The transgenic Cnp-Cre and Tcf7l2^fl/fl (exon11 flanked by loxP sites) mice were provided by Dr Hui Fu (Department of Anatomy, Basic Medical School of Wuhan University, Wuhan, China). Male and female mice were used in the current study. Tcf7l2^fl/fl and Cnp-cre. C57BL/6 served as the genetic background for the Tcf7l2^fl/fl mice. All animal experiments were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH publications no. 8023, revised 1978) and were approved by Institutional Animal Care and Use Committee at Wuhan University (Wuhan, China).

Sections containing the spinal cord (25.4×76.2 mm) were hybridized overnight with a labeled RNA probe (0.8–1.2 µg/ml) at 65°C. The sections were washed in 2X saline-sodium citrate (SSC) at 67°C, incubated with RNase (1 µg/ml, 2X SSC) at 37°C, washed in 0.2X SSC at 67°C, blocked in 1X phosphate-buffered saline (PBS) with 10% lamb sera, and incubated in alkaline phosphatase-labeled anti-digoxigenin antibody (catalog no. 11093274910; Roche Diagnostics GmbH-Mannheim, Germany; 1:2,000, 10% lamb sera) overnight at 4°C. Sections were washed and stained with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate or BM purple (Roche Diagnostics GmbH). Staining was terminated following visual inspection using a light microscope. Finally, sections were washed in 1X PBS three times, fixed in 4% paraformaldehyde and coverslipped with glycerol.

Slides were incubated with antibodies (anti-CNP, catalog no. NE1020, 1:200, Calbiochem; anti-APC, catalog no. OP80, 1:60, EMD Millipore, Billerica, MA, USA; anti-PDGFRA, catalog no. APA5, 1:300, BD Bioscience, Franklin Lakes, NJ, USA) overnight at 4°C. Sections were then washed three times with 1X PBS, incubated with Alexa-488- or Alexa-555-conjugated secondary antibodies (goat anti-mouse IgG-Alexa Flour 555, catalog no. A21422, 1:500, Cell Signaling Technology, Inc., Danvers, MA, USA; Goat anti-rabbit IgG-Alexa Flour 488, catalog no. 4412s, 1:500, Cell Signaling Technology, Inc.) and fluorescent images were obtained using a Nikon epifluorescence microscope (Nikon Corporation, Tokyo, Japan). Antibodies used in the study were as follows: Anti-APC, WNT signaling pathway regulator (APC; 1:60; cat. no. OP80; EMD Millipore), anti-2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNP; 1:200; cat. no. NE1020; EMD Millipore), anti-myelin basic protein (MBP; 1:200; cat. no. NE1018; EMD Millipore,) and anti-platelet-derived growth factor receptor α (PDGFRA; 1:300; cat. no. APA5; BD Biosciences).

For western blot analysis, whole cell lysates were prepared from the corpus callosum at the thirtieth day after birth (P30). using RIPA buffer (Applygen Technologies, Inc., Beijing, China), phenylmethylsulfonyl fluoride (Biosharp, Shanghai, China) and PhosSTOP (Roche Diagnostics GmbH). The tissue samples were minced using an electric homogenizer. The protein concentrations in the centrifugation-clarified cell lysates were measured using a bicinchoninic acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and equal quantities of protein were separated on 10% SDS-PAGE gel and transferred (100 V for 2 h) to Hybond polyvinylidene fluoride membranes (GE Healthcare Life Sciences, Shanghai, China). For Western blotting, primary antibodies against CNP (1:100), MBP (1:1,000), GAPDH (1:1,000), β-actin (1:10,000) were used. Signals were developed using horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse antibody conjugated to horse radish peroxidase, 1:5,000, catalog no. AS1106, Aspen Biotechnology Co., Ltd., Hubei, China); goat anti-rabbit antibody conjugated to horse radish peroxidase, 1:5,000, catalog no. AS1107, Aspen Biotechnology Co., Ltd.) and an ECL kit (GE Healthcare Life Science).

Slides were washed in 1X PBS three times, and dyed with eriochrome cyanine (catalog no. B100835-5G, Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China) for 30 min, and then washed three times with 1X PBS. Sections were differentiated by 10% iron alum (catalog no. 10009218, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 8 min, and washed three times with 1X PBS, prior to coverslipping with neutral gum (catalog no. 1000416, Sinopharm Chemical Reagent Co., Ltd.).

The rotarod performance test is based on a rotating rod with forced motor activity applied by a mouse. The test is used to measure parameters, such as riding time (sec) or endurance. In the rotarod apparatus (IITC Life Science, Woodland Hills, CA, USA), mice were placed individually on a horizontally oriented, rotating cylinder suspended above a cage floor, which is low enough not to injure the animal, but high enough to induce avoidance of fall. The fall off time of the mouse from the rotating rod was recorded. The difference in fall off times from the rotating rod between the two groups was taken as an index of muscle relaxation and motor coordination activity.

The TCF7L2 cKO and control mice (age, 8–10 weeks) were fed with a mixture of 0.2% cuprizone (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in ground chow for up to 6 weeks. Cuprizone is a copper chelator that induces demyelination of the corpus callosum if administered orally to adult mice. A normal diet was provided for 2 weeks after cuprizone treatment. The demyelination peaked in the fifth week. The remyelination occurred thereafter and was allowed to develop for two weeks during the current study.

Quantification was performed on data from at least three independent experiments, and the data were presented as means ± standard error of the mean in the graphs. Student's t-test was used for comparisons between two sets of data. ImageJ version 2.0 (National Institutes of Health, Bethesda, MD, USA) was used for quantitative analysis of the mRNA and protein expression levels of MBP, proteolipid protein 1 (PLP1), CNP, APC and PDGFRA. P<0.05 was considered to indicate statistically significant differences.

The Cre-loxP system was used to conditionally knock out TCF7L2 in oligodendroglial lineage cells, and avoid the lethality of TCF7L2-null newborns (14). Cnp-cre and Tcf7l2^fl/fl (exon11 is flanked by loxP sites) mice were used to delete the TCF7L2 exon11 sequence in the OPCs, which is the DNA binding domain (15).

Knockout of the TCF7L2 exon 11 sequence in the oligodendroglial lineage cells (Fig. 1) demonstrated that the mRNA levels of MBP and PLP1 in the spinal cords were significantly lower when compared with those of the control group at P0 (P<0.001; Fig. 1A and C), and a similar reduction in the spinal cords was observed at P7 (MBP, P<0.01; PLP, P<0.05; Fig. 1B and D) indicating that TCF7L2 positively regulates OL differentiation. Furthermore, corpus callosum samples were obtained, and protein samples from the Cnp-cre-mediated TCF7L2 KO mice and littermate control mice were extracted at P30. Western blot analysis revealed markedly decreased expression levels of CNP and MBP in the TCF7L2 cKOs compared with the controls (Fig. 2A and B). Quantitative analysis demonstrated that the expression levels of CNP and MBP were significantly lower than those of the control mice (P<0.01; Fig. 2C). In order to exam motor coordination activity, the rotarod performance test demonstrated that the riding time on the rotating rod was significantly shorter in the TCF7L2 cKO group when compared with the control mice at P30, P45 and P60 (P<0.05; Fig. 3). The riding time of the TCF7L2 cKO group mice was decreasing at P90 and P120 (P=0.131 and P=0.087, respectively). Together, these data indicate that TCF7L2 promotes OL differentiation and myelination during developmental myelination.

Various previous studies have proposed that TCF7L2 may inhibit OL differentiation and oligodendrogenesis during myelin formation (16,17). There are few studies that have revealed the role of TCF7L2 in remyelination. Based on the current data, it was hypothesized that TCF7L2 promotes OL differentiation during remyelination. Among the various animal and cell models available to investigate remyelination, the cuprizone model stands out due to its reproducibility, simplicity to induce demyelination and low mortality rates (18). In the present study, the 0.2% cuprizone demyelination/remyelination model was used to determine the role of Tcf7l2 in remyelination.

After feeding mice 0.2% cuprizone for 6 weeks, myelin histochemistry with Eriochrome cyanine revealed that there was almost no myelin in the corpus callosum samples between the two groups (Fig. 4A). The magnified view demonstrated further that the corpus callosum samples were fully demyelinated in the two groups (Fig. 4A).

After returning to normal chow feed for 2 weeks, the corpus callosum samples were almost completely remyelinated in the control mice (Fig. 4B and C). By contrast, remyelination did not occur in the TCF7L2 cKO mice (Fig. 4B and C). Furthermore, immunofluorescence demonstrated that the expression levels of APC and CNP in the control group were significantly higher than the TCF7L2 cKO group (Fig. 5A and B) and almost the same as normal expression levels of APC and CNP (data not shown). Quantitative analysis further indicated that the expression levels of APC and CNP were significantly lower than those of the control mice (P<0.001; Fig. 5C). In addition, the mRNA expression level of MBP observed by in situ hybridization was significantly higher in the control group (data not shown). These data indicate that TCF7L2 cKO inhibits remyelination, whereas it promotes OL differentiation during remyelination.

To determine whether OPCs were perturbed in TCF7L2 cKO mice, the expression level of PDGFRA, an OL precursor cell marker, was examined during remyelination. After returning to feeding with normal chow for 2 weeks, the expression level of PDGFRA was almost the same in the two groups during remyelination (Fig. 6Aa and Ab). Quantitative analysis further indicated that there was no significant alteration in terms of OPCs between the two groups during remyelination (P>0.05; Fig. 6B). These data indicate that TCF7L2 cKO does not affect OPCs during remyelination.