SOX2 acts with LHX2 and HOX factors to specify regionalized gene expression. (A) Overview of GFP reporter experiments in zebrafish embryos. (B–D) Representative examples of resulting transgenic zebrafish embryos after the injection of GFP reporters containing enhancer elements commonly (B) or specifically bound by SOX2 in the mouse cortex (C) or spinal cord (D). Spatial activity of transgenic enhancers is reported by GFP expression (white staining). Tracks depict SOX2 ChIP-seq reads in the cortex (blue) and spinal cord (red). (E) Activity of a transgenic enhancer bound by SOX2 in the mouse cortex upon mutation of SOX or LHX motifs (stars). (F) Transgenic activity of an enhancer bound by SOX2 in the mouse spinal cord upon mutation of SOX or HOX motifs (stars). (G) Activity of GFP reporters containing either a wild-type enhancer active in the forebrain, its synthetic version in which the nucleotide sequence, apart from the intact LHX and SOX motifs, have been randomized, or a version in which the LHX motif was swapped for HOX, PBX, and MEIS motifs. (H) Activity of GFP reporters containing either a wild-type enhancer active in the caudal neural tube, its synthetic version in which the nucleotide sequence, apart from the intact HOX, PBX, MEIS, and SOX motifs, has been randomized, or an enhancer version in which its HOX, PBX, and MEIS motifs were swapped for LHX motifs. (I,J) Luciferase reporter assay in P19 cells. Luciferase reporters containing either wild-type enhancers active in the forebrain (I) or in the caudal neural tube (J) were cotransfected with different combinations of vectors expressing SOX2, LHX2, HOXB6, PBX3, or MEIS1 proteins (I,J). Other enhancers examined consisted of variants in which either the transcription factor binding motifs or their flanking sequences were mutated, or variants in which the LHX motifs and HOX, PBX, and MEIS motifs had been interchanged. (*) 0.05 > P > 0.01; (**) 0.01 > P > 0.001.
