Expression And Function Analysis Of DjPreb And DjStag Gene From Planarian Dujesia Japonica

Planarians are bilateral symmetry and triploblastic, which are members of the phylum Platyhelminthes. Planarians have the ability to regenerate their whole body after being cut into many segments. Because of their special evolution status and strong regeneration ability, planarians have become an excellent model system for studying evolution of the animals, embryonic development and the biology of regeneration. The PREB protein has similarities to a subset of proteins belonging to the family of WD-repeat proteins that plays a role as a gene regulator. In higher eukaryotes, two cohesin isoforms exist, each including the subunits Smcl, Smc3, Sccl, and one of the Scc3 orthologs STAG1/SA1 or STAG2/SA2. STAG is not only involved in chromosome segregation but also in transcriptional co-activation via a mechanism involving protein-protein interactions with transcription factors. However, little is known about Preb and Stag genes in this important animal. Therefore, the objectives of this study were to characterize a PREB-related gene DjPreb and a STAG-related gene DjStag from the planarians Dugesia japonica and to examine their expression patterns and functions during the embryonic development as well as in intact and regenerating planarians. This will contribute to the understanding of the characterization of the gene Preb and Stag and providing the experimental reference for the exploration of the mechanism of animal regeneration.First, DjPreb and DjStag were identified from the cDNA library of planarian Dugesia japonica. Then whole mount in situ hybridization, Real-Time PCR detection, RNA interference and other methods were carried out to study the expression and function of the gene. Bioinformatics was also carried out to analyze the evolutionary relationship of DjPreb and DjStag. These studies provide a better understanding of the Preb and Stag. 1. Characterization and expression of DjPreb gene in the planarian Dugesia japonicaThe planarian DjPreb cDNA is comprised of 1101 bp and contains a 972 bp open reading frame encoding a deduced protein of 323 amino acids with a 69 bp 5'-UTR and a 60 bp 3'-UTR. BLAST analysis showed that DjPreb is PREB/PREB-like members. Genomic analysis reveals that DjPreb gene consisted of 2 exons spaced by 1 intron.The expression of DjPreb mRNA was not observed in the embryos before stage 3 by whole-mount in situ hybridization. DjPreb positive signal was first found in stage 4. It was expressed in some peripheral cells in the embryo. The number of DjPreb-positive embryonic cells was increased in stage 5. DjPreb was expressed in the dorsolateral regions and part of anterior regions in stage 6. In stage 7, DjPreb positive signals were detected in dorsolateral regions along the A-P axis. From stage 8 to stage juvenile, DjPreb mRNA was strongly expressed not only in the differentiating tissues of the anterior and posterior regions, but also in the parenchyma of the dorsolateral regions, and generating gradient in the head of juvenile. These results of DjPreb expression pattern suggested that DjPreb may play an essential role in the regulation of head and tail formation and anterior/posterior patterning.We also examined its spatial and temporal expression in both intact and regenerating planarians by relative quantitative real-time PCR and whole-mount in situ hybridization. The analysis indicated that DjPreb exhibited a gradient expression pattern with highest levels being in the anterior and posterior regions and lowest levels in central regions in intact and regenerating planarians. During regeneration the expression of DjPreb was upregulated. Strong expression of DjPreb was observed in the anterior and posterior blastemas. These results suggest that DjPreb may play a role in planarian regeneration and participate in head and tail formation.2. Characterization and expression of DjStag gene in the planarian Dugesia japonicaThe planarian DjStag cDNA is comprised of 1362 bp and contains a 489 bp open reading frame coding for a deduced protein of 162 amino acids with a 170 bp 5'-UTR and a 703 bp 3'-UTR. BLAST analysis shows that DjStag is a STAG/STAG-like member. We examined the expression pattern of DjStag in planarian during the embryonic development by whole-mount in situ hybridization. DjStag was detected in the embryonic cells in the germ band at early embryo stage. The number of DjStag-positive embryonic cells was increased in stage 5. Later, it was mainly expressed in parenchyma of the lateral region. In the juvenile, the most extensive expression of DjStag was not only observed in the head and tail regions, but also in the parenchyma between the epidermis and the gastrodermis. These results suggest that DjStag is expressed in both the differentiating cells and the cellular subset which will become the neoblast cells of the adult worm.We showed the spatial and temporal expression of DjStag of planarian Dugesia japonica in both intact and regenerating planarians by whole-mount in situ hybridization and relative quantitative real-time PCR. All results including those from regeneration from head, tail and trunk pieces showed that DjStag transcripts were first detected in the blastemas at 3 day after amputation. Examination of many preparations indicated that the maximal level of expression of DjStag transcripts occured at 5 days after cutting. After regeneration for 7 days, DjStag was weakly expressed. A similar decrease occurs independently of the orientation of cut. The expression pattern does not show significant differences in the different types of regeneration. Relative quantitative real-time PCR analysis of DjStag mRNA indicated that the expression of DjStag mRNA was increased after amputation compared to normal intact planarians, and the maximal level of expression of DjStag transcripts occurred at 5 days after amputation. All results suggest that DjStag is involved in planarian regeneration, possibly playing a role in maintaining the ability of pluripotent stem cells to regenerate lost tissue in planarians.3. Function of DjPreb and DjStag gene in the planarian Dugesia japonicaThe cDNAs for DjPreb and DjStag were cloned into the L4440 vector and then expressed in the bacterial strain HT115 to make dsRNA. The worms were fed with RNAi food directly. The animals were then amputated anterior and posterior of the pharynx and allowed to regenerate. The morphologic changes were observed during the regeneration with stereomicroscope. Relative quantitative real-time PCR was used to monitor the quantitative expression of DjPreb and DjStag gene in regenerated planarians fed with RNAi food.When DjPreb and DjStag RNAi animals were amputated into three fragments post-RNAi, regeneration was severely impaired. Regenerating fragments displayed profound regeneration defects at the end of the regeneration, including indented posterior blastema, caudal regeneration defects, the whole body started to appear thicker and shrunken, inability to regenerate photoreceptors and pharynx. Relative quantitative real-time PCR analysis indicated that DjPreb and DjStag expression dramatically decreased after DjPreb and DjStag RNAi in regenerates when compared to normal regenerating planarians.