Fig. S1. Constructs used to identify the minimal sequences required for the notochord activity of the Ci-tune CRM. Schematic of the most informative plasmids that were created to identify the minimal sequences required for the notochord activity of the Ci-tune CRM. Black horizontal lines represent genomic fragments. The red region with an arrow depicts the Ci-FoxA-a basal promoter region contained in the pFBΔSP6 vector (see Materials and methods). For simplicity, neither the basal promoter nor the lacZ reporter gene is drawn to scale. Putative transcription factor binding sites are indicated by the following symbols: red rectangle, T-box binding site; blue oval, Fox binding site. Sites that were ablated by mutations are crossed in black. Notochord activity, or lack thereof, is symbolized by either a ‘+’ or a ‘−’ on the right side. The construct ‘155-bp syn’, in bold font, is the minimal Ci-tune notochord CRM.

Fig. S2. Interspecific conservation of the 155-bp notochord CRM. (A) Alignment of the C. intestinalis 155-bp notochord CRM sequence to the homologous region of the C. savignyi genome, obtained from the VISTA whole-genome alignment of the two species (http://pipeline.lbl.gov/cgi-bin/gateway2). T-box binding sites in the C. intestinalis CRM sequence are highlighted in red, the Fox binding site is highlighted in blue. Putative T-box and Fox binding sites in the C. savignyi sequence are highlighted in pink and light blue, respectively. Coordinates for the alignment are: C. intestinalis v.2.0 chr_09q:3619536-3619690; C. savignyi Sep. 2005 reftig_58:1341574-1341732. (B) Low-magnification micrograph of a group of C. savignyi mid-tailbud embryos electroporated at the one-cell stage with the 1655-bp XPA16474 construct. Notochord staining is seen in ∼30% of the fully developed embryos. (C,D) Individual C. savignyi mid-tailbud embryos showing staining in trunk endoderm, endodermal strand and in a few notochord cells. (E) Low-magnification micrograph of a group of C. savignyi mid-tailbud embryos electroporated at the one-cell stage with the 155-bp Ci-tune CRM. Notochord staining is seen in ∼11% of the fully developed embryos.

Fig. S3. Low-magnification micrographs of transgenic embryos treated with cytochalasin B. (A) Lineage map of the vegetal half of the Ciona embryo at the 110-cell stage. Notochord precursors are labeled in red, muscle precursors in orange, trunk mesenchyme in purple, neural precursors in light blue. Precursors of two different lineages are labeled with two colors. (B-H) Low-magnification microphotographs of Ciona embryos electroporated at the one-cell stage with the constructs indicated underneath each panel. Binding sites that have been mutated are covered by an ‘X’. Embryos were cleavage arrested with cytochalasin B at the 110-cell stage.

Fig. S4. Effects of the reversal of the Ci-FoxA-a binding site on the notochord activity of the Ci-tune CRM. (A-F) Low-magnification micrographs of late-tailbud embryos that were electroporated at the one-cell stage with the constructs indicated at the bottom left of each panel, then fixed and stained to detect reporter activity. In F, red arrowheads indicate embryos showing notochord staining. T1r, T-box site 1 reversed; T2r, T-box 2 site reversed; T1m, T-box site 1 mutant; T2m, T-box site 2 mutant; Foxr, Fox site reversed.

Fig. S5. Genome-wide identification of clusters of Ci-Bra and Ci-FoxA-a sites related to those found in the Ci-tune CRM. (A) Schematics of the 155-bp Ci-tune CRM in either the ‘syn’ (i.e. 5′-3′) orientation with respect to the basal promoter (top) or the ‘anti’ (i.e. 3′-5′) configuration. The Ci-Bra binding site with the 5′-3′ orientation (T-box site 1) is represented by a red rectangle, the Ci-Bra binding site with the 3′-5′ orientation (T-box site 2) is represented by a beige rectangle. The FoxA-a binding site is depicted as a blue oval. Small arrows indicate the orientation of each site, with 5′-3′ sequences pointing to the right. Presence or absence of notochord activity is symbolized by either a ‘+’ or a ‘−’, respectively, in the right column. (B) Various combinations of two Ci-Bra and one Ci-FoxA-a binding sites with core sequences identical to those found in the 155-bp Ci-tune CRM within a 70-bp interval were identified by the GUFEE program, PCR-amplified and tested in vivo by electroporation into Ciona zygotes.