Alteration of programmed cell death and gene expression by 5-bromodeoxyuridine during limb development in mice

Some chemicals are known to induce limb malformations in mice. The occurrence of limb abnormality induced by chemical reagents is due to changes in the programmed cell death (PCD). 5-Bromodeoxyuridine (BrdU) is known as a potent teratogen and has been reported to induce polydactyly and other limb malformations in rodents (DiPaolo, Science 145, 501-503, 1964). Here, we undertook the morphological and genetic analyses of fetuses with limb malformations in BrdU-treated mice, in order to investigate an alteration of gene expression that resembles that of mutant mice with similar limb malformations. The fetuses of the BrdU-treated mice exhibited preaxial polydactyly and preaxial triphalangism of the hindlimb at a high incidence. Our observations showed that the PCD in the preaxial necrotic zone was found to be delayed or absent on day 11 of pregnancy. Histological analyses of these fetuses showed that the preaxial apical ectodermal ridge (AER) of the hindlimb was hyperplastic and consisted of several irregular layers. In observation of the whole-mount in situ hybridization, we detected the anterior-extended overexpression of Hoxd-11 and Hoxd-13 genes in the mesenchyme cells and the overexpression of Fgf4 and Fgf8 genes in the anterior region of the AER of hindlimbs of BrdU-treated fetuses. Our study shows that the injection of BrdU changed the PCD and gene expression during limb development and induced time-specific limb malformations during fetal development. This examination of the changes of the PCD and gene expression will be useful markers for the investigation of toxicities and teratologieties of other chemicals now present in the world environment.