Comparative molecular pathology of cadmium- and all-trans-retinoic acid-induced postaxial forelimb ectrodactyly

Cadmium chloride (CdCl2) and all-trans-retinoic acid (RA) induce postaxial forelimb ectrodactyly in C57BL/6N mice when administered during early limb development, and co-administration yields a synergistic response suggesting a common final pathway to the defect. In the current study, forelimb buds from embryos given high maternal teratogenic doses of CdCl2 or RA, or the combination of both agents at low doses were collected at various time points after treatment on GD 9.5 and examined for cellular apoptosis, proliferation, and patterning genes. Some cellular perturbations detected in the developing limb bud were similar for both teratogens, whereas other alterations were unique to each agent. For example, at 12 and 18 h, CdCl2 treatment increased apoptotic cells in the mesenchyme underneath the apical ectodermal ridge (AER), whereas RA caused apoptosis in the AER and proximal mesenchyme. Further, the combined low-dose treatment increased cell death synergistically in all three regions. CdCl2 and the low-dose combined treatment inhibited mesenchymal proliferation at 12 h, which was associated with induction of p21cip1 and inhibition of phospho-c-Jun. In contrast, RA did not inhibit mesenchymal proliferation and did not induce p21cip1 expression or change c-Jun phosphorylation. All three treatment groups showed a delay in the patterning of distal chondrogenesis centers as indicated by Sox9 expression. There was also common inhibition in the expression of AER markers, Fgf8 and Fgf4, and the mesenchymal marker Msx1 involved in the maintenance of epithelial–mesenchymal interactions. Collectively, a model is hypothesized where limb patterning can be perturbed by insults to both ectoderm and mesoderm.