| Abstract |
The main goal in the first part of this thesis was to develop a site specific recombination
system in the arthropod crustacean Parhyale hawaiensis in order to exploit the recently developed
transformation and gene trapping technology for functional comparative studies in arthropods. For
that reason, I chose the PhiC31 integration system, which has been successfully used in
Drosophila and in human cell lines. The PhiC31 bacteriophage inserts its genome into the host’s
DNA by using specific recombination between two sites: attB (bacterial attachment point) and attP
(phage attachment point).The recognition and recombination of these sites is catalysed by a site
specific integrase, encoded by the phage’s genome.
First, I created transgenic amphipods carrying the attP site and tested the possibility of
introducing a heat inducible reporter gene through site specific recombination. After confirming that
specific recombination occurs in Parhyale, I replaced the fluorescent marker gene of a gene trap.
The conversion of a DsRed trap (distal) to a GFP expressing trap was highly efficient, suggesting
that the integrase system could be used as a means of routinely converting gene traps into various
genetic tools in Parhyale hawaiensis.
The gene trapping and trap conversion constructs used in this study incorporate elements
with proven activity in different species (3xP3, fluorescent proteins, PhiC31 integrase, splice
acceptor sequence). Therefore, these same constructs could serve as a universal platform for
gene trapping and trap conversion in diverse organisms.
In the second part of the thesis, done in collaboration with the postdoctoral researcher Dr.
Tasos Pavlopoulos from the University of Cambridge, we studied the role of the homeotic gene
Ubx on appendage development and specialisation in crustaceans. The diversity of appendage
morphology seen in crustaceans today, is the outcome of the evolution of an ancient homogenous
trunk into specialised regions adapted for distinct functions. Various genetic mechanisms that
could produce this diversity have been proposed, but functional evidence to test many of these
hypotheses is still missing. The evolution of maxillipeds, which are thoracic limbs differentiated for
feeding in crustaceans, represents one such example. Based on the correlation between the
absence of the homeotic gene Ubx and the development of maxillipeds in several crustacean
species, Averof and Patel proposed in 1997 that a shift of Ubx expression from anterior thoracic
segments could serve as a mechanism for the specification of the maxilliped fate.
The establishment of heat inducible gene expression in Parhyale hawaiensis gave us the
opportunity to test this hypothesis directly in the amphipod by ectopically expressing Ubx in the first
thoracic appendages, that normally develop into maxillipeds. Our results show that Ubx is sufficient
to induce the transformation of maxillipeds into thoracic limbs, while complementary experiments
of Ubx repression (from the laboratory of NH Patel) indicate its necessity to define thoracic identity.
To explain the unexpected transformation of the maxillary appendages into maxillipeds by
ectopic expression of Ubx, we studied the effect of Ubx on another homeotic gene Sex combs
reduced (Scr), showing that this transformation was an indirect effect of the Ubx missexpression.
Finally, this study allowed us to describe a large range of partial transformations of the maxillary
appendages towards the thoracic fate, indicating that changes in the expression of a homeotic
gene (like Ubx) can provide a great morphological diversity for natural selection to act upon.
|
Ανάπτυξη και εφαρμογή γενετικών εργαλείων στο αρθρόποδο καρκινοειδές Parhyale hawaiensis
