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doi: 10.1242/10.1242/dev.00185

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Six3 and Six6 activity is modulated by members of the groucho family

Javier López-Ríos1, Kristin Tessmar2,*, Felix Loosli2, Joachim Wittbrodt2,{dagger} and Paola Bovolenta1,{dagger}

1 Instituto Cajal, CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain
2 Developmental Biology Programme, EMBL, Meyerhofstrasse 1, 69012 Heidelberg, Germany
* Present address: Department of Biochemistry and Biophysics, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA



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  		Fig. 1. SIX6 and SIX3 interact with TLE1 and AES. (A) Schematic diagram of the domain organisation of human SIX6, SIX3, TLE1 and AES. All vertebrate TLE proteins have the same organisation, as illustrated for TLE1. Besides the QD and WDR domains, these proteins have GP, CcN and SP domains, which have been shown to be involved in transcriptional repression, nuclear localisation and protein interactions. (B) High stringency two-hybrid analysis of the interactions between SIX3, SIX6, SIX1, mSix2, mSix4 and Optix with AES, TLE and Groucho proteins. Constructs containing the full-length or specific domains of SIX and Groucho/TLE human genes were used to map the interactions between these two classes of molecules. (C) Low stringency two-hybrid analysis of the interactions of SIX proteins with the WDR domain of TLE1 and TLE3. (D) Western blot analysis of pull-down experiments using GST::AES (lane 2) and GST::TLE1 proteins (lanes 3, 4) and in vitro synthesised Flag-tagged SIX proteins. Lane 1 shows the respective SIX proteins translated by TnT (input). Lane 5 shows control pull-downs with GST alone. SD, Six domain; HD, Homeo-domain; Ct, C-terminal domain; Gly, glycine-rich region; QD, glutamine-rich domain; GP, glycine-proline rich region; CcN, casein kinase II/cdc2 kinase site/nuclear localisation domain region; SP, serine-proline rich region; WDRD, WD-40 repeats domain.

 


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  		Fig. 2. Comparison of the expression domains of Six3, Six6 and Gro/Tle genes in medaka embryos. Whole-mount in situ hybridisations at different developmental stages as indicated at the top of each colum. All embryos are dorsalview, anterior to the left. Embryos were hybridised with probes to Six3 (A-C), Tle1 (D-F), Tle3 (G-I), Tle4 (J-L), Aes (M-O) and Six6 (P-R). Note how Tle1 and Aes are expressed in the eye field from early stages. Arrowhead in H indicates the lens vesicle; arrowhead in K, the optic stalk; arrows in O, Q and R, the ventral diencephalon. ov, optic vesicle; ey, eye; mb, midbrain; hb, hindbrain. Scale bars 0.1 mm.

 


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  		Fig. 3. TLE1 over-expression enlarges the eye field in medaka embryos. Dorsal (A-D,G,H) and ventral (E,F) views (anterior to the left) of TLE1-injected embryos. (A) Over-expression of TLE1 (inset shows expression of the co-injected GFP mRNA) causes a visible enlargement of the optic vesicles and bulging of the midbrain (arrowhead). Whole-mount in situ hybridisations demonstrate that expression of Six3 (B) and Rx2 (D) are expanded (arrows) compared to control embryos (C, and Fig. 2B). Note that TLE1 injections lead to the expansion of the posterior domain of Pax6 and to the appearance of ectopic Pax6 expression (arrowhead in F), as compared to controls (E). Otx2 expression was similar to that of controls (G,H). ey, eye; mb, midbrain; hb, hindbrain; wt, wild type. Scale bar: 0.1 mm.

 


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  		Fig. 6. AES abrogates SIX3- and SIX6-induced phenotypes. Dorsal views of stage 24 injected embryos hybridised with Rx2 probe. Embryos injected with 50 ng/µl of either SIX3 (A) or SIX6 (C) show ectopic Rx2 expression in the midbrain (open arrows). This phenotype is inhibited by co-injections of AES mRNA (B,D). Scale bar: 0.1 mm.

 


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  		Fig. 4. TLE1 synergizes with SIX3 and SIX6 in over-expression assays. Dorsal views of stage 24 embryos injected with either SIX3 (A,B,E,F) or SIX6 (C,D) mRNA alone (A,C,E) or in combination with TLE1 (B,D,F). Concentrations are indicated in the panels. Embryos were hybridised to detect Rx2 expression. Note that 10 ng/µl of SIX3 or SIX6 are not effective in inducing ectopic Rx2 expression (A,C). Co-injection with TLE1, clearly boosts SIX3 and SIX6 activity and induces ectopic Rx2-positive tissue in the midbrain (arrows in B and D). The phenotype induced by higher doses of SIX3 (E, ectopic midbrain expression. arrow) was also enhanced by TLE1 co-injections (F), leading to the striking appearance of additional separate patches of ectopic Rx2 expression (arrowheads in F). Scale bar: 0.1 mm.

 


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  		Fig. 5. AES over-expression reduces the eye size in medaka embryos. Dorsal (except E, ventral) views of embryos at stage 24 (A,C,G,H,I), stage 32 (B,D,E) and stage 20 (F) injected with AES. Anterior is to the left. Dotted white lines indicate the extent of the eye domains. Embryos show a unilateral (A) or bilateral (D) loss of the eye(s). (A inset) Expression of the co-injected GFP mRNA. (B) The same embryos as in A but at a later stage of development, showing that the failure of eye formation (arrowhead) is permanent. Whole-mount in situ hybridisations demonstrate that the reduction or absence of the eye(s) is accompanied by a decrease of the expression domain of Six3 (arrows in C,E) and Rx2 (arrows in F,G). Apart from the reduced domain of the affected eye (arrow in H), Pax6 (H) and Otx2 (I) expression domains were similar to those of wild-type embryos. Occasionally, ectopic expression of Otx2 was observed in the hindbrain (arrow in I). mb, midbrain. Scale bars: 0.1 mm.

 




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