Fig. 1. Lrp5/6 mutant alleles and expression of Lrp5 and Lrp6 in early embryos. (A, top) Genomic structure and restriction map of the region surrounding exon 1 (black box) of the Lrp5 gene. Primers used to amplify the wild-type allele, Lrp5-U1 and Lrp5-D1, are indicated by arrows. (A, bottom) Targeting vector used to replace part of exon 1 of Lrp5 with the IRES-ß-galactosidase (ß-gal) reporter and MC1-neomycin phosphotransferase (neo) selection cassette by homologous recombination. Primers used to amplify the mutant allele, neo and Lrp5-D1 are indicated by arrows (see Materials and methods for further details). IRES, internal ribosome entry site; TK, thymidine kinase. (B, top) Genomic structure of part of the Lrp6 gene. Primers used to amplify the wild-type allele, Lrp6-U1 and Lrp6-D1 are indicated by arrows. Exons are indicated by black boxes and the arrowhead shows the position of the gene-trap insertion in intron 5. (B, bottom) Genomic structure of the mutant Lrp6 allele. The gene trap vector contains the splice acceptor (SA) of the mouse engrailed 2 gene (En2), the transmembrane domain of rat CD4 and the ß-galactosidase-neo (ß-geo) reporter. Primers used to amplify the mutant allele, Lrp6-U1 and en2 are indicated by arrows. (C) PCR genotyping of Lrp5 and Lrp6 mutations. Ethidium bromide-stained 1.5% agarose gel of DNA products produced by PCR amplification of genomic DNA using the genotyping primers shown in A and B. Numbers on the left indicate the size of the molecular weight markers in base pairs. (D) Northern blot analysis of 20 µg of total RNA isolated from adult kidneys of Lrp5^+/+ and Lrp5^-/- mice probed with the 3'UTR of Lrp5 (top) and reprobed with actin cDNA (bottom) as a loading control. (E-I) Expression of Lrp6-ß-galactosidase reporter fusion protein. Anterior is towards the left in all panels. Control embryo (E) and Lrp6^+/- embryos (F-I) were stained with X-gal. Lrp6 is widely expressed at 6.5 dpc (F) and 7.5 dpc (G-I). Line in G indicates the plane of section in H. Transverse (H) and sagittal (I) sections of 7.5 dpc Lrp6^+/- embryos showing ß-galactosidase expression in the embryonic ectoderm (ee), mesoderm (me) and definitive endoderm (de) emerging from the primitive streak (ps). (J-N) Whole-mount RNA in situ analysis of Lrp5 expression in wild-type embryos. Anterior is towards the left in all panels. Embryos were stained with sense control (J) and antisense (K-N) riboprobes. At 6.5 dpc (K), Lrp5 is expressed throughout the ectoderm. At 7.5 dpc (L-N) Lrp5 expression is highest in the visceral endoderm overlying the extra-embryonic, but not embryonic region. Line in L indicates the plane of transverse section shown in M. Transverse (M) and sagittal (N) sections of stained embryos showing Lrp5 expression in the visceral endoderm (ve), embryonic ectoderm (ee), but not in nascent mesoderm (me) or definitive endoderm (de) emerging from the primitive streak (ps).