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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Simeone,A., Acampora,D., Massino,G., Stornainolo,A. & Boncinelli,E. Nested expression domains of four homeobox genes in developing rostral brain. Nature 358, 687–690 (1992).
Acampora,D. & Simeone,A. Understanding the roles of Otx1 and Otx2 in the control of brain morphogenesis. Trends Neurosci. 22, 116–122 (1999).
Lumsden,A. & Krumlauf,R. Patterning the vertebrate neuraxis. Science 274, 1109–1115 (1996).
Beddington,R. S. P. & Robertson,E. J. Anterior patterning in mouse. Trends Genet. 14, 277–284 (1998).
Ang, S.-L., Conlon,R. A., Jin,O. & Rossant,J. Positive and negative signals from mesoderm regulate the expression of mouse Otx2 in ectoderm explants. Development 120, 2979–2989 (1994).
Wassarman,K. M. et al. Specification of the anterior hindbrain and establishment of a normal mid/hindbrain organizer is dependent on Gbx2 gene function. Development 124, 2923–2934 (1997).
McMahon,A. P., Joyner,A. L., Bradley,A. & McMahon,J. A. The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell 69, 581–595 (1992).
Martinez,S., Crossley,P. H., Cobos,I., Rubenstein,J. L. R. & Martin,G. R. FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression. Development 126, 1189–1200 (1999).
Bally-Cuif,L. & Wassef,M. Determination events in the nervous system of the vertebrate embryo. Curr. Opin. Genet. Dev. 5, 450–458 (1995).
Joyner,A. L. Engrailed, Wnt and Pax genes regulate midbrain-hindbrain development. Trends Genet. 12, 15–20 (1996).
Wassef,M. & Joyner,A. L. Early mesencephalon/metencephalon patterning and development of the cerebellum. Prospect. Dev. Neurobiol. 5, 3–16 (1997).
Martinez,S., Wassef,M. & Alvarado-Mallart,R. M. Induction of amesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en. Neuron 6, 971–981 (1991).
Martinez,S., Marin,F., Nieto,M. A. & Puelles,L. Induction of ectopic engrailed expression and fate change in avian rhombomeres: intersegmental boundaries as barriers. Mech. Dev. 51, 289–303 (1995).
Meinhardt,H. Cell determination boundaries as organizing regions for secondary embryonic fields. Dev. Biol. 96, 375–385 (1983).
Davis,C. A. & Joyner,A. L. Expression patterns of the homeobox containing genes En1 and En2 and the proto-oncogene int-1 diverge during mouse development. Gen. Dev. 2, 1736–1744 (1988).
Schwenk,F., Baron,U. & Rajewsky,K. A cre-transgenic mouse strain for ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucl. Acids Res. 24, 5080–5081 (1995).
Millet,S., Bloch-Gallego,E., Simeone,A. & Alvarado-Mallart, R.-M. The caudal limit of Otx2 gene expression as a marker of the midbrain/hindbrain boundary: a study using in situ hybridisation and chick–quail homotopic grafts. Development 122, 3785–3797 (1996).
Marin,F. & Puelles,L. Morphological fate of rhombomeres in quail/chick chimeras: a segmental analysis of hindbrain nuclei. Eur. J. Neurosci. 7, 1714–1738 (1995).
Danielian,P. S. & McMahon,A. P. Engrailed-1 as a target of the Wnt-1 signalling pathway in vertebrate midbrain development. Nature 383, 332–334 (1996).
Lee,S. M. K., Danielian,P. S., Fritzsch,B. & McMahon,A. P. Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain. Development 124, 959–969 (1997).
Meyers,E. N., Lewandoski,M. & Martin,G. R. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nature Genet. 18, 136–141 (1998).
Reifers,F. et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125, 2381–2395 (1998).
Lun,K. & Brand,M. A series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signalling events in development of the midbrain-hindbrain boundary. Development 125, 3049–3062 (1998).
Wurst,W., Auerbach,A. B. & Joyner,A. Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum. Development 120, 2065–2075 (1994).
Schwarz,M., Alvarez-Bolado,G., Urbánek,P., Busslinger,M. & Gruss,P. Conserved biological function between Pax-2 and Pax-5 in midbrain and cerebellum development: Evidence from targeted mutation. Proc. Natl Acad. Sci. USA 94, 14518–14523 (1997).
Acampora,D., Avantaggiato,V., Tuorto,F. & Simeone,A. Genetic control of brain morphogenesis through Otx gene dosage requirement. Development 124, 3639–3650 (1997).
Suda,Y., Matsuo,I. & Aizawa,S. Cooperation between Otx1 and Otx2 genes in developmental patterning of rostral brain. Mech. Dev. 69, 125–141 (1997).
Hanks,M., Wurst,W., Anson-Cartwright,L., Auerbach,A. B. & Joyner,A. L. Rescue of the En1 mutant phenotype by replacement of En1 with En2. Science 269, 679–682 (1995).
Mallamaci,A., Di Blas,E., Briata,P., Boncinelli,E. & Corte,G. OTX2 homeoprotein in the developing central nervous system and migratory dells of the olfactory area. Mech. Dev. 58, 165–178 (1996).
Flenniken,A. M., Gale,N. W., Yancopoulos,G. D. & Wilkinson,D. G. Distinct and overlapping expression patterns of ligands for Eph-related receptor tyrosine kinases during mouse embryogenesis. Dev. Biol. 179, 382–401 (1996).
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.).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Broccoli, V., Boncinelli, E. & Wurst, W. The caudal limit of Otx2 expression positions the isthmic organizer. Nature 401, 164–168 (1999). https://doi.org/10.1038/43670
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/43670
This article is cited by
-
Enhanced production of mesencephalic dopaminergic neurons from lineage-restricted human undifferentiated stem cells
Nature Communications (2023)
-
The embryonic patterning gene Dbx1 governs the survival of the auditory midbrain via Tcf7l2-Ap2δ transcriptional cascade
Cell Death & Differentiation (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.