Abstract
Abstract
Otx genes in Vertebrates are homologs of the Drosophila gene orthodenticle (otd) and play a very important role in the specification of the
anterior Central Nervous System. Studies on these genes have been performed in mice, where Otx1 and Otx have been well characterized. Subsequently Otx3 has been isolated in Zebrafish, and Xotx1, Xotx2, Xotx4 and Xotx5 in Xenopus
laevis. In mammals otx5 does not exist, but there is another otx family gene,
named crx (cone-rod homeobox), for which the relation with otx5 has not been definitely cleared. Crx is involved in eye development and mutations in this gene lead to several retina diseases, that include Leber congenital amaurosis (a severe childhood degeneration that affects cones and rods), cone and rod dystrophy (an adult onset disease) and retinitis pigmentosa.
In this thesis work we concentrate our attention on Xotx2 and Xotx5 genes in Xenopus in a research project aimed at explaining their role in eye development. Like all otd/otx class genes, Xotx2 and Xotx5 contain a bicoid class homeodomain, characterized by a specific Lysin residue in position 50, (corresponding to position 9 on the DNA binding helix) that is crucial for their DNA binding specificity. Experiments in mice have shown that Otx2 is expressed in the whole epiblast of the early embryo. Later on, Otx2 expression is restricted to most anterior regions that include the neuroectoderm corresponding to telencephalic, diencephalic and mesencephalic regions. This
gene is very important, and in fact Otx2-/- knockout mice die early in
embryogenesis because of total loss of the anterior CNS structures and general malformations on the body plan (caused by abnormal movements during gastrulation). In Xenopus embryogenesis, Xotx2 expression is similar to Otx2 expression in mice; Otx2 and this gene is expressed in the Spemann’s Organizer region and later in CNS rostral regions, including the eye.
Abstract
Xotx5 expression in the early embryo is similar to that of Xotx2. It is
expressed in the Spemann’s Organizer region at early gastrula and later also in anterior neuroectoderm. After the neural tube closure its expression is found only in the eye e in the pineal gland, both photorecepting structures. Xotx5 overexpression experiments have shown effects similar to Xotx2 overexpression, producing reduction of posterior structures and anterior and neural tissue induction in the whole embryo.
The expression of these genes in eye development led to new experiments that have shown the key role of these genes in eye differentiation. In fact Xotx5 expression, during eye development, becomes more restricted to both bipolar and photoreceptor cells as Xotx2 expression becomes restricted to bipolar cells only. Lipotransfection experiments of retinal progenitors with constructs for the two genes have shown that Xotx2 leads progenitors through a bipolar cell fate, while Xotx5 leads through photoreceptor cell fate. The aim of this thesis has been to analyze the importance of the C-Terminal portions of these transcription factors. In order to do this, we created by PCR various C-terminal deleted Xotx2 and Xotx5 constructs. Then their mRNA was then transcribed “in vitro” and microinjected in Xenopus embryos at 4 cells stage. Embryos were then characterized by the observed phenotype and analyzed with appropriate molecular markers to evidentiate ectopic structures induced by the microinjection. These constructs analysis is preliminary to further retinal progenitor lipotransfection experiments; the aim is to identify which are the essential functional domains of Xotx2 and Xotx5 for their action on cell type specification in retina.
