Linking the embryonic clock with temporal collinearity of Hox gene activation

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Somitogenesis is a crucial process in vertebrate embryo development, whereby mesodermal-derived structures, called somites, are repeatedly formed along the anteriorposterior(AP) body axis. A molecular clock, first evidenced by cyclic expression of the hairy1 gene, sets the pace of somite formation. Although somites are morphologically identical, they are actually molecularly different. The identity of each embryonic segment is determined by combinatorial expression of hox genes. Previous studies showed that Hairy1 protein interacts with the mesodermal identity protein Brachyury, which is also required for hox gene activation. To assess if the embryonic clock is timing hox gene activation, hairy1 and brachyury were cloned into an expression vector containing a fluorescent reporter gene. The resulting plasmids were subsequently electroporated in chick embryos at stage 3HH-4HH and the embryos incubated for further development. Phenotype analysis of electroporated embryos suggests that over-expression of either hairy1 or Brachyury genes delays or even prevents somite formation, to approximately the same extent. Furthermore, when the C-terminal domain of Hairy1, capable of interacting with Brachyury, was over-expressed, a dosedependent phenotype was obtained, suggesting that it resulted from Brachyury protein titration. These results strongly suggest that cyclic Hairy1 - Brachyury dimer formation is required for somite segmentation.