| Embryonic stem cells mainly are refered to a class of cells isolated from the inner cell mass(ICM)stage of mammalian embryonic development,which has self-renewal ability(Self-Renewal)and pluripotency.Embryonic stem cells have a wide range of application prospects and can be used to produce human organ transplantation materials,produce cloned animals,genetic breeding,etc.Previous studies on embryonic stem cells have mostly focused on pluripotency signaling pathways and transcriptional networks,however,the functional study of endoplasmic reticulum related genes in embryonic stem cell development is not comprehensive,among which Atl3(Atlastin GTPase 3)is a member of the GTPase family encoding similar dynamic membranes,mainly localized to highly curved ER membranes,and Atl3-related mutations(Y192C and P338R)can induce type I hereditary sensory and autonomic neuropathy.Atl3 mutations have a significant impact on cell ER morphogenesis,which in turn disturbs normal endoplasmic reticulum dynamics and affects the distribution of ER in cells.Studying the effect of Atl3 on embryonic stem cell development helps to understand the role of endoplasmic reticulum-related genes in the regulation of embryonic fate.In this study,the resident protein Atl3 gene on the endoplasmic reticulum membrane and embryonic stem cells were combined to study the effect of Atl3 on the maintenance and loss of pluripotency in mESCs(Mouse Embryonic Stem Cells).First,we explored the effect of overexpression of Atl3 on the maintenance of R1-ESCs pluripotency,and found that overexpression of Atl3 had no significant effect on the maintenance of R1-ESCs pluripotency.We then constructed a shRNA vector for Atl3 and found that after R1-ESCs and OG2-ESCs knocked down Atl3 during the pluripotency maintenance phase,the cell morphology changed significantly: the clone was flattened as a whole,cell-cell adhesions were reduced,the boundaries of individual cells in the clumped clones were clear,and the phenotype remained the same after passage,and there were no cell line differences.The Epithelial-Mesenchymal Transition(EMT)that occurs during embryonic development contributes to cell movement in the embryo,and the reduction of cell-to-cell adhesions in R1-ESCs and OG2-ESCs after knocking down Atl3 has not been mediated by EMT.We found that knocking down Atl3 during the pluripotency maintenance stage slightly increased the expression of Nanog and Oct4 of R1-ESCs at the m RNA and protein levels.The expression levels of na(?)ve marker genes Esrrb and Stella,which represent better pluripotent states of mESCs,were also elevated.According to the morphological phenotype of R1-ESCs and OG2-ESCs after knocking down Atl3,knocking down Atl3 is likely to promote the differentiation of R1-ESCs in the direction of nerve cells in the pluripotency maintenance stage,and the mutually confirmed q-PCR and WB results confirmed that the expression levels of the signature genes Pax6 and Nestin of nerve cells were indeed increased,and R1-ESCs could promote their differentiation in the direction of nerve cells after knocking down Atl3.Studies have pointed out that Pax6 overexpression can inhibit the proliferation of mESCs and induce cell cycle arrest,so we then tested the cell cycle and apoptosis of R1-ESCs after knocking down Atl3 in the pluripotency maintenance stage,and found that the cells of R1-ESCs in the G1 phase of knocking down Atl3 increased;cytopenia in the G2/M phase;There was no significant change in apoptosis levels,suggesting that the decrease in the total number of R1-ESCs that knocked down Atl3 was due to changes in the cell cycle.Next,we explored the transcription level of Atl3 during R1-ESCs differentiation,cultured EB spheres in a hanging drop with mES medium,and found that the transcription level of Atl3 gradually increased as the EB differentiation progressed.In the EB differentiation stage of R1-ESCs,after knocking down Atl3,the edges of the cell mass are blurred and irregular and the surrounding cells become flattened when D6,with strong light transmission.Through q-PCR detection,it was found that the expression of mesoderm gene was generally inhibited in D6,and the ectoderm gene Pax6 was significantly promoted in D6.Pax6 is a signature gene of the neuroectoderm and is involved in the expression of neuron-specific genes.The expression levels of R1-ESCs knocked down Atl3 were upregulated during the pluripotency maintenance and EB differentiation stages,indicating that knocking down Atl3 would affect the differentiation of ESCs in the neural direction.We will continue to explore the specific effects of knocking down Atl3 during the directed neural differentiation of R1-ESCs in our next experiments.Finally,we explored the mechanism by RNA-Seq on R1-ESCs that knocked down Atl3 during the pluripotency maintenance stage.Through GO analysis,it was found that the signaling pathways involved in the downregulated genes in the differential analysis were TGF-β signaling pathways,and the signaling pathways involved in the upregulated genes were mainly c AMP signaling pathways,and I will continue to explore how Atl3 affects the specific molecular regulatory mechanism of cell fate determination by regulating the dynamic changes of the endoplasmic reticulum.In summary,overexpression of Atl3 had no obvious effect on the maintenance of pluripotency of R1-ESCs in the pluripotency maintenance stage.After R1-ESCs and OG2-ESCs knock down Atl3,the cell morphology is flat and free and loose,and the reduction of cell-to-cell junctions is not mediated by EMT;Increased expression levels of R1-ESCs’ pluripotency gene and na(?)ve marker gene;The cell cycle was changed,the G1 phase cells increased,the G2/M phase cells decreased,and the cell proliferation ability was weakened;It also promoted the differentiation of R1-ESCs in the direction of nerve cells;In the EB differentiation stage,the transcription level of Atl3 gradually increased with the advancement of EB differentiation process.Mesoderm expression of R1-ESCs was inhibited after knockdown Atl3. |
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