GATA6 binds to regulatory regions that control the expression of the mouse Shh gene and blocks expression of reporters driven by this sequence. Conditional deletion of Shh from the mouse limb bud rescues polydactyly induced by conditional loss of GATA6 in the mouse hindlimbs.

(A) GATA6 binding sites identified by EMSA in the conserved region (ShhE) of the mouse Shh limb bud enhancer ZRS. Position of GATA6 binding sites are indicated relative to the first HindIII site in the ZRS sequence [16]. Oligos 7 and 8 used to perform EMSA are indicated. Positions of qPCR primers used in CHIP experiments are shown (P4–P6), P6 is a control pair of primers located ∼6 kb upstream of the ShhE sequence. (B) Electrophoretic mobility shift assays of in vitro translated GATA6 with oligos from the ShhE region of the ZRS enhancer. Arrows point to the position of either the GATA6-oligo complexes or to the unbound labeled probes. (C) Chromatin IP to assay GATA6 interaction with the conserved region (ShhE) of the mouse Shh limb bud enhancer (ZRS) in limb buds isolated from either E11.5 Prx1-Cre; ROSA26-rtTA; GATA6-Tg animals, that had been pulsed with doxycycline from E4.5 until harvest, versus limb buds isolated from E11.5 Prx1-Cre; GATA6^fl/fl mice. Positions of primers on the ShhE enhancer are displayed in A. Chromatin was sheared to between 500–1000 bp prior to ChIP with anti-GATA6 antibody. **p<0.01; Student's test. Error bar indicates standard deviation, n = 3. (D) GATA6 and FOG2 cooperatively block Hand2/Hoxd13-mediated induction of a luciferase reporter driven by the conserved region of the ZRS enhancer (ShhE) that drives Shh expression in the limb bud. NIH3T3 cells were co-transfected with an ShhE-firefly luciferase reporter plus expression vehicles encoding either Hand2, Hoxd13, GATA6-WT, GATA6-DNA binding domain mutant (GATA6DBDmut), or FOG2, as indicated. Firefly luciferase units were normalized to the expression of co-transfected SV40-renilla luciferase to obtain Relative Luciferase Units (RLU). *p<0.05, **p<0.01, ***p<0.001; Student's test. Error bar indicates standard deviation, n = 3. (E) Alizarin Red/Alcian blue staining of the hindlimbs of P0 mice of the indicated genotypes. At least 5 animals of each genotype were analyzed; representative samples are shown. (F) Whole mount in situ hybridization analysis of Gli1 expression in E11.5 Prx1-Cre; GATA6^fl/fl; Shh^fl/fl embryos vs. E11.5 Prx1-Cre; GATA6^fl/fl; Shh^fl/+ embryos. Four limbs of each genotype were analyzed; representative images are shown. Ectopic Gli1 expression is noted by the red arrow.