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Fig. 5. In vivo requirement for cis sites and direct interaction between Ey and So. (A-D,F,G) Detection of LacZ mRNA by in situ hybridization; in all cases, the staining reaction was carried out for 3 hours. (A) 3'ato³⁴⁸-ßgal reporter gene expression. (B) The introduction of several base-pair changes in the Ey-binding site results in loss of reporter gene expression in 3'ato^348Pax6MUT-ßgal. (C) The introduction of several base-pair changes in the So-binding site results in loss of reporter gene expression in 3'ato^348SoMUT- ßgal. (D) The introduction of multiple base-pair changes in both So- and Ey-binding sites results in loss of reporter gene expression in 3'ato^{1.2Pax6-SoMUT}-ßgal. A faint signal as a stripe and/or along the margins of the eye disc was detected in some of the transgenic lines after overnight ß-gal staining (not shown). (E) Ey and So can directly interact at the protein level. A GST-So^SixHD fusion protein (containing the Six and homeobox domains) binds full-length Ey protein. GST alone does not bind Ey, and GST-So^SixHD does not bind luciferase. 10% of the ³⁵S-labelled proteins and 100% of the pulled-down yields are shown. (F) Insertion of three As between the So- and Ey/Pax6-binding sites in 3'ato^348+3A- ßgal results in a reduction in reporter gene expression. (G) Insertion of six As between the So- and Ey/Pax6-binding sites in 3'ato^348+6A-ßgal severely affects reporter gene expression. A weak signal was detected in some of the transgenic lines after overnight ß-gal staining (not shown).

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