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

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

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

Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm

In this study we describe the morphological and genetic analysis of the Drosophila mutant gurtelchen (gurt). gurt was identified by screening an EMS collection for novel mutations affecting visceral mesoderm development and was named after the distinct belt shaped visceral phenotype. Interestingly, determination of visceral cell identities and subsequent visceral myoblast fusion is not affected in mutant embryos indicating a later defect in visceral development. gurt is in fact a new huckebein (hkb) allele and as such exhibits nearly complete loss of endodermal derived structures. Targeted ablation of the endodermal primordia produces a phenotype that resembles the visceral defects observed in huckebein(gurtelchen) (hkb(gurt)) mutant embryos. It was shown previously that visceral mesoderm development requires complex interactions between visceral myoblasts and adjacent tissues. Signals from the neighbouring somatic myoblasts; play an important role in cell type determination and are a prerequisite for visceral muscle fusion. Furthermore, the visceral mesoderm is known to influence endodermal migration and midgut epithelium formation. our analyses of the visceral phenotype of hkb(gurt) mutant embryos reveal that the adjacent endoderm plays a critical role in the later stages of visceral muscle development, and is required for visceral muscle elongation and outgrowth after proper myoblast fusion. (C) 2009 Elsevier Ireland Ltd. All rights reserve