(A–L) Etv4 and Etv5 expression in E10.75 (A–H) or E11.5 (I–L) limb buds. Arrowheads indicate anterior boundary of Fgfr-inactivated domain as identified both by downregulation of Fgfr1 and FGF readout Spry4 expression (data not shown) (Verheyden et al., 2005; Verheyden and Sun, 2008).

(A) Etv5 targeting strategy where loxP sites flank exons 10 and 11. Germline FLP or cre mice were used to generate Etv5^fl or Etv5⁻ allele, respectively. The majority of Etv5^−/− animals die shortly after birth. This lethality was not reported in an existing mutant of Etv5 (Chen et al., 2005), suggesting that our Etv5⁻ is a more severe loss-of-function allele. (B) Luciferase (Turo-luc) assay. Reporter fold induction is normalized to activity when empty vector is transfected (column 1). Expression of mutant ETV5 alone does not lead to a change (column 2 versus 1, n=3, p=0.498). Expression of mutant ETV5 in the presence of wild-type ETV5 does not lead to downregulation of reporter activity compared to wild-type ETV5 alone (column 4 versus 3, n=3, p=0.0483). (C) qRT-PCR of Etv5 in prospective hindlimb field of E9.5 embryos to detect intact, but not mutated transcripts. The level of transcripts in mutant tissue is 2.5±1.8(SD)% (n=3) of that in Etv5^fl/+control (n=3). (D) RT-PCR of Etv5 and β-actin in E10.0 limb buds. Two-tailed student t-test was used and error bars in all figures represent standard deviation (SD).

(A–E) Expression of Mkp3 and Spry2 is normal in Tcre;Etv mutant limb buds as assayed by RNA in situ hybridization in E11 (A–D) or qRT-PCR in E10.75 (37–39 somite stage) hindlimb buds (E)(p=0.49 for Mkp3, n=3; p=0.46 for Spry2, n=3). (F–H) Postnatal day 1 Tcre;Etv hindlimbs are normal along the P-D axis as confirmed by measurements of the three segments (n=6; p=0.24, 0.17 and 0.32 for S, Z and A′, respectively). (I–N) Tcre;Etv hindlimbs exhibit triphalangeal extra digits anteriorly (asterisks), and frequent digit 1 transformation from biphalangeal to triphalangeal (1′, n=8/12). Anterior digits in I, K and M are magnified in J, L and N, respectively. (O,P) Sox9 expression in E12.5 limb buds indicates extra digit (arrow) and enlarged digit 1′ primordium. (Q–T) Lysotracker Red staining. Arrows indicate normal cell death. Abbreviations: autopod (A′), metatarsal (mt), stylopod (S) and zeugopod (Z).

(A–N) Gene expression in E11.5 hindlimb buds. Arrowheads indicate ectopic anterior expression. B, D are anterior views of limb buds in A, C, respectively. (O–R) Non-overlapping Fgf4 (AER) and Gli1 (mesenchyme) expression at indicated somite (so) stages. Arrowhead indicates ectopic Gli1. (S–V) Hindlimb skeleton. Boxed regions in S and T are magnified in U and V. (W) Changes of Gli1 and Ptch1 expression in cultured anterior limb mesenchyme cells as assayed by qRT-PCR and normalized to their levels without culture (n≥3 for each time point and condition).