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

Heinz-Georg Belting^1,*, Giselbert Hauptmann^1,*, Dirk Meyer¹, Salim Abdelilah-Seyfried², Ajay Chitnis³, Cathrin Eschbach¹, Iris Söll¹, Christine Thisse⁴, Bernard Thisse⁴, Kristin B. Artinger⁵, Karen Lunde¹ and Wolfgang Driever^1,

¹ Albert-Ludwigs-Universität Freiburg, Institut für Biologie I, Hauptstrasse 1, D-79104 Freiburg, Germany
² Department of Physiology, UCSF, Third and Parnassus Avenue, San Francisco, CA 94143-0725, USA
³ Unit on Vertebrate Neural Development, NIH/NICHD Lab of Molecular Genetics, Building 6B, Room 3B-315, 9000 Rockville Pike, Bethesda MD 20892, USA
⁴ IGBMC, 1, rue Laurent Fries, BP163, 67404 Illkirch Cedex, France
⁵ Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
^* These authors contributed equally to this work

Author for correspondence (e-mail: driever{at}uni-freiburg.de)

Accepted August 1, 2001

The vertebrate midbrain-hindbrain boundary (MHB) organizes patterning and neuronal differentiation in the midbrain and anterior hindbrain. Formation of this organizing center involves multiple steps, including positioning of the MHB within the neural plate, establishment of the organizer and maintenance of its regional identity and signaling activities. Juxtaposition of the Otx2 and Gbx2 expression domains positions the MHB. How the positional information is translated into activation of Pax2, Wnt1 and Fgf8 expression during MHB establishment remains unclear. In zebrafish spiel ohne grenzen (spg) mutants, the MHB is not established, neither isthmus nor cerebellum form, the midbrain is reduced in size and patterning abnormalities develop within 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 primordium are reduced. We show that spg mutants have lesions in pou2, which encodes a POU-domain transcription factor. Maternal pou2 transcripts are distributed evenly in the blastula, and zygotic expression domains include the midbrain and hindbrain primordia during late gastrulation. Microinjection of pou2 mRNA can rescue pax2.1 and wnt1 expression in the MHB of spg/pou2 mutants without inducing ectopic expression. This indicates an essential but permissive 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 that pou2 is required for the establishment of the normal expression domains of wnt1 and pax2.1 in the MHB primordium.

Key words: Hindbrain, MHB, Midbrain, Isthmus, engrailed, fgf8, gbx2, otx2, pax2.1, spg, wnt1, POU domain, Danio rerio

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