The Identification and the Functional Validation of Eye Development and Regeneration Genes in Schmidtea Mediterranea

Abstract

[eng] Discovering the master genes necessary to build the eye in an invertebrate model such as S. mediterranea could help us to understand numerous retinopathies and age-related degeneration of the human eye. The aim of this study was to select and determine the functional activity of genes involved in the regeneration and development of the S. mediterranea eye. Gene ontology was the tool used to select the genes; while RNA interference and RNA hybridization provided the first approach towards establishing possible roles in the eye development process. From 45 genes studied, silencing of five opsins, Smed-rhodopsin1, Smed-rhodopsin2, Smed-rhodopsin10, Smed-melanopsin2 and Smed-melanopsin3; four transcription factors, Smed-exd (-pbx), Smed-hox2, Smed-mitf2 and Smed-mitf3; and two ABC transporters, Smed-white and Smed-mrp1-2, specifically inhibited normal eye regeneration in S. mediterranea. This suggested they are relevant for or involved in phototransduction, eye pigment biosynthesis, photoreceptor microtubule transport or eye developmental processes. Smed-melanopsin2 and Smed-melanopsin3 RNAi affected the regeneration of pigment cells and skin pigmentation. Smed-melanopsin3 RNAi produced misrouted photoreceptor axonal fibres and fused pigment cups. 13 out of the 14 opsin or light sensor genes studied had ubiquitous mRNA expression throughout the body, which might be explained by the presence of a dermal light sense, as observed in other flatworms. These proteins might serve as photoentrainment regulators or as photolyases responsible for the repair of photodamaged DNA. Smed-hox1 RNAi inhibited the proper development of anterior structures. Smed-VATP synthase, Smed-kinase, Smed-dye and Smed-centrin transiently inhibited eye regeneration before the inhibition of whole body regeneration. This was followed by death, which might indicate the existence of a photoreceptor ATP synthesis and microvilli transport specificity; or also the importance of these genes in cell survival. Smed-peropsin1, Smed-yy1, Smed-hmt, Smed-tpr1, Smed-tpr2, Smed-kinase, Smed-mrp1-2 and Smed-hox1 have eye expression patterns. Additionally, Smed-hox1 and Smed-mrp1-2 also present inhibition of anterior structures. Smed-white RNAi produced hypopigmented, fused eye cups and photoreceptor cell size reduction. This was probably induced via the damaged pigment cells, due to the dependence between these two cell types. Smed-melanopsin1, Smed-ver and Smed-hox2 have brain lobes-like expression patterns. These RNA sequences might be signalling neuronal cells. Smed-tpr1 mRNA injection induced rapid pharynx autotomy, also provoked by Smed-ver and Smed-white-sf2. All three are melanin biosynthesis-related genes. This ejection suggests the existence of an additional natural mechanism of tissue release, probably provoked for the action of a small amino acid. For the first time, the rabdomeres of the Schmidtea mediterranea eye have been photographed using electron microscopy. These structures are very similar to their homologues in other flatworms.

[spa] El objetivo de este estudio ha sido encontrar genes implicados en la regeneración y por consiguiente en el desarrollo de los ojos de S. mediterranea. Para ello se han usado herramientas informáticas de selección de genes homólogos de otras especies, la interferencia con RNA mensajero y la hibridación in situ de mRNA. De los 45 genes estudiados, el RNAi de cinco opsinas Smed-rhodopsin1, Smed-rhodopsin2, Smed-rhodopsin10, Smed-melanopsin2 y Smed-melanopsin3; cuatro factores de transcripción Smed-exd (-pbx), Smed-hox2, Smed-mitf2 y Smed-mitf3; junto con dos transportadores ABC Smed-white y Smed-mrp1-2, inhiben la regeneración normal de los ojos de S. mediterranea. Aunque ninguno de estos genes se expresa en los ojos, la expresión ubicua del mRNA de las opsinas por todo el cuerpo de la planaria podría indicar la presencia de un sentido fotodermal previamente observado en otras planarias. Los mRNA de los genes Smed-peropsin1, Smed-yy1, Smed-hmt, Smed-tpr1, Smed-tpr2, Smed-kinase, Smed-hox1 y Smed-mrp1-2 se expresan en los ojos. La interferencia con RNA de los tres últimos provoca inhibición de las estructuras anteriores. Los genes Smed-kinase, Smed-VATP synthase, Smed-dye y Smed-centrin inhiben la regeneración de los ojos antes de provocar una destrucción celular masiva que desemboca en la muerte del animal. El RNAi de los genes Smed-white, Smed-melanopsin2 y Smed-melanopsin3 afecta a la pigmentación de la piel y en Smed-melanopsin3, además, a las rutas que los axones de los fotoreceptores siguen hasta llegar a los ganglios cerebrales. En los RNAi de Smed-white las células pigmentarias no desaparecen pero cambian de posición, se fusionan en el centro de la cabeza. Smed-melanopsin1, Smed-ver y Smed-hox2 tienen patrones de expresión parecidos a los ganglios cerebrales. RNAi de tres genes implicados con la biosíntesis de pigmentos, Smed-tpr1, Smed-ver y Smed-white-sf2, inducen la autotomía de la faringe. Efecto que sugiere la existencia de un mecanismo natural de expulsión de este órgano. Por primera vez los rabdómeros del ojo de Schmidtea mediterranea han sido fotografiados con microscopía electrónica, siendo éstos similares a los de otras planarias.