Fig. 3. hoxa3-IRESCre ablates Fgf8 function in the third and fourth PA endoderm and ectoderm. (A) 11 kb of murine hoxa3 genomic sequence extending from a 5' Sau3AI site to an EcoRI site 3' of the stop codon in exon 2 was used to create the targeting vector. (B) The targeted allele contains the IRESCre cassette (red box) inserted in an ApaI site four bases 3' of the stop codon. Function of the hoxa3-IRESCre allele assayed with the Rosa26^lacZ reporter. (C) Whole-mount and sectioned preparations of Rosa26^lacZ/+; hoxa3^IRESCre/+ embryos stained for ß-galactosidase activity (blue staining) at the 10, 16, 20, 23 and 26-28 ss reveal the caudal-to-rostral progression of hoxa3-IRESCre activity and anterior limit at the rhombomere 4/5 boundary and the anterior border of PA3. The relatively dorsal plane of the coronal sections through the PAs is demonstrated by the black line labeled d in whole-mount panel 20. These results demonstrate hoxa3-IRESCre activity in all three tissue layers of developing PAs 3-6. PAs are numbered; ss are noted in lower right corner of each panel. 2p, PA2 pouch; en, endoderm. (D,E) Functionally relevant recombination of Fgf8^GFP in the developing PA epithelia by hoxa3-IRESCre occurs throughout the Fgf8 PA3-6 epithelial expression domains. (D) Coronal sections through the PAs of different stage Fgf8^GFP/+; hoxa3^IRESCre/+ embryos were assayed for GFP using fluorescent immunohistochemistry. Somite stages (ss) are labeled in the lower right corner of each panel followed by a letter indicating relative plane of each section within the arch of interest; v, ventral; d, dorsal (plane illustrated in Fig. 3C, whole-mount panel 20, black lines labeled v or d). PAs are numbered; 3p, PA3 pouch, etc. Fgf8^GFPR expression is initially detected throughout the epithelia of developing PA3 and then is lost from the rostral endoderm (yellow arrowheads, sections 24v, d and 27v, d). Because the Rosa26^lacZ reporter studies in Fig. 3C indicate that hoxa3-IRESCre is active throughout the endoderm of PA3 from the 20 ss, this loss of Fgf8^GFPR expression in rostral endoderm reflects a change in the Fgf8 expression domain, not failure of hoxa3-IRESCre expression in these cells. (E) GFP expression was assayed in coronal sections of Fgf8^GFP/+; deleterCre embryos. Stage-matched embryos to those shown in D were assayed for GFP. Note that the expression of GFP from the globally recombined Fgf8^GFPR allele entirely recapitulates that seen in the PAs of Fgf8^GFP/+; hoxa3^IRESCre/+ embryos, including the loss of expression at later stages in the rostral endoderm of PA3 (yellow arrowheads, sections 24v,d and 27v,d). These studies confirm that hoxa3-IRESCre ablates Fgf8 function throughout its PA epithelial expression domains from at least 20 ss. Labels are as noted for D.