No abstract

Studies in cultured cells and in vitro have identified many actin regulators and begun to define their mechanisms of action. Among these are Enabled (Ena)/VASP proteins, anti-Capping proteins that influence fibroblast migration, growth cone motility, and keratinocyte cell adhesion in vitro. However, partially redundant family members in mammals and maternal Ena contribution in Drosophila previously prevented assessment of the roles of Ena/VASP proteins in embryonic morphogenesis in flies or mammals. We used several approaches to remove maternal and zygotic Ena function, allowing us to address this question. We found that inactivating Ena does not disrupt cell adhesion or epithelial organization, suggesting its role in these processes is cell type-specific. However, Ena plays an important role in many morphogenetic events, including germband retraction, segmental groove retraction and head involution, whereas it is dispensable for other morphogenetic movements. We focused on dorsal closure, analyzing mechanisms by which Ena acts. Ena modulates filopodial number and length, thus influencing the speed of epithelial zippering and the ability of cells to match with correct neighbors. We also explored filopodial regulation in cultured Drosophila cells and embryos. These data provide new insights into developmental and mechanistic roles of this important actin regulator.

Gene product distribution is often used to infer developmental similarities and differences in animals with evolutionarily diverse body plans. However, to address commonalties of developmental mechanisms, what is really needed is a method to assess and compare gene function in divergent organisms. This requires mutations eliminating gene function. Such mutations are often difficult to obtain, even in organisms amenable to genetic analysis. To address this issue we have investigated the use of double-stranded RNA interference to phenocopy null mutations. We show that RNA interference can be used to phenocopy mutations of the Deformed orthologues in Drosophila and Tribolium. We discuss the possible use of this technique for comparisons of developmental mechanisms in organisms with differing ontogenies.

During development, cells craft an impressive array of actin-based structures, mediating events as diverse as cytokinesis, apical constriction, and cell migration. One challenge is to determine how cells regulate actin assembly and disassembly to carry out these cell behaviors. During Drosophila oogenesis diverse cell behaviors are seen in the soma and germline. We used oogenesis to explore developmental roles of two important actin regulators: Enabled/VASP proteins and Capping protein. We found that Enabled plays an important role in cortical integrity of nurse cells, formation of robust bundled actin filaments in late nurse cells that facilitate nurse cell dumping, and migration of somatic border cells. During nurse cell dumping, Enabled localizes to barbed ends of the nurse cell actin filaments, suggesting its mechanism of action. We further pursued this mechanism using mutant Enabled proteins, each affecting one of its protein domains. These data suggest critical roles for the EVH2 domain and its tetramerization subdomain, while the EVH1 domain appears less critical. Enabled appears to be negatively regulated during oogenesis by Abelson kinase. We also explored the function of Capping protein. This revealed important roles in oocyte determination, nurse cell cortical integrity and nurse cell dumping, and support the idea that Capping protein and Enabled act antagonistically during dumping. Together these data reveal places these actin regulators shape oogenesis.

SUMMARYThe planar cell polarity (PCP; non-canonical Wnt) pathway is required to orient the cells within the plane of an epithelium. Here, we show that cofilin 1 (Cfl1), an actin-severing protein, and Vangl2, a core PCP protein, cooperate to control PCP in the early mouse embryo. Two aspects of planar polarity can be analyzed quantitatively at cellular resolution in the mouse embryo: convergent extension of the axial midline; and posterior positioning of cilia on cells of the node. Analysis of the spatial distribution of brachyury + midline cells shows that the Cfl1 mutant midline is normal, whereas Vangl2 mutants have a slightly wider midline. By contrast, midline convergent extension fails completely in Vangl2 Cfl1 double mutants. Planar polarity is required for the posterior positioning of cilia on cells in the mouse node, which is essential for the initiation of left-right asymmetry. Node cilia are correctly positioned in Cfl1 and Vangl2 single mutants, but cilia remain in the center of the cell in Vangl2 Cfl1 double mutants, leading to randomization of left-right asymmetry. In both the midline and node, the defect in planar polarity in the double mutants arises because PCP protein complexes fail to traffic to the apical cell membrane, although other aspects of apical-basal polarity are unaffected. Genetic and pharmacological experiments demonstrate that F-actin remodeling is essential for the initiation, but not maintenance, of PCP. We propose that Vangl2 and cofilin cooperate to target Rab11 + vesicles containing PCP proteins to the apical membrane during the initiation of planar cell polarity.

Genetic background effects contribute to the phenotypic consequences of mutations and are pervasive across all domains of life that have been examined, yet little is known about how they modify genetic systems. In part this is due to the lack of tractable model systems that have been explicitly developed to study the genetic and evolutionary consequences of background effects. In this study we demonstrate that phenotypic expressivity of the scalloped E3 (sd E3 ) mutation of Drosophila melanogaster is background dependent and is the result of at least one major modifier segregating between two standard lab wild-type strains. We provide evidence that at least one of the modifiers is linked to the vestigial region and demonstrate that the background effects modify the spatial distribution of known sd target genes in a genotype-dependent manner. In addition, microarrays were used to examine the consequences of genetic background effects on the global transcriptome. Expression differences between wild-type strains were found to be as large as or larger than the effects of mutations with substantial phenotypic effects, and expression differences between wild type and mutant varied significantly between genetic backgrounds. Significantly, we demonstrate that the epistatic interaction between sd E3 and an optomotor blind mutation is background dependent. The results are discussed within the context of developing a complex but more realistic view of the consequences of genetic background effects with respect to mutational analysis and studies of epistasis and cryptic genetic variation segregating in natural populations.

No abstract

Axin proteins are key negative regulators of the canonical Wnt signal transduction pathway. Although Axin2 null mice are viable, we identified an unusual ENU-induced recessive allele of Axin2 , canp , that causes midgestation lethality in homozygotes. We show that the Axin2 canp mutation is a V26D substitution in an invariant N-terminal sequence motif and that the Axin2 canp protein is more stable than wild type. As predicted for an increased level of a negative regulator, the Axin2 canp mutation leads to decreased Wnt signaling in most tissues, and this can account for most of the morphological phenotypes of Axin2 canp mutants. In contrast, there is a paradoxical increase in canonical Wnt activity in the late primitive streak of all Axin2 canp mutant embryos that is associated with the formation of an ectopic tail in some mutants. Treatment of wild-type embryos with an inhibitor of Tankyrase that stabilizes Axin proteins also causes inhibition of Wnt signaling in anterior regions of the embryo and a gain of Wnt signaling in the primitive streak. The results indicate that although increased stability of Axin2 leads to a loss of canonical Wnt signaling in most tissues, stabilized Axin2 enhances Wnt pathway activity in a specific progenitor population in the late primitive streak.