hindlimb development. Here was observed that down-regulation of Irx1 and Irx2 expression in interdigital tissue coincided with the commencement of cell death. However, an interesting finding is that in the interdigital tissue, RA inhibited Irx1 and Irx2 expression before the first signs of active caspase 3 were observed. The role of RA in programmed cell death control during hindlimb development is well known, as well as its effects mediated by BMP signaling. In the present study, RA inhibited Irx1 and Irx2 expression by a BMP-independent mechanism. On this basis, the present study suggests that Irx1 and Irx2 expression may play a protective role against cell death. It follows that downregulation of Irx genes in the interdigital tissue by RA may be a pre-requisite to promote the molecular cascade that ends in cell death, since Irx genes may repress the expression of genes involved in the cell death process, as has been suggested for the Msx gene in the boundary between body wall and wing in Drosophila, and for 21 h of TGFb treatment, 23446639 while Irx2 did so at 19 h. Remarkably, the Irx1 and Irx2 expression pattern induced by TGFb seemed to be limited away and around the bead resembling a perichondrium as PF-562271 web compared with Sox9 expression. To ensure that the formation of the ectopic digit in the interdigital tissue is due to TGFb, gene expression of Sox9 and ColII was evaluated. Results confirmed that TGFb-treatment triggers the chondrogenesis molecular cascade because it was able to induce the onset of Sox9 expression and ColII, 30 min and 16 h after treatment, respectively, and ended in the formation of an ectopic digit as previously reported. In our hands, the implantation of beads soaked in PBS never gave rise to ectopic digits, suggesting that simply wounding into interdigital tissue did not trigger their formation. As the Irx1 and Irx2 expression pattern observed in the interdigital tissue is inhibited or induced around cartilage condensations by TGFb depending on the developmental stage, and they are expressed at the edge of skeletal elements, it is possible to speculate that Irx1 and Irx2 expression is established once the wavefront of cartilage differentiation is finished. To test this hypothesis, beads soaked in TGFb at different concentrations were placed at the interdigital tissue, and after 21 h it was observed that expression of Irx genes was established further away from the bead, in a concentration-dependent manner, although similar results were obtained at 50 and 75 ng/ml TGFb. On this basis, the following experiment was done to promote chondrogenesis on the tip of digits and then evaluate whether Irx genes would be expressed at the boundary between the new cartilage at the tip of digit and non-cartilage tissue. Beads soaked Regulation of Irx Genes during Limb Development Bmp4 during Xenopus development. This protector effect of Irx against cell death is probably lost in the limbs of fusedtoes mutant mice, which show massive cell death and upregulation of Bmp4 and Dkk1, as well as down-regulation of Fgf8 9346307 and Fgf10. Interestingly, Irx3, Irx5 and Irx6 are expressed in the interdigital tissue of 
the mouse limb as occurs for the Irx1 and Irx2 genes in the chick embryo. So, it is possible that Irx genes may be involved in cell death control. Supporting this hypothesis, in other systems the deletion of Irx genes results in cell death, for instance, Irx1a, Irx4a or Irxl1 morphants show extensive cell death in the rostral region of the ze
