Qisheng Zuo

Crucial Genes and Pathways in Chicken Germ Stem Cell Differentiation

Background: Germ cells are critical for any species that multiplies through sexual reproduction. Results: We found 173 candidate key genes and 18 key signaling pathways that are differentially activated. Conclusion: Our results showed the crucial genes and pathways involved in the regulation of chicken male germ cell differentiation. Significance: This study narrows the range of functional genes and pathways during ESC differentiation. Male germ cell differentiation is a subtle and complex regulatory process. Currently, its regulatory mechanism is still not fully understood. In our experiment, we performed the first comprehensive genome and transcriptome-wide analyses of the crucial genes and signaling pathways in three kinds of crucial cells (embryonic stem cells, primordial germ cell, and spermatogonial stem cells) that are associated with the male germ cell differentiation. We identified thousands of differentially expressed genes in this process, and from these we chose 173 candidate genes, of which 98 genes were involved in cell differentiation, 19 were involved in the metabolic process, and 56 were involved in the differentiation and metabolic processes, like GAL9, AMH, PLK1, and PSMD7 and so on. In addition, we found that 18 key signaling pathways were involved mainly in cell proliferation, differentiation, and signal transduction processes like TGF-β, Notch, and Jak-STAT. Further exploration found that the candidate gene expression patterns were the same between in vitro induction experiments and transcriptome results. Our results yield clues to the mechanistic basis of male germ cell differentiation and provide an important reference for further studies.

CRISPR/Cas9 mediated chicken Stra8 gene knockout and inhibition of male germ cell differentiation

An efficient genome editing approach had been established to construct the stable transgenic cell lines in the domestic chicken (Gallus gallus domesticus) at present. Our objectives were to investigate gene function in the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells(SSCs). Three guides RNA (gRNAs) were designed to knockout the Stra8 gene, and knockout efficiency was evaluated in domestic chicken cells using cleavage activity of in vitro transcription of gRNA, Luciferase-SSA assay, T7 endonuclease I assay(T7E1) and TA clone sequence. In addition, the Cas9/gRNA plasmid was transfected into ESCs to confirm the function of Stra8. SSA assay results showed that luciferase activity of the vector expressing gRNA-1 and gRNA- 2 was higher than that of gRNA-3. TA clone sequencing showed that the knockdown efficiency was 25% (10/40) in DF-1 cells, the knockdown efficiency was 23% (9/40) in chicken ESCs. T7E1 assay indicated that there were cleavage activity for three individuals, and the knockdown efficiency was 12% (3/25). Cell morphology, qRT-PCR, immunostaining and FCS indicated that Cas9/gRNA not only resulted in the knockout of Stra8 gene, but also suggested that the generation of SSCs was blocked by the Stra8 gene knockdown in vitro. Taken together, our results indicate that the CRISPR/Cas9 system could mediate stable Stra8 gene knockdown in domestic chicken’s cells and inhibit ECSs differentiation into SSCs.

Site-Directed Genome Knockout in Chicken Cell Line and Embryos can Use CRISPR/Cas Gene Editing Technology

The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens.

Regulatory mechanism of protein metabolic pathway during the differentiation process of chicken male germ cell

We explored the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro. We sequenced the transcriptome of embryonic stem cells, primordial germ cells, and spermatogonial stem cells with RNA sequencing (RNA-Seq), bioinformatics analysis methods, and detection of the key genes by quantitative reverse transcription PCR (qRT-PCR). Finally, we found 16 amino acid metabolic pathways enriched in the biological metabolism during the differentiation process of embryonic stem cells to primordial germ cells and 15 amino acid metabolic pathways enriched in the differentiation stage of primordial germ cells to spermatogonial stem cells. We found three pathways, arginine-proline metabolic pathway, tyrosine metabolic pathway, and tryptophan metabolic pathway, significantly enriched in the whole differentiation process of embryonic stem cells to spermatogonial stem cells. Moreover, for these three pathways, we screened key genes such as NOS2, ADC, FAH, and IDO. qRT-PCR results showed that the expression trend of these genes were the same to RNA-Seq. Our findings showed that the three pathways and these key genes play an important role in the differentiation process of embryonic stem cells to male germ cells. These results provide basic information for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro.

Regulation of Hedgehog Signaling in Chicken Embryonic Stem Cells Differentiation into Male Germ Cells (Gallus gallus)

The study aims to analyze the key signaling pathways in regulating the process of embryonic stem cells (ESCs) differentiation into spermatogonial stem cells (SSCs). Based on RNA Sequencing result, we further explored the specific regulating mechanisms of Hedgehog (HH) signaling in this process. HH signaling was found to be a crucial signaling pathway participating in the differentiation process of ESCs to SSCs. In Retinoic acid (RA) induced in vitro differentiation assay, the expression of two germ cell marker genes, integrin α6, and integrin β1, was observed to significantly increase, while it decreased dramatically when IHH was knocked down. Fluorescence activated cell sorting analysis showed that the proportion of integrin α6+ and integrin β1+ cells in the RA group was significantly higher than that in the RA + siRNA‐ Indian Hedgehog (IHH) group. In in vivo situations, siRNA‐IHH could stably express in fertilized chicken embryos and significantly down‐regulate the IHH expression. With real‐time quantitative PCR and western blot, we identified that integrin α6 and integrin β1 expression was significantly suppressed along with IHH inhibition in vivo. We concluded that Hedgehog signaling pathway positively regulates the differentiation of ESCs to male germ cells through signal transduction by IHH. J. Cell. Biochem. 118: 1379–1386, 2017.

Study on the role of JAK/STAT signaling pathway during chicken spermatogonial stem cells generation based on RNA-Seq

Abstract Spermatogonial stem cells (SSCs) form the foundation for spermatogenesis and sustain male fertility. To explore the regulatory mechanisms of chicken SSCs generation, we obtained highly purified chicken embryonic stem cells (ESCs), primordial germ cells (PGCs) and SSCs by fluorescence-activated cell sorting (FACS). High-throughput analysis methods (RNA-Seq) were used to sequence the transcriptome level of these cells. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment were used to analyze RNA-Seq results. BMP4 was used to induce chicken ESCs differentiation to SSCs-like cells in vitro. The quantitative real-time (qRT)-PCR was used to detect the expression changes of the key genes. The results showed that 22 relevant critical pathways were found by RNA-Seq, one of them was the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. Total of 103 related genes were detected in this pathway. Protein-protein interactions analysis found that 87 proteins were significantly related to 19 key proteins in this pathway. These 87 proteins were enriched in 21 biological processes and 18 signaling pathways. Moreover, during the differentiation of chicken ESCs to SSCs-like cells induced by BMP4 in vitro , JAK2 and STAT3 were activated. The qRT-PCR results showed that the expression trends of JAK2 and STAT3 were basically the same as in vivo. We concluded that JAK/STAT signaling pathway plays an important role in the process of chicken SSCs generation both in vivo and in vitro ; it may achieve its function through multiple biological processes and other related pathways.

CRISPR/Cas9-Mediated Deletion of C1EIS Inhibits Chicken Embryonic Stem Cell Differentiation Into Male Germ Cells (Gallus gallus)

We previously found that C1EIS is preferentially expressed in Chicken spermatogonial stem cells (SSCs) by RNA sequencing (RNA‐seq), so our current study focused on C1EISs role in Chicken embryonic stem cells (ESCs) differentiation into male germ cells. We constructed a CRISPR/Cas9 vector targeting C1EIS. T7 endonuclease I (T7EI) digestion method and sequencing of TA cloning were used to detect the knock‐out efficiency of the Single guide RNA (sgRNA) after the cas9/gRNA vector transfected into D fibroblasts 1(DF‐1), ESCs, and Chicken embryos. The results showed that CRISPR/Cas9 gene knockout efficiency is about 40%. Differentiation of the targeted ESCs into SSCs was inhibited at the embryoid body stage due to C1EIS deficiency. Immunofluorescent staining revealed that the mutagenized ESCs (RA (Retinoic Acid) with C1EIS Knock out) expressed lower levels of integrin α6 and integrin β1 compared to wild type cells. Quantitative real‐time PCR (QRT‐PCR) revealed Oct4 and Sox2 expression significantly increased, contrarily integrin β1 and Stra8 expression significantly decreased than RA induced group and RA with C1EIS Overexpression. During retinoic acid‐induced differentiation, knockout of C1EIS in ESCs inhibited formation of SSC‐like cells, suggesting C1EIS plays a vital role in promoting differentiation of avian ESCs to SSCs by regulating expression of multiple pluripotency‐related genes. J. Cell. Biochem. 118: 2380–2386, 2017.

Study on the regulatory mechanism of the lipid metabolism pathways during chicken male germ cell differentiation based on RNA-seq.

Here, we explore the regulatory mechanism of lipid metabolic signaling pathways and related genes during differentiation of male germ cells in chickens, with the hope that better understanding of these pathways may improve in vitro induction. Fluorescence-activated cell sorting was used to obtain highly purified cultures of embryonic stem cells (ESCs), primitive germ cells (PGCs), and spermatogonial stem cells (SSCs). The total RNA was then extracted from each type of cell. High-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. Gene Ontology (GO) analysis and the KEGG database were used to identify lipid metabolism pathways and related genes. Retinoic acid (RA), the end-product of the retinol metabolism pathway, induced in vitro differentiation of ESC into male germ cells. Quantitative real-time PCR (qRT-PCR) was used to detect changes in the expression of the genes involved in the retinol metabolic pathways. From the results of RNA-seq and the database analyses, we concluded that there are 328 genes in 27 lipid metabolic pathways continuously involved in lipid metabolism during the differentiation of ESC into SSC in vivo, including retinol metabolism. Alcohol dehydrogenase 5 (ADH5) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) are involved in RA synthesis in the cell. ADH5 was specifically expressed in PGC in our experiments and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) persistently increased throughout development. CYP26b1, a member of the cytochrome P450 superfamily, is involved in the degradation of RA. Expression of CYP26b1, in contrast, decreased throughout development. Exogenous RA in the culture medium induced differentiation of ESC to SSC-like cells. The expression patterns of ADH5, ALDH1A1, and CYP26b1 were consistent with RNA-seq results. We conclude that the retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation.

miR-31 Regulates Spermatogonial Stem Cells Meiosis via Targeting Stra8†

Stra8 (stimulated by retinoic acid gene 8) is a specific gene that is expressed in mammalian germ cells during transition from mitosis to meiosis and plays a key role in the initiation of meiosis in mammals and birds. So, the evaluation of the Stra8 pathway in cSSCs may provide a deeper insight into mammalian spermatogenesis. miRNA was also an important regulating factor for meiosis of SSCs. However, there is currently no data indicating that miRNA regulate the meiosis of SSCs via Stra8. Here, we predicted the prospective miRNA targeting to Stra8 using the online Bioinformatics database‐Targetscan, and performed an analysis of the dual‐luciferase recombinant vector, pGL3‐CMV‐LUC‐MCS‐Stra8‐3′UTR. miR‐31 mimics (miR‐31m), miR‐31 inhibitors (miR‐31i), Control (NC, scrambled oligonucleotides transfection) were transfected into cSSCs; Stra8 and miRNA were analyzed by RT‐qPCR, immunofluorescence, and Western blot. The detection of haploid was conducted by flow cytometry. The results showed that miR‐31 regulates meiosis of cSSCs via targeting Stra8 in vitro and in vivo. Our study identifies a new regulatory pathway that miR‐31 targets Stra8 and inhibits spermatogenesis. J. Cell. Biochem. 118: 4844–4853, 2017.

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

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.