Norma T. Takaesu

dSno Facilitates Baboon Signaling in the Drosophila Brain by Switching the Affinity of Medea Away From Mad and Toward dSmad2

A screen for modifiers of Dpp adult phenotypes led to the identification of the Drosophila homolog of the Sno oncogene (dSno). The dSno locus is large, transcriptionally complex and contains a recent retrotransposon insertion that may be essential for dSno function, an intriguing possibility from the perspective of developmental evolution. dSno is highly transcribed in the embryonic central nervous system and transcripts are most abundant in third instar larvae. dSno mutant larvae have proliferation defects in the optic lobe of the brain very similar to those seen in baboon (Activin type I receptor) and dSmad2 mutants. This suggests that dSno is a mediator of Baboon signaling. dSno binds to Medea and Medea/dSno complexes have enhanced affinity for dSmad2. Alternatively, Medea/dSno complexes have reduced affinity for Mad such that, in the presence of dSno, Dpp signaling is antagonized. We propose that dSno functions as a switch in optic lobe development, shunting Medea from the Dpp pathway to the Activin pathway to ensure proper proliferation. Pathway switching in target cells is a previously unreported mechanism for regulating TGFβ signaling and a novel function for Sno/Ski family proteins.

DNA-binding domain mutations in SMAD genes yield dominant-negative proteins or a neomorphic protein that can activate WG target genes in Drosophila.

Mutations in SMAD tumor suppressor genes are involved in approximately 140,000 new cancers in the USA each year. At this time, how the absence of a functional SMAD protein leads to a tumor is unknown. However, clinical and biochemical studies suggest that all SMAD mutations are loss-of-function mutations. One prediction of this hypothesis is that all SMAD mutations cause tumors via a single mechanism. To test this hypothesis, we expressed five tumor-derived alleles of human SMAD genes and five mutant alleles of Drosophila SMAD genes in flies. We found that all of the DNA-binding domain mutations conferred gain-of-function activity, thereby falsifying the hypothesis. Furthermore, two types of gain-of-function mutation were identified – dominant negative and neomorphic. In numerous assays, the neomorphic allele SMAD4100T appears to be capable of activating the expression of WG target genes. These results imply that SMAD4100T may induce tumor formation by a fundamentally different mechanism from other SMAD mutations, perhaps via the ectopic expression of WNT target genes – an oncogenic mechanism associated with mutations in Adenomatous Polyposis Coli. Our results are likely to have clinical implications, because gain-of-function mutations may cause tumors when heterozygous, and the life expectancy of individuals with SMAD4100T is likely to be different from those with other SMAD mutations. From a larger perspective, our study shows that the genetic characterization of missense mutations, particularly in modular proteins, requires experimental verification.

Fat facets deubiquitylation of Medea/Smad4 modulates interpretation of a Dpp morphogen gradient

The ability of secreted Transforming Growth Factor β (TGFβ) proteins to act as morphogens dictates that their influence be strictly regulated. Here, we report that maternally contributed fat facets (faf; a homolog of USP9X/FAM) is essential for proper interpretation of the zygotic Decapentaplegic (Dpp) morphogen gradient that patterns the embryonic dorsal-ventral axis. The data suggest that the loss of faf reduces the activity of Medea (a homolog of Smad4) below the minimum necessary for adequate Dpp signaling and that this is likely due to excessive ubiquitylation on a specific lysine. This study supports the hypothesis that the control of cellular responsiveness to TGFβ signals at the level of Smad4 ubiquitylation is a conserved mechanism required for proper implementation of a morphogen gradient.

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.

α/β Hydrolase2, a predicated gene adjacent to mad in Drosophila melanogaster, belongs to a new global multigene family and is associated with obesity

The experimental validation of genes predicted from genomic sequence and the identification of functions for these genes is an increasingly important task. We report a multidisciplinary analysis of CG3488, a predicted gene adjacent to Mothers against dpp in Drosophila melanogaster. We cloned and sequenced a cDNA corresponding to CG3488 and we show that it is expressed in embryos. A computational analysis shows that CG3488 contains a number of conserved domains present in enzymes capable of lipid hydrolysis. A phylogenetic analysis shows that CG3488 is the homolog of human α/β hydrolase2 and that these genes belong to a novel multigene family with members in animals, plants, fungi, and bacteria. A genetic analysis shows that heterozygosity for a chromosomal deletion that removes CG3488 dominantly enhances the excess lipid phenotype associated with a mutation in adipose, an uncloned obesity gene. Further, overexpression of a CG3488 transgene rescues this obesity phenotype. Overall, the data suggests that CG3488 functions as a lipase and that analyses of its homologs will provide unique insights into lipid metabolism in many species.

Drosophila CORL is required for Smad2-mediated activation of Ecdysone Receptor expression in the mushroom body.

CORL proteins (FUSSEL/SKOR proteins in humans) are related to Sno/Ski oncogenes but their developmental roles are unknown. We have cloned Drosophila CORL and show that its expression is restricted to distinct subsets of cells in the central nervous system. We generated a deletion of CORL and noted that homozygous individuals rarely survive to adulthood. Df(4)dCORL adult escapers display mushroom body (MB) defects and Df(4)dCORL larvae are lacking Ecdysone Receptor (EcR-B1) expression in MB neurons. This is phenocopied in CORL-RNAi and Smad2-RNAi clones in wild-type larvae. Furthermore, constitutively active Baboon (type I receptor upstream of Smad2) cannot stimulate EcR-B1 MB expression in Df(4)dCORL larvae, which demonstrates a formal requirement for CORL in Smad2 signaling. Studies of mouse Corl1 (Skor1) revealed that it binds specifically to Smad3. Overall, the data suggest that CORL facilitates Smad2 activity upstream of EcR-B1 in the MB. The conservation of neural expression and strong sequence homology of all CORL proteins suggests that this is a new family of Smad co-factors.

Use of restriction fragment length polymorphisms to study the origin of human aneuploidy

Aneuploidy is the most common class of chromosome abnormality in humans, occurring in at least 5% of all clinically recognizable pregnancies.’ Some forms of aneuploidy are compatible with live birth, approximately 0.3% of newborn infants having an additional or missing chromosome.Z Furthermore, no less than 35% of all spontaneous abortions are attributable to the two commonest types of aneuploidy, namely, autosomal trisomy and sex chromosome mono~omy.~ Despite the frequency with which they occur, relatively little is known about the parental origin of monosomy or most trisomies. Previous studies of parental origin have relied on one of two approaches, inheritance of the X-linked Xg blood group or inheritance of chromosome heteromorphisms. However, technical factors limit the efficiency of both approaches, with most studies reporting successful determinations of origin in only a minority of cases. In addition, most autosomes do not have cytologically detectable variable regions or appropriate biochemical markers and consequently abnormalities involving these chromosomes have not yet been studied. The recent identification of restriction fragment length polyrnorphisms (RFLPs) on all human chromosomes provides a new approach to the study of the origin of de novo chromosome abnormalities. In this paper we summarize the results of previous studies of parental origin using Xg and chro-

Characterization of three VNTR systems at D21S112

D21S112 is a highly polymorphic marker on the long arm of chromosome 21. Our analysis of this locus indicated the presence of three VNTR systems. We estimated the heterozygosity of each system and sequenced one of the repetitive regions. Utilizing PCR, we demonstrated that the sequenced VNTR is responsible for the system with the highest level of heterozygosity. Combining data from the three systems makes D21S112 one of the most informative loci on the chromosome.

CORL Expression and Function in Insulin Producing Neurons Reversibly Influences Adult Longevity in Drosophila

CORL proteins (known as SKOR in mice, Fussel in humans and fussel in Flybase) are a family of CNS specific proteins related to Sno/Ski oncogenes. Their developmental and adult roles are largely unknown. A Drosophila CORL (dCORL) reporter gene is expressed in all Drosophila insulin-like peptide 2 (dILP2) neurons of the pars intercerebralis (PI) of the larval and adult brain. The transcription factor Drifter is also expressed in the PI in a subset of dCORL and dILP2 expressing neurons and in several non-dILP2 neurons. dCORL mutant virgin adult brains are missing all dILP2 neurons that do not also express Drifter. This phenotype is also seen when expressing dCORL-RNAi in neurosecretory cells of the PI. dCORL mutant virgin adults of both sexes have a significantly shorter lifespan than their parental strain. This longevity defect is completely reversed by mating (lifespan increases over 50% for males and females). Analyses of dCORL mutant mated adult brains revealed a complete rescue of dILP2 neurons without Drifter. Taken together, the data suggest that dCORL participates in a neural network connecting the insulin signaling pathway, longevity and mating. The conserved sequence and CNS specificity of all CORL proteins imply that this network may be operating in mammals.

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.