Dynamic expression and regulatory mechanism of TGF-β signaling in chicken embryonic stem cells differentiating into spermatogonial stem cells

Abstract

The present study investigated the dynamic expression and regulatory mechanism of transforming growth factor β (TGF-β) signaling involved in embryonic stem cells (ESCs) differentiation into male germ cells. Candidate genes involved in TGF-β signaling pathway were screened from RNA-sequencing (RNA-seq), which were further validated by quantitative real-time PCR (qRT-PCR). Bone morphogenetic protein 4 (BMP4) was used to induce differentiation of ESCs in vitro Inhibition of TGF-β signaling pathway was reflected by Western blot of SMAD2 and SMAD5 expression. Differentiating efficiency of germ cells was evaluated by immunofluorescence and fluorescence-activated cell sorting (FACS). Germ cell marker genes were assessed by qRT-PCR in the differentiation process, with activation or inhibition of TGF-β signaling pathway. In the process of in vitro induction, SMAD2 and SMAD5 were found to significantly up-regulated in BMP4 group versus the control and inhibition groups after 4 and 14 days. Expression of CKIT, CVH, DAZL, STRA8, and INTEGRIN α6 were significantly increased in the BMP4 group compared with the control group, while down-regulated in the inhibition groups. The proportion of germ cell-like cells was decreased from 17.9% to 2.2% after 4 days induction, and further decreased from 14.1% to 2.1% after 14 days induction. Correspondingly, expression of marker genes in germ cells was significantly lower. In vivo inhibition of TGF-β signaling pathway reduced germ cells formation from 5.5% to 1.6%, and down-regulated the expression of CKIT, CVH, DAZL, STRA8, and INTEGRIN α6 In conclusion, our study reveals the mechanism regulating spermatogonial stem cells (SSCs) and lays the basis for further understanding of the regulatory network.

Keywords: Embryonic stem cells; Primordial germ cells; RNA-seq; Spermatogonial stem cells; TGF-β signaling.

Figure 1. Results are shown for cell isolation, culture, sorting, and identification

(A) The sex of chicken ESCs and PGCs was determined. Samples 1, 3, 5, 7, 8, and 10 were females of genotype ZW with two DNA fragments at 600 and 450 bp. Samples 2, 4, 6, and 9 were males of genotype ZZ with only one fragment at 600 bp (M: 100 bp marker). (B) ESC, PGC, and SSC clones are shown. ESC clones resemble a bird’s nest with clear edges; PGC clones are larger than ESCs with more obvious nuclei and clearly visible areas around the cells; SSC clones are large and clump into a mass that resembles a bunch of grapes, scale bar: 68.8 μm. (C) Gene expression in ESCs, PGCs, and SSCs was detected by qRT-PCR. (D) Cell sorting results are shown for chicken ESCs, PGCs, and SSCs.

Figure 2. Venny analysis of TGF-β signaling pathway genes involved in the process of ESCs differentiation into SSCs

(A) Cluster analysis of the differential expression of genes (DEGs) in ESCs, PGCs, and SSCs and the DEGs in the key signaling pathway (P<0.05). (B) Venny analysis of signaling pathways involved in the process of ESCs differentiation into SSCs. (C) Twenty-six DEGs in the TGF-β signaling pathway are shown, red represents down-regulated genes, and green represents up-regulated genes. (D) Venny analysis for DEG in ESC, PGC, and SSC.

Figure 3. Dynamic expression patterns of the molecules involved in TGF-β signaling pathway during ESCs differentiation into SSCs

Major molecules in the TGF-β signaling pathway were classified based on their functions. The dynamic expression of TGF-β signaling ligands (A), receptors (B), regulators (C), and downstream molecules (D) involved in ESCs differentiation into SSCs was derived from the RNA-seq data. Black represents ESCs, Gray represents PGCs, and White represents SSCs.

Figure 4. qRT-PCR validation of key TGF-β signaling genes expressed in ESCs, PGCs, and SSCs

Fourteen genes with major expression differences were selected for qRT-PCR validation. (A) Ratio of relative expression value of the gene in PGCs versus SSCs. (B) The ratio of relative expression values of the genes in PGCs versus SSCs.

Figure 5. Inhibition efficiency of TGF-β signaling in vitro

Western blot and qRT-PCR were performed to evaluate the inhibition efficiency of TGF-β signaling in vitro. (A) Smad2 and Smad5 expression in control and inhibition groups on differentiation days 4 and 14 (CON: control group; LY-100༚100 nM TGF-β subgroup inhibitor LY2109761; LDN-100: 100 nM BMP4 subgroup inhibitor LDN193189). (B) Quantitative evaluation of Smad5 expression in different groups on differentiation days 4 and 14. Statistical difference was assessed by comparing the BMP4 group with the control group, and the sample treated with H₂O was regard as Blank Control (*P<0.05, **P<0.01).

Figure 6. Morphology and cell marker identification of the BMP4-induced male germ cells differentiation

(A) Morphological changes in the cells under BMP4 induction with or without TGF-β signaling inhibitors (LY: TGF-β subgroup inhibitor LY2109761; LDN: BMP4 subgroup inhibitor LDN193189; DOUBLE: two inhibitors were used). Toluidine Blue was used to stain the mast cells in the DOUBLE inhibition group, scale bar: 68.8 μm. (B) Immunocytochemical staining of the germ cell markers was used to identify the germ cells. Integrin α6, integrin β1, DAZL, and C-kit were used as the germ cells markers.

Figure 7. Evaluation of the germ cell marker genes expression in vitro by qRT-PCR and FACS

(A) Expression of germ cell marker genes, including C-Kit, CVH, DAZL, STRA8, integrin α6, and integrin β1 in different treatment groups was evaluated by qRT-PCR. Expression relative to β-actin is presented. (B) The PGC marker C-kit and the SSC marker integrin α6 were used to identify the male germ cells with FACS. A positive rate was shown in each group. (C) Quantification of the C-kit positive rate in each group based on FACS analysis. (D) Quantification of the integrin α6 positive rate in each group based on FACS analysis. Statistical significance was assessed by comparing the BMP4 group with the control group, and the other groups with the BMP4 group (*P<0.05, **P<0.01). The expression of genes in BMP4 group was regarded as positive Control, and the sample treated with H₂O were regarded as Blank Control.

Figure 8. Haploid generation on day 14 during in vitro induction

The result of haploid generation efficiency detection showed that the percentage of haploid in BMP4 group from 21.7% (BMP group) down to 14.9% (LY-100 group), 10.5 % (LDN-100 group), and 8.17% (DOUBLE group), when LY-100 and LDN-100 were added into the induced system. Quantitative evaluation of haploid efficiency in different groups on differentiation day 14 (capital letters represent high significant differences).

Figure 9. Inhibition efficiency of TGF-β signaling in vivo. qRT-PCR and Western blot were performed to evaluate the inhibition efficiency of TGF-β signaling in vivo

(A) The expression of Smad2 and Smad5 in different groups on embryo development days 5.5 and 18 by Western blot. (B) Quantitative evaluation of the expression of Smad2 and Smad5 in different groups on embryo development days 5.5 and 18. Statistical significance was assessed by comparing each group with the control group (*P<0.05, **P<0.01). The expression of Smad2 and Smad5 in Normal incubation process were regarded as Control, and the sample treated with H₂O was regarded as Blank Control.

Figure 10. Evaluation of the expression of germ cells marker genes in vivo by qRT-PCR and FACS

(A) Expression of germ cell marker genes, including C-Kit, CVH, DAZL, STRA8, integrin α6, and integrin β1, in different treatment groups was evaluated by qRT-PCR. Their expression relative to β-actin is presented. The expression of genes in Normal incubation process was regarded as Control, and the sample treated with H₂O was regard as Blank Control (B). The PGC marker C-kit and the SSC marker integrin α6 were used to identify the male germ cells with FACS. The positive rate was shown in each group. (C) Quantification of the C-kit positive rate in each group based on FACS analysis. (D) Quantification of the integrin α6 positive rate in each group based on FACS analysis. Statistical significance was assessed by comparing each group with the control group (*P<0.05, **P<0.01).