| Autophagy is a protein degradation pathway that occurs in all eukaryotic cells.It degrades cytoplasmic components to produce macromolecules for energy metabolism in response to changing nutritional conditions.Autophagy can also remove excess and damaged organelles to maintain homeostasis,which plays a dual regulatory role in the process of cell growth and death.In addition,autophagy is also believed to be a regulatory factor of cell migration,which can promote cell movement.Autophagy protein ATG1 is a serine/threonine protein kinase,and its kinase complex with ATG13 plays an important role in autophagy initiation.ATG1 can interact with a number of proteins that perform autophagy,and many signaling pathways that regulate autophagy converge in ATG1.Therefore,ATG1 may be a key regulatory point in the regulation of autophagy pathway.Many studies have shown that the autophagy gene Atg1 plays an important role in the regulation of neurogenesis of animal central nervous system(CNS),but the specific molecular mechanism remains unknown.Planarians are well known for their powerful regenerating abilities,and they can regenerate a new head in a week following in decapitation.Therefore,planarians are an excellent animal model for studying CNS regeneration.CNS regeneration involves many cellular processes such as proliferation,differentiation and migration,and autophagy may play an important role in this process.However,the role of autophagy in CNS regeneration in planarian has not been reported.In this investigation,RNAi interference,in situ hybridization,immunofluorescence,q PCR and other experimental techniques were used to explore the role of Atg1 in cephalic ganglia regeneration of planarians,and the effects of RNAi-Atg1 on the expression of stem cell-related genes were investigated.The results are as follows:(1)Identification and homology analysis of Atg1(DjAtg1)gene in planarian Dugesia japonicaScreening in the transcriptome database of Dugesia japonica,the c DNA sequence of Atg1 was identified.The full-length of DjAtg1 c DNA is 2625 bp,which contains a open reading frame(ORF)of 2445bp encoding 814 amino acids polypeptide.Multi-sequence alignment analysis of amino acid sequences between DjAtg1 and serine/threonine kinase proteins of other species revealed that DjAtg1 contains the functional domain S-TKC of the serine/threonine kinase protein family,which suggests that it belongs to the serine/threonine kinase protein family.The similarity between DjATG1 and other ATG1 from Caenorhabditis elegans,Mus musculus,Danio rerio,Drosophila melanogaster and Homo sapiens is 46.15%.(2)The spatial and temporal expression pattern of DjAtg1The results of in situ hybridization showed that DjAtg1was mainly expressed in the cephalic ganglion,but weakly expressed in the intestinal tissue.During the regeneration of planarian,the hybridization signal of DjAtg1 was mainly concentrated on the newly formed blastema in the anterior region of the trunk fragment,which revealed a brain structure with an inverted"U"shape.The hybridization signal of DjAtg1was much stronger on the day 5 of regeneration than that of other time point of regeneration.The q PCR results showed that the expression level of DjAtg1 was significantly up-regulated during the regeneration following the cut,and reached maximum high level on day 5 of regeneration.The spatiotemporal expression pattern of DjAtg1 indicates that DjAtg1 may be necessary for planarian regeneration.(3)Effects of RNAi-DjAtg1 on the expression of neural-related-genesImmunofluorescence and WISH(Whole In Situ Hybridization)were performed on anti-SYNORF1 and anti-Ptyr neuroantibodies,as well as a series of neural-related markers,including pc2?Chat?Th?Gad?Otx?Netrin1?Net R?Coe and Sox P2.The results showed that the newly regenerated brain strucrture in RNAi-anmials was abnormal.Compared with the control animals,the two cephalic ganglias were significantly smaller and shorter,the anterior commissure connecting them was significantly thickened,and the lateral neuronal branches projecting from the cephalic ganglion to the lateral were short.In addition,compared with the control animals,the regenerated neurons of planarian in RNAi-animals were significantly abnormal,the number of positive cells of neuron-related marker was significantly reduced,and the hybridization signal of neural transcription factors was significantly weaker,or even almost no signal.Based on the above results,we hypothesized that DjAtg1 gene may be a key factor in planarian CNS-regeneration.(4)Effects of RNAi-DjAtg1 on the expression of planarian stem cell marker related genesImmunofluorescenc showed that the numbers of H3P positive cells in RNAi-animals were not significantly different from that in the control animals.However,the hybridization signal of Djwi A(stem cell marker)in the whole planarians was significantly stronger than that of the control group in the anterior pharyngeal and posterior enation regions.But the numbers of stem cells in the head region in RNAi-animals were significantly less than that in the control animals.The hybridized signal pattern of stem cell marker H2B in the regenerated planarian was consistent with that of Djwi A in the whole planarian.The hybridization signal of H2B was significantly stronger than that in the control group,but the number of H2B+cells in the head region was significantly less than that in the control group.In addition,the expression level of Djwi A in whole planarians was up-regulated by 2.8-fold,and the expression level of H2B in regenerated planarians was up-regulated by 3.4-fold.In addition,in situ hybridization of NB21.11 and AGAT(markers of early and late epidermal cells in the regenerated planarians)showed that the positive signals of NB21.11 and AGAT were much weak in the head region RNAi-aminals compared to the control animals.The above results suggest that,RNAi-DjAtg1 may not affect the self-renewal and proliferation of stem cells,but it may affect stem cells the migration and differentiation.(5)Analysis of RNAi-DjAtg1 on the whole gene expression by RNAseqComparative transcriptome sequencing revealed that RNAi-DjAtg1 caused 1041 transcripts differently expressed,of which 399 transcripts were down-regulated.Go Top 30 analysis revealed most down-regulated transcripts are related to cell adhension,extracellular matrix and matrix metalloproteinases.Numerous literatures have addressed that genes related to cell adhension,extracellular matrix and matrix metalloproteinases are involved in cell migration and cell differentiation.Therefore,it is deduced that RNAi-DjAtg1 affects stem cell migration and differentiation,which further leads to the abnormal regeneration of planarian cephalic ganglions.Summary:(1)The protein encoded by DjAtg1 gene contains the S-TKC functional domain and belongs to the serine/threonine protein kinase family;(2)DjAtg1 is expressed in the cephalic ganglia of planarians,RNAi-DjAtg1 caused abnormal cephalic ganglia regeneration in planarians,suggesting that DjAtg1 is a key regulator of planarian brain regeneration;(3)RNAi-DjAtg1 does not affect the proliferation of stem cells,but affects the distribution pattern of stem cells;(4)RNAi-DjAtg1 caused the down-regulation of genes related to cell migration and differentiation in planarians.Conclusion:DjAtg1 is an important regulator in planarian brain regeneration.RNAi-DjAtg1 may affect the migration and differentiation of stem cells,thus affecting the normal formation of planarian cephalic ganglia. |
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