The Wnt family of secreted molecules functions in cell-fate determination and morphogenesis during development in both vertebrates and invertebrates (reviewed in ref. 1). Drosophila Wingless is a founding member of this family, and many components of its signal transduction cascade have been identified, including the Frizzled class of receptor. But the mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. Here we describe a gene that is necessary for all Wingless signalling events in Drosophila. We show that arrow gene function is essential in cells receiving Wingless input and that it acts upstream of Dishevelled. arrow encodes a single-pass transmembrane protein, indicating that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, our data suggests a new and conserved function for this LRP subfamily in Wingless/Wnt signal reception.

Summary The ability of adult stem cells to maintain their undifferentiated state depends upon residence in their niche. While simple models of a single self-renewal signal are attractive, niche-stem cell interactions are likely to be more complex. Many niches have multiple cell types, and the Drosophila testis is one such complex niche with two stem cell types, germline stem cells (GSCs) and somatic cyst progenitor cells (CPCs). These stem cells require chemokine activation of Jak/STAT signaling for self-renewal. We identified the transcriptional repressor Zfh-1 as a presumptive somatic target of Jak/STAT signaling, demonstrating that it is necessary and sufficient to maintain CPCs. Surprisingly, sustained zfh-1 expression or intrinsic STAT activation in somatic cells caused neighboring germ cells to self-renew outside their niche. In contrast, germline-intrinsic STAT activation was insufficient for GSC renewal. This data reveals unexpected complexity in cell interactions in the niche, implicating CPCs in GSC self-renewal.

For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae , that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type–related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

A temperature-sensitive DNA topoisomerase II mutant of the yeast Saccharomyces cerevisiae has been identified. Genetic analysis shows that a single recessive nuclear mutation is responsible for both temperature-sensitive growth and enzymatic activity. Thus, topoisomerase II is essential for viability and the mutation is most probably in the structural gene. Experiments with synchronized mutant cells show that at the nonpermissive temperature cells can undergo one, and only one, round of DNA replication. These cells are arrested at medial nuclear division. Analysis of 2-,um plasmid DNA from these cells shows it to be in the form of multiply intertwined catenated dimers. The results suggest that DNA topoisomerase II is necessary for the segregation of chromosomes at the termination of DNA replication.DNA topoisomerases are enzymes that catalyze the concerted breakage and rejoining of DNA backbone bonds (1). Topoisomerases can be divided into several categories, depending on their source and mode of action (reviewed in ref.2). Eukaryotic type 2 topoisomerases, the subject of this paper, can catalyze several different DNA isomerization reactions, including the relaxation, catenation, decatenation, knotting, and unknotting of closed double-stranded DNA circles (3-5). Although the eukaryotic type 2 DNA topoisomerases are fairly well characterized in vitro, nothing is known about their in vivo roles. It has been suggested that these enzymes might be involved in initiation of DNA replication (3) or in the segregation of daughter DNA molecules at the termination of DNA replication (6, 7).The properties of the yeast topoisomerases are quite similar to their counterparts from mammalian cells (8,9 MATa adel ade2 ura3-52, top2-1(ts), a segregant from the last backcross, was used for phenotype studies.Yeast Growth Conditions. Medium for growth of yeast was YPD (11) or YM-5 (12). The in vivo uniform labeling experiments were carried out essentially as described (12), except that [5,6-3H]uracil at 3 tCi/ml (41 Ci/mmol; 1 Ci = 37 GBq) was used. Cells were synchronized with a-factor pheromone as described (13). Nuclear staining was carried out as described (14), except that Hoechst 33258 dye was used instead of DAPI. Progress through the yeast cell cycle was monitored morphologically by phase-contrast microscopy (13).Topoisomerase Assays. Cells were grown in 25 ml of YPD medium at 250C. During exponential growth cultures were shifted to 370C for 20 min, chilled, centrifuged, washed with cold H20, and recentrifuged. The cell pellet was resuspended in 0.5 ml of yeast lysis buffer (20 mM Tris HCl, pH 7.5/1 mM Na2EDTA/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/500 mM KCl/10% glycerol). Onethird volume of glass beads (Sigma, type IV,tm) was added and the cells were lysed by brief sonication. The lysate was centrifuged for 10 min in a desk top centrifuge.One microliter of the supernatant (undiluted or diluted in yeast lysis buffer plus 100 ,ug of bovine serum albumin per ml) was used for topoisomerase assays. D...

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Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).

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Activation of the Wnt signaling cascade provides key signals during development and in disease. Here we provide evidence, by designing a Wnt receptor with ligand-independent signaling activity, that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. We have uncovered a branch of the Wnt pathway in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3beta/Zw3, the kinase normally required for controlling beta-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. We propose that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo.