Person:

Binari, Richard

In Drosophila, widely-used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the Twin Spot Generator technique (TSG) and demonstrate that through mitotic recombination, TSG generates green and red twin spots in internal fly tissues, visible even as single cells. We discuss the wide applications of TSG to lineage and genetic mosaic studies.

Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4–8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes.

The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site–driven RNA interference constructs have been inserted into the genome randomly using P-element–mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.

Signaling proteins often form dynamic protein-protein interaction (PPI) complexes to achieve multi-functionality. Methods to abrogate a subset of PPI interfaces without depleting the full-length protein will be valuable for structure-function relationship annotations. Here, we describe the use of Peptide Aptamer Interference (PAPTi) approach for structure-function network studies. We identified peptide aptamers against Dishevelled (Dsh) and β-catenin (β-cat) to target the Wnt signaling pathway and demonstrate that these FN3-based MONOBODYs (FNDYs) can be used to perturb protein activities both in vitro and in vivo. Further, to investigate the crosstalk between the Wnt and Notch pathways, we isolated FNDYs against the Notch Ankyrin (ANK) region and demonstrate that perturbing the ANK domain of Notch increases the inhibitory activity of Notch towards Wnt signaling. Altogether, these studies demonstrate the power of the PAPTi approach to dissect specific PPI interactions within signaling networks.

Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)–Ras–extracellular signal–regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification–mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type– and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine–protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.

To facilitate the genetic analysis of muscle assembly and maintenance, we have developed a method for efficient RNA interference (RNAi) in Drosophila primary cells using double-stranded RNAs (dsRNAs). First, using molecular markers, we confirm and extend the observation that myogenesis in primary cultures derived from Drosophila embryonic cells follows the same developmental course as that seen in vivo. Second, we apply this approach to analyze 28 Drosophila homologs of human muscle disease genes and find that 19 of them, when disrupted, lead to abnormal muscle phenotypes in primary culture. Third, from an RNAi screen of 1140 genes chosen at random, we identify 49 involved in late muscle differentiation. We validate our approach with the in vivo analyses of three genes. We find that Fermitin 1 and Fermitin 2, which are involved in integrin-containing adhesion structures, act in a partially redundant manner to maintain muscle integrity. In addition, we characterize CG2165, which encodes a plasma membrane Ca2+-ATPase, and show that it plays an important role in maintaining muscle integrity. Finally, we discuss how Drosophila primary cells can be manipulated to develop cell-based assays to model human diseases for RNAi and small-molecule screens.

Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.

We describe the characterization of the Drosophila gene, hopscotch (hop), which is required maternally for the establishment of the normal array of embryonic segments. In hop embryos, although expression of the gap genes appears normal, there are defects in the expression patterns of the pair-rule genes even-skipped, runt, and fushi tarazu, as well as the segment-polarity genes engrailed and wingless. We demonstrate that the effect of hop on the expression of these genes is stripe-specific. The hop gene encodes a putative nonreceptor tyrosine kinase of the Janus kinase family, based on an internal duplication of the catalytic domain. We present a model in which the Hop tyrosine kinase is involved in the control of pair-rule gene transcription in a stripe-specific manner. Our results provide the first evidence for stripe-specific regulation of pair-rule genes by a tyrosine kinase.

Chimaeras, fanciful beasts that drew their force from being composed of parts of disparate animals, have stimulated our collective imagination for centuries. In modern terms, chimaeras are composite animals consisting of genetically distinct cell populations and are called "mosaics" if the different cell types have emerged from the same zygote. Phenotypic studies of chimeric animals formed from invertebrates, amphibians, birds, and mammals have provided many fundamental insights into biological processes, most notably in developmental biology. Many methods for generating both chimaeras and a range of markers for tracing their lineages have been developed over the years. Our laboratory has been intimately involved in the development of methods that facilitate the creation of genetic mosaics in Drosophila. Here, we review our contributions to the development of this field and discuss a number of approaches that will improve further the tool kit for generating mosaic animals.

Adult stem cells or residential progenitor cells are critical to maintain the structure and function of adult tissues (homeostasis) throughout the lifetime of an individual. Mis-regulation of stem cell proliferation and differentiation often leads to diseases including cancer, however, how wildtype adult stem cells and cancer cells respond to cellular damages remains unclear. We find that in the adult Drosophila midgut, intestinal stem cells (ISCs), unlike tumor intestinal cells, are resistant to various cellular damages. Tumor intestinal cells, unlike wildtype ISCs, are easily eliminated by apoptosis. Further, their proliferation is inhibited upon autophagy induction, and autophagy-mediated tumor inhibition is independent of cas-pase-dependent apoptosis. Interestingly, inhibition of tumorigenesis by autophagy is likely through the sequestration and degradation of mitochondria, as compromising mitochondria activity in these tumor models mimics the induction of autophagy and increasing the production of mitochondria alleviates the tumor-suppression capacity of autophagy. Together, these data demonstrate that wildtype adult stem cells and tumor cells show dramatic differences in sensitivity to cellular damages, thus providing potential therapeutic implications targeting tumorigenesis.