Gland as compared with the infundibulum and the JSI124 site magnum (Figure 1A). Further, quantitative PCR analysis revealed that WNT4 mRNA levels in the 1418741-86-2 web isthmus and the shell gland were 3.59- and 3.29-fold (P,0.01) greater, respectively, than for the infundibulum, and its expression decreased 0.16-fold in the magnum (Figure 1B). To determine localization of WNT4 mRNA in the chicken oviduct, in situ hybridization analysis was performed (Figure 1C). The WNT4 mRNA was most abundant in stromal cells and luminal epithelia (LE) of the isthmus and the shell gland, respectively. However, little or no mRNA was detected in the infundibulum and the magnum of the chick oviduct.expression of WNT4 mRNA in the chicken oviduct in the present study. As illustrated in Figure 2A and 2B, expression of WNT4 mRNA increased in DES-treated oviducts as compared with untreated oviducts. Further, quantitative PCR analysis confirmed that WNT4 expression increased 1.6-fold (P,0.05) in DES-treated as compared to control oviducts (Figure 2C). In addition, DES treatment stimulated 4.1- and 12.3-fold increases (P,0.001) in WNT4 mRNA in the isthmus and the shell gland, respectively (Figure 2D). To determine localization of WNT4 mRNA in chick oviducts treated with DES, in situ hybridization analysis was used to reveal that WNT4 mRNA is expressed predominantly expressed in GE of the isthmus and the shell gland (Figure 2E). There was little or no detectable WNT4 mRNA in the infundibulum and magnum.Post-transcriptional regulation of microRNA affecting WNTTo demonstrate the possibility that expression of WNT4 is affected through the post-transcriptional regulation by miRNAs, we performed a miRNA target validation assay. We identified potential miRNA binding sites within the 39-UTR of the WNT4 gene using the miRNA target prediction database (miRDB; http://mirdb.org/miRDB/) which revealed only one putative binding site for miR-1786. Therefore, we determined whether miR1786 influenced expression of the WNT4 gene via its 39-UTR. As illustrated in Figures 3C and 3D, the expression level of GFPexpressing cells decreased 33.5 (P,0.05) in the presence of miR1786, as compared with control values based on FACS and fluorescence microscopy analyses. In addition, miR-1786 expression was reduced 75 (P,0.01) in the DES-treated oviducts as compared to untreated oviducts of chicks through miRNA-specific quantitative RT-PCR analysis (Figure 3E). These results reveal that miR-1786 regulates WNT4 expression post-transcriptionally in vivo by binding directly to the WNT4 transcript.Expression and localization of WNT4 in the chicken oviduct at different stages of the laying cycleWe previous reported spatial and temporal changes in gene expression in the oviduct of laying hens at different stages of the laying cycle [8]. In order to detect cell-specific localization of WNT4 mRNA in the chicken oviduct between 3 h and 20 h after ovulation, RT-PCR, quantitative PCR and in situ hybridization analyses were performed. As illustrated in Figure 1D, RT-PCR analysis detected the highest level of WNT4 mRNA expression at 3 h post-ovulation in the shell gland and lowest expression at 20 h post-ovulation in the shell gland, but little or no detectable WNT4 mRNA in the magnum at either time point. In addition, quantitative PCR analysis revealed that expression of WNT4 mRNA was 3.32-fold (P,0.001) at 3 h than at 20 h post-ovulation in the shell gland, but changes in expression of WNT4 mRNA were not different between 3 h an.Gland as compared with the infundibulum and the magnum (Figure 1A). Further, quantitative PCR analysis revealed that WNT4 mRNA levels in the isthmus and the shell gland were 3.59- and 3.29-fold (P,0.01) greater, respectively, than for the infundibulum, and its expression decreased 0.16-fold in the magnum (Figure 1B). To determine localization of WNT4 mRNA in the chicken oviduct, in situ hybridization analysis was performed (Figure 1C). The WNT4 mRNA was most abundant in stromal cells and luminal epithelia (LE) of the isthmus and the shell gland, respectively. However, little or no mRNA was detected in the infundibulum and the magnum of the chick oviduct.expression of WNT4 mRNA in the chicken oviduct in the present study. As illustrated in Figure 2A and 2B, expression of WNT4 mRNA increased in DES-treated oviducts as compared with untreated oviducts. Further, quantitative PCR analysis confirmed that WNT4 expression increased 1.6-fold (P,0.05) in DES-treated as compared to control oviducts (Figure 2C). In addition, DES treatment stimulated 4.1- and 12.3-fold increases (P,0.001) in WNT4 mRNA in the isthmus and the shell gland, respectively (Figure 2D). To determine localization of WNT4 mRNA in chick oviducts treated with DES, in situ hybridization analysis was used to reveal that WNT4 mRNA is expressed predominantly expressed in GE of the isthmus and the shell gland (Figure 2E). There was little or no detectable WNT4 mRNA in the infundibulum and magnum.Post-transcriptional regulation of microRNA affecting WNTTo demonstrate the possibility that expression of WNT4 is affected through the post-transcriptional regulation by miRNAs, we performed a miRNA target validation assay. We identified potential miRNA binding sites within the 39-UTR of the WNT4 gene using the miRNA target prediction database (miRDB; http://mirdb.org/miRDB/) which revealed only one putative binding site for miR-1786. Therefore, we determined whether miR1786 influenced expression of the WNT4 gene via its 39-UTR. As illustrated in Figures 3C and 3D, the expression level of GFPexpressing cells decreased 33.5 (P,0.05) in the presence of miR1786, as compared with control values based on FACS and fluorescence microscopy analyses. In addition, miR-1786 expression was reduced 75 (P,0.01) in the DES-treated oviducts as compared to untreated oviducts of chicks through miRNA-specific quantitative RT-PCR analysis (Figure 3E). These results reveal that miR-1786 regulates WNT4 expression post-transcriptionally in vivo by binding directly to the WNT4 transcript.Expression and localization of WNT4 in the chicken oviduct at different stages of the laying cycleWe previous reported spatial and temporal changes in gene expression in the oviduct of laying hens at different stages of the laying cycle [8]. In order to detect cell-specific localization of WNT4 mRNA in the chicken oviduct between 3 h and 20 h after ovulation, RT-PCR, quantitative PCR and in situ hybridization analyses were performed. As illustrated in Figure 1D, RT-PCR analysis detected the highest level of WNT4 mRNA expression at 3 h post-ovulation in the shell gland and lowest expression at 20 h post-ovulation in the shell gland, but little or no detectable WNT4 mRNA in the magnum at either time point. In addition, quantitative PCR analysis revealed that expression of WNT4 mRNA was 3.32-fold (P,0.001) at 3 h than at 20 h post-ovulation in the shell gland, but changes in expression of WNT4 mRNA were not different between 3 h an.
