src64 and tec29 are required for microfilament contraction during Drosophila cellularization

Formation of the Drosophila cellular blastoderm involves both membrane invagination and cytoskeletal regulation. Mutations in src64 and tec29 reveal a novel role for these genes in controlling contraction of the actin-myosin microfilament ring during this process. Although membrane invagination still proceeds in mutant embryos, its depth is not uniform, and basal closure of the cells does not occur during late cellularization. Double-mutant analysis between scraps, a mutation in anillin that eliminates microfilament rings, and bottleneck suggests that microfilaments can still contract even though they are not organized into rings. However, the failure of rings to contract in the src64 bottleneck double mutant suggests that src64 is required for microfilament ring contraction even in the absence of Bottleneck protein. Our results suggest that src64-dependent microfilament ring contraction is resisted by Bottleneck to create tension and coordinate membrane invagination during early cellularization. The absence of Bottleneck during late cellularization allows src64-dependent microfilament ring constriction to drive basal closure.     

Actomyosin contraction during cellularization is regulated in part by Src64 control of Actin 5C protein levels

Src64 is required for actomyosin contraction during cellularization of the Drosophila embryonic blastoderm. The mechanism of actomyosin ring constriction is poorly understood even though a number of cytoskeletal regulators have been implicated in the assembly, organization, and contraction of these microfilament rings. How these cytoskeletal processes are regulated during development is even less well understood. To investigate the role of Src64 as an upstream regulator of actomyosin contraction, we conducted a proteomics screen to identify proteins whose expression levels are controlled by src64. Global levels of actin are reduced in src64 mutant embryos. Furthermore, we show that reduction of the actin isoform Actin 5C causes defects in actomyosin contraction during cellularization similar to those caused by src64 mutation, indicating that a relatively high level of Actin 5C is required for normal actomyosin contraction and furrow canal structure. However, reduction of Actin 5C levels only slows down actomyosin ring constriction rather than preventing it, suggesting that src64 acts not only to modulate actin levels, but also to regulate the actomyosin cytoskeleton by other means.     

Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization

Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti-Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.     

The Maternal-to-Zygotic Transition Targets Actin to Promote Robustness during Morphogenesis

Robustness is a property built into biological systems to ensure stereotypical outcomes despite fluctuating inputs from gene dosage, biochemical noise, and the environment. During development, robustness safeguards embryos against structural and functional defects. Yet, our understanding of how robustness is achieved in embryos is limited. While much attention has been paid to the role of gene and signaling networks in promoting robust cell fate determination, little has been done to rigorously assay how mechanical processes like morphogenesis are designed to buffer against variable conditions. Here we show that the cell shape changes that drive morphogenesis can be made robust by mechanisms targeting the actin cytoskeleton. We identified two novel members of the Vinculin/α-Catenin Superfamily that work together to promote robustness during Drosophila cellularization, the dramatic tissue-building event that generates the primary epithelium of the embryo. We find that zygotically-expressed Serendipity-α (Sry-α) and maternally-loaded Spitting Image (Spt) share a redundant, actin-regulating activity during cellularization. Spt alone is sufficient for cellularization at an optimal temperature, but both Spt plus Sry-α are required at high temperature and when actin assembly is compromised by genetic perturbation. Our results offer a clear example of how the maternal and zygotic genomes interact to promote the robustness of early developmental events. Specifically, the Spt and Sry-α collaboration is informative when it comes to genes that show both a maternal and zygotic requirement during a given morphogenetic process. For the cellularization of Drosophilids, Sry-α and its expression profile may represent a genetic adaptive trait with the sole purpose of making this extreme event more reliable. Since all morphogenesis depends on cytoskeletal remodeling, both in embryos and adults, we suggest that robustness-promoting mechanisms aimed at actin could be effective at all life stages.     

Maternal cyclin B levels "Chk" the onset of DNA replication checkpoint control in Drosophila

In many animals, early development of the embryo is characterized by synchronous, biphasic cell divisions. These cell divisions are controlled by maternally inherited proteins and RNAs. A critical question in developmental biology is how the embryo transitions to a later pattern of asynchronous cell divisions and transfers the prior maternal control of development to the zygotic genome. The most-common model regarding how this transition from maternal to zygotic control is regulated posits that this is a consequence of the limitation of maternal gene products, due to their titration during early cell divisions. Here we discuss a recent article by Crest et al.1 that instead proposes that the balance of Cyclin-dependent Kinase 1 and Cyclin B (Cdk1-CycB) activity relative to that of the Drosophila checkpoint kinase Chk1 determines when asynchronous divisions begin.     

Characterization of anillin mutants reveals essential roles in septin localization and plasma membrane integrity

Anillin is a conserved component of the contractile ring that is essential for cytokinesis, and physically interacts with three conserved cleavage furrow proteins, F-actin, myosin II and septins in biochemical assays. We demonstrate that the Drosophila scraps gene, identified as a gene involved in cellularization, encodes Anillin. We characterize defects in cellularization, pole cell formation and cytokinesis in a series of maternal effect and zygotic anillin alleles. Mutations that result in amino acid changes in the C-terminal PH domain of Anillin cause defects in septin recruitment to the furrow canal and contractile ring. These mutations also strongly perturb cellularization, altering the timing and rate of furrow ingression. They cause dramatic vesiculation of new plasma membranes, and destabilize the stalk of cytoplasm that normally connects gastrulating cells to the yolk mass. A mutation closer to the N terminus blocks separation of pole cells with less effect on cellularization, highlighting mechanistic differences between contractile processes. Cumulatively, our data point to an important role for Anillin in scaffolding cleavage furrow components, directly stabilizing intracellular bridges, and indirectly stabilizing newly deposited plasma membrane during cellularization.     

Genetic analysis of the cellularization of the Drosophila embryo.

The synchronous cellularization of the Drosophila embryo at the blastoderm stage provides a unique system for studying the molecular mechanisms involved in cytokinesis, using genetical and biochemical approaches. The cellularization process requires the major components of the embryonic cytoskeleton that are deposited into the egg during oogenesis. Genetical analysis indicates that it requires also the products of additional maternally-acting genes, as well as that of a limited set of zygotically-acting genes. The cellularization defective phenotypes associated with small deficiencies uncovering these latter loci reveal specific steps within this complex process. The molecular analysis of these genes will ultimately provide meaningful insights into the normal process of cellularization. Among them, the serendipity alpha gene encodes a membrane-associated protein, which is exclusively accumulated during cellularization, and is required for the reorganization of the microfilaments as the onset of cellularization.     

Evidence for functional differentiation among Drosophila septins in cytokinesis and cellularization

The septins are a conserved family of proteins that are involved in cytokinesis and other aspects of cell-surface organization. In Drosophila melanogaster, null mutations in the pnut septin gene are recessive lethal, but homozygous pnut mutants complete embryogenesis and survive until the pupal stage. Because the completion of cellularization and other aspects of early development seemed likely to be due to maternally contributed Pnut product, we attempted to generate embryos lacking the maternal contribution in order to explore the roles of Pnut in these processes. We used two methods, the production of germline clones homozygous for a pnut mutation and the rescue of pnut homozygous mutant flies by a pnut(+) transgene under control of the hsp70 promoter. Remarkably, the pnut germline-clone females produced eggs, indicating that stem-cell and cystoblast divisions in the female germline do not require Pnut. Moreover, the Pnut-deficient embryos obtained by either method completed early syncytial development and began cellularization of the embryo normally. However, during the later stages of cellularization, the organization of the actin cytoskeleton at the leading edge of the invaginating furrows became progressively more abnormal, and the embryos displayed widespread defects in cell and embryo morphology beginning at gastrulation. Examination of two other septins showed that Sep1 was not detectable at the cellularization front in the Pnut-deficient embryos, whereas Sep2 was still present in normal levels. Thus, it is possible that Sep2 (perhaps in conjunction with other septins such as Sep4 and Sep5) fulfills an essential septin role during the organization and initial ingression of the cellularization furrow even in the absence of Pnut and Sep1. Together, the results suggest that some cell-division events in Drosophila do not require septin function, that there is functional differentiation among the Drosophila septins, or both.     

Zygotic activity of the nullo locus is required to stabilize the actin-myosin network during cellularization in Drosophila

Cellularization of the Drosophila embryo requires the establishment of a hexagonal network of actin and myosin filaments that are interconnected around the nuclei in the cortex of the syncytial blastoderm. This cytoskeletal network provides the framework and possibly the contractile force for the membrane invaginations that synchronously subdivide the syncytial embryo into individual cells. Zygotic expression of the nullo locus is essential for the preservation of an intact actin-myosin network. Embryos deleted for the nullo locus have a disrupted network, resulting in the formation of many multinucleate cells. We show that nullo is not required for the initial formation of the actin-myosin network, but is necessary for the maintenance of its hexagonal shape during cellularization. The phenotype of embryonic mosaics is nonautonomous, indicating that nullo does not have to be expressed in every nucleus for proper cellularization. Examination of nullo mutant clones in adults reveals that nullo activity is not required for cell division in imaginal discs. Furthermore, germline clone experiments suggest that maternal expression of the nullo locus is not essential for either germline proliferation or the cellularization of progeny. We propose a model in which nullo functions specifically at cellularization to stabilize the actin-myosin network during contraction.     

The egg came first, of course! Anterior-posterior pattern formation in Drosophila embryogenesis and oogenesis

The anterior-posterior body pattern of the Drosophila embryo is initiated through the action of maternal gene products. In particular, three groups of maternally acting genes (the anterior, posterior and terminal groups) have been shown to direct the synthesis and spatial restriction of the three major organizing activities in the egg. The initial spatial localizations of the maternal organizing activities are established during oogenesis. After fertilization these activities regulate zygotic gene activity along the anterior-posterior axis of the egg.