Konstanz, Univ., Diss., 2004.

Techn. Universiẗat, Diss., 2005--München.

Drosophila insulin like peptides (DILPs) and their human homolog insulin act as messengers to control many physiological processes in the body. Fields in which insulin signaling is crucial are e.g. growth, stress responses and aging. Consequently, many diseases are caused by disturbed insulin signaling, of which diabetes is the most prominent. During the last decades the functions of insulins and their signaling pathways have been studied in detail; what remains less well understood is how the production of insulin and insulin like peptides is regulated. The family of Neprilysins (Neps) belongs to the M13-zinc ion binding metallopeptidases. Neprilysins cleave peptides that regulate a wide range of cellular processes and are therefore linked to a variety of diseases like cancer, analgesia, hypertension or Alzheimer’s disease. In the fruit fly Drosophila melanogaster, five Neprilysins are expressed; but their in vivo substrates have not yet been identified. One of the Drosophila Neprilysins, Nep4, is expressed in the CNS, in muscle tissue, in cardiac tissue and in male reproductive organs. Nep4 is expressed in two isoforms, Nep4A and Nep4B. Isoform A is composed of a short intracellular domain, a transmembrane domain and a large extracellular domain containing the catalytically active center, whereas soluble Nep4B only consists of the extracellular domain. This thesis reveals that overexpression of catalytically active Nep4A in muscle tissue leads to animals with impaired insulin expression, decreased size and weight, affected feeding behavior and reduced locomotion speed. Further phenotypes are an impaired energy metabolism and larval lethality. Knockdown of the whole enzyme or knockout of its catalytic activity also interferes with feeding and locomotion speed and, in addition, causes pupal lethality. As an explanation for the phenotypes, Nep4 mediated hydrolysis of different short neuropeptide F (sNPF) species, which were identified as novel substrates of the peptidase, is proposed. sNPF is known to regulate insulin signaling and knockdown of sNPF phenocopies the Nep4 overexpression phenotypes, which suggests that Nep4 mediated hydrolysis of sNPF regulates insulin expression in the fly. Based on these results additional regulatory peptides were identified as novel Nep4 substrates. Among them are peptides that do not only regulate insulin signaling, but also feeding behavior (Hallier et al., 2016). These findings represent good evidence that muscle bound Nep4 is key to regulate homeostasis of distinct hemolymph circulating peptide hormones. Nep4 localizing to the surface of the central nervous system is likely necessary to ensure effective ligand clearance and thus proper regulation of corresponding peptide receptors.

No description available.

Many organs and structures formed during the embryonic morphogenesis of animals derive from epithelia. Epithelia are made up of apicobasally polarized cells which adhere to and communicate with each other, allowing for epithelial integrity and plasticity. During embryonic morphogenesis, epithelia change their shape and migrate in a coordinated manner. How these epithelial processes are regulated is still not fully understood. In a forward genetic screen using the embryo of the fruit fly Drosophila melanogaster, candidate genes influencing the morphogenesis of epithelial structures were identified. Three genes, CG17364, CG17362 and CG9040 were identified as possible regulators of lumen stability in the salivary glands, tubular organs deriving from the embryonic epithelium. Furthermore, the gene Cdep was found to play a crucial role in epithelial sheet migration during dorsal closure of the embryo. Embryos carrying genomic insertions that could affect the expression of CG17364, CG17362 and CG9040 show a luminal penotype of the embryonic salivary glands characterized by alternating bloated and seemingly closed sections. Therefore, one of these genes or a combination of them likely plays a role in stabilizing the salivary gland lumen. However, neither CG17364 nor CG17362 or CG9040 contain any known protein domains, hence their molecular roles remain unknown. Cdep (Chondrocyte-derived ezrin-like protein) is a member of the FERM-FA subclass of proteins. Proteins of the FERM family have been shown to interact with the plasma membrane and membrane-bound proteins as well as cytoskeleton components. Accordingly, they have been implicated in stabilizing the cell cortex, and some of them are involved in signal transduction mechanisms. In addition to a FERM domain, Cdep also contains a RhoGEF domain, although is still not clear whether it actually exerts GEF activity. Genomic insertions in the Cdep locus cause defects in embryonic dorsal closure and atypical migratory behaviour in epithelial tubes. In order to study the molecular role of Cdep, the CRISPR/Cas9 system was employed to establish loss-of-function mutants of Cdep. The mutants show aberrations in germ band retraction, dorsal closure and head involution. Moreover, I found that two mutants carrying a premature STOP codon in the Cdep ORF, CdepE16X and CdepG17X, rescue the defects observed in embryonic cuticles mutant for two other FERM-FA members yurt (yrt) and coracle (cora). A deletion of the full Cdep ORF did not rescue those defects. I hypothesize that CdepE16X and CdepG17X encode Cdep variants with increased activity, which compensates for the loss of yrt or cora function, respectively. In conclusion, this leads to a model in which Cdep acts in parallel to Yrt and Cora during Drosophila embryonic morphogenesis. Many of the defects described in this study are reminiscent of phenotypes found in embryos mutant for components and downstream effectors of the Jun-N-terminal Kinase (JNK) pathway. Hence, my work supports an earlier hypothesis according to which a mouse homologue of Cdep, Farp2, acts as an upstream activator of the JNK pathway during epithelial cell migration in vitro (Miyamoto et al., 2003) The data provided here shows that Cdep plays a role in the morphogenesis of a great number of epithelia-derived organs and structures in vivo. My study therefore elucidates a missing link between cell migration cues and JNK pathway activation.

No description available.

Morphogens are secreted signalling molecules that are expressed in restricted groups of cells within the developing tissue. From there, they are secreted and travel throughout the target field and form concentration gradients. These concentration profiles endow receiving cells with positional information. A number of experiments in Drosophila demonstrated that the morphogen Decapentaplegic (Dpp) forms activity gradients by inducing the expression of several target genes above distinct concentration thresholds at different distances from the source. This way, Dpp contributes to developmental fates in the target field such as the Drosophila wing disc. Although the tissue distribution as well as the actual shape and size of the Dpp morphogen concentration gradient has been visualized, the cell biological mechanisms through which the morphogen forms and maintains a gradient are still a subject of debate. Two hypotheses as to the dominant mechanism of movement have been proposed that can account for Dpp spreading throughout the Drosophila wing imaginal target tissue: extracellular diffusion and planar transcytosis, i. e. endocytosis and resecretion of the ligand that is thereby transported through the cells. Here, I present data indicating that implications of a theoreticalanalysis of Dpp spreading, where Dpp transport through the target tissue is solely based on extracellular diffusion taking into account receptor binding and subsequent internalization, are inconsistent with experimental results. By performing Fluorescence Recovery After Photobleaching (FRAP) experiments, I demonstrate a key role of Dynamin-mediated endocytosis for Dpp gradient formation. In addition, I show that most of GFP-Dpp traffics through endocytic compartments at the receiving epithelial cells, probably recycled through apical recycling endosomes (ARE). Finally, a Dpp recycling assay based on subcellular photouncage of ligand is presented to address specifically the Dpp recycling event at the receiving cells.

Morphogens are signaling molecules that play a key role in animal development. They spread from a restricted source into an adjacent target tissue forming a concentration gradient. The fate of cells in the target tissue is determined by the local concentration of such morphogens. Morphogen transport through the tissue has been studied in experiments which lead to the suggestion of several transport mechanisms. While diffusion in the extracellular space contributes to transport, recent experiments on the morphogen Decapentaplegic (Dpp) in the fruit fly Drosophila provide evidence for the importance of a cellular transport mechanism that was termed "planar transcytosis". In this mechanism, morphogens are transported through cells by repeated rounds of internalization and externalization. Starting from a microscopic theoretical description of these processes, we derive systems of nonlinear transport equations which describe the interplay of transcytosis and passive diffusion. We compare the results of numerical calculations based on this theoretical description of morphogen transport to recent experimental data on the morphogen Dpp in the Drosophila wing disk. Agreement with the experimental data is only achieved if the parameters entering the theoretical description are chosen such that transcytosis contributes strongly to transport. Analyzing the derived transport equations, we find that transcytosis leads to an increased robustness of the created gradients with respect to morphogen over-expression. Indications for this kind of robustness have been found in experiments. Furthermore, we theoretically investigate morphogen gradient formation in disordered systems. Here, an important question is how the position of concentration thresholds can be defined with high precision in the noisy environment present in typical developing tissues. Among other things, we find that the dimensionality of the system in which the gradient is formed plays an important role for the precision. Comparing gradients formed by transcytosis to those formed by extracellular diffusion, we find substantial differences that may result in a higher precision of gradients formed by transcytosis. Finally, we suggest several experiments to test the theoretical predictions of this work.

No description available.

No description available.