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 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.

A major goal of evolutionary developmental biology is to explore mechanisms and events underlying evolution of the myriad body plan morphologies expressed both genetically and phenotypically within the animal kingdom. Arthropods exhibit an astounding array of morphological diversity both within and between representative sub-phyla, thus providing an ideal phylum through which to address questions of body plan innovation and diversification. Major arthropod groups are recognised and defined by the distinct form and number of articulated appendages present along the antero-posterior axis of their segmented bodies. A great deal is known about the developmental genetics of limb development in the model insect Drosophila melanogaster, added to which, much comparative gene expression data and a growing body of functional genetic data is emerging for other arthropod species. Arthropod limb primordia are consistently marked by expression of the homeobox gene Distal-less (Dll), and the focus of this thesis is to compare signalling mediated by early Dll regulatory genes activity along antero-posterior and dorso-ventral embryonic axes during limb specification in Drosophila, with the activity of their orthologs in the widely disparate chelicerate, the spider mite Tetranychus urticae - interpreting new data with that available for other arthropods. Having made a detailed study of spider mite embryonic (and post-embryonic) development, to provide a basis for understanding mRNA transcription and protein activity patterns, I confirmed typical expression of Tetranychus Dll in prosomal limb primordia. I obtained limited results for the candidate antero-posterior positioning genes wingless and engrailed, although one of the two engrailed paralogs I identified is reportedly expressed in posterior segmental compartments, consistent with possible conservation of Engrailed-Wingless interactions in metameric patterning and positive regulation of Dll in arthropod limb specification. In Drosophila, wingless-dependent Dll transcription is restricted along the dorso-ventral axis by dorsal Dpp-mediated and ventral EGFR-mediated signalling gradients. Based on data from Tetranychus and other arthropods, neither dorsal nor ventral signalling regimes appear conserved outside the Drosophila system. Dll suppression in fly abdominal segments occurs due to powerful Hox (Ubx/AbdA) repression of the early Dll cis-regulatory element; this is discussed in relation to the independently evolved limbless chelicerate opisthosoma, informed by hypothetical scenarios of cis (regulatory DNA) and trans (coding sequence) evolution. Given practical difficulties and limitations encountered while working with spider mites, I offer a final assessment of the place of Tetranychus urticae as a non-model, and yet still valuable chelicerate species to consider carrying into the exciting future of evolutionary developmental biology.