Organogenesis in vertebrates requires the tight control of cell proliferation and differentiation. The homeobox-containing transcription factor Six3 plays a pivotal role1,2 in the proliferation of retinal precursor cells. In a yeast two-hybrid screen, we identified the DNA replication-inhibitor geminin as a partner of Six3. Geminin inhibits cell-cycle progression3 by sequestering Cdt1 (refs 4, 5), the key component for the assembly of the pre-replication complex6. Here, we show that Six3 efficiently competes with Cdt1 directly to bind to geminin, which reveals how Six3 can promote cell proliferation without transcription. In common with Six3 inactivation2,7, overexpression of the geminin gene (Gem; also known as Gmn) in medaka (Oryzias latipes) induces specific forebrain and eye defects that are rescued by Six3. Conversely, loss of Gem (in common with gain of Six3 (ref. 1)) promotes retinal precursor-cell proliferation and results in expanded optic vesicles, markedly potentiating Six3 gain-of-function phenotypes. Our data indicate that the transcription factor Six3 and the replication-initiation inhibitor geminin act antagonistically to control the balance between proliferation and differentiation during early vertebrate eye development.
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We acknowledge F. Loosli and J. Martinez for their critical input and discussion; P. Bovolenta and J. Lopez-Rios for the human Six3 construct and antibody; Z. Lygerou, T. Nishimoto, A. Mansouri, A. Nebreda and M. Mechali for providing plasmids and cells; and A. Akhtar and V. Neubrand for methodological advice. We thank C. Grabher, M. Rembold and W. Norton for critically reading the manuscript, E. Grzebisz for fish husbandry and A. Krone for technical assistance. We would like to thank M. Kessel for communicating unpublished results and for critical input. This work was supported by grants from the Deutsche Forschungsgemeinschaft, Collaborative Research Centre 488, the EU and HFSPO to J.W.
The authors declare that they have no competing financial interests.
Subcellular localization. (JPG 30 kb)
Mapping of the interacting domains of Geminin. (JPG 29 kb)
Luciferase assay indicates inhibitory effect of Geminin on Six3 transcriptional activity. (JPG 23 kb)
Geminin overxpression results in reduction of anterior neural marker expression. (JPG 75 kb)
a) Antisense morpholino oligonucleotide blocks translation of medaka Geminin; b) addition of MoGFP, MoMiss or MoGem at 0.1mM to in vitro transcription/translation system reactions; c) Western Blot analysis of gemUTR-GFP expression in injected embryos. (JPG 45 kb)
Geminin depletion and Six3 overexpression synergize in inducing retinal hyperplasia. (JPG 28 kb)
Concentration dependent effect upon injection of geminin mRNA and phenotypic rescue after coinjection with Six3 or Six3W216R mRNAs. (DOC 23 kb)
Morpholino injection experiments. (DOC 32 kb)
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Bene, F., Tessmar-Raible, K. & Wittbrodt, J. Direct interaction of geminin and Six3 in eye development. Nature 427, 745–749 (2004). https://doi.org/10.1038/nature02292
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