Homeobox-containing genes in the nemertean "Lineus" : key players in the antero-posterior body patterning and in the specification of the visual structures

One of the most important breakthroughs in the field of developmental biology
has been the discovery of the homeobox and of its widespread phylogenetic
conservation. Many homeobox-containing genes encode transcription factors that
regulate gene expression during important developmental processes, such as patterning
and cell differentiation. Not only their sequences, but often also their expression
patterns and their functions are conserved throughout bilaterian animals. Despite
specific knowledge from selected model organisms, which belong to the
Deuterostomia and the Ecdysozoa, an unified view about the evolutionary conserved
developmental mechanisms requires more investigations from the Lophotrochozoa,
the third clade of Bilateria, which has been neglected, so far.
We have worked with nermerteans, also called ribbonworms, which are
members of the Lophotrochozoa. Because of its evolutionary position, its relative
simplicity and impressive developmental plasticity, Lineus sanguineus, a marine
ribbonworm from the class Anopla, is an attractive system to investigate the
specification of the body plan and the mechanism by which differentiated cells
maintain or reprogram their identity in a context-dependent manner. In this thesis,
Lineus was used as a model system in an attempt to reveal to which extent the rostral/
caudal specification of the antero-posterior axis and the eye specification network are
conserved throughout the Bilateria.
Although the Hox genes play important roles in the antero-posterior
specification of the bilaterian body, the most rostral and the most caudal regions of
the embryo are specified by orthodenticle-like (Otx) and caudal-like (Cdx),
respectively. To test whether this is also the case in Lophotrochozoa, we first have
characterized the full-length Ls-Otx and Ls-Cdx genes. Then, we have shown that
expression patterns in developing and adult Lineus suggest an involvement of Otx in
the development, the specification and the maintenance of the anterior sensory
structures and anterior brain regions. This is in good agreement with the proposed
conserved functions of Otx among Bilateria. Similarly, the restriction of Ls-Cdx
expression at the posterior extremity of the developing Lineus larva suggest that the
presumed conserved role of Cdx in the specification of the posterior end of bilaterian
embryos could be conserved in Lineus. Additionally, we have studied both, the
expression patterns and the variation of expression levels of Ls-Otx and Ls-Cdx
during regeneration. This has revealed that Ls-Cdx is specifically up-regulated, during
posterior regeneration, only, whereas Ls-Otx is up-regulated during both, anterior and
posterior regeneration. The Ls-Otx expression becomes restricted to the anterior
regenerating blastema only one week after the onset of regeneration. As it has been
suggested that the CNS plays a crucial role in nemertean regeneration and as Ls-Otx is
specifically expressed at the tip of the sectioned nerve cord of the early regenerating
stages, we propose that Ls-Otx could be part of a signaling network responsible for
the onset of regeneration. Additional information has been obtained from Lineus
lacteus, a close relative of Lineus sanguineus, which does not exhibit the same
regeneration capacities. In the light of the expression pattern of Otx in amputated
Lineus lacteus, we propose that the differences in regeneration capacities between
nemertean species could rely on the differences in the capacity of their differentiated
cells to de-differentiate in response to signals emitted from Otx expressing cells of the
nerve cord, rather than in the capacity to emit the signals leading to the onset of
regeneration.
In a second project, we have investigated the specification of the visual
structures in L.sanguineus. Studies in Drosophila and vertebrates have revealed that a
combinatorial expression of members of the evolutionary conserved “eye
specification network” specify the eye field. The key members of this eye
specification network are the Pax-6, Six, Eyes absent and Dachshund genes. We
wanted to know whether this network is involved in the development, maintenance
and regeneration of the Lineus eyes. At the beginning of this PhD work, it was already
known that LsPax-6 is expressed in the developing eye field in Lineus. In addition, we
had reported that its inactivation by RNA-mediated gene interference (RNAi) in an
adult L.sanguineus leads to the disappearance of the adult eyes. To further investigate
the specification of the Lineus eyes, we have characterized three Six genes, LsSix1/2,
LsSix3/6 and LsSix4/5. Their expressions, especially the one of LsSix1/2, suggest an
involvement in the development and the regeneration of the Lineus eyes. In addition,
we have observed a cross-reaction of a Drosophila antibody anti-dachshund with the
developing Lineus eyes. Taken together, these data support the idea that the “eye
specification network” could be conserved in nemerteans. This molecular unity
underlying eye specification in all bilaterian clades strongly supports the hypothesis
of a monophyletic origin of the eyes.