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.

Résumé confidentiel / Confidential abstract

The oncogene Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine Kinase (RTK) and was initially discovered as the fusion protein NPM (nucleophosmin)-ALK in a subset of Anaplastic Large Cell Lymphomas (ALCL). Since then more fusion proteins have been identified in a variety of cancers. Further, overexpression of ALK due to gene amplification has been observed in many malignancies, amongst others neuroblastoma, a pediatric cancer. Lately, activating point mutations in the kinase domain of ALK have been described in neuroblastoma patients and neuroblastoma cell lines. In contrast, the physiological function of ALK is still unclear, but ALK is suggested to play a role in the normal development and function of the nervous system. By employing cell culture based approaches, including a tetracycline-inducible PC12 cell system and the in vivo D. melanogaster model system, we aimed to analyze the downstream signalling of ALK and its role in neuroblastoma. First, we wished to analyze whether ALK is able to activate the small GTPase Rap1 contributing to differentiation/proliferation processes. Activated ALK recruits a complex of the GEF C3G and CrkL and activates C3G by tyrosine phosphorylation. This activated complex is able to activate Rap1 resulting either in neurite outgrowth in PC12 cells or proliferation of neuroblastoma cells suggesting a potential role in the oncogenesis of neuroblastoma driven by gain-of-function mutant ALK. Next, we could show that seven investigated ALK mutations with a high probability of being oncogenic (G1128A, I1171N, F1174L, F1174S, R1192P, F1245C and R1275Q), are true gain-of-function mutations, respond differently to ALK inhibitors and have different transforming ability. Especially the F1174S mutation correlates with aggressive disease development. However, the assumed active germ line mutation I1250T is in fact a kinase dead mutation and suggested to act as a dominant-negative receptor. Finally, ALK mutations are most frequently observed in MYCN amplified tumours correlating with a poor clinical outcome. Active ALK regulates mainly the initiation of MYCN transcription in human neuroblastoma cell lines. Further, ALK gain-of-function mutants and MYCN synergize in transforming NIH3T3 cells. Overall, somatic mutations appear to be more aggressive than germ line mutations, implying a different impact on neuroblastoma. Further, successful application of ALK inhibitors suggests a promising future for the development of patient-specific treatments for neuroblastoma patients.