“GENETIC CONTROL OF PINEAL ORGAN DEVELOPMENT: functional analysis of the homeobox gene Xbsx”

The pineal organ of all Vertebrates is capable of producing and releasing melatonin rhythmically. In non-mammalian vertebrates such as fish, reptiles, and amphibia, the pineal complex is a photosensitive structure and it serves as a central pacemaker able to translate directly light information into endogenous signals. This property is due to pineal photoreceptors that express photopigments and contain all the molecular components of the clock, representing thus a circadian system. This project focused on the molecular pathways involved in pineal complex development. Our purpose was to cast light on the factors essential to its development and to investigate their relationship with the day and night cycle using the frog Xenopus laevis as model system. To study the genetic network implicated in the control of pineal organ development we isolated an homeobox gene named Brain Specific homeobox (Bsx). This gene is evolutionarily conserved, and it has been isolated in Drosophila, human, mouse and frogs. Specifically, we observed that in Xenopus laevis, the Xbsx gene can be considered a very early marker of the neural pineal fate commitment, being expressed as early as the pineal territory is defined. At later stages of development, Xbsx is not expressed in proliferating cells but only in differentiated pineal photoreceptors with a cyclic rhythm of expression, displaying high levels during the dark and very low levels during the light period. However, the absence of light seems to prevent the Xbsx expression, which remains low and similar to its normal day-time levels. We found that Xbsx plays a role both in control the cell proliferation rate during pineal development and in pinealocytes differentiation during embryonic pineal organ development. In fact, our data indicate that, in the absence of Xbsx, photoreceptor precursors fail to exit cell cycle and eventually die. On the contrary, Xbsx overexpression promotes cell cycle exit, generating a higher number of photoreceptors. Finally, we investigated if embryonic pineal organ development was completely uncoupled from the day–night cycle, since circadian rhythms of cell cycle have been reported, in several tissues, such as oral and intestinal epithelia, bone marrow and in zebrafish skin from the larval to adult stages. In agreement with these findings indicating a circadian control of cell cycle, we observed that pineal cell proliferation during embryonic development is characterized by cyclic fluctuations in Xenopus embryos, reaching a maximum rate of proliferating cells toward the end of the light period. This mechanism would allow to coordinate proliferation and differentiation of neurons in the pineal organ. As a support to this hypothesis, we observed that the functional ablation of Xbsx is able to affect this rhythm of pineal cell cycle progression exclusively in a restricted temporal window, in particular only during the dark period. These data suggest that Xbsx, being a gene expressed rhythmically in a LD cycle and necessary to pineal photoreceptor differentiation, could be also necessary to allow cell proliferation and differentiation to occur at a certain time of the day