Then we asked whether lack of ALX3 increases the vulnerability of cells to death by oxidative stress. Initially, using DAPI, we determined that exposure to a relatively high concentration of the oxidative stress-inducing agent tert-butyl-hydroperoxide (t-BOOH; 300 μM) resulted in a significant increase in the number of non viable primary MEM cells prepared from wild type embryos (Supplementary Fig. S2). Notably, the proportion of non-viable MEM cells prepared from Alx3-deficent embryos was significantly higher compared to that of wild type cells (Supplementary Fig. S2). To confirm these results, we treated MEM cells with a sub-lethal concentration of t-BOOH (100 μM) and determined the proportion of cell death in wild type and Alx3-deficient primary MEM cells using propidium iodide. In this case, exposure to t-BOOH only resulted in significantly increased death in the case of cells derived from Alx3-deficient embryos, whereas cells from wild type embryos were unaffected (Fig. 3H and Supplementary Fig. S2). These experiments indicate that embryonic mesenchyme cells are more vulnerable to oxidative stress in the absence of ALX3.