No Evidence that Wnt Ligands Are Required for Planar Cell Polarity in Drosophila.

The frizzled (fz) and dishevelled (dsh) genes are highly conserved members of both the planar cell polarity (PCP) pathway and the Wnt signaling pathway. Given these dual functions, several studies have examined whether Wnt ligands provide a tissue-scale orientation cue for PCP establishment during development, and these studies have reached differing conclusions. Here, we re-examine this issue in the Drosophila melanogaster wing and notum using split-Gal4 co-expression analysis, multiplex somatic CRISPR, and double RNAi experiments. Pairwise loss-of-function experiments targeting wg together with other Wnt genes, via somatic CRISPR or RNAi, do not produce PCP defects in the wing or notum. In addition, somatic CRISPR against evi (aka wntless), which is required for the secretion of Wnt ligands, did not produce detectable PCP phenotypes. Altogether, our results do not support the hypothesis that Wnt ligands contribute to PCP signaling in the Drosophila wing or notum.

Large-Scale Transgenic Drosophila Resource Collections for Loss- and Gain-of-Function Studies.

The Transgenic RNAi Project (TRiP), a Drosophila melanogaster functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNA interference (RNAi) fly stocks. To date, it has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express single guide RNAs targeting upstream of a gene transcription start site. Gene activation is triggered by coexpression of catalytically dead Cas9 fused to an activator domain, either VP64-p65-Rta or Synergistic Activation Mediator. TRiP-KO stocks express one or two single guide RNAs targeting the coding sequence of a gene or genes. Cutting is triggered by coexpression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated >5000 TRiP-OE or TRiP-KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

Monocyte Production of IFN-γ Is Interleukin-12 Dependent in a Model of Mevalonate Kinase Deficiency.

Mevalonate Kinase Deficiency (MKD) is an autoinflammatory disease caused by mutations in the mevalonate kinase gene, which produces an enzyme responsible for the production of isoprenoids in the mevalonate pathway. Patient data indicate that MKD is a multicytokine disease with increased plasma levels of cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and interferon (IFN)-γ. To study the mechanisms responsible for these changes, the mevalonate pathway was inhibited with lovastatin in peripheral blood mononuclear cells (PBMCs) and monocytes isolated from the blood of healthy donors followed by stimulation with lipopolysaccharide (LPS) to induce an inflammatory response. Lovastatin treatment resulted in increased levels of IL-6, IL-12p40, and IFN-γ mRNA in both PBMCs and monocytes following LPS stimulation compared with control cells. An IL-12 neutralizing antibody blocked the increased levels of IFN-γ mRNA following lovastatin treatment in PBMCs indicating that this effect is dependent on IL-12. Flow cytometry experiments indicated that monocytes, not lymphocytes or granulocytes, are the source of increased IFN-γ and that both classical and nonclassical/intermediate monocytes express IFN-γ. These results indicate that blocking IL-12 or IFN- γ may be therapeutic options for MKD patients.

The ultrastructural organization of actin and myosin II filaments in the contractile ring: new support for an old model of cytokinesis.

Despite recent advances in our understanding of the components and spatial regulation of the contractile ring (CR), the precise ultrastructure of actin and myosin II within the animal cell CR remains an unanswered question. We used superresolution light microscopy and platinum replica transmission electron microscopy (TEM) to determine the structural organization of actin and myosin II in isolated cortical cytoskeletons prepared from dividing sea urchin embryos. Three-dimensional structured illumination microscopy indicated that within the CR, actin and myosin II filaments were organized into tightly packed linear arrays oriented along the axis of constriction and restricted to a narrow zone within the furrow. In contrast, myosin II filaments in earlier stages of cytokinesis were organized into small, discrete, and regularly spaced clusters. TEM showed that actin within the CR formed a dense and anisotropic array of elongate, antiparallel filaments, whereas myosin II was organized into laterally associated, head-to-head filament chains highly reminiscent of mammalian cell stress fibers. Together these results not only support the canonical "purse-string" model for contractile ring constriction, but also suggest that the CR may be derived from foci of myosin II filaments in a manner similar to what has been demonstrated in fission yeast.