Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation in the mouse

Lim1 is a homeobox gene expressed in the extraembryonic anterior visceral e ndoderm and in primitive streak-derived tissues of early mouse embryos. Mic e homozygous for a targeted mutation of Lim1 lack head structures anterior to rhombomere 3 in the hindbrain, To determine in which tissues Lim1 is req uired for head formation and its mode of action, we have generated chimeric mouse embryos and performed tissue layer recombination explant assays. In chimeric embryos in which the visceral endoderm was composed of predominant ly wild-type cells, we found that Lim1(-/-) cells were able to contribute t o the anterior mesendoderm of embryonic day 7.5 chimeric embryos but that e mbryonic day 9.5 chimeric embryos displayed a range of head defects, In add ition, early somite stage chimeras generated by injecting Lim1(-/-) embryon ic stem cells into wild-type tetraploid blastocysts lacked forebrain and mi dbrain neural tissue. Furthermore, in explant recombination assays, anterio r mesendoderm from Lim1(-/-) embryos was unable to maintain the expression of the anterior neural marker gene Otx2 in wild-type ectoderm. In complemen tary experiments, embryonic day 9.5 chimeric embryos in which the visceral endoderm was composed of predominantly Lim1(-/-) cells and the embryo prope r of largely wild-type cells, also phenocopied the Lim1(-/-) headless pheno type, These results indicate that Lim1 is required in both primitive streak -derived tissues and visceral endoderm for head formation and that its inac tivation in these tissues produces cell non-autonomous defects. We discuss a double assurance model in which Lim1 regulates sequential signaling event s required for head formation in the mouse.