Novel role for SOX2 in the development of the zebrafish epithalamus
The sex determining region Y-box 2 (sox2) gene is one of the most important transcription factors during development, particularly the development of the central nervous system (CNS). It is expressed in embryonic stem cells and later in neural stem cells, where it modulates their maintenance and differentiation. In humans, heterozygous mutations are associated with eye malformations, including anophthalmia and severe microphthalmia. Also, a subset of patients has extra-ocular phenotypes, such as hearing loss, seizures and pituitary hypoplasia. Although the roles of sox2 in embryonic stem cells and eye development are well studied, the function of sox2 in brain development and disease is still elusive. The aim of this project was to characterize a novel role for sox2 in the development of zebrafish epithalamus, which was identified from an in silico screen previously performed in our laboratory. The zebrafish epithalamus, located in the dorsal diencephalon, consists of three main structures: the pineal gland, the parapineal organ and the habenular nuclei. The pineal gland, also known as epiphysis, is a photoreceptive (in zebrafish) and neuroendocrine organ that detects light and rhythmically produces melatonin in order to regulate the circadian rhythms. The parapineal organ is located to the left side of the pineal gland and is important for the elaboration of the asymmetries observed between the left and right habenular nuclei. Finally, the bilateral habenulae are part of the dorsal diencephalic conduction system that links the forebrain with the mid- and hindbrain. The left and right habenulae show both molecular and neuroanatomical asymmetries, including differences in neuropil organization, in levels of gene expression and in the morphology and connectivity of their neurons’ projections. The relatively simple architecture of the pineal gland and the asymmetric character of the habenulae provide a useful tool for studying cell-fate determination, cell migration and establishment of brain asymmetries. In this study, we used zebrafish as a model to dissect the novel functions of sox2 in the development of the epithalamus. We showed that sox2 works synergistically with Notch pathway to negatively regulate neurogenesis within the pineal gland. The pineal gland consists of only two cell types: the photoreceptors and the projection neurons. Previous studies showed that the Notch and BMP pathways are important for the proper specification of these cells. Here, we show that sox2 normally inhibits the photoreceptor cell fate, whereas it has no effect on the number of projection neurons. Therefore, sox2 complements Notch and BMP pathways in cell-fate determination within the pineal gland. In addition, downregulation of sox2 results in abnormal parapineal organ development and disruption of the asymmetric architecture of the habenulae. A subset of sox2 morphant embryos develops right-sided parapineal organs, which is consistent with abnormal bilateral expression of the Nodal gene, pitx2 (paired-like homeodomain transcription factor 2). Also, timelapse experiments showed that migration of the parapineal cells is defective, resulting in scattered cells. The aberrant parapineal development leads to disorganization of the habenular nuclei, as shown by the abnormal neuropil arrangement and the expression of the asymmetric marker kctd12.1 (potassium channel tetramerisation domain containing 12.1).