Fig. 1. Retroviral insertional mutation of CNBP resulted in anterior patterning and craniofacial defects. (A) Morphology of newborn wild-type mouse. (B) A heterozygote newborn mouse with a short snout and lacking eyes. (C,D) Heterozygotes with a smaller lower jaw (C) and missing eyes (D). (E) A homozygote lacking rostral head structures, including the entire forebrain. (F,G) As early as E7.5, a homozygous mutant embryo is smaller than its wild-type littermate. A constriction is observed between embryonic and extra-embryonic regions in Cnbp^-/- mutants (arrow in G). (H,I) By E8.5, forebrain truncation is evident in mutant embryo (arrow in I). (J,K) At E9.5, Cnbp^-/- embryos were smaller with forebrain truncations. (L) The integration site of the provirus in the Cnbp gene locus. The flanking sequence, 5' fA8, was cloned by inverse PCR. The Cnbp gene was cloned and characterized by using 5' fA8 as probe. The positions of the 5' fA8 probe and primers 1, 2 and 3 (P1, P2, and P3) for genotyping are shown. (M) Genotype analysis of Cnbp mutant mice by Southern blot. The presence of a single 4 kb fragment represents wild-type allele, while a larger 8 kb fragment, a result of the proviral insertion, represents a mutant allele. (N) Genotype analysis of E6.5 embryos by PCR demonstrates the recovery of wild-type (lanes 1, 5), heterozygous (lanes 3, 4, 6, 8) and homozygous embryos (lanes 2, 7). Primers P1 and P3 amplify a 500 bp wild-type fragment, whereas primers P2 (a Neo insertion-specific primer) and P3 together amplify a 300 bp mutant fragment. (O) Northern blot analysis of total RNA isolated from E9.5 whole embryos derived from Cnbp-heterozygous mutant parents. A 1.65 kb mRNA was detected in the wild-type and heterozygous embryos but was undetectable in the homozygous embryos using Cnbp cDNA as a probe. (P,Q) Immunostaining in tissue sections was performed, using an anti-CNBP polyclonal antibody, to examine CNBP protein levels in E7.25 wild-type and mutant embryos. CNBP protein was localized to the ANE and ADE of a wild-type embryo (brown staining in P) but was absent in the Cnbp^-/--mutant embryo (Q).