Supplementary Figure 9: Shh expression in the zli is codependent on the SBE1 and SBE5 enhancers.
From: Cis-regulatory architecture of a brain signaling center predates the origin of chordates
(a) Genomic map of the Shh gene and a 1-Mb region upstream showing the position of SBE1 and SBE5 (blue ovals), as well as ten other previously described Shh enhancers50,57-59. The 228-kb deletion in the mouse Del(C1-Z) Tracer line (mm9 chr. 5: 29,413,901–29,642,246; referred to herein as ShhΔSBE5) is outlined in red. (b–f) Whole-mount in situ hybridization for Shh on wild-type (WT), ShhΔSBE5/ΔSBE5, ShhΔSBE1ΔSBE5/ΔSBE1ΔSBE5, ShhP1; ShhΔSBE1ΔSBE5/ΔSBE1ΔSBE5 and ShhΔSBE5/SBE5Δ2kb embryos at E10.5. The extent of Shh expression along the length of the zli is highlighted (red bracket). The ShhP1 transgene expresses Shh under the influence of SBE1 and Shh floor plate enhancers 1 and 2 (SFPE1 and SFPE2) and restores Shh expression in the ventral midbrain, ventroposterior diencephalon and zli of SBE1/SBE5 double mutants. ShhP1 embryos also display ectopic Shh expression in the otic vesicle (ov). The loss of Shh expression in the fore and hindlimb buds (fl, hl) of SBE5 mutants is attributed to deletion of the zone of polarizing regulatory sequence (ZRS)60. Because the ZRS is not included on the ShhP1 transgene, Shh limb bud expression is not restored in e. Scale bar, 1 mm. (g) Quantification of the spatial distribution of Shh expression in the zli normalized to head size at E10.5. A smaller (2-kb) deletion of SBE5 generated by CRISPR/Cas9 (SBE5Δ2kb) had the same effect on Shh zli expression as the larger (228-kb) SBE5 deletion allele (ΔSBE5). ***P < 0.0001, n = 7, two-sided Student’s t test.
