ARPP19, but not ENSA, is essential for embryogenesis. (A) The knockout first (KO-first) allele (Tm1a allele) was converted into a conditional allele that does not harbor the selection cassette by mating heterozygous Arpp19^{tm1a(KOMP)Mbp} mice with Gt(ROSA)26Sor^{tm1(FLP1)Dym/Wtsi} mice. Exon 4 of the murine Arpp19 gene was then flanked by LoxP sequences (violet triangles), and Cre-mediated recombination resulted in Arpp19-null alleles. (B) Representative PCR products of WT Arpp19 and floxed alleles (Lox). (C) Number (percentage) of live births and embryos recovered at the indicated developmental stages from heterozygous crosses between Arpp19^Wt/Δ mice. (D) Statistics of the different phenotypes observed for the indicated mouse genotypes. Representative images of E8.5 and E10.5 Arpp19^Lox/Lox and Arpp19^Δ/Δ embryos from heterozygous crosses confirmed by genotyping. Histological sections from the indicated embryos were stained with hematoxylin-eosin, and images were acquired with a transmitted light microscope. Magnifications are indicated in the images. (E) Immunohistochemical analysis of mitotic (M), phosphorylated histone H3 signal (pH3), and interphasic (I) cells in the epidermal basal layer of E17.5 Arpp19^Wt/Δ and Arpp19^Δ/Δ embryos. Data are the mean ± SD of two different experiments (five embryos for each genotype/experiment). Bar, 10 µm. Bar magnifications, 5 µm. (F) Representative images of E18 Arpp19^Δ/Lox and Arpp19^Δ/Δembryos in which Arpp19 was deleted in embryos by tamoxifen injection of pregnant Arpp19^Lox/Wt PolII Cre ERT2 females at E7.5 after mating with Arpp19^Lox/Lox RNApolII Cre ERT2^Ki/Ki males (three experiments). (G) Schematic representation of the Ensa KO-first allele (Tm1a) that encodes LacZ instead of the ENSA protein under the control of its endogenous promoter. (H) Distribution of genotyped embryos obtained from crossing two heterozygous Ensa KO-first mice (results from three independent intercrosses). Of note, although all embryos were normal at E8.5, the genotype distribution does not follow the Mendelian ratio owing to failure of genotyping three embryos. (I) Representative light microscopy images of Ensa WT, heterozygous, and knockout E8.5 embryos obtained from mating Ensa heterozygous Tm1a mice. Magnification is indicated in the figure.