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Fig. 1. Cripto expression and gene targeting. (A-C) Expression of Cripto in nascent mesoderm, node and axial mesendoderm of a wild-type late-streak stage embryo; frontal section in B is counterstained with Methyl Green. (C) High-power view of boxed region in B shows expression in midline mesendodermal cells, but not in epiblast or visceral endoderm cells being displaced during gastrulation. (D,E) Widespread ß-galactosidase staining in a heterozygous Cripto^lacZ/+ embryo at the neural plate (late allantoic bud) stage; transverse section in E shows staining in the nascent mesoderm and definitive endoderm, with most intense staining in the axial mesendoderm; no expression is detected in the primitive streak or neuroectoderm. (F-H) Generation of hypomorphic and null alleles of Cripto. (F) The targeting strategy for the Cripto^3loxP allele inserts a floxed PGK-neo cassette into the intron between Cripto exons 5 and 6. Following germline transmission, the PGK-neo cassette was deleted by crossing the Cripto^3loxP/+ mice with EIIa-Cre transgenic mice; complete excision results in the null allele Cripto^del. (G) Southern blot detection of the targeted allele in ES genomic DNA digested with XbaI. An 8 kb fragment is detected by probe A for the wild-type allele, and a 5.5 kb fragment (arrow) is detected for the Cripto^3loxP allele. (H) Southern blot detection of the targeted allele using probe B in ES genomic DNA digested with HindIII; 11 kb (wild-type) and 6.8 kb (targeted) fragments are detected. Positions of molecular standards at 10 and 5 kb are indicated. Scale bars: 100 µm in A-D; 50 µm in E. ame, axial mesendoderm; B, BamHI; de, definitive endoderm; E, EcoRI; epi, epiblast; H, HindIII; mes, nascent mesoderm; nd, node; ne, neuroectoderm; ps, primitive streak; ve, visceral endoderm; X, XhoI; Xb, XbaI.

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