Characterization of SIMO wild-type and mutant enhancer by reporter gene assays in chick and zebrafish.

(A, B) Schematic view of reporter constructs used for in ovo electroporation of chick embryos. Reporter constructs carry wild-type or mutant mouse SIMO element upstream of hsp68 minimal promoter and β-galactosidase open reading frame. In mutant SIMO Meis binding sites were abolished by introduction of specific single-point mutations changing Meis recognition sequence TGACAG/A into TcACAG/A. (C–F) Whole-mount view or histological sections through the eye of β-galactosidase–stained chick embryos of stage HH21-22 electroporated either with (C, E) wild-type or with (D, F) mutant SIMO fragment. Positive X-gal staining correlates with the activity of reporter constructs. Wild-type SIMO fragment supports reporter construct expression in lens but not the mutant SIMO fragment. (G, H) Schematic view of reporter constructs used for transgenesis in zebrafish. Reporter constructs carry wild-type or mutant zebrafish SIMO element upstream of zebrafish gata2a minimal promoter and EGFP open reading frame. In mutant zebrafish SIMO Meis binding sites were abolished by introduction of specific single-point mutations changing Meis recognition sequence TGACAG/A into TcACAG/A. In order to control for transgenesis efficiency in vivo the reporter genes contain a second cassette composed of a cardiac actin promoter driving the expression of a red fluorescent protein (DsRed). EGFP and DsRed transcriptional units are separated by an insulator. (I-L) Wild-type SIMO enhancer activity is detected at 48 hpf (n = 160, 68% EGFP of DsRed positive), (I, J), but not for the mutant construct (n = 36, 89% EGFP negative of DsRed positive) (K, L). LE—lens, NR—neural retina.