Functional analysis of Tcl1 using Tcl1-deficient mouse embryonic stem cells

Abstract

Tcl1 is highly expressed in embryonic stem (ES) cells, but its expression rapidly decreases following differentiation. To assess Tcl1's roles in ES cells, we generated Tcl1-deficient and -overexpressing mouse ES cell lines. We found that Tcl1 was neither essential nor sufficient for maintaining the undifferentiated state. Tcl1 is reported to activate Akt and to enhance cell proliferation. We found that Tcl1 expression levels correlated positively with the proliferation rate and negatively with the apoptosis of ES cells, but did not affect Akt phosphorylation. On the other hand, the phosphorylation level of β-catenin decreased in response to Tcl1 overexpression. We measured the β-catenin activity using the TOPflash reporter assay, and found that wild-type ES cells had low activity, which Tcl1 overexpression enhanced 1.8-fold. When the canonical Wnt signaling is activated by β-catenin stabilization, it reportedly helps maintain ES cells in the undifferentiated state. We then performed DNA microarray analyses between the Tcl1-deficient and -expressing ES cells. The results revealed that Tcl1 expression downregulated a distinct group of genes, including Ndp52, whose expression is very high in blastocysts but reduced in the primitive ectoderm. Based on these results, we discuss the possible roles of Tcl1 in ES cells.

Figure 1. Targeted disruption of the murine Tcl1 gene.

(A) Tcl1 gene structure and targeting vector. Arrows represent the forward and reverse primers (P1 and P2) used to screen the targeted ES clones, and arrowheads (H1 and H2) represent the primers used to identify homozygous knockout clones. (B) Identification of Tcl1+/+, +/−, and −/− ES cell clones by genomic PCR using H1 and H2. (C) Western blot analysis of Tcl1 and β-actin in wild-type (WT), Tcl1^−/− (KO) #2 and #4, Tcl1^−/−(CAG-Tcl1) #1 and #3, and Tcl1^−/−(CAG-EGFP) #5 ES cells. Tcl1^−/−(CAG-Tcl1) #1 and #3, and Tcl1^−/−(CAG-EGFP) #5 were derived from Tcl1^−/− (KO) #4.

Figure 2. Effects of Tcl1 deficiency and overexpression on ES-cell differentiation.

The expression of representative stem and differentiation markers was examined by real time PCR in ES cells grown in LIF(+) culture (left half of each panel) and with EB formation (right half of each panel). For EB formation, trypsinized ES cells were seeded into a bacterial grade dish, and cultured for 12 days. Values are expressed as mean ± SEM of three technical replicates. ∗P<0.05 and ∗∗P<0.01 by Student’s t-test. Tcl1^−/− (KO) vs. wild-type (WT) or Tcl1^−/−(CAG-Tcl1) cells.

Figure 3. Effect of Tcl1 expression on ES cell growth.

(A) Cell proliferation assay. Cell proliferation between 24 and 48 hours of culture was analyzed by MTT assay. The proliferation rate of Tcl1^−/− (KO) was significantly lower than that of wild-type (WT) or Tcl1^−/−(CAG-Tcl1) cells. Values are expressed as means ± SD (left panel, n = 12 each; right panel, n = 8 each). Difference from Tcl1^−/− (KO) ES cells: ∗∗P<0.01. (B) The percentage of cleaved caspase 3-positive cells was calculated for 9–13 areas selected at random (left panel, n = 9–13, 6312–14211 nuclei per cell line; right panel, n = 10, 3329–4538 nuclei per cell line). The percentage of cleaved caspase 3-positive apoptotic cells in Tcl1^−/− (KO) cells was significantly higher than that in wild-type (WT) or Tcl1^−/−(CAG-Tcl1) cells. Values are expressed as means ± SD. Difference from Tcl1^−/− (KO) ES cells: ∗P<0.05, ∗∗P<0.01. (C) Teratoma formation of wild-type (WT) (n = 9), Tcl1^−/− (KO) #4 (n = 6), Tcl1^−/−(CAG-EGFP) #5 (n = 3), and Tcl1^−/−(CAG-Tcl1) #1 (n = 3) and #3 (n = 5) ES cells. The weight of each teratoma is shown in grams. Horizontal bars indicate the mean of each sample. ∗P<0.03. (D) Histological analysis of the teratomas derived from Tcl1^−/− (KO) #4 and #5 and Tcl1^−/−(CAG-Tcl1) #1 and #3 ES cells. No histological differences were recognizable between Tcl1^−/− (KO) and Tcl1^−/−(CAG-Tcl1) ES cells.

Figure 4. Analysis of Akt and Wnt/β-catenin signaling in Tcl1-deficient and -overexpressing ES cells.

(A) Western blot analysis of GSK, Akt, and β-catenin in wild-type (WT), Tcl1^−/− (KO) #2 and #4, Tcl1^−/−(CAG-Tcl1) #1 and #3, and Tcl1^−/−(CAG-EGFP) #5 ES cells. (B) Western blot analysis of active β-catenin, Oct3/4, and HSP90 in the cytoplasmic (C) and nuclear (N) fractions of wild-type (WT), Tcl1^−/− (KO) #2 and #4, and Tcl1^−/−(CAG-Tcl1) #10 and #1 ES cells. Tcl1^−/−(CAG-Tcl1) #10 and #1 were derived from Tcl1^−/− (KO) #2 and #4, respectively. Proper fractionation was confirmed by western blotting of Oct3/4 and HSP90, which localize to the nucleus and cytoplasm, respectively. Be8cause active β-catenin levels in the nuclear fractions were much lower than those in the cytoplasmic fractions, active β-catenin in the nuclear fractions was detected by approximately two-fold longer exposure compared with that in the cytoplasmic fractions. (C) TOPflash assay. P values of wild-type ES cells (WT) compared with Tcl1^−/−(CAG-Tcl1) #1 and #3 ES cells were less than 0.01. P values of Tcl1^−/− (KO) #4 and #5 ES cells compared with Tcl1^−/−(CAG-Tcl1) #1 and #3 ES cells were less than 0.02.

Figure 5. Differentially expressed genes in Tcl1 ^−/− ES cells compared with Tcl1 ^−/−(CAG-Tcl1) and wild-type ES cells.

(A) Real time PCR analysis of 2 genes that were shown to be upregulated by Tcl1 expression in DNA microarray analysis. (B) Real time PCR analysis of 16 genes that were shown to be downregulated by Tcl1 expression in DNA microarray analysis. Expression of these genes were compared among wild-type (WT), Tcl1^−/− (KO) #5, and Tcl1^−/−(CAG-Tcl1) #11 and #14 ES cells. Tcl1^−/−(CAG-Tcl1) #11 and #14 were derived from Tcl1^−/− (KO) #5. Values are expressed as means ± SEM of three technical replicates. Difference from Tcl1^−/− (KO) ES cells: ∗P<0.05, ∗∗P<0.01.