Investigation of Prx1 protein expression provides evidence for conservation of cardiac‐specific posttranscriptional regulation in vertebrates

Abstract

Gene targeting experiments have defined that the homeobox gene Prx1 is essential for normal craniofacial, limb, and vascular development. Although its RNA expression pattern is well established, Prx1 protein expression in the developing embryo has not been examined. A novel Prx1 antibody was produced to define the normal Prx1 protein expression pattern in the developing mouse embryo. In craniofacial and limb mesenchyme, Prx1 protein expression is consistent with previously published data on RNA localization. However, a remarkable discrepancy was found in cardiac tissue. Prx1 protein is undetectable in the murine embryonic and adult heart, despite the presence of Prx1 transcripts. These data demonstrate that Prx1 expression is posttranscriptionally regulated. This discrepancy between the presence of Prx1 transcript and the absence of detectable protein was also observed in embryonic chick heart, suggesting conservation of the regulatory mechanism in vertebrates. This observation provides a new explanation of why the Prx null mice lack cardiac malformations.

Comparative analysis of Prx1 and Prx2 expression in mice provides evidence for incomplete compensation

“…5A and C). This lack of detectable Prx2 protein in the heart is consistent with data from our expression analysis of Prx1, in which we were unable to detect Prx1 protein in the heart despite the presence of RNA transcripts (Chesterman et al, 2001). This data is also consistent with the lack of a cardiac phenotype in the Prx gene-targeted mice (Bergwerff et al, 2000).…”

“…Cells lysates, protein gels, and protein transfer were processed as previously described (Chesterman et al, 2001). The blots were blocked in 5% milk plus 30 mg histidine.…”

“…Transient transfection, fixation, and processing were performed as previously described (Chesterman et al, 2001). Cells were incubated with anti-Prx2 antibody at a concentration of 5 g/ml, and then with goat anti-rabbit FITC-conjugated secondary antibody (Jackson ImmunoResearch) diluted 1:100.…”

“…Timed matings, embryo harvesting, embryo processing, and diaminobenzidine (DAB) staining were performed as previously described (Chesterman et al, 2001). For comparison, adjacent sections were stained with a previously characterized anti-Prx1 antibody (Chesterman et al, 2001).…”

“…For comparison, adjacent sections were stained with a previously characterized anti-Prx1 antibody (Chesterman et al, 2001). The anti-Prx2 antibody was used at a concentration of 5 g/ml, and the anti-Prx1 antibody at 1 g/ml.…”