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The  Iroquois  homeobox ( Irx ) genes play a crucial role in the regionalization and patterning of tissues and organs during metazoan development. The  Irx1  and  Irx2  gene expression pattern during hindlimb development has been investigated in different species, but its regulation during hindlimb morphogenesis has not been explored yet. The aim of this study was to evaluate the gene expression pattern of  Irx1  and  Irx2  as well as their regulation by important regulators of hindlimb development such as retinoic acid (RA), transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signaling during chick hindlimb development.  Irx1  and  Irx2  were coordinately expressed in the interdigital tissue, digital primordia, joints and in the boundary between cartilage and non-cartilage tissue. Down-regulation of  Irx1  and  Irx2  expression at the interdigital tissue coincided with the onset of cell death. RA was found to down-regulate their expression by a bone morphogenetic protein-independent mechanism before any evidence of cell death. Furthermore, TGFβ protein regulated  Irx1  and  Irx2  in a stage-dependent manner at the interdigital tissue, it inhibited their expression when it was administered to the interdigital tissue at developing stages before their normal down-regulation. TGFβ administered to the interdigital tissue at developing stages after normal down-regulation of  Irx1  and  Irx2  evidenced that expression of these genes marked the boundary between cartilage tissue and non-cartilage tissue. It was also found that at early stages of hindlimb development FGF signaling inhibited the expression of  Irx2 . In conclusion, the present study demonstrates that  Irx1  and  Irx2  are coordinately expressed and regulated during chick embryo hindlimb development as occurs in other species of vertebrates supporting the notion that the genomic architecture of  Irx  clusters is conserved in vertebrates.

Irx1 and Irx2 Are Coordinately Expressed and Regulated by Retinoic Acid, TGFβ and FGF Signaling during Chick Hindlimb Development