The caudal limit of Otx2 expression positions the isthmic organizer

Abstract

The homeobox gene Otx2 is expressed in the anterior neural tube with a sharp limit at the midbrain/hindbrain junction (the isthmic organizer)1. Otx2 inactivation experiments have shown that this gene is essential for the development of its expression domain2. Here we investigate whether the caudal limit of Otx2 expression is instrumental in positioning the isthmic organizer and in specifying midbrain versus hindbrain fate, by ectopically expressing Otx2 in the presumptive anterior hindbrain using a knock-in strategy into the En1 locus. Transgenic offspring display a cerebellar ataxia. Morphological and histological studies of adult transgenic brains reveal that most of the anterior cerebellar vermis is missing, whereas the inferior colliculus is complementarily enlarged. During early neural pattern formation expression of the midbrain markers Wnt1 and Ephrin-A5, the isthmic organizer markers Pax2 and Fgf-8 and the hindbrain marker Gbx2 are shifted caudally in the presumptive hindbrain territory. These findings show that the caudal limit of Otx2 expression is sufficient for positioning the isthmic organizer and encoding caudal midbrain fate within the mid/hindbrain domain.

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Figure 1: Knock-in strategy of Otx2IRESlacZ (Otx2lacZ) into the En1 locus.
Figure 2: Ectopic expression of Otx2 results in phenotypic alterations of midbrain and cerebellum.
Figure 3: Ectopic expression of Otx2 results in phenotypic alterations at E15.
Figure 4: RNA in situ hybridization of mid/hindbrain-specific markers in wild-type (En1+/lacZ; a, c, e, f, i) and mutants (En1+/Otx2lacZ; b, d, g, h, j) in a lateral (a–d) and dorsal view (e–j) at E10.5.
Figure 5: RNA in situ hybridization of mid/hindbrain-specific markers in control En1+/lacZ (a, c, e, g) and mutant En1+/Otx2lacZ (b, d, f, h) embryos at E9.5.
Figure 6: Genetic interactions leading to isthmic organizer (IsO) formation and mid/hindbrain specification.

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Acknowledgements

We thank L. Bally-Cuif, J. Favor and M. Wassef for critically reading the manuscript; A. Nagy for R1 ES cells; G. Corte, R. Hawkes, A. Joyner, G. Martin, A. Mallamaci, A. Simeone and D. Wilkinson for antibodies and probes; K. Rajewski for the cre-expressing transgenic mouse line; P. Westphal, A. Drexler-Kurz and B. Klädtke for technical support; and S. Rengsberger and A. Maier for secretarial assistance and artwork. This work was supported by EU Biotech (W.W.), EU Biomed (E.B. and W.W.), HSFP (W.W.) and the Deutsche Forschungsgemeinschaft (SFP 190, W.W.).

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Correspondence to Wolfgang Wurst.

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