Fig. 1. Expression pattern and targeted homologous recombination of the Chrd gene. (A-F) Whole mount in situ hybridization with Chrd probe. (A) At E7, the expression domain of Chrd extends from the rostral limit of the notochord to the node. (B,C) As gastrulation progresses and the node moves posteriorly, Chrd expression in the newly formed axial mesendoderm is maintained. (D,E) After the embryo turns, Chrd transcripts are present in the dorsal endoderm adjacent to the notochord; the arrowhead indicates the level of the section in E. (F) By late E 8.5, the axial expression of Chrd has disappeared from most of the trunk and tail, but it is still strong in the pharynx, chordoneural hinge of the tailbud and postanal gut. (G) Schematic representation of the chordin locus (top), targeting construct (middle) and mutant allele (bottom). The approximate location of the four cysteine-rich repeats is indicated by boxes. The position of the primers used in the genotyping reactions (A,A',B,B') is indicated by arrows. The thick black bars indicate the location of the probe used to distinguish between recombination events occurring 5' or 3' of the stop codons placed at the SfiI site. (H) Southern blot analysis of genomic DNA from two targeted cell lines (E2 and D9) and wild-type, heterozygous and homozygous mutant embryos digested with EcoRV. The 17 kb band corresponds to the wild type allele. The 3 kb band results from an homologous recombination event 5' of the stop codons inserted at SfiI. (I) PCR analysis of embryos obtained from matings between heterozygous mice. (A,A') PCR amplifications used in the original ES cell screening. (B,B') Amplifications used to genotype the animals during the study. (J) In situ hybridization analysis of Chrd and Bmp4 expression in wild-type and Chrd^-/- embryos. Both antisense probes were transcribed from full-length cDNA clones. The arrowhead indicates the lack of Chrd transcripts in the node of the mutants. The maintenance of normal Bmp4 expression in the extra-embryonic region of the embryos serves as an internal control for the in situ procedure.

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