Characterization of the Hox patterning genes in acoel flatworms

[eng] One of the main issues in animal evolution deal with the transition from radial organisms (Cnidaria and Ctnenophora), with only one axis of symmetry, the Oral-Aboral (OA) axis, to bilateral organism (Bilateria), bearing two orthogonal body axes, the Antero-posterior (AP) and the Dorso-ventral (DV) axis. Finding the extant bilateral organism closest to the bilaterian ancestor is the first and necessary step to open new ways of analysis. Recent molecular phylogenies have convincingly shown that the acoel flatworms, traditionally classified within the turbellarian Platyhelminthes, are the sister group of the remaining Bilateria, branching out before the common ancestor of protostomes, and deuterostomes. Hox and ParaHox genes encode for transcriptional regulators involved in the control the AP body axis in all bilateral animals. Hox genes are usually organized in clusters in Bilateria, which have been originated by means of several gene tandem duplications from an original ProtoHox gene. In addition, Hox genes show a collinear correspondence between gene order within the cluster and the body levels at which these genes are expressed. On the contrary, in the phylogenetic sister group of Bilateria, the Cnidaria, Hox genes are not linked in a single cluster and do not seem to play a similar role for patterning the OA axis. Since it is still unclear when in the evolutionary history of bilaterians the Hox system first conferred positional identity along the AP-axis, the comparative study of the patterning genes Hox and ParaHox in acoel flatworms, could be crucial to understand the origin of the Hox-ParaHox axial patterning system and how the morphological transition from radial to bilateral animals took place. In this Thesis, we report on the cloning, genomic arrangement, and expression domains of Hox genes in the acoel species Symsagittifera roscoffensis. Three Hox genes were detected: one from each of the major groups of Hox genes, which are anterior, central, and posterior, named SrHox1, SrHox5 and SrHoxPost respectively. All acoel species studied to date contain the same minimal complement of three Hox genes and one Cdx ParaHox gene, suggesting that the last common bilaterian ancestor (or Urbilateria) had a simple Hox gene complement, composed of only 3 or 4 genes. In bacterial artificial chromosome cloning, sequencing, and chromosomal fluorescence in situ hybridization, Hox genes were not observed as being clustered in a unique genomic region in S. roscoffensis. Nevertheless, despite its dispersion within the genome, Hox genes are expressed in nested domains along the AP axis in the juvenile worm. The basic set of Hox genes in acoels and their coarse nested spatial deployment might be the first indicators of the role of Hox genes in the evolution of bilateral symmetry and AP positional identity from a hypothetical radial ancestor. In order to understand how the AP axis has been established over evolutionary time, the execution of functional analyses is essential. With this purpose, we have performed the knockdown of the posterior Hox, IpHoxPost, during the postembryonic development, regeneration and adulthood of the acoel species Isodiametra pulchra, using RNA interference technologies. The analysis has been done for the first time in acoels, and we demonstrate that the functions of this gene are restricted to the posterior region of the animal, within the muscular and neural tissues. We conclude, therefore, that the posterior Hox genes were used to specify and maintain defined anatomical regions within the AP axis of animals since the beginning of bilaterian evolution.[spa] "Caracterización de los genes Hox en el acelo Symsagittifera roscoffensis". Los genes Hox codifican factores de transcripción que regionalizan el eje antero-posterior durante el desarrollo embrionario en todos los animales bilaterales estudiados. Los animales radiales (cnidarios y ctenóforos) poseen genes Hox, pero estos no desempeñan un rol similar al de sus homólogos en Bilateria, por lo que el sistema de regionalización Hox puede ser considerado una innovación de los Bilateria. Recientes análisis filogenéticos han demostrado que Acoelomorpha (acelos y nemertodermátidos), un grupo de gusanos clasificados tradicionalmente como platelmintos, divergieron antes del último antecesor común de protóstomos y deuteróstomos. En consecuencia, representan el grupo de organismos bilaterales idóneo para estudiar la evolución del sistema Hox entre cnidarios y bilaterales. Por este motivo, el objetivo principal de esta tesis ha sido analizar el sistema Hox en acelos. Encontramos un complemento simple de 3 genes Hox en las 2 especies de acelos estudiadas: Symsagittifera roscoffensis e Isodiametra pulchra. Estos genes no están ligados en el genoma de S. roscoffensis pero se expresan de forma colinear durante el desarrollo postembrionario, lo que representa el primer ejemplo de expresión colinear de genes Hox en Bilateria, indicando que la colinearidad estuvo presente en el ancestro de todos los Bilateria. Las funciones del Hox posterior fueron analizadas mediante RNA de interferencia en I. pulcra. El fenotipo knockdown indica que IpHoxPost está implicado en la regulación del establecimiento de las estructuras morfológicas en la parte posterior, especialmente de los músculos bucales y los situados alrededor de los aparatos copuladores; así como en el proceso de maduración de los huevos y la proliferación celular. Esto indica que el rol del Hox posterior en la regulación del desarrollo y diversificación del mesodermo postembrionario y la musculatura ha surgido tempranamente durante la evolución de los Bilateria