ignaling. Although expression of these genes has been previously reported in the development of the chick hindlimb, the present study demonstrates that Irx1 and Irx2 are coordinately expressed and regulated during chick embryo hindlimb development as occurs in mouse, Xenopus and zebrafish embryos lending support to the notion that the genomic architecture of Irx clusters is conserved in vertebrates. 31 HH, 
down-regulation of both genes persisted in the first and second interdigital Vatalanib tissues and eventually disappeared in all interdigits and the presence of active caspase 3 is evident. In accordance with other reports, Irx1 and Irx2 were expressed in the prospective and presumptive joint sites and in the boundary between cartilage and non-cartilage tissue. RA Down-Regulates Irx1 and Irx2 Expression Before the Onset of Cell Death In order to determine whether down-regulation of Irx genes was associated with the onset of cell death in interdigital tissue, here was evaluated the role of RA and BMP on promotion of cell death and on regulation of Irx genes. RA and BMPs are potent promoters of cell death during interdigital regression. Beads soaked in the pro-apoptotic factors RA and BMP7 were placed in the third interdigit at stage 27 HH. It was observed that after 8 h, RA-treatment began to inhibit Irx1 and Irx2 expression in 9 out of 12 experimental cases. Remarkably, this inhibition occurred before the appearance of the first signs of cell death, which were first observed after 12 h of RA-treatment. In contrast, BMP7 or NOGGIN did not regulate Irx1 or Irx2 at 8 h neither at longer treatments. As control of functionality of the proteins, it was observed that cell death was induced by BMP7 or inhibited by NOGGIN, at 8 and 12 h. To confirm that BMP signaling was not involved in Irx1 and Irx2 regulation induced by RA, one bead soaked in RA and another in NOGGIN were simultaneously placed in the third interdigit. Results showed that under these conditions NOGGIN at 8 h or up to 24 h never repressed the inhibitory effect of RA on Irx1 and Irx2 expression, as control of functionality of the protein it was observed that cell death promoted by RA was inhibited by NOGGIN at 8 and 12 h, indicating that protein was functionally active. Control beads never induced cell death. The expression of Irx1 and Irx2 was observed in skeletal primordia and as RA is known to inhibit chondrogenesis and promote cell death, the role of RA on regulation of Irx1 and Irx2 at the digital rays was evaluated. Results showed that Irx1 and Irx2 expression began to be inhibited from 4 h posttreatment before cell death induction that was evident from 12 h post-treatment, correlating with Sox9 down-regulation. TGFb Regulates Irx1 and Irx2 Expression during Chondrogenesis On the basis that RA and TGFb have antagonistic functions in the control of chondrogenesis and cell death, and that in the present study the concomitant down-regulation of Irx1, Irx2 and Sox9 expression occurred after RA treatment, the role of TGFb in the regulation of Irx1 and Irx2 was 9346307 evaluated at the interdigital tissue during ectopic digit formation by performing two experiments. The first experiment examined if TGFb regulated Irx1 and Irx2 expression at developmental stages before normal down-regulation of these genes in the interdigital tissue. Thus, beads soaked 10422886 in TGFb were placed in the interdigital tissue of hindlimbs at stage 27 HH. Results showed that inhibition of Irx1 and Irx2 expression began at 4
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