| Abstract |
Molecular genetic analysis in model organisms, including nematodes ( Caenorhabditis), insects ( Drosophila) and vertebrates (mouse), have revealed that key aspects of body patterning are regulated by similar developmental genes and genetic circuits in these diverse animals. The developmental function and evolution of these conserved genes and regulatory pathways has provided a framework for studying morphological evolution. Arthropods (insects, crustacea, myriapods, chelicerates) are favored as testing ground for evolutionary-developmental comparisons, because of their incredible diversity, our in-depth knowledge of Drosophila and their relatively well-known phylogenies. Such studies have relied so far on the isolation and comparison of homologous genes and their expression patterns among different species, together with their functional comparison, albeit exclusively in Drosophila. This approach has produced valuable information concerning the molecular mechanisms that underlie morphological diversity. However, it does not allow functional analysis in non-model species, thus precluding the isolation of new genes or the identification of new gene functions. The establishment of broadly applicable genetic transformation systems allows manipulations in species, that have not been genetically tractable in the past. The first part of this thesis describes the application of transgenic technology to two non-model arthropod species, the red flour beetle Tribolium castaneum and, for the first time, to a crustacean species, the amphipod Parhyale hawaiensis. The genetic transformation of these species was achieved by using the transposable element Minos, combined with a set of versatile genetic tools including a selection of marker genes and transposase sources. A number of quantitative and qualitative parameters were examined, that confirmed the specificity of Minos transposition mechanism and its utility as a transformation vector. Although the protocols for microinjections and breeding varied considerably, depending on the lifestyle and mode of development of each organism, transformation efficiencies were high in both species. The second part of the thesis describes the use of transgenic technology for the functional comparison of cis-regulatory elements from the Drosophila Distal-less gene in Tribolium and Parhyale. Given that Distal-less displays a conserved role in the formation of animal limbs, this study aimed to decipher the molecular changes that are responsible for the evolution of arthropod limb number. In addition, the latter section describes the isolation and characterization of active cis-regulatory sequences from Parhyale hsp70 genes, that will be used in the near future to conditionally mis-express genes of interest in the amphipod.
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Ανάπτυξη και Εφαρμογή της Διαγονιδιακής Τεχνολογίας στην Εξελικτική Αναπτυξιακή Βιολογία Αρθροπόδων
