The Rap1 Guanine Nucleotide Exchange Factor C3G Is Required for Preservation of Larval Muscle Integrity in Drosophila melanogaster

C3G is a guanine nucleotide exchange factor (GEF) and modulator of small G-protein activity, which primarily acts on members of the Rap GTPase subfamily. Via promotion of the active GTP bound conformation of target GTPases, C3G has been implicated in the regulation of multiple cellular and developmental events including proliferation, differentiation and apoptosis. The Drosophila C3G orthologue exhibits a domain organization similar to that of vertebrate C3G. Through deletion of the C3G locus, we have observed that loss of C3G causes semi-lethality, and that escaping adult flies are characterized by a reduction in lifespan and general fitness. In situ hybridization reveals C3G expression in the developing embryonic somatic and visceral muscles, and indeed analysis of C3G mutants suggests essential functions of C3G for normal body wall muscle development during larval stages. C3G mutants display abnormal muscle morphology and attachment, as well as failure to properly localize βPS integrins to muscle attachment sites. Moreover, we show that C3G stimulates guanine nucleotide exchange on Drosophila Rap GTPases in vitro. Taken together, we conclude that Drosophila C3G is a Rap1-specific GEF with important functions in maintaining muscle integrity during larval stages

Validation of a Drosophila model of wild-type and T315I mutated BCR-ABL1 in chronic myeloid leukemia: an effective platform for treatment screening

Chronic myeloid leukemia (CML) is caused by a balanced chromosomal translocation resulting in the formation of BCR-ABL1 fusion gene encoding a constitutively active BCR-ABL1 tyrosine kinase, which activates multiple signal transduction pathways leading to malignant transformation. Standard treatment of CML is based on tyrosine kinase inhibitors (TKI); however, some mutations have proven elusive particularly the T315I mutation. Drosophila melanogaster is an established in vivo model for human diseases including cancer. The targeted expression of chimeric human/fly and full human BCR-ABL1 in Drosophila eyes has been shown to result in detrimental effects. In this study, we expressed human BCR-ABL1p210 and the resistant BCR-ABL1p210/T315I fusion oncogenes in Drosophila eyes. Expression of BCR-ABL1p210/T315I resulted in a severe distortion of the ommatidial architecture of adult eyes with a more prominent rough eye phenotype compared to milder phenotypes in BCR-ABL1p210 reflecting a stronger oncogenic potential of the mutant. We then assessed the efficacy of the currently used TKI in BCR-ABL1p210 and BCR-ABL1p210/T315I expressing flies. Treatment of BCR-ABL1p210 expressing flies with potent kinase inhibitors (dasatinib and ponatinib) resulted in the rescue of ommatidial loss and the restoration of normal development. Taken together, we provide a CML tailored BCR-ABL1p210 and BCR-ABL1p210/T315I fly model which can be used to test new compounds with improved therapeutic indices

Midgut and muscle development in Drosophila melanogaster

The fully developed and functional Drosophila midgut comprises two layers, the visceral mesoderm and the endoderm. The visceral muscle of the midgut is formed by the fusion of founder cells with fusion competent cells to form the muscle syncytia. The specification of these cells and thus the fusion and the formation of the midgut muscle is dependent on the  Receptor tyrosine kinase (RTK) Alk (Loren et al., 2003). The endoderm underlies the visceral muscle and is formed from cells that originate from the anterior and the posterior parts of the embryo. These cells use the visceral mesoderm as a substrate for their migration. Using Alk mutant animals, we have studied endoderm migration during embryonic development. While the initial migration of the endoderm is not affected in the absence of the visceral mesoderm, we observe that the later dorsal-ventral endodermal migration does not take place. The development of the visceral muscle and its dependence on the endoderm is poorly understood.  We have analysed gürtelchen (gurt) mutant animals, originally identified in a genetic screen for mutations affecting visceral muscle formation. Gurt mutants are so named due to their belt-like phenotype of the visceral muscle (gürtelchen is German for belt). Mapping of the genomic locus identified gurt as a mutation in a previously described gene - huckebein (hkb) which is known to have an important function in endoderm development. Gurt (hkb) mutants were used to further study the interaction between the endoderm and the visceral muscle during development. The initial specification of founder cells and fusion competent myoblasts as well as fusion events are unaffected in gurt (hkb) mutants, however, the elongation and stretching of the visceral muscle does not proceed as normal. Moreover, ablation of the visceral mesoderm disrupts endoderm migration, while ablation of the endoderm results in a delayed disruption of visceral muscle formation. Signaling between the two tissues was investigated in detail. Since Alk is a critical player in visceral muscle development, we employed Alk mutant embryos for this task. In addition to the role of Alk in specifying the founder cells and initiating the visceral muscle fusion, we have shown that Alk mediated signaling has a role in the induction of the midgut constriction process by regulating dpp expression in the developing embryonic gut.  Finally, we wished to identify genes in the founder cells/fusion competent myoblasts that might be regulated by Alk. C3G is a gunaine nucleotide exchange factor expressed in the visceral muscle founder cells. Deletion of the Drosophila C3G locus resulted in the generation of null mutants in C3G which are viable, but display decreased longevity, fitness and are semi-lethal. Further analysis of C3G mutants indicated that C3G is essential for normal larval musculature development, in part by regulating integrin localization at muscle attachment sites

The Leukemic Fly: Promises and Challenges

Leukemia involves different types of blood cancers, which lead to significant mortality and morbidity. Murine models of leukemia have been instrumental in understanding the biology of the disease and identifying therapeutics. However, such models are time consuming and expensive in high throughput genetic and drug screening. Drosophila melanogaster has emerged as an invaluable in vivo model for studying different diseases, including cancer. Fruit flies possess several hematopoietic processes and compartments that are in close resemblance to their mammalian counterparts. A number of studies succeeded in characterizing the fly’s response upon the expression of human leukemogenic proteins in hematopoietic and non-hematopoietic tissues. Moreover, some of these studies showed that these models are amenable to genetic screening. However, none were reported to be tested for drug screening. In this review, we describe the Drosophila hematopoietic system, briefly focusing on leukemic diseases in which fruit flies have been used. We discuss myeloid and lymphoid leukemia fruit fly models and we further highlight their roles for future therapeutic screening. In conclusion, fruit fly leukemia models constitute an interesting area which could speed up the process of integrating new therapeutics when complemented with mammalian models

Ten-eleven translocation proteins and their role beyond DNA demethylation – what we can learn from the fly

Ten-eleven Translocation (TET) proteins have emerged as a family of epigenetic regulators that are important during development and have been implicated in various types of cancers. TET is a highly conserved protein that has orthologues in almost all multicellular organisms. Here, we review recent literature on the novel substrate specificity of this family of DNA 5-methylcytosine demethylases on DNA 6-methyladenine and RNA 5-methylcytosine that were first identified in the invertebrate model Drosophila. We focus on the biological role of these novel epigenetic marks in the fruit fly and mammals and highlight TET proteins’ critical function during development specifically in brain development

Phenotypic and transcriptomic impact of expressing mammalian TET2 in the Drosophila melanogaster model

Ten-Eleven Translocation (TET) proteins have recently come to light as important epigenetic regulators conserved in multicellular organisms. TET knockdown studies in rodents have highlighted the critical role of these proteins for proper brain development and function. Mutations in mammalian mTET proteins and mTET2 specifically are frequent and deregulated in leukaemia and glioma respectively. Accordingly, we examined the role of mTET2 in tumorigenesis in larval haemocytes and adult heads in Drosophila melanogaster. Our findings showed that expression of mutant and wild type mTET2 resulted in general phenotypic defects in adult flies and accumulation of abdominal melanotic masses. Notably, flies with mTET2-R43G mutation at the N-terminus of mTET2 exhibited locomotor and circadian behavioural deficits, as well as reduced lifespan. Flies with mTET2-R1261C mutation in the catalytic domain, a common mutation in acute myeloid leukaemia (AML), displayed alterations affecting haemocyte haemostasis. Using transcriptomic approach, we identified upregulated immune genes in fly heads that were not exclusive to TET2 mutants but also found in wild type mTET2 flies. Furthermore, inhibiting expression of genes that were found to be deregulated in mTET2 mutants, such as those involved in immune pathways, autophagy, and transcriptional regulation, led to a rescue in fly survival, behaviour, and hemocyte number. This study identifies the transcriptomic profile of wild type mTET2 versus mTET2 mutants (catalytic versus non-catalytic) with indications of TET2 role in normal central nervous system (CNS) function and innate immunity

A transgenic Drosophila melanogaster model to study Human T-Lymphotropic Virus oncoprotein Tax-1-driven transformation in vivo.

Affiliations ECOFECTInternational audienceHuman T-cell lymphotropic virus type 1 (HTLV-1)-induced adult T-cell leukemia/lymphoma is an aggressive malignancy. HTLV-2 is genetically related to HTLV-1 but does not cause any malignant disease. HTLV-1 Tax transactivator (Tax-1) contributes to leukemogenesis via NF-κB. We describe transgenic Drosophila models expressing Tax in the compound eye and plasmatocytes. We demonstrate that Tax-1 but not Tax-2 induces ommatidial perturbation and increased plasmatocyte proliferation and that the eye phenotype is dependent on Kenny (IKKγ/NEMO), thus validating this new in vivo model

Epstein-Barr Virus DNA Enhances Diptericin Expression and Increases Hemocyte Numbers in Drosophila melanogaster via the Immune Deficiency Pathway

Infection with the Epstein-Barr virus (EBV) is associated with several malignancies and autoimmune diseases in humans. The following EBV infection and establishment of latency, recurrences frequently occur resulting in potential viral DNA shedding, which may then trigger the activation of immune pathways. We have previously demonstrated that levels of the pro-inflammatory cytokine IL-17, which is associated with several autoimmune diseases, are increased in response to EBV DNA injection in mice. Whether other pro-inflammatory pathways are induced in EBV DNA pathobiology remains to be investigated. The complexity of mammalian immune systems presents a challenge to studying differential activities of their intricate immune pathways in response to a particular immune stimulus. In this study, we used Drosophila melanogaster to identify innate humoral and cellular immune pathways that are activated in response to EBV DNA. Injection of wild-type adult flies with EBV DNA induced the immune deficiency (IMD) pathway resulting in enhanced expression of the antimicrobial peptide diptericin. Furthermore, EBV DNA increased the number of hemocytes in flies. Conditional silencing of the IMD pathway decreased diptericin expression in addition to curbing of hemocyte proliferation in response to challenge with EBV DNA. Comparatively, upon injecting mice with EBV DNA, we detected enhanced expression of tumor necrosis factor-α (TNFα); this enhancement is rather comparable to IMD pathway activation in flies. This study hence indicates that D. melanogaster could possibly be utilized to identify immune mediators that may also play a role in the response to EBV DNA in higher systems