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The prepattern transcription factor Irx2, a target of the FGF8/MAP kinase cascade, is involved in cerebellum formation

Nature Neuroscience volume 7, pages 605612 (2004) | Download Citation



The cerebellum develops from the rhombic lip of the rostral hindbrain and is organized by fibroblast growth factor 8 (FGF8) expressed by the isthmus. Here we report characterization of Irx2, a member of the Iroquois (Iro) and Irx class of homeobox genes, that is expressed in the presumptive cerebellum. When Irx2 is misexpressed with Fgf8a in the chick midbrain, the midbrain develops into cerebellum in conjunction with repression of Otx2 and induction of Gbx2. During this event, signaling by the FGF8 and mitogen-activated protein (MAP) kinase cascade modulates the activity of Irx2 by phosphorylation. Our data identify a link between the isthmic organizer and Irx2, thereby shedding light on the roles of Iro and Irx genes, which are conserved in both vertebrates and invertebrates.

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  1. 1.

    & The role of the rhombic lip in avian cerebellum development. Development 126, 4395–4404 (1999).

  2. 2.

    , & Inductive signal and tissue responsiveness defining the tectum and the cerebellum. Development 128, 2461–2469 (2001).

  3. 3.

    et al. Identification of the vertebrate Iroquois homeobox gene family with overlapping expression during early development of the nervous system. Mech. Dev. 69, 169–181 (1997).

  4. 4.

    , , & Expression of two novel mouse iroquois homeobox genes during neurogenesis. Mech. Dev. 91, 317–321 (2000).

  5. 5.

    et al. Xiro3 encodes a Xenopus homolog of the Drosophila Iroquois genes and functions in neural specification. EMBO J. 17, 191–203 (1998).

  6. 6.

    , , & Xiro, a Xenopus homolog of the Drosophila Iroquois complex genes, controls development at the neural plate. EMBO J. 17, 181–190 (1998).

  7. 7.

    , , , & A role for iro1 and iro7 in the establishment of an anteroposterior compartment of the ectoderm adjacent to the midbrain-hindbrain boundary. Development 129, 2317–2327 (2002).

  8. 8.

    , & Xiro-1 controls mesoderm patterning by repressing Bmp-4 expression in the Spemann organizer. Dev. Dyn. 222, 368–376 (2001).

  9. 9.

    & The Wnt-activated Xiro-1 gene encodes a repressor that is essential for neural development and downregulates Bmp-4. Development 128, 551–560 (2001).

  10. 10.

    , , & A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101, 435–445 (2000).

  11. 11.

    et al. Early subdivisions in the neural plate define distinct competence for inductive signals. Development 129, 83–93 (2002).

  12. 12.

    , , , & mirror encodes a novel PBX-class homeoprotein that functions in the definition of the dorsal-ventral border in the Drosophila eye. Genes Dev. 11, 1073–1082 (1997).

  13. 13.

    & araucan and caupolican provide a link between compartment subdivisions and patterning of sensory organs and veins in the Drosophila wing. Genes Dev. 10, 2935–2946 (1996).

  14. 14.

    , , & Compartments and organising boundaries in the Drosophila eye: the role of the homeodomain Iroquois proteins. Development 126, 4933–4942 (1999).

  15. 15.

    , , & Araucan and caupolican, two members of the novel iroquois complex, encode homeoproteins that control proneural and vein-forming genes. Cell 85, 95–105 (1996).

  16. 16.

    , , & Organization of mouse iroquois homeobox genes in two clusters suggests a conserved regulation and function in vertebrate development. Genome Res. 10, 1453–1462 (2000).

  17. 17.

    et al. Cloning and chromosomal mapping of human and chicken Iroquois (Irx) genes. Cytogenet. Cell. Genet. 92, 320–325 (2001).

  18. 18.

    & c-Irx2 expression reveals an early subdivision of the neural plate in the chick embryo. Mech. Dev. 87, 203–206 (1999).

  19. 19.

    , & Neuroepithelial co-expression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer. Mech. Dev. 101, 111–118 (2001).

  20. 20.

    , & Midbrain development induced by FGF8 in the chick embryo. Nature 380, 66–68 (1996).

  21. 21.

    , , , & FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression. Development 126, 1189–1200 (1999).

  22. 22.

    , & FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate. Development 126, 4827–4838 (1999).

  23. 23.

    , & Fgf8 and Gbx2 induction concomitant with Otx2 repression is correlated with midbrain-hindbrain fate of caudal prosencephalon. Development 126, 3191–3203 (1999).

  24. 24.

    & Expression of Gbx-2 during early development of the chick embryo. Mech. Dev. 76, 157–159 (1998).

  25. 25.

    , & The caudal limit of Otx2 expression positions the isthmic organizer. Nature 401, 164–168 (1999).

  26. 26.

    et al. A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer. Nature 401, 161–164 (1999).

  27. 27.

    In vivo electroporation: a new frontier for gene delivery and embryology. Differentiation 70, 163–171 (2002).

  28. 28.

    et al. Sequential roles for Fgf4, En1 and Fgf8 in specification and regionalisation of the midbrain. Development 126, 945–959 (1999).

  29. 29.

    , , & Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain. Development 124, 959–969 (1997).

  30. 30.

    , & Iroquois: a prepattern gene that controls the formation of bristles on the thorax of Drosophila. Mech. Dev. 59, 63–72 (1996).

  31. 31.

    , , & Compartments and organising boundaries in the Drosophila eye: the role of the homeodomain Iroquois proteins. Development 126, 4933–4942 (1999).

  32. 32.

    & Granule cell raphes and parasagittal domains of Purkinje cells: complementary patterns in the developing chick cerebellum. J. Neurosci. 18, 9342–9353 (1998).

  33. 33.

    & Sonic hedgehog regulates the growth and patterning of the cerebellum. Development 126, 3089–3100 (1999).

  34. 34.

    , , , & FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression. Development 126, 1189–1200 (1999).

  35. 35.

    , , , & Distinct regulators control the expression of the mid-hindbrain organizer signal FGF8. Nat. Neurosci. 4, 1175–1181 (2001).

  36. 36.

    , & An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat. Genet. 18, 136–141 (1998).

  37. 37.

    & Early anterior/posterior patterning of the midbrain and cerebellum. Annu. Rev. Neurosci. 24, 869–896 (2001).

  38. 38.

    , & Changing requirements for Gbx2 in development of the cerebellum and maintenance of the mid/hindbrain organizer. Neuron 36, 31–43 (2002).

  39. 39.

    , , , & The isthmic neuroepithelium is essential for cerebellar midline fusion. Development 130, 5319–5330 (2003).

  40. 40.

    & Tight transcriptional control of the ETS domain factors Erm and Pea3 by Fgf signaling during early zebrafish development. Mech. Dev. 107, 105–117 (2001).

  41. 41.

    , , , & Sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish. Development 128, 2175–2186 (2001).

  42. 42.

    , , & Identification of Sef, a novel modulator of FGF signalling. Nat. Cell Biol. 4, 165–169 (2002).

  43. 43.

    , , , & Sef is a feedback-induced antagonist of Ras/MAPK-mediated FGF signalling. Nat. Cell Biol. 4, 170–174 (2002).

  44. 44.

    , , & Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos. Development 128, 4153–4164 (2001).

  45. 45.

    , & The homeoprotein Xiro1 is required for midbrain-hindbrain boundary formation. Development 129, 1609–1621 (2002).

  46. 46.

    et al. The Iroquois homeobox gene Irx2 is not essential for normal development of the heart and midbrain-hindbrain boundary in mice. Mol. Cell. Biol. 23, 8216–8225 (2003).

  47. 47.

    , & Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).

  48. 48.

    et al. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265, 966–970 (1994).

  49. 49.

    & Identification of a developmentally regulated protein-tyrosine kinase by using anti-phosphotyrosine antibodies to screen a cDNA expression library. Proc. Natl. Acad. Sci. USA 86, 5449–5453 (1989).

  50. 50.

    , , , & The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif. Mol. Cell. Biol. 18, 710–720 (1998).

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We thank K. Kitamura, J. Aruga, and A. Leutz for probes; and J.L. Gomez-Skarmeta and C.C. Hui for comments. We also thank T. Shimizu and A. Saitoh for assistance. This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas (C) and Creative Basic Research from the Ministry of Education, Science Sports and Culture of Japan, and by the Toray Science Foundation.

Author information


  1. Department of Developmental Neurobiology, Institute of Development, Aging and Cancer, Tohoku University 4-1, Seiryo, Aoba, Sendai 980-8575, Japan.

    • Ken Matsumoto
    • , Shigeki Nishihara
    • , Mika Kamimura
    • , Keiko Ogura
    •  & Toshihiko Ogura
  2. Nara Institute of Science and Technology, Graduate School of Biological Sciences, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan.

    • Tomoki Shiraishi
    • , Takao Otoguro
    • , Masayuki Uehara
    •  & Yukiko Maeda
  3. MRC Centre for Developmental Neurobiology, King's College London, 4th Floor New Hunts House, Guy's Hospital, London SE1 1UL, UK.

    • Andrew Lumsden


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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Toshihiko Ogura.

Supplementary information

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  1. 1.

    Supplementary Note 1

    When Irx2 was misexpressed in the right midbrain at stage 9, both Otx2 and Gbx2 were expressed normally on the electroporated side (Exp.) at E4, as compared with the control sides of the same brains (Cont.) (83%, n=11). In contrast, Pax2 was induced weakly, and En1 was also induced rostrally on the right side of the electroporated embryos with rostral expansion of the midbrain (arrowheads), implying that Irx2 transforms the diencephalons to midbrain, a similar effect observed in Fgf8a misexpression. Less frequently, Fgf8 was also induced rostrally, yet this induction was weak. GFP fluorescence derived from co-electroporated pCAGGS-GFP is shown on the right (GFP). When Fgf8a was misexpressed, as for Irx2 misexpression, neither Gbx2 nor Otx2 were affected (Exp.), as compared with control sides (Cont.) (100%, n=15 and 10 for Gbx2 and Otx2, respectively). GFP fluorescence derived from the co-electroporated pCAGGS-GFP is shown on the right (GFP). Irx2 expression was not affected by Fgf8a misexpression. From these data, we concluded that the enlarged tectum resulted from the ectopic expression of Pax2/En1 and FGF8. En2 was induced by Fgf8a (data not shown), implying that the induced En2 caused the enlarged tectum. Irx2 expression was not affected by misexpression of Fgf8a.

  2. 2.

    Supplementary Note 2

    When Fgf8b alone was misexpressed in the midbrain at a low concentration of 0.05 μg/μl, expression of Gbx2 and Otx2 was not affected. In contrast, when Irx2 was misexpressed together with such a low concentration of Fgf8b, induction of Gbx2 and repression of Otx2 were observed. Changes of expression patterns were obtained in 90% of cases for Otx2 and 73% for Gbx2.

  3. 3.

    Supplementary Note 3

    (a) We further dissected Irx2 by fusing its amino and carboxyl portions to the GAL4 DNA binding domain —GAL4-Irx2(1-110) and GAL4-Irx2(183-477), respectively. Then we inserted these fusion genes into the pCMX expression vector. The resultant plasmids [pCMX-GAL4-Irx2 (1-110) and pCMX-GAL4-Irx2 (183-477)] were introduced into NIH3T3 cells along with a GAL4 reporter construct (4xUAS-TK-luciferase), and the expressed luciferase activity was measured (white bars). GAL4-Irx2 (1-110) alone did not affect the luciferase activity. In contrast, GAL4-Irx2 (183-477) acted by itself as a strong repressor, suppressing the luciferase activity about 6 fold. In the presence of Wt-Mek1 (gray bars), GAL4-Irx2 (1-110) became a weak activator, and GAL4-Irx2 (183-477) diminished its repressor activity. More drastic changes were observed when the constitutively active R4F-Mek1 was co-expressed (dark bars). In the presence of active R4F-Mek1, GAL4-Irx2 (1-110) acquired a strong activator function, whereas GAL4-Irx2 (183-477) lost its repressor activity significantly. This strongly suggests that the Mek1-mediated phosphorylation converts Irx2 from a repressor to an activator, depending on the signaling context of MAP kinase. Since Mek1 MAP kinase kinase lies downstream of the FGF cascade, our data strongly suggest that FGF signaling switches on and off the activity of Irx2 in vivo. (b) Alignment of amino acid sequences of human, mouse, chick, Fugu and Xenopus Irx2 proteins. In this alignment, only amino terminal parts are shown. Conserved residues are boxed. Serine residues of the putative MAP kinase phosphorylation sites are shown in red. Positions of serine 46 and 65 of chick Irx2 are indicated by red asterisks. Schematic illustration of chick Irx2 protein and the putative phosphorylation sites is also shown. HD:Irx2 homeodomain.

  4. 4.

    Supplementary Note 4

    Schematic representation of GST-Irx2 fusion proteins. The Iro domain, which is conserved among members of the Irx/Iro family, is indicated by a blue box. The 2/5 domain, which is conserved in Irx2 and Irx5, is shown by a red box. Putative MAP kinase phosphorylation sites (positions 294, 326, and 331) are shown. The carboxyl terminal part of Irx2 (183-477) was subdivided into four subdomains, and each subdomain was fused to the carboxyl terminus of the GST gene to make a fusion construct. Fusion proteins were expressed in E. coli, then purified for in vitro kinase assays.

  5. 5.

    Supplementary Note 5

    We misexpressed Irx2-S46/65/326A mutant, in which three serine residues were substituted to alanine. Since MAP kinase does not phosphorylate this mutant, misexpression of this mutant along with Fgf8a would further confirm that Irx2 indeed functions downstream of FGF8 signaling. As expected, Irx2S46/65/326A failed to repress Otx2 expression even when misexpressed with Fgf8a. Irx2S46/65/326A alone did not alter Otx2 expression (data not shown).

  6. 6.

    Supplementary Note 6

    Repression of Fgf8 expression by Irx2-EnR was not evident until 18 hours after electroporation. Both En1 and Pax2 were repressed 24 hours after electroporation, then Gbx2 was repressed and Otx2 was induced 30 hours after electroporation.

  7. 7.

    Supplementary Note 7

    Pax2, En1 and Fgf8 were induced 12 hours after misexpression of Irx2(1-306). Repression of Otx2 became evident 18 hours after electroporation. Gbx2 was induced 24 hours after electroporation of Irx2(1-306).

  8. 8.

    Supplementary Note 8

    Expression patterns of Irx2, Fgf8, Gbx2 and Otx2 at stage 10. The rostral limit of Irx2 expression coincides with the rostral limits of Fgf8 and Gbx2 expression (black arrowheads) and the caudal limit of Otx2 expression (red arrowhead). Note that the expression domains of Irx2 and Fgf8 overlap at this stage.

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