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Fig. 1. Targeted mutation of Mixl. (A) Genomic organization of the Mixl1 locus and structure of the targeting vector. Boxes indicate exons, coding sequence is filled, with the homeodomain indicated by lighter shading. A mutant allele (Mixl1–) is produced by replacing exon 1 downstream of the ATG with an EGFP loxP-MC1-neo-loxP cassette. Excision of the floxed MC1neo sequence by Cre-recombinase generates the allele Mixl1{Delta}neo. Positions of 5' and 3' genomic DNA probes and PCR primers are indicated. The positions of restriction enzymes are shown, together with the sizes of diagnostic restriction fragments (HIII, HindIII; RI, EcoRI). (B) Southern blot analysis of HindIII-digested DNA purified from wild-type, Mixl1+/– and Mixl1+/{Delta}neo mice, probed with the 3' probe. The 11 kb band corresponds to the wild-type Mixl1 allele. The 4.7 kb band identifies the Mixl1 allele after homologous recombination with the targeting construct. The targeted allele, after CRE-mediated excision of the neomycin resistance cassette, gives rise to a 3.7 kb band. WT, wild type. (C-F) GFP expression in Mixl1+/– (+/–) (C,E) and Mixl1–/– (–/–) (D,F) 7.5 dpc embryos visualized by confocal microscopy. High levels of GFP expression are found in the primitive streak and lateral mesoderm of the Mixl1–/– embryo. By contrast, a lower and more uniform expression of GFP in the mesoderm is observed in the Mixl1+/– 7.5 dpc embryo. (E) Transverse section of a Mixl1+/– 7.5 dpc embryo through the middle level of the primitive streak, showing GFP expression in the mesoderm. (F) Transverse section of a Mixl1–/– embryo reveals GFP expression in the ingressing cells (arrowhead) in the thickened primitive streak (ps) and the nascent mesoderm (lm) located lateral to the primitive streak.