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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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.
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.
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.
(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.
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.
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).
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.
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).
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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Neural Development (2012)