Dissercting the activities of Capicua, Dorsal and Groucho in Drosophila dorsoventral patterning

Abstract

During early development of bilaterian animals, specific body structures are formed and body axes are established. The establishment of body axes requires the precise and coordinated function of signalling pathways and transcription factors that induce changes at the level of genetic material. The formation of the dorsoventral (DV) axis in the early embryo of Drosophila melanogaster is controlled mainly by the nuclear factor Dorsal (Dl), which forms a ventral to dorsal gradient and both activates and represses genes along the presumptive DV axis. In this thesis we have focused on the transcriptional repression occuring during DV axis establishment. Through genetic and biochemical analysis we have dissected the function of an additional nuclear factor, Capicua (Cic) in the repression of Dl target genes. Cic had been previously suggested to be involved in the repression of Dl targets, however this possible function was obscured by its function in DV patterning during oogenesis. During oogenesis, Cic acts as a member of the signalling pathway that establishes the formation of the Dl gradient in the embryo, thus affects both Dl-dependent activation and repression. We have found that, independently of its function in the ovary, Cic is involved in repression occuring DV patterning of the embryo, and that these two functions are carried by distinct isoforms of the protein. The main embryonic isoform, which was previously characterized, participates in DV patterning of the embryo, while the function of oogenesis is exerted by a different, yet uncharacterized isoform. Repression of genes in the DV axis depends on short enhancer elements (VRE elements), that contain Dl binding sites and AT-rich sequences that highly resemble the consensus binding site for Cic in its other targets in the early embryo. We have detected a direct interaction between the embryonic Cic protein and the AT-rich sequences adjacent to the Dl binding sites in the VRE elements of two genes repressed by Dl: zerknüllt (zen) and tolloid (tld). This interaction is of low affinity and, as a consequence, depends on the presence of Dl in the nuclei. Therefore, repression occurs only in ventral regions, where both Cic and Dl are active, distinguishing repression of DV genes from other Cic targets. Furthermore, we have shown that Cic is the key factor for the recruitment of the Groucho (Gro) corepressor to the regulatory sequences of zen. Finally, we have shown that Cic acts as a sensor of the Torso RTK pathway during repression of genes along the DV axis.

Durante el desarrollo temprano de los animales bilaterales se forman estructuras específicas y se establecen ejes corporales. El establecimiento de los ejes corporales requiere el funcionamiento preciso y coordinado de vías de señalización y factores de transcripción que producen cambios a nivel genético. En esta tesis, nos hemos centrado en la formación del eje dorsoventral (DV) en el embrión temprano de Drosophila melanogaster, que esta regulado mayoritariamente por el factor nuclear Dorsal (Dl). Nos hemos enfocado en la represión transcripcional que ocurre durante dicho proceso. Mediante análisis genéticos y bioquímicos hemos descrito el papel de otro factor nuclear, el represor Capicua (Cic) en la represión de genes dianas de Dl. Nuestro estudio demuestra una implicación directa de Cic en la represión de los genes diana de Dl. Hemos visto una unión directa de Cic en cis-elementos que están adjuntos a los sitios de unión de Dl en los promotores de los genes diana. La unión de Cic a estos sitios ocurre con baja afinidad y, como consecuencia, depende de la presencia de Dl en el núcleo. Además, hemos demostrado que Cic es el elemento clave para el reclutamiento del corepresor Groucho (Gro) a las secuencias reguladoras de los genes reprimidos en el eje DV. Por último, hemos visto que la regulación de Cic por la vía de señalización RTK Torso es el mecanismo principal de modulación de la represión de los genes diana de Dl en las extremidades del embrión.