(A–I’) Genome-edited mESCs with various genotypes were differentiated into nEBs and stained for ventral markers Nkx2.2 (Cyan), Olig2 (Magenta) and Isl1/2 (Red), indicating Hh pathway activation, and the dorsal marker Pax7 (Green) indicating Hh pathway inactivity. (J–R) Nkx2.2, Isl1/2, Olig2 or Pax7 staining quantification (box and whiskers). WTnEBs have a quiescent Hh response (A,A’,J). Loss of Shh (Ptch1+/L (B,B’,K) or Smo (Smo-/-) (C,C’,L) in cells with intact Ptch1/2 function (Ptch1LacZ is null) increases Pax7. nEBs without Ptch1 (Ptch1LacZ/LacZ, D,D’,M) have an activated Hh response. Loss of Shh (Ptch1LacZ/LacZ;Shh-/-, E,E’,N) decreases the response. Ptch1LacZ/LacZ;Smo-/- nEBs acquire Pax7 (F,F’,O) and are devoid of all three ventral markers. nEBs lacking all Ptch1/2 activity (Ptch1LacZ/LacZ;Ptch2-/-) have an activated Hh response pathway (G,G’,P). Ptch1LacZ/LacZ;Ptch2-/-;Shh-/- nEBs retain similar ventral identity (H,H’,Q). Ptch1LacZ/LacZ;Ptch2-/-;Smo-/- nEBs lose ventral identity and express Pax7 (I,I’,R). Scale bar is 100 µm. (S) Nkx2.2 and Isl1/2 expression in Ptch1LacZ/LacZ;Ptch2-/- nEBs is quantified (box and whisker) in the presence of 0–300 nM cyclopamine. (T) Ptch1+/LacZ;Shh-/- and Ptch1LacZ/LacZ;Shh-/-, but not Ptch1LacZ/LacZ;Ptch2-/-;Shh-/- retain their ability to respond to exogenously supplied ShhN by inducing Nkx2.2 expression. (U) Fibroblast-like cells derived from Ptch1LacZ/LacZ;Ptch2-/- mESCs were transfected with a Gli:luciferase construct alone or together with Ptch1. Independently, Ptch1LacZ/LacZ;Ptch2-/- cells were mock transfected or transfected with ShhN. Gli:Luciferase is quantified in co-cultures. Ptch1LacZ/LacZ;Ptch2-/- cells expressing Ptch1 can respond to ShhN supplied in co-cultured cells. p-value is indicated, n = 6. Variance is s.e.m. in A–U.