Differential effects of Cbl isoforms on Egfr signaling in Drosophila

The Cbl family of proteins downregulate epidermal growth factor receptor (Egfr) signaling via receptor internalization and destruction. These proteins contain two functional domains, a RING finger domain with E3 ligase activity, and a proline rich domain mediating the formation of protein complexes. The Drosophila cbl gene encodes two isoforms, D-CblS and D-CblL. While both contain a RING finger domain, the proline rich domain is absent from D-CblS. We demonstrate that expression of either isoform is sufficient to rescue both the lethality of a D-cbl null mutant and the adult phenotypes characteristic of Egfr hyperactivation, suggesting that both isoforms downregulate Egfr signaling. Interestingly, targeted overexpression of D-CblL, but not D-CblS, results in phenotypes characteristic of reduced Egfr signaling and suppresses the effect of constitutive Egfr activation. The level of D-CblL was significantly correlated with the phenotypic severity of reduced Egfr signaling, suggesting that D-CblL controls the efficiency of downregulation of Egfr signaling. Furthermore, reduced dynamin function suppresses the effects of D-CblL overexpression in follicle cells, suggesting that D-CblL promotes internalization of activated receptors. D-CblL is detected in a punctate cytoplasmic pattern, whereas D-CblS is mainly localized at the follicle cell cortex. Therefore, D-CblS and D-CblL may downregulate Egfr through distinct mechanisms.     

Genetic link between beta-sarcoglycan and the Egfr signaling pathway

Sarcoglycans are a multimeric, integral membrane protein complex that is part of the dystrophin glycoprotein complex. Previous findings suggest that the dystrophin glycoprotein complex plays roles not only in maintaining the mechanical structure of the cell membrane but also in signal transduction. To evaluate the functions of sarcoglycans, we here took advantage of Drosophila, which is useful for screening genetic interactions. Morphological aberrancy was observed in the adult compound eyes of Drosophila beta-sarcolgycan (dscgbeta) knockdown flies. We also detected genetic interactions between dscgbeta and Egfr related genes, such as rhomboid-1, rhomboid-3, and mirror. Furthermore two extra cell types with strong expression of Rhomboid were found in the ommatidia of dscgbeta knockdown pupal retina. These cells exhibited phosphorylation of ERKA, suggesting that Egfr signaling is activated via Rhomboid. Through these in vivo analyses, we conclude that dscgbeta negatively regulates the Egfr signaling pathway.     

Structure-function analysis of the Drosophila retinal determination protein Dachshund

Dachshund (Dac) is a highly conserved nuclear protein that is distantly related to the Ski/Sno family of corepressor proteins. In Drosophila, Dac is necessary and sufficient for eye development and, along with Eyeless (Ey), Sine oculis (So), and Eyes absent (Eya), forms the core of the retinal determination (RD) network. In vivo and in vitro experiments suggest that members of the RD network function together in one or more complexes to regulate the expression of downstream targets. For example, Dac and Eya synergize in vivo to induce ectopic eye formation and they physically interact through conserved domains. Dac contains two highly conserved domains, named DD1 and DD2, but no function has been assigned to either of them in an in vivo context. We performed structure-function studies to understand the relationship between the conserved domains of Dac and the rest of the protein and to determine the function of each domain during development. We show that only DD1 is essential for Dac function and while DD2 facilitates DD1, it is not absolutely essential in spite of more than 500 million years of conservation. Moreover, the physical interaction between Eya and DD2 is not required for the genetic synergy between the two proteins. Finally, we show that DD1 also plays a central role for nuclear localization of Dac.     

Characterization of DIP1, a novel nuclear protein in Drosophila melanogaster

We have recently identified in Drosophila melanogaster a new gene encoding a nuclear protein, DIP1. Here we report the developmental expression and the finding that DIP1 subcellular localization is in the nucleus and at the nuclear periphery during interphase in embryos. Interestingly, in humans, DIP1 antibody identified signals in nuclei from cultured cells and reacted with a rough 30kDa protein in Western blotting experiments, demonstrating evolutionary conservation.     

Yantar, a conserved arginine-rich protein is involved in Drosophila hemocyte development

To identify novel factors involved in Drosophila hematopoiesis, we screened a collection of lethal recessive mutations that also affected normal hemocyte composition in larvae. We present the characterization of the gene yantar (ytr) for which we isolated null and hypomorphic mutations that were associated with severe defects in hemocyte differentiation and proliferation; ytr is predominantly expressed in the hematopoietic tissue during larval development and encodes an evolutionary conserved protein which is predominantly localized in the nucleus. The hematopoietic phenotype in ytr mutants is consistent with a defect or block in differentiation of precursor hemocytes: mutant larvae have enlarged lymph glands (LGs) and have an excess of circulating hemocytes. In addition, many cells exhibit both lamellocyte and crystal cell markers. Ytr function has been preserved in evolution as hematopoietic specific expression of the Drosophila or mouse Ytr proteins rescue the differentiation defects in mutant hemocytes.     

The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

Expression of a beetle, Dendroides canadensis, antifreeze protein in Drosophila melanogaster

Antifreeze protein 1 (DAFP-1), from the beetle Dendroides canadensis, was expressed in Drosophila melanogaster. Mean thermal hysteresis values (the difference between freezing and melting points), indicative of antifreeze protein activity, in the hemolymph of transgenic flies were found to be as high as 6.23+/-0.10 degrees C (using the nanoliter osmometer). Direct comparisons of the capillary and nanoliter osmometer techniques for measuring THA were made, illustrating the much higher values obtained by the latter. Transgenic Drosophila had supercooling points, both in contact with ice and not, that were slightly, but significantly, lower than wild-type controls (1.5-2.0 degrees C and 2.0-4.0 degrees C, respectively). The results indicate functionality of DAFP-1 in Drosophila melanogaster (the ability of DAFP-1 to inhibit both inoculative freezing across the cuticle and freezing initiated by endogenous ice nucleators). The much larger effects of DAFPs in inhibiting inoculative freezing and ice nucleation in Dendroides canadensis relative to the transgenic Drosophila may partially result from the lower DAFP concentrations and activities in Drosophila, however the absence of multiple types of DAFPs and absence of tissue specific expression may also contribute. Transgenic Drosophila were also able to live significantly longer than controls at 0 degrees C and 4 degrees C, indicating that DAFP-1 is able to increase cold tolerance at above freezing temperatures.     

Regulation of gene expression in the distal region of the Drosophila leg by the Hox11 homolog, C15

The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IV and V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and B are mutually antagonistic resulting in exclusion of B expression from the pretarsus. Although Al is necessary, it is not sufficient to repress B, indicating another factor is required. Here, this factor is identified as the product of the C15 gene, which is another homeodomain protein, a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al and, together, C15 and Al appear to directly repress B. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl- border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap.     

Evidence that nervy, the Drosophila homolog of ETO/MTG8, promotes mechanosensory organ development by enhancing Notch signaling

In the imaginal tissue of developing fruit flies, achaete (ac) and scute (sc) expression defines a group of neurally-competent cells called the proneural cluster (PNC). From the PNC, a single cell, the sensory organ precursor (SOP), is selected as the adult mechanosensory organ precursor. The SOP expresses high levels of ac and sc and sends a strong Delta (Dl) signal, which activates the Notch (N) receptor in neighboring cells, preventing them from also adopting a neural fate. Previous work has determined how ac and sc expression in the PNC and SOP is regulated, but less is known about SOP-specific factors that promote SOP fate. Here, we describe the role of nervy (nvy), the Drosophila homolog of the mammalian proto-oncogene ETO, in mechanosensory organ formation. Nvy is specifically expressed in the SOP, where it interacts with the Ac and Sc DNA binding partner Daughterless (Da) and affects the expression of Ac and Sc targets. nvy loss- and gain-of-function experiments suggest that nvy reinforces, but is not absolutely required for, the SOP fate. We propose a model in which nvy acts downstream of ac and sc to promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP.     

Drosophila crooked-neck protein co-fractionates in a multiprotein complex with splicing factors

The Drosophila crooked neck (crn) gene encodes an unusual TPR-containing protein whose function is essential for embryonic development. Homology with other TPR-proteins involved in cell cycle control, initially led to the proposal that Crn might play a critical role in regulation of embryonic cell divisions. Here, we show that Crn does not have a cell cycle function in the embryo. By using specific antibodies we also show that the Crn protein is a nuclear protein which localizes in "speckles" which could correspond to preferential localization of several other splicing factors. Fractionation of nuclear extracts on sucrose gradients revealed Crn in a 900 kDa multiproteic complex together with snRNPs, suggesting that Crn participates in the assembly of the splicing machinery in vivo.     

Immunohistochemical analysis of a novel dehydroepiandrosterone sulfotransferase-like protein in Drosophila neural circuits

Sulfotransferase (ST)-catalyzed sulfation plays an important role in various neuronal functions such as homeostasis of catecholamine neurotransmitters and hormones. Drosophila is a popular model for the study of memory and behavioral manifestations because it is able to mimic the intricate neuroregulation and recognition in humans. However, there has been no evidence indicating that cytosolic ST(s) is(are) present in Drosophila. The aim of this study is to investigate whether or not cytosolic ST(s) is(are) expressed in the Drosophila nervous system. Immunoblot analysis demonstrated the presence of dehydroepiandrosterone (DHEA) ST-like protein in Drosophila brain and a sensitive fluorometric assay revealed its sulfating activity toward DHEA. Immunohistochemical staining demonstrated this DHEA ST-like protein to be abundant in specific neurons as well as in several bundles of nerve fibers in Drosophila. Clarification of a possible link between ST and a neurotransmitter-mediated effect may eventually aid in designing approaches for alleviating neuronal disorders in humans.     

Decapentaplegic head capsule mutations disrupt novel peripodial expression controlling the morphogenesis of the Drosophila ventral head

Drosophila adult structures derive from imaginal discs, which are sacs with apposed epithelial sheets, the disc proper (DP) and the peripodial epithelium (PE). The Drosophila TGF-beta family member decapentaplegic (dpp) contributes to the development of adult structures through expression in all imaginal discs, driven by enhancers from the 3' cis-regulatory region of the gene. In the eye/antennal disc, there is 3' directed dpp expression in both the DP and PE associated with cell proliferation and eye formation. Here, we analyze a new class of dpp cis-regulatory mutations, which specifically disrupt a previously unknown region of dpp expression, controlled by enhancers in the 5' regulatory region of the gene and limited to the PE of eye/antennal discs. These are the first described Drosophila mutations that act by solely disrupting PE gene expression. The mutants display defects in the ventral adult head and alter peripodial but not DP expression of known dpp targets. However, apoptosis is observed in the underlying DP, suggesting that this peripodial dpp signaling source supports cell survival in the DP.     

Mutations in Nop60B,the Drosophila homolog of human dyskeratosis congenita 1, affect the maintenance of the germ-line stem cell lineage during spermatogenesis

Spermatogenesis in Drosophila is maintained by germ-line stem cells. These cells undergo self-renewing divisions and also generate daughter gonial cells, whose function is to amplify the germ cell pool. Gonial cells subsequently differentiate into spermatocytes that undergo meiosis and generate haploid gametes. To elucidate the circuitry that controls progression through spermatogenic stem cell lineages, we are identifying mutations that lead to either excess germ cells or germ cell loss. From a collection of male sterile mutants, we identified P-element-induced hypomorphic alleles of nop60B, a gene encoding a pseudouridine synthase. Although null mutations are lethal, our P element-induced alleles generate viable, but sterile flies, exhibiting severe testicular atrophy. Sterility is reversed by P-element excision, and the atrophy is rescued by a Nop60B transgene, confirming identity of the gene. Using cell-type-specific markers, we find that testicular atrophy is due to severe loss of germ cells, including stem cells, but much milder effects on the somatic cells, which are themselves maintained by a stem cell lineage. We show that Nop60B activity is required intrinsically for the maintenance of germ-line stem cells. The relationship of these phenotypes to the human syndrome Dyskeratosis congenita, caused by mutations in a Nop60B homolog, is discussed.