Long Noncoding RNA LncPGCR Mediated by TCF7L2 Regulates Primordial Germ Cell Formation in Chickens.

Although lncRNAs have been identified as playing critical roles in the development of germ cells, their potential involvement in the development of PGCs in chickens remains poorly understood. Differentially expressed lncRNAs (DELs) from previous RNA-seq of embryonic stem cells (ESCs), PGCs, and spermatogonial stem cells (SSCs) were analyzed by K-means clustering, from which a key candidate, lncRNA (lncRNA PGC regulator, LncPGCR) was obtained. We confirmed that LncPGCR plays a positive role in the development of PGCs by increasing the expression of the PGC marker gene (Cvh and C-kit), while downregulating the pluripotency-associated gene (Nanog) in vitro and in vivo. The activation and expression of LncPGCR are regulated by histone acetylation, and transcription factor TCF7L2. Mechanistically, a rescue assay was performed to further confirm that LncPGCR contributed to the development of PGCs by regulating the gga-miR-6577-5p/Btrc signaling pathway. Adsorption of gga-miR-6577-5p activated the WNT signaling cascade by relieving the gga-miR-6577-5p-dependent inhibition of Btrc expression. Taken together, our study discovered the growth-expedited role of LncPGCR in PGCs development, showing the potential LncPGCR/miR-6577-5p/Btrc pathway. The results and findings provide a novel insight into the development of PGCs.

Nanos2 promotes differentiation of male germ cells basing on the negative regulation of Foxd3 and the treatment of 5-Azadc and TSA.

The transcription factor positioning in promoter regions relate to gene regulation, and the level of DNA methylation and histone acetylation also impact the promoter activity. In this study, we tested and verified the core promoter region and key transcription factor of Nanos2 which is a male-critical gene in the differentiation of embryonic stem cells to male germ cells, meanwhile, epigenetic effects by mean of 5-Aza-2'-deoxycytidine (5-Azadc) and Trichostin A (TSA) on the activity of Nanos2 promoter were detected. The results reveal that key transcription factor Foxd3 is a negative regulator of Nanos2, which suggests that loss-of-function of Foxd3 causes strong expression of Nanos2 responsive to large amounts of primordial germ cells and spermatogonial stem cells,whereas its overexpression causes the opposite effect. Furthermore, both 5-Azadc and TSA can provoke responses of Nanos2, but the combination effect of the two is better.

Acetyl-coenzyme A acyltransferase 2 promote the differentiation of sheep precursor adipocytes into adipocytes.

The acetyl CoA acyltransferase 2 (ACAA2) is a key enzyme of the fatty acid oxidation pathway, catalyzing the last step of the mitochondrial beta oxidation, thus playing an important role in the fatty acid metabolism. The purpose of this study was to investigate the effect of knocking out ACAA2 on the expression of genes lipoprteinlipase (LPL), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase, fat mass and obesity-associated gene, adipocyte fatty acid-binding protein (AP2) in precursor adipocytes and their differentiation into adipocytes. The knockout vector was constructed using CRISPR-Cas RNA-guided nuclease technology with an efficiency of 23.80%, and the vector was transfected into precursor adipocyte cells, while an overexpression vector of the ACAA2 gene was also transfected in another group of preadipocytes. Quantitative polymerase chain reaction showed that the expression of the PPAR-γ, LPL, and AP2 was significantly lower in the knockout compared with the overexpression group, while there was no difference in cell growth. After induction of adipocyte precursor cells into adipocytes using dexamethasone, insulin, and IBMX, oil red staining showed a significantly different number of lipid droplets in the knockout group. These results provide a preliminary indication for a possible involvement of the ACAA2 gene in adipocyte differentiation in vitro.

RXRG associated in PPAR signal regulated the differentiation of primordial germ cell.

Primitive germ cells (PGCs) are the "ancestor" of sex germ cells. However, their regulation mechanism is not clear at present. The aim of this study is to investigate the effect of PPAR signaling pathway on the differentiation of chicken embryonic stem cells (ESCs) into PGCs, providing theoretical support for the further study of the mechanism of regulation of chicken PGCs. Based on the results of RNA-Seq analysis, the key signal pathway PPAR and the key signal molecule RXRG in the pathway were successfully screened. Also, we made the PPAR signal transduction pathway be activated in vivo and in vitro. The results showed that the expression of FABP3 and SCD-1 was up-regulated by high expression of RXRG, which significantly up-regulated the expression of Cvh, C-kit, and the ratio of Cvh + C-kit+ cells was higher than that of control group. The formation of PGCs was blocked by interfering with PPAR signaling pathway. We conclude that RXRG positively regulates the PPAR signaling pathway and promotes differentiation of chicken ESCs into PGCs. Thus, gene RXRG can be used to study the regulation of PGCs differentiation in chickens.

Regulation of fibroblast growth factor 8 (FGF8) in chicken embryonic stem cells differentiation into spermatogonial stem cells.

Fibroblast growth factors (FGFs) are essential in regulating the formation of spermatogonial stem cells (SSCs). Here, we explored the effect of FGF8 on chicken SSCs formation by knockdown or overexpression of FGF8 in chicken embryonic stem cells (ESCs) both in vitro and in vivo. Our results showed that knockdown of FGF8 could facilitate the differentiation of ESCs into SSCs, overexpression of FGF8 could promote PGCs self-renewal, inhibit SSCs formation. This study further revealed the positive correlation between the expression level of FGF8 and MAPK/ERK signal. In the absence of FGF8, the expression of downstream genes such as FGFR2, GRB2, RAS, BRAF, RAF1, and MEK2 was not maintained, while overexpressing FGF8 enhances them. Thus, our study demonstrated that FGF8 can regulate germ cell fate by modulating the dynamic equilibrium between differentiation and self-renewal, which provides a new idea for the study of germ cell regulatory network.

MAPK8 regulates chicken male germ cell differentiation through JNK signaling pathway.

The study aims to analyze the key signaling pathways in regulating the process of embryonic stem cells (ESCs) differentiation into spermatogonial stem cells (SSCs). We explored the specific regulating mechanisms of C-Jun amino-terminal kinase (JNK) signaling in this process. Interference/overexpression of MAPK8 allows the JNK signaling pathway to be blocked/activated. In Retinoic acid (RA) induced in vitro differentiation assays, the expression of germ cell marker genes, cvh, c-kit, integrin α6 and integrin ß1, was observed to upregulate while activating JNK signaling significantly. Fluorescence Activated Cell Sorting (FACs) analysis showed that the proportion of cvh+ and integrin α6+ cells in the overexpression group was significantly higher than which in the RA + shRNA-MAPK8 group. In in vivo situations, shRNA-MAPK8 could stably express in chicken embryos and significantly down-regulate expression of MAPK8 and downstream genes in JNK signaling pathway. With PAS stain, we found that PGCs (primordial germ cells) was significantly decreased after inhibiting MAPK8. With real-time quantitative PCR (qRT-PCR) and Western Blot, we identified that reproductive related genes expression was significantly suppressed after inhibiting MAPK8 in vivo. We preliminarily concluded that knockdown/ overexpression of MAPK8 could affect differentiation of ESC by inhibiting/activating JNK signal.

Hedgehog-Gli1 signaling regelates differentiation of chicken (Gallus gallus) embryonic stem cells to male germ cells.

Gli1 is an important signaling molecular in Hedgehog signaling pathway. In our study, we explored the adjustment effect of Hedgehog-Gli1 signaling pathway on chicken male germ cells differentiation based on the transcriptome-wide analyses of chicken ESCs, primordial germ cells (PGCs) and spermatogonia stem cells (SSCs) that were associated with male germ cell differentiation. We screened out Hedgehog signaling pathway and identified 8 candidated differentially expressed genes (DEGs), Wnt3a, Wnt16, Wnt8a, HHIPL1, Gli1, BMP6, BMP7 and TTLL4. These DEGs expression change trend among blastoderm, genital ridge and testes, from which ESCs, PGCs and SSCs were isolated was the same as RNA-Seq data with quantitative RT-PCR evaluation. Based on retinoic acid (RA) induction of ESCs to SSCs in vitro, Gli1 overexpression has the ability to induce ESCs differentiation and SSCs-like cells formation and high expression of related reproductive genes, like Cvh, C-kit, Blamp1, Prmd14, Stra8, Dazl, integrin α6 and integrin ß1 and so on in vitro. While RNAi knockdown of Gli1 can protect ESCs from differentiating into SSCs and correspondingly reduce the expression of the associated reproductive gene in vivo and vitro. Immunochemistry results showed that Gli1 overexpression could increase the expression of PGCs markers Cvh and C-kit and SSCs markers integrin α6 and integrin ß1 in vivo, while Gli1 knockdown can have the opposite effect in vivo and in vitro. PAS stain and flow cytometry (FCM) evaluation results indicated the quantity of germ cells is decrease or increase with Gli1 knockdown or overexpression. Collectively, these results uncovered a novel function of Gli1 and demonstrated Hedgehog-Gli1 signaling pathway involved in chicken male germ cell differentiation, where it acts as a facilitator.

CYP1A1 based on metabolism of xenobiotics by cytochrome P450 regulates chicken male germ cell differentiation.

This study aimed to explore the regulatory mechanism of metabolism of xenobiotics by cytochrome P450 during the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) and consummate the induction differentiation system of chicken embryonic stem cells (cESCs) into SSCs in vitro. We performed RNA-Seq in highly purified male ESCs, male primordial germ cells (PGCs), and SSCs that are associated with the male germ cell differentiation. Thereinto, the metabolism of xenobiotics by cytochrome P450 was selected and analyzed with Venny among male ESC vs male PGC, male PGC vs SSC, and male ESC vs SSC groups and several candidates differentially expressed genes (DEGs) were excavated. Finally, quantitative real-time PCR (qRT-PCR) detected related DEGs under the condition of retinoic acid (RA) induction in vitro, and the expressions were compared with RNA-Seq. By knocking down CYP1A1, we detected the effect of CYP1A1-mediated metabolism of xenobiotics by cytochrome P450 on male germ cell differentiation by qRT-PCR and immunocytochemistry. Results showed that 17,742 DEGs were found during differentiation of ESCs into SSCs and enriched in 72 differently significant pathways. Thereinto, the metabolism of xenobiotics by cytochrome P450 was involved in the whole differentiation process of ESCs into SSCs and several candidate DEGs: CYP1A1, CYP3A4, CYP2D6, ALDH3B1, and ALDH1A3 were expressed with the same trend with RNA-Seq. Knockdown of CYP1A1 caused male germ cell differentiation under restrictions. Our findings showed that the metabolism of xenobiotics by cytochrome P450 was significantly different during the process of male germ cell differentiation and was persistently activated when we induced cESCs to differentiate into SSCs with RA in vitro, which illustrated that the metabolism of xenobiotics by cytochrome P450 played a crucial role in the differentiation process of ESCs 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.