| PART ONETranscription factor 7-like 2 regulates oligodendrocyte differentiation during normal developmental myelinationObjective:To study the influence to the developmental myelination in the central nervous system (CNS) by transcription factor 7-like 2 through conditional knockout Tcf7l2.Methods:We used Cnp-Cre/Tcf7l2fl/tl and Olig2-Cre/Tcf7l2n/fl/tl transgenic mice as experimental group and Tcf7l2fl/tlas control group to study the influence to the developmental myelination in the CNS by Tcf7l2 at different time points (P0, P7, P14, P21, P30, P45, P60, P90 and P120). In our study, we used rotarod behavior test, in situ hybridization (ISH), western-blot, immunofluorescence (IF) and transmission electron microscopy (TEM) to compare differences of behavior, morphology, the mRNA and protein level of myelin-associated genes and myelin ultrastructure between the two groups.Results:(1) Rotarod test. The riding time on the rotarod in Cnp-Cre/Tcf7l2fl/tl mice was significantly shorter than the control group at P30, P45 and P60 (P< 0.05); and the riding time on the rotarod in Tcf7l2 cKO group was decreasing compared with control group at P90 (P= 0.131) and P120 (P= 0.087).(2) In situ hybridization. The mRNA expression level of myelin-associated genes Mbp and Plp in spinal cord was significantly reduced compared with the control group at different developmental time points (P0, P7) using Cnp-Cre / Tcf7l2fl/tl and Tcf7l2fl/tl mice (P< 0.05). And the mRNA expression level of Plp in spinal cord was also significantly reduced compared with the control group at different developmental time points (P0, P21) using Olig2-Cre/Tcf7l2tl/tl and Tcf7l2fl/fl mice.(3) Western-blot. Proteins in corpus callosum were extracted from Cnp-Cre/ Tcf7l2fl/tl and littermate control mice at P30, Myelin-associated proteins Cnp and Mbp decreased significantly compared with the control group (P< 0.01).(4) Transmission electron microscopy. Cervical spinal cords were taken at P21 using Olig2-Cre/Tcf7l2fl/tl and littermate control mice. Myelin sheath was significantly thinner compared with the control group. Quantitative analysis of the value of g-ratio (axon diameter/axon+myelin sheath diameter) increased significantly more than the control group (P< 0.001).(5) Immunofluorescence. There was no significant difference between the two groups in NG2 (an OPC marker) expression (P> 0.05), there was no significant difference in the proliferation of oligodendrocytes (OL) between the two groups (P> 0.05), there were also no significant difference astrocytes (GFAP, an astrocyte marker) and neurons (NeuN, a neuron marker) between the two groups (P> 0.05).Conclusions:Tcf7l2 can promote oligodendrocyte differention and myelination. It is not due to the abnormalities in the number of oligodendrocyte precursor cells, it docs not affect the proliferation of OLs, and it also does not affect the number of astrocytes and neurons.PART TWOTranscription factor 7-like 2 regulates oligodendrocyte differentiation during remyelinationObjective:To study the influence of remyelination by conditional knockout transcription factor 7-like 2.Methods:We used Cnp-Cre/Tcf7l2fl/tl transgenic and Tcf7l2fl/tl control mice to study the influence to the myelin regeneration in the CNS by Tcf7l2. In our study,8-10 weeks old Tcf7l2 cKO and control mice were fed with 0.2% dicyclohexanoneoxaly dihydrazone (cuprizone) for 6 weeks to induce demyelination, and then established demyelinated animal models. After 6 weeks, mice from the two groups returned to normal diet. We used rotarod behavior test, eriochrome cyanide staining, ISH, western-blot and IF to compare differences of behavior, morphology, myelin-associated proteins and mRNA expression level of myelin-associated genes between the two groups in order to observe the process of remyelination and repair after demyclination which was caused by 0.2% cuprizone.Results:(1) Rotarod test. Tcf7l2 cKO mice stayed on the rotarod apparatus significantly shorter than the control group at 3 weeks after feeding with 0.2% cuprizone (P<0.05). After returning to normal diet for 2 weeks (at 7 and 8 weeks), the control group mice stayed on the rotarod apparatus significantly longer than the Tcf7l2 cKO group (P< 0.05), whereas experimental group had no significant recovery.(2) Eriochrome cyanide staining. Corpus callosum were completely demyelinated in Tcf7l2 cKO and control groups after feeding 0.2% cuprizone for 6 weeks, corpus callosums in both groups were not stained. After returning to normal diet for 2 weeks, corpus callosum was significantly stained in control group, whereas it was also not stained in Tcf7l2 cKO group.(3) Immunofluorescence. The expression of myelin-associated protein Ape in both Tcf7l2 cKO and control group almost disappeared after feeding 0.2%cuprizone for 6 weeks. After returning to normal diet for 2 weeks, Tcf7l2 cKO mice expressed significantly less Cnp, Mbp and Ape in corpus callosum than those in the control group. There was no significant difference in the expression of Pdgfra (an OPC marker) between the two groups (P> 0.05).(4) In situ hybridization. The mRNA expression level of Mbp significantly increased in the control group mice after returning to normal diet for 2 weeks, whereas there was no significant change in Tcf7l2 cKO group.Conclusions:Tcf7l2 positively regulates OL differentiation and remyelination. And it is not due to the abnormalities in the number of OPCs.PART THREEThe mechanism of Transcription factor 7-like 2 regulating myelination and remyelinationObjective:To explore the molecular mechanism of Tcf7l2 regulating myelination and remyelination.Methods:We used Cnp-Cre/Tcf7l2fl/tl transgenic and Tcf7l2tl/tl control mice. In our study, we did in vivo and in vitro experiments to further explore the mechanism of Tcf7l2 to myelin formation and remyelination respectively. In vivo study, we extracted protein of corpus callosum from Tcf7l2 cKO and control mice at Pl4, and then did western-blot. In vitro study, we performed primary OL culture at P0?P2 neonates from the corticals of Cnp-Cre/Tcl7l2fl/tl and Iettermate control mice, and extracted RNA of Tcf7l2 cKO and control OLs during early and late OL differentiation respectively, we then did RNA-sequence, and RT-qPCR to compare the differences of myelin-associated genes, target genes of Tcf7l2 related canonical Wnt/?-catenin and PI3K signaling pathway between the two groups.Results:(1) RT-qPCR in vivo. About 75% Tcf7l2 were knockout in Tcf7l2 cKO group; the mRNA expression level of myelin associated genes Cnp, Mbp and Mag largely decreased compared with control group(P< 0.01); there was no significant difference in pdgfra mRNA expression between the two groups (P> 0.05); the target genes Wnt5b, Wnt7b, Fosll, Gpc4 and Nkx2.2 of canonical Wnt/(3-catenin signaling pathway were significantly reduced in Tcf7l2 cKO mice (P< 0.001); and the target genes Thbs2, Sppl, Csflr, Thbsl and Lparl of PI3K signaling pathway were also significantly decreased in Tcf7l2 cKO group (P< 0.001).(2) Western-blot in vivo. In vivo study, P-Akt expression of corpus callosum in Tcf7l2 cKO mice was significantly reduced compared with the control group at P14 (P<0.001). There was no significant difference in Akt expression between the two groups (P> 0.05).(3) Immunofluorescence. In vitro study, the expression of Cnp was significantly decreased in Tcf7l2 cKO group during early OL differentiation. The expression of Cnp, Mbp and Nkx2.2 were significantly decreased in Tcf7l2 cKO group during late OL differentiation. There were no significant differences in the expression of Pdgfra, Olig2 and Ki67 between the two groups.(4) RNA-Sequence. The target genes Wnt5b, Wnt7b, Fosll and CD44 of canonical Wnt/?-catenin signaling pathway were significantly reduced in Tcf7l2 cKO OLs; and the target genes Epha2, Tnr, Itga3, Lparl, Itga7, Vwf, Thbs2, Cdknla and Thbsl of PI3K signaling pathway were also significantly decreased in Tcf7l2 cKO OLs during early OL differentiation. RNA-Sequence also revealed that target genes Lama2, Col11a2, Col6a5, Angpt2 and Csflr of PI3K signaling pathway were significantly reduced in the Tcf7l2 cKO group; the target gene Hes5 of Notch signaling pathway was significantly higher in the control group; and the target genes BMP4, Gli2, Gli3 and Shh of Hedgehog signaling pathway were significantly higher in the control group during late OL differentiation.(5) RT-qPCR in vitro. During early OL differentiation, about 80% Tcf7l2 were knockout, Tcf711 and Lefl which were TCF/LEF1 family members were significantly higher in Tcf7l2 cKO group compensatory (P< 0.001); the mRNA expression level of myelin associated genes Cnp and Mbp largely decreased compared with control group (Cnp, P< 0.05; Mbp, P< 0.01); there was no significant difference in pdgfra mRNA expression between the two groups (P> 0.05); the target genes Wnt5b, Wnt7b, Fosll and Gpc4 of canonical Wnt/?-catenin signaling pathway were significantly reduced in Tcf7l2 cKO mice (P< 0.001, except for Wnt5b, P< 0.05); and the target genes Thbs2, Sppl, Csflr, Thbs1, Lpar1, Jak3 and Itga3 of PI3K signaling pathway were also significantly decreased in Tcf7l2 cKO group (P< 0.001, except for Thbsl, P< 0.05). During late OL differentiation, RT-qPCR experiments also showed that about 75% Tcf7l2 were knockout, Lefl which was a TCF/LEF1 family member was significantly higher in Tcf7l2 cKO group compensatory(P< 0.001); the mRNA expression level of myelin associated genes Cnp, Mbp and Mag largely decreased compared with control group (Cnp and Mbp, P< 0.01; Mag, P< 0.001), there was no significant difference in pdgfra mRNA expression between the two groups (P> 0.05); the target gene Hes5 of Notch signaling pathway was significantly higher in the control group (P< 0.001); and the target genes BMP4, Gli2, Gli3 and Shh of Hedgehog signaling pathway were significantly higher in the control group (P< 0.001). The target genes Smad1, Smad2, Smad7 and Id2 of BMP4 signaling pathway were almost similar between the two groups (P> 0.05). The results of RNA-Sequence and RT-qPCR were almost consistent.Conclusions:Tcf7l2 promotes OL differentiation and myelin formation by activating canonical Wnt//?-catenin and PI3K signaling pathway during the early OL differentiation. During late OL differentiation, Tcf7l2 inhibits Notch and Hedgehog signaling pathways which inhibit OL differentiation and myelination, thereby promoting OL differentiation and myelination. |
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