spiel ohne grenzen/pou2 is required during establishment of the zebrafish midbrain-hindbrain boundary organizer

The vertebrate midbrain-hindbrain boundary (MHB) organizes patterning and n euronal differentiation in the midbrain and anterior hindbrain. Formation o f this organizing center involves multiple steps, including positioning of the MHB within the neural plate, establishment of the organizer and mainten ance of its regional identity and signaling activities. Juxtaposition of th e Otx2 and Gbx2 expression domains positions the MHB. How the positional in formation is translated into activation of Pax2, Wnt1 and Fgf8 expression d uring MHB establishment remains unclear. In zebrafish spiel ohne grenzen (s pg) mutants, the MHB is not established, neither isthmus nor cerebellum for m, the midbrain is reduced in size and patterning abnormalities develop wit hin the hindbrain. In spg mutants, despite apparently normal expression of otx2, gbx1. and fgf8 during late gastrula stages, the initial expression of pax2.1, wnt1 and eng2, as well as later expression of fgf8 in the MHB prim ordium are reduced. We show that spg mutants have lesions in pou2, which en codes a POU-domain transcription factor. Maternal pou2 transcripts are dist ributed evenly in the blastula, and zygotic expression domains include the midbrain and hindbrain primordia during late gastrulation. Microinjection o f pou2 mi can rescue pax2.1 and wnt1 expression in the MHB of spg/pou2 muta nts without inducing ectopic expression. This indicates an essential but pe rmissive role for pou2 during MHB establishment. pou2 is expressed normally in noi/pax2.1 and ace/fgf8 zebrafish mutants, which also form no MHB. Thus , expression of pou2 does not depend on fgf8 and pax2.1. Our data suggest t hat pou2 is required for the establishment of the normal expression domains of wntl and pax2.1 in the MHB primordium.