| Tissue homeostasis requires a good balance between the removal of dead cells and the generation of new cells.Because ingested food,pathogens and toxins can damage the epithelium,the gastrointestinal tract relies on constant self-renewal to maintain homeostasis.Adult intestinal stem cells(ISCs)produce all mature cell types of the gut epithelium,and any imbalance in the process can lead to the onset of disease,such as cancer.In the adult mammalian small intestine,the ISC cell population is near the crypt of crypts.Each crypt produces about 300 cells per day.This process is supported by four to six ISCs per crypt,which migrate into the crypts and undergo translocation and amplification to divide and differentiate into intestinal absorptive cells the major component of the villi.Many signaling pathways,including Notch,BMP and Wnt,have been shown to play key roles in in this proliferating population.However,until now it has been difficult to accurately identify and study this cell population due to the lack of specific stem cell markers.Drosophila and mammalian gut have great similarities in development,cell composition and genetic control,with considerable conservation between gut pathophysiology and regeneration and the signaling pathways that control them.The discovery of adult Drosophila intestinal stem cells provides a new model system for studying ISC biology.ISCs can be identified by their small nuclear size and expression of the Notch ligand Delta(Dl).ISC self-renews to produce a new ISC and an undifferentiated mature EB cell.Both ISCs and EB cells express the transcription factor escargot(esg)of the Snail / Slug family.EB cells differentiate into ECs and EE cells.ECs identifiable by their large endoreplicating nuclei and Pdm1(Nubbin)expression,and EE cells can be labeled with Prospero.Studies on the self-renewal and differentiation regulatory mechanisms of Drosophila enteric stem cells may provide some clues to the study on the proliferation and differentiation of gut stem cells in humans or other vertebrates.The current research reveals that the regulation of Wnt,Notch,EGFR and JAK-STAT conserved pathways on the proliferation and differentiation of intestinal stem cells under physiological and regenerative conditions,and Steven X.Hou et al.screened 405 protein-coding genes involved in the regulation of Drosophila intestinal stem cell proliferation and differentiation of genes through RNAi lines.So consider whether there are other genes involved in the regulation of proliferation and differentiation of intestinal stem cells.Long non-coding RNAs have received widespread attention over the years and thousands of lncRNAs have been discovered that play an important regulatory role in epigenetic,transcriptional and post-transcriptional aspects.Studies have shown that lncRNA Pnky can regulate the differentiation of embryonic neurons and post-developmental neural stem cells,so we think lncRNA is also involved in the regulation of intestinal stem cell proliferation and differentiation.Using 82 lncRNAs knockout Drosophila strains constructed by the laboratory of Prof.Gao Guanjun at Tsinghua University,we integrated them into a Drosophila by genetic crosses with esg-Gal4 flies.Esg can drive the expression of GFP or DsRed in ISC / EB cells.In the context of lncRNA mutations,the number of GFP or DsRed-labeled ISCs / EBs is observed.We obtained a total of 70 strains of integrated Drosophila,X chromosome 5,? chromosome 36,? chromosome 29.Among them,15 lncRNAs were found in the RNA-seq database of flyBase website,including 2 X chromosomes,8 II chromosomes,and 5 III chromosomes.Under the physiological conditions,70 strains of Drosophila melanogaster were screened and all of lncRNAs did not affect the proliferation of intestinal stem cells.The prospero antibody screened 15 strains of Drosophila melanogaster and they did not affect the differentiation of intestinal stem cells into EE cells.DSS(dextran sulphate sodium)is a polyanionic derivative of dextran,feeding drosophila can cause intestinal epithelial damage,promote the proliferation of intestinal stem cells to replenish damaged cells.Using 5% DSS to treat Drosophila,we screened 15 strains of integrated Drosophila and found that the number of intestinal stem cells of CR43282 knock-out Drosophila after DSS treatment decreased compared with that of the control,indicates that CR43282 affects the proliferation of intestinal stem cells under the condition of intestinal regeneration in Drosophila melanogaster.The CR43282 knockout Drosophila strain was correct by single fly PCR and gene sequencing.According to the principle of lncRNA knockout Drosophila construction chromosome mapping map,CR43282 mutant Drosophila knockout CR43282 and CR46040,To reverse-verify the function of CR43282,we need transgenic plasmids that overexpress CR43282,CR46040,and construct CR43282,CR46040 plasmid respectively.On the other hand,a decrease in the number of ISC / EB cells in the CR43282 knockout Drosophila strain was found by PH3 staining due to a decrease in ISC proliferation,shows that CR43282 participates in the process of ISCs proliferation during intestinal regeneration.The regulation of lncRNA on intestinal stem cells has not been reported yet.In this study,the screening of lncRNAs that affect the proliferation and differentiation of intestinal stem cells in Drosophila melanogaster would be helpful to reveal the regulatory mechanism of intestinal stem cell proliferation and differentiation. |
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