PCNA in situ hybridization:: a novel and reliable tool for detection of dynamic changes in proliferative activity

In order to investigate developmental processes, several methods have been established that allow the visualization of local proliferation zones and to follow their dynamics during morphogenesis. In this study we present a detailed description of transitory and continuous proliferation zones in the developing chick embryo. By tracing the S-phase marker proliferating cell nuclear antigen (PCNA) at the mRNA level we were able to identify the initiation and termination of proliferation programs. This approach provides additional information in comparison to the well-known BrdU incorporation or the PCNA immunostaining, which exclusively labels cells that contain PCNA protein. By means of PCNA in situ hybridization we analyzed the normal expression pattern in the 2- to 5-day-old chick embryo. We furthermore monitored the effects on PCNA expression after various manipulations such as removal of the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the surface ectoderm. In addition, we applied morphogens, such as fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and retinoic acid (RA), and subsequently analyzed changes in the pattern of PCNA expression. While ablation of ZPA, AER, or ectoderm are known to reduce cell proliferation and were paralleled by loss of PCNA expression, neither BMP-2 nor BMP-4 affected PCNA expression. Upregulation of PCNA expression could be achieved by application of RA or FGFs, factors known to induce cell proliferation during limb bud outgrowth. The PCNA in situ hybridization data presented here clearly show that this method offers a novel, very sensitive tool for tracing cell proliferation and for visualizing the dynamic patterns arising due to the initiation and termination of the proliferation program.