocleavage furrow formation by actin organization in the syncytial Drosophila embryo

    Coordinated membrane and cytoskeletal remodeling activities are required for membrane extension in processes such as cytokinesis and syncytial nuclear division cycles in Drosophila. Pseudocleavage furrow membranes in the syncytial Drosophila blastoderm embryo show rapid extension and retraction regulated by actin-remodeling proteins. The F-BAR domain protein Syndapin (Synd) is involved in membrane tubulation, endocytosis, and, uniquely, in F-actin stability. This study reports a role for Synd in actin-regulated pseudocleavage furrow formation. Synd localized to these furrows, and its loss resulted in short, disorganized furrows. Synd presence was important for the recruitment of the septin Peanut and distribution of Diaphanous and F-actin at furrows. Synd and Peanut were both absent in furrow-initiation mutants of RhoGEF2 and Diaphanous and in furrow-progression mutants of Anillin. Synd overexpression in rhogef2 mutants reversed its furrow-extension phenotypes, Peanut and Diaphanous recruitment, and F-actin organization. It is concluded that Synd plays an important role in pseudocleavage furrow extension, and this role is also likely to be crucial in cleavage furrow formation during cell division (Sherlekar, 2016).

    Cleavage furrow formation during cell division requires a highly conserved set of cytoskeletal and membrane-trafficking proteins. Their positioning and initiation involves microtubules and the centralspindlin complex. Rho-GTPase-activating protein (RacGAP50C) of this complex positions Rho-GTP exchange factor (RhoGEF) Pebble at contractile rings, and another RhoGEF2 functions in pseudocleavage furrows to activate Rho1 for furrow initiation. Rho1 recruits formins that assemble an actin scaffold for contractile-ring formation and/or furrow initiation. Formin activity also depends on the presence of a scaffold protein, Anillin, at the contractile ring. RacGAP50C also accumulates Anillin at the furrow, which is responsible for both septin and myosin II association at the contractile ring. Cytokinesis failure increases in Caenorhabditis elegans when embryos are depleted of both Rho kinase and Anillin/septins, implying that they work together for robust furrow formation (Sherlekar, 2016).

    The cell division cycle is accompanied by drastic changes in cell shape that necessitate dynamic interplay between the membrane and actin cytoskeleton. In the Drosophila syncytial embryo, nuclear division cycles 10-13 are rapid and involve dynamic pseudocleavage furrow ingression and retraction between adjacent dividing nuclei. These furrows serve to prevent spindle cross-talk across compartments during metaphase of each cycle and organize the embryo into discrete polarized functional units. Furrow positioning and initiation at this stage requires RhoGEF2 for recruiting Rho1 and the formin Diaphanous (Dia). Microtubules are required for furrow positioning, while furrow ingression involves dynamic growth of actin filaments through Profilin and the action of anticapping proteins (like Ena/VASP). The syncytial cycles are followed by massive elongation of furrows to form individual cells in a process called cellularization, during which membrane extension is fueled by flattening of apical microvilli and Rab11-mediated endocytosis and driven by an actomyosin contractile ring that, apart from actin and myosin II, also comprises Anillin, septins, RhoGEF2, and Dia. Although contractile rings first form only during cellularization in early developing Drosophila embryos, the syncytial pseudocleavage furrows contain most of the proteins present in the contractile ring such as Rho1, RhoGEF2, Dia, Anillin, and septins (Sherlekar, 2016).

    F-BAR domain-containing proteins link membrane and cytoskeleton in various processes, including endocytosis, cell shape and polarity, cell motility, and cytokinesis. The yeast orthologues of F-BAR protein Cip4 are known to recruit formins and influence their nucleation and elongation activities. In addition, Hof1 (Cip4 in Saccharomyces cerevisiae) coiled-coil domain binds Septin (Cdc10) and localizes it to the bud neck. Drosophila Cip4, however, is not essential for formin Dia recruitment to cellularization furrows, and its loss does not result in a defect in cellularization but its overexpression shows dia loss-of-function phenotypes. The F-BAR domain protein, Syndapin/Pacsin (Synd), initially identified as a binding partner for Dynamin and neuronal Wiscott-Aldrich syndrome protein (N-Wasp) via its SH3 domain, participates in endocytosis and actin remodeling. Mammalian Synd1 binds to the actin nucleator Cordon bleu (Cobl)and mediates its interaction with Arp2/3 to affect actin nucleation during neuromorphogenesis. Synd, unlike other F-BAR proteins, directly binds and stabilizes F-actin and, unlike any N- or F-BAR protein, can generate a range of membrane curvatures much greater than its own intrinsic curvature. Drosophila Synd promotes expansion of the subsynaptic reticulum, which also requires actin-remodeling. Drosophila Synd also binds to Anillin via its myosin-binding domain in vitro, localizes at the cytokinetic furrow (earlier than Drosophila Cip4) in D.Mel-2 cells, and is important for cytokinesis during male meiosis in primary spermatocytes. Together these studies suggest a role for Synd in coordinated membrane and actin remodeling during cleavage furrow formation. However, no analysis of its recruitment dynamics or functional analysis in organization of actin or actin-remodeling proteins with respect to furrow initiation or extension machinery has been carried out so far. This study reports that Synd is important for syncytial Drosophila pseudocleavage furrow extension; septin Peanut (Pnut) recruitment; and distinct Dia, Anillin, and actin localization. Most significantly, Synd can recruit actin remodeling proteins, organize actin, and result in furrow extension during pseudocleavage furrow formation in rhogef2-depleted embryos (Sherlekar, 2016).

    Syndapins belong to the family of highly conserved F-BAR-domain containing proteins with diverse roles in membrane tubulation, Clathrin-mediated and bulk endocytosis, and actin remodeling and cytokinesis. Synd is thus poised to play a role in processes like furrow formation, which needs orchestrated remodeling of both the membrane and the cytoskeleton. Furrow elongation in syncytial Drosophila embryos is an excellent model system to study the role of proteins that drive its formation. Previous studies show that furrow formation involves membrane addition by trafficking and membrane extension by remodeling of the actin meshwork. This study has conclusively demonstrated that Synd functions to promote furrow formation by organization and elongation of F-actin structures. Synd is essential for recruitment and distribution of Pnut and Dia on the membrane. In turn, Pnut and Dia also affect Synd distribution on the membrane. RhoGEF2/Dia and Anillin/Pnut have been previously shown to regulate F-actin architecture at cleavage and cellularization furrows, and loss of Synd in synd mutants therefore affects actin both directly and through its influence on Pnut and Dia localization. As with other actin-regulated processes, even though a linear pathway of association/regulation of these actin-remodeling proteins to the furrow membrane is unlikely, the data imply that Synd is a key component in the RhoGEF2-Dia-Anillin/Pnut pathway during actin-driven furrow elongation. Synd2 can bind and inhibit Rac1 via its SH3 domain, thus reducing Arp2/3 activity, and may therefore be able to potentiate Dia activity by increasing RhoA levels. Such a mechanism can explain increased Dia function when Synd is overexpressed in RhoGEF2 knockdown embryos, which, along with recruitment of Pnut to the membrane, can help organize actin and elongate cleavage furrows (Sherlekar, 2016).

    Actin stabilization into continuous structures reversed the furrow length defect in synd mutant embryos. Jasplakinolide (Jasp) blocks actin turnover at the contractile ring and affects cleavage furrow invagination while preserving furrow integrity, and hence showed fewer punctae in synd and rhogef2 mutant embryos. CytoD, on the other hand, allows actin polymerization, and as a result, synd and rhogef2 mutant embryos treated with CytoD displayed more organized actin structures and elongated furrows. This provides mechanistic insight into how Synd functions in regulating actin polymerization and may be further investigated through kinetic studies of actin polymerization (Sherlekar, 2016).

    Overexpression of Synd and not Pnut in the rhogef2RNAi-containing embryos partially reversed pseudocleavage furrow recruitment and morphology defects seen in rhogef2RNAi and increased the furrow length compared with wild type. Synd activity is thus needed at the pseudocleavage furrow for extension, and some as yet uncharacterized proteins play a role in furrow limitation. It is interesting to compare the functions of F-BAR domain proteins, Synd with Cip4 in furrow elongation and Dia recruitment. Cip4 antagonizes Dia function, and its overexpression has dia loss-of-function phenotypes like missing furrows. It is possible that opposing activities of F-BAR proteins Synd and Cip4 with respect to Dia are in a balance, and future experiments can test whether this function plays a role in limiting the growth of pseudocleavage furrows (Sherlekar, 2016).

    Because Synd's SH3 domain interacts with Dynamin, and Dynamin has a role in endocytosis and furrow extension in syncytial divisions and cellularization, it remained to be investigated whether Clathrin-dependent endocytosis defects in synd mutants also affect furrow elongation. This study shows that synd mutant embryos have defects in cleavage furrow-tubule length and Rab5 vesicle numbers. Decrease in Rab5 vesicle numbers is also seen in rhogef2 mutant embryos. However, Synd-GFP overexpression in rhogef2 mutant embryos is able to reverse the furrow-extension defect without rescuing the Rab5 endocytic vesicle defect. Taken together, these data show that Synd has a role in endocytosis, but the reversal of furrow phenotypes in rhogef2 mutant embryos is due to the ability of Synd to recruit and organize actin and proteins of the actin-remodeling machinery such as Dia and Pnut (Sherlekar, 2016).

    This analysis of membrane architecture and pseudocleavage furrow length in rhogef2, pnut, and synd mutants found that shorter furrows in each of these mutants were also loose/unstable and had slow lateral movement during the nuclear cycle. Septins brace the plasma membrane against aberrant cell-shape deformation and are able to tubulate phosphatidylinositol-4,5-bisphosphate liposome membranes when treated with a brain extract. It is probable that Septin-mediated membrane tubulation activity and cell-shape effects are dependent on the presence of F-BAR proteins like Synd. Sept7 mutants in Xenopus show unstable and undulating membranes during gastrulation. This substantiates Synd’s role in maintenance of membrane integrity and shape by affecting actin organization and Pnut recruitment (Sherlekar, 2016).

    Overall mutant and epistatic analyses presented in this study find a significant role for the F-BAR domain protein Synd in mediating pseudocleavage furrow extension. This study favors a model in which Synd, along with Anillin and RhoGEF2, provide a platform for recruitment of Dia and Pnut to allow persistent and stable growth of actin to promote furrow elongation. Further experiments combining protein interactions and deduction of the biophysical nature of Synd-Pnut-actin association with the plasma membrane will elucidate the molecular mechanism that makes Synd an important component of pseudocleavage furrow-extension or contractile-ring assembly at large (Sherlekar, 2016).

    Number of nuclear divisions in the Drosophila blastoderm controlled by onset of zygotic transcription

    The cell number of the early Drosophila embryo is determined by exactly 13 rounds of synchronous nuclear divisions, allowing cellularization and formation of the embryonic epithelium. The pause in G2 in cycle 14 is controlled by multiple pathways, such as activation of DNA repair checkpoint, progression through S phase, and inhibitory phosphorylation of Cdk1, involving the genes grapes, mei41, and wee1. In addition, degradation of maternal RNAs and zygotic gene expression are involved. The zinc finger Vielfaltig (Vfl) controls expression of many early zygotic genes, including the mitotic inhibitor fruhstart. The functional relationship of these pathways and the mechanism for triggering the cell-cycle pause have remained unclear. This study shows that a novel single-nucleotide mutation in the 3' UTR of the RNA polymerase RNPII215 gene leads to a reduced number of nuclear divisions that is accompanied by premature transcription of early zygotic genes and cellularization. The reduced number of nuclear divisions in mutant embryos depends on the transcription factor Vfl and on zygotic gene expression, but not on grapes, the mitotic inhibitor Fruhstart, and the nucleocytoplasmic ratio. It is proposed that activation of zygotic gene expression is the trigger that determines the timely and concerted cell-cycle pause and cellularization (Sung, 2012).

    Embryos from germline clones of the lethal mutation X161 (in the following, designated as mutant embryos) showed a reduced cell number but otherwise developed apparently normally until at least gastrulation stage. Cell specification along the anterior-posterior and dorsoventral axes proceeded as in wild-type, as demonstrated by the seven stripes of eve expression, mesoderm invagination, and cephalic furrow formation. The reduced cell number can be due to a lower number of nuclear divisions prior to cellularization or to loss of nuclei in the blastoderm. To distinguish these possibilities, time-lapse recordings were performed of mutant embryos in comparision to wild-type. To measure the cell-cycle length, the nuclei in these embryos were fluorescently labeled. Three types of embryos were observed: (1) with 13 nuclear divisions with an extended interphase 13 (28 min versus 21 min in wild-type), (2) with 12 nuclear divisions, and (3) with partly 12 and partly 13 nuclear divisions with an extended interphase 13. Because a severe nuclear fallout phenotype was not observed, it is concluded that the reduced cell number in gastrulating embryos is due to the reduced number of nuclear divisions. Consistent with these observations, the number of centromeres and centrosomes was normal in mutant embryos (Sung, 2012).

    In wild-type embryos, interphase 14 is different from the preceeding interphases, in that the plasma membrane invaginates to enclose the individual nuclei into cells. In X161 embryos with patches in nuclear density, furrow markers showed more advanced furrows in the part with a lower number of divisions, indicating a premature onset of cellularization. Furthermore, in time-lapse recordings, the speed of membrane invagination was measured, with no obvious difference found between X161 and wild-type embryos. Additionally, cellularization was investigated by live imaging with moesin-GFP labeling F-actin. Clear accumulation of F-actin at the furrow canals was observed in wild-type embryos after about 20 min in interphase 14, but not in interphase 13. In X161 embryos with 12 nuclear divisions, a comparable reorganization was observed already in interphase 13 after about 25 min. This analysis shows that both the cell-cycle pause and cellularization are initiated in X161 embryos earlier than in wild-type embryos (Sung, 2012).

    To identify the mutated gene in X161, the lethality and blastoderm phenotype was mapped. The X161 gene was separated from associated mutations on the chromosome by meiotic recombination and mapped to a region of four genes by complementation analysis with duplications and deficiencies. Sequencing of the mapped region and complementation tests with two independent RPII215 loss-of-function alleles, RPII215(1) and RPII215[G0040], and a transgene comprising the RPII215 locus revealed the large subunit of the RNA polymerase II as the mutated gene. A single point mutation was identified in the 3' UTR of RPII215 about 40 nt downstream of the stop codon. This region in the 3' UTR is not conserved and does not show any obvious motifs (Sung, 2012).

    To test whether the mutation in the noncoding region affects transcript or protein expression, mRNA levels were quantified by reverse transcription and quantitative PCR and protein levels by whole-mount staining and immunoblotting with extracts of manually staged embryos. mRNA levels were found to be the same in wild-type and X161. In contrast, immunohistology and immunoblotting revealed reduced RPII215 protein levels. In summary, the analysis shows that the X161 point mutation within the 3' UTR affects mainly RPII215 protein levels. The precocious onset of cellularization raised the hypothesis that the timing of zygotic gene expression may be affected in the X161 embryos. To establish the expression profiles of selected maternal and zygotic genes, nCounter NanoString technology was used with embryos staged by the nuclear division cycle. Embryos expressing histone 2Av-RFP were manually selected 3 min after anaphase of the previous mitosis or at midcellularization (Sung, 2012).

    Expression of ribosomal proteins was analyzed. They did not change much and were not different in wild-type and mutant embryos, confirming the robustness of the method. Zygotic genes, whose expression strongly increases during the syncytial cycles, showed an earlier upregulation in X161 than in wild-type embryos. Comparing the profiles by plotting the ratio of the expression levels, a clear difference was revealed in cycle 12, with a factor of up to ten, indicating that zygotic genes are precociously expressed in X161 embryos. The premature expression of early zygotic genes was confirmed by whole-mount in situ hybridization for slam and frs mRNA (Sung, 2012).

    Next, expression profiles were analyzed of RNAs subject to RNA degradation. Transcripts representative for the two classes of degradation were selected, depending on zygotic gene expression, and on egg activation. Degradation of string, twine, and smaug transcripts in interphase 14 depends of zygotic gene expression. In X161 mutants, the mRNA of these three genes was degraded already in cycle 13, slightly sooner than in wild-type. The profiles of string and twine RNA were confirmed by RNA in situ hybridization. Consistent with the precocious RNA degradation in X161, Twine and String protein levels decreased already in interphase 13 of X161 embryos. Finally, the profile was analyzed of mRNAs whose degradation depends on egg activation. No consistent pattern or clear difference was detected between the profiles of wild-type and X161 mutants. The data show that zygotic gene expression starts earlier in X161 than in wild-type and that degradation of mRNAs follows zygotic gene expression (Sung, 2012).

    The cell cycle may be paused prematurely by altered levels of maternal factors, such as CyclinB, grapes, and twine, or by precociously expressed zygotic genes, such as frs and trbl. To distinguish these two options, mutant embryos with suppressed zygotic gene expression were analyzed. Embryos injected with the RNA polymerase II inhibitor α-amanitin develop until mitosis 13 but then fail to cellularize and may undergo an additional nuclear division, depending on injection conditions. Using this assay, whether zygotic genes are required for the reduced number of nuclear divisions was tested in X161 mutants. If the precocious cell-cycle pause were due, for example, to reduced levels of CyclinB mRNA, α-amanitin injection should not change the reduced number of divisions. All injected mutant embryos passed through at least 13 nuclear divisions, similar to injected wild-type embryos, whereas injection of water resulted in a mixed phenotype of 12 and 13 nuclear divisions, comparable to uninjected X161 embryos. This experiment demonstrates that the reduced division number in X161 embryos requires zygotic gene expression (Sung, 2012).

    The expression of many early zygotic genes is controlled by the zinc-finger protein Vfl (also called Zelda). Tests were performed to see whether the precocious cell-cycle pause in X161 mutants is mediated by vfl-dependent genes. Analysis of X161 vfl double-mutant embryos revealed that, in contrast to X161 mutants, the cell cycle undergoes at least 13 divisions. Activation of zygotic gene expression was further analyzed by staining for Vfl and activated RPII21. Staining of both in presyncytial stages of X161 mutants was detected already in cycle 5. No specific staining for the activated RPII215 was detected in X161 vfl double-mutant embryos, and no difference in Vfl staining in syncytial embryos was detected in wild-type and X161 embryos. These findings show that the genes relevant for the precocious cell-cycle pause in X161 mutants are vfl target genes. A zygotic gene involved in cell-cycle control is frs, which is sufficient to induce a pause of the cell cycle. Analysis of X161 frs double-mutant embryos showed, however, that the number of nuclear divisions was not changed as compared to X161 single mutants. This indicates that frs is not the only cell-cycle inhibitor expressed in the early embryo. Proteins mediating the DNA repair checkpoint, such as Grapes/Chk1, are required for the cell-cycle pause. Passing normally through the nuclear division cycles, the cell cycle shows striking abnormalities in nuclear envelope formation and chromosome condensation in interphase 14 in embryos from grapes females. Tests were performed to see whether the timing of the transition in cell-cycle behavior in grapes embryos depends on the onset of zygotic transcription by analyzing X161 grapes double-mutant embryos. Some of the X161 grapes double mutants were found to show the defects in nuclear envelope formation and chromatin condensation already in interphase 13, indicating that the requirement of grapes for chromatin structure shifted from interphase 14 to 13. These data suggest that the activation of grapes and the DNA checkpoint depends on the onset of zygotic gene expression (Sung, 2012).

    A factor controlling the number of nuclear divisions is the ploidy of the embryo, given that haploid embryos undergo 14 instead of 13 nuclear divisions prior to cellularization. Based on this and on related observations, it has been proposed that the nucleocytoplasmic (N/C) ratio controls the trigger for MBT. To address the functional relationship of X161 and the N/C ratio, haploid X161 embryos were analyzed. A mixture was observed in the number of nuclear divisions between 12 and 14 in fixed embryos. Embryos were even observed containing three patches with nuclear densities corresponding to 12, 13, and 14 nuclear divisions. About half of the embryos underwent 12 nuclear divisions, similar to X161 embryos. These data suggest that ploidy acts independently of general onset of zygotic transcription, which is consistent with the observation that only a subset of zygotic genes are expressed with a delay in haploid embryos. Consistent with this report, cellularization starts for a first time temporarily in interphase 14 in haploid embryos and for a second time in interphase 15. These observations suggest that the N/C ratio in Drosophila specifically affects cell-cycle regulators such as frs, for example, but not general zygotic genome activation and onset of cellularization (Sung, 2012).

    In summary, the data support the model that activation of the zygotic genome controls the timing of the MBT. First, onset of MBT is sensitive to changes in RNA polymerase II activity. Second, the changes in zygotic gene expression in X161 embryos occur earlier than the changes in zygotic RNA degradation, Cdc25 protein destabilization, or activation of grapes. Third, the X161 mutant phenotype depends on zygotic transcription and on the transcription factor Vfl, showing that the precocious cell-cycle pause and onset of cellularization cannot be due to changes in maternal factors, such as higher expression of CyclinB. Although the altered levels of RNA polymerase II in X161 mutants probably affect expression of many genes during oogenesis, these changes seem not to matter in functional terms, given the overall normal morphology and specific mutant phenotype. It is conceivable that transcriptional repressors are expressed or translated in eggs in lower levels. In the embryo, such lower levels of repressors would allow the trigger for onset of zygotic gene expression to reach the threshold earlier than in wild-type embryos. The first signs of zygotic transcription are detected already during the presyncytial stages, before nuclear cycle 8/9. This may be the time when the trigger for MBT is activated (Sung, 2012).

    Calpain A controls mitotic synchrony in the Drosophila blastoderm embryo

    The beautiful mitotic waves that characterize nuclear divisions in the early Drosophila embryo have been the subject of intense research to identify the elements that control mitosis. Calcium waves in phase with mitotic waves suggest that calcium signals control this synchronized pattern of nuclear divisions. However, protein targets that would translate these signals into mitotic control have not been described. This study investigated the role of the calcium-dependent protease Calpain A in mitosis. Impaired Calpain A function was shown to result in loss of mitotic synchrony and ultimately halted embryonic development. The presence of defective microtubules and chromosomal architecture at the mitotic spindle during metaphase and anaphase and perturbed levels of Cyclin B indicate that Calpain A is required for the metaphase-to-anaphase transition. The results suggest that Calpain A functions as part of a timing module in mitosis, at the interface between calcium signals and mitotic cycles of the Drosophila embryo (Vieira, 2016).

    Co-activation of microRNAs by Zelda is essential for early Drosophila development

    Transcription factors and microRNAs (miRNAs) are two important classes of trans-regulators in differential gene expression. Transcription factors occupy cis-regulatory motifs in DNA to activate or repress gene transcription, whereas miRNAs specifically pair with seed sites in target mRNAs to trigger mRNA decay or inhibit translation. Dynamic spatiotemporal expression patterns of transcription factors and miRNAs during development point to their stage- and tissue-specific functions. Recent studies have focused on miRNA functions during development; however, much remains to explore regarding how the expression of miRNAs is initiated and how dynamic miRNA expression patterns are achieved by transcriptional regulatory networks at different developmental stages. This study has focused on the identification, regulation and function of miRNAs during the earliest stage of Drosophila development, when the maternal-to-zygotic transition (MZT) takes place. Eleven miRNA clusters comprise the first set of miRNAs activated in the blastoderm embryo. The transcriptional activator Zelda is required for their proper activation and regulation, and Zelda binding observed in genome-wide binding profiles is predictive of enhancer activity. In addition, other blastoderm transcription factors, comprising both activators and repressors, the activities of which are potentiated and coordinated by Zelda, contribute to the accurate temporal and spatial expression of these miRNAs, which are known to function in diverse developmental processes. Although previous genetic studies showed no early phenotypes upon loss of individual miRNAs, this analysis of the mir-1; miR-9a double mutant revealed defects in gastrulation, demonstrating the importance of co-activation of miRNAs by Zelda during the MZT (Fu, 2014).

    Similar to protein-coding genes, miRNA genes are regulated by sophisticated spatial and temporal signals to ensure their proper production in specific cell types. The muscle-specific transcription factors Twist (Twi) and Mef2 are key activators of mir-1 in Drosophila. Genomic studies have also identified regulators of miRNAs, such as Dorsal (Dl), c-Myc (Diminutive -- FlyBase) and Ecdysone. However, for many miRNAs, particularly those differentially expressed across developmental stages, the regulatory networks that control their transcription remain unknown. This study examined the gene network that regulates miRNA functions during the maternal-to-zygotic transition (MZT), a time when developmental control is transferred from maternal products preloaded into the egg to the embryo's own genome, which in Drosophila is activated ~1 hour after fertilization. During the MZT, thousands of maternal RNAs are degraded and hundreds of newly synthesized RNAs appear; thus, the MZT represents a major reprogramming event of the early transcriptome. Previous studies have reported that the zinc-finger transcription factor Zelda (Vielfaltig -- FlyBase) plays a key role during the MZT in Drosophila, collectively activating batteries of genes involved in early developmental processes, such as sex determination, cellularization and axis patterning. Interestingly, Zelda also activates the miR-309 cluster of eight miRNAs, which is involved in the clearance of many maternally loaded mRNAs. Since Zelda plays such an extensive role in zygotic genome activation, possibly as a pioneer factor to prime genes for transcriptional activation, this study investigated the possibility that Zelda activates the miRNAs expressed during the MZT (Fu, 2014).

    This study identified a group of miRNAs (11 clusters) that are zygotically expressed in cellular blastoderm embryos; Zelda was shown to regulate all 11. The enhancers of several miRNAs were localized by virtue of Zelda ChIP binding; Zelda binding sites, also known as CAGGTAG sites or TAGteam sites, in these enhancers were shown to be essential for proper activation. It was further shown that anteroposterior (AP) and dorsoventral (DV) patterning factors work together with Zelda to ensure timely and robust transcriptional activation of these miRNAs, contributing to their accurate spatial expression patterns. The reduced and disrupted miRNA expression seen in Zelda mutants affects their downstream functions in maternal mRNA degradation, cell death gene repression and Hox gene regulation. Ventral midline defects were observed during gastrulation in mir-1; miR-9a double mutants, that were not seen in either single mutant, suggesting that the coordinated activation of miRNAs by Zelda is crucial for their combinatorial function. This analysis offers a systems-level view and understanding of the early gene network. Zelda sits as a major hub in the network, globally activating both protein-coding and non-coding genes, thereby orchestrating the early developmental processes (Fu, 2014).

    This study identified the set of miRNAs expressed in 2- to 3-h Drosophila embryos, a time when the MZT is well underway and the fate map of the embryo is being established. These early expressed miRNAs are regulated globally by Zelda, both directly via binding to cis-regulatory enhancers and indirectly by affecting the expression of additional transcriptional regulators. Together with previously published data on specific miRNAs, it was possible to integrate the early miRNAs, their upstream regulators and downstream targets into the early gene network (Fu, 2014).

    Using expression profiling data from 2- to 3-h wild-type and zelda mutant embryos, blastoderm-specific pri-miRNA transcription units, which included seven intergenic and four intronic miRNAs (clusters) were identified. Since the expression levels of all 11 miRNAs were affected in Zelda mutants, it was possible to better distinguish blastoderm-specific isoforms, particularly in the case of intronic miRNAs, such asmir-11, which resides in an intron of E2f . Moreover, maternal E2f expression could be differentiated from zygotic expression by observing the intronic signal, which was clearly downregulated in zelda mutants (Fu, 2014).

    The early miRNAs exhibit strikingly different expression patterns, and it is noteworthy that, similar to the protein-coding targets of Zelda, two different strategies are used to regulate these miRNAs. Some miRNAs, such as miR-9a, were completely abolished in Zelda mutants, indicating that Zelda is their sole activator, whereas others, such as mir-1, were affected temporally and/or spatially, indicating that Zelda works together with other factors to establish robust and precise domains of expression. For example, mir-1 downregulation in Zelda mutants is likely to be due to the cumulative effect of loss of direct inputs from Zelda and the delayed expression of twi that occurs in Zelda mutants. Thus, the effect onmir-1 is the result of a breakdown in the Zelda-Twi-mir-1 feedforward loop (Fu, 2014).

    Cis-regulatory modules/enhancers of miRNAs have been predicted based on the presence of transcription factor binding or specific chromatin marks, and verified in only some cases. For example, two regions upstream of mir-1 that bind Twi/Dl were shown to drive a mir-1-like expression pattern. It was reasoned that it could be possible to locate enhancers of all early miRNAs by simply looking for Zelda-bound regions upstream of the pri-miRNA transcription units, especially since Zelda is a global activator during the MZT. This approach worked well; eight of nine enhancers recapitulated endogenous-like expression. Mutation of Zelda binding sites in enhancers further demonstrated direct Zelda input. As proof of principle, enhancers of two genes, miR-9a and mir-1, were analyzed and it was shown that mutation of the Zelda binding sites had the same effect as eliminating Zelda in trans. These results indicate that Zelda directly regulates the early expressed miRNAs, often in conjunction with other transcription factors, many of which are also regulated by Zelda. Zelda is a major hub in the early network, establishing multiple feedforward loops and closely linking the transcription factors and miRNAs expressed in this stage (Fu, 2014).

    The MZT is a key event during the development of an organism, whereby the transcriptome is reprogrammed in the first few hours of development. This requires the clearance of previous information (maternal mRNA degradation) and the initiation of a new program (zygotic genome activation). The maternal mRNA degradation machinery comprises both maternally derived and zygotically derived pathways. In Drosophila, Smaug (Smg), a maternally loaded RNA-binding protein, is central to the mRNA clearance pathway. By recruiting the CCR4-NOT deadenylation complex, Smg destabilizes two-thirds of the maternal mRNAs that undergo degradation (i.e. that are unstable) upon egg activation. By contrast, miR-309 is a key component of the zygotically derived pathway to clear mRNA. When analyzing the maternal RNAs upregulated in zelda mutants, it was noted in this study that 81% of them (434) depend on zygotic degradation pathways; 125 of the 434 genes are also upregulated in miR-309 mutants, indicating that Zelda, by activating miR-309, is involved in maternal RNA degradation. Therefore, Zelda plays important roles in both of the hallmark events of the MZT. Interestingly, the miR-309 targets account for only ~30% of the unstable maternal RNAs upregulated in Zelda mutants, and another 14% are putative targets of the other early miRNAs, indicating that Zelda might activate additional zygotic pathways to mediate maternal mRNA degradation (Fu, 2014).

    Several miRNAs, in addition to miR-309, have been shown to target specific mRNAs in the early embryo; for example, miR-iab-4 and miR-iab-4as target Hox genes. However, although each of the miRNAs is predicted to target hundreds of genes, in many cases the individual miRNA loss-of-function phenotypes are relatively mild. There are several explanations for this phenomenon: (1) the miRNA does not function at the time that it is expressed, but might function later; (2) miRNAs 'fine-tune' the expression levels of their target genes, which might not be reflected in obvious phenotypes when they are mutated; and (3) miRNA functions are redundant, such that knockdown of one miRNA may be compensated by another (Fu, 2014).

    To better address the functions of miRNAs, investigators have used several genetic approaches: gain-of-function assays, using sensitized genetic backgrounds, and assaying double mutants. For example, the miR-6; mir-11 double mutant exhibits increased apoptosis, leading to lower survival rates compared with either of the single miRNA mutants. Using a similar approach, this study observed fully penetrant gastrulation defects in mir-1; miR-9a double mutants. Neither single mutant is embryonic lethal, nor shows any sign of ventral furrow defects; however, mir-1 mutants are larval lethal and display muscle defects, while miR-9a mutants show wing margin defects in adulthood. Importantly, the double-mutant phenotype is the earliest phenotype seen for any known miRNA, or combination of miRNAs, thus far tested. These results support the idea that co-activation of miRNAs by Zelda is required for normal development (Fu, 2014).

    The mir-1; miR-9a double-mutant phenotype resembles, to some extent, the ventral furrow defects observed in RhoGEF2 loss-of-function mutant. Rho signaling is involved in the cell shape changes associated with ventral furrow invagination, and loss of Rho signaling results in very disorganized invagination. Curiously, mir-1 and miR-9a are both predicted to target RhoGAP68F, a negative regulator of Rho signaling. RhoGAP68F is maternally loaded and cleared during the MZT. It is possible that the gastrulation phenotype of the mir-1; miR-9a double mutant is caused in part by excess activity of RhoGAP68F. Although no obvious upregulation of RhoGAP68F transcripts were seen in mir-1; miR-9a mutant embryos by in situ hybridization, it is possible that subtle upregulation of RhoGAP68F, combined with effects on other predicted targets, all contribute to the gastrulation defects observed in the double mutant (Fu, 2014).

    The co-activation of groups of miRNAs by master regulators such as Zelda may be crucial for miRNA activity during development, as revealed by the severe gastrulation phenotype of the mir-1; miR-9a double mutant. Such coordinated activation of miRNAs might also occur at later stages in development in tissues in which Zelda is expressed, such as the central nervous system. In the future, various combinations of mutations in miRNA genes that are co-regulated by Zelda, or other key factors, might unveil additional functions of miRNAs across development stages (Fu, 2014).

    Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila

    The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, this study refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. 523 proteins were determined to be high-confidence RBPs, half of which have not been previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development (Sysoev, 2015).

    Drak is required for actomyosin organization during Drosophila cellularization

    The generation of force by actomyosin contraction is critical for a variety of cellular and developmental processes. Nonmuscle myosin II is the motor that drives actomyosin contraction, and its activity is largely regulated by phosphorylation of myosin regulatory light chain. During the formation of the Drosophila cellular blastoderm, actomyosin contraction drives constriction of microfilament rings, modified cytokinesis rings. This study found that Death-associated protein kinase related (Drak) is necessary for most of the phosphorylation of myosin regulatory light chain during cellularization. Drak was shown to be required for organization of myosin II within the microfilament rings. Proper actomyosin contraction of the microfilament rings during cellularization also requires Drak activity. Constitutive activation of myosin regulatory light chain bypasses the requirement for Drak, suggesting that actomyosin organization and contraction are mediated through Drak's regulation of myosin activity. Drak also is involved in the maintenance of furrow canal structure and lateral plasma membrane integrity during cellularization. Together, these observations suggest that Drak is the primary regulator of actomyosin dynamics during cellularization (Chougule, 2016).

    Tight regulation of actomyosin is likely critical for many cellular processes, but how this is accomplished is as yet poorly understood. A key input to the regulation of myosin II is through phosphorylation of the Serine-19, or the Serine-19 and Threonine-18 residues of MRLC (Spaghetti squash). The variety of MRLC kinases might allow different specific aspects of actomyosin dynamics, such as localization, organization and contraction to be regulated independently. Such a system would provide greater flexibility and control than either a single kinase, or multiple kinases acting in concert, regulating all of these functions. drak was found to be required for the organization of myosin II into contractile rings, but is not required for localization of myosin to the cellularization front. Since the majority of Sqh phosphorylation during cellularization is dependent on drak activity, Drak either regulates most aspects of myosin II dynamics during cellularization, or Drak-regulated myosin II organization is required for further function of myosin II, such as contraction (Chougule, 2016).

    Myosin II is somewhat less disorganized and Sqh phosphorylation is slightly increased during late cellularization in drak mutants, suggesting that phosphorylation of myosin II by other kinases occurs during late cellularization. Thus other kinases might act synergistically with Drak to regulate actomyosin organization during late cellularization. For example, Drak function has been shown to be partially redundant with Rok function during later development. An alternative possibility is that other kinases that do not normally function in myosin II organization in the microfilament rings might phosphorylate Sqh to some degree and lead to some organization of myosin II in the absence of Drak activity (Chougule, 2016).

    Myosin II has been implicated in actin bundling and F-actin organization in some contexts. Since F-actin appears to be organized normally within drak mutant microfilament rings during early cellularization, it is concluded that myosin II does not play a role in initially organizing F-actin within the microfilament rings during cellularization. F-actin is somewhat disorganized during late cellularization in drakdel mutant embryos, but not as severely as myosin II, nor does the pattern of F-actin distribution fit the pattern of myosin II distribution in drakdel mutant embryos. These observations suggest that F-actin disorganization is an indirect consequence of Drak regulation of myosin II activity, and that F-actin disorganization might be due to actomyosin contraction defects or furrow canal structural defects (Chougule, 2016).

    Anillin is required for the organization of actomyosin contractile rings during cellularization and cytokinesis. scraps (scra, anillin) mutant embryos have a myosin II organization defect somewhat similar to that of drak mutant embryos: myosin II is found in discrete bars in the actomyosin network. Despite this similarity, myosin II defects differ between scra and drak mutant embryos. Myosin II becomes more disorganized during late cellularization in scra mutant embryos. Myosin II becomes slightly better organized during late cellularization in drak mutant embryos. This organizational difference is likely caused by actomyosin contraction during microfilament ring constriction occurring in a highly disorganized cytoskeleton in scra mutant embryos, and occurring in a disorganized cytoskeleton that has slightly improved during constriction in drak mutant embryos. Anillin only interacts with myosin II when MRLC is phosphorylated. Together with these results, this suggests that Drak phosphorylation of Sqh might be necessary for Anillin-mediated myosin II organization within the contractile ring (Chougule, 2016).

    Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 leads to the unfolding of inactive myosin II hexamers into an open conformation that allows assembly of bipolar myosin II filaments and their association with F-actin to form actomyosin filaments. This is likely how Drak organizes myosin II. Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 also leads to the activation of the Mg2+-ATPase activity of myosin II that slides actin filaments past each other, causing actomyosin contraction. Three aspects of the drak mutant phenotype support the requirement for Drak in actomyosin contraction: wavy cellularization fronts caused by non-uniform furrow canal depths, abnormal microfilament ring shapes, and failure of microfilament rings to constrict during late cellularization. These are the same defects that suggest an actomyosin contraction defect in src64 mutant embryos. However, src64 mutant embryos do not show myosin II organization defects. Because effective actomyosin contraction likely requires properly organized actomyosin filaments within the contractile ring apparatus, it is unclear whether Drak directly regulates actomyosin contraction or whether Drak only enables actomyosin contraction through proper organization of myosin II within the microfilament rings. One possibility is that phosphorylation of Sqh by Drak both organizes actomyosin filaments into a contractile ring apparatus and directs actomyosin contraction. An alternative possibility is that Drak is directly responsible for organizing actomyosin filaments into a contractile ring by phosphorylating Sqh, but Drak is not directly involved in its contraction and different kinases that phosphorylate Sqh regulate actomyosin contraction. Thus, Drak could be an early regulator of myosin II activity during cellularization, such that further phosphorylation of Sqh and myosin II-driven contraction is dependent on Drak-mediated organization of myosin II. At some level the regulation of actomyosin contraction diverges from the regulation of actomyosin filament organization: Src64 is required for contraction, but has no role in myosin II organization (Chougule, 2016).

    Rescue of myosin II organization, actomyosin contraction and F-actin distribution defects in drak mutant embryos by the mono-phosphorylated SqhE21 phosphomimetic suggests that Drak-mediated mono-phosphorylation of Sqh at Serine-21 is sufficient for regulation of actomyosin dynamics during cellularization. Although the diphosphorylated SqhE20E21 phosphomimetic also rescues myosin II organization and actomyosin contraction defects, it does not rescue F-actin distribution defects in drak mutant embryos. These results are consistent with Drak primarily phosphorylating Sqh at Serine-21, and are consistent with reports that DAPK family members phosphorylate MRLC mainly at Serine-19 (Chougule, 2016).

    The normal teardrop shape of the furrow canals in early cellularization is likely caused by actomyosin contraction in the microfilament rings. In drak mutant embryos, unexpanded early cellularization furrow canals and failure of many late cellularization furrow canals to expand further suggest that Drak is required for proper furrow canal structure. Some of the furrow canal structural defects in drak mutant embryos are similar to those of nullo mutant embryos: collapsed furrow canals and blebbing. However, nullo mutant embryos, as well as RhoGEF2 or dia mutant embryos, have other, more severe furrow canal defects: missing or regressing furrow canals and compromised lateral membrane-furrow canal compartment boundaries. Furthermore, cytochalasin treatment causes similar defects, suggesting that reduced F-actin levels in the furrow canals are responsible for these defects. Thus Nullo, RhoGEF2 and Dia regulate F-actin and its levels in furrow canals. These observations suggest that Drak regulates myosin II and thereby regulates actomyosin organization and contraction, and that these are necessary for structural integrity and expansion of the furrow canals, but not for their continued existence (Chougule, 2016).

    The furrow canals of drak mutant embryos during late cellularization show extensive blebbing into the lumens. This is consistent with a defect in furrow canal membrane or cortex integrity. Blebs can be formed by local rupture of the cortical cytoskeleton or detachment of the plasma membrane from the cortical actomyosin cytoskeleton. Actomyosin contraction has been implicated in bleb formation. Therefore, it is proposed that blebbing in furrow canals is caused by aberrant localized actomyosin contraction during late cellularization in the disorganized actomyosin cytoskeleton of drak mutant embryos. Contraction is presumably driven by phosphorylation of Sqh by kinases other than Drak. Since actomyosin contraction occurs in a disorganized actomyosin cytoskeleton, it does not lead to uniform constriction of the microfilament rings, but instead leads to localized contraction that produces cytoplasmic blebs. However, other causes for furrow canal defects are possible. Plasma membrane attachment sites might not form or function properly in the disorganized furrow canal cytoskeleton in drak mutant embryos. The disorganized cytoskeleton might inhibit vesicle trafficking. Vesicle trafficking itself might be defective: mammalian DAPKs have been shown to be involved in membrane trafficking and in phosphorylation of syntaxin A1. Vesiculated lateral plasma membrane in drak mutant embryos during late cellularization suggests that the plasma membrane breaks down. Intriguingly, scra mutant embryos have lines of vesicles where the closely apposed lateral plasma membranes would have been. However in scra mutant embryos, vesiculation is observed during early cellularization, but to a lesser extent than during late cellularization. drak mutant embryos do not show lateral plasma membrane vesiculation defects until late cellularization. drak mutant defects in both the furrow canal membrane and the lateral plasma membrane might reflect a general defect in membrane integrity. It will be interesting to investigate the potential role of myosin II organization in furrow canal structure and plasma membrane integrity (Chougule, 2016).

    The Smaug RNA-binding protein is essential for microRNA synthesis during the Drosophila maternal-to-zygotic transition

    Metazoan embryos undergo a maternal-to-zygotic transition (MZT) during which maternal gene products are eliminated and the zygotic genome becomes transcriptionally active. During this process RNA-binding proteins (RBPs) and the microRNA-induced silencing complex (miRISC) target maternal mRNAs for degradation. In Drosophila, the Smaug (SMG), Brain tumor (BRAT) and Pumilio (PUM) RBPs bind to and direct the degradation of largely distinct subsets of maternal mRNAs. SMG has also been shown to be required for zygotic synthesis of mRNAs and several members of the miR-309 family of microRNAs (miRNAs) during the MZT. This study carried out global analysis of small RNAs both in wild type and in smg mutants. It was found that 85% all miRNA species encoded by the genome are present during the MZT. Whereas loss of SMG has no detectable effect on Piwi-interacting RNAs (piRNAs) or small interfering RNAs (siRNAs), zygotic production of more than 70 species of miRNAs fails or is delayed in smg mutants. SMG is also required for the synthesis and stability of a key miRISC component, Argonaute 1 (AGO1), but plays no role in accumulation of the Argonaute-family proteins associated with piRNAs or siRNAs. In smg mutants, maternal mRNAs that are predicted targets of the SMG-dependent zygotic miRNAs fail to be cleared. BRAT and PUM share target mRNAs with these miRNAs but not with SMG itself. The study hypothesizes that SMG controls the MZT, not only through direct targeting of a subset of maternal mRNAs for degradation but, indirectly, through production and function of miRNAs and miRISC, which act together with BRAT and/or PUM to control clearance of a distinct subset of maternal mRNAs (Luo, 2016).

    To identify small RNA species expressed during the Drosophila MZT and to assess the role of SMG in their regulation 18 small-RNA libraries were produced and sequenced: nine libraries from eggs or embryos produced by wild-type females and nine from smg-mutant females. The 18 libraries comprised three biological replicates each from the two genotypes and three time-points: (1) 0-to-2 hour old unfertilized eggs, in which zygotic transcription does not occur and thus only maternally encoded products are present; (2) 0-to-2 hour old embryos, the stage prior to large-scale zygotic genome activation; and (3) 2-to-4 hour old embryos, the stage after to large-scale zygotic genome activation. After pre-alignment processing, a total of ~144 million high quality small-RNA reads was obtained and 110 million of these perfectly matched the annotated Drosophila genome (Luo, 2016).

    Loss of SMG had no significant effect on piRNAs and siRNAs, or on the Argonaute proteins associated with those small RNAs: Piwi, Aubergine (AUB), AGO3, and AGO2, respectively. In contrast, loss of SMG resulted in a dramatic, global reduction in miRNA populations during the MZT as well as reduced levels of AGO1, the miRISC-associated Argonaute protein in Drosophila (Luo, 2016).

    A pre-miRNA can generate three types of mature miRNA: (1) a canonical miRNA, which has a perfect match to the annotated mature miRNA; (2) a non-canonical miRNA, which shows a perfect match to the annotated mature miRNA but with additional nucleotides at the 5'- or 3'- end that match the adjacent primary miRNA sequence, and (3) a miRNA with non-templated terminal nucleotide additions (an NTA-miRNA), which has nucleotides at its 3'-end that do not match the primary miRNA sequence (Luo, 2016).

    In these libraries a total of 364 distinct miRNA species were identified that mapped to miRBase, comprising 85% (364/426) of all annotated mature miRNA species in Drosophila. Thus, the vast majority of all miRNA species encoded by the Drosophila genome are expressed during the MZT. Overall, in wild type, an average of 75% of all identified miRNAs fell into the canonical category. The remaining miRNAs were either non-canonical (10%) or NTA-miRNAs (15%) (Luo, 2016).

    To validate these sequencing results, those mature miRNA species identified in the data that perfectly matched the Drosophila genome sequence (i.e., canonical and non-canonical) were compared with a previously published miRNA dataset from 0 to 6 hour old embryos. To avoid differences caused by miRBase version, data sets from previous study were remapped to miRBase Version 19 and f99% of their published miRNA species were found to be on the miRNA list (176/178 mature miRNA species comprising 161 canonical miRNA s and 94 non-canonical miRNA s) . There were an additional 181 mature miRNA species in the library that had not been identified as expressed in early embryos in the earlier study (Luo, 2016).

    As a second validation, the list of maternally expressed miRNA species (those present in the 0-to-2 hour wild-type unfertilized egg samples) were compared with the most recently published list of maternal miRNAs, which had been defined in the same manner. 99% of the 86 published maternal miRNA species were on this study's maternal miRNA list (85/86). An additional 144 maternal miRNA species in the library were identified that had not been observed in the previous study. Identification of a large number of additional miRNA species in unfertilized eggs and early embryos can be attributed to the depth of coverage of the current study. The current dataset, therefore, provides the most complete portrait to date of the miRNAs present during the Drosophila MZT (Luo, 2016).

    Next, global changes in miRNA species during the MZT were analyzed in wild-type embryos. A dramatic increase was observed in the proportion of miRNAs relative to other small RNAs that was due to an increase in absolute miRNA amount rather than a decrease in the amount of other types of small RNAs. In wild-type 0-to-2 hour unfertilized eggs, the proportion of the small RNA libraries comprised of canonical and non-canonical miRNAs was 12.8%. These represent maternally loaded miRNAs since unfertilized eggs do not undergo zygotic genome activation. The proportion of small RNAs represented by miRNAs increased dramatically during the MZT, reaching 50.7% in 2-to-4 hour embryos. The other abundant classes of small RNAs underwent either no change or relatively minor changes over the same time course. It is concluded that there is a large amount of zygotic miRNA synthesis during the MZT in wild-type embryos (Luo, 2016).

    For more detailed analysis of the canonical, non-canonical and NTA isoforms focus was placed on 154 miRNA species that possessed an average of > 10 reads per million (RPM) for all three isoform types in one or more of the six sample sets. A focus was placed on changes in wild type. Among all miRNAs, in wild type the proportion of canonical isoforms increased over the time-course from 69% to 83%, the proportion of non-canonical miRNAs remained constant (from 9% to 10%) , and the proportion of the NTA-miRNAs decreased (from 22% to 7%). These results derive from the fact that, during the MZT, the vast majority of newly synthesized miRNAs were canonical, undergoing a more than seven-fold increase from 103,105 to 744,043 RPM; that non-canonical miRNAs underwent a comparable, nearly seven-fold, increase from 13,902 to 92,199; whereas NTA-miRNAs underwent a less than two-fold increase, from 32,840 to 63,847, thus decreasing in relative proportion (Luo, 2016).

    Whereas the proportion of the small-RNA population that was comprised of miRNAs increased fourfold over the wild-type time-course, concomitant with increases in overall miRNA abundance, there was no such increase in the smg mutant embryos: 21.9% of the small RNAs were miRNAs in 0-to-2 hour unfertilized smg mutant eggs (mean RPM = 203,415) and 20.5% (mean RPM = 196,110) were miRNAs in 2-to-4 hour smg mutant embryos (Luo, 2016).

    This difference between wild type and smg mutants could have resulted from the absence of a small number of extremely highly expressed miRNA species in the mutant. Alternatively, it may have been a consequence of a widespread reduction in the levels of all or most zygotically synthesized miRNAs in smg mutants. To assess the cause of this difference, canonical miRNA reads were graphed in scatter plots. These showed that a large number of miRNA species had significantly reduced expression levels in 0-to-2 and in 2-to-4 hour smg-mutant embryos relative to wild type. Most of the down-regulated miRNA species exhibited a more than four-fold reduction in abundance relative to wild type. Furthermore, this reduction occurred for miRNA species expressed over a wide range of abundances in wild type (Luo, 2016).

    Box plots were then used to analyze the canonical, non-canonical and NTA isoforms of the 154 miRNA species identified in the previous section. These showed that, in wild type, the median abundance of canonical, non-canonical and 3' NTA miRNAs increased significantly in 0-to-2 and in 2-to-4 hour embryos relative to 0-to-2 hour unfertilized eggs. In contrast, there was no significant increase in the median abundance of any of the three isoforms of miRNAs in the smg-mutant embryos. Also for all three isoform types, when each time point was compared between wild type and smg mutant, there was no difference between wild type and mutant in 0-to-2 hour unfertilized eggs but there was a highly significant difference between the two genotypes at both of the embryo time-points. Whereas the abundance of miRNAs differed between wild-type and mutant embryos, there was no difference in length or first-nucleotide distribution of canonical miRNAs, nor in the non-templated terminal nucleotides added to NTA-miRNAs (Luo, 2016).

    As described above, during the wild-type MZT canonical miRNAs comprised the major isoform that was present (69% to 83% of miRNAs). It was next asked whether miRNA species could be categorized into different classes based on their expression profiles during the wild-type MZT. 131 canonical miRNA species that had > 10 mean RPM in at least one of the six datasets were analyzed. Hierarchical clustering of their log 2 RPM values identified five distinct categories of canonical miRNA species during the MZT. The effects of smg mutations on each of these classes were analyzed (Luo, 2016).

    The data are consistent with a model in which SMG degrades its direct targets without the assistance of miRNAs whereas a large fraction of the indirectly affected maternal mRNAs in smg mutants fails to be degraded by virtue of being targets of zygotically produced miRNA species that are either absent or present at significantly reduced levels in smg mutants. Thus, SMG is required both for early, maternally encoded decay and for late, zygotically encoded decay. In the former case SMG is a key specificity component that directly binds to maternal mRNAs; in the latter case SMG is required for the production of the miRNAs (and AGO1 protein) that are responsible for the clearance of an additional subset of maternal mRNAs (Luo, 2016).

    In Drosophila, the stability of miRNAs is enhanced by AGO1 and vice versa. Since miRNA levels are dramatically reduced in smg mutants, Ago1 mRNA and AGO1 protein levels were assessed during the MZT both in wild type and in smg mutants. In wild type, AGO1 levels were low in unfertilized eggs and 0-to-2 hour embryos but then increased substantially in 2-to-4 hour embryos. These western blot data are consistent with an earlier, proteomic, study that reported a more than three-fold increase in AGO1 in embryos between 0-to-1.5 hours and 3-to-4.5 hours. In contrast to AGO1 protein, it was found using RT-qPCR that Ago1 mRNA levels remained constant during the MZT. Taken together with a previous report that Ago1 mRNA is maternally loaded, the increase in AGO1 protein levels in the embryo is, therefore, most likely to derive from translation of maternal Ago1 mRNA rather than from newly transcribed Ago1 mRNA (Luo, 2016).

    Next, AGO1, AGO2, AGO3, AUB and Piwi protein levels were analyzed in eggs and embryos from mothers carrying either of two smg mutant alleles: smg1 and smg47. The smg mutations had no effect on the expression profiles of AGO2, AGO3, AUB or Piwi. In contrast, in smg-mutant embryos, the amount of AGO1 protein at both 0-to-2 and 2-to-4 hours was reduced relative to wild type and this defect was rescued in embryos that expressed full-length, wild-type SMG from a transgene driven by endogenous smg regulatory sequences. The reduction of AGO1 protein levels in smg mutants was not a secondary consequence of reduced Ago1 mRNA levels since Ago1 mRNA levels in both the smg-mutant and the rescued-smg-mutant embryos were very similar to wild type (Luo, 2016).

    A plausible explanation for the decrease in AGO1 levels in smg mutants is the reduced levels of miRNAs, which would then result in less incorporation of newly synthesized AGO1 into functional miRISC and consequent failure to stabilize the AGO1 protein. To assess this possibility, a time-course in wild-type unfertilized eggs was analyzed in which zygotic genome activation and, therefore, zygotic miRNA synthesis, does not occur. It was found that AGO 1 levels were reduced in 2-to-4 hour wild-type unfertilized eggs compared with wild-type embryos of the same age. This result is consistent with a requirement for zygotic miRNAs in the stabilization of AGO1 protein (Luo, 2016).

    Next, wild-type unfertilized egg and smg-mutant unfertilized egg time-courses were compared, and AGO1 levels were found to be further reduced in the smg mutant relative to wild type. This suggests that SMG protein has an additional function in the increase in AGO1 protein levels that is independent of SMG's role in zygotic miRNA production (since these are produced in neither wild-type nor smg-mutant unfertilized eggs) (Luo, 2016).

    To assess whether this additional function derives from SMG's role as a post-transcriptional regulator of mRNA, smg1 mutants were rescued either with a wild-type SMG transgene driven by the Gal4:UAS system (SMGWT) or a GAL4:UAS-driven transgene encoding a version of SMG with a single amino-acid change that abrogates RNA-binding (SMGRBD) and, therefore, is unable to carry out post-transcriptional regulation of maternal mRNAs. It was found that, whereas AGO1 was detectable in both unfertilized eggs and embryos from SMGWT-rescued mothers, AGO1 was undetectable in unfertilized eggs from SMGRBD-rescued mothers and was barely detectable in embryos from these mothers. Thus, SMG's RNA-binding ability is essential for its non-miRNA-mediated role in regulation of AGO1 levels during the MZT (Luo, 2016).

    Since the abundance of SMGWT and SMGRBD proteins is very similar, the preceding result excludes the possibility that it is physical interaction between SMG and AGO1 that stabilizes the AGO1 protein. It was previously shown that the Ago1 mRNA is not bound by SMG. Thus, SMG must regulate one or more other mRNAs whose protein products, in turn, affect the synthesis and/or stability of AGO1 protein. It is known that turnover of AGO1 protein requires Ubiquitin-activating enzyme 1 (UBA1) and is carried out by the proteasome . It was previously shown that the Uba1 mRNA is degraded during the MZT in a SMG-dependent manner and that both the stability and translation of mRNAs encoding 19S proteasome regulatory subunits are up-regulated in smg-mutant embryos. It is speculated that increases in UBA1 and proteasome subunit levels in smg mutants contribute to a higher rate of AGO1 turnover and, thus, lower AGO1 abundance than in wild type (Luo, 2016).

    AGO1 physically associates with BRAT. It is not known whether AGO1 interacts with PUM but it has been reported that, in mammals and C. elegans , Argonaute-family proteins interact with PUM/PUF-family proteins. Recent studies identified direct target mRNAs of the BRAT and PUM RBPs in early Drosophila embryos and showed through analysis of brat mutants that, during the MZT, BRAT directs late (i.e., after zygotic genome activation) decay of a subset of maternal mRNAs. These data permitted asking whether the maternal mRNAs that are predicted to be indirectly regulated by SMG via its role in miRISC production might be co-regulated by BRAT and/or PUM (Luo, 2016).

    A highly significant overlap was found between the predicted miRNA-dependent indirect targets of SMG and both BRAT-and PUM-bound mRNAs in early embryos. This suggests that BRAT and PUM might function together with miRISC during the MZT to direct decay of maternal mRNAs (Luo, 2016).

    Given that BRAT and PUM bind to largely non-overlapping sets of mRNAs during the MZT, there are three types of hypothetical BRAT-PUM-miRISC-containing complexes: one with both BRAT and PUM, one with BRAT only, one with PUM only. To assess this possibility for a specific set of zygotically produced miRNAs, the lists of mRNAs stabilized in 2-to-3 hour old embryos from miR-309 deletion mutants were compared to the lists of BRAT and PUM direct-target mRNAs. There was no significant overlap of PUM-bound mRNAs with those up-regulated in miR-309 mutants. However, there was a highly significant overlap of mRNAs up-regulated in miR-309-mutant embryos with BRAT-bound mRNAs. These results lead to the hypothesis that BRAT (but not PUM) co-regulates clearance of miR-309-family miRNA target maternal mRNAs during the MZT (Luo, 2016).

    Differentially-dimensioned furrow formation by zygotic gene expression and the MBT

    Despite extensive work on the mechanisms that generate plasma membrane furrows, understanding how cells are able to dynamically regulate furrow dimensions is an unresolved question. This study presents an in-depth characterization of furrow behaviors and their regulation in vivo during early Drosophila morphogenesis. The deepening in furrow dimensions with successive nuclear cycles is largely due to the introduction of a new, rapid ingression phase (Ingression II). Blocking the midblastula transition (MBT) by suppressing zygotic transcription through pharmacological or genetic means causes the absence of Ingression II, and consequently reduces furrow dimensions. The analysis of compound chromosomes that produce chromosomal aneuploidies suggests that multiple loci on the X, II, and III chromosomes contribute to the production of differentially-dimensioned furrows, and the X-chromosomal contribution was tracked to furrow lengthening to the nullo gene product. Checkpoint proteins are required for furrow lengthening; however, mitotic phases of the cell cycle are not strictly deterministic for furrow dimensions, as a decoupling of mitotic phases with periods of active ingression occurs as syncytial furrow cycles progress. Finally, the turnover of maternal gene products was examined, and this was found to be a minor contributor to the developmental regulation of furrow morphologies. These results suggest that cellularization dynamics during cycle 14 are a continuation of dynamics established during the syncytial cycles and provide a more nuanced view of developmental- and MBT-driven morphogenesis (Xie, 2018).

    Essential function of the serine hydroxymethyl transferase (SHMT) gene during rapid syncytial cell cycles in Drosophila

    Many metabolic enzymes are evolutionary highly conserved and serve a central function for catabolism and anabolism of cells. The serine hydroxymethyl transferase (SHMT) catalysing the conversion of serine and glycine and vice versa feeds into the tetrahydrofolate mediated C1 metabolism. This study identified a Drosophila mutation in SHMT (CG3011) in a screen for blastoderm mutants. Embryos from SHMT mutant germline clones specifically arrest the cell cycle in interphase 13 at the time of the mid blastula transition (MBT) and prior to cellularisation. The phenotype is due to a loss of enzymatic activity as it cannot be rescued by an allele with a point mutation in the catalytic center but by an allele based on the SHMT coding sequence from E. coli. Onset of zygotic gene expression and degradation of maternal RNAs in SHMT mutant embryos are largely similar to wild type embryos. The specific timing of the defects in SHMT mutants indicates that at least one of the SHMT-dependent metabolites becomes limiting in interphase 13, if it is not produced by the embryo. These data suggest that mutant eggs contain maternally provided and SHMT-dependent metabolites in amounts which suffice for early development until interphase 13 (Winkler, 2017).

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    Maternally transcribed genes

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