Expression And Function Analysis Of Djp53 And Djp53 Signaling Pathway From Planarian Dujesia Japonica

Planarians (Platyhelminthes, class Turbellaria) are bilaterian, triploblastic free-living flatworms best known for their impressive ability to regenerate lost body parts, which are excellent models for studying evolution of the animals, embryonic development, molecular mechanism of stem cell and regeneration. P53 of transcription factor is best known for its role as a tumor suppressor but also has functions in diverse cellular processes including the DNA damage response, cell proliferation, senescence and apoptosis. Therefore, the objectives of this study were to characterize Djp53 gene from the planarians Dugesia japonica and to examine their expression patterns and functions during the embryonic development, intact and regenerating planarians, using Northern blotting, whole-mount in situ hybridization (WISH) with digoxigenin-labelled RNA probes and RT-PCR. L4440-Djp53 interference vector was also constructed to speculate the potential biological functions of Djp53 gene in planarians.K228 clone was obtained from the cDNA library of Dugesia japonica. The sequence analysis showed that the largest ORF consisting 849 bp which encoded a deduced protein of 283 amino acid, and contained an activation domain, a sequence-specific DNA domain, two nuclear localization sequences and other conserved domains of P53 protein. We named Djp53 gene in this study.Whole-mount in situ hybridization revealed that the expression of Djp53 mRNA was not observed as early as in stage 2 embryos during the embryonic development. Djp53 positive signal was first found in stage 3. It was expressed in some peripheral cells in the embryo. Then, the number of Djp53-positive embryonic cells was increased, expressed in the nervous system regions, pharynx and strongly expressed in the anterior regions. From stage 8 to juvenile, Djp53 gene even dispersed throughout the parenchyma. These results of Djp53 expression pattern suggested that Djp53 may play an essential role in the nervous system formation.We also examined the expression in both intact and regenerating planarians. The results showed that Djp53 gene strongly expressed in the blastema at 1,3,5 days after amputation, and the highest expression level at 3 days after amputation. However, the gene expressed less throughout the parenchyma of adult intact and regenerated at 7,10 days, and the signals were consistent with the spatial location of stem cell.Djp53 was silenced by RNAi, the relative expression level of Djp53 in intact planarians decreased significantly, which showed Djp53 have been successfully suppressed. Loss of the Djp53 planarians were failed to regenerate normally, and had a rough eye phenotype at 15 days after amputation. The results indicate that Djp53 is essential for planarian regeneration and effect blastema formation at early stages by regulating neoblasts migration, proliferation and differentiation, which required for stem cell maintenance at late regeneration and adult planarian.The dsRNA of p53,14-3-3, RhoA was synthesized and then injected in the intact planarians.The injection schedule was:once injection with 50 ng of dsRNA, every 2 days for three injections. Total RNA was extracted in 2 days after the last injection.The expression of these genes was detected by Realtime-PCR after RNAi, the results showed the relative expression level of these genes was decreased or increased, indicating that the relationship among p53,14-3-3, RhoA and MLC exists in planarian.