Fig. 1. The presence of the neo^r gene in the 3'untranslated region (UTR) of the Fgf8 gene has transcriptional and morphological consequences. (A) The Fgf8^H hypomorphic and Fgf8^– alleles. The neomycin phosphotransferase gene (neo^r, blue arrow) and alkaline phosphatase reporter gene (AP, yellow block arrowhead) are located in the 3'UTR of Fgf8. PCR primers used to amplify exon 5 containing reverse transcription (RT) products are shown as black arrowheads (P1 and P2). LoxP sites are shown as red arrowheads. The null allele lacks Fgf8 exon 5 as previously described (Moon and Capecchi, 2000). If recombined by Cre recombinase, the Fgf8^H allele produces the AP reporter regulated by the Fgf8 promoter. (B) Semi-quantitative RT/PCR of cDNAs prepared by reverse transcription of mRNA isolated from E9.5 embryos. The amount of exon 5 containing Fgf8 mRNA is decreased in Fgf8 mutants (Fgf8^H/–, white arrowheads) when compared with wild type (Fgf8^+/+), null heterozygote (Fgf8^+/–) or hypomorph allele heterozygote (Fgf8^H/+) littermate controls. The results displayed show the amplification products obtained with amplification cycles of X or X+1 to assure that the amplification was performed within the linear range for each product and that the signal obtained was not saturated. The control panel shows RT-PCR products using GAPDH primers and confirms that specimens from the Fgf8^H/– mutant embryos contain comparable amounts of total mRNA with controls, although the amount of Fgf8 mRNA in Fgf8^H/– compound heterozygous, hypomorphic mutants is markedly decreased. (C) Newborn Fgf8^H/– mutant and Fgf8^H/+ control. The mutant is smaller, cyanotic and edematous with obvious craniofacial abnormalities (arrowheads). All of the mutants had CNS and mandibular malformations, 30% had a cleft palate. (D-G) Skeleton preps of Fgf8^H/– mutant and control. (E) The mutant exhibits micrognathia, fusion of the maxilla (mx), jugal bone (jg) and mandible (mb), and absence of the tympanic ring (t, yellow arrowhead). (G) Cleft palate (arrowhead) in a mutant resulting from failure of fusion of the palatine shelves (pl) with the maxillary shelves (ms).