Genetic control of brain morphogenesis through Otx gene dosage requirement

Understanding the genetic mechanisms that control patterning of the vertebrate brain represents a major challenge for developmental neurobiology. Previous data suggest that Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, might both contribute to brain morphogenesis. To gain insight into this possibility, the level of OTX proteins was modified by altering in vivo the Otx gene dosage. Here we report that Otx genes may cooperate in brain morphogenesis and that a minimal level of OTX proteins, corresponding either to one copy each of Otx1 and Otx2, or to only two copies of Otx2, is required for proper regionalization and subsequent patterning of the developing brain. Thus, as revealed by anatomical and molecular analyses, only Otx1-/-; Otx2+/- embryos lacked mesencephalon, pretectal area, dorsal thalamus and showed an heavy reduction of the Ammon's horn, while the metencephalon was dramatically enlarged occupying the mesencencephalic area. In 8.5 days post coitum (d.p.c.) Otx1-/-; Otx2+/- embryos, the expression patterns of mesencephalic-metencephalic (mes-met) markers such as En-1 and Wnt-1 confirmed the early presence of the area fated to give rise to mesencephalon and metencephalon while Fgf-8 transcripts were improperly localized in a broader domain. Thus, in Otx1-/-; Otx2+/- embryos, Fgf-8 misexpression is likely to be the consequence of a reduced level of specification between mes-met primitive neuroepithelia that triggers the following repatterning involving the transformation of mesencephalon into metencephalon, the establishment of an isthmic-like structure in the caudal diencephalon and, by 12.5 d.p.c., the telencephalic expression of Wnt-1 and En-2. Taken together these findings support the existence of a molecular mechanism depending on a precise threshold of OTX proteins that is required to specify early regional diversity between adjacent mes-met territories and, in turn, to allow the correct positioning of the isthmic organizer.