Fig 3. The function of individual Ci-Bra binding sites can be modulated by either an (AC) microsatellite or a flanking sequence.

a,c: Schematic representations of wild-type (WT) and mutant CRMs, as described and colored in Fig 2; the (AC) microsatellite sequence is schematized as a segmented brown rectangle. b: Mutational series of the area boxed in orange in the 253-bp construct. Red and blue nucleotides correspond to the Ci-Bra and Ci-Fox sites, respectively, and orange nucleotides indicate the bases changed in each mutant plasmid. The (AC)₆ microsatellite sequence is boxed in green. The relative ability of each construct to direct notochord gene expression is shown by plus signs at the right of each sequence. d-i: Photos of embryos electroporated with the constructs depicted in a,b,c; arrowheads are color-coded as in Fig 1. j: Quantification of notochord-stained embryos harboring the constructs in a,c. Error bars indicate standard deviation from the mean. k: Identification of an extended CTAM sequence (colored) shared by a subset of individually-acting Ci-Bra binding sites. l-w: Microphotographs of embryos carrying wild-type CRMs (l,p,t) compared to embryos carrying various mutant versions of Ci-CRM109 (m-o) Ci-CRM99 (q-s) and Ci-CRM86 (u-w). Core Ci-Bra binding sites are capitalized. Mutations are depicted in red. Abbreviations: FSM: “frame-shift” mutation, LSM: linker scanning mutation. See also S3 Fig.