Sections were cut 5 mm thick and stained with hematoxylin and eosin or Pirosirius Red

h of TGFb treatment. Similarly, the second experiment was performed in ectopic digit at 29 HH to explore if TGFb was able to regulate Irx1 and Irx2 expression, which normally disappeared from the interdigital tissue. Results showed that Irx1 expression began after Results Down-Regulation of Irx1 and Irx2 in Interdigital Tissue Coincides with the Onset of Cell Death Expression of Irx1 and Irx2 was evaluated during hindlimb development from stages 2431 HH. In this study both genes were coordinately expressed at stage 2428 HH first in the posterior region and uninterruptedly in both prospective digital and interdigital regions. Here, down-regulation of Irx1 and Irx2 in the third interdigit began at stage 29 HH coinciding with the onset of cell death. At stage 30 Regulation of Irx Genes during Limb Development 3 Regulation of Irx Genes during Limb Development in 50 ng/ml TGFb were placed at the tip of digits at stage 27 HH. We preferred to use this concentration because it had similar effects that 75 ng/ml TGFb. Under these conditions, the expression of Sox9 in the phalanx was enlarged showing a rounded appearance. Irx1 and Irx2 10501907 expression seemed inhibited over the bead from 8 h, while below it the expression was noted as a transversal line in the phalanx. After 24 h of TGFb-treatment, Irx1 and Irx2 expression appeared around the enlarged and rounded cartilage that is Sox9 positive, resembling a perichondrium. Irx1 and Irx2 Expression is Regulated by FGF Signaling An observation obtained from the Irx1 and Irx2 gene expression pattern is that they were not expressed in the AIC316 undifferentiated zone beneath AER. It is known that this zone is dependent on FGF signaling. Because in this study Irx1 and Irx2 are coordinately expressed and regulated by RA and TGFb, we solely evaluated the expression of Irx2 in the undifferentiated zone beneath AER region to determine whether non-expression of Irx2 is a consequence of inhibition by FGF signaling. Beads soaked in the FGFR-selective inhibitor SU5402 were placed in the undifferentiated zone beneath AER of hindlimbs at stage 24 HH. This treatment was not able to induce Irx2 expression at 2, 4 h, or later, instead it resulted in activation of cell death that was evident at low level from 2 h but it extended from 4 h. Because this analysis did not allow to determine whether the inhibition of FGF signaling regulate Irx2 expression, we determined whether beads soaked in FGF8 or FGF10, placed in posterior-distal region of hindlimbs at 9305921 stage 24 HH, have effects on Irx2 expression. Results showed that its expression began to be inhibited by FGF8 and FGF10 from 4 h and 8 h, respectively. Discussion Findings reported by McDonald et al show the expression of Irx1 present in the digit-forming region and later in the digits, while Irx2 is highly expressed throughout the digital plate of the chick embryo. In contrast with that study, the present study showed that Irx1 and Irx2 were coordinately expressed during hindlimb development, as shown in other animal models. The two genes were expressed as a continuous label comprising well-defined areas of mesodermal tissue of both prospective digital and interdigital areas, and later in digital and interdigital regions. In fact, the Irx2 probe used in the present study was also used in the mentioned report. Probably, the high concentrations of proteinase K and the elimination of the Iro and homeo box from both probes allowed us to obtain a specific expression of both genes in