Fig. 2. The Ap2-IRESCre driver ablates FGF8 in the developing pharyngeal arch (PA) ectoderm. (A) A 12 kb genomic fragment containing exons 6, 7, the 3' UTR and polyadenylation signal from the Ap2 locus was used to generate the targeting vector for homologous recombination in ES cells. (B) The targeted allele contains the IRESCre cassette (see Materials and methods), positioned 198 bp 3' of the translation stop in an engineered AscI site. (C) AP2-IRESCre was tested for Cre activity by crossing to the Rosa26^lacZ reporter strain. 12, 21 and 35 somite stage embryos, with the genotype AP2^IRESCre/+; Rosa26^lacZ/+, were assayed for ß-galactosidase activity (blue staining). Somite stages (ss) are labeled in the lower right corner of each panel and PAs are numbered. Blue staining, and both yellow and red arrowheads denote regions of Cre activity in the ectoderm of the PAs as they develop; Cre activity in the caudal ectoderm that will form PAs 3-6 is highlighted by the large red arrowhead in the 12 and 21 ss embryos. (D) Functionally relevant recombination of the Fgf8^AP conditional reporter allele in the developing PA ectoderm by the AP2-IRESCre driver. A whole-mount, 21 ss Fgf8^AP/+;AP2^-IRESCre/+ embryo is shown in the left panel after assaying for alkaline phosphatase activity. The black line indicates the plane of the coronal section shown in the right panel. The ectoderm of developing PA 3, and that of PAs 1 and 2, are stained violet because of Fgf8^APR activity. Although AP2-IRESCre is expressed in neural crest, the ectomesenchyme of the PAs is not stained because Fgf8 is not expressed in these cells. (E) AP2-IRESCre ablates Fgf8 function throughout its expression domains in the PA ectoderm. Expression of Fgf8^GFPR after recombination with the AP2-IRESCre (left panel) versus universal `deleter' Cre driver (right panel) (Schwenk et al., 1995), was assessed by whole-mount anti-GFP immunohistochemistry of 22 ss, stage-matched embryos (ss in lower right corner). The domains of Fgf8 inactivation resulting from the AP2-IRESCre driver are the same ectodermal domains detected with the universal `deleter' Cre. Fgf8^GFPR is expressed in caudal ectoderm that will form PAs 3-6. (F,G) Coronal sections through PAs 1 and 2, and the developing third arch region of a 20 ss embryo. Fgf8^GFP/+;AP2^IRESCre/+ embryo (G) reveals Fgf8^GFPR expression throughout the ectoderm of the developing third arch and cleft.