New resources for the Drosophila 4th chromosome : FRT101F enabled mitotic clones and Bloom syndrome helicase enabled meiotic recombination

Genes on the long arm of the Drosophila melanogaster 4th chromosome are difficult to study because the chromosome lacks mitotic and meiotic recombination. Without recombination numerous standard methods of genetic analysis are impossible. Here, we report new resources for the 4th. For mitotic recombination, we generated a chromosome with an FRT very near the centromere in 101F and a derivative that carries FRT101F with a distal ubiquitously expressed GAL80 transgene. This pair of chromosomes enables both unmarked and MARCM clones. For meiotic recombination, we demonstrate that a Bloom syndrome helicase and recombination defective double mutant genotype can create recombinant 4th chromosomes via female meiosis. All strains will be available to the community via the Bloomington Drosophila Stock Center. Additional resources for studies of the 4th are in preparation and will also be made available. The goal of the 4th Chromosome Resource Project is to accelerate the genetic analysis of protein-coding genes on the 4th, including the 44 genes with no demonstrated function. Studies of these previously inaccessible but largely conserved genes will close longstanding gaps in our knowledge of metazoan development and physiology.Peer reviewe

Distinct Molecular Evolutionary Mechanisms Underlie the Functional Diversification of the Wnt and TGFβ Signaling Pathways

The canonical Wnt pathway is one of the oldest and most functionally diverse of animal intercellular signaling pathways. Though much is known about loss-of-function phenotypes for Wnt pathway components in several model organisms, the question of how this pathway achieved its current repertoire of functions has not been addressed. Our phylogenetic analyses of 11 multigene families from five species belonging to distinct phyla, as well as additional analyses employing the 12 Drosophila genomes, suggest frequent gene duplications affecting ligands and receptors as well as co-evolution of new ligand–receptor pairs likely facilitated the expansion of this pathway’s capabilities. Further, several examples of recent gene loss are visible in Drosophila when compared to family members in other phyla. By comparison the TGFβ signaling pathway is characterized by ancient gene duplications of ligands, receptors, and signal transducers with recent duplication events restricted to the vertebrate lineage. Overall, the data suggest that two distinct molecular evolutionary mechanisms can create a functionally diverse developmental signaling pathway. These are the recent dynamic generation of new genes and ligand–receptor interactions as seen in the Wnt pathway and the conservative adaptation of ancient pre-existing genes to new roles as seen in the TGFβ pathway. From a practical perspective, the former mechanism limits the investigator’s ability to transfer knowledge of specific pathway functions across species while the latter facilitates knowledge transfer

The Sno Oncogene Antagonizes Wingless Signaling during Wing Development in Drosophila

The Sno oncogene (Snoo or dSno in Drosophila) is a highly conserved protein and a well-established antagonist of Transforming Growth Factor-β signaling in overexpression assays. However, analyses of Sno mutants in flies and mice have proven enigmatic in revealing developmental roles for Sno proteins. Thus, to identify developmental roles for dSno we first reconciled conflicting data on the lethality of dSno mutations. Then we conducted analyses of wing development in dSno loss of function genotypes. These studies revealed ectopic margin bristles and ectopic campaniform sensilla in the anterior compartment of the wing blade suggesting that dSno functions to antagonize Wingless (Wg) signaling. A subsequent series of gain of function analyses yielded the opposite phenotype (loss of bristles and sensilla) and further suggested that dSno antagonizes Wg signal transduction in target cells. To date Sno family proteins have not been reported to influence the Wg pathway during development in any species. Overall our data suggest that dSno functions as a tissue-specific component of the Wg signaling pathway with modest antagonistic activity under normal conditions but capable of blocking significant levels of extraneous Wg, a role that may be conserved in vertebrates

The TGF-β Family: Signaling Pathways, Developmental Roles, and Tumor Suppressor Activities

Intercellular communication is a critical process for all multicellular organisms, and communication among cells is required for proper embryonic development and adult physiology. Members of the Transforming Growth Factor-β (TGF-β) family of secreted proteins communicate information between cells via a complex signaling pathway, and family members are capable of inducing a wide range of cellular responses. The purpose of this review is to provide the reader with a broad introduction to our current understanding of three aspects of the TGF-β family. These are the molecular mechanisms utilized by TGF-β signaling pathways, the developmental roles played by TGF-β family members in a variety of species, and the growing list of cancers in which various TGF-β signaling pathways display tumor suppressor activity

Informatics approaches to understanding TGFβ pathway regulation

In recent years, informatics studies have predicted several new ways in which the transforming growth factor β (TGFβ) signaling pathway can be post-translationally regulated. Subsequently, many of these predictions were experimentally validated. These approaches include phylogenetic predictions for the phosphorylation, sumoylation and ubiquitylation of pathway components, as well as kinetic models of endocytosis, phosphorylation and nucleo-cytoplasmic shuttling. We review these studies and provide a brief `how to' guide for phylogenetics. Our hope is to stimulate experimental tests of informatics-based predictions for TGFβ signaling, as well as for other signaling pathways, and to expand the number of developmental pathways that are being analyzed computationally

dSmad2 differentially regulates dILP2 and dILP5 in insulin producing and circadian pacemaker cells in unmated adult females.

Much is known about environmental influences on metabolism and systemic insulin levels. Less is known about how those influences are translated into molecular mechanisms regulating insulin production. To better understand the molecular mechanisms we generated marked cells homozygous for a null mutation in the Drosophila TGF-β signal transducer dSmad2 in unmated adult females. We then conducted side-by-side single cell comparisons of the pixel intensity of two Drosophila insulin-like peptides (dILP2 and dILP5) in dSmad2- mutant and wild type insulin producing cells (IPCs). The analysis revealed multiple features of dSmad2 regulation of dILPs. In addition, we discovered that dILP5 is expressed and regulated by dSmad2 in circadian pacemaker cells (CPCs). Outcomes of regulation by dSmad2 differ between dILP2 and dILP5 within IPCs and differ for dILP5 between IPCs and CPCs. Modes of dSmad2 regulation differ between dILP2 and dILP5. dSmad2 antagonism of dILP2 in IPCs is robust but dSmad2 regulation of dILP5 in IPCs and CPCs toggles between antagonism and agonism depending upon dSmad2 dosage. Companion studies of dILP2 and dILP5 in the IPCs of dCORL mutant (fussel in Flybase and SKOR in mammals) and upd2 mutant unmated adult females showed no significant difference from wild type. Taken together, the data suggest that dSmad2 regulates dILP2 and dILP5 via distinct mechanisms in IPCs (antagonist) and CPCs (agonist) and in unmated adult females that dSmad2 acts independently of dCORL and upd2

Drosophila TGIF Proteins Are Transcriptional Activators

The information carried by transforming growth factor β (TGF-β) signaling molecules induces profound responses in target cells. To restrict this information to appropriate cells, TGF-β signaling pathways are tightly regulated by dynamic interactions with transcriptional activators and repressors. Numerous cross-species experiments have shown that TGF-β family members and their signal transduction machinery (receptors and Smad signal transducers) are functionally conserved between vertebrates and invertebrates. TG-interacting factor (TGIF) is a homeodomain-containing transcriptional corepressor of TGF-β-dependent gene expression in mammals that is associated with holoprosencephaly in humans. Here we report a biochemical analysis of TGIF from zebra fish and Drosophila. Our study reveals an unprecedented role reversal between vertebrate and invertebrate TGIF proteins. Zebra fish TGIF, like its mammalian relative, interacts with general corepressors and represses TGF-β-responsive gene expression. We identified a tandem duplication of TGIF genes in Drosophila. In contrast to vertebrate TGIFs, both Drosophila TGIFs strongly activate transcription. We also demonstrate that Drosophila TGIF proteins physically interact with both Mad and dSmad2, suggesting a role in Dpp and activin signaling. Thus, dTGIF may be the first transcription factor in the Drosophila activin pathway. Overall, our study suggests that assumptions about the functional equivalence of conserved proteins must be validated experimentally

CORL Expression in the Drosophila Central Nervous System Is Regulated by Stage Specific Interactions of Intertwined Activators and Repressors

CORL proteins (SKOR in mice and Fussel in humans) are a subfamily of central nervous system (CNS) specific proteins related to Sno/Ski oncogenes. Their developmental and homeostatic roles are largely unknown. We previously showed that Drosophila CORL (dCORL; fussel in Flybase) functions between the Activin receptor Baboon and Ecdysone Receptor-B1 (EcR-B1) activation in mushroom body neurons of third instar larval brains. To better understand dCORL regulation and function we generated a series of reporter genes. We examined the embryonic and larval CNS and found that dCORL is regulated by stage specific interactions between intertwined activators and repressors spanning numerous reporters. The reporter AH.lacZ, which contains sequences 7-11kb upstream of dCORL exon1, reflects dCORL brain expression at all stages. Surprisingly, AH.lacZ was not detected in EcR-B1 expressing mushroom body neurons. In larvae AH.lacZ is coexpressed with Elav and the transcription factor Drifter in dILP2 insulin producing cells of the pars intercerebralis. The presence of dCORL in insulin producing cells suggests that dCORL functions non-autonomously in the regulation of EcR-B1 mushroom body activation via the modulation of insulin signaling. Overall, the high level of sequence conservation seen in all CORL/SKOR/Fussel family members and their common CNS specificity suggest that similarly complex regulation and a potential function in insulin signaling are associated with SKOR/Fussel proteins in mammals