| Planarians are unique animals,showing powerful regenerating abilities.Therefore,they have become the ideal animals for studying regeneration.Autophagy is a conserved molecular mechanism evolved in eukaryotic cells,and plays an important roles in body remodeling during animals development and keeping cellular homeostasis.Planarians need to regulate body shape continuously to maintain well-proportioned body sizes,this process may involved the autophagy and autophagic cell death.In order to studying the role of autophagy and autopahgic cell death during planarian body remodeling,the full-length cDNA of autohoagy-related gene 7(DjAtg7)from planarian Dugesia japonica was cloned,the morphological features of autophagy was observed by Transmission Electron Microscope(TEM),and the function of DjAtg7 was analyzed by whole-mount in situ hybridization,RNA interference and qPCR.The results of the study are as follows:(1)Cloning and bioinformatics analysis of DjAtg7 gene of Dugesia japonica.The full-length cDNA of DjAtg7 gene is 2272 bp containing the open reading frame(ORF)2082 bp encoded 693 amino acids.Bioinformatics analysis revealed that it is a hydrophilic protein with multiple phosphorylation sites,and no transmembrane region,no transmembrane region,no nuclear export signals and no signal peptide.SMART analysis showed that it contains two conserved domains of E1 enzyme-ATP binding site and catalytic activity site in ATG7 homologues.Evolutionary analysis of DjATG7 shows that planarian is closer to vertebrates than to arthropods.(2)Analysis of DjAtg7 expression patterns during planarian regeneration: Detection of DjAtg7 expression was detected by qPCR at 0,1,3,5,7,and 10 days follwoing planarian amputation.The results showed that the expression level of DjAtg7 increased apparently at 3 day of regeneration,which was 1.33-fold compared to the control level.The expression of DjAtg7 remained relatively high level at 5,7 and 10 day of regeneration.The up-expression of DjAtg7 during planarian regeneration indicated that DjAtg7 might be related to planarian regeneration.The whole mount in situ hybridization(WISH)revealed that the transcripts of DjAtg7 were mainly expressed in intestinal tissues in intact animals.On the 3,5 and 7 day of regeneration,the strong hybridization signals appeared in the blastema.But the strong hybridization signals gradually disappeared from the blastema and appeared in the distal branches of the old intestinal tissues on 10 day of regeneration.The results indicate that DjAtg7 gene may be involved in the planarian intestine regeneration and re-construction of intestinal structure.Fluorescence in situ hybridization(FISH)co-localization of DjAtg7 and Djpk-1 further confirmed that DjAtg7 was mainly expressed in intestinal tissues of planarians and highly expressed in newly regenerated intestinal branches.DjAtg7 does not show colocalization with Djpiwi-1 in the blastema,indicating that the newly regenerated intestinal cells were not the neoblasts or direct neoblast progeny.The expression pattern of DjAtg7 indicates that this gene plays an important role in the intestinal regeneration process in the early stage of planarian regeneration.(3)Observation of RNAi-DjAtg7 phenotype and detecting of autophagy-related genes: RNAi-DjAtg7 animals can regenerate normal planarians with no more apparent difference compared to the control animals,indicates that RNAi-DjAtg7 has no effects on planarian regeneration.After15 days of regeneration,in RNAi-DjAtg7 animals can take the food,suggesting that RNAi-DjAtg7 does not affect the regeneration of the intestinal tract or the physiological function of the intestinal system.qPCR results revealed that the endogenous DjAtg7 expression in RNAi-DjAtg7 animals decreased significantly,suggesting that the efficiency of RNAi was valid.qPCR results also revealed that DjAtg5,DjAtg8 and DjAtg12,being related to classcial autophagy pathway,did not change significantly in RNAi-DjAtg7 animals.Otherwise,the expression levels of DjAtg1,DjPi3 k and DjAtg6,being related to alternative autophagy pathway increased significantly in RNAi-DjAtg7 animals.Especially,the expression of DjRab9 A increased by 3.3-fold in RNAi-DjAtg7 animals,which is necessary for the alternative autophagy pathway.These results suggest that there may exist an Atg7-independent autophagy pathway in planarians.(4)Observation of submicroscopic structure during the process of planarians body remodeling: TEM was used to observe the planarians on the 7 and 10 day of regeneration.It was found that in normal cells,the nucleus remained round and there were no autophagic vesicles in the cytoplasm.At the early stage of autophagy,autophagic vesicles begin to appear in the cytoplasm.In the middle stage of autophagy,a large number of autophagic vesicles accumulate in the cytoplasm and form autophagosome.At the late stage of autophagy,cells exhibit typical characteristics of autophagic cell death.In addition to the nucleus,the cytoplasm is automatically digested.In the final stage of autophagy,chromatin is highly concentrated and degraded into fragmented structures,surrounded by multiple membranes.It is also observed that large vesicles containing vesicles are released from inside the cell to outside the cell.This emptying of the cytoplasm is consistent with the hypothesis that autophagic cell death can provide nutrients for the survival of other cells.It was also found that RNAi-DjAtg7 did not affect the autophagosome formation in planarians.Conclusion:(1)In this work,the full-length cDNA sequences were first cloned,and the encoding protein belongs to the member of super E1-activating enzymes;(2)qPCR and WISH revealed that DjAtg7 may involve in planarian intestinal regeneration and re-construction of intestinal structure;(3)TEM revealed that autophagy and autophagic cell death exist in planarian body remodeling.Autophagy and autophagic cell death may provide raw materials for planarian regeneration;(4)RNAi-DjAtg7 does not affect planarian regeneration and the autophagosome formation,suggesting that molecular mechanism is more complex than it is expected. |
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