Integrity of the midbrain region is required to maintain the diencephalic-mesencephalic boundary in zebrafish no isthmus/pax2.1 mutants.

Initial anterior-posterior patterning of the neural tube into forebrain, midbrain, and hindbrain primordia occurs already during gastrulation, in response to signals patterning the gastrula embryo. After the initial establishment, further development within each brain part is thought to proceed largely independently of the others. However, mechanisms should exist that ensure proper delineation of brain subdivisions also at later stages; such mechanisms are, however, poorly understood. In zebrafish no isthmus mutant embryos, inactivation of the pax2.1 gene leads to a failure of the midbrain and isthmus primordium to develop normally from the gastrula stage onward (Lun and Brand [1998] Development 125:3049-3062). Here, we report that, after the initially correct establishment during gastrulation stages, the neighbouring forebrain primordium and, partially, the hindbrain primordium expand into the misspecified midbrain territory in no isthmus mutant embryos. The expansion is particularly evident for the posterior part of the diencephalon and less so for the first rhombomeric segment, the territories immediately abutting the midbrain/isthmus primordium. The nucleus of the posterior commissure is expanded in size, and marker genes of the forebrain and rhombomere 1 expand progressively into the misspecified midbrain primordium, eventually resulting in respecification of the midbrain primordium. We therefore suggest that the genetic program controlled by Pax2.1 is not only involved in initiating but also in maintaining the identity of midbrain and isthmus cells to prevent them from assuming a forebrain or hindbrain fate.