Fig. 2. Endodermal WNT-β-catenin signaling is required for GT initiation. (A-C) SEM analysis showing an absence of GT outgrowth in the Shh^cre/Gfp;β-Cat^c/c embryo (B) and a larger GT in the Shh^cre/Gfp;β-Cat^loxEx3 embryo (C). (D-F) β-Catenin indirect immunoflouresence showing that the protein was detected mainly on the cell membranes of both GT ectoderm and UE, as well as weakly in the mesechyme (D). Complete removal of β-catenin in the UE was confirmed in Shh^cre/Gfp;β-Cat^c/c GT (arrows, E), and ectopic accumulation of β-catenin was observed in Shh^cre/Gfp;β-Cat^loxEx3 endoderm (arrow, F). (G-I) Whole-mount Fgf8 in situ hybridization showing expression in the distal cloacal endoderm in control embryos (G), but not in Shh^cre/Gfp;β-Cat^c/c embryos (arrows, H); in Shh^cre/Gfp;β-Cat^loxEx3 GT, Fgf8 expression is ectopically expanded (I). (J,K) Whole-mount Bmp4 in situ hybridization showing a reduction in Shh^cre/Gfp;β-Cat^c/c cloacal mesenchyme (K). (M-O) TUNEL analysis showing increased cell death in Shh^cre/Gfp;β-Cat^c/c cloacal endoderm and ectopic apoptotic cells in the surrounding mesenchyme (arrows in N). (P-R) PHH3 immunostaining revealing markedly reduced cell proliferation in E10.5 Shh^cre/Gfp;β-Cat^c/c cloacal endoderm. (S-U,W,X) Fgf8 in situ hybridization showing an absence of expression in E10.5 Msx2-Cre;β-Cat^c/c limbs (T) and ectopic expression in the flank ectoderm and dorsal limb ectoderm in Msx2-cre;β-Cat^loxEx3 embryos (U). (V) Note that ectopic Fgf8 expression appears to correspond to Msx2-Cre expression (arrows in U,V). At E12.5, ectopic outgrowth was observed in the inter-limb region of Msx2-Cre;β-Cat^loxEx3 embryos (arrows, X). Scale bars: 100 µm in A-F; 50 µm in M,N,P,Q.